Low-passage Cell Lines Research Articles

Selected GRIN2A mutations in melanoma cause oncogenic effects that can be modulated by extracellular glutamate

GRIN2A mutations are frequent in melanoma tumours but their role in disease is not well understood. GRIN2A encodes a modulatory subunit of the N-methyl-d-aspartate receptor (NMDAR). We hypothesized that certain GRIN2A mutations increase NMDAR function and support melanoma growth through oncogenic effects. This hypothesis was tested using 19 low-passage melanoma cell lines, four of which carried novel missense mutations in GRIN2A that we previously reported. We examined NMDAR expression, function of a calcium ion (Ca2+) channel and its contribution to cell growth using pharmacological modulators; findings were correlated with the presence or absence of GRIN2A mutations. We found that NMDAR expression was low in all melanoma cell lines, independent of GRIN2A mutations. In keeping with this, NMDAR-mediated Ca2+ influx and its contribution to cell proliferation were weak in most cell lines. However, certain GRIN2A mutations and culture media with lower glutamate levels enhanced NMDAR effects on cell growth and invasion. The main finding was that G762E was associated with higher glutamate-mediated Ca2+ influx and stronger NMDAR contribution to cell proliferation, compared with wild-type GRIN2A and other GRIN2A mutations. The pro-invasive phenotype of mutated cell lines was increased in culture medium containing less glutamate, implying environmental modulation of mutation effects.In conclusion, NMDAR ion channel function is low in cultured melanoma cells but supports cell proliferation and invasion. Selected GRIN2A mutations, such as G762E, are associated with oncogenic consequences that can be modulated by extracellular glutamate. Primary cultures may be better suited to determine the role of the NMDAR in melanoma in vivo.

Side population cells from long-term passage non-small cell lung cancer cells display loss of cancer stem cell-like properties and chemoradioresistance.

The side population (SP) assay is a widely used method for isolating stem cell-like cells from cancer cell lines and primary cells. The cancer cells used in different laboratories have been passaged for different generations. Emerging evidence revealed that repeated passaging of cell lines for multiple generations frequently leads to change of characteristics. Thus, it is worth investigating the effects of repeated passaging on the biological and functional properties of the enriched SP fraction from early- and late-passage cells. The present study reports that the cancer stem cell (CSC) characteristics, including increased frequency of tumor-initiating and self-renewal capacity, and resistance to the chemotherapy agent doxorubicin and ionizing radiation, was diminished in SP cells from late-passage non-small cell lung cancer (NSCLC) cells. This finding revealed that the SP from long-term passage NSCLC cells was not consistently enriched for stem cell-like cancer cells, and low-passage cell lines and primary cancer cells are therefore recommended in the CSCs field.

Abstract 3347: Establishment of four novel HPV-16 positive cell lines of invasive cervical carcinoma of Indian origin and characterization of CD133 positive cancer stem cells

Abstract Background and Aim: Cancer of the cervix is the 2nd most common cancer in Indian women. Cell lines are essential in vitro models in cancer research. Till date, available cell lines in cervical cancer were highly passaged, developed from Caucasian, Black or Asian origin; hence, establishment of a low-passage cervical cancer derived cell line was the first aim of this study. Cancer stem cells (CSCs) are proposed to be responsible for metastasis and therapy resistance. Previously, we reported CD133 to be a putative marker for CSC, and they were further characterized. Materials and Methods: 25 women were recruited after informed consent and approval from the Institute Ethics committee. A small portion of the biopsy was collected in DMEM/F12 media and subjected to primary culture. Successful propagation was possible in 4 cases. Their morphology, epithelial nature and HPV status were evaluated. Variable Number Tandem Repeat (VNTR) assay, cell cycle and kinetic studies were performed. Tissue expression of CD133 and CD49f stem cell markers was evaluated by direct immunofluorescence on frozen tumor samples. Adherent cells were sorted into CD133+ vs CD133- tumor bulk from all 4 cell lines by FACS. These two populations were evaluated for differences in tumorosphere formation, chemosensitivity to cisplatinum, expression of stemness (SOX2, OCT4, NANOG) and EMT (SNAIL, SLUG, TWIST, VIMENTIN and E-CADHERIN) markers at the transcript level. Observations: Four cell lines designated RSBS-9, RSBS-14, RSBS-23 derived from non-keratinizing squamous cell carcinoma and RSBS-43 derived from adenocarcinoma cervix were established and passaged up to 50 times. The epithelial nature of cell lines was confirmed by cytokeratin and epithelial membrane antigen positivity. VNTR assay confirmed derivation of cell lines from respective parental tissue sample. All 4 cell lines were HPV-16positive. The cell doubling time was approximately 48 hours. Immunofluorescence on tissue samples revealed diffuse positivity for CD49f indicating that it may not represent a CSC marker as previously reported whereas CD133 staining showed scattered positive cells. No particular location of CD133+ CSC was observed. Comparison of CD133+ with CD133- bulk population cells revealed increased tumorosphere formation; however, no significant difference in the chemosensitivity to cisplatinum was seen. Real time quantitation of transcripts of stemness and EMT markers revealed CD133+ cells showing upregulation of EMT markers VIMENTIN, SNAIL, SLUG in the RSBS-23 cell line and of VIMENTIN and TWIST in RSBS-9 cell line whereas there was no significant difference in the RSBS-14 and RSBS-43 cell lines for any of the markers studied. Conclusion: Low passage cell lines are useful model systems for understanding the biology of cervical cancer. CD133+ Cancer Stem cells exhibit some EMT markers in 2 of the 4 cell lines established. Citation Format: Shifa Javed, Bal Krishan Sharma, Sanjeev Kumar Sharma, Swati Sood, Rashmi Bagga, Shalmoli Bhattacharyya, Radhika Srinivasan. Establishment of four novel HPV-16 positive cell lines of invasive cervical carcinoma of Indian origin and characterization of CD133 positive cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3347.

Abstract LB-131: Birinapant and carboplatin co-therapy can effectively target platinum resistant ovarian cancer initiating cells

Abstract High-grade serous ovarian cancers (HGSOCs) contain CA125-negative cancer-initiating cells resistant to standard platinum therapy. High expression of cellular inhibitors of apoptosis proteins (cIAPs) in these platinum resistant HGSOC initiating cells causes resistance to apoptosis despite platinum-induced DNA damage. cIAPs can be degraded with the addition of birinapant, a SMAC-mimetic, sensitizing cancer initiating cells to carboplatin and resulting in their elimination. Using in vitro drug assays, we demonstrate that more than half of human HGSOCs (13/23 independent samples) can be targeted with birinapant and carboplatin co-therapy. Importantly, treatment with this co-therapy eliminated tumor cells irrespective of their clinically-defined platinum sensitivity suggesting that this therapeutic approach could be efficacious with patient who have platinum sensitive and/or resistant disease. Pre-clinical trials in mice bearing intra-peritoneal low-passage human-derived HGSOC cell lines demonstrate that birinapant and carboplatin co-therapy significantly improves overall survival. A vast majority of co-therapy treated mice are still alive, 5 months after all animals treated with vehicle or carboplatin monotherapy have succumbed to tumor. Given that co-therapy was efficacious in approximately 50% of HGOSCs, evaluation of biomarkers that can predict this response are clinically relevant. The expression of cIAP levels were quantified using western blot and then immunohistochemistry in the CA125 negative fraction of our 23 clinical samples. We found that expression of ≥24ng of cIAP in the CA125-negative HGSOC cell lysate could accurately predict response to co-therapy with 100 percent accuracy. Detection and quantification of cIAP in CA125 negative cells by immunohistochemistry (IHC) could also predict a response to co-therapy with 95 percent accuracy. We report on careful analysis of two sets of biomarkers specifically measuring druggable targets in ovarian cancer initiating cells that could predict a response to co-therapy with birinapant and carboplatin. This therapeutic approach, that can overcome platinum resistance in ovarian cancer initiating stem cells, will be tested in an upcoming clinical trial alongside our proposed companion diagnostic biomarkers of response. Citation Format: Vincent La, Rachel Fujikawa, Deanna Janzen, Liat Bainvoll, Sanaz Memarzadeh. Birinapant and carboplatin co-therapy can effectively target platinum resistant ovarian cancer initiating cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-131.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Integrity of genome-wide genotype data from low passage lymphoblastoid cell lines

We compared genotype data from the HumanExomeCore Array in peripheral blood mononuclear cells and low passage lymphoblastoid cell lines from the same 24 individuals to test for genotypic errors caused by the Epstein–Barr Virus transformation process. Genotype concordance across the 24 comparisons was 99.57% for unfiltered genotype data, and 99.63% following standard genotype quality control filters. Mendelian error rates and levels of heterozygosity were not significantly different between lymphoblastoid cell lines and their parent peripheral blood mononuclear cells. These results show that at low passage numbers, genotype discrepancies are minimal even before stringent quality control, and extend current evidence qualifying the use of low-passage lymphoblastoid cell lines as a reliable DNA source for genotype analysis.

Characterization of novel low passage primary and metastatic colorectal cancer cell lines.

In vitro models are essential to understanding the molecular characteristics of colorectal cancer (CRC) and the testing of therapies for CRC. Many efforts to establish and characterize primary CRC cell lines have been published, most describing a small number of novel cell lines. However, there remains a lack of a large panel of uniformly established and characterized cell lines. To this end we established 20 novel CRC cell lines, of which six were derived from liver metastases. Genetic, genomic and transcriptomic profiling was performed in order to characterize these new cell lines. All data are made publically available upon publication.By combining mutation profiles with CNA and gene expression profiles, we generated an overall profile of the alterations in the major CRC-related signaling pathways. The combination of mutation profiles with genome, transcriptome and methylome data means that these low passage cell lines are among the best characterized of all CRC cell lines. This will allow researchers to select model cell lines appropriate to specific experiments, facilitating the optimal use of these cell lines as in vitro models for CRC. All cell lines are available for further research.

The Combination of Belinostat with Zidovudine for Treatment of HTLV-I Related Adult T-Cell Leukemia-Lymphoma

Adult T-cell leukemia-lymphoma (ATLL) is an aggressive malignancy with poor prognosis that is incurable by conventional drugs. Histone deacetylase (HDAC) inhibitors (HDIs) are broadly active anti-neoplastic agents that can be cautiously exploited in the treatment of ATLL. The HTLV-I provirus is clonally integrated in virtually all ATLL cells. The HTLV-I 5'LTR promoter is trans -activated by the viral protein Tax through binding of CREB and p300/CBP. Tax binding enhances p300/CBP histone acetyl transferase (HAT) activity resulting in histone acetylation, chromatin unwinding, and transcription of the viral genome. HAT activity is countered by HDACs, and also by the viral-encoded protein HTLV-I bZip factor (HBZ), which is transcribed from the negative proviral strand at the 3' end. The 5' LTR is often mutated or deleted in ATLL cells thus HBZ is the only viral gene consistently expressed in ATLL. We hypothesized that HDAC inhibitors, which can be potent anti-neoplastic drugs, would reactivate HTLV-I in ATLL tumors containing intact provirus, thus killing virus-infected cells and provoking an immune response in vivo. Supporting this notion, adding the old generation HDI valproic acid (VPA) to AZT/IFNa in one subject suffering from aggressive acute-type ATLL resulted in HTLV-I proviral load reduction, followed by a complete molecular and prolonged clinical remission that lasted several years. We recently tested other more potent HDIs on primary ATLL cells and low-passage cell lines, which are rare and difficult to establish. In general, HDIs induced dose-dependent Tax expression and apoptosis in ATLL cells. Belinostat, a pan HDI, caused H3 acetylation, blocked HBZ protein expression, and induced Tax protein expression in ATLL cells resulting in concomitant apoptosis. ATLL cells exhibit high constitutive expression of nuclear factor kappa B (NF-kB), which is known to be activated by Tax and to play a major role in HTLV-I mediated T-cell transformation and ATLL development. Paradoxically, while belinostat induced Tax, it also inhibited NF-kB nuclear activity resulting in apoptosis. Further, adding AZT to belinostat augmented ATLL cell death. By contrast, IFNα diminished apoptosis induced by belinostat in freshly isolated ATLL cells from some of our patients. Based on these preclinical data, we have developed a clinical trial using belinostat in combination with AZT as consolidation therapy for ATLL. Our primary goal using this approach is to eradicate residual ATLL cells and HTLV-I infected reservoirs that normally persist regardless of treatment. DisclosuresNo relevant conflicts of interest to declare.

Preclinical efficacy of belinostat in combination with zidovudine in adult T-cell leukemia-lymphoma

Adult T-cell leukemia-lymphoma (ATLL) is an aggressive malignancy that is incurable by conventional drugs. Histone deacetylase (HDAC) inhibitors (HDIs) are broadly active anti-neoplastic agents that can be cautiously exploited in the treatment of ATLL. The HTLV-I provirus is clonally integrated in virtually all ATLL cells. The viral promoter is trans-activated by Tax protein through binding of CREB and p300/CBP at the 5'LTR. Tax binding enhances p300/CBP histone acetyl transferase (HAT) activity resulting in chromatin unwinding and transcription of the viral genome. Such HAT activity is countered by HDACs, and also by HBZ, which unlike Tax is the only viral gene consistently transcribed from the negative 3’ proviral strand end in ATLL. We recently conducted a clinical trial using the old generation HDI valproic acid (VPA) combined with zidovudine (AZT) and interferon alpha (IFNα) during maintenance therapy in patients with ATLL. We hypothesized that the HDI would reactivate HTLV-I thus provoking an immune response against circulating ATLL cells that normally persist after AZT/IFNα therapy. Supporting this notion adding VPA to AZT/IFNα resulted in HTLV-I proviral load reduction, followed by a molecular and prolonged clinical remission in one subject. We recently compared and tested more potent HDIs on primary ATLL cells and low-passage cell lines. In general, HDIs induced dose dependent Tax expression and apoptosis in ATLL cells, and provoked an intracellular innate immune response evidenced by the upregulation of IFN-B and IRF-7. The pan HDI belinostat caused H3 acetylation, blocked HBZ protein expression, and induced Tax protein expression in ATLL cells, while paradoxically inhibiting NF-κB nuclear activity, resulting in concomitant apoptosis. Further, adding AZT to belinostat augmented ATLL cell death. In contrast, IFNα treatment diminished apoptosis induced by belinostat. Based on these preclinical data, we proposed using belinostat in combination with AZT as consolidation therapy for ATLL. A pilot clinical trial is underway at our institution.

AAV9 delivering a modified human Mullerian inhibiting substance as a gene therapy in patient-derived xenografts of ovarian cancer

To improve ovarian cancer patient survival, effective treatments addressing chemoresistant recurrences are particularly needed. Mullerian inhibiting substance (MIS) has been shown to inhibit the growth of a stem-like population of ovarian cancer cells. We have recently engineered peptide modifications to human MIS [albumin leader Q425R MIS (LRMIS)] that increase production and potency in vitro and in vivo. To test this novel therapeutic peptide, serous malignant ascites from highly resistant recurrent ovarian cancer patients were isolated and amplified to create low-passage primary cell lines. Purified recombinant LRMIS protein successfully inhibited the growth of cancer spheroids in vitro in a panel of primary cell lines in four of six patients tested. Adeno-associated virus (AAV) -delivered gene therapy has undergone a clinical resurgence with a good safety profile and sustained gene expression. Therefore, AAV9 was used as a single i.p. injection to deliver LRMIS to test its efficacy in inhibiting growth of palpable tumors in patient-derived ovarian cancer xenografts from ascites (PDXa). AAV9-LRMIS monotherapy resulted in elevated and sustained blood concentrations of MIS, which significantly inhibited the growth of three of five lethal chemoresistant serous adenocarcinoma PDXa models without signs of measurable or overt toxicity. Finally, we tested the frequency of MIS type II receptor expression in a tissue microarray of serous ovarian tumors by immunohistochemistry and found that 88% of patients bear tumors that express the receptor. Taken together, these preclinical data suggest that AAV9-LRMIS provides a potentially well-tolerated and effective treatment strategy poised for testing in patients with chemoresistant serous ovarian cancer.

Inhibitors of pan-PI3K Signaling Synergize with BRAF or MEK Inhibitors to Prevent BRAF-Mutant Melanoma Cell Growth.

BRAF and MEK inhibitors have improved outcomes for patients with BRAF-mutant melanoma, but their efficacy is limited by both intrinsic and acquired resistances. Activation of the PI3K pathway can mediate resistance to these agents, providing a strong rationale for combination therapy in melanoma. Here, a panel of nine low-passage human metastatic melanoma cell lines with BRAF mutations was tested in cell proliferation and protein expression assays for sensitivity to inhibitors of MEK (selumetinib) and BRAF (vemurafenib) as single agents and in combination with inhibitors of pan-PI3K (ZSTK474), pan-PI3K/mTOR (BEZ235), individual PI3K isoforms (p110α, A66; p110β, TGX-221; p110γ, AS-252424; p110δ, idelalisib), or mTORC1/2 (KU-0063794). Selumetinib and vemurafenib potently inhibited cell proliferation in all cell lines, especially in those that expressed low levels of phosphorylated AKT (pAKT). ZSTK474 and BEZ235 also inhibited cell proliferation in all cell lines and enhanced the antitumor activity of selumetinib and vemurafenib in the majority of lines by either interacting synergistically or additively to increase potency or by inducing cytotoxicity by significantly increasing the magnitude of cell growth inhibition. Furthermore, ZSTK474 or BEZ235 combined with selumetinib to produce robust inhibition of pERK, pAKT, and pS6 expression and synergistic inhibition of NZM20 tumor growth. The inhibitors of individual PI3K isoforms or mTORC1/2 were less effective at inhibiting cell proliferation either as single agents or in combination with selumetinib or vemurafenib, although KU-0063794 synergistically interacted with vemurafenib and increased the magnitude of cell growth inhibition with selumetinib or vemurafenib in certain cell lines. Overall, these results suggest that the sensitivity of BRAF-mutant melanoma cells to BRAF or MEK inhibitors is at least partly mediated by activation of the PI3K pathway and can be enhanced by combined inhibition of the BRAF/MEK and PI3K/mTOR signaling pathways.

Using HDAC Inhibitors to Eradicate Adult T-Cell Leukemia-Lymphoma

Adult T-cell leukemia-lymphoma (ATLL) is an aggressive malignancy with a poor prognosis caused by HTLV-I, which is endemic in Japan, the Caribbean, and South America. ATLL cannot be cured with conventional chemotherapy thus urging the development of new therapeutic strategies. Histone deacetyalse (HDAC) inhibitors are broadly active anti-neoplastic agents, which have shown efficacy in T-cell lymphomas. At our institution we encounter a relatively high number of ATLL cases as compared to other regions in the U.S. We have conducted a pilot trial using AZT/interferon-α(IFNα) plus the HDAC inhibitor valproic acid (VPA) during maintenance therapy in acute (leukemia-type) ATLL. We hypothesized that VPA would reactivate HTLV-I leading to a cytotoxic T-cell immune response followed by reduction of residual blood circulating ATLL clones that normally persist during AZT/IFNα therapy despite its suppressive effects. Supporting this notion, the addition of VPA to AZT/IFNα resulted in reduction of HTLV-I proviral loads in treated patients and a sustained molecular response in one subject who is still alive >4.5 years later. More recently, we have tested newly available and more potent HDAC inhibitors using fresh primary leukemic ATLL cells and low-passage cell lines. Through HDAC inhibition, these agents reactivated HTLV-I Tax gene expression and induced apoptosis in a dose-dependent manner. Belinostat, which is currently FDA-approved for the treatment of relapsed or refractory peripheral T-cell lymphoma, augmented ATLL cell death when added to AZT/IFNα at nanomolar concentrations. Therefore, we have proposed a new pilot clinical trial using belinostat for the treatment of ATLL during post-induction therapy. The role of HDAC inhibitors in ATLL, pre-clinical laboratory studies involving these agents, and clinical trial design will be discussed at the meeting DisclosuresOff Label Use: The use of HDAC inhibitors in adult T-cell Leukemia-lymphoma.

Establishment of highly tumorigenic human colorectal cancer cell line (CR4) with properties of putative cancer stem cells.

BackgroundColorectal cancer (CRC) has the third highest mortality rates among the US population. According to the most recent concept of carcinogenesis, human tumors are organized hierarchically, and the top of it is occupied by malignant stem cells (cancer stem cells, CSCs, or cancer-initiating cells, CICs), which possess unlimited self-renewal and tumor-initiating capacities and high resistance to conventional therapies. To reflect the complexity and diversity of human tumors and to provide clinically and physiologically relevant cancer models, large banks of characterized patient-derived low-passage cell lines, and especially CIC-enriched cell lines, are urgently needed.Principal FindingsHere we report the establishment of a novel CIC-enriched, highly tumorigenic and clonogenic colon cancer cell line, CR4, derived from liver metastasis. This stable cell line was established by combining 3D culturing and 2D culturing in stem cell media, subcloning of cells with particular morphology, co-culture with carcinoma associated fibroblasts (CAFs) and serial transplantation to NOD/SCID mice. Using RNA-Seq complete transcriptome profiling of the tumorigenic fraction of the CR4 cells in comparison to the bulk tumor cells, we have identified about 360 differentially expressed transcripts, many of which represent stemness, pluripotency and resistance to treatment. Majority of the established CR4 cells express common markers of stemness, including CD133, CD44, CD166, EpCAM, CD24 and Lgr5. Using immunocytochemical, FACS and western blot analyses, we have shown that a significant ratio of the CR4 cells express key markers of pluripotency markers, including Sox-2, Oct3/4 and c-Myc. Constitutive overactivation of ABC transporters and NF-kB and absence of tumor suppressors p53 and p21 may partially explain exceptional drug resistance of the CR4 cells.ConclusionsThe highly tumorigenic and clonogenic CIC-enriched CR4 cell line may provide an important new tool to support the discovery of novel diagnostic and/or prognostic biomarkers as well as the development of more effective therapeutic strategies.

Biological and molecular effects of small molecule kinase inhibitors on low-passage human colorectal cancer cell lines.

Low-passage cancer cell lines are versatile tools to study tumor cell biology. Here, we have employed four such cell lines, established from primary tumors of colorectal cancer (CRC) patients, to evaluate effects of the small molecule kinase inhibitors (SMI) vemurafenib, trametinib, perifosine, and regorafenib in an in vitro setting. The mutant BRAF (V600E/V600K) inhibitor vemurafenib, but also the MEK1/2 inhibitor trametinib efficiently inhibited DNA synthesis, signaling through ERK1/2 and expression of genes downstream of ERK1/2 in BRAF mutant cells only. In case of the AKT inhibitor perifosine, three cell lines showed a high or intermediate responsiveness to the drug while one cell line was resistant. The multikinase inhibitor regorafenib inhibited proliferation of all CRC lines with similar efficiency and independent of the presence or absence of KRAS, BRAF, PIK3CA, and TP53 mutations. Regorafenib action was associated with broad-range inhibitory effects at the level of gene expression but not with a general inhibition of AKT or MEK/ERK signaling. In vemurafenib-sensitive cells, the antiproliferative effect of vemurafenib was enhanced by the other SMI. Together, our results provide insights into the determinants of SMI efficiencies in CRC cells and encourage the further use of low-passage CRC cell lines as preclinical models.

Evidence That GRIN2A Mutations in Melanoma Correlate with Decreased Survival

Previous whole-exome sequencing has demonstrated that melanoma tumors harbor mutations in the GRIN2A gene. GRIN2A encodes the regulatory GluN2A subunit of the glutamate-gated N-methyl-d-aspartate receptor (NMDAR), involvement of which in melanoma remains undefined. Here, we sequenced coding exons of GRIN2A in 19 low-passage melanoma cell lines derived from patients with metastatic melanoma. Potential mutation impact was evaluated in silico, including within the GluN2A crystal structure, and clinical correlations were sought. We found that of 19 metastatic melanoma tumors, four (21%) carried five missense mutations in the evolutionarily conserved domains of GRIN2A; two were previously reported. Melanoma cells that carried these mutations were treatment-naïve. Sorting intolerant from tolerant analysis predicted that S349F, G762E, and P1132L would disrupt protein function. When modeled into the crystal structure of GluN2A, G762E was seen to potentially alter GluN1–GluN2A interactions and ligand binding, implying disruption to NMDAR functionality. Patients whose tumors carried non-synonymous GRIN2A mutations had faster disease progression and shorter overall survival (P < 0.05). This was in contrast to the BRAF V600E mutation, found in 58% of tumors but showing no correlation with clinical outcome (P = 0.963). Although numbers of patients in this study are small, and firm conclusions about the association between GRIN2A mutations and poor clinical outcome cannot be drawn, our results highlight the high prevalence of GRIN2A mutations in metastatic melanoma and suggest for the first time that mutated NMDARs impact melanoma progression.

Designing personalised cancer treatments

The concept of personalised medicine for cancer is not new. It arguably began with the attempts by Salmon and Hamburger to produce a viable cellular chemosensitivity assay in the 1970s, and continues to this day. While clonogenic assays soon fell out of favour due to their high failure rate, other cellular assays fared better and although they have not entered widespread clinical practice, they have proved to be very useful research tools. For instance, the ATP-based chemosensitivity assay was developed in the early 1990s and is highly standardised. It has proved useful for evaluating new drugs and combinations, and in recent years has been used to understand the molecular basis of drug resistance and sensitivity to anti-cancer drugs.Recent developments allow unparalleled genotyping and phenotyping of tumours, providing a plethora of targets for the development of new cancer treatments. However, validation of such targets and new agents to permit translation to the clinic remains difficult. There has been one major disappointment in that cell lines, though useful, do not often reflect the behaviour of their parent cancers with sufficient fidelity to be useful. Low passage cell lines — either in culture or xenografts are being used to overcome some of these issues, but have several problems of their own. Primary cell culture remains useful, but large tumours are likely to receive neo-adjuvant treatment before removal and that limits the tumour types that can be studied. The development of new treatments remains difficult and prediction of the clinical efficacy of new treatments from pre-clinical data is as hard as ever. One lesson has certainly been that one cannot buck the biology — and that understanding the genome alone is not sufficient to guarantee success. Nowhere has this been more evident than in the development of EGFR inhibitors. Despite overexpression of EGFR by many tumour types, only those with activating EGFR mutations and an inability to circumvent EGFR blockade have proved susceptible to treatment.The challenge is how to use advanced molecular understanding with limited cellular assay information to improve both drug development and the design of companion diagnostics to guide their use. This has the capacity to remove much of the guesswork from the process and should improve success rates.

Culture of low passage colorectal cancer cells and demonstration of variation in selected tumour marker expression.

There is increasing evidence that a tumour comprises of heterogeneous population of cells. Thus, studying homogenous cell lines in vitro may yield results that are not reflective of the true situation in a tumour and studying low passage cell lines maintained in a heterogeneous population before they transform away from the original state may provide a more complete picture of colorectal cancer. A method was developed to isolate and establish low passage colorectal cancer cell lines from tumour biopsies. The media contents, combination of antimicrobials and specimen collection and transport conditions employed, successfully eliminated microbial contamination which is frequently present in samples obtained from the gastrointestinal tract. A variety of growth forms indicating a heterogeneous mixture of cells was seen in the initial cultures. Using fluorescence immunocytochemistry, primary tumour cultures were shown to variably express selected tumour markers, carcinoembryonic antigen and C2 antigen. These low passage cell lines growing in a heterogeneous environment would more closely reflect the characteristics of the cells of the original tumour.

Development of a Colon Cancer GEMM-Derived Orthotopic Transplant Model for Drug Discovery and Validation

Effective therapies for KRAS-mutant colorectal cancer (CRC) are a critical unmet clinical need. Previously, we described genetically engineered mouse models (GEMM) for sporadic Kras-mutant and non-mutant CRC suitable for preclinical evaluation of experimental therapeutics. To accelerate drug discovery and validation, we sought to derive low-passage cell lines from GEMM Kras-mutant and wild-type tumors for in vitro screening and transplantation into the native colonic environment of immunocompetent mice for in vivo validation. Cell lines were derived from Kras-mutant and non-mutant GEMM tumors under defined media conditions. Growth kinetics, phosphoproteomes, transcriptomes, drug sensitivity, and metabolism were examined. Cell lines were implanted in mice and monitored for in vivo tumor analysis. Kras-mutant cell lines displayed increased proliferation, mitogen-activated protein kinase signaling, and phosphoinositide-3 kinase signaling. Microarray analysis identified significant overlap with human CRC-related gene signatures, including KRAS-mutant and metastatic CRC. Further analyses revealed enrichment for numerous disease-relevant biologic pathways, including glucose metabolism. Functional assessment in vitro and in vivo validated this finding and highlighted the dependence of Kras-mutant CRC on oncogenic signaling and on aerobic glycolysis. We have successfully characterized a novel GEMM-derived orthotopic transplant model of human KRAS-mutant CRC. This approach combines in vitro screening capability using low-passage cell lines that recapitulate human CRC and potential for rapid in vivo validation using cell line-derived tumors that develop in the colonic microenvironment of immunocompetent animals. Taken together, this platform is a clear advancement in preclinical CRC models for comprehensive drug discovery and validation efforts.

Abstract 4155: Personalized chemosensitivity profiling of cancers isolated from genetically-engineered mice.

Abstract BACKGROUNDHigh-throughput functional chemosensitivity testing is a potentially effective way to customize personalized chemotherapy treatment. This testing requires low-passage patient-specific cancer cell lines, however, generating such cell lines from patients’ tumors is difficult, due in large part, to contaminating fibroblasts that often overgrow the malignant cells. METHODSWe constructed a series of hypoxanthine phosphoribosyl transferase (hprt) null immunodeficient mice. These include nude hprt-null, SCID hprt-null and NOD-SCID hprt-null mice. During growth of xenografted human cancers in these mice, hprt-null murine stromal cells replace their human counterparts. Tumors from patients were implanted and expanded in these mice. Explanted tumors were then grown in tissue culture flasks with selection media (hypoxanthine, aminopterin, thymidine; HAT media) to eliminate the mouse biochemically-defective fibroblasts and produce pure human cancer cell lines. We screened one cell line against the Johns Hopkins Drug Library containing ∼3,100 drugs. Selected potent drugs were used for in vivo xenograft tumor inhibition studies. RESULTSWe successfully established several pancreatic cancer cell lines from patients using the hprt null immunodeficient mice. We performed chemosensitivity profiling on one of the pancreatic cancer cell lines using 3,100 drugs. From the profiling, we selected potent drugs, which are FDA-approved but are not currently used for pancreatic cancer. We chose two drugs that demonstrated greater in vitro chemosensitivity compared to control cell lines. The two drugs were tested against mouse xenografts where both drugs demonstrated superior in vivo efficacy to the sensitive cell line tumors compared to control tumors. CONCLUSIONSChemotherapy for cancer may be personalized using low passage cell lines derived from patients through biochemically selectable mice. Further tests are needed to determine whether in vitro chemosensitivity testing of low-passage patient-specific cancer cell lines can predict clinical patient response. Citation Format: Hirohiko Kamiyama, Sherri Rauenzahn, Joong Sup Shim, Jun O. Liu, Anirban Maitra, James R. Eshleman. Personalized chemosensitivity profiling of cancers isolated from genetically-engineered mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4155. doi:10.1158/1538-7445.AM2013-4155

Personalized Chemotherapy Profiling Using Cancer Cell Lines from Selectable Mice

High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult because contaminating stromal cells overgrow the malignant cells. We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts. Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified 77 U.S. Food and Drug Administration (FDA)-approved drugs with activity, and two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs. Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice.