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In Vitro Diffuse Large B-Cell Lymphoma Cell Line Models as Tools to Investigate Novel Immunotherapeutic Strategies

Matylda kubacz.

1 Department of Immunology, Medical University of Warsaw, 02-097 Warsaw, Poland

Aleksandra Kusowska

2 Doctoral School, Medical University of Warsaw, 02-091 Warsaw, Poland

Magdalena Winiarska

3 Laboratory of Immunology, Mossakowski Medical Research Institute, Polish Academy of Sciences, 02-106 Warsaw, Poland

Małgorzata Bobrowicz

Simple summary.

Despite a range of emerging immunotherapeutic strategies, the aggressive nature of DLBCL poses an ongoing clinical challenge. Therefore, there is a need to pursue more effective treatment modalities based on monoclonal antibodies, antibody-drug conjugates and CAR-T cell therapy. Up to now, the above-mentioned immunotherapeutic options have been extensively studied with the aid of established cell line models, which have significantly facilitated proceeding from preclinical to clinical investigations and led to improvement of DLBCL treatment. However, there are still several important challenges associated with faithful recapitulation of the aggressive nature of DLBCL. Therefore, the current review discusses means in which cell line models fulfill an essential tool leading to greater understanding DLBCL biology and development of novel immunotherapeutic strategies.

Despite the high incidence of diffuse large B-cell lymphoma (DLBCL), its management constitutes an ongoing challenge. The most common DLBCL variants include activated B-cell (ABC) and germinal center B-cell-like (GCB) subtypes including DLBCL with MYC and BCL2/BCL6 rearrangements which vary among each other with sensitivity to standard rituximab (RTX)-based chemoimmunotherapy regimens and lead to distinct clinical outcomes. However, as first line therapies lead to resistance/relapse (r/r) in about half of treated patients, there is an unmet clinical need to identify novel therapeutic strategies tailored for these patients. In particular, immunotherapy constitutes an attractive option largely explored in preclinical and clinical studies. Patient-derived cell lines that model primary tumor are indispensable tools that facilitate preclinical research. The current review provides an overview of available DLBCL cell line models and their utility in designing novel immunotherapeutic strategies.

1. Introduction

The most common non-Hodgkin lymphoma (NHL) subtype, diffuse large B-cell lymphoma (DLBCL), accounts for up to 40% of all lymphoid tumors [ 1 ]. Its management remains an ongoing challenge with up to 50% of patients relapsing or developing resistance (r/r patients) [ 2 ]. The treatment is most often based on a combination of rituximab (RTX) with cyclophosphamide, vincristine, doxorubicin and prednisone (R-CHOP) [ 3 ]. To this date, several factors behind R-CHOP resistance have been identified, with high genetic and clinical heterogeneity as one of the most significant [ 4 ].

In 2000, Alizadeh et al. identified two main subtypes: germinal center B-cell (GCB)-like and activated B-cell (ABC)-like subtype, with the latter being characterized with significantly worse prognosis [ 5 ]. Discovery of several unique DLBCL subtypes based on cell of origin (COO) as well as molecular features has complemented International Prognostic Index (IPI) in identifying high-risk disease and predicting therapy efficacy [ 3 ]. However, there is no universally accepted way in integrating various prognostic factors in DLBCL [ 6 ].

A subgroup of DLBCL with MYC, BCL2 and BCL6 rearrangement can be identified which is linked to worse prognosis [ 7 , 8 ]. Development of DLBCL may additionally occur as a life-threatening complication, named as Richter transformation (Richter syndrome, RT) [ 9 ]. RT is development of an aggressive lymphoma on the background of chronic lymphocytic leukemia (CLL) and occurs in approximately 2–10% CLL’s patients [ 10 , 11 ]. High DLBCL heterogeneity (reviewed by Yanguas-Casás et al. [ 12 ]) is reflected at the molecular level by DLBCL model cell lines [ 12 ]. The cell lines are relatively easy to harvest so they can be used at almost any moment to analyze their features in flow cytometry and colorimetric or enzymatic assays [ 13 ]. Those advantages make cell line models a useful tool for investigation of novel therapies, which have recently heralded a new era in DLBCL management [ 14 ].

2. Established Cell Lines 2-Dimenstional (2D) Models as Tools to Study DLBCL Biology

2.1. cell line establishement from primary cell culture.

Since establishment of the first leukemia-lymphoma (LL) cell lines in 1964 [ 15 ] a vast number of cellular models have been characterized [ 16 ]. Factors behind frequent failure in cell line establishment are varied and not fully understood; however, it has been reported that cells derived from r/r patients with unfavorable prognostic features possess enhanced growth potential in vitro, indicating a higher success rate for cell line establishment [ 17 ]. Isolation and propagation of the cells are illustrated in Figure 1 and thoroughly described in [ 18 , 19 ].

Establishment of patient-derived cell line models. Created with BioRender.com, accessed on 30 October 2022.

2.2. Challenges in Establishment of Cell Lines Faithfully Recapitulating DLBCL

Immortalized LL cell lines offer a repeatable and reliable diagnostic tool to model primary tumor in the clinical arena around the world [ 20 ]. However, using non-authenticated cell lines is associated with a risk of about 1:6 for choosing a false, contaminated cell line [ 21 ]. In 1999, when up to 14.8% of human hematopoietic lines were false cell cultures due to cross-contamination, Drexler et al. raised awareness on a recurring problem of overgrowth of cell lines by other cellular models and mycoplasma contamination [ 21 ]. Furthermore, a percentage of the established cell lines, the so-called B-lymphoblastoid cell lines (referred to as EBV+ B-LCLs), resulted from unintended immortalization of non-malignant B cells by “passenger” EBV. EBV+ B-LCLs carry genetic aberrations, which may mimic malignancy-associated features and lead to misidentification as malignant cells [ 16 , 21 ]. Authentication testing is of utmost importance to ensure a panel of non-contaminated, high-quality cell line models with stabile phenotypic characteristics [ 22 ]. In depth molecular characterization and validation of leukemia and lymphoma cell lines in LL-100 panel provided valuable data on the genetic variability of these cancer models facilitating the understanding of LL pathogenesis [ 23 ].

Noteworthy, cell line models often arise from patients with end-stage, non-nodal, and leukemic phase lymphomas that possess a wide-ranging repertoire of biased mutational sequences [ 24 ]. Therefore, Caesar et al. optimized a strategy to purify and facilitate ex vivo expansion of non-malignant human B cells at germinal center stage, that can be genetically modified to enable combinatorial expression of putative tumor suppressor genes [ 24 , 25 ].

It is also common to perform experiments using cell line models representative of other B-cell malignancies and extrapolating them to DLBCL as certain features overlap between them [ 26 , 27 ]. The most frequently used ones are BL cell lines [ 28 ], since BL tumors double in size in approximately 25 h [ 29 ] and therefore the established BL models possess a high growth potential, which makes them relatively easy to harvest and sustain in vitro. A panel of BL lines includes Raji, the first continuous line of hematopoietic origin [ 15 ], Daudi, Ramos and BJAB [ 28 ]. FL and/or MCL lines are also frequently used [ 28 ].

2.3. DLBCL Cell Line Models Available to Study Biology of the Aggressive B-Cell Lymphoma

DLBCL biology is studied with highly heterogeneous models representative of specific subtypes [ 28 ]. Cell line models established in two research centers: Stanford University (SU) and Ontario Cancer Institute (OCI) have dominated DLBCL research [ 30 , 31 , 32 ]. Epstein et al. established three EBV-negative SU-DHL cell lines (SU-DHL-1, -2 from pleural effusion; SU-DHL-3 from peritoneal effusion), followed by seven next ones (from pleural effusion: SU-DHL-7, SU-DHL-8, SU-DHL-9, and SU-DHL-10; from peritoneal effusion: SU-DHL-4, SU-DHL-6; from a lymph node: SU-DHL-5), which comprise a group of highly diverse malignancies arising from B lymphocytes [ 30 ]. Phenotypic characterization and histological description of EBV-negative cell lines of B-lineage including OCI-Ly-1, OCI-Ly-2, OCI-Ly-3, OCI-Ly-4, OCI-Ly-7, OCI-Ly-8, and OCI-Ly-18 have been reported by Tweeddale et al. [ 31 ] and Chang et al. [ 32 ]. They were established either at diagnosis (OCI-4, OCI-8, OCI-18) or during a patient’s relapse (OCI-1, OCI-2, OCI-3, OCI-7). Interestingly, OCL-Ly-3 serves as an example of cell line dependent on growth factors as it undergoes self-regulation by IL-6 [ 33 ].

2.3.1. Panel of ABC-DLBCL Models

ABC-DLBCL, which constitutes around 30% of DLBCL cases, is characterized with a clinically unfavorable outcome as compared to GCB-DLBCL [ 34 ]. Several ABC-DLBCL representing cell lines, including Ri-1 (Riva), SU-DHL-2, SU-DHL-9, OCI-Ly18, HBL-1, RC-K8, U-2946, or TDM8 are available.

Unique features of the available cell lines allow for the choice of tailored preclinical models. For instance, RC-K8 with dysregulated Rel/NF-κB pathway is an appealing candidate for testing novel immunotherapeutic agents for ABC subtype [ 35 ]. Moreover, ULA serves as a model of multidrug resistance as it originated from the patient who had resisted several chemotherapy courses [ 26 ]. Lastly, U-2946 is an example of a cell line with MCL1 (member of BCL2 family) overexpression, which is a recurrent feature in ABC-DLBCL that promotes drug resistance and overall cancer cell survival [ 36 ].

2.3.2. Panel of GCB-DLBCL Models

GCB-DLBCL constitutes up to 50% of DLBCL cases and has a GC phenotype defined as CD10+, BCL6+ [ 34 ]. Although its management has been more successful than of the ABC subtype, the high genetic heterogeneity can lead to unexpected clinical outcome in a number of patients [ 6 , 34 , 37 ]. Cell line models of the GCB subtype include RL, SU-DHL-4, SU-DHL-6, SU-DHL-8, SU-DHL-10, OCI-Ly1, OCI-Ly3, OCI-Ly7 or Karpas422.

Interestingly, Karpas422, bearing both t(14;18) and t(4;11) along with several other abnormalities, is an attractive model to study chemotherapy-resistant NHL as it originated from DLBCL patient unresponsive to consecutive chemotherapeutic schemes [ 38 ].

One of the hallmarks of GCB-DLBCL, present in up to 30% of cases, is t(14;18) translocation, which results in Bcl-2 overexpression [ 39 , 40 ]. Additionally, t(14;18) in GCB-DLBCL is associated with significantly worse prognosis compared to GC-type DLBCL without the translocation (29 to 63% 2-year survival, respectively) [ 40 ]. GCB-DLBCL cell line models containing t(14;18) include for example SU-DHL4, SU-DHL6, and OCI-Ly1.

2.3.3. Cell Line Models with Unique Features Representative of RS-DLBCL and Secondary DLBCL

Unique cell line representatives of RS-DLBCL formed on CLL background and secondary DLBCL formed on Hodgkin lymphoma (HL) background have also been developed. RS-DLBCL represents a group of mainly CD5+ and CD23+ large blast-like neoplastic B cells with small nucleoli with diffuse growth pattern and high proliferative potential [ 41 ]. Up to 95% of RS-DLBCL cases represent the more aggressive ABC-subtype with a variable BCL6 expression, MUM1+ and CD10- [ 42 ]. Despite its unique features, current recommendations for RS management are the same as for aggressive NHLs or de novo DLBCL [ 43 ]. Importantly, high expression of programmed death (PD-1) and programmed death ligand (PD-L1) observed in RS patients [ 44 ] make them appealing targets for immunotherapy (reviewed by Iannello et al. [ 45 ]).

Generally, aggressive NHL has a worse prognosis in comparison to HL [ 46 , 47 ]. However, survival of HL patients with a relapse, especially after chemotherapy, is comparable to patients with primary aggressive NHL [ 48 , 49 ]. The first heterogenic HL-NHL cell line, established by Amini et al. [ 50 ], U-2932, was further shown to be composed of two different subpopulations with unique phenotypic features [ 51 ]. Additionally, the expression levels of CD20 and CD38 antigens in both populations were demonstrated to change through the 100 days of culturing, raising questions about the stability of the U-2932 model, and hence its reliability [ 52 ]. Furthermore, Sambade at al established a cell line with chromosomal rearrangements similar to recurrent aberrations occurring in both HL and NHL (including microsatellite instability), recognized as U-2940 [ 53 ]. Initially, U-2940 was reported to arise from DLBCL [ 53 ]; however, further research ascribed it as a model of primary mediastinal B-cell lymphoma (PMBL) [ 54 , 55 ].

Table 1 provides a concise summary of thoroughly characterized DLBCL cell lines.

Overview of the cell lines used to study DLBCL.

3. 3-Dimensional (3D) Models

Despite being an indispensable tool within cancer research, most of the cell lines fail to reliably recapitulate the importance of genetic and microenvironmental factors of tumors in disease progression [ 65 ]. Thus, 3-dimensional (3D) culture systems that provide an insight into pathophysiology of tumor microenvironment and allow to monitor cell functions (such as proliferation, differentiation, motility, and metabolism) [ 66 ] offer a testbed for therapeutic agents prior to in vivo studies [ 67 ].

Duś-Szachniewicz et al. established 3D spheroid models based on Ri-1 and Raji cells, which were used to test cytotoxicity of doxorubicin (DOX) and ibrutinib (IBR) on B-NHLs [ 68 ]. Co-culturing of lymphoma cells with stromal cells led to a reduced IBR-induced apoptosis in comparison to the 3D monoculture, which recapitulates the significance of stromal cells in tumor pathophysiology. Lara et al. investigated the effect of different RTX isotypes generated by recombinant DNA technology on 2D and 3D-cultured B-cell lymphoma lines (Raji, Daudi, BJAB and Granta-519) and observed considerable differences in potency of RTX-mediated complement-dependent cytotoxicity (CDC) with respect to antibody isotype and model structure, with 3D models limiting penetration of RTX and limiting its cytotoxic activity [ 69 ]. The promising results encourage further investments in the generation of faithful 3D model systems investigating other B-cell malignancies, including DLBCL.

4. Significance of Cell Line Models Resistant to Therapeutic Agents

Years of RTX employment as well as recent findings on mechanisms of insensitivity to a range of immunotherapies indicate that developing resistance is an unavoidable side effect of the regimens [ 70 ]. Generating cellular models of acquired resistance is based on repeated incubations of cells with increasing concentrations of a cytotoxic agent ( Figure 2 ) [ 71 ].

Generation of cell line models resistant to therapeutic agents. Created with BioRender.com, accessed on 30 October 2022.

RTX-resistant cell lines (RRCLs) were generated by Czuczman et al. from Raji, SU-DHL-4, RL and U-2932 [ 72 ]. The cells acquired resistance mainly via CDC, due to medium supplementation with human serum, and additionally to antibody dependent cell-mediated cytotoxicity (ADCC) [ 72 ]. Additionally, CD20 underwent downregulation and expression of pro-apoptotic members of the BCL-2 family (Bax and Bak) was reduced [ 72 , 73 ]. RRCLs hold potential to be used in investigations of immunotherapeutic strategies that aim to overcome the RTX resistance phenomenon.

The mechanisms of potential insensitivity to CD37-targeting agents can also be investigated in cell line models. Accordingly, Arribas et al. generated two DLBCL cell line models (SU-DHL-2 and SU-DHL-4), which were resistant to IMGN529/DEBIO1562 (an anti-CD37 ADC) and demonstrated different phenotypic changes within the models [ 74 ]. SU-DHL-2 (ABC-DLBCL model) carried CD37 loss and 25 mutations in kinases or transcription factors, SU-DHL-4 sustained CD37 expression but carried 48 shared mutations in genes encoding for cytokines, kinases, oncogenes, and transcription factors. Melhus et al. presented work exploring expression characteristics and intrinsic factors associated with sensitivity of 55 lymphoma cell lines (including 20 GCB subtype and 7 ABC subtype) to 177^Lu-lilotomab satetraxetan, anti-CD37 radioimmunoconjugate [ 75 ]. Intrinsic treatment resistance to 177^Lu-lilotomab satetraxetan is evident; however, only in a subset of cell lines and is independent of genetic lymphoma hallmarks including TP53, BCL2, and MYC.

5. Investigating Immunotherapeutic Strategies

5.1. exploring potential of monoclonal antibodies (mabs).

The availability of various lymphoma cellular models is vital for testing novel antibodies in preclinical trials. Several B-cell antigens e.g., CD19, CD22, CD37, CD40, CD47, CD79b, and CD80 have been proposed as targets for mAbs mainly for r/r patients [ 76 ].

Gehlert et al. used B-ALL cell lines (SEM, Jurkat, CEM, MOLT-16, and Nalm-6 cells) to test efficacy of CD19-targeting mAb, tafasitamab [ 77 ]. The mAb was optimized in a way to improve antibody hexamerization, which enhanced CDC but had no influence on antibody-dependent cellular phagocytosis (ADCP) or ADCC. Additionally, combination of tafasitamab and RTX improved cytotoxicity against B-ALL cell lines in vitro [ 78 ]. Although the study did not include DLBCL cell lines, preclinical studies performed on B-ALL models encouraged investigation of tafasitamab efficacy (in monotherapy or in combination with lenalidomide) in r/r DLBCL patients who cannot be qualified for ASCT.

As demonstrated in a range of preclinical studies with cellular models, the CD37 antigen is widely expressed across multiple types of B-cell lymphoid neoplasms [ 79 ], which prompted extensive development of CD37-targeting mAbs, as recently reviewed [ 80 ]. Lately, a panel of DLBCL models (OCI-Ly7, OCI-Ly19, RC-K8, Ri-1, SU-DHL-4, SU-DHL-8, WSU-DLCL-2, and U-2932) were used to confirm efficacy of bispecific CD37 antibody (DuoHexaBody-CD37), which triggered potent ADCC, ADCP and superior CDC to other tested CD37-targeting mAbs [ 81 ].

Since it has been shown that CD38 is an important prognostic marker also in DLBCL [ 82 ], preclinical studies evaluating efficacy of anti-CD38 mAbs like daratumumab in DLBCL are undertaken. In in vitro and in vivo models of DLBCL (cell lines: Toledo, WSU-DLC2, SU-DHL-4, SU-DHL-6), MCL and FL potent daratumumab-mediated ADCC and ADCP was demonstrated independently of CD38 expression [ 83 ].

Furthermore, Bouwstra et al. demonstrated that high expression of CD47, the so-called “don’t eat me” immune checkpoint, correlated with detrimental effect on OS in non-GCB DLBCL patients after R-CHOP therapy [ 84 ]. Further studies on DLBCL cell lines (OCI-Ly3, U-2932, SU-DHL-2, SU-DHL-4, SU-DHL-6, SU-DHL-10) demonstrated increased therapeutic effect of RTX after blocking CD47 in non-GCB DLBCL model [ 84 ]. Moreover, B-NHL co-cultures (DLBCL lines: Pfeiffer and Karpas422; BL; Raji, Daudi; FL: RL) showed that ADCC and ADCP induced by a CD47- and CD19-targeting bispecific antibody (TG-1801) was enhanced when it was used in a “U2-regimen” (with anti-CD20-mAb: ubilituximab; and PI3Kδ/CK1e inhibitor: umbralisib) than in monotherapy [ 85 ].

By now, it has been discovered that PD-L1 is aberrantly expressed on HLs and little is known about its role in NHLs [ 86 ]. Analysis performed on a panel of several NHL cell lines including 28 DLBCL models revealed that PD-L1 expression was confined to only 3 models (HBL-1, OCI-Ly-10 and RC-K8) [ 87 ]. Astonishingly, as shown with the aid of various human DLBCL cell lines (including OCI-Ly-3, TDM8, SU-DHL-4), the PD-L1 levels can be upregulated by vincristine administration improving efficacy of the PD-L1 blockade therapy [ 88 ]. An extensive testing of immune checkpoint inhibitors in preclinical investigations have inspired clinical trials that eventually led to FDA’s approval of nivolumab and pembrolizumab for certain types or r/r lymphomas [ 89 , 90 ].

5.2. Antibody-Drug Conjugates (ADCs) and Targeted-Drug Delivery

Antibody-drug conjugates (ADCs) are monoclonal antibodies bound to cytotoxic payload that is delivered directly to the tumor cells [ 91 ]. Immunologic functions of ADC such as CDC and ADCP are weakened to strengthen the antitumor effect of the molecule, which induces killing of the cells [ 92 ].

Approval of polatuzumab vedotin (Pola) in 2019, CD79b-targeting ADC, benefited a significant percentage of r/r DLBCL patients [ 93 ]. Nonetheless, insensitivity to ADCs may appear as in other immunotherapeutic options. DLBCL cell line panel consisting of Pola-sensitive (DB, STR-428, SU-DHL10, SU-DHL-4, NU-DUL-1, U-2932) and Pola-resistant (SU-DHL-8, HT, SU-DHL-2, RC-K8) cell lines served as a tool to investigate resistance mechanisms to Pola, which included low CD79b expression, high expression of anti-apoptotic Bcl-xL and ABC transporters [ 94 ]. Interestingly, exposition of SU-DHL-8, SU-DHL-2 and HT (DLBCL cell lines) resistant to anti-CD79b ADC (Pola) leads to CD20 upregulation and enhances RTX sensitivity via CDC and ADCC [ 95 ]. This justifies the use of combination therapy for Pola with RTX and bendamustine regimen.

Moreover, sensitivity of 27 commonly used DLBCL cell lines of ABC and GCB subtypes to MMAE-conjugated ADCs: anti-CD22 (pinatuzumab vedotin) and anti-CD79B ADCs (Pola) was assessed [ 96 ]. Although majority of the cells were sensitive to both ADCs, Farage, HS445 and HT responded only to anti-CD22; OCI-Ly3, HBL-1, and Pfeifer models responded only to anti-CD79b. Only SU-DHL-2 and WSU-NHL were resistant to both drugs.

Furthermore, a potent in vitro activity against multiple B cell lines (including Ramos, Raji, Daudi, Farage, and RL) via ADCC, ADCP, and CDC was exerted by naratuximab emtansine (IMGN529), an investigational CD37-targeting ADC conjugated to DM1 [ 97 ]. The results were confirmed on a similar panel of DLBCL models (U-2932, SU-DHL-4, DOHH-2, OCI-Ly18, OCI-Ly7, and Farage) by Hicks et al. who further demonstrated increased apoptosis and cell death of those cellular models as a consequence of synergistic anti-tumor potency of naratuximab ematansine and anti-CD20 agents, especially RTX [ 75 ]. In a similar pattern, U-RT-1 cell line was used to demonstrate high antitumor activity of anti-CD37 alfa-amanitin-conjugated antibodies [ 98 ].

5.3. CAR-T Cell Therapy

Introduction of T cells modified with chimeric antigen receptors (CARs) has led to a significant advancement in management of multiple malignancies, including lymphoid neoplasms [ 99 ]. By now, three CAR-T cells (axicabtagene ciloleucel, lisocabtagene maraleucel and tisagenlecleucel) have been registered in treatment of r/r DLBCL. Surprisingly, majority of preclinical studies testing efficacy of CARs were performed on B-ALL cell lines, not on DLBCL models. However, the existing ones, provide important directions in optimizing CAR T-cells.

The great success of anti-CD19 CAR-T cells in B-ALL encouraged preclinical studies on their utility against DLBCL. Interestingly, improved killing of B-cell lymphoma lines, including OCI-Ly2 and OCI-Ly19, Raji, Daudi, DEL (anaplastic large cell lymphoma model), Granta-519 and Jeko-1 (MCL), was triggered by the combination of anti-CD19-CAR-T cells with an anti-CD20-IFN fusion protein indicating that antibody-targeted IFN could improve the CAR-T cell therapy [ 100 ]. Noteworthy, in Raji and U-2932 anti-CD19-CAR-T demonstrated higher efficacy when combined with ibrutinib than in monotherapy [ 101 ]. Although no pathway responsible for synergistic effect was identified [ 101 ], the experiment suggests that this approach might offer a benefit to B-cell lymphoma patients.

Furthermore, promising findings on anti-CD37 mAbs also encouraged exploration of CD37 as a target for CAR-T cells. The panel of several B-cell lymphoma models (BL-41, Daudi, Granta-519, K422, K562, Jeko-1, Jurkat, Maver-1, MINO, Raji, Ramos, ROS-50, SC-1, SU-DHL-6, SU-DHL-4, Oci-Ly3, Oci-Ly7, and Oci-Ly10) and two xenograft mice models were constructed to analyze the cytotoxic effect of CD37CAR [ 102 ].

Despite the promising results on CD19 CAR-T cells, around half of high-grade lymphoma patients develops resistance after receiving such treatment [ 103 ]. Unfortunately, resistance mechanisms to CAR-T cell therapy in DLBCL are not well-understood yet, as most of the observations arise primarily from ALL studies [ 70 ]. By now, there is also a lack of existing cellular models with developed insensitivity to CAR-T cell therapies that could be used to optimize therapeutic approaches.

Both Table 2 and Figure 3 provide a concise summary of utility of cellular models in exploring targeted immunotherapies for DLBCL.

Targeted immunotherapies in DLBCL investigated on cellular models. ( A ) Monoclonal antibodies (mAbs); ( B ) Antibody-drug conjugates (ADCs); ( C ) chimeric antigen receptor T cells (CAR T-cells). Created with BioRender.com, accessed on 30 October 2022.

Overview of immunotherapeutic strategies and cell lines used in evaluation of their efficacy.

6. Xenograft Mouse Models

Animal models are a powerful tool to test novel immunotherapeutic options prior to clinical investigations [ 104 ]. In essence, xenograft mouse models of human lymphoma cells can be established by implantation of stable cell lines of primary tumor samples into immunosuppressed recipient or humanized mice. Serial passaging of the engrafted tumors may be necessary to achieve high xenotransplantation efficiency. Transplantation occurs into tail vein (disseminated model) or subcutaneously (orthotropic model). In case of disseminated models the transplanted cells are most often genetically modified to express luciferase and tumor growth is measured using bioluminescence [ 105 ]. In orthotropic models bioluminescence imaging and measurement by calipers can be applied [ 106 ]. By mimicking genetic alterations found in the human disease, the utilized models (e.g., genetically engineered mouse models (GEMMs) have allowed the detailed in vivo investigation of several lymphoma-associated oncogenes and tumor suppressors, shedding light on their role in normal B cell development and tumorigenesis. However, it has to be said that the following approach is associated with specific advantages and disadvantages; for instance GEMMs cannot reproduce the genetic complexity and the heterogeneity of the human tumors, an aspect especially important when aiming at discovery and pre-clinical testing of novel therapeutics. Additionally, a few other important challenges accompany the utility of xenograft models, including the lack of an immune response against the tumor and lack of physiological microenvironment [ 104 , 107 ]. Nonetheless, animal research is an indispensable step before proceeding to clinical trials.

Murine models are commonly utilized in testing efficacy and safety of mAbs, ADCs and CAR-T cells. Xenograft models derived from different cell lines injected subcutaneously (BL: Raji, Ramos, Namalwa; DLBCL: SU-DHL-6; acute lymphoblastic leukemia: SUP-B15) demonstrated significant lymphoma inhibition by XmAb5574, an anti-CD19 mAb with an Fc-engineered domain for effector function improvement. [ 108 ]. Furthermore, the growth of each NHL xenograft model, obtained by cell lines’ (BL: Raji, Ramos; MCL; FL and DLBCL cell lines) implantation above the right flank, was inhibited by highly cytotoxic anti-CD37 agent, recognized as AGS67E [ 109 ]. Importantly, murine models lessen efforts associated with designing strategies, which aim at overcoming RTX-resistance. A panel of human lymphoma cells (BL: Raji, Ramos; DLBCL: RL, U2932, SU-DHL-5; lymphoblastic lymphoma: U-698-M; RRCLs: Raji 2R, Raji 4RH, and RL 4RH; ofatumumab-exposed cell lines: Raji, U-2932, RL) inoculated via tail vein injection to generate respective xenograft models enabled comparison of efficacy of two anti-CD20 mAbs: RTX and ofatumumab [ 110 ]. In vivo studies, performed on xenografted tumors after subcutaneous injection of Pola-sensitive (e.g., SU-DHL-4, U-2932) and Pola-resistant (e.g., SU-DHL-2, RC-K8) cell lines, showed that Pola increased CD20 expression in Pola-resistant xenograft models and had an enhanced antitumor activity when combined with RTX [ 94 ]. Since extrapolating findings based on BL models to DLBCL is a widely accepted and justified practice, it comes as no surprise that numerous in vivo studies utilize Raji xenograft model for testing CAR-T efficacy. In particular, potency of CD19 CAR constructs, observed in both disseminated and orthotropic models [ 111 ], or synergistic effect of CD19 CAR combined with ibrutinib, analyzed with the aid of a disseminated model [ 112 ], have been thoroughly investigated in Raji-derived xenograft models. Studies on MCL disseminated xenograft models (Mino, JEKO-1) demonstrated additive effect of CD19 CAR combined with ibrutinib [ 113 ] and identified bispecific CD79b/CD19 [ 114 ]. CAR as strategies to improve standard-of-care therapies against MCL. In addition, profound efficacy of CD19 CAR-based therapies against B-ALL in clinical settings prompted the establishment of leukemic humanized mice with human immune system as a valuable pre-clinical model [ 115 ].

7. Conclusions

The current review has summarized available cell lines used as powerful tools in investigating DLBCL biology as well as in evaluating therapeutic efficacy of antitumor agents. Despite the wide arsenal of cellular DLBCL models representative of ABC and GCB subtypes, only several are repeatedly used, including SU-DHL-4, SU-DHL-6, OCI-Ly-3, TDM8, or U-2932, to optimize available immunotherapies. To ensure reliable modeling of the primary tumor it is crucial to analyze a genetic background of each model and ensure it remains uncontaminated with foreign material. Importantly, several lines have been authenticated and included in the LL-100 panel (ABC-DLBCL: NU-DHL-1, OCI-Ly3, Ri-1, U-2932, U-2946; GCB-DLBCL: DOHH-2, OCI-Ly7, OCI-LY19, SU-DHL-4, SU-DHL-6, and WSU-DLCL2).

Effective employment of cell line models encourages other research teams to utilize the same model systems, which on the one hand facilitates the reproducibility of the generated data among research centers yet limits the scope of investigations. After all, DLBCL remains one of the most heterogenic lymphoid malignancies and requires multiple cellular models to study its biology. Simultaneously, it is crucial to report failures associated with culturing of specific cell lines to allow for verification and perhaps modifications of the models to overcome culturing challenges. Surprisingly, U-2932 remains a frequently used cell line in DLBCL research regardless of the existing controversies on its complex phenotype, which might perhaps serve as an advantage in development of more universal immunotherapeutics. Furthermore, it remains a common strategy to use other B-cell lymphoma models, especially BT cell lines (Raji, Daudi, Ramos) to yield results and extrapolate them to DLBCL.

Importantly, the wide arsenal of the existing cellular models contributed significantly to the acceleration of the preclinical testing of various immunotherapeutics, which has been observed in the past decade along with approval of a range of mAbs, ADCs, and CAR T. However, years of immunotherapy employment demonstrated that it inevitably leads to development of resistance, which remains an ongoing challenge to overcome. Consequently, an urge to expand even wider arsenal of commonly available patient-derived cell lines and to develop agent-resistant cell lines persists. Furthermore, investment in 3D systems is required to provide insight into efficacy of anti-tumor molecules before proceeding with animal testing. The attempts aiming at optimizing modeling of the primary tumor will benefit in improved optimization of (immuno)therapeutic strategies.

Funding Statement

This work was supported by the Ministry of Education and Science within “Regional Initiative of Excellence” in years 2019–2022, program 013/RID/2018/19, project budget 12,000,000 PLN. This project was supported by European Research Council 805038/STIMUNO/ERC-2018-STG (M.W.), Polish National Science Centre 2019/35/D/NZ5/01191 (M.B.) and Medical University of Warsaw 1M19/1/M/MG/N/21/21 (M.K.).

Author Contributions

Conceptualization, M.K. and M.B.; methodology, M.K. and M.B.; investigation, M.K., A.K., M.B. and M.W.; writing—original draft preparation, M.K., A.K., M.B. and M.W.; writing—review and editing, M.K., A.K., M.B. and M.W.; visualization, A.K. and M.B.; supervision, M.W.; funding acquisition, M.B. and M.W. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Fish cell line: depositories, web resources and future applications

• Published: 27 November 2023
• Volume 76 , pages 1–25, ( 2024 )

Cite this article

• Murali S. Kumar 1 ,
• Vijay Kumar Singh 1 ,
• Akhilesh Kumar Mishra 1 ,
• Basdeo Kushwaha 1 ,
• Ravindra Kumar 1 &
• Kuldeep Kumar Lal 1  

Cell lines are important bioresources to study the key biological processes in the areas like virology, pathology, immunology, toxicology, biotechnology, endocrinology and developmental biology. Cell lines developed from fish organs are utilized as a model in vitro system in disease surveillance programs, pharmacology, drug screening and resolving cases of metabolic abnormalities. During last decade, there were consistent efforts made globally to develop new fish cell lines from different organs like brain, eye muscles, fin, gill, heart, kidney, liver, skin, spleen, swim bladder, testes, vertebra etc. This increased use and development of cell lines necessitated the establishment of cell line depositories to store/preserve them and assure their availability to the researchers. These depositories are a source of authenticated and characterized cell lines with set protocols for material transfer agreements, maintenance and shipping as well as logistics enabling cellular research. Hence, it is important to cryopreserve and maintain cell lines in depositories and make them available to the research community. The present article reviews the current status of the fish cell lines available in different depositories across the world, along with the prominent role of cell lines in conservation of life on land or below water.

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Abdul NA, Seepoo AM, Gani T, Sugumar V, Selvam S, Allahbagash B, Abdul Kuthoos AN, Palsamy RK, Kishore MP, Rajwade J M, Azeez SSH (2022) Development and characterization of five novel cell lines from Snubnose pompano , Trachinotus blochii (Lacepede, 1801), and their application in gene expression and virological studies. J Fish Dis 45:121–139. https://doi.org/10.1111/jfd.13542

Article   CAS   PubMed   Google Scholar  

Abe T, Ishikawa T, Masuda T, Mizusawa K, Tsukamoto T, Mitani H, Yanagisawa T, Todo T, Iigo M (2006) Molecular analysis of Dec1 and Dec2 in the peripheral circadian clock of zebrafish photosensitive cells. Biochem Biophys Res Commun 351:1072–1077. https://doi.org/10.1016/j.bbrc.2006.10.172

Ahmed VPI, Chandra V, Vijayakumar P, Venkatesan C, Shukla R, Bhonde RR, Sahul Hameed AS (2008) A new epithelial-like cell line from eye muscle of catla, Catla catla (Hamilton): development and characterization. J Fish Biol 72:2026–2038

Article   CAS   Google Scholar  

Ahmed VPI, Chandra V, Sudhakaran R, Kumar SR, Sarathi M, Babu SV, Bhonde RR, Sahul Hameed AS (2009) Development and characterization of cell lines derived from rohu, Labeo rohita (Hamilton), and catla, Catla catla (Hamilton). J Fish Dis 32:211–218

Ahne W (1979) Fish cell culture: a fibroblastic line (PG) from ovaries of juvenile pike ( Esox lucius ). In Vitro 15:839–840

Article   Google Scholar  

Ahne W (1985) Use of fish cell cultures for toxicity determination in order to reduce and replace the fish tests. Zentralbl Bakteriol Mikrobiol Hyg B 180:480–504

CAS   PubMed   Google Scholar  

Anonymous (2001) Contamination of cell lines- a conspiracy of silence. Lancet Oncol 2:393. https://doi.org/10.1016/S1470-2045(00)00402-2

Arat S, Caputcu AT, Akkoc T, Pabuccuoglu S, Sagarika H, Cirit U et al (2011) Using cell banks as a tool in conservation programmes of native domestic breeds: the production of the first cloned Anatolian grey cattle. Reprod Fertil Dev 23:1012–1023

Article   PubMed   Google Scholar  

Babich HJ, Rosenberg DW, Borenfreund E (1991) In vitro cytotoxicity studies with the fish hepatoma cell line, PLHC-1 ( Poeciliopsis lucida ). Ecotoxicol Environ Saf 21:327–336

Babu VS, Nambi K, Chandra V, Ahmed VPI, Bhonde R, Hameed AS (2011) Establishment and characterization of a fin cell line from Indian walking catfish, Clarias batrachus (L.). J Fish Dis 34:355–364

Babu SV, Chandra V, Nambi KSN, Majeed SA, Taju G, Patole MS, Hameed ASS (2012) Development and characterization of novel cell lines from Etroplus suratensis and their applications in virology, toxicology and gene expression. J Fish Biol 80:312–334

Bairoch A (2018) The cellosaurus, a cell line knowledge resource. J Biomol Tech 29:25–38. https://doi.org/10.7171/jbt.18-2902-002

Article   PubMed   PubMed Central   Google Scholar  

Bejar J, Borrego JJ, Alvarez MC (1997) A continuous cell line from the cultured marine fish gilt-head seabream ( Sparus aurata L.). Aquaculture 150:143–153

Benjaminson MA, Gilchriest JA, Lorenz M (2002) In vitro edible muscle protein production system (MPPS): Stage 1, fish. Acta Astronaut 51:879–889

Ben-Nun IF, Montague SC, Houck ML, Tran HT, Garitaonandia I, Leonardo TR, Wang YC, Laurent LC, Ryder OA, Loring JF (2011) Induced Pluripotent stem cells from highly endangered species. Nat Methods 8:829–831

Bols NC, Lee LEJ (1991) Technology and uses of cell culture from tissues and organs of bony fish. Cytotechnology 6:163–187

Bols NC, Barlian A, Chirino Trejo M, Caldwell SJ, Goegan P, Lee LEJ (1994) Development of a cell line from primary cultures of rainbow trout, Oncorhynchus mykiss (Walbaum), gills. J Fish Dis 17:601–611

Bols NC, Pham PH, Dayeh VR, Lee LEJ (2017) Invitromatics, invitrome, and invitroomics: introduction of three new terms for in vitro biology and illustration of their use with the cell lines from rainbow trout. In Vitro Cell Dev Biol Ani 53:383–405. https://doi.org/10.1007/s11626-017-0142-5

Bols NC, Lee LEJ, Dowd GC (2023) Distinguishing between ante factum and post factum properties of animal cell lines and demonstrating their use in grouping ray-finned fish cell lines into invitromes. In Vitro Cell Dev Biol Anim 59:41–62

Bowser PR, Plumb JA (1980) Fish cell lines: establishment of a line from ovaries of channel catfish. In Vitro 16:365–368

Bowser DH, Frenkel K, Zelikoff JT (1994) Effects of in vitro nickel exposure on the macrophage-mediated immune functions of rainbow trout ( Oncorhynchus mykiss ). Bull Environ Contam Toxicol 52:367–373

Bradford CS, Sun L, Collodi P, Barnes DW (1994) Cell cultures from zebrafish embryos and adult tissues. J Tissue Cult Methods 16:99–107

Bradford CS, Miller AE, Toumadje A, Nishiyama K, Shirahata S, Barnes DW (1997) Characterization of cell cultures derived from Fugu, the Japanese pufferfish. Mol Mar Biol Biotechnol 6:279–288

Cabane OF (2019). The 2020 Global Alternative Food Awards: alternative dairy. https://static1.squarespace.com/static/5b9f712ff93fd4ab389d7b82/t/5e45e2c745646d18abb40458/1581638350283/NewProteinV.2.8.png .

Capes-Davis A, Theodosopoulos G, Atkin I, Drexler HG, Kohara A, MacLeod RAF, Masters JR, Nakamura N, Reid YA, Reddel RR (2010) Check your cultures! A list of cross-contaminated or misidentified cell lines. Int J Cancer 127:1–8. https://doi.org/10.1002/ijc.25242

Chang N, Sun C, Gao L, Zhu D, Xu X, Zhu X, Xiong JW, Xi JJ (2013) Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res 23:465–472

Article   CAS   PubMed   PubMed Central   Google Scholar  

Chaudhary DK, Sood N, Pradhan PK, Singh A, Punia P, Agarwal NK, Rathore G (2012) Establishment of a macrophage cell line from adherent peripheral blood mononuclear cells of Catla catla . In Vitro Cell Dev Biol Ani 48:340–348

Chaudhary DK, Sood N, Swaminathan TR, Rathore G, Pradhan PK, Agarwal NK, Jena JK (2013) Establishment and characterization of an epithelial cell line from thymus of Catla catla . Gene 512:546–553

Chaudhary DK, Sood N, Rathore G, Pradhan PK, Punia P, Agarwal NK, Jena JK (2014) Establishment and characterization of macrophage cell line from thymus of Catla catla (Hamilton, 1822). Aquacult Res 45:299–311. https://doi.org/10.1111/j.1365-2109.2012.03227.x

Chen SN, Kou GH (1981) A cell line derived from Japanese eel ( Anguilla japonica ) ovary. Fish Pathol 16:129–137

Choi MS, Kim YJ, Kwon EY, Ryoo JY, Kim SR, Jung UJ (2015) High-fat diet decreases energy expenditure and expression of genes controlling lipid metabolism, mitochondrial function and skeletal system development in the adipose tissue, along with increased expression of extracellular matrix remodelling-and inflammation-related genes. Br J Nutr 113:867–877

Collet B, Boudinot P, Benmansour A, Secombes CJ (2004) An Mx1 promoter–reporter system to study interferon pathways in rainbow trout. Dev Comp Immunol 28:793–801. https://doi.org/10.1016/j.dci.2003.12.005

Collodi P, Kamei Y, Ernst T, Miranda C, Buhler DR, Barnes DW (1992) Culture of cells from zebrafish ( Brachydanio rerio ) embryo and adult tissues. Cell Biol and Toxicol 8:43–61

Dannevig BH, Falk K, Namork E (1995) Isolation of the causal virus of infectious salmon anaemia (ISA) in a long-term cell line from Atlantic salmon head kidney. J Gen Virol 76:1353–1359

Dehler CE, Boudinot P, Martin SA, Collet B (2016) Development of an efficient genome editing method by CRISPR/Cas9 in a fish cell line. Mar Biotechnol 18:449–452. https://doi.org/10.1007/s10126-016-9708-6

Devold M, Krossøy B, Aspehaug V, Nylund A (2000) Use of RT-PCR for diagnosis of infectious salmon anaemia virus (ISAV) in carrier sea trout Salmo trutta after experimental infection. Dis Aquat Organ 40:9–18. https://doi.org/10.3354/dao040009

Dharmaratnam A, Kumar R, Valaparambil BS, Sood N, Pradhan PK, Das S, Thangaraj RS (2020) Establishment and characterization of fantail goldfish fin (FtGF) cell line from goldfish, Carassius auratus  for in vitro propagation of cyprinid herpes virus-2 (CyHV-2). PeerJ 8:e9373

Diago ML, Lopez Fierro P, Razquin BE, Villena AJ (1995) Establishment and characterization of a pronephric stromal cell line (TPS) from rainbow trout, Oncorhynchus mykiss W. Fish Shellfish Immunol 5:441–457

Driever W, Rangini Z (1993) Characterization of a cell line derived from zebrafish ( Brachydanio rerio ) embryos. In Vitro Cell Dev Biol Anim 29A:749–754

Dubey A, Goswami M, Yadav K, Sharma BS (2014) Development and characterization of a cell line WAF from freshwater shark Wallago attu . Mol Biol Rep 41:915–924

Dubey A, Goswami M, Yadav K, Chaudhary D (2015a) Oxidative stress and nano-toxicity induced by TiO 2 and ZnO on WAG cell line. PLoS ONE 10:e0127493

Dubey A, Goswami M, Yadav K, Mishra A, Kumar A (2015b) Establishment of a novel muscle cell line from Wallago attu for in vitro study of pesticide toxicity. Gene Cell Tissue 2:e25568

Etoh H, Hyodo Taguchi Y, Aoki K, Murata M, Matsudaira H (1983) Incidence of chromatoblastomas in aging goldfish ( Carassius auratus ). J Natl Cancer Inst 70:523–528

Etoh H, Suyama I, Hyodo Taguchi Y, Matsudaira H (1988) Establishment and characteristics of various cell lines from medaka (Teleostei). In: Kuroda Y, Kurstak E, Maramorosch K (eds) Invertebrate and fish tissue culture. Springer, Berlin

Google Scholar  

Faber MN, Sojan JM, Saraiva M, van West P, Secombes CJ (2021) Development of a 3D spheroid cell culture system from fish cell lines for in vitro infection studies: evaluation with Saprolegnia parasitica . J Fish Dis 44:701–710

Faisal M, Ahne W (1990) A cell line (CLC) of adherent peripheral blood mononuclear leucocytes of normal common carp Cyprinus carpio . Dev Comp Immunol 14:255–260

FAO (2017) The future of food and agriculture—trends and challenges. http://www.fao.org/3/a-i6583e.pdf

Farrukh A, Paez JI, Campo AD (2018) 4D biomaterials for light-guided angiogenesis. Adv Func Mater 29:1807734

Fijan N, Sulimanovic D, Bearzotti M, Muzinic D, Zwillenberg LO, Chilmonczyk S, Vautherot JF, de Kinkelin P (1983) Some properties of the Epithelioma papulosum cyprini (EPC) cell line from carp Cyprinus carpio . Ann Inst Pasteur Virol 134:207–220

Article   PubMed Central   Google Scholar  

Ford L, Subramanium K, Waltzek TB, Bowser PR, Hanson L (2021) Cytochrome oxidase gene sequencing reveals channel catfish ovary cell line is contaminated with brown bullhead cells. J Fish Dis 44:119–122

Frerichs GN, Morgan D, Hart D, Skerrow C, Roberts RJ, Onions DE (1991) Spontaneously productive C-type retrovirus infection of fish cell lines. J Gen Virol 72:2537–2539

Fryer JL, Lannan CN (1994) Three decades of fish cell culture: a current listing of cell lines derived from fishes. J Tissue Cult Methods 16:87–94

Fryer JL, Yusha A, Pilcher KS (1965) The in vitro cultivation of tissue and cells of Pacific salmon and steelhead trout. Ann N Y Acad Sci 126:566–586

Fryer JL, Mccain BB, Leong LC (1981) A cell line derived from rainbow trout ( Salmo gairdneri ) Hepatoma. Fish Path 15:193–200. https://doi.org/10.3147/jsfp.15.193

Ganassin RC, Sanders SM, Kennedy CJ, Joyce EM, Bols NC (1999) Development and characterization of a cell line from Pacific herring, Clupea harengus pallasi , sensitive to both naphthalene cytotoxicity and infection by viral hemorrhagic septicemia virus. Cell Biol Toxicol 15:299–309. https://doi.org/10.1023/a:1007615818427

Gardell AM, Qin Q, Rice RH, Li J, Kultz D (2014) Derivation and osmotolerance characterization of three immortalized tilapia ( Oreochromis mossambicus ) cell lines. PLoS ONE 9:e95919

George GA, Sobhana KS, Sunny SM, Sreedevi S (2018) Evaluation of various tissues of the Caerulean damsel , Pomacentrus caeruleus for initiating in vitro cell culture systems. Proc Natl Acad Sci India, b, Biol Sci 88:293–303. https://doi.org/10.1007/s40011-016-0751-x

Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S et al (2013) Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol 87:1315–1530

Goswami M, Sharma BS, Tripathi AK, Yadav K, Bahuguna SN, Nagpure NS, Lakra WS, Jena JK (2012) Development and characterization of cell culture systems from Puntius (Tor) chelynoides (McClelland). Gene 500:140–147

Goswami M, Sharma BS, Bahuguna SN, Nagpure NS, Lakra WS (2013a) A SRCF cell line from Schozothorax richarsonii : development and characterization. Tissue Cell 45:219–226

Goswami M, Yadav K, Dubey AK, Sharma BS, Kanwar R, Kumar R, Nagpure NS (2013b) In vitro cytotoxicity assessment of two heavy metal salts in RF cell line. Drug Chem Toxicol 37:48–54

Graf M, Hartmann N, Reichwald K, Englert C (2013) Absence of replicative senescence in cultured cells from the short-lived killifish Nothobranchius furzeri . Exp Gerontol 48:17–28

Gratacap RL, Regan T, Dehler CE, Martin SAM, Boudinot P, Collet B, Houston RD (2020) Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system. BMC Biotechnol 20:35. https://doi.org/10.1186/s12896-020-00626-x

Gravell M, Malsberger RG (1965) A permanent cell line from the fathead minnow ( Pimephales promelas ). Ann N Y Acad Sci 126:555–565

Grunow B, Mohamet L, Shiels HA (2015) Generating an in vitro 3D cell culture model from zebrafish larvae for heart research. J Exp Biol 218:1116–1121

PubMed   PubMed Central   Google Scholar  

Halpern BS, Maier J, Lahr HJ, Blasco G, Costello C, Cottrell RS, Deschenes O, Ferraro DM, Froehlich HE, McDonald GG, Millage KD, Weir MJ (2021) The long and narrow path for novel cell-based seafood to reduce fishing pressure for marine ecosystem recovery. Fish Fish 22:652–664

Hameed ASS (2010) Development and application of fish cell lines: a review. Indian J Ani Sci 80:125–134

Hameed ASS, Vijayakumar P, Shukla R, Bright Singh IS, Thirunavukkarasu AR, Bhonde RR (2006) Establishment and characterization of India’s first marine fish cell line (SISK) from the kidney of sea bass ( Lates calcarifer ). Aquaculture 257:92–103

Hedrick RP, Gilad O, Yun S, Spangenberg JV, Marty GD, Nordhausen RW, Kebus MJ, Bercovier H, Eldar A (2000) A herpesvirus associated with mass mortality of juvenile and adult koi, a strain of common carp. J Aquat Ani Health 12:44–57

Hodgson P, Ireland J, Grunow B (2018) Fish, the better model in human heart research? Zebrafish Heart aggregates as a 3D spontaneously cardiomyogenic in vitro model system. Prog Biophys Mol Biol 138:132–141

Horbach SPJM, Halffman W (2017) The ghosts of HeLa: How cell line misidentification contaminates the scientific literature. PLoS ONE 12:e0186281

Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD et al (2013) Efficient in vivo genome editing using RNA-guided nucleases. Nat Biotechnol 31:227–229. https://doi.org/10.1038/nbt.2501

Iwamoto T, Nakai T, Mori K, Arimoto M, Furusawa I (2000) Cloning of the fish cell line SSN-1 for piscine nodaviruses. Dis Aquat Organ 43:81–89

Jimeno-Romero A, Gwinner F, Müller M, Mariussen E, Soto M, Kohl Y (2021) Sea bass primary cultures versus RTgill-W1 cell line: influence of cell model on the sensitivity to nanoparticles. Nanomaterials 11:3136. https://doi.org/10.3390/nano11113136

Kadim IT, Mahgoub O, Baqir S, Faye B, Purchas R (2015) Cultured meat from muscle stem cells: a review of challenges and prospects. J Integr Agric 14:222–233

Kaur G, Dufour JM (2012) Cell lines: valuable tools or useless artifacts. Spermatogenesis 2:1–5

Kawano A, Haiduk C, Schirmer K, Hanner R, Lee LEJ, Dixon B, Bols NC (2011) Development of a rainbow trout intestinal epithelial cell line and its response to lipopolysaccharide. Aquac Nutr 2011:17. https://doi.org/10.1111/j.1365-2095.2010.00757.x

Kelly RK, Souter BW, Miller HR (1978) Fish cell lines: comparisons of CHSE-214, FHM, and RTG-2 in assaying IHN and IPN viruses. J Fish Res Board Can 35:1009–1011. https://doi.org/10.1139/f78-164

Kelly RK, Miller HR, Nielsen O, Clayton JW (1980) Fish cell culture: characteristics of a continuous fibroblastic cell line from walleye ( Stizostedion vitreum vitreum ). Can J Fish Aquat Sci 37:1070–1075

Kimura T, Yoshimizu M, Gorie S (1986) A new rhabdovirus isolated in Japan from cultured hirame (Japanese flounder) Paralichthys olivaceus and ayu Plecoglossus altivelis . Dis Aquat Organ 1:209–217

Kuhn C, Vielkind U, Anders F (1979) Cell cultures derived from embryos and melanoma of poeciliid fish. In Vitro 15:537–544

Kumar A, Singh N, Goswami M, Srivastava JK, Mishra AK, Lakra WS (2016) Establishment and characterization of a new muscle cell line of zebrafish ( Danio rerio ) as an in vitro model for gene expression studies. Anim Biotechnol 27:166–173

Kumar MS, Soni P, Singh N, Kumar R, Srivastava S, Mishra AK, Singh VK, Kushwaha B (2020) Establishment and characterization of eye muscle cell line from Snow Trout, Schizothorax richardsonii (Gray, 1832), a vulnerable coldwater fish, for in vitro studies. Turk J Fish Aquat Sci 21:95–105. https://doi.org/10.4194/1303-2712-v21_2_05

Kumar MS, Soni P, Kumar R, Singh N, Srivastava S, Mishra AK, Singh VK, Kushwaha B (2021) Development of caudal fin cell line from hill trout Barilius bendelisis (Hamilton, 1807) for cytotoxicity and transfection studies. Croat J Fish 79:15–24

Lai YS, John JA, Lin CH, Guo IC, Chen SC, Fang K, Lin CH, Chang CY (2003) Establishment of cell lines from a tropical grouper, Epinephelus awoara (Temminck & Schlegel), and their susceptibility to grouper irido- and noda-viruses. J Fish Dis 26:31–42

Laizé V, Rosa JT, Tarasco M, Cancela ML (2022) Status, challenges, and perspectives of fish cell culture-Focus on cell lines capable of in vitro mineralization. In: Fernández I, Fernandes J (eds) Cellular and molecular approaches in fish biology. Academic Press, Cambridge

Lakra WS, Goswami M, Thangaraj SR, Rathore G (2010a) Development and characterization of two new cell lines from common carp, Cyprinus carpio (Linn). Biol Res 43:385–392

Lakra WS, Thangraj RS, Rathore G, Goswami M, Yadav K, Kapoor S (2010b) Development and characterization of three new diploid cell lines from Labeo rohita (Ham.). Biotechnol Prog 26:1008–1013

Lakra WS, Goswami M, Yadav K, Gopalakrishnan A, Patiyal RS, Singh M (2011a) Development and characterization of two cell lines PDF and PDH from Puntius denisonii (Day 1865). In Vitro Cell Dev Biol Ani 47:89–94

Lakra WS, Thangaraj RS, Joy KP (2011b) Development, characterization, conservation and storage of fish cell lines: a review. Fish Physiol Biochem 37:1–20

Lammel T, Tsoukatou G, Jellinek J, Sturve J (2019) Development of three-dimensional (3D) spheroid cultures of the continuous rainbow trout liver cell line RTL-W1. Ecotoxicol Environ Saf 167:250–258

Langhans SA (2018) Three-dimensional in vitro cell culture models in drug discovery and drug repositioning. Front Pharmacol 9:6. https://doi.org/10.3389/fphar.2018.00006

Lannan CN, Winton JR, Fryer JL (1984) Fish cell lines: establishment and characterization of nine cell lines from salmonids. In Vitro 20:671–676

Lester K, Hall M, Urquhart K, Gahlawat S, Collet B (2012) Development of an in vitro system to measure the sensitivity to the antiviral Mx protein of fish viruses. J Virol Methods 182:1–8. https://doi.org/10.1016/j.jviromet.2012.01.014

Liu Q, Yuan Y, Zhu F, Hong Y, Ge R (2018) Efficient genome editing using CRISPR/Cas9 ribonucleoprotein approach in cultured Medaka fish cells. Biol Open 7(8):bio035170. https://doi.org/10.1242/bio.035170

Majeed AS, Nambi KS, Taju G, Hameed ASSAS (2013a) Development, characterization and application of a new fibroblastic-like cell line from kidney of a freshwater air breathing fish Channa striatus (Bloch, 1793). Acta Trop 127:25–32

Majeed AS, Nambi KS, Taju G, Sundar Raj N, Madan N, Sahul Hameed AS (2013b) Establishment and characterization of permanent cell line from gill tissue of Labeo rohita (Hamilton) and its application in gene expression and toxicology. Cell Biol Toxicol 29:59–73

Majeed AS, Nambi KS, Taju G, Sarath Babu V, Farook MA, Sahul Hameed AS (2014) Development and characterization of a new gill cell line from air breathing fish Channa striatus (Bloch 1793) and its application in toxicology: direct comparison to the acute fish toxicity. Chemosphere 96:89–98

Majeed AS, Nambi KSN, Taju G, Sahul Hameed AS (2015) Isolation, propagation, characterization, cryopreservation, and application of novel cardiovascular endothelial cell line from Channa striatus (Bloch, 1793). Cell Biochem Biophys 71:601–616. https://doi.org/10.1007/s12013-014-0240-x

Marques CL, Rafael MS, Cancela ML, Laize V (2007) Establishment of primary cell cultures from fish calcified tissues. Cytotechnology 55:9–13

Masters JRW (2010) Cell line misidentification: the beginning of the end. Nat Rev Cancer 10:441–448

Matsumoto J, Ishikawa T, Masahito P, Takayama S (1980) Permanent cell lines from erythrophoromas in goldfish ( Carassius auratus ). J Natl Cancer Inst 64:879–890

Minghetti M, Drieschner C, Bramaz N, Schug H, Schirmer K (2017) A fish intestinal epithelial barrier model established from the rainbow trout ( Oncorhynchus mykiss ) cell line. RTgutGC Cell Biol Toxicol 33:539–555

Minghetti M, Schirmer K (2019) Interference of silver nanoparticles with essential metal homeostasis in a novel enterohepatic fish in vitro system. Environ Sci Nano 6:1777–1790. https://doi.org/10.1039/c9en00310j

Nagpure NS, Kumar A, Dubey A, Mishra AK, Kumar R, Goswami M (2013) Establishment of National repository of fish cell lines (NRFC) at NBFGR. Lucknow Fishing Chimes 33:33–38

Nagpure NS, Mishra AK, Ninawe AS, Rasal A, Dubey A, Kumar A, Goswami M, Kumar R, Jena JK (2016) Molecular and cytogenetic characterization of fish cell lines and its application in aquatic research. Natl Acad Sci Lett 39:11–16

Nambi KSN, Majeed AS, Taju SG, Sivasubbu S, Sarath Babu V, Sahul Hameed AS (2017) Effects of nicotine on zebrafish: a comparative response between a newly established gill cell line and whole gills. Comp Biochem Physiol 195C:68–77

Nanthini R, Majeed SA, Vimal S, Taju G, Sivakumar S, Santhosh Kumar S, Sahul Hameed AS (2019) In vitro propagation of tilapia lake virus in cell lines developed from Oreochromis mossambicus . J Fish Dis 42:1543–1552

Neimark J (2015) Line of attack. Science 347:938–940

Neukirch M, Haenen OLM (2004) Susceptibility of CCB cell line to different fish viruses. Bull Eur Assoc Fish Pathol 24:209–211

Noga EJ, Hartmann JX (1981) Establishment of walking catfish ( Clarias batrachus ) cell lines and development of a channel catfish ( Ictalurus punctatus ) virus vaccine. Can J Fish Aquat Sci 38:925–930

Ostrander GK, Blair JB, Stark BA, Marley GM, Bales WD, Veltri RW, Hinton DE, Okihiro M, Ortego LS, Hawkins WE (1995) Long-term primary culture of epithelial cells from rainbow trout ( Oncorhynchus mykiss ) liver. In Vitro Cell Dev Biol Anim 31:367–378

Parameswaran V, Shukla R, Bhonde RR, Sahul Hameed AS (2006) Splenic cell line from sea bass, Lates calcarifer : establishment and characterization. Aquaculture 261:43–53

Parameswaran V, Ahmed VPI, Shukla R, Bhonde RR, Hameed ASS (2007) Development and characterization of two new cell lines from milkfish ( Chanos chanos ) and grouper ( Epinephelus coioides ) for virus isolation. Mar Biotechnol 9:281–291

Pasquariello R, Verdile N, Pavlovic R, Panseri S, Schirmer K, Brevini TAL, Gandolfi E (2021) New stable cell lines derived from the proximal and distal intestine of Rainbow Trout ( Oncorhynchus mykiss ) retain several properties observed in vivo . Cells 10:1555. https://doi.org/10.3390/cells10061555

Paw BH, Zon LI (1999) Primary fibroblast cell culture. Methods Cell Biol 59:39–43

Peng WC, Logan CY, Fish M, Anbarchian T, Aguisanda F, Álvarez-Varela A, Wu P, Jin Y, Zhu J, Li B, Grompe M, Wang B, Nusse R (2018) Inflammatory cytokine TNFα promotes the long-term expansion of primary hepatocytes in 3D culture. Cell 175:1607–1619

Perez-Prieto SI, Rodriguez-Saint-Jean S, Garcia-Rosado E, Castro D, Alvarez MC, Borrego JJ (1999) Virus susceptibility of the fish cell line SAF-1 derived from gilt-head seabream. Dis Aquat Organ 35:149–153

Perry GM, McDonald GJ, Ferguson MM, Ganassin RC, Bols NC (2001) Characterization of rainbow trout cell lines using microsatellite DNA profiling. Cytotechnology 37:143–151. https://doi.org/10.1023/A:1020516804173

Pham PH, Misk E, Papazotos F, Jones G, Polinski MP, Contador E, Russel S, Garver KA, Lumsden JS, Bols NC (2020) Screening of fish cell lines for piscine orthoreovirus-1 (PRV-1) amplification: identification of the non-supportive PRV-1 invitrome. Pathogens 9:833. https://doi.org/10.3390/pathogens9100833

Post MJ, Levenberg S, Kaplan DL, Genovese N, Fu J, Bryant CJ, Negowetti N, Verzijden K, Moutsatsou P (2020) Scientific, sustainability and regulatory challenges of cultured meat. Nature Food 1:403–415

Potter G, Alec ST, Smith NTK, Vo JM, Weston W, Bertero A, Maves L, Mack DL, Rostain A (2020) A more open approach is needed to develop cell-based fish technology: it starts with zebrafish. One Earth 3:54–64

Rastogi A, Yadav MK, Joaquin MPC, Verma DK, Thangaraj RS, Kushwaha B, Paria A, Pradhan PK, Sood N (2022) Development of cell lines from brain, spleen and heart of ornamental blood parrot cichlid and their susceptibility to Tilapia tilapinevirus . Aquaculture 561:738711. https://doi.org/10.1016/j.aquaculture.2022.738711

Rodd AL, Messier NJ, Vaslet CA, Kane AB (2017) A 3D fish liver model for aquatic toxicology: morphological changes and Cyp1a induction in PLHC-1 microtissues after repeated benzo(a)pyrene exposures. Aquat Toxicol 186:134–144

Romano P, Manniello A, Aresu O, Armento M, Cesaro M, Parodi B (2009) Cell line data base: structure and recent improvements towards molecular authentication of human cell lines. Nucleic Acids Res 37:D925–D932

Rubio N, Datar I, Stachura D, Kaplan D, Krueger K (2019) Cell-based fish: a novel approach to seafood production and an opportunity for cellular agriculture. Front Sustain Food Syst 3:43

Saad MK, Yuen JSK Jr, Joyce CM, Li X, Lim T, Wolfson TL, Wu J, Laird J, Vissapragada S, Calkins OP, Ali A, Kaplan DL (2023) Continuous fish muscle cell line with capacity for myogenic and adipogenic-like phenotypes. Sci Rep 13:5098. https://doi.org/10.1038/s41598-023-31822-2

Schirmer K, Fischer M, Eszter S, Andersen S, Kunz P, Lillicrap A (2021) Validation report for the test guideline 249 on fish cell line acute toxicity - the RTgill-W1 cell line assay (Series on testing and assessment, Report No.: 334). doi: https://doi.org/10.1787/c66d5190-en

Shamir ER, Ewald AJ (2014) Three-dimensional organotypic culture: experimental models of mammalian biology and disease. Nat Rev Mol Cell Biol 15:647–664

Singh N, Soni P, Kushwaha B, Kumar MS, Srivastava JK, Srivastava S, Mishra AK, Kumar R (2021) Establishment of a testis cell line from Clarias magur : a potential resource for in-vitro applications. Nucleus 64:211–217. https://doi.org/10.1007/s13237-020-00345-w

Soni P, Pradhan PK, Thangaraj RS, Sood N (2018) Development, characterization and application of a new epithelial cell line from caudal fin of Pangasianodon hypophthalmus (Sauvage 1878). Acta Trop 182:215–222

Sood N, Chaudhary DK, Pradhan PK, Verma DK, Thangaraj RS, Kushwaha B, Punia P, Jena JK (2015) Establishment and characterization of a continuous cell line from thymus of striped snakehead, Channa striatus (Bloch 1793). In Vitro Cell Dev Biol Ani 51:787–796

Stulberg CS, Coriell LL, Kniazeff AJ, Shannon JE (1970) The animal cell culture collection. In Vitro 5:1–16

Suryakodi S, Majeed SA, Taju G, Vimal S, Sivakumar S, Ahmed AN, Shah FA, Bhat SA, Sarma D, Begum A, Sahul Hameed AS (2021) Development and characterization of novel cell lines from kidney and eye of rainbow trout, Oncorhynchus mykiss, for virological studies. Aquaculture 532:736027. https://doi.org/10.1016/j.aquaculture.2020.736027

Suyama I, Etoh H (1979) A cell line derived from the fin of the goldfish, Carassius auratus . Dobutsugaku Zasshi 88:321–324

Suyama I, Etoh H (1988) Establishment of a cell line from Umbra limi (Umbridae; Pisces). In: Kuroda Y, Kurstak E, Maramorosch K (eds) Invertebrate and fish tissue culture. Springer, Berlin

Taju G, Majeed AS, Nambi KS, Sahul Hameed AS (2013) Development and characterization of cell line from the gill tissue of Catla catla (Hamilton, 1822) for toxicological studies. Chemosphere 90:2172–2180

Taju G, Majeed AS, Nambi KS, Farook MA, Vimal S, Sahul Hameed AS (2014a) In vitro cytotoxic, genotoxic and oxidative stress of cypermethrin on five fish cell lines. Pestic Biochem Physiol 113:15–24

Taju G, Majeed AS, Nambi KSN, Sahul Hameed AS (2014b) In vitro assay for the toxicity of silver nanoparticles using heart and gill cell lines of Catla catla and gill cell line of Labeo rohita . Com Biochem Phys C61:41–52

Thangaraj RS, Basheer VS, Gopalakrishnan A, Rathore G, Chaudhary DK, Kumar R, Jena JK (2013) Establishment of caudal fin cell lines from tropical ornamental fishes Puntius fasciatus and Pristolepis fasciata endemic to the Western Ghats of India. Acta Trop 128:536–541

Thangaraj RS, Lakra WS, Gopalakrishnan A, Basher VS, Kushwaha B, Sajeela KA (2010) Development and characterization of a new epithelial cell line PSF from caudal fin of Green chromide,  Etroplus suratensis  (Bloch, 1790). In Vitro Cell Dev Biol Animal 46:647–656. https://doi.org/10.1007/s11626-010-9326-y

Thangaraj RS, Basheer VS, Kumar R, Kathirvelpandian A, Sood N, Jena JK (2015) Establishment and characterization of fin-derived cell line from ornamental carp, Cyprinus carpio koi , for virus isolation in India. In Vitro Cell Dev Biol Anim 51:705–713

Thangaraj RS, Basheer VS, Gopalakrishnan A, Sood N, Pradhan PK (2016a) A new epithelial cell line, HBF from caudal fin of endangered yellow catfish, Horabagrus brachysoma (Gunther, 1864). Cytotechnology 68:515–523

Thangaraj RS, Kumar R, Jency PM, Charan R, Syamkrishnan MU, Basheer VS, Sood N, Jena JK (2016b) A new fish cell line derived from the caudal fin of freshwater angelfish Pterophyllum scalare , development and characterization. J Fish Biol 89:1769–8110

Thangaraj RS, Ravi C, Kumar R, Dharmaratnam A, Saidmuhammed BV, Pradhan PK, Sood N (2018) Derivation of two tilapia ( Oreochromis niloticus ) cell lines for efficient propagation of Tilapia Lake Virus (TiLV). Aquaculture 492:206–214

Thangaraj RS, Dharmaratnam A, Raja SA, Raj NS, Lal KK (2020) Establishment and cryopreservation of a cell line derived from caudal fin of endangered catfish Clarias dussumieri Valenciennes, 1840. J Fish Biol 96:722–730

Thangaraj RS, Narendrakumar L, Geetha PP, Shanmuganathan AR, Dharmaratnam A, Nithianantham SR (2021) Comprehensive update on inventory of finfish cell lines developed during the last decade (2010–2020). Rev Aquacult. https://doi.org/10.1111/raq.12566

Thangaraj RS, Nithianantham SR, Narendrakumar L, Johny TK, Sood N, Pradhan PK, Lal KK (2022) Cichlids endemic to India are not susceptible to Tilapia Lake virus infection. Aquaculture 548:737589. https://doi.org/10.1016/j.aquaculture.2021.737589

Verma PJ, Sumer H (2015) Cell reprogramming: methods and protocols, methods in molecular biology. Humana Press, New York. https://doi.org/10.1007/978-1-4939-2848-4_10

Book   Google Scholar  

Villena AJ (2003) Applications and needs of fish and shellfish cell culture for disease control in aquaculture. Rev Fish Biol Fisher 13:111–140

Vo NGK (2022) The sine qua non of the fish invitrome today and tomorrow in environmental radiobiology. Int J Radiat Biol 98:1025–1033. https://doi.org/10.1080/09553002.2020.1812761

Winton J, Batts W, deKinkelin P, LeBerre M, Bremont M, Fijan N (2010) Current lineages of the epithelioma papulosum cyprini (EPC) cell line are contaminated with fathead minnow, Pimephales promelas, cells. J Fish Dis 8:701–704. https://doi.org/10.1111/j.1365-2761.2010.01165.x

Wolf K, Ahne W (1982) Fish cell culture. Advances in Cell Culture 2:305–328

Wolf K, Quimby MC (1962) Established eurythermic line of fish cells in vitro. Science 135:1065–1066

Wolf K, Bullock GL, Dunbar CE, Quimby MC (1968) Tadpole edema virus: a viscerotropic pathogen for anuran amphibians. J Infect Dis 118:253–262

Wolf K, Mann JA (1980) Poikilotherm vertebrate cell lines and viruses: a current listing for fishes. In Vitro 16(2):168–179

Yadav K, Lakra WS, Sharma J, Goswami M, Singh A (2012) Development and characterization of a cell line TTCF from endangered mahseer Tor tor (Ham.). Fish Physiol Biochem 38:1035–1045

Yadav MK, Rastogi A, Joaquin MPC, Verma DK, Rathore G, Thangaraj RS, Paria A, Pradhan PK, Sood N (2021) Establishment and characterization of a continuous cell line from heart of Nile tilapia Oreochromis niloticus and its susceptibility to tilapia lake virus. J Virol Methods 287:113989. https://doi.org/10.1016/j.jviromet.2020.113989

Yadav MK, Rastogi A, Verma DK, Paria A, Kushwaha B, Rathore G, Thangaraj RS, Pradhan PK, Sood N (2022) Establishment and characterization of a continuous cell line from caudal fin of Labeo calbasu (Hamilton, 1822). Cell Biol Int 46:1299–1304

Yip JH, Bols NC (1982) The fusion of trout spermatozoa with Chinese hamster fibroblasts. J Cell Sci 53:307–321

Zeng WR, Beh SJ, Bryson-Richardson RJ, Doran PM (2017) Production of zebrafish cardiospheres and cardiac progenitor cells in vitro and three-dimensional culture of adult zebrafish cardiac tissue in scaffolds. Biotechnol Bioeng 114:2142–2148

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The authors are grateful to the Secretary, DARE and Director General, ICAR, Ministry of Agriculture and Farmers’ Welfare, New Delhi; Dr. J K Jena, DDG (Fy.), ICAR, New Delhi, India; and Director, ICAR-NBFGR, Lucknow, for their support, encouragement and guidance. The authors are also thankful to the Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi, for the financial support to execute the National Fish Cell Line Repository (Phase I and II) in project modes at ICAR-NBFGR, Lucknow, India.

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Kumar, M.S., Singh, V.K., Mishra, A.K. et al. Fish cell line: depositories, web resources and future applications. Cytotechnology 76 , 1–25 (2024). https://doi.org/10.1007/s10616-023-00601-2

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ORIGINAL RESEARCH article

Derivation of breast cancer cell lines under physiological (5%) oxygen concentrations.

• 1 Auckland Cancer Society Research Centre, University of Auckland, Auckland, New Zealand
• 2 Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand
• 3 Univ Lyon, Claude Bernard University, Cancer Research Center of Lyon, INSERM 1052, CNRS5286, Faculty of Pharmacy, Lyon, France
• 4 Hospices Civils de Lyon, Molecular Biology of Tumors, GHE Hospital, Bron, France
• 5 Auckland City Hospital—Oncology, Grafton, Auckland, New Zealand

Background: Most human breast cancer cell lines currently in use were developed and are cultured under ambient (21%) oxygen conditions. While this is convenient in practical terms, higher ambient oxygen could increase oxygen radical production, potentially modulating signaling pathways. We have derived and grown a series of four human breast cancer cell lines under 5% oxygen, and have compared their properties to those of established breast cancer lines growing under ambient oxygen.

Methods: Cell lines were characterized in terms of appearance, cellular DNA content, mutation spectrum, hormone receptor status, pathway utilization and drug sensitivity.

Results: Three of the four lines (NZBR1, NZBR2, and NZBR4) were triple negative (ER-, PR-, HER2-), with NZBR1 also over-expressing EGFR. NZBR3 was HER2+ and ER+ and also over-expressed EGFR. Cell lines grown in 5% oxygen showed increased expression of the hypoxia-inducible factor 1 (HIF-1) target gene carbonic anhydrase 9 ( CA9 ) and decreased levels of ROS. As determined by protein phosphorylation, NZBR1 showed low AKT pathway utilization while NZBR2 and NZBR4 showed low p70S6K and rpS6 pathway utilization. The lines were characterized for sensitivity to 7-hydroxytamoxifen, doxorubicin, paclitaxel, the PI3K inhibitor BEZ235 and the HER inhibitors lapatinib, afatinib, dacomitinib, and ARRY-380. In some cases they were compared to established breast cancer lines. Of particular note was the high sensitivity of NZBR3 to HER inhibitors. The spectrum of mutations in the NZBR lines was generally similar to that found in commonly used breast cancer cell lines but TP53 mutations were absent and mutations in EVI2B, LRP1B , and PMS2 , which have not been reported in other breast cancer lines, were detected. The results suggest that the properties of cell lines developed under low oxygen conditions (5% O 2 ) are similar to those of commonly used breast cancer cell lines. Although reduced ROS production and increased HIF-1 activity under 5% oxygen can potentially influence experimental outcomes, no difference in sensitivity to estrogen or doxorubicin was observed between cell lines cultured in 5 vs. 21% oxygen.

Introduction

The humidified atmosphere used in most human tumor cell culture systems includes carbon dioxide and ambient oxygen (21%). This is considerably higher than that in vivo , which is thought to average 5% (range 1–8%) as a consequence of oxygen transport, consumption in tissues and diffusion from the blood supply to the tissues ( 1 , 2 ). The difference raises the question of whether the oxygen concentration in air, for instance by generating increased reactive oxygen species (ROS), might modulate signaling pathways in “regular” cell culture conditions and thus alter responses to therapeutic agents ( 3 , 4 ). In our previous studies, human melanoma specimens were cultured in an atmosphere of 5% O 2 , 5% CO 2 , 90% N 2 to minimize oxygen-mediated damage ( 5 ) and in subsequent studies, these conditions were used in the development of more than 100 melanoma lines ( 6 , 7 ) and more than 50 carcinoma lines ( 8 ). In this report, we describe four human breast cancer cell lines that were developed under these conditions, to ascertain whether their properties, including receptor status, signaling pathway utilization, mutations and drug responses, were comparable to those of established cell lines.

Materials and Methods

Cell culture.

Culture conditions have been described ( 9 , 10 ) for MCF-7, SKBr3, MDA-MB-231, BT474, and ZR75.1 cells [purchased from the American Type Culture Collection (ATCC)]. Cell lines were grown in α-modified minimal essential medium (αMEM; Invitrogen) supplemented with 5 μg/mL insulin, 5 μg/mL transferrin and 5 ng/mL sodium selenite (ITS; Roche Applied Sciences), 100 U/mL penicillin, 100 μg/mL streptomycin, and 5% fetal bovine serum (FBS). NZBR1, NZBR2, NZBR3 and NZBR4 cell lines were grown under low oxygen conditions (5% O 2 , 5% CO 2 , 90% N 2 ) to mimic physiologically oxygen levels in tumors. The cell lines of ATCC provenance were cultured in an atmosphere of 5% CO 2 in air at 37°C.

For estrogen response, MCF-7 and NZBR3 cell lines were cultured in estrogen deprived media (phenol red-free RPMI 1640 containing 10% charcoal-stripped fetal bovine serum from ICPbio International Ltd., Auckland, NZ and Invitrogen, Auckland, NZ), and penicillin/streptomycin (10 U/ml and 10 μg/ml, respectively) for the 2 days before 17β-estradiol treatment. Estrogen-deprived medium was used for all assays related to estrogen stimulation.

As described previously ( 8 ), tumor tissue taken from four patients undergoing surgery for breast cancer was sent to the histopathologist immediately after surgery, where a portion was placed into growth medium without serum for laboratory studies. Formal consent was obtained from all patients, using guidelines approved by the Northern A Health and Disability Ethics Committee.

Solid tumor specimens were disaggregated either immediately or after overnight storage at 4°C. Normal, adipose, or grossly necrotic material was removed and the tumor tissue was minced finely using crossed scalpels. Tissue was reduced to small clumps by passage through a 0.65-mm stainless steel sieve. The size of the aggregates varied greatly (aggregates of 5–100 cells). Material containing larger aggregates was pipetted into tubes, collected by low-speed centrifugation to remove blood cells, necrotic material, and debris (30 × g, 2 min), and then washed twice (30 × g, 2 min) in growth medium. Preparations were monitored by phase contrast microscopy, and cytospins of cell suspensions were stained by hematoxylin/eosin and examined by a pathologist to ensure that they contained tumor cells. Cultures were set up in growth medium supplemented with 5% FBS under an atmosphere of 5% O 2 , 5% CO 2 , and 90% N 2 in a Tri-Gas Forma incubator. Tissue culture plates or flasks had previously been coated with a thin layer of agarose to prevent the growth of fibroblasts ( 5 ).

Early passage cell lines (< passage 33) used in this study were developed in this laboratory. All cell lines were tested negative for mycoplasma contamination.

Chemicals and Reagents

Everolimus, afatinib, dacomitinib, lapatinib and ARRY-380 was purchased from Selleck Chemicals (Houston, USA). 17β-estradiol, tamoxifen, paclitaxel and doxorubicin were purchased from Sigma (Auckland, NZ). NVP-BEZ235 ( 11 , 12 ) was synthesized according to published protocols.

Short Tandem Repeat Profiling

The NZBR cell lines were typed by short tandem repeat profiling by DNA Diagnostics (Auckland, New Zealand) (Table S1 ). The combination of markers selected was consistent with the National Institute of Standards and Technology database recommendations for identity testing.

Cell Proliferation Assay

As described previously ( 9 , 10 , 13 , 14 ), proliferation was measured using a thymidine incorporation assay. Cells were seeded (3,000 per well) in 96 well plates in the presence of varying concentrations of inhibitors for 3 days. 3 H-thymidine (0.04 μCi per well) was added to each well and cultures were incubated for 6 h; cells were harvested on glass fiber filters using an automated TomTec harvester. Filters were incubated with Betaplate Scint and thymidine incorporation measured in a Trilux/Betaplate counter. Effects of inhibitors on the incorporation of 3 H-thymidine into DNA were determined relative to the control (non-drug-treated) cells.

For the growth response to 17β-estradiol in 5 and 21% oxygen culture conditions, cells were seeded at 2,500 per well in 96 well plates in the presence or absence of 50 nM 17β-estradiol for 3 days. For the growth response to doxorubicin in 5 and 21% oxygen culture conditions, cells were seeded at 1,000 per well in 96 well plates in the presence of varying concentrations of inhibitors for 5 days.

Measurement of Reactive Oxygen Species (ROS)

Intracellular ROS were detected with the cell-permeable fluorescent probe 2',7'- dichlorofluorescein diacetate (DCFDA) (ABCAM) according to the manufacturer's instruction. Cells (1 × 10 5 ) were harvested, incubated with 20 μM DCFDA in medium for 60 min in the dark at 37°C. Cells were analyzed in a BD Accuri™ Flow Cytometer.

Reverse Transcription, cDNA Synthesis, and Quantitative PCR

As described in detail ( 15 ), oligo-dT and random primers were used to reverse transcribe RNA with qScript Flex cDNA kit (Quantabio) according to the manufacturer's instructions. To investigate whether estrogen exposure can lead to increased expression of estrogen response genes and whether cell lines grown in 5% oxygen conditions can induce carbonic anhydrase IX ( CA9 ), which is the most well established target of HIF, quantitative RT-PCR (qRT-PCR) was performed using gene-specific primers (Table S2 ) and Sybr Green Master Mix (Life Technologies), and expression values were normalized relative to HPRT1 RNA expression. The 2 ∧ (–delta delta CT) method was used to analyze the relative changes in gene expression.

Western Blotting

As described ( 9 , 10 , 16 ), breast cancer cell lines were grown to log-phase, washed twice with ice-cold PBS, and lysed in SDS lysis buffer according to the manufacturer's protocol (Cell Signaling Technology, Danvers, MA). Protein concentration was quantified using the bicinchoninic acid reagent (Sigma). Cell lysates containing 25 μg of protein were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to PVDF membranes (Millipore). Membranes were immunoblotted with antibodies against phospho-AKT (S473), total AKT, phospho-p70S6K (T389), total p70S6K, phospho-rpS6 (S235/ 236), total rpS6, phospho-ERK (T202/Y204), total ERK (all from Cell Signaling Technology), tubulin (Sigma), and actin (Millipore). Bound antibody was visualized using SuperSignal West Pico (Thermo Scientific, Waltham, MA) or ECL plus (GE Healthcare, Auckland, NZ) and the chemiluminescence detection system by Fujifilm Las-3000.

Genomic Analysis

For whole exome sequencing (WES), 250 ng of genomic DNA from each cell line was sheared using the EpiShear™ Multi-Sample Sonicator (Active Motif). The quantity and fragment size of the sheared DNA were assessed on a Tapestation 2200 (Agilent) with the high sensitivity D1000 tape. Sheared DNA (100 ng) was used for the preparation of the whole exome libraries (WELs). WELs were prepared using the SureSelect XT2 (SSXT2) reagent kit and the SureSelect Clinical Research Exome V2 exome enrichment kit following the manufacturer's instructions (Agilent Technologies). The WELs were sequenced on a NextSeq500 (NCS v2.0, Illumina Inc.) to obtain around 40 to 44 million paired end reads (2 × 150 bp, 12 to 13 Gbp) per exome.

The quality of the sequences was assessed using Fastqc ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). The reads were aligned to the human reference genome (hg19) with BWA (bwa 0.7.12) ( 17 ). The resulting sam files were converted to bam and then the bam files were sorted using the samtools (Samtools-1.3.1) ( 18 ). Mpileup files were generated (Samtools-1.3.1) with the following parameters: maximum depth (-d) 500, minimum base quality (-Q) 15 and minimum mapping quality (-q) 10. Varscan v2.3.9 ( 19 ) was used to call variants and to generate VCF (variant call format) files ( 20 ). The variants in the vcf files were annotated with information from various SNP databases (dbSNP138 etc) using ANNOVAR ( 21 ) followed by the annotation for the variants' effect with SnpEffect ( 22 ). Variants present in the 1000genome_Oct2014 database were excluded. Variants predicted to have a “High” (e.g., nonsense) or “Moderate” (missense) impact in genes in the two breast cancer gene lists (see Results Section) were selected using SnpSift ( 23 ).

Data Analysis

T -tests or Mann-Whitney Rank Sum Tests was used for comparison of groups. Correlation analysis was performed with Spearman Rank Order correlation coefficient (R) and statistical significance (P) using SigmaPlot (Systat Software, Inc.). Values of P < 0.05 were considered to be statistically significant.

Initial Characterization of NZ Breast Cancer Cell Lines

The cell lines were characterized by cellular DNA content, hormone receptor expression and tamoxifen sensitivity. The lines were all aneuploid and three of the four lines (NZBR1, NZBR2, and NZBR4) were triple-negative with no expression of estrogen receptor, progesterone receptor and HER2 (Table 1 ; Figure 1A ). The ER+ NZBR3 cell line was sensitive to tamoxifen with an IC50 value of 60 nM (Figure 1B ). The triple-negative cell lines were relatively resistant to tamoxifen with IC50 values of >1000, 390, and >1000 nM, respectively.

Table 1 . Source, clinical and pathological features of tumors used to derive the New Zealand Breast Cancer cell lines, and DNA ploidy.

Figure 1 . Receptor expression, and growth inhibitory concentrations for NZBR cell lines exposed to tamoxifen. (A) Relative expression of estrogen receptor (ER), progesterone receptor (PR), HER2 and EGFR in NZBR1, NZBR2, NZBR3, and NZBR4 breast cancer cell lines. Actin is shown as loading control. (B) IC50 values (50% inhibition of growth) for tamoxifen, shown as the mean ± standard error of triplicate experiments. The highest tamoxifen concentration used (1,000 nM) is indicated where 50% growth inhibition was not reached.

Sensitivity of ER+ MCF-7 and NZBR3 to Estrogen

A [ 3 H]-thymidine incorporation assay was used to assess the effect of estrogen on cell proliferation when the cells were cultured under 5 and 21% oxygen conditions. Both MCF-7 and NZBR3 cell lines displayed significant growth stimulation by estrogen and MCF-7 cells showed increased response to estrogen as compared to NZBR3. However, the two oxygen concentrations did not have distinguishable effects on the growth response to estrogen (Figure 2A ).

Figure 2 . Sensitivity of ER + MCF-7 and NZBR3 cells to estrogen. (A) Relative growth of MCF-7 and NZBR3 cells in response to 17β-estradiol was measured using thymidine incorporation when cells were cultured under 5 or 21% oxygen conditions. (B) Comparison of estrogen response gene ( GREB1, CCND1 , and TFF1 ) expression measured by quantitative RT-PCR in breast cancer cells exposed to 17β-estradiol (50 nM) for 24 h. Results are shown as the mean ± standard error of triplicate experiments. * p < 0.05.

We next examined the expression of three estrogen-responsive genes in these two cell lines; namely Growth Regulation By Estrogen In Breast Cancer 1 ( GREB1 ), Cyclin D1 ( CCND1 ) and Trefoil Factor 1 ( TTF1 ) ( 24 ). Both GREB1 and TFF1 showed significant upregulation with estrogen stimulation (Figure 2B ).

Comparison of Reactive Oxygen Species (ROS) Production in Cells Cultured Under 5 and 21% Oxygen Condition

As ROS production can be affected by the oxygen concentration during culture, we measured intracellular ROS levels by using the hydrogen peroxide-sensitive dye 2′,7′ –dichlorofluorescein diacetate (DCFDA) in cells cultured under 5 and 21% oxygen concentrations. A significant increase in ROS concentrations was observed in cells cultured in 21% oxygen (pairwise T - test, p < 0.05) (Figure 3A ).

Figure 3 . Differential response to culture conditions of 5 and 21% oxygen. (A) Relative ROS concentration in cells cultured under 5 or 21% oxygen conditions as measured by DCFDA fluorescence. (B) Comparison of hypoxia target gene CA9 expression measured by quantitative RT-PCR in breast cancer cells under 5 or 21% oxygen conditions. Results are shown as the mean ± standard error of triplicate experiments. * p < 0.05.

Expression of Hypoxia Inducible Factor 1-Regulated Carbonic Anhydrase IX ( CA9 ) Under 5 and 21% Oxygen Conditions

Oxygen concentration can affect the expression of hypoxia-inducible factor 1 (HIF-1)-regulated genes, including carbonic anhydrase IX ( CA9 ) ( 25 ). Cell lines were cultured at oxygen concentrations of 5 and 21% for over 2 weeks before testing for the expression of CA9 . Apart from NZBR1, which had no measurable transcript by RT-qPCR, all cell lines showed decreased CA9 expression in 21% compared with 5% oxygen (Figure 3B ).

mTOR Pathway Signaling and Sensitivity to mTOR Inhibitor Everolimus and Dual PI3K and mTOR Inhibitor BEZ235

The phosphorylation status of AKT, p70S6K, rpS6, and ERK in the NZBR lines was examined (Figure 4A ). NZBR1 showed the lowest degree of phosphorylated AKT expression, yet both NZBR1 and NBZR3 had comparable levels of phosphorylated p70S6K (mTOR pathway effector). On the other hand, both NZBR2 and NZBR4 contained low levels of phosphorylated p70S6K, indicating that they had low mTOR pathway utilization. NZBR2 and NZBR4 showed a low degree of phosphorylated rpS6, whereas NZBR1 had low levels of phosphorylated ERK.

Figure 4 . Signaling pathway usage and growth inhibitory concentrations for NZBR cell lines exposed to different drugs. (A) Signaling pathway usage as measured by phosphorylation of AKT, p70S6K, rpS6 and ERK. Immunoblots with antibodies specific for phosphorylated proteins and their respective total protein are indicated. Tubulin is the loading control. IC50 values for (B) everolimus, and (C) NVP-BEZ235, shown as the mean ± standard error of triplicate experiments. Cell lines are identified as estrogen receptor (ER) positive, HER2 positive and triple negative (TN) (orange, here and below).

We have shown that estrogen receptor positive (ER+) breast cancer cell lines generally are more sensitive to everolimus than are receptor negative lines ( 9 ). However, ER expression is not a requirement for an everolimus response since some ER negative breast cancer cell lines are sensitive to growth inhibition by everolimus ( 9 ), as exemplified here by the TN NZBR2 cells, which are exquisitely sensitive to everolimus (Figure 4B ). The degree of signaling via the PI3K/AKT/mTOR pathway did not correlate with growth inhibitory responses to everolimus or BEZ235 treatment (Figures 4B,C ).

Two out of the three triple-negative NZBR cell lines were resistant to everolimus, with IC50 values of over 500 nM (Figure 4B ) in a 3-day 3 H-thymidine incorporation assay. The ER+ HER2+ NZBR3 cells showed high sensitivity to everolimus (IC50 11.5 nM), yet the triple-negative NZBR2 with low mTOR pathway utilization also showed increased everolimus sensitivity (IC50 1.1 nM) (Figure 4B ). Significant differences ( p < 0.05) between NZBR2 and the other three cell lines in BEZ235 sensitivity were also observed.

Sensitivity to Paclitaxel and Doxorubicin

The sensitivities of the NZBR cell lines, and the ER+ MCF-7, HER2+ SKBR3 and triple-negative MDA-MB-231 cell lines to the therapeutic agents doxorubicin (a topoisomerase II poison) and paclitaxel (a microtubule poison) were tested (Figure 5 ). The ER+ HER2+ NZBR3 cell line was least sensitive of all the cell lines to both paclitaxel and doxorubicin.

Figure 5 . Sensitivity of NZBR cell lines to cytotoxic drugs. IC50 values for (A) paclitaxel and (B) doxorubicin, shown as the mean ± standard error of triplicate experiments.

Since doxorubicin-induced release of free radicals may cause oxidative stress ( 26 ), we compared the sensitivity to doxorubicin when the cell lines were cultured under 5 vs. 21% oxygen conditions. No significant difference (pairwise T -test, p > 0.05) in sensitivity to doxorubicin was observed when the cell lines were cultured in 5% vs. 21% oxygen (Figure S1 ).

Sensitivity to HER2 Inhibitors Lapatinib, Afatinib, Dacomitinib and ARRY-380

We compared the sensitivity of the NZBR cell lines and other breast cancer cell lines with or without ER or HER2 to the HER2 inhibitors lapatinib, afatinib, dacomitinib and ARRY-380 (ONT-380; irbinitinib; tuncatinib). As expected, drug resistance was observed in the HER2- cell lines; however, the HER2+ cell line ZR75.1 also showed similar resistance (Figure 6 ). HER2+ NZBR3 cells showed high sensitivity to all HER2 inhibitors tested. These data indicate that HER2 expression is necessary but not sufficient for augmented sensitivity to HER2 inhibitors. HER2 inhibitor sensitivity was not dependent on co-expression of ER.

Figure 6 . Sensitivity of breast cancer cell lines exposed to kinase inhibitors. IC50 values for afatinib, dacomitinib, lapatinib and ARRY-380, shown as the mean ± standard error of triplicate experiments. The highest inhibitor concentration used (1,000 nM) is indicated where 50% growth inhibition was not reached.

Effects of ARRY-380 on Signal Transduction in Four HER2+ Breast Cancer Cell Lines

The effects of ARRY-380 on HER2, AKT, and ERK activation were examined in four HER2+ breast cancer cell lines, which showed a range of sensitivities to ARRY-380. After overnight exposure to 100 nM or 1000 nM of ARRY-380, we observed no effect on total HER2, AKT and ERK expression in the cell lines. ARRY-380 inhibited HER2 phosphorylation in all lines, and ERK phosphorylation in cell lines with detectable phosphorylation (Figure 7 ). Of interest, the HER2 inhibitor-resistant ZR75.1 cell line showed minimal suppression of AKT phosphorylation by ARRY-380 as compared to the other three HER2 inhibitor sensitive lines (Figures 6 , 7 ). Even though ZR75.1 cells showed loss of HER2 phosphorylation in response to ARRY-380, this was not accompanied by any diminution of the phosphorylation of ERK.

Figure 7 . Sensitivity of signaling pathways in ZR75.1, SKBR3, BT474 and NZBR3 breast cancer cell lines to ARRY-380. Cells were treated with ARRY-380 for 24 h and signaling pathway usage was measured by phosphorylation of HER2, AKT, and ERK. Immunoblots with antibodies specific for phosphorylated proteins are indicated. Actin is the loading control.

To examine the mutation status of these cell lines, the following genes ( AKT1, ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, FAM175A, FANCC, MEN1, MRE11A, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, ALB2, PIK3CA, PMS1, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53 , and XRCCC2 ) were selected from the hereditary breast cancer panel of AmbryGenetics, GeneDx, and University of Washington. The somatic mutations gene list ( GATA3, ESR1, TBX3, RUNX1, NCOR1, KMT2C, SPEN, ARID1A, NOTCH2, MAP2K4, RB1, MTOR, MED12, and LRP1B ) was selected from somatic mutations in the cancer (COSMIC) database. Several sequence variants that result in missense or nonsense mutations were detected in genes associated with breast cancer (Table 2 ). Since germline DNA samples for these cell lines were not available, it is not known whether these mutations were of somatic or germline origin. We detected mutations in BRCA2, AKT1, NF1, EVI2B, MRE11A, ATM, BRCA2, MLH1, PMS2 , and LRP1B (Table 2 ).

Table 2 . Nine genes with mutations in the New Zealand breast cancer cell lines.

Most mutated genes found in the NZBR cell lines had also been found to be mutated in other breast cancer cell lines (Table 3 ). The mutation frequencies detected in comparison to those of previously characterized breast cancer cell lines and breast carcinomas are listed in Table S3 . The spectrum of mutations was generally similar to that found in commonly used breast cancer cell lines, although EVI2B, LRP1B , and PMS2 mutations were not found in other breast cancer cell lines. (Table 3 ). Since the EVI2B (Ectopic viral integration site 2B protein homolog) gene lies within an intron of the NF1 (Neurofibromatosis type 1) gene and is transcribed in the opposite direction to the NF1 gene ( 28 ), we compared NF1 and EVI2B RNA expression levels in relation to those of the estrogen receptor gene ESR1 in the genome-wide RNA transcript profile from TCGA (breast invasive carcinoma gene expression) using the RNAseq data set (TCGA_BRCA_exp_HiSeqV2-2017-09-08; in 1,218 tumor tissue samples). A weak but significant positive correlation was observed in the expression of ESR1 and NF1 (Spearman's order coefficient r = 0.313, p = 0.0000002) while EVI2B and ESR1 expression were negatively correlated ( r = −0.178, p = 4.4 × 10 −10 ). No significant correlation of NF1 and EVI2B was observed ( r = −0.002, p = 0.927).

Table 3 . Mutated genes of New Zealand breast cancer cell lines found in other breast cancer cell lines.

Our findings show that the behavior of the four breast cancer cell lines developed under low oxygen conditions (5% O 2 ) is generally similar to that of commercially available breast cancer cell lines. It is of interest that the hormone receptor status of the new lines covers the main classes of breast cancer, with three of the four having triple negative receptor status. Proliferation of the ER+ NZBR3 cell line was sensitive to estrogen stimulation, and expression of the estrogen responsive genes GREB1 and TFF1 was increased after 24 h of estrogen exposure (Figure 2 ). As the cyclin D1 response decreased to background levels by 24 h, the lack of upregulation of CCDN1 in our study is consistent with reports by others ( 29 ).

Apart from NZBR1, the remaining three NZBR cell lines, MCF-7, SKBR3, and MDA-MB-231 showed higher expression of hypoxia-inducible factor-regulated gene CA9 ( 30 ) when cultured under 5% oxygen conditions. Significantly increased ROS levels were also found in cell lines cultured under 21% oxygen conditions (Figure 3A ). It has been suggested that abundant ROS and associated oxidative stress in cells cultured at high oxygen levels could affect the physiology of cells in culture ( 3 , 31 ), which could potentially influence cell phenotypes and experimental outcomes.

The genomic mutation frequency in the breast cancer cell lines is significantly higher than that in breast carcinoma in vivo ( T test; p < 0.002) (Table S3 ), but the study size is too small to make conclusions about the relative mutation frequencies of the low oxygen-derived lines and ambient oxygen-derived lines. The spectrum of mutations in the NZBR lines is similar to that found in commonly used breast cancer cell lines, except that the EVI2B, LRP1B and PMS2 mutations have not been reported in breast cancer lines (Table 3 ). The availability of sequence data for these cell lines allows investigation of relationships to drug sensitivity. A surprising finding was that no TP53 mutations were identified. TP53 mutations were identified in 18/32 lines (56%) of a cell line cohort ( 32 ) and in 23% of the breast cancer samples ( 33 ). However, the number of cell lines generated in this study is too small to conclude that the frequency of TP53 mutations is lower in cell lines derived under low oxygen conditions.

The triple negative line NZBR1 is of interest because of the relative absence of gene mutations in the genome sequence. One possible explanation is that this cell line possesses a CpG island methylator phenotype (CIMP) ( 14 – 16 ) that contributes to its neoplastic properties ( 34 , 35 ). NZBR1 expresses EGFR, but it may not depend on the EGFR pathway for survival since it is insensitive to all the HER2 inhibitors tested and these inhibitors also inhibit the EGFR. NZBR1 is generally resistant to targeted agents but is moderately sensitive to the cytotoxic drugs doxorubicin and paclitaxel.

The triple negative line NZBR2 carries a BRCA2 mutation as well as an AKT1 mutation. The latter differs from the hot spot mutation AKT1 E12K that confers sensitivity to the dual mTOR/PI3K inhibitor BEZ235 ( 36 ); and NZBR2 shows only moderate sensitivity to BEZ235. NZBR2 shows high sensitivity to everolimus, which is approved for treating advanced hormone receptor-positive, HER2-negative breast cancer. NZBR2 is sensitive to everolimus despite its low level of p70S6K phosphorylation. This result extends our previous study indicating that everolimus sensitivity does not require ER signaling ( 9 ).

The ER+ NZBR3 cell line expresses HER2 and is highly sensitive to tamoxifen, but is relatively resistant to the cytotoxic drugs paclitaxel and doxorubicin. It shows high phospho-AKT expression, suggesting sensitivity to AKT inhibitors ( 37 ). NZBR3 is also highly responsive to the HER2 inhibitors lapatinib, afatinib, dacomitinib, and ARRY-380. Mutations for EVI2B, LRP1B , and NF1 have also been identified (Table 2 ). EVI2B is a transmembrane protein ( 28 ) while NF1 is a tumor suppressor gene ( 38 ). NF1 loss is associated with resistance to BRAF inhibitors ( 39 ) and EGFR inhibitors ( 40 ). The EVI2B gene lies within an intron of the NF1 gene and is transcribed in the opposite direction to the NF1 gene, but their expression is not correlated, suggesting that they are independently regulated. NF1 mutations are associated with increased ERK phosphorylation ( 41 ), RAF-MEK-ERK signaling and phosphoinositide 3-kinase (PI3K)/mTOR pathway utilization ( 42 ). Patients whose cancers, including breast cancers, possess NF1 mutations have worse survival ( 43 , 44 ). This may be consistent with the relative resistance of NZBR3 cells to paclitaxel and doxorubicin (Figure 5 ). However, these cells are sensitive to HER2 inhibitors, suggesting that they depend more on AKT signaling (which is sensitive to HER2 inhibitors) than on RAS/NF1-ERK signaling (which is less sensitive; Figure 7 ). LRP1B is a putative tumor suppressor and a member of the low-density lipoprotein receptor family ( 45 ); gene mutation has been reported in several cancer types but not previously in breast cancer ( 46 – 48 ).

The triple negative NZBR4 line is interesting in that it has a number of gene mutations associated with DNA repair including BRCA2 , the double strand break repair nuclease homolog A ( MRE11A ), mismatch repair system component homolog 2 ( PMS2 ), MutL homolog 1 ( MLH1 ) and Ataxia Telangiectasia Mutated serine/threonine kinase ( ATM ). BRCA2 , a tumor suppressor that is essential for the repair of double-strand DNA breaks by homologous recombination, is associated with risk of developing breast cancer ( 49 – 52 ). MRE11A is part of the DNA double-strand break repair MRE11/RAD50/NBS1 complex that is required for non-homologous joining of DNA ends which associates with breast cancer risk ( 53 ). Heterozygous germline mutations in the mismatch repair genes including MLH1 and PMS2 can cause Lynch syndrome, an autosomal dominant cancer predisposition syndrome conferring a high risk of colorectal, endometrial, and other cancers in adulthood ( 54 , 55 ). Mutations of MLH1 and PMS2 contribute to microsatellite instability and increased mutation rates in cancer cells ( 56 ). The ATM gene encodes a protein kinase that activates the response to double strand DNA breaks. Pathological variants of this gene showed a significantly increased risk of breast cancer with a penetrance that appears similar to that conferred by germline mutations in BRCA2 ( 57 ). It is possible that mutations of genes associated with the DNA repair pathways may render NZBR4 cells responsive to PARP inhibitors ( 58 ).

The drug sensitivity profiles reported here include comparative data for a series of HER2 inhibitors. Lapatinib is an EGFR and HER2 inhibitor with IC50s of 10.8 and 9.2 nM in cell-free assays. Afatinib is an inhibitor of EGFR (wt), EGFR (L858R), EGFR (L858R/T790M) and HER2 with IC50s of 0.5, 0.4, 10, and 14 nM respectively in cell-free assays. Dacomitinib is a potent, irreversible pan-ErbB inhibitor, mostly of EGFR with an IC50 value of 6 nM in a cell-free assay. ARRY-380 is a potent and selective HER2 inhibitor with IC50 of 8 nM, equipotent against truncated p95-HER2, 500-fold more selective for HER2 over EGFR (Selleckchem.com; all enzyme inhibition data). ARRY-380 is under clinical investigation for treating HER2+ metastatic breast cancer patients. Since ARRY-380 is HER2 specific, it has the potential to block HER2 signaling without causing the toxicities of EGFR inhibition ( 59 ). In comparing the responses of the NZBR cell lines to HER2 inhibitors, we have included data for four established lines, MCF-7, SKBR3, MDA-MB-231, and ZR75.1. ARRY-380, the most effective of the HER inhibitors tested, is not active against the HER2+ cell line ZR75.1, which is also resistant to other HER2-directed inhibitors (Figure 6 ). However, ZR75.1 cells have high levels of phosphorylated AKT and low levels of phosphorylated HER2, and ARRY-380 exposure (up to 1 μM) failed to suppress the relative abundance of phosphorylated AKT. Since the HER2 pathway is weakly active, the results suggest that an alternative signaling pathway can contribute to growth and survival. ZR75.1 cells are also highly sensitive to the mTOR inhibitor everolimus ( 9 ) and it is possible that combination of everolimus with a HER2 inhibitor could reduce the rate of acquired drug resistance. Understanding the mechanisms that contribute to HER2 inhibitor resistance, including identification of predictive biomarkers such as HER2 phosphorylation status, is important for progress in the use of this type of therapy.

In summary, our results suggest that the behavior of cell lines developed under low oxygen conditions (5% O 2 ) is generally similar to that of commonly used breast cancer cell lines developed under 21% oxygen conditions. Although no difference in sensitivity to estrogen or doxorubicin was observed between cell lines cultured in 5% vs. 21% oxygen, reduced ROS and upregulation of the hypoxia response, as indicated by CA9 expression, were observed in cells cultured under 5% oxygen conditions. Therefore we cannot exclude the possibility that culture of cells under 5 vs. 21% oxygen conditions could have consequences for the effectiveness of drug treatment in cell lines as predictors of therapy responses in patients.

Ethics Statement

Ethics approval and consent to participate: This study was carried out in accordance with the recommendations of New Zealand Health and Disability Ethics Committee guidelines–ref AKL/2000/184/AM06, New Zealand Health and Disability Ethics Committee. The protocol was approved by the Northern A Health and Disability Ethics Committee. All subjects gave written informed consent in accordance with the Declaration of Helsinki.

Author Contributions

EYL and BCB designed this study. EYL, CF-P, WRJ, EM, PMK, DCS and MEA-A carried out the study. EYL, CF-P, SKB, RJB, DCS and MEA-A assisted with the data analysis. EYL drafted the manuscript. RJB, GJF, DCS, CF-P, MEA-A, SKB and BCB assisted with the manuscript preparation. All authors read and approved the final manuscript.

This work is supported by the Cancer Society New Zealand—Auckland, New Zealand Breast Cancer Foundation, Lottery Health Grant and the Auckland Cancer Society Research Centre.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2018.00425/full#supplementary-material

Figure S1 Sensitivity of breast cancer cell lines exposed to doxorubicin at 5 or 21% oxygen conditions. IC50 values for doxorubicin shown as the mean ± standard error of triplicate experiments.

Table S1 Short Tandem Repeat profiling for the NZBR cell lines.

Table S2 List of primers used in the experiments.

Table S3 Mutation frequency in breast carcinoma and breast cancer cell lines.

Abbreviations

ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2; EGFR, epidermal growth factor receptor; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; TCGA, The Cancer Genome Atlas; GREB1, Growth Regulation By Estrogen In Breast Cancer 1; CCND1, Cyclin D1; TTF1, Trefoil Factor 1; HIF, Hypoxia-inducible factor.

1. Sullivan M, Galea P, Latif S. What is the appropriate oxygen tension for in vitro culture? Mol Hum Reprod. (2006) 12:653. doi: 10.1093/molehr/gal081

PubMed Abstract | CrossRef Full Text | Google Scholar

2. Hammond EM, Asselin MC, Forster D, O'Connor JP, Senra JM, Williams KJ. The meaning, measurement and modification of hypoxia in the laboratory and the clinic. Clin. Oncol. (2014) 26:277–88. doi: 10.1016/j.clon.2014.02.002

3. Halliwell B. Cell culture, oxidative stress, and antioxidants: avoiding pitfalls. Biomed J. (2014) 37:99–105. doi: 10.4103/2319-4170.128725

4. Hemnani T, Parihar MS. Reactive oxygen species and oxidative DNA damage. Indian J Physiol Pharmacol. (1998) 42:440–52.

PubMed Abstract | Google Scholar

5. Marshall ES, Finlay GJ, Matthews JH, Shaw JH, Nixon J, Baguley BC. Microculture-based chemosensitivity testing: a feasibility study comparing freshly explanted human melanoma cells with human melanoma cell lines. J Natl Cancer Inst. (1992) 84:340–5. doi: 10.1093/jnci/84.5.340

6. Marshall ES, Holdaway KM, Shaw JH, Finlay GJ, Matthews JH, Baguley BC. Anticancer drug sensitivity profiles of new and established melanoma cell lines. Oncol Res. (1993) 5:301–9.

7. Marshall ES, Matthews JH, Shaw JH, Nixon J, Tumewu P, Finlay GJ, et al. Radiosensitivity of new and established human melanoma cell lines: comparison of [3H]thymidine incorporation and soft agar clonogenic assays. Eur J Cancer (1994) 30A:1370–6. doi: 10.1016/0959-8049(94)90188-0

CrossRef Full Text | Google Scholar

8. Marshall ES, Baguley BC, Matthews JH, Jose CC, Furneaux CE, Shaw JH, et al. Estimation of radiation-induced interphase cell death in cultures of human tumor material and in cell lines. Oncol Res. (2004) 14:297–304. doi: 10.3727/096504003773994833

9. Leung EY, Askarian-Amiri M, Finlay GJ, Rewcastle GW, Baguley BC. Potentiation of growth inhibitory responses of the mTOR inhibitor everolimus by dual mTORC1/2 inhibitors in cultured breast cancer cell lines. PLoS ONE (2015) 10:e0131400. doi: 10.1371/journal.pone.0131400

10. Leung EY, Kim JE, Askarian-Amiri M, Rewcastle GW, Finlay GJ, Baguley BC. Relationships between signaling pathway usage and sensitivity to a pathway inhibitor: examination of trametinib responses in cultured breast cancer lines. PLoS ONE (2014) 9:e105792. doi: 10.1371/journal.pone.0105792

11. Garcia-Morales P, Hernando E, Carrasco-Garcia E, Menendez-Gutierrez MP, Saceda M, Martinez-Lacaci I. Cyclin D3 is down-regulated by rapamycin in HER-2-overexpressing breast cancer cells. Mol Cancer Ther. (2006) 5:2172–81. doi: 10.1158/1535-7163.MCT-05-0363

12. Stowasser F, Baenziger M, Garad SD. Preparation of Salts Crystalline Forms of 2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydroimidazo[4,5-c]quinolin-1-yl)-Phenyl]propionitrile Its Use as a Drug. PCT Int Publ. No:WO 2008/064093 A3 (2008). Available online at: https://patentimages.storage.googleapis.com/5c/86/e5/423ced14ca0929/WO2008064093A3.pdf

13. Leung E, Kim JE, Askarian-Amiri M, Finlay GJ, Baguley BC. Evidence for the existence of triple-negative variants in the MCF-7 breast cancer cell population. Biomed Res Int. (2014) 2014: 836769. doi: 10.1155/2014/836769

14. Leung E, Kim JE, Rewcastle GW, Finlay GJ, Baguley BC. Comparison of the effects of the PI3K/mTOR inhibitors NVP-BEZ235 and GSK2126458 on tamoxifen-resistant breast cancer cells. Cancer Biol Ther. (2011) 11:938–46. doi: 10.4161/cbt.11.11.15527

15. Leung EY, Askarian-Amiri ME, Sarkar D, Ferraro-Peyret C, Joseph WR, Finlay GJ, et al. Endocrine therapy of estrogen receptor-positive breast cancer cells: early differential effects on stem cell markers. Front Oncol. (2017) 7:184. doi: 10.3389/fonc.2017.00184

16. Leung EY, Kim JE, Askarian-Amiri M, Joseph WR, McKeage MJ, Baguley BC. Hormone resistance in two MCF-7 breast cancer cell lines is associated with reduced mtor signaling, decreased glycolysis, and increased sensitivity to cytotoxic drugs. Front Oncol. (2014) 4:221. doi: 10.3389/fonc.2014.00221

17. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics (2009) 25:1754–60. doi: 10.1093/bioinformatics/btp324

18. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics (2009) 25:2078–9. doi: 10.1093/bioinformatics/btp352

19. Koboldt DC, Zhang Q, Larson DE, Shen D, McLellan MD, Lin L, et al. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res. (2012) 22:568–76. doi: 10.1101/gr.129684.111

20. Danecek P, Auton A, Abecasis G, Albers CA, Banks E, DePristo MA, et al. The variant call format and VCFtools. Bioinformatics (2011) 27:2156–8. doi: 10.1093/bioinformatics/btr330

21. Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. (2010) 38:e164. doi: 10.1093/nar/gkq603

22. Cingolani P, Patel VM, Coon M, Nguyen T, Land SJ, Ruden DM, et al. Using drosophila melanogaster as a model for genotoxic chemical mutational studies with a new program, SnpSift. Front Genet. (2012) 3:35. doi: 10.3389/fgene.2012.00035

PubMed Abstract | CrossRef Full Text

23. Cingolani P, Platts A, Wang LL, Coon M, Nguyen T, Wang L, et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w(1118); iso-2; iso-3. Fly (2012) 6:80–92. doi: 10.4161/fly.19695

24. Won Jeong K, Chodankar R, Purcell DJ, Bittencourt D, Stallcup MR. Gene-specific patterns of coregulator requirements by estrogen receptor-alpha in breast cancer cells. Mol Endocrinol. (2012) 26:955–66. doi: 10.1210/me.2012-1066

25. Bhadury J, Einarsdottir BO, Podraza A, Bagge RO, Stierner U, Ny L, et al. Hypoxia-regulated gene expression explains differences between melanoma cell line-derived xenografts and patient-derived xenografts. Oncotarget (2016) 7:23801–11. doi: 10.18632/oncotarget.8181

26. Yang F, Teves SS, Kemp CJ, Henikoff S. Doxorubicin, DNA torsion, and chromatin dynamics. Biochim Biophys Acta (2014) 1845:84–9. doi: 10.1016/j.bbcan.2013.12.002

27. The Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature (2012) 490:61–70. doi: 10.1038/nature11412

28. Cawthon RM, Andersen LB, Buchberg AM, Xu GF, O'Connell P, Viskochil D, et al. cDNA sequence and genomic structure of EV12B, a gene lying within an intron of the neurofibromatosis type 1 gene. Genomics (1991) 9:446–60. doi: 10.1016/0888-7543(91)90410-G

29. Castro-Rivera E, Samudio I, Safe S. Estrogen regulation of cyclin D1 gene expression in ZR-75 breast cancer cells involves multiple enhancer elements. J Biol Chem. (2001) 276:30853–61. doi: 10.1074/jbc.M103339200

30. Wykoff CC, Beasley NJ, Watson PH, Turner KJ, Pastorek J, Sibtain A, et al. Hypoxia-inducible expression of tumor-associated carbonic anhydrases. Cancer Res. (2000) 60:7075–83. Available online at: http://cancerres.aacrjournals.org/content/60/24/7075.long

31. Halliwell B. Oxidative stress in cell culture: an under-appreciated problem? FEBS Lett. (2003) 540:3–6. doi: 10.1016/S0014-5793(03)00235-7

32. Saunus JM, Smart CE, Kutasovic JR, Johnston RL, Kalita-de Croft P, Miranda M, et al. Multidimensional phenotyping of breast cancer cell lines to guide preclinical research. Breast Cancer Res Treat. (2018) 167:289–301. doi: 10.1007/s10549-017-4496-x

33. Walerych D, Napoli M, Collavin L, Del Sal G. The rebel angel: mutant p53 as the driving oncogene in breast cancer. Carcinogenesis (2012) 33:2007–17. doi: 10.1093/carcin/bgs232

34. Siah SP, Quinn DM, Bennett GD, Casey G, Flower RL, Suthers G, et al. Microsatellite instability markers in breast cancer: a review and study showing MSI was not detected at 'BAT 25' and 'BAT 26' microsatellite markers in early-onset breast cancer. Breast Cancer Res Treat. (2000) 60:135–42. doi: 10.1023/A:1006315315060

35. Halpern N, Goldberg Y, Kadouri L, Duvdevani M, Hamburger T, Peretz T, et al. Clinical course and outcome of patients with high-level microsatellite instability cancers in a real-life setting: a retrospective analysis. Onco Targets Ther. (2017) 10:1889–96. doi: 10.2147/OTT.S126905

36. Rudolph M, Anzeneder T, Schulz A, Beckmann G, Byrne AT, Jeffers M, et al. AKT1 (E17K) mutation profiling in breast cancer: prevalence, concurrent oncogenic alterations, and blood-based detection. BMC Cancer (2016) 16:622. doi: 10.1186/s12885-016-2626-1

37. Yndestad S, Austreid E, Svanberg IR, Knappskog S, Lonning PE, Eikesdal HP. Activation of Akt characterizes estrogen receptor positive human breast cancers which respond to anthracyclines. Oncotarget (2017) 8:41227–41. doi: 10.18632/oncotarget.17167

38. Pemov A, Li H, Patidar R, Hansen NF, Sindiri S, Hartley SW, et al. The primacy of NF1 loss as the driver of tumorigenesis in neurofibromatosis type 1-associated plexiform neurofibromas. Oncogene (2017) 36:3168–77. doi: 10.1038/onc.2016.464

39. Gibney GT, Smalley KS. An unholy alliance: cooperation between BRAF and NF1 in melanoma development and BRAF inhibitor resistance. Cancer Discov. (2013) 3:260–3. doi: 10.1158/2159-8290.CD-13-0017

40. de Bruin EC, Cowell C, Warne PH, Jiang M, Saunders RE, Melnick MA, et al. Reduced NF1 expression confers resistance to EGFR inhibition in lung cancer. Cancer Discov. (2014) 4:606–19. doi: 10.1158/2159-8290.CD-13-0741

41. Cui Y, Costa RM, Murphy GG, Elgersma Y, Zhu Y, Gutmann DH, et al. Neurofibromin regulation of ERK signaling modulates GABA release and learning. Cell (2008) 135:549–60. doi: 10.1016/j.cell.2008.09.060

42. See WL, Tan IL, Mukherjee J, Nicolaides T, Pieper RO. Sensitivity of glioblastomas to clinically available MEK inhibitors is defined by neurofibromin 1 deficiency. Cancer Res. (2012) 72:3350–9. doi: 10.1158/0008-5472.CAN-12-0334

43. Uusitalo E, Kallionpaa RA, Kurki S, Rantanen M, Pitkaniemi J, Kronqvist P, et al. Breast cancer in neurofibromatosis type 1: overrepresentation of unfavourable prognostic factors. Br J Cancer (2017) 116:211–7. doi: 10.1038/bjc.2016.403

44. Uusitalo E, Rantanen M, Kallionpaa RA, Poyhonen M, Leppavirta J, Yla-Outinen H, et al. Distinctive cancer associations in patients with neurofibromatosis type 1. J Clin Oncol. (2016) 34:1978–86. doi: 10.1200/JCO.2015.65.3576

45. Liu CX, Li Y, Obermoeller-McCormick LM, Schwartz AL, Bu G. The putative tumor suppressor LRP1B, a novel member of the low density lipoprotein (LDL) receptor family, exhibits both overlapping and distinct properties with the LDL receptor-related protein. J Biol Chem. (2001) 276:28889–96. doi: 10.1074/jbc.M102727200

46. Lu YJ, Wu CS, Li HP, Liu HP, Lu CY, Leu YW, et al. Aberrant methylation impairs low density lipoprotein receptor-related protein 1B tumor suppressor function in gastric cancer. Genes Chromosomes Cancer (2010) 49:412–24. doi: 10.1002/gcc.20752

47. Prazeres H, Torres J, Rodrigues F, Pinto M, Pastoriza MC, Gomes D, et al. Chromosomal, epigenetic and microRNA-mediated inactivation of LRP1B, a modulator of the extracellular environment of thyroid cancer cells. Oncogene (2011) 30:1302–17. doi: 10.1038/onc.2010.512

48. Tabouret E, Labussiere M, Alentorn A, Schmitt Y, Marie Y, Sanson M. LRP1B deletion is associated with poor outcome for glioblastoma patients. J Neurol Sci. (2015) 358:440–3. doi: 10.1016/j.jns.2015.09.345

49. Meeks HD, Song H, Michailidou K, Bolla MK, Dennis J, Wang Q, et al. BRCA2 polymorphic stop codon K3326X and the risk of breast, prostate, and ovarian cancers. J Natl Cancer Inst. (2016) 108: djv315. doi: 10.1093/jnci/djv315

50. Petrucelli N, Daly MB, Pal T. BRCA1- and BRCA2-Associated hereditary breast and ovarian cancer. In: Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews(R). Seattle, WA: University of Washington (1993).

Google Scholar

51. Narod SA, Foulkes WD. BRCA1 and BRCA2: 1994 and beyond. Nat Rev Cancer (2004) 4:665–76. doi: 10.1038/nrc1431

52. Narod SA, Salmena L. BRCA1 and BRCA2 mutations and breast cancer. Discov Med. (2011) 12:445–53.

53. Hsu HM, Wang HC, Chen ST, Hsu GC, Shen CY, Yu JC. Breast cancer risk is associated with the genes encoding the DNA double-strand break repair Mre11/Rad50/Nbs1 complex. Cancer Epidemiol Biomarkers Prev. (2007) 16:2024–32. doi: 10.1158/1055-9965.EPI-07-0116

54. Baris HN, Barnes-Kedar I, Toledano H, Halpern M, Hershkovitz D, Lossos A, et al. Constitutional mismatch repair deficiency in israel: high proportion of founder mutations in mmr genes and consanguinity. Pediatr Blood Cancer (2016) 63:418–27. doi: 10.1002/pbc.25818

55. Win AK, Lindor NM, Jenkins MA. Risk of breast cancer in Lynch syndrome: a systematic review. Breast Cancer Res. (2013) 15:R27. doi: 10.1186/bcr3405

56. Chen W, Swanson BJ, Frankel WL. Molecular genetics of microsatellite-unstable colorectal cancer for pathologists. Diagn Pathol. (2017) 12:24. doi: 10.1186/s13000-017-0613-8

57. Goldgar DE, Healey S, Dowty JG, Da Silva L, Chen X, Spurdle AB, et al. Rare variants in the ATM gene and risk of breast cancer. Breast Cancer Res. (2011) 13:R73. doi: 10.1186/bcr2919

58. Livraghi L, Garber JE. PARP inhibitors in the management of breast cancer: current data and future prospects. BMC Med. (2015) 13:188. doi: 10.1186/s12916-015-0425-1

59. Moulder SL, Borges VF, Baetz T, McSpadden T, Fernetich G, Murthy RK, et al. Phase I Study of ONT-380, a HER2 Inhibitor, in Patients with HER2(+)-advanced solid tumors, with an expansion cohort in HER2(+) metastatic breast cancer (MBC). Clin Cancer Res. (2017) 23:3529–36. doi: 10.1158/1078-0432.CCR-16-1496

Keywords: breast cancer, PI3K mTOR inhibitor BEZ235, estrogen receptor, HER2 inhibitors, 5% and 21% oxygen, CA9, ROS, Triple negative breast cancer (TNBC)

Citation: Leung EY, Askarian-Amiri ME, Singleton DC, Ferraro-Peyret C, Joseph WR, Finlay GJ, Broom RJ, Kakadia PM, Bohlander SK, Marshall E and Baguley BC (2018) Derivation of Breast Cancer Cell Lines Under Physiological (5%) Oxygen Concentrations. Front. Oncol . 8:425. doi: 10.3389/fonc.2018.00425

Received: 04 April 2018; Accepted: 11 September 2018; Published: 12 October 2018.

Reviewed by:

Copyright © 2018 Leung, Askarian-Amiri, Singleton, Ferraro-Peyret, Joseph, Finlay, Broom, Kakadia, Bohlander, Marshall and Baguley. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Euphemia Y. Leung, [email protected] Bruce C. Baguley, [email protected]

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

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Tips for running western blots, why it’s important to include sample identification when optimizing lab protocols, how identification systems can help preserve research integrity, scientists have identified an entirely new form of life in the gut: obelisks, 5 features that every modern lims should have, going green: does using -70°c affect sample storage, 4 tips for shipping samples from your lab, 2023 labtag year in review, 4 sophisticated uses for ink technologies in healthcare & biomedical research, revolutionizing histology labeling with patented histobar histology cassettes, breakthrough: ga international announces the development of snappeel technology, the history of cell lines in biological research.

Cell lines are a necessary tool in the arsenal of almost everyone who studies human diseases. Though it remains debatable whether these cell lines accurately represent how cells in vivo respond and operate, they remain an essential model for screening pharmaceuticals, testing new genetic modification techniques, exploring new signaling pathways, and developing new strategies to analyze and target human disorders, from cancer to inflammatory and neurological disorders. It’s, therefore, worth exploring where these commonly used cell lines came from and addressing the legacy they leave behind.

The beginnings of cell culture

Cell culture dates back to the 1800s when Wilhelm Roux was first able to culture chicken embryos using a saline solution for only a few days. It really took off a bit later in the early 1900s, when primary cell culture was first utilized by Ross Granville Harrison, who was able to culture nerve cells from frogs using the hanging drop method. This method typically involves using an inverted watch glass to suspend drops of liquid containing cells instead of spreading them across a petri dish and potentially squishing them. Dr. Harrison would take frog embryonic tissue submerged in lymph solution on cover slides and turn it upside down, where he could observe new fibers develop from the tissue. This technique was not performed under sterile conditions, so while it was a great first start, cell culture didn’t truly become widely adopted until aseptic techniques were introduced, ultimately leading to the development of the first cancer cell lines. 1

The first cell line

The name Henrietta Lacks carries a lot of weight when it comes to scientific achievement and controversy. A mother of five and only 31 at the time of her cervical cancer diagnosis, she had undergone radium treatment and had a biopsy of her tumor sent to Dr. George Gey’s lab. Dr. Gey, who had tried and failed to re-grow cervical cancer samples from various patients, soon found that Lack’s tumor cells were capable of continually proliferating. These cells ultimately led to the first widely-used cancer cell line, HeLa. Interestingly, they also helped shed light on the role of human papillomaviruses (HPV) in promoting cervical cancer, having identified HPV 18 as the strain that was responsible for HeLa cells’ ability to rapidly reproduce. 2,3

Unfortunately, while the contribution of these cells to biological research as a whole has been immense, some controversy has arisen due to the process by which the cells were obtained. Henrietta Lacks never consented to the use of her cells in biological research, which led to several lawsuits from her family accusing companies of unfairly profiting from the cells. 2 Though some of her descendants have called for a reduction in the use of HeLa cells in research, others are now leading a campaign called #HELA100, which hopes to celebrate her life and legacy and bring attention to the incredible role her contributions have made to science over the last 70 years. 4

Origins of other important lines

HeLa cells aren’t the only ones with a fascinating story. Another widely used cell line, HEK293, dates back nearly 50 years, when Dr. Frank Graham worked with Dr. Alex van der Eb in the Netherlands, introducing DNA via calcium phosphate transfection. This technique, still widely used today to transfect cells, allowed Dr. Graham to generate a HEK293 cell line expressing Adenovirus 5-transforming genes. He eventually brought the cell line back to Canada and, in collaboration with McMaster University, developed Ad5-based viral vectors for gene transfer protocols and recombinant vaccines. 5

Perhaps the most widely used breast cancer cell line ever, MCF-7, dates back to 1973 in a lab run by Dr. Soule at the Michigan Cancer Foundation. These cells were isolated from the pleural effusion, or build-up of fluid between the lungs, of a 69-year-old woman with metastatic disease. This cell line has particular relevance to the discovery of hormone responsiveness in breast cancer; in vitro testing was carried out on the cultured cells when they were first harvested, with the anti-estrogen drug tamoxifen able to inhibit MCF-7 cell growth, an effect that was reversed by adding back estrogen. Though anti-estrogen therapy would be studied in more detail later, the cells represented, at the time, the main building block to showing that estrogen could directly stimulate breast cancer cell growth. 6

Though cell lines may not have the standing they once did in the scientific community, they have still played a massive role in furthering research from the bottom up safely and efficiently. New cellular models are continuously being designed to better mimic human disorders compared with cell lines, such as organs on a chip and induced pluripotent stem cells ( iPSCs ), themselves a new kind of cell line. Without cell lines like HeLa and MCF-7, however, we would never have been able to achieve such lofty heights, and many life-saving treatments would have never been discovered in the first place.

LabTAG by GA International is a leading manufacturer of high-performance specialty labels a nd a supplier of identification solutions used in research and medical labs as well as healthcare institutions.

References:

• Richter M, et al. From Donor to the Lab: A Fascinating Journey of Primary Cell Lines. Front Cell Dev Biol. 2021;9:711381.
• Science Alert. What Are HeLa Cells? A Cancer Biologist Explains The Controversy That Cannot Die. Available at: https://www.sciencealert.com/expert-explains-how-the-controversial-hela-cells-gained-their-immortality.
• John Hopkins Medicine. The Legacy of Henrietta Lacks. Available at: https://www.hopkinsmedicine.org/henriettalacks/.
• Henrietta Lacks: science must right a historical wrong. Available at: https://www.nature.com/articles/d41586-020-02494-z.
• Government of Canada. Recognizing the scientific impact of Dr. Frank Graham’s HEK 293 cell line.
• Comşa Ş, et al. The Story of MCF-7 Breast Cancer Cell Line: 40 years of Experience in Research. Anticancer Res. 2015;35(6):3147-3154.

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Theses/dissertations from 2023 2023.

Exploring strain variation and bacteriophage predation in the gut microbiome of Ciona robusta , Celine Grace F. Atkinson

Distinct Nrf2 Signaling Thresholds Mediate Lung Tumor Initiation and Progression , Janine M. DeBlasi

Thermodynamic frustration of TAD2 and PRR contribute to autoinhibition of p53 , Emily Gregory

Utilization of Detonation Nanodiamonds: Nanocarrier for Gene Therapy in Non-Small Cell Lung Cancer , Allan E. Gutierrez

Role of HLA-DRB1 Fucosylation in Anti-Melanoma Immunity , Daniel K. Lester

Targeting BET Proteins Downregulates miR-33a To Promote Synergy with PIM Inhibitors in CMML , Christopher T. Letson

Regulated Intramembrane Proteolysis by M82 Peptidases: The Role of PrsS in the Staphylococcus aureus Stress Response , Baylie M. Schott

Histone Deacetylase 8 is a Novel Therapeutic Target for Mantle Cell Lymphoma and Preserves Natural Killer Cell Cytotoxic Function , January M. Watters

Theses/Dissertations from 2022 2022

Regulation of the Heat Shock Response via Lysine Acetyltransferase CBP-1 and in Neurodegenerative Disease in Caenorhabditis elegans , Lindsey N. Barrett

Determining the Role of Dendritic Cells During Response to Treatment with Paclitaxel/Anti-TIM-3 , Alycia Gardner

Cell-free DNA Methylation Signatures in Cancer Detection and Classification , Jinyong Huang

The Role Of Eicosanoid Metabolism in Mammalian Wound Healing and Inflammation , Kenneth D. Maus

A Holistic Investigation of Acidosis in Breast Cancer , Bryce Ordway

Characterizing the Impact of Postharvest Temperature Stress on Polyphenol Profiles of Red and White-Fruited Strawberry Cultivars , Alyssa N. Smith

Theses/Dissertations from 2021 2021

Multifaceted Approach to Understanding Acinetobacter baumannii Biofilm Formation and Drug Resistance , Jessie L. Allen

Cellular And Molecular Alterations Associated with Ovarian and Renal Cancer Pathophysiology , Ravneet Kaur Chhabra

Ecology and diversity of boletes of the southeastern United States , Arian Farid

CircREV1 Expression in Triple-Negative Breast Cancer , Meagan P. Horton

Microbial Dark Matter: Culturing the Uncultured in Search of Novel Chemotaxonomy , Sarah J. Kennedy

The Multifaceted Role of CCAR-1 in the Alternative Splicing and Germline Regulation in Caenorhabditis elegans , Doreen Ikhuva Lugano

Unraveling the Role of Novel G5 Peptidase Family Proteins in Virulence and Cell Envelope Biogenesis of Staphylococcus aureus , Stephanie M. Marroquin

Cytoplasmic Polyadenylation Element Binding Protein 2 Alternative Splicing Regulates HIF1α During Chronic Hypoxia , Emily M. Mayo

Transcriptomic and Functional Investigation of Bacterial Biofilm Formation , Brooke R. Nemec

A Functional Characterization of the Omega (ω) subunit of RNA Polymerase in Staphylococcus aureus , Shrushti B. Patil

The Role Of Cpeb2 Alternative Splicing In TNBC Metastasis , Shaun C. Stevens

Screening Next-generation Fluorine-19 Probe and Preparation of Yeast-derived G Proteins for GPCR Conformation and Dynamics Study , Wenjie Zhao

Theses/Dissertations from 2020 2020

Understanding the Role of Cereblon in Hematopoiesis Through Structural and Functional Analyses , Afua Adutwumwa Akuffo

To Mid-cell and Beyond: Characterizing the Roles of GpsB and YpsA in Cell Division Regulation in Gram-positive Bacteria , Robert S. Brzozowski

Spatiotemporal Changes of Microbial Community Assemblages and Functions in the Subsurface , Madison C. Davis

New Mechanisms That Regulate DNA Double-Strand Break-Induced Gene Silencing and Genome Integrity , Dante Francis DeAscanis

Regulation of the Heat Shock Response and HSF-1 Nuclear Stress Bodies in C. elegans , Andrew Deonarine

New Mechanisms that Control FACT Histone Chaperone and Transcription-mediated Genome Stability , Angelo Vincenzo de Vivo Diaz

Targeting the ESKAPE Pathogens by Botanical and Microbial Approaches , Emily Dilandro

Succession in native groundwater microbial communities in response to effluent wastewater , Chelsea M. Dinon

Role of ceramide-1 phosphate in regulation of sphingolipid and eicosanoid metabolism in lung epithelial cells , Brittany A. Dudley

Allosteric Control of Proteins: New Methods and Mechanisms , Nalvi Duro

Microbial Community Structures in Three Bahamian Blue Holes , Meghan J. Gordon

A Novel Intramolecular Interaction in P53 , Fan He

The Impact of Myeloid-Mediated Co-Stimulation and Immunosuppression on the Anti-Tumor Efficacy of Adoptive T cell Therapy , Pasquale Patrick Innamarato

Investigating Mechanisms of Immune Suppression Secondary to an Inflammatory Microenvironment , Wendy Michelle Kandell

Posttranslational Modification and Protein Disorder Regulate Protein-Protein Interactions and DNA Binding Specificity of p53 , Robin Levy

Mechanistic and Translational Studies on Skeletal Malignancies , Jeremy McGuire

Novel Long Non-Coding RNA CDLINC Promotes NSCLC Progression , Christina J. Moss

Genome Maintenance Roles of Polycomb Transcriptional Repressors BMI1 and RNF2 , Anthony Richard Sanchez IV

The Ecology and Conservation of an Urban Karst Subterranean Estuary , Robert J. Scharping

Biological and Proteomic Characterization of Cornus officinalis on Human 1.1B4 Pancreatic β Cells: Exploring Use for T1D Interventional Application , Arielle E. Tawfik

Evaluation of Aging and Genetic Mutation Variants on Tauopathy , Amber M. Tetlow

Theses/Dissertations from 2019 2019

Investigating the Proteinaceous Regulome of the Acinetobacter baumannii , Leila G. Casella

Functional Characterization of the Ovarian Tumor Domain Deubiquitinating Enzyme 6B , Jasmin M. D'Andrea

Integrated Molecular Characterization of Lung Adenocarcinoma with Implications for Immunotherapy , Nicholas T. Gimbrone

The Role of Secreted Proteases in Regulating Disease Progression in Staphylococcus aureus , Brittney D. Gimza

Advanced Proteomic and Epigenetic Characterization of Ethanol-Induced Microglial Activation , Jennifer Guergues Guergues

Understanding immunometabolic and suppressive factors that impact cancer development , Rebecca Swearingen Hesterberg

Biochemical and Proteomic Approaches to Determine the Impact Level of Each Step of the Supply Chain on Tomato Fruit Quality , Robert T. Madden

Enhancing Immunotherapeutic Interventions for Treatment of Chronic Lymphocytic Leukemia , Kamira K. Maharaj

Characterization of the Autophagic-Iron Axis in the Pathophysiology of Endometriosis and Epithelial Ovarian Cancers , Stephanie Rockfield

Understanding the Influence of the Cancer Microenvironment on Metabolism and Metastasis , Shonagh Russell

Modeling of Interaction of Ions with Ether- and Ester-linked Phospholipids , Matthew W. Saunders

Novel Insights into the Multifaceted Roles of BLM in the Maintenance of Genome Stability , Vivek M. Shastri

Conserved glycine residues control transient helicity and disorder in the cold regulated protein, Cor15a , Oluwakemi Sowemimo

A Novel Cytokine Response Modulatory Function of MEK Inhibitors Mediates Therapeutic Efficacy , Mengyu Xie

Novel Strategies on Characterizing Biologically Specific Protein-protein Interaction Networks , Bi Zhao

Theses/Dissertations from 2018 2018

Characterization of the Transcriptional Elongation Factor ELL3 in B cells and Its Role in B-cell Lymphoma Proliferation and Survival , Lou-Ella M.m. Alexander

Identification of Regulatory miRNAs Associated with Ethanol-Induced Microglial Activation Using Integrated Proteomic and Transcriptomic Approaches , Brandi Jo Cook

Molecular Phylogenetics of Floridian Boletes , Arian Farid

MYC Distant Enhancers Underlie Ovarian Cancer Susceptibility at the 8q24.21 Locus , Anxhela Gjyshi Gustafson

Quantitative Proteomics to Support Translational Cancer Research , Melissa Hoffman

A Systems Chemical Biology Approach for Dissecting Differential Molecular Mechanisms of Action of Clinical Kinase Inhibitors in Lung Cancer , Natalia Junqueira Sumi

Investigating the Roles of Fucosylation and Calcium Signaling in Melanoma Invasion , Tyler S. Keeley

Synthesis, Oxidation, and Distribution of Polyphenols in Strawberry Fruit During Cold Storage , Katrina E. Kelly

Investigation of Alcohol-Induced Changes in Hepatic Histone Modifications Using Mass Spectrometry Based Proteomics , Crystina Leah Kriss

Off-Target Based Drug Repurposing Using Systems Pharmacology , Brent M. Kuenzi

Investigation of Anemarrhena asphodeloides and its Constituent Timosaponin-AIII as Novel, Naturally Derived Adjunctive Therapeutics for the Treatment of Advanced Pancreatic Cancer , Catherine B. MarElia

The Role of Phosphohistidine Phosphatase 1 in Ethanol-induced Liver Injury , Daniel Richard Martin

Theses/Dissertations from 2017 2017

Changing the Pathobiological Paradigm in Myelodysplastic Syndromes: The NLRP3 Inflammasome Drives the MDS Phenotype , Ashley Basiorka

Modeling of Dynamic Allostery in Proteins Enabled by Machine Learning , Mohsen Botlani-Esfahani

Uncovering Transcriptional Activators and Targets of HSF-1 in Caenorhabditis elegans , Jessica Brunquell

The Role of Sgs1 and Exo1 in the Maintenance of Genome Stability. , Lillian Campos-Doerfler

Mechanisms of IKBKE Activation in Cancer , Sridevi Challa

Discovering Antibacterial and Anti-Resistance Agents Targeting Multi-Drug Resistant ESKAPE Pathogens , Renee Fleeman

Functional Roles of Matrix Metalloproteinases in Bone Metastatic Prostate Cancer , Jeremy S. Frieling

Disorder Levels of c-Myb Transactivation Domain Regulate its Binding Affinity to the KIX Domain of CREB Binding Protein , Anusha Poosapati

Role of Heat Shock Transcription Factor 1 in Ovarian Cancer Epithelial-Mesenchymal Transition and Drug Sensitivity , Chase David Powell

Cell Division Regulation in Staphylococcus aureus , Catherine M. Spanoudis

A Novel Approach to the Discovery of Natural Products From Actinobacteria , Rahmy Tawfik

Non-classical regulators in Staphylococcus aureus , Andy Weiss

Theses/Dissertations from 2016 2016

In Vitro and In Vivo Antioxidant Capacity of Synthetic and Natural Polyphenolic Compounds Identified from Strawberry and Fruit Juices , Marvin Abountiolas

Quantitative Proteomic Investigation of Disease Models of Type 2 Diabetes , Mark Gabriel Athanason

CMG Helicase Assembly and Activation: Regulation by c-Myc through Chromatin Decondensation and Novel Therapeutic Avenues for Cancer Treatment , Victoria Bryant

Computational Modeling of Allosteric Stimulation of Nipah Virus Host Binding Protein , Priyanka Dutta

Cell Cycle Arrest by TGFß1 is Dependent on the Inhibition of CMG Helicase Assembly and Activation , Brook Samuel Nepon-Sixt

Gene Expression Profiling and the Role of HSF1 in Ovarian Cancer in 3D Spheroid Models , Trillitye Paullin

VDR-RIPK1 Interaction and its Implications in Cell Death and Cancer Intervention , Waise Quarni

Regulation of nAChRs and Stemness by Nicotine and E-cigarettes in NSCLC , Courtney Schaal

Targeting Histone Deacetylases in Melanoma and T-cells to Improve Cancer Immunotherapy , Andressa Sodre De Castro Laino

Nonreplicative DNA Helicases Involved in Maintaining Genome Stability , Salahuddin Syed

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• Published: 13 September 2023

miR-92a-3p regulates cisplatin-induced cancer cell death

• Romain Larrue 1   na1 ,
• Sandy Fellah 1   na1 ,
• Nihad Boukrout 1 ,
• Corentin De Sousa 1 ,
• Julie Lemaire 1 ,
• Carolane Leboeuf 1 ,
• Marine Goujon 1 ,
• Michael Perrais 1 ,
• Bernard Mari 2 ,
• Christelle Cauffiez 1 ,
• Nicolas Pottier   ORCID: orcid.org/0000-0001-8913-6286 1   na2 &
• Cynthia Van der Hauwaert   ORCID: orcid.org/0000-0001-9549-3722 1   na2  

Cell Death & Disease volume  14 , Article number:  603 ( 2023 ) Cite this article

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• Non-coding RNAs
• Non-small-cell lung cancer

Non-small cell lung cancer is characterized by a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Therefore, the identification of new molecular determinants underlying sensitivity of cancer cells to existing therapy is of particular importance to develop new effective combinatorial treatment strategy. MicroRNAs (miRNAs), a class of small non-coding RNAs, have been established as master regulators of a variety of cellular processes that play a key role in tumor initiation, progression and metastasis. This, along with their widespread deregulation in many distinct cancers, has triggered enthusiasm for miRNAs as novel therapeutic targets for cancer management, in particular in patients with refractory cancers such as those harboring KRAS mutations. In this study, we performed a loss-of-function screening approach to identify miRNAs whose silencing promotes sensitivity of lung adenocarcinoma (LUAD) cells to cisplatin. Our results showed in particular that antisense oligonucleotides directed against miR-92a-3p, a member of the oncogenic miR-17 ~ 92 cluster, caused the greatest increase in the sensitivity of KRAS-mutated LUAD cells to cisplatin. In addition, we demonstrated that this miRNA finely regulates the apoptotic threshold and the proliferative capacity of various tumor cell lines with distinct genetic alterations. Collectively, these data suggest that targeting miR-92a-3p may serve as an effective strategy to overcome treatment resistance of solid tumors.

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Introduction.

Lung cancer is the leading cause of cancer-related mortality worldwide. Lung adenocarcinoma (LUAD) is the most common subtype of lung cancers and accounts for approximately 40% of all cases [ 1 , 2 ]. The last 20 years have witnessed considerable progresses in the understanding of the pathogenesis of lung cancers including LUAD, with the advent of genomic technologies, the generation of several genetically engineered mouse models of lung cancers, and the construction of large databases characterizing the molecular features of respiratory tumors [ 3 , 4 , 5 , 6 ]. Altogether, these multidisciplinary approaches have transformed our view of LUAD from histopathological subtypes to precise molecular and genetic entities that can now be resolved at the single-cell level. Indeed, a vast array of genomic and epigenetic alterations has been reported in LUAD, in particular in genes coding for signaling proteins critical for maintaining normal cellular proliferation and survival [ 6 , 7 , 8 , 9 ]. Consequently, key genetic determinants are now routinely used to inform disease classification, prognostic stratification, and to support treatment decisions. For example, molecularly targeted therapies have remarkably improved treatment for patients whose tumors harbor somatically activated oncogenes such as mutant EGFR or translocated ALK , RET or ROS [ 10 ]. Nevertheless, most LUADs either lack an identifiable driver oncogene or carry mutations that are not currently clinically actionable such as those affecting KRAS , and are therefore still treated with conventional chemotherapy [ 11 , 12 , 13 ].

Despite progress in the understanding of the molecular and cellular basis and mechanisms leading to treatment failure, drug resistance continues to be the main limiting factor to achieve cures in patients with advanced cancer. Indeed, resistance of cancer cells is the major cause of tumor recurrence or relapse and affects not only conventional treatments but also targeted and immunological therapies [ 14 , 15 ]. Treatment failure often results from multiple genetic and epigenetic alterations and the subsequent perturbation of critical gene and protein networks regulating tumor cell fate [ 16 , 17 ]. Thus, targeting entire oncogenic networks may achieve deeper and prolonged therapeutic response than targeting individual genes or proteins within a pathway.

MicroRNAs (miRNAs), a class of small non-coding RNAs (ncRNAs) acting as negative regulators of genes involved in fundamental cellular pathways, are implicated in virtually all oncogenic processes [ 18 , 19 ]. Aberrant miRNA expression is a hallmark of cancer and restoring normal miRNA levels in neoplastic cells exerts significant anti-tumor properties. The ability of miRNAs to regulate multiple genes either within a molecular oncogenic pathway or belonging to distinct but interconnected carcinogenic pathways makes them excellent drug candidates for the development of novel anti-cancer therapeutics [ 20 ]. Indeed, developing miRNA-based therapeutics is likely to be more comprehensive and efficient than targeting individual genes or proteins, especially as a limited number of miRNAs are usually dysregulated in cancer, compared to the large expression changes characterizing cancer cell transcriptome and proteome [ 21 ].

In this study, we performed a functional miRNA screening to analyze the effects of individual miRNA inhibitors on cell viability and sensitivity to cisplatin using a LUAD cell line harboring KRAS mutation. Moreover, our results uncovered miR-92a-3p, a member of the miR-17 ~ 92 cluster, as an important regulator of the apoptotic process in various cancer cell lines including pancreatic cancer.

Silencing of miR-92a-3p increases sensitivity of lung adenocarcinoma cells to cisplatin

As the most advanced miRNA-based therapeutic strategy relies on miRNA silencing, we designed a loss-of-function approach to uncover miRNAs whose decreased expression is functionally associated with increased sensitivity to cisplatin. For this, we first profiled A549 cells to identify the most highly expressed miRNAs (Supplementary Table 1 ), which were identified by small RNA seq, and then performed individual knockdown experiments for each of them using 138 LNA (Locked Nucleic Acid)-based anti-miRNAs (Fig. 1A ). Indeed, the LUAD cell line A549 harbors KRASG12S point mutation and is widely used as a representative lung cancer cell model with KRAS mutation [ 22 ]. Our loss-of-function data identified miR-92a-3p as one of the best miRNAs whose knockdown is associated with cisplatin sensitivity (Fig. 1B ).

A Library of LNA-based miRNA inhibitors against the most expressed miRNAs in A549 was transfected in A549 cell line. Cells were then exposed to cisplatin at 30 µM for 72 h. Viability was measured using the CellTiterGlo assay. B miR-92a-3p (red dot) is the miRNA whose down-regulation is the most associated with cell death after cisplatin exposure at the indicated concentration. n  = 2 independent experiments; *** p  < 0.001.

Modulation of miR‐92a-3p influences cisplatin-induced apoptosis of A549 cells through BIM targeting

The BH3-only BIM protein, a bona fide target of miR-92a-3p [ 23 ], plays a major role in the initial molecular events of the intrinsic apoptotic pathway, in particular in response to anticancer drugs [ 24 ]. Indeed, tumoral expression of BIM has been shown to play a critical role in determining the response of cancer cells to not only conventional cytotoxic drugs, but also to a vast array of targeted agents [ 24 ]. Therefore, we investigated whether modulation of miR-92a-3p in LUAD cells influences their sensitivity to cisplatin through the targeting of BIM. For this, we first confirmed that siRNA-mediated silencing of BIM is sufficient to induce cisplatin resistance of LUAD cells. As depicted in Fig. 2A , siRNA-mediated silencing of BIM in A549 cells significantly reduced the cleavage of caspase 3, a reliable marker of cell apoptosis [ 25 ]. Then, we showed that modulation of miR-92a-3p inversely affects BIM expression. Indeed, overexpression of miR-92a-3p in A549 cells resulted in a strong decrease of BIM protein expression, whereas its silencing led to the induction of BIM protein expression (Fig. 2B, D ). Finally, we performed gain and loss of function experiments to assess the impact of miR-92a-3p modulation on cisplatin-induced cell apoptosis. We showed that overexpression of miR-92a-3p impaired cisplatin cytotoxic effects on A549 cells, whereas its silencing exacerbated cisplatin apoptotic effects (Fig. 2C, E ). As miRNAs usually regulate multiple functionally-related RNA targets to exert their function, we also investigated whether inhibiting miR-92a-3p also promotes cisplatin-induced apoptosis in BIM-depleted cells. As depicted in Fig. S1 , our results show that the regulation of apoptosis by miR-92a-3p in LUAD cells is not restricted to the targeting of BIM but instead likely involves additional targets.

A Western blot showing the effect of BIM inhibition on the caspase 3 apoptotic response induced by cisplatin (30 µM for 24 h). B – E Western blots showing the effect of ( B , D ) the modulation of miR-92a-3p on BIM expression and ( C , E ) on the caspase 3 apoptotic response induced by cisplatin (30 µM for 24 h). F , G Proliferation measured by cell viability 3 days after transfection of cells with ( F ) a premiR negative control and a premiR-92a-3p or ( G ) a LNA negative control and a LNA-miR-92a-3p. H , I Immunofluorescence showing the effect of ( H ) miR-92a-3p overexpression or ( I ) inhibition on Ki-67 expression. Quantitative data were obtained by measuring co-localization of DAPI staining with Ki-67 positive areas using ImageJ software. Data are presented as the mean +/− SEM. J , K Colony formation assay stained with crystal violet in cells transfected ( J ) with a premiR negative control and a premiR-92a-3p or ( K ) with a LNA negative control and a LNA-miR-92a-3p 4 and 7 days following transfection. L Western blot showing the effect of PTEN inhibition on the PTEN/AKT axis and on the caspase 3 apoptotic response induced by cisplatin (30 µM for 24 h). M , N Western blots showing the effect of the modulation of miR-92a-3p on the PTEN/AKT axis. Data are shown as mean +/− SEM. n  = 3 independent experiments. Representative Western blots are shown along with their quantification. * p  < 0.05; ** p  < 0.01; *** p  < 0.001. CTL control, LNA Locked Nucleic Acid.

Altogether, these results suggest that miR-92a-3p influences LUAD cell sensitivity to cisplatin by regulating BIM expression.

Modulation of miR‐92a-3p expression affects the proliferation of LUAD cells by targeting PTEN

To further characterize the oncogenic properties of miR-92a-3p, we investigated whether modulation of miR-92a-3p expression also impacts LUAD cell proliferation. To explore this, we first analyzed whether changes in miR-92a-3p expression affects cell growth using the CellTiterGlo cell-based assay. Our results showed that ectopic expression of miR-92a-3p in A549 cells performed three days after transfection led to an increase in cell viability that likely reflects enhanced cell growth, whereas antisense-mediated reduction of miR-92a-3p expression had the opposite effect (Fig. 2F, G ). To strengthen these findings, we then performed Ki-67 staining, a reliable method for evaluating cell proliferation, using A549 cells in which miR-92a-3p is either silenced or overexpressed. As shown in Fig. 2H, I , transfection of miR-92a-3p mimics increased the number of proliferating cells compared to control cells, whereas its knockdown negatively influenced cell growth. To determine the effect of miR-92a-3p on tumor-initiating capacity, a clonogenic assay was also performed. This showed that mimic miR-92a-3p-transfected A549 cells gave rise to significantly more colonies than control cells at the indicated time points following transfection (Fig. 2J ). Conversely, silencing of miR-92a-3p expression resulted in a reduced number of colonies (Fig. 2K ). Finally, as miR-92a-3p has been previously reported to directly target PTEN, the main regulator of the pro-proliferative and survival PI3K/AKT signaling pathway [ 26 , 27 ], we evaluated whether miR-92a-3p also influences the PTEN/AKT axis in LUAD (Fig. 2L–N ). As shown in Fig. 2M , overexpression of miR-92a-3p was sufficient to reduce PTEN expression and to increase phosphorylation of AKT, whereas silencing of miR-92a-3p decreased pAKT expression (Fig. 2N ). Overall, these results demonstrate that miR-92a-3p contributes to the malignant phenotype of LUAD by regulating cell proliferation.

MiR-92a-3p also modulates the sensitivity of other LUAD cell lines with distinct molecular alterations to cisplatin

To evaluate the importance of targeting miR-92a-3p in LUAD, we performed additional experiments using two other LUAD cell lines exhibiting distinct mutational profiles (PC9 and H1975, which harbor TP53 and EGFR mutations). Figure 3A–D showed that similarly, inhibition of miR-92a-3p increased BIM expression and the cleavage of caspase 3 following cisplatin treatment. Moreover, transfection with miR-92a-3p mimic increased cell growth and Ki-67 positive cells whereas transfection with miR-92a-3p inhibitor decreased cell proliferation in both cell lines (Fig. 3E–L ). Altogether, these results suggest that miR-92a-3p influences LUAD cell sensitivity to cisplatin and proliferation independently of their mutational status.

A – D Western blots showing the effect of the inhibition of miR-92a-3p on ( A , C ) BIM expression and ( B , D ) the caspase 3 apoptotic response induced by cisplatin (30 µM for 24 h) in both cell lines. E – H Proliferation measured by cell viability 3 days after transfection in both cell lines with ( E , G ) a premiR negative control and a premiR-92a-3p or with ( F , H ) a LNA negative control and a LNA-miR-92a-3p. I – L Immunfluorescence showing the effect of miR-92a-3p ( I , K ) overexpression or ( J , L ) inhibition on Ki-67 expression. Quantitative data were obtained by measuring co-localization of DAPI staining with Ki-67 positive areas using ImageJ software. Data are presented as the mean +/− SEM. n  = 3 independent experiments. Representative Western blots are shown along with their quantification. * p  < 0.05; ** p  < 0.01; *** p  < 0.001. CTL control, LNA Locked Nucleic Acid.

Concomitant altered pulmonary expression of both BIM and miR-92a-3p in the KRASG12D-mediated lung cancer mouse model

To further strengthen our in vitro findings, expression levels of miR-92a-3p and BIM were assessed in the KRASG12D-mediated lung adenocarcinoma mouse model, which closely resembles the genetic and pathophysiological features of human lung adenocarcinoma. Compared with control, these analyses showed an increased expression of miR-92a-3p in tumor tissue, 15 weeks after tumor induction, along with a reduced tumoral BIM protein expression compared to adjacent non-tumor tissue (Fig. 4 ).

A Relative expression of miR-92a-3p in lung tissues of mice treated with tamoxifen ( n  = 10) or vehicle ( n  = 6). B Representative images of lung tissue sections assessed by hematoxylin and eosin staining and immunohistochemical analysis of BIM. *** p  < 0.001). VEH vehicle, TMX tamoxifen, HE hematoxylin and eosin.

miR-92a-3p also influences response of pancreatic cancer cells to cisplatin

As mutation of KRAS is the most frequent genetic alteration in pancreatic adenocarcinoma as well as in precancerous lesions such as PanIN (Pancreatic Intraepithelial neoplasia) [ 28 ], we evaluated whether miR-92a-3p also influences response of KRAS-mutated pancreatic cancer cells to cisplatin by performing loss of function experiments in PANC1 cells, a human epithelioid carcinoma cell line harboring the KRASG12D mutation. This showed that downregulation of miR-92a-3p led to the induction of BIM expression and to the increase of caspase 3 cleavage after cisplatin exposure (Fig. 5A, B ). Moreover, silencing of miR-92a-3p decreased PANC1 cell proliferation as shown in Fig. 5C .

A , B Western blot showing the effect of miR-92a-3p inhibition in PANC1 cells on ( A ) BIM expression and ( B ) the caspase 3 apoptotic response induced by cisplatin (70 µM for 24 h). Representative Western blots are shown along with their quantification. C Immunofluorescence showing the effect of miR-92a-3p inhibition on Ki-67 expression. Quantitative data were obtained by measuring co-localization of DAPI staining with Ki-67 areas using ImageJ software. Data are presented as the mean +/− SEM. n  = 3 independent experiments. D Relative expression of miR-92a-3p in tumoral tissues from the KRASG12D-mediated pancreatic cancer mouse model with or without caerulein injections and compared with corresponding wild-type mice. E Representative images of mouse pancreatic sections stained with hematoxylin and eosin and immunohistochemical analysis of BIM. Data are presented as the mean +/− SEM. n  = at least four mice per group. * p  < 0.05, *** p  < 0.001. WT wild type.

Expression of miR-92a-3p was also assessed in tumoral tissues from the KRASG12D-mediated pancreatic cancer mouse model and compared with corresponding wild-type animals (Fig. 5D ). Figure 5E indicates that mice exhibiting PanIN lesions or invasive pancreatic ductal adenocarcinoma show an increased miR-92a-3p expression and a downregulation of BIM expression.

Targeting the polycistronic lncRNA MIR17HG has no effect on the sensitivity of LUAD cells to cisplatin

As miR-92a-3p belongs to the polycistronic miRNA cluster miR-17 ~ 92 encoded by MIR17HG, which gives rise to six oncogenic miRNAs grouped into four different families (miR-17, miR-18, miR-19, and miR-92) based on their seed regions [ 29 ], we reasoned that directly targeting nuclear MIR17HG would provide a much more efficient approach to promote cisplatin sensitivity by simultaneously inducing the downregulation of all members of the miR-17 ~ 92 cluster. Interestingly, this strategy was recently successfully applied in multiple myeloma using GapmeRs, which consist of single-stranded antisense oligonucleotides able to induce RNase-H cleavage of the targeted transcript especially in the nucleus [ 30 ]. We first confirmed the nuclear localization of MIR17HG by RNA-Fish® in A549 cells and its increased expression in the LUAD mouse model (Fig. S2A–B ).

We then designed ten distinct GapmeRs directed against human MIR17HG and tested their efficacy in vitro using A549 and PC9 cell lines. As shown in Fig. S2C , among the ten tested GapmeRs, eight were able to efficiently repress nuclear MIR17HG 48 h after transfection. Nevertheless, two of them exhibited strong cellular toxicity likely due to off-target effects and were thus excluded. We then assessed whether the six selected GapmeRs significantly alter the expression level of the miRNAs including in the miR-17 ~ 92 cluster. As depicted in Fig. S2C , each of the six selected GapmeRs reproducibly but modestly induced the downregulation of all members of the cluster 72 h after transfection in A549 cells (Fig. S2C ) and in PC9 cells (data not shown). However, in contrast to the study from Morelli et al. [ 31 ], none of the selected GapmeRs was able to affect BIM expression or cisplatin sensitivity (Fig. S2D ).

Cancer therapy usually relies on the simultaneous or sequential administration of chemotherapeutic agents with non-overlapping mechanisms of action to achieve greater treatment efficacy and to prevent the selection of drug resistant clones as well as the subsequent early regrowth of tumors [ 32 , 33 ]. Nevertheless, despite remarkable successes in some forms of lymphoma, breast and testicular cancers, most patients receiving polychemotherapy inevitably relapse [ 34 ]. Therefore, new insights into the molecular determinants of tumor sensitivity to chemotherapy are critical to optimize current treatment regimens and to develop new effective combination therapies. This is particularly true for mutant KRAS-driven lung tumors, which are usually refractory to first-line platinum-based chemotherapy [ 35 ]. In this study, we reasoned that miRNA-based combination therapy represents an attractive therapeutic strategy to increase cisplatin response of KRAS-driven lung cancer. Indeed, the ability of miRNAs to modulate multiple transcripts from the same or interconnected oncogenic pathways represents a steady advantage over traditional single gene approaches [ 36 ].

Functional genetic screening is a powerful unbiased strategy for assessing the role of specific genes in different neoplastic phenotypes, and the rapid identification of novel drug targets [ 37 , 38 ]. Indeed, this approach has the great advantage of enabling the establishment of a causative link between gene expression and a specific oncogenic phenotype such as response to anticancer agents, in contrast to many other profiling high-throughput methods that are mainly correlative [ 37 ]. In line with this, we designed a miRNA loss-of-function drug sensitization screen to uncover miRNAs whose loss-of-function silencing reduces the viability of KRAS mutated A549 cells in the presence of a sublethal concentration of cisplatin. Among the various miRNAs investigated, we focused our attention on miR-92a-3p, whose decreased expression has the greatest impact on cisplatin sensitivity. Indeed, miR-92a-3p is a well-characterized small oncogenic ncRNA whose pro-tumoral activity has been characterized in a variety of distinct human malignancies [ 39 , 40 , 41 ]. In particular, this ncRNA is almost invariably overexpressed in tumors and represents an important regulator of numerous tumorigenic processes including cell proliferation and apoptosis through various molecular mechanisms that can be shared by cancer cells or unique to a given tumor type [ 42 , 43 , 44 ]. For example, miR-92a-3p has been shown to inhibit apoptosis and tumor growth in many distinct malignancies by targeting the BH3-only protein BIM and PTEN respectively [ 23 , 27 ], while promoting cell proliferation by cancer-type specific mechanisms involving targeting of genes such as BTG2 in breast cancer or FBXW7 in renal cancer [ 39 , 45 ]. In lung cancer, previous reports have notably shown the diagnostic/prognostic value of miR-92a-3p as well as a significant association between expression of this miRNA and response to radiotherapy [ 46 , 47 , 48 , 49 ]. Nevertheless, although several in vitro evidence support an oncogenic role of miR-92a-3p in lung cancer cells [ 50 ], its precise molecular function remains unclear and little is known about the role played by miR-92a-3p in chemoresistance. In this study, we showed that miR-92a-3p, using gain- and loss-of-function approaches, is an important determinant of LUAD sensitivity to cisplatin not only in cells in which KRAS is mutated but also in those harboring alteration of TP53 . This is indeed of particular interest, as TP53 somatic mutations frequently occur in LUAD patients and are usually associated with resistance to therapy including cisplatin-based treatment regimens [ 51 ].

Mechanistically, we showed that miR-92a-3p influences, at least in part, cisplatin sensitivity by fine-tuning the BH3 only protein BIM, a central activator of apoptosis [ 52 ]. This is consistent with a previous study that linked loss of expression of BIM with cisplatin resistance in ovarian cancer [ 52 ]. Interestingly, we also showed that silencing of miR-92a-3p also increases BIM protein expression and sensitivity to cisplatin in another KRAS-driven tumor cell line model distinct from LUAD, the pancreatic ductal adenocarcinoma. As this malignancy is also usually refractory to therapies, our results warrant additional investigations to evaluate whether the targeting of miR-92a-3p could represent a new therapeutic option, especially as cisplatin is not used as first-line treatment in this disease. Finally, our results also showed that modulation of miR-92a-3p expression not only influences apoptosis but also cancer cell proliferation likely by regulating the PTEN/AKT signaling pathway. Thus, miR-92a-3p is involved in the regulation of distinct biological cell processes essential for the initiation and maintenance of the malignant phenotype [ 53 ].

As miR-92a-3p belongs to the polycistronic miRNA cluster miR-17 ~ 92 encoded by MIR17HG , we hypothesized that directly targeting MIR17HG using a single GapmeR, would represent a better strategy to promote cisplatin sensitivity by simultaneously inducing the downregulation of the six miRNAs encoded by miR-17 ~ 92. Indeed, it is well-established that the oncogenic activity of the cluster primarily relies on its cognate miRNAs that efficiently modulate multiple oncogenic pathways by regulating a repertoire of targets that can be shared by members of this cluster or specific to an individual miRNA. However, in contrast to Morelli et al. (2018) who recently successfully applied this approach in multiple myeloma, none of the GapmeRs tested was able to modulate BIM expression or promote the sensitivity of LUAD to cisplatin [ 31 ]. This may be explained by an addiction of multiple myeloma-derived cells to MIR17HG or an off-target effect of the GapmeR designed by Morelli et al. (2018) [ 31 ]. Therefore, additional works should be performed to better understand these discrepancies.

In conclusion, our results support a fundamental role of miR-92a-3p in the regulation of the apoptotic process which may be therapeutically exploited to promote the sensitivity of cancer cells to cisplatin.

Cell culture

Human non-small cell lung cancer (NSCLC) A549 cells (ATCC, LGC Standards S.A.R.L, Molsheim, France) and human pancreatic epithelial carcinoma PANC1 cells (ATCC) were cultured in Glutamax-containing DMEM (Thermofisher Scientific, Illkirch-Graffenstaden, France) medium supplemented with 10% FBS (Thermofisher Scientific) and 1% Penicillin/Streptomycin (Thermofisher Scientific). Human NSCLC PC9 (ATCC) and H1975 (ATCC) cells were cultured in Glutamax-containing RPMI (Thermofisher Scientific) supplemented as above. Cells were cultured at 37 °C in a humidified atmosphere of 5% CO 2 in T75 flasks and seeded into 6-, 24- or 96-well plates 24 h before the experiments.

Functional screening

A library of LNA-based miRNA inhibitors (Qiagen, miRbase version 10, Les Ulis, France) against the most highly expressed miRNAs in A549 cells was used in this study (Supplemental Table 1 ), which were identified by small RNA seq on SOLID 5500 WF (Thermofisher Scientific). Each LNA inhibitor (10 nM) was transfected in triplicate in A549 cells using Lipofectamine RNAiMAX (Thermofisher Scientific) in 96-well plates. After 48 hours, cells were exposed to a sub-apoptotic dose of cisplatin (30 μM for three days). Viability was assessed using the CellTiterGlo assay (Promega, Charbonnières-les-Bains, France). Normalization was carried out by dividing each sample value by the median of all samples on the plate (the majority of sample wells will thus serve as a reference). miRNA candidates were selected for further analysis based on statistical significance ( p  < 0.01) and degree of sensibility induced (assessed by viability after drug exposure, normalized viability below 3).

Small RNA-Seq

Small RNA-Seq was performed as described in [ 54 ]. Briefly, 500 ng of total RNAs were ligated, reverse transcribed and amplified (18 cycles) with the reagents from the NextFlex small RNAseq kit V3 (Bioo scientific, Villebon-sur-Yvette, France). Amplified libraries were quantified with the Bioanalyzer High Sensitivity DNA Kit (Agilent, Les Ulis, France), pooled and size-selected from 140 nt to 170 nt with the LabChip XT DNA 300 Assay Kit (Caliper Lifesciences, Villepinte, France). Libraries were then sequenced on an Illumina Nextseq 500 Mid Flowcell with 75 pb reads for a total of 147 M reads.

Transfection and cisplatin exposure

Cells were transfected at 30 to 40% of confluency in 6-, 24- or 96-well plates, using Lipofectamine RNAiMAX (Thermofisher Scientific) with pre-miRNAs and control miRNA (Thermofisher Scientific) or LNA inhibitors and LNA negative control (Qiagen) at a final concentration of 10 nM. Antisens oligonucleotides GapmeRs, directed against MIR17HG (Supplementary Table 2 ; Qiagen) and siRNA directed against BIM or PTEN (ThermoFisher Scientific) were transfected as described above at a final concentration of 25 nM and 10 nM respectively.

Forty-eight hours after transfection, cells were exposed to a sub-apoptotic dose of cisplatin at 30 μM or 70 µM for 24 h for NSCLC cell lines and PANC1 respectively.

Protein extraction and immunoblotting

Cells were lysed in RIPA (Radioimmunoprecipitation assay) buffer (Sigma-Aldrich, Saint-Quentin-Fallavier, France) containing protease and phosphatase inhibitors (Roche, Meylan, France). Lysates were quantified using the BCA (Bicinchoninic acid) protein assay kit (Thermofisher Scientific). Proteins were separated by SDS-polyacrylamide gel and transferred onto nitrocellulose membranes (Biorad, Marnes-la-Coquette, France). The membranes were blocked with 5% fat free milk or Bovine Serum Albumin (BSA, in case of phospho protein) in Tris-buffered Saline (TBS) containing 0.1% Tween-20 (TBS-T) and subsequently incubated with their respective primary antibodies overnight at 4 °C. After washing with TBS-T, membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies for 45 min, followed by washing with TBS-T. Protein bands were visualized with Amersham ECL substrates (GE Healthcare, Buc, France).

The following antibodies were used: goat anti-HSP60 (sc-1052, Santa Cruz Biotechnology Inc., Heidelberg, Germany), rabbit anti-cleaved Caspase 3 (Asp175) (#9661, Cell Signaling Technology, Saint-Cyr-L’École, France), rabbit anti-Bim (#2933, Cell Signaling Technology), rabbit anti-PTEN (#9559, Cell Signaling Technology), rabbit anti-pAKT (#4058, Cell Signaling Technology), rabbit anti-AKT (#9272, Cell Signaling Technology).

Immunofluorescence analysis

Cells were grown on a Round Glass Coverslip Ø 16 mm (Thermofisher Scientific) placed inside a 24-well plate. Coverslips were washed in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 15 min, then cells were permeabilized using 0.1% Triton X-100 (Agilent Technologies, Les Ulis, France) for 10 min and blocked with 3% BSA in TBS for 30 min. Incubation with primary antibody rabbit anti-Ki-67 (#9129, Cell Signaling Technology) was performed in a blocking solution BSA (1%) at 37 °C for 1 h. After three washes with TBS, cells were incubated with secondary antibodies for 45 min at 37 °C. Coverslips were then fixed on microscope slides using ProLong Gold Antifade Reagent with DAPI (Thermofisher Scientific). Fluorescence was detected with a Spinning Disk confocal microscope (Zeiss, Rueil Malmaison, France). Quantitative data were obtained by measuring co-localization of DAPI staining with Ki-67 areas using ImageJ software (National Institute of Health).

Cell proliferation

Cells were grown in 6-well plates and then transfected. After 24 h, cells were seeded into 96-well plates. Cell proliferation was evaluated after three days using the CellTiterGlo kit (Promega).

Clonogenicity

Cells were grown in 6-well plates and then transfected. After 24 h, 4000 cells were seeded into 6-well plates for four and seven days. Cells were fixed with cold methanol and stained with 0.5% crystal violet (Sigma-Aldrich). Excess stain was removed by washing repeatedly with water. For colony quantification, the crystal violet staining was solubilized using 10% acetic acid and the absorbance was measured at a wavelength of 590 nm.

Custom Stellaris® FISH Probes were designed against MIR17HG using the Stellaris® RNA FISH Probe Designer (Biosearch Technologies, Hoddesdon, United-Kingdom) available online at https://www.biosearchtech.com .

A549 cells were grown in μ-Dish 35 mm glass bottom (Ibidi, Nanterre, France). After 48 h of culture, cells were permeabilized using 70% ethanol and then hybridized with the MIR17HG Stellaris RNA FISH Probe set labeled with CAL Fluor Red 590 (Biosearch Technologies), following the manufacturer’s instructions at the final concentration of 500 nM. Acquisition was performed using an inverted confocal microscope LSM 710 (Zeiss) with the high-resolution module AiryScan (Zeiss) and a 63x/1.4 oil immersion lens. Images were processed using Zen software (Zeiss).

Mouse models of cancer

All animal care and experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Lille University (Protocol Number: APAFIS#17098.05 and APAFIS#00422.02). Sample size was chosen empirically based on our previous experiences in the calculation of experimental variability; no statistical method was used to predetermine sample size and no samples, mice or data points were excluded from the reported analyses. Experiments were performed non-blinded using treatment group randomized mice. Manipulators carried out all experimental protocols under strict guidelines to ensure careful and consistent handling of the mice.

CCSP-Cre; LSL-KRAS G12D lung adenocarcinoma mouse model

CCSP-Cre and LSL-KRAS G12D mice were purchased from Jackson laboratory (L’Arbresle, France). To induce lung tumors, tamoxifen (0.25 mg/g) (Sigma Aldrich) was injected in CCSP-Cre; LSL-KRAS G12D mice (8 to 12 weeks old males) for 5 days. Fifteen weeks after tumor initiation, mice were sacrificed by cervical dislocation. For histological analysis, lungs were perfused with 10% formalin (Sigma-Aldrich) and then included in paraffin. For molecular biology analysis, lungs were stored in RNAlater solution (Thermofisher Scientific).

Pdx1-Cre; LSL-KRASG12D pancreatic cancer mouse model

Pdx1-Cre; LSL-KRASG12D mice were a generous gift from Dr Nicolas Jonckheere (UMR9020 CNRS – U1277 Inserm).

To induce pancreatic tumors, intraperitoneal injections of 37.5 µg/mL caerulein (Sigma-Aldrich) solution were performed using 6-month-old transgenic mice following a two-step protocol. First, an acute treatment consisting of an injection every hour for 6 h (1st day) followed by a chronic treatment consisting of caerulein injections 5 days a week for 59 days. After sacrifice, pancreas were collected from 6-month-old KRASG12D males and WT control mice, fixed and embedded in paraffin for histological analysis or flash-frozen for RNA extraction.

RNA extraction

Total RNAs were extracted from tissue or cell samples with the miRNeasy Mini kit (Qiagen), according to the supplier’s recommendations.

Quantitative RT-PCR

For cell samples, miRNA retro-transcription was performed using RT miRCURY LNA kit (Qiagen). Quantitative PCR was performed on a StepOnePlus TM Real-Time PCR System (Thermofisher Scientific) with miRCURY LNA Probe PCR kit (Qiagen) and with the following primers: miR-17-5p (YP02119304), miR-18a-5p (YP00204207), miR-19a-3p (YP00205862), miR-19b-3p (YP00204450), miR-20a-5p (YP00204292), miR-92a-3p (YP00204258). For normalization, transcript levels of RNU44 (YP00203902) were used as endogenous control for miRNA expression. For mouse samples, miRNA retro-transcription was performed using the TaqMan™ MicroRNA Reverse Transcription Kit (Thermofisher Scientific). Quantitative PCR was performed on a StepOnePlus TM Real-Time PCR System using Universal Master Mix (Thermofisher Scientific) and the following TaqMan assays: miR-92a-3p (assay ID 000430), snoRNA202 (assay ID 001232) and snoRNA251 (assay ID 001236).

mRNA retro-transcription was performed using the High Capacity cDNA reverse transcription kit (Thermofisher Scientific). Expression levels of genes (primers are listed in Supplementary Table 3 ) were performed on a StepOnePlus TM Real-Time PCR System (Thermofisher Scientific) with Fast SYBR Green Master Mix (Thermofisher Scientific). For normalization, transcript levels of PPIA were used as endogenous control for gene expression.

Relative expression levels of mRNAs and miRNAs were assessed using the comparative threshold cycle method (2 −ΔΔCT ) [ 55 ].

5-μm paraffin-embedded sections were mounted and stained with hematoxylin and eosin (Sigma-Aldrich). Acquisition was performed using a DMi8 microscope (Leica, Nanterre, France) at 20x magnification.

Immunohistochemistry

5-μm paraffin-embedded sections were sequentially incubated in xylene (5 min twice), 100% ethyl alcohol (5 min twice), 95% ethyl alcohol (5 min twice), and 80% ethyl alcohol (5 min). After washing with water, the sections were antigen-retrieved using citrate buffer (pH 6.0; DAKO, Les Ulis, France) in a domestic microwave oven for 20 min and cooled to ambient temperature. Sections were then washed with TBS-T and quenched with 3% hydrogen peroxide in TBS for 10 min, blocked for avidin/biotin activity, blocked with serum-free blocking reagent, and incubated with primary antibody rabbit anti BIM (#C34C5, Cell Signaling Technology). Immunohistochemical staining was developed using the DAB (3,3’-Diaminobenzidine) substrate system (DAKO). Acquisition was performed using a DMi8 microscope (Leica) at 20x magnification.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software. Results are given as mean ± SEM. Two-tailed Mann-Whitney test was used for single comparisons; one-way ANOVA followed by Bonferroni post hoc test was used for multiple comparisons. p -value less than 0.05 was considered statistically significant.

Data availability

All data generated or analyzed during this study are available from the corresponding author on reasonable request.

Zappa C, Mousa SA. Non-small cell lung cancer: current treatment and future advances. Transl Lung Cancer Res. 2016;5:288–300.

Article   CAS   PubMed   PubMed Central   Google Scholar  

Schulze AB, Evers G, Kerkhoff A, Mohr M, Schliemann C, Berdel WE, et al. Future options of molecular-targeted therapy in small cell lung cancer. Cancers. 2019;11:690.

Dutt A, Wong KK. Mouse models of lung cancer. Clin Cancer Res Off J Am Assoc Cancer Res. 2006;12:4396s–402s.

Article   CAS   Google Scholar  

Ocak S, Sos ML, Thomas RK, Massion PP. High-throughput molecular analysis in lung cancer: insights into biology and potential clinical applications. Eur Respir J. 2009;34:489–506.

Kwon MC, Berns A. Mouse models for lung cancer. Mol Oncol. 2013;7:165–77.

Pikor LA, Ramnarine VR, Lam S, Lam WL. Genetic alterations defining NSCLC subtypes and their therapeutic implications. Lung Cancer Amst Neth. 2013;82:179–89.

Article   Google Scholar  

Shim HS, Choi YL, Kim L, Chang S, Kim WS, Roh MS, et al. Molecular testing of lung cancers. J Pathol Transl Med. 2017;51:242–54.

Article   PubMed   PubMed Central   Google Scholar  

Herbst RS, Morgensztern D, Boshoff C. The biology and management of non-small cell lung cancer. Nature. 2018;553:446–54.

Article   CAS   PubMed   Google Scholar  

Imyanitov EN, Iyevleva AG, Levchenko EV. Molecular testing and targeted therapy for non-small cell lung cancer: current status and perspectives. Crit Rev Oncol Hematol. 2021;157:103194.

Article   PubMed   Google Scholar  

Hirsch FR, Scagliotti GV, Mulshine JL, Kwon R, Curran WJ, Wu YL, et al. Lung cancer: current therapies and new targeted treatments. Lancet Lond Engl. 2017;389:299–311.

Lemjabbar-Alaoui H, Hassan OU, Yang YW, Buchanan P. Lung cancer: Biology and treatment options. Biochim Biophys Acta. 2015;1856:189–210.

CAS   PubMed   PubMed Central   Google Scholar  

Kato S, Fujiwara Y, Hong DS. Targeting KRAS: crossroads of signaling and immune inhibition. J Immunother Precis Oncol. 2022;5:68–78.

Seguin L, Durandy M, Feral CC. Lung adenocarcinoma tumor origin: a guide for personalized medicine. Cancers. 2022;14:1759.

Amable L. Cisplatin resistance and opportunities for precision medicine. Pharmacol Res. 2016;106:27–36.

Lin JJ, Shaw AT. Resisting resistance: targeted therapies in lung cancer. Trends Cancer. 2016;2:350–64.

Galluzzi L, Vitale I, Michels J, Brenner C, Szabadkai G, Harel-Bellan A, et al. Systems biology of cisplatin resistance: past, present and future. Cell Death Dis. 2014;5:e1257.

Liu WJ, Du Y, Wen R, Yang M, Xu J. Drug resistance to targeted therapeutic strategies in non-small cell lung cancer. Pharmacol Ther. 2020;206:107438.

Landau DA, Slack FJ. MicroRNAs in mutagenesis, genomic instability, and DNA repair. Semin Oncol. 2011;38:743–51.

Peng Y, Croce CM. The role of MicroRNAs in human cancer. Signal Transduct Target Ther. 2016;1:15004.

Reda El Sayed S, Cristante J, Guyon L, Denis J, Chabre O, Cherradi N. MicroRNA therapeutics in cancer: current advances and challenges. Cancers. 2021;13:2680.

Christopher AF, Kaur RP, Kaur G, Kaur A, Gupta V, Bansal P. MicroRNA therapeutics: discovering novel targets and developing specific therapy. Perspect Clin Res. 2016;7:68–74.

Leung ELH, Luo LX, Liu ZQ, Wong VKW, Lu LL, Xie Y, et al. Inhibition of KRAS-dependent lung cancer cell growth by deltarasin: blockage of autophagy increases its cytotoxicity. Cell Death Dis. 2018;9:1–15.

Niu H, Wang K, Zhang A, Yang S, Song Z, Wang W, et al. miR-92a is a critical regulator of the apoptosis pathway in glioblastoma with inverse expression of BCL2L11. Oncol Rep. 2012;28:1771–7.

Sionov RV, Vlahopoulos SA, Granot Z. Regulation of Bim in health and disease. Oncotarget. 2015;6:23058–134.

Crowley LC, Waterhouse NJ. Detecting cleaved caspase-3 in apoptotic cells by flow cytometry. Cold Spring Harb Protoc. 2016;2016.

Yu JSL, Cui W. Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination. Development. 2016;143:3050–60.

Lu C, Shan Z, Hong J, Yang L. MicroRNA-92a promotes epithelial-mesenchymal transition through activation of PTEN/PI3K/AKT signaling pathway in non-small cell lung cancer metastasis. Int J Oncol. 2017;51:235–44.

Waters AM, Der CJ. KRAS: the critical driver and therapeutic target for pancreatic cancer. Cold Spring Harb Perspect Med. 2018;8:a031435.

Ota A, Tagawa H, Karnan S, Tsuzuki S, Karpas A, Kira S, et al. Identification and characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma. Cancer Res. 2004;64:3087–95.

Amodio N, Stamato MA, Juli G, Morelli E, Fulciniti M, Manzoni M, et al. Drugging the lncRNA MALAT1 via LNA gapmeR ASO inhibits gene expression of proteasome subunits and triggers anti-multiple myeloma activity. Leukemia. 2018;32:1948–57.

Morelli E, Biamonte L, Federico C, Amodio N, Di Martino MT, Gallo, et al. Therapeutic vulnerability of multiple myeloma to MIR17PTi, a first-in-class inhibitor of pri-miR-17-92. Blood. 2018;132:1050–63.

Pritchard JR, Lauffenburger DA, Hemann MT. Understanding resistance to combination chemotherapy. Drug Resist Updat Rev Comment Antimicrob Anticancer Chemother. 2012;15:249–57.

CAS   Google Scholar  

Yap TA, Parkes EE, Peng W, Moyers JT, Curran MA, Tawbi HA. Development of immunotherapy combination strategies in cancer. Cancer Discov. 2021;11:1368–97.

Chatterjee N, Bivona TG. Polytherapy and Targeted Cancer Drug Resistance. Trends Cancer. 2019;5:170–82.

Xie M, Xu X, Fan Y. KRAS-Mutant Non-Small Cell Lung Cancer: An Emerging Promisingly Treatable Subgroup. Front Oncol. 2021;11:672612.

Garzon R, Marcucci G, Croce CM. Targeting microRNAs in cancer: rationale, strategies and challenges. Nat Rev Drug Discov. 2010;9:775–89.

Iorns E, Lord CJ, Turner N, Ashworth A. Utilizing RNA interference to enhance cancer drug discovery. Nat Rev Drug Discov. 2007;6:556–68.

Haley B, Roudnicky F. Functional genomics for cancer drug target discovery. Cancer Cell. 2020;38:31–43.

Jinghua H, Qinghua Z, Chenchen C, Lili C, Xiao X, Yunfei W, et al. MicroRNA miR-92a-3p regulates breast cancer cell proliferation and metastasis via regulating B-cell translocation gene 2 (BTG2). Bioengineered. 2021;12:2033–44.

Wang Y, Chen A, Zheng C, Zhao L. miR-92a promotes cervical cancer cell proliferation, invasion, and migration by directly targeting PIK3R1. J Clin Lab Anal. 2021;35:e23893.

Yang J, Hai J, Dong X, Zhang M, Duan S. MicroRNA-92a-3p enhances cisplatin resistance by regulating Krüppel-Like factor 4-mediated cell apoptosis and epithelial-to-mesenchymal transition in cervical cancer. Front Pharmacol. 2021;12:783213.

Olive V, Jiang I, He L. mir-17-92, a cluster of miRNAs in the midst of the cancer network. Int J Biochem Cell Biol. 2010;42:1348–54.

Zhang X, Li Y, Qi P, Ma Z. Biology of MiR-17-92 cluster and its progress in lung cancer. Int J Med Sci. 2018;15:1443–8.

Zhao W, Gupta A, Krawczyk J, Gupta S. The miR-17-92 cluster: Yin and Yang in human cancers. Cancer Treat Res Commun. 2022;33:100647.

Zeng R, Huang J, Sun Y, Luo J. Cell proliferation is induced in renal cell carcinoma through miR-92a-3p upregulation by targeting FBXW7. Oncol Lett. 2020;19:3258–68.

Huang YF, Liu MW, Xia HB, He R. Expression of miR-92a is associated with the prognosis in non-small cell lung cancer: an observation study. Medicine (Baltimore). 2022;101:e30970.

Reis PP, Drigo SA, Carvalho RF, Lopez Lapa RM, Felix TF, Patel D, et al. Circulating miR-16-5p, miR-92a-3p, and miR-451a in plasma from lung cancer patients: potential application in early detection and a regulatory role in tumorigenesis pathways. Cancers. 2020;12:2071.

Vykoukal J, Fahrmann JF, Patel N, Shimizu M, Ostrin EJ, Dennison JB, et al. Contributions of circulating microRNAs for early detection of lung cancer. Cancers. 2022;14:4221.

Zeng L, Zeng G, Ye Z. Bioinformatics analysis for identifying differentially expressed MicroRNAs derived from plasma exosomes associated with radiotherapy resistance in non-small-cell lung cancer. Appl Bionics Biomech. 2022;2022:9268206.

Alcantara KMM, Garcia RL. MicroRNA‑92a promotes cell proliferation, migration and survival by directly targeting the tumor suppressor gene NF2 in colorectal and lung cancer cells. Oncol Rep. 2019;41:2103–16.

Mogi A, Kuwano H. TP53 mutations in nonsmall cell lung cancer. BioMed Res Int. 2011;2011:e583929.

Google Scholar  

Wang J, Zhou JY, Wu GS. Bim protein degradation contributes to cisplatin resistance. J Biol Chem. 2011;286:22384–92.

Taieb J, Prager GW, Melisi D, Westphalen CB, D’Esquermes N, Ferreras A, et al. First-line and second-line treatment of patients with metastatic pancreatic adenocarcinoma in routine clinical practice across Europe: a retrospective, observational chart review study. ESMO Open. 2020;5:e000587.

Savary G, Dewaeles E, Diazzi S, Buscot M, Nottet N, Fassy J, et al. The long noncoding RNA DNM3OS is a reservoir of FibromiRs with major functions in lung fibroblast response to TGF-β and pulmonary fibrosis. Am J Respir Crit Care Med. 2019;200:184–98.

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods San Diego Calif. 2001;25:402–8.

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Acknowledgements

We thank the Plateformes Lilloises en Biologie et Santé (PLBS) – UMS 2014 – US 41 (Sophie Crespin): the animal care facility (PLBS UAR 2014 - US 41); the BioImaging Center Lille (Meryem Tardivel and Antonino Bongiovanni), Edmone Dewaeles and Nicolas Jonckheere for their technical assistance. This work was supported by the Ligue Nationale contre le Cancer - Comité Septentrion and by the Contrat de Plan Etat-Région CPER Cancer 2015-2020 (to CC, NP and MP). The Canther Laboratory is part of the ONCOLille Institute.

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These authors contributed equally: Romain Larrue, Sandy Fellah.

These authors jointly supervised this work: Nicolas Pottier, Cynthia Van der Hauwaert.

Authors and Affiliations

University Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, UMR9020-U1277 - CANTHER - Cancer Heterogeneity Plasticity and Resistance to Therapies, 59000, Lille, France

Romain Larrue, Sandy Fellah, Nihad Boukrout, Corentin De Sousa, Julie Lemaire, Carolane Leboeuf, Marine Goujon, Michael Perrais, Christelle Cauffiez, Nicolas Pottier & Cynthia Van der Hauwaert

Université Côte d’Azur, CNRS UMR7275, IPMC, FHU-OncoAge, IHU RespiERA, 06560, Valbonne, France

Bernard Mari

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Contributions

CC, NP conceptualized the study. CVDH, NP, CC designed the methodology. RL, SF, NB, CDS, JL, CL, MG, CVDH performed the experiments. RL, SF, NB, CL, BM, MP, CC, NP, CVDH performed data analysis. CVDH, CC, NP wrote the manuscript. All Authors read and approved the final manuscript.

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Correspondence to Cynthia Van der Hauwaert .

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Larrue, R., Fellah, S., Boukrout, N. et al. miR-92a-3p regulates cisplatin-induced cancer cell death. Cell Death Dis 14 , 603 (2023). https://doi.org/10.1038/s41419-023-06125-z

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Received : 30 March 2023

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Accepted : 05 September 2023

Published : 13 September 2023

DOI : https://doi.org/10.1038/s41419-023-06125-z

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The Dow is set to surge 50% by 2030 as the 'roaring 20s' are alive and well for stocks, market vet says

• With the stock market trading at record highs, the "Roaring 20s" thesis is alive and well.
• That's according to Ed Yardeni, who expects the Dow and S&P 500 to soar 50% by 2030.
• "That target could be achieved with a forward P/E of 20 and forward earnings at $400 per share," Yardeni said.

With stocks trading at record highs, the "Roaring 20's" bull thesis remains intact, according to market veteran Ed Yardeni.

Yardeni said in a recent note that his roaring 20s thesis, which is based on the idea that AI will help unleash a productivity boom in the economy, will help drive the stock market 50% higher by 2030, with the Dow Jones Industrial Average and S&P 500 rising to 60,000 and 8,000, respectively. 

Yardeni said his 2030 targets are based on continued earnings growth and a simple 6% compounded annual growth rate, which is slightly lower than the stock market's historical average annual return of 7% net of inflation.

"That target could be achieved with a forward P/E of 20 and forward earnings at $400 per share, up 60% from an estimated $250 per share this year. We think that's possible in our Roaring 2020s scenario," Yardeni said. 

Forward S&P 500 earnings per share hit $257.20 last week, and analysts currently estimate that S&P 500 EPS will rise to $278 in 2025 and $313 in 2026. 

"These estimates suggest that $400 by 2030 is quite possible," Yardeni said.

Helping fuel those earnings, according to Yardeni, is continued consumer resilience, which will be driven by tens of millions of baby boomers that are set to spend their nest egg on all kinds of goods and services over the next few decades.

In an interview with CNBC on Tuesday, Yardeni Research chief market strategist Eric Wallerstein outlined the firm's broad outlook for stocks over the next few years.

"This whole roaring 2020s scenario right now is our highest probability outcome. We attribute a 60% likelihood of that. We have a 20% scenario of a meltup in the stock market, and if the Fed preliminary cuts, we can see that. Meltups are fine you just have to know when to get out," Wallerstein said.

"And then there's that 20% scenario where there's another revival in inflation. But for now we see productivity growth really being a strong driver of real incomes and for the next several years driving the market higher."

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