Unique Cell Line Research Articles

Detection of glioblastoma intratumor heterogeneity in radiosensitivity using patient-derived neurosphere cultures.

Glioblastoma (GBM) is characterized by extensive clonal diversity suggesting the presence of tumor cells with varying degrees of treatment sensitivity. Radiotherapy is an integral part of glioblastoma treatment. Whether GBMs are comprised of spatially distinct cellular populations with uniform or varying degrees of radiosensitivity has not been established. Spatially distinct regions of three GBMs (J3, J7 and J14) were resected and unique cell lines were derived from each region. DNA from cell lines, corresponding tumor fragments, and patient blood was extracted for whole exome sequencing. Variants, clonal composition, and functional implications were compared and analyzed with superFreq and IPA. Limiting dilution assays were performed on cell lines to measure intrinsic radiosensitivity. Based on WES, cell lines generated from different regions of the same tumor were more closely correlated with their tumor of origin than the other GBMs. Variant and clonal composition comparisons showed that cell lines from distinct tumors displayed increasing levels of ITH with J3 and J14 having the lowest and highest, respectively. The radiosensitivities of the cell lines generated from the J3 tumor were similar as were those generated from the J7 tumor. However, the radiosensitivities of the 2 cell lines generated from the J14 tumor (J14T3 and J14T6) were significantly different with J14T6 being more sensitive than J14T3. Data suggest a tumor dependent ITH in radiosensitivity. The existence of ITH in radiosensitivity may impact not only the initial therapeutic response but also the effectiveness of retreatment protocols.

Abstract 3419: Lymph node colonization promotes distant tumor metastasis through the induction of tumor-specific immunosuppression

Abstract The majority of cancer-associated deaths result from distant organ metastasis rather than the primary tumor, yet the mechanisms that enable this process remain poorly understood. For most solid tumors, colonization of regional or distant lymph nodes (LNs) typically precedes the formation of distant organ metastases, yet it remains unclear whether LN metastasis plays a functional role in disease progression. LNs are education hubs of the adaptive immune system wherein antigens derived from pathogens or malignancies are presented to lymphocytes to elicit an adaptive immune response. Nonetheless, LN metastasis, which is typically attributed to passive drainage of tumor cells through lymphatics, frequently does not lead to the generation of anti-tumor immunity, but instead correlates with further disease progression. Here, we find that LN metastasis represents a critical step in tumor progression through the capacity of such metastases to induce tumor-specific immunosuppression in a manner that promotes further dissemination of tumors to distant organs. Using an in vivo passaging approach of a non-metastatic syngeneic melanoma, we generated 300 unique cell lines exhibiting varying degrees of LN metastatic capacity. Transcriptional profiling of the lines reveals a conserved enrichment for immune-related programs. We show that the presence of these LN metastases enables distant organ seeding of metastases in a manner that the parental tumor cannot, and this differential seeding is eliminated in mice lacking an adaptive immune response. Furthermore, this promotion of distant seeding by LN metastases is tumor specific. Using mass cytometry to perform organism-wide immune profiling, we identify multiple cellular mediators of tolerance. In particular, we find that LN metastases have the capacity to both resist NK cell cytotoxicity and induce regulatory T cells (Tregs) in vitro. Furthermore, depletion of NK cells in vivo enables non-metastatic tumors to disseminate to LNs, and ablation of Tregs using FoxP3-DTR mice eliminates the occurrence of lymphatic metastases. Through the use of whole exome sequencing, we show that neither the metastatic proclivity nor immunosuppression evolve through the acquisition of driver mutations, loss of neoantigens, loss of MHC class I presentation, or decreases in melanoma antigen expression. Rather, by RNA-seq and ATAC-seq, we show that a conserved interferon signaling axis is upregulated in LN metastases and is rendered stable through epigenetic regulation of chromatin accessibility. Furthermore, using CRISPR/Cas9, we find that these pathways are required for LN metastatic seeding, and validate their conserved significance in additional mouse models of pancreatic ductal adenocarcinoma and head and neck squamous cell carcinoma (HNSCC), along with RNA-seq analysis of malignant populations sorted from HNSCC patients. Together, these findings demonstrate a critical role for LN metastasis in promoting tumor-specific immunosuppression. Citation Format: Nathan E. Reticker-Flynn, Pamela A. Basto, Weiruo Zhang, Maria M. Martins, Serena Chang, Andrew J. Gentles, John B. Sunwoo, Sylvia K. Plevritis, Edgar G. Engleman. Lymph node colonization promotes distant tumor metastasis through the induction of tumor-specific immunosuppression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3419.

Mid-Infrared Imaging Is Able to Characterize and Separate Cancer Cell Lines

Malignancies are still responsible for a large share of lethalities. Macroscopical evaluation of the surgical resection margins is uncertain. Big data based imaging approaches have emerged in the recent decade (mass spectrometry, two-photon microscopy, infrared and Raman spectroscopy). Indocianine green labelled MS is the most common approach, however, label free mid-infrared imaging is more promising for future practical application. We aimed to identify and separate different transformed (A-375, HT-29) and non-transformed (CCD986SK) cell lines by a label-free infrared spectroscopy method. Our approach applied a novel set-up for label-free mid-infrared range classification method. Transflection spectroscopy was used on aluminium coated glass slides. Both whole range spectra (4000–648 cm−1) and hypersensitive fingerprint regions (1800–648 cm−1) were tested on the imaged areas of cell lines fixed in ethanol. Non-cell spectra were possible to be excluded based on mean transmission values being above 90%. Feasibility of a mean transmission based spectra filtering method with principal component analysis and linear discriminant analysis was shown to separate cell lines representing different tissue types. Fingerprint region resulted the best separation of cell lines spectra with accuracy of 99.84% at 70–75 mean transmittance range. Our approach in vitro was able to separate unique cell lines representing different tissues of origin. Proper data handling and spectra processing are key steps to achieve the adaptation of this dye-free technique for intraoperative surgery. Further studies are urgently needed to test this novel, marker-free approach.

Abstract PR04: Lymph node colonization promotes distant tumor metastasis through the induction of tumor-specific immunosuppression

Abstract The majority of cancer-associated deaths result from distant organ metastasis rather than the primary tumor, yet the mechanisms that enable this process remain poorly understood. For most solid tumors, colonization of regional or distant lymph nodes (LNs) typically precedes the formation of distant organ metastases, yet it remains unclear whether LN metastasis plays a functional role in disease progression. LNs are education hubs of the adaptive immune system wherein antigens derived from pathogens or malignancies are presented to lymphocytes to elicit an adaptive immune response. Nonetheless, LN metastasis, which is typically attributed to passive drainage of tumor cells through lymphatics, frequently does not lead to the generation of an antitumor immune response but instead correlates with further disease progression. Here, using a novel mouse model, we find that LN metastasis represents a critical step in tumor progression through the capacity of such metastases to induce tumor-specific immunosuppression in a manner that promotes further dissemination of tumors to distant organs. We use an in vivo passaging approach of a nonmetastatic syngeneic melanoma to generate 300 unique cell lines of known phylogenetic relationships that exhibit varying degrees of LN metastatic capacity. Transcriptional profiling of the lines reveals a conserved enrichment for immune-related programs and constitutive activation of an interferon signaling axis, and using ATAC-seq, we find that these programs are rendered stable through alterations in chromatin accessibility. We show that the presence of these LN metastases enables distant organ seeding of metastases in a manner that the parental tumor cannot, and this differential seeding is eliminated in mice lacking an adaptive immune response. Using mass cytometry to perform organism-wide immune profiling, we identify multiple cellular mediators of tolerance. In particular, we find that LN metastases have the capacity to both resist NK cell cytotoxicity and induce regulatory T cells (Tregs) in vitro. Furthermore, depletion of NK cells in vivo enables nonmetastatic tumors to disseminate to LNs, and ablation of Tregs using FoxP3-DTR mice eliminates the occurrence of lymphatic metastases. Through the use of whole-exome sequencing, we show that neither the metastatic proclivity nor immunosuppression evolves through the acquisition of driver mutations, loss of neoantigens, loss of MHC class I presentation, or decreases in melanoma antigen expression. Rather, using CRISPR/Cas9, we find that key interferon-induced molecules are required for LN metastatic seeding, and using additional mouse models of pancreatic ductal adenocarcinoma and head and neck squamous cell carcinoma (HNSCC), along with RNA-seq analysis of malignant populations sorted from HNSCC patients, we find that these effects are conserved across multiple malignancies and in humans. Together, these findings demonstrate a critical role for LN metastasis in promoting tumor immune tolerance. This abstract is also being presented as Poster B25. Citation Format: Nathan E. Reticker-Flynn, Pamela A. Basto, Weiruo Zhang, Alborz Bejnood, Justin A. Kenkel, Maria M. Martins, Serena Chang, Andrew J. Gentles, John B. Sunwoo, Sylvia K. Plevritis, Edgar G. Engleman. Lymph node colonization promotes distant tumor metastasis through the induction of tumor-specific immunosuppression [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR04.

Dual TGF-β and AURKA targeting enhances chemosensitivity in triple-negative breast cancer.

e13106 Background: Chemotherapy resistance remains a significant barrier in the effective treatment of patients with triple negative breast cancer (TNBC). TGF-β signaling is a well characterized oncogenic pathway in TNBC that promotes chemoresistance by inducing epithelial to mesenchymal transition (EMT) and tumor stemness. Aurora-A kinase (AURKA) is a serine/threonine kinase responsible for chromosomal stability during mitosis. Significantly, aberrant expression of AURKA induces breast cancer progression. We hypothesized that aberrant activation of TGF-β and AURKA signaling pathways contributes to chemoresistance in TNBC by promoting EMT and tumor stemness and that dual-targeting of these oncoproteins will enhance chemosensitivity. Methods: RNA-Seq data were analyzed from patient derived xenografts (PDx) established from patients with Stage I-III TNBC who received pre-operative taxane and anthracycline based chemotherapy. Chemoresistance was defined as an RCB score of 1-3, and chemosensitivity was defined as an RCB score of 0. Unique TNBC cell lines developed from brain metastasis PDxs (TNBC M14, TNBC M25) were treated in vitro with 10nM docetaxel (DTX), 50nM LY2157299 (TGF-β inhibitor) and 50nM MLN8237 (AURKA inhibitor). EMT reprogramming was determined by measuring the expression of vimentin, ALDH activity and mammosphere growth. Apoptotic cells following drug treatment were labeled with Red Annexin-V marker and monitored in real time using the IncuCyte instrument. Results: RNA-Seq revealed that there was no baseline difference in expression of AURKA between chemoresistant and chemosensitive TNBC PDxs. However, there was a 2.7-fold increase in AURKA expression in the post-treatment PDx models compared to pre-treatment PDx models. In vitro treatment of the M14 and M25 cell lines with DTX demonstrated no significant increase in apoptotic cells compared to control, whereas treatment with the combination of DTX, LY2157299 and MLN8237 resulted in a two-fold increase in apoptosis compared to treatment with DTX alone (p = 0.0068 M14, p = 0.003 M25). Dual-targeting with LY2157299 and MLN8237 reduced the expression of vimentin and ALDH activity. Conclusions: TGF-β and AURKA play a central role inducing EMT and a stem-cell-like phenotype in TNBC that confers chemoresistance. AURKA is up-regulated after exposure to chemotherapy in chemoresistant TNBC PDx models. LY2157299 and MLN8237 reduce TNBC stemness in M14 and M25 cell lines and enhance DTX chemosensitivity. Dual-targeting of TGF-β and AURKA is a potentially promising approach in chemoresistant TNBC.

OR06-06 Pituitary Tumors and Immortalized Cell Lines Generated by Cre-Inducible Expression of SV40 T-Antigen

Pituitary and hypothalamic cell lines have been developed by targeted oncogenesis. This involved using cell-specific transcriptional regulatory sequences to drive expression of large and small SV40 T-antigens in transgenic mice. Invariably, tumors develop in some of the mice, and the cells in these tumors can sometimes be adapted to grow in culture into stable, immortalized cell lines that maintain some of the features of differentiated cells. Cell lines that represent pre-gonadotropes (αT3-1), gonadotropes (LβT2), precursors to the POU1F1 lineage (GHFT1, Pit1-zero), differentiated cells of the POU1F1 lineage (Pit1-triple, TaT1, and Pit1-PRL), and GnRH neurons (GT1-1) have been made by this approach. Tumors often develop early and cause infertility or death. To increase the opportunity for generating cell lines and to make it feasible to follow the process of tumorigenesis, we developed a mouse strain that expresses SV40 T-antigens in response to cre-recombinase. Using CRISPR/Cas9 we inserted an 8 kb cassette with coding sequences for SV40 T-antigens and IRES-GFP into the Rosa26 locus, downstream from a stop sequence flanked by loxP sites: Rosa26LSL-SV40-GFP. 30% of the progeny born from hybrid zygotes injected with template DNA, CRISPR/Cas9, and sgRNA had correctly targeted the Rosa26 locus. These mice were mated with previously established Prop1-cre and Tshb-cre transgenic lines. The majority of Rosa26LSL-SV40-GFP/+; Prop1-cre and Rosa26LSL-SV40-GFP/+; Tshb-cre mice developed dwarfism and large tumors by 4 wks. The pituitaries of Rosa26LSL-SV40-GFP/+; Tshb-cre mice appear grossly normal at birth, but they are enlarged and showing evidence of increased vascularization by 2 wks. Flow-sorted GFP-positive cells from Rosa26LSL-SV40-GFP/+; Prop1-cre and Rosa26LSL-SV40-GFP/+; Tshb-cre mice express Prop1 and TSH, respectively. Tumors from Rosa26LSL-SV40-GFP/+; Tshb-cre mice were adapted to growth in cell culture. We have established a thyrotrope-like cell line that expresses Cga and Pou1f1. These studies demonstrate the utility of the novel, Rosa26LSL-SV40-GFP mouse line for reliable targeted oncogenesis and development of unique cell lines.The authors have nothing to disclose.

Evaluation of immune checkpoints in small cell lung cancer: implications for immunotherapy

Abstract Introduction The FDA recently approved Atezolizumab, a programmed death-ligand 1 (PD-L1) inhibitor, for first-line combinatory treatment in patients with extensive-stage small cell lung cancer. Across several cancer types, immunotherapy has been shown to provide durable benefit in patients with metastatic disease. However, Atezolizumab only improves overall survival by 2 months. This could be due to the fact that <20% of patients express >1% tumor PD-L1 positivity. We assert that a better understanding of this immune contexture – defined as abundance and type of checkpoint molecules in SCLC – may provide important criteria for patient stratification. Objectives In these studies, we will test the hypothesis that immune checkpoints in addition to the PD-1/PD-L1 axis play a role in immune evasion in SCLC. Methods To evaluate expression of immune checkpoint molecules, we acquired bulk RNA-Seq data of SCLC cell lines and primary tumor specimens. Further, we generated tissue-microarrays containing >20 unique cell lines and >200 unique patient specimens with fully annotated clinical data to confirm expression of these molecules by immunohistochemistry. Results Preliminary RNA-Seq analyses show low PD-L1 expression in SCLC cell lines and primary tumors. Further, ubiquitously high expression of certain checkpoint molecules (B7-H3) is seen. Future analysis will confirm abundance and type of checkpoint molecules as well as capture interpatient heterogeneity. Conclusions Understanding the checkpoint molecules present in SCLC could further understanding of SCLC immune suppression, ultimately leading to optimal single-agent immunotherapy deployment and rational, effective combination therapy for patients with SCLC.

Dysfunctional Natural Killer cells expanded from a liver with Hepatocellular Carcinoma show reduced killing against a unique HCC cell line

BackgroundThe liver has one of the largest reserves of NK cells in the body. NK cells in the liver are key defenses against viral hepatitis, liver fibrosis, and tumorigenesis. In the healthy liver, they exhibit higher cytotoxicity and secrete more cytokines compared to those isolated from peripheral blood. Hepatocellular carcinoma (HCC) is one of the most rapidly increasing cancers worldwide. Previously, NK cells have been shown to be dysfunctional in HCC. Additionally, NK cells are reduced in number intratumorally and peripherally and there is a decrease in the cytotoxic CD56dim subpopulation. Many studies have shown a downregulation of key signaling molecules involved in peripheral NK cell cytotoxic activity. We aimed to further investigate the level of dysfunction of NK cells isolated from HCC livers to document the nature of dysfunction of liver‐resident NK cells in eliminating HCC cell lines.MethodsBriefly, samples of tissue were collected from explanted livers and made into single cell suspensions. Next, lymphocytes were isolated using density‐based centrifugation. The resulting fraction was placed into RPMI‐1640 with IL‐2 and IL‐15. After two weeks, samples were taken to determine the number and purity of NK cells by flow cytometry. Target cell lines were labeled with chromium‐51 to test the killing capacities of NK cells isolated from healthy donor PBMC’s or HCC patient livers. NK cells were prepared in serial dilutions and in triplicate to generate a killing curve. After 4 hours of coincubation of effector NK cells with target cells, the released radioactive particles were measured and analyzed.ResultsTwo cell lines, Hep3B (ATCC) and HCO2 (generated in our lab), were used as target cells. Figures 1 and 2 depict the results of separate killing assays using the different target HCC cell lines. The effector cells in each assay consist of NK cells sourced from either healthy donor PBMC’s, cirrhotic patient PBMC’s, or HCC liver. Notably, the results show that even under the constant stimulation of IL‐2 and IL‐15, which are known interleukins that are involved in the proliferation and activation of NK cells and T‐cells, there is a large dysfunction in NK cells sourced from diseased livers. More strikingly, our results show that there is a specific inability of NK cells sourced from HCC livers to eliminate HCO2 cells which have been previously shown in our lab to be sorafenib‐resistant. Our hypothesis is that there is an innate dysfunction that is present in NK cells isolated from diseased livers that is not recoverable even when cultured in the presence of activating cytokines. Additionally, HCO2 cells possess a unique resistance to killing by NK cells isolated and expanded from an HCC liver.ConclusionHere we show that NK cells can be isolated and expanded, ex vivo, from human diseased livers. Additionally, we show that despite being stimulated for over 2 weeks with cytokines, expanded NK cells still show a marked reduction in killing capacity against a variety of HCC cell lines compared to NK cells expanded from PBMC’s from healthy and diseased donors. Our results confirm that there is an innate dysfunction that is present within NK cells isolated from diseased livers that is not recoverable even when cultured in the presence of activating cytokines.HCC cell lines (Hep3B, in circles, HCO2, in triangles) were labeled with Chromium‐51 for 2 hours. Target cells were rinsed with media and cocultured with NK cells at various effector‐to‐target ratios. NK cells were sourced from either PBMC’s from a cirrhotic patient (PBNK, in blue) or a liver with hepatocellular carcinoma (LNK, in green) and expanded ex vivo with IL‐2 and IL‐15.Figure 1

Characterizing the pharmacogenome using molecular inversion probes for targeted next-generation sequencing.

Aim: This study assesses the technical performance and cost of a targeted next-generation sequencing (NGS) multigene pharmacogenetic (PGx) test. Materials & methods: A genetic test was developed for 21 PGx genes using molecular inversion probes to generate library fragments for NGS. Performance of this test was assessed using 53 unique reference control cell lines from the Genetic Testing Reference Materials Coordination Program (GeT-RM). Results: 93.7% of variants were successfully called and the repeatability rate was 99.9%. Reference calls were available for 78.4% of diplotype calls resulting from PGx testing, and concordance for the test was 85.7%. Cost per sample was $32-$56. Conclusion: A targeted NGS assay using molecular inversion probe technology is able to characterize the pharmacogenome efficiently.

Abstract 5097: Clinical trial outcomes for drug combination therapies can be predicted by modeling independent action using cancer cell line screens

Abstract Background: In many cancer settings, it is evident that combination drug therapies show increased efficacy compared to monotherapies. However, the development of new combination therapies is slowed by the fact that it is infeasible to experimentally evaluate the vast number of possible drug combinations. In an effort to overcome this problem, we developed a computational method which uses monotherapy screening data in cell lines to estimate the efficacy of combinations of two or more chemotherapy drugs. When validated against published phase 3 clinical cancer trial results, our method is capable of predicting clinical trial success or failure with a high degree of sensitivity and specificity. Methods and Results: We processed existing high-throughput in vitro drug screening datasets (i.e. GDSC and CTRPv2) to obtain fitted dose response curves for more than 600,000 drug-cell line pairs, including data for 695 unique compounds and 1347 unique cell lines. We then used this dataset to develop a method of estimating the efficacy of combinations of two or more drugs. While previous efforts to predict drug combination efficacy have largely focused on dose-agnostic estimates of drug synergy, we modeled dose-dependent estimates of independent drug action, where each patient treated with a drug combination only derives benefit from the single drug in the combination to which they are most sensitive. For method validation, we systematically searched clinicaltrials.gov to identify completed, phase 3 cancer clinical trials that evaluated drug combinations for which we could estimate efficacy and which had at least 50 patients per trial arm. Of the >9,000 clinical trials considered, 37 trials (11 successful and 26 unsuccessful) met our inclusion criteria. Using clinically determined plasma concentrations for each drug, we then predicted relative efficacy for each of the experimental drug combinations versus the control group therapies in these trials. These predictions were clustered into two groups (predicted high benefit vs low benefit) using k-means clustering, and these clusters were compared to whether or not the clinical trials were considered a success by the original study investigators. Our method correctly classified 84% of the trials, with a sensitivity of 73% and a specificity of 88%. Furthermore, efficacy predictions for combinations containing erlotinib were dependent on EGFR mutation status, suggesting that this method may be useful for identifying molecular subtypes which are sensitive to a given combination therapy. Conclusion: Clinically meaningful predictions of drug combination efficacy can be generated from single-agent, in vitro drug screens under the assumption of independent drug action. This enables efficacy predictions to be made for >150,000 2-drug combinations and millions of 3- and 4-drug combinations using the existing GDSC and CTRPv2 datasets. Citation Format: Alexander Ling, R. Stephanie Huang. Clinical trial outcomes for drug combination therapies can be predicted by modeling independent action using cancer cell line screens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5097.

Abstract 2703: Lymph node colonization promotes distant tumor metastasis through the induction of systemic immune tolerance

Abstract The majority of cancer-associated deaths are the result of distant organ metastasis, an event that is typically preceded by metastasis to regional or distant lymph nodes (LNs). LNs are education hubs of the adaptive immune system wherein antigens derived from pathogens or malignancies are presented to lymphocytes in a manner that facilitates elimination of the threat. Nonetheless, LN metastasis, which is typically attributed to passive drainage of tumor cells through lymphatics, frequently does not lead to the generation of an anti-tumor immune response, but instead correlates with poor prognosis and further disease progression. Here, we find that LN metastasis represents a critical step in tumor progression through the capacity of such metastases to induce systemic immune tolerance in a manner that promotes further dissemination of tumors to distant organs. Through serial in vivo passaging of a syngeneic melanoma in mice, we generate nearly 300 unique cell lines that exhibit an enhanced capacity to metastasize to LNs. Transcriptional profiling of these lines reveals increased expression of immune-related programs. We show that the presence of these LN metastases enables distant organ seeding of metastases in a manner that the parental tumor cannot, and this differential seeding is eliminated in mice that lack an adaptive immune response. To query the effects of the LN metastases on the systemic immune response, we perform organism-wide immune profiling by mass cytometry and identify a number of cellular mediators of tolerance. In particular, we find that LN metastases have the capacity to both resist NK cell cytotoxicity and induce regulatory T cells (Tregs) in vitro. Furthermore, depletion of NK cells in vivo enables non-metastatic tumors to disseminate to LNs, and ablation of Tregs using FoxP3-DTR mice eliminates the occurrence of lymphatic metastases. We further identify an interferon signaling axis that is constitutively activated within the LN metastases in the absence of exogenous interferon signaling. Through the use of ATAC-seq, we find that this program is conferred through epigenetic regulation of chromatin accessibility. Knockout of key interferon-induced genes using CRISPR/Cas9 in the LN-metastatic cells reveals that this program is required for enhanced LN metastatic seeding in vivo, and their overexpression increases LN metastasis of the non-metastatic cells. Using additional mouse models of pancreatic ductal adenocarcinoma and head and neck squamous cell carcinoma (HNSCC), we show that these findings are conserved across multiple malignancies. Additionally, we perform RNA-seq on sorted malignant populations from node-positive and node-negative HNSCC patients and confirm that these differences in transcriptional profiles extend to the human disease. Together, these findings demonstrate a critical role for LN metastasis in promoting tumor immune tolerance. Citation Format: Nathan E. Reticker-Flynn, Maria M. Martins, Pamela A. Basto, Weiruo Zhang, Alborz Bejnood, Andrew J. Gentles, John B. Sunwoo, Sylvia K. Plevritis, Edgar G. Engleman. Lymph node colonization promotes distant tumor metastasis through the induction of systemic immune tolerance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2703.

Discovery of a novel natural killer cell line with distinct immunostimulatory and proliferative potential as an alternative platform for cancer immunotherapy

BackgroundHuman natural killer (NK) cell lines serve as an attractive source for adoptive immunotherapy, but NK-92 remains the only cell line being assessed in the clinic. Here, we established a novel NK cell line, NK101, from a patient with extra-nodal natural killer/T-cell lymphoma and examined its phenotypic, genomic and functional characteristics.MethodsSingle cell suspensions from lymphoma tissue were expanded with anti-NKp46/anti-CD2-coated beads in the presence of IL-2. A continuously growing CD56+ cell clone was selected and designated as NK101. Flow cytometry and RNA sequencing were used to characterize phenotypic and genomic features of NK101. In vitro cytotoxicity and IFN-γ/TNF-α secretion were measured by flow cytometry-based cytotoxicity assay and enzyme-linked immunosorbent assay, respectively, after direct co-culture with tumor cells. Immunomodulatory potential of NK101 was assessed in an indirect co-culture system using conditioned medium. Finally, in vivo antitumor efficacy was evaluated in an immunocompetent, syngeneic 4T1 mammary tumor model.ResultsNK101 displayed features of CD56dimCD62L+ intermediate stage NK subset with the potential to simultaneously act as a cytokine producer and a cytotoxic effector. Comparative analysis of NK101 and NK-92 revealed that NK101 expressed lower levels of perforin and granzyme B that correlated with weaker cytotoxicity, but produced higher levels of pro-inflammatory cytokines including IFN-γ and TNF-α. Contrarily, NK-92 produced greater amounts of anti-inflammatory cytokines, IL-1 receptor antagonist and IL-10. Genome-wide analysis revealed that genes associated with positive regulation of leukocyte proliferation were overexpressed in NK101, while those with opposite function were highly enriched in NK-92. The consequence of such expressional and functional discrepancies was well-represented in (i) indirect co-culture system where conditioned medium derived from NK101 induced greater proliferation of human peripheral blood mononuclear cells and (ii) immunocompetent 4T1 tumor model where peritumoral injections of NK101 displayed stronger anti-tumor activities by inducing higher tumor-specific immune responses. In a manufacturing context, NK101 not only required shorter recovery time after thawing, but also exhibited faster growth profile than NK-92, yielding more than 200-fold higher cell numbers after 20-day culture.ConclusionNK101 is a unique NK cell line bearing strong immunostimulatory potential and substantial scalability, providing an attractive source for adoptive cancer immunotherapy.

Our Unique Cell Lines Derived Serially After Injection With Human T24 HRAS Transformed NIH/3T3 Mouse Cells (GhrasT‐NIH/3T3) Showed The Highly Tumorigenic T1‐A Cells Cultured From The First Primary Tumor Had A Six‐Fold Higher LD50 For Resveratrol Killing Than The LD50 For Highly Metastatic Lung T4‐PA Cells Derived After Three Subsequent In Vivo / In Vitro Serial Manipulations

Our new cell lines demonstrating progressively increasingly metastatic behavior as they were derived in a series of in vivo/in vitro manipulations (Figure 1) provide the opportunity to evaluate in vivo and in vitro compounds and methods to kill primary tumor cells and various forms of increasingly metastatic cells derived from the same primary tumor (Figure 2) (Ray, D.B., et. al., Experimental Cell Research, 2016 Jan; 340 (1): pp. 1–11). We determined the dose of the anti‐cancer natural phytoalexin resveratrol to kill 50% (LD50s) of our various cells that represent progressive cancer stages. Cell counts were recorded daily, over the course of 3 to 6 days, using gridded plates and photomicrographs of six pre‐determined 2 mm square sections of each plate. An average percent change in cell number from each individual grid was then calculated. P‐values were determined between each treatment time and dose and significance was determined. The NIH Swiss and NIH/3T3 cells were not significantly affected by 3–300 μM after 3 days. After 3 or 4 days the LD50s for the tumorigenic cells were GhrasT‐NIH/3T3 cells: LD50 = 3.5+/− 1.0 μM; T1‐A cells: LD50 = 45 +/− 5.0 μM; T4‐PA cells: LD50 = 7.0 +/− 1.5 μM. Thus, unexpectantly, the highly tumorigenic T1‐A cells were significantly more difficult to kill than the highly metastatic T4‐PA cells. This pattern may mimic the epithelial to mesenchymal transition (EMT) seen in breast and other cancer progression. EMT enhances mobility, invasion, and resistance to apoptotic stimuli as tumor cells acquire stem cell properties and exhibit marked therapeutic resistance. RNA‐Seq, genome sequencing (WGS) and spectral karyotyping (SKY) analysis of these cells demonstrating increasingly aggressive metastatic cancer tumorigenesis that progressed from a single nuclear genotype and mitochondrial haplotype should help contribute to the understanding of cancer progression. These cells are available by contacting our laboratory.Support or Funding InformationSupported in part by the National Science Foundation grant # 8602787, the Grove City College Research Fund, the Grove City College Swezey Research Fund and the Grove City College Janicki Foundation Research Fund.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P2-06-25: A phenotypic screening and machine learning platform efficiently identifies triple negative breast cancer-selective and readily druggable targets

Abstract Identifying effective oncogenic targets is challenged by the complexity of genetic alterations in cancer and their poorly understood relation to cell function and survival. This, combined with the fact that the majority of the proteome is predicted to be undruggable, may explain why genomic approaches have had only moderate success in identifying and translating oncogenic drug targets. There is a need for methods that rapidly and accurately identify “pharmacologically effective” targets without the requirement for priori knowledge of complex signaling networks. We developed an approach that uses machine learning to relate results from unbiased phenotypic screening of kinase inhibitors to their biochemical activity data. This process, which we call idTRAX (Identification of Drug TaRgets and Anti-targets by Cellular and Molecular Cross-referencing), identifies targets that are pharmacologically responsive and readily druggable. Additionally, the identified targets are not typically overcome by the robustness of signaling networks, because only targets that effectively induce a phenotype upon pharmacological engagement are selected. We applied this methodology to triple negative breast cancer, which still lacks targeted therapy. We screened 19 breast cancer cell lines with ˜500 small-molecule kinase inhibitors with annotated kinase activity data and identified cell-line specific kinase targets, i.e. kinases whose inhibition causes cell line-specific cytotoxicity or cytostaticity. We were able to identify various unique cell line selective kinase addictions along with well-known driver kinase addictions, for example, dependence on FGFR2 in the MFM-223 cell line and AKT kinases in MFM-223, CAL-148, and others. We also found that triple negative breast cancer cell lines exhibit heterogeneous target patterns, indicating the need of personalized medicine approach to tackle them. We further compared candidate targets identified by idTRAX with those reported from RNAi- and CRISPR-based screens. Strikingly, the correlation between targets identified by the pharmacological approach and those identified by knockdown/knockout-based screens was low [average rank correlation (ρ) = 0.0273- 0.1074], suggesting that gene-silencing approaches may not be the most efficient at identifying targets for small-molecule drug discovery or repurposing. Our approach provides a platform for rapidly identifying sample-specific drug targets and potentially guiding personalised therapy regimens. Citation Format: Gautam P, Jaiswal A, Aittokallio T, Hassan A-A, Wennerberg K. A phenotypic screening and machine learning platform efficiently identifies triple negative breast cancer-selective and readily druggable targets [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-25.

Establishment of a New Scirrhous Gastric Cancer Cell Line with FGFR2 Overexpression, OCUM-14.

The prognosis of scirrhous gastric carcinoma (SGC), which is characterized by rapid infiltration and proliferation of cancer cells accompanied by extensive stromal fibrosis, is extremely poor. In this study, we report the establishment of a unique SGC cell line from a gastric cancer patient in whom an autopsy was performed. A new SGC cell line, OCUM-14, was established from malignant ascites of a male patient with SGC. A postmortem autopsy was performed on the patient. Characterization of OCUM-14 cells was analyzed by microscopic examination, reverse transcription polymerase chain reaction, fluorescence in situ hybridization analysis, immunohistochemical examination, CCK-8 assay, and in vivo assay. OCUM-14 cells grew singly or in clusters, and were floating and round-shaped. Most OCUM-14 cells had many microvilli on their surfaces. The doubling time was 43.1h, and the subcutaneous inoculation of 1.0 × 107 OCUM-14 cells into mice resulted in 50% tumor formation. mRNA expressions of fibroblast growth factor receptor 2 (FGFR2) and human epidermal growth factor receptor 2 (HER2) were observed in OCUM-14 cells. FGFR2, but not HER2, overexpression was found in OCUM-14 cells. The heterogeneous overexpression of FGFR2 was also found in both the primary tumor and metastatic lesions of the peritoneum, lymph node, bone marrow, and lung of the patient. The FGFR2 inhibitors AZD4547 and BGJ398 significantly decreased the growth of OCUM-14 cells, while paclitaxel and 5-fluorouracil significantly decreased the proliferation of OCUM-14 cells, but cisplatin did not. A new gastric cancer cell line, OCUM-14, was established from SGC and showed FGFR2 overexpression. OCUM-14 might be useful for elucidating the characteristic mechanisms of SGC and clarifying the effect of FGFR2 inhibitors on SGC.

In Vitro Murine Hematopoiesis Supported by Signaling from a Splenic Stromal Cell Line.

There are very few model systems which demonstrate hematopoiesis in vitro. Previously, we described unique splenic stromal cell lines which support the in vitro development of hematopoietic cells and particularly myeloid cells. Here, the 5G3 spleen stromal cell line has been investigated for capacity to support the differentiation of hematopoietic cells from progenitors in vitro. Initially, 5G3 was shown to express markers of mesenchymal but not endothelial or hematopoietic cells and to resemble perivascular reticular cells in the bone marrow through gene expression. In particular, 5G3 resembles CXCL12-abundant reticular cells or perivascular reticular cells, which are important niche elements for hematopoiesis in the bone marrow. To analyse the hematopoietic support function of 5G3, specific signaling pathway inhibitors were tested for the ability to regulate cell production in vitro in cocultures of stroma overlaid with bone marrow-derived hematopoietic stem/progenitor cells. These studies identified an important role for Wnt and Notch pathways as well as tyrosine kinase receptors like c-KIT and PDGFR. Cell production in stromal cocultures constitutes hematopoiesis, since signaling pathways provided by splenic stroma reflect those which support hematopoiesis in the bone marrow.

Abstract A29: The CUTO panel of patient-derived NSCLC cell lines reveals unique molecular characteristics and responses to targeted therapies

Abstract Cell lines generated from human tumors have been an invaluable tool in dissecting the underlying molecular mechanisms of cancer biology and for cancer drug development. The large library of original human lung cancer cell lines established by Minna and Gazdar, though instrumental in investigating various aspect of lung cancer biology, lacks examples of some of the diverse mutations responsible for this cancer. The CUTO (Colorado University Thoracic Oncology) cell line series, initiated in 2011, had the goal of curating a new panel of cells derived from NSCLC patients with distinct oncogenic drivers. By generating multiple, unique cell lines representing each oncogene driver subset, we believe that these lines would allow study of interpatient variability that underlie differential patient responses and duration of response to targeted therapies. The current CUTO panel consist of 33 cell lines with either 1) gene rearrangements in ALK, RET, ROS1 or NTRK1; 2) activating mutations in the ERBB gene family including exon 20 insertions in EGFR and ERBB2 (HER2) as well as rare mutations in EGFR; and 3) inactivation of the NF1 gene either as a concurrent mutation with other oncogenic alterations or as the sole driver mutation. For cells harboring gene rearrangements we have amassed four EML4-ALK lines, two KIF5B-RET cell lines, five ROS1 fusion lines (3 CD74-ROS1, 1 SDC4-ROS1, and 1 TPM3-ROS1), and one MPRIP-NTRK1 cell line. Our ERBB mutant lines include one cell line with HER2 exon 20 insertion, three lines with different exon 20 insertions in EGFR, and two EGFR mutant cell lines with compound mutations. We present our characterization of these cell lines in terms of their proliferation profiles and molecular signaling pathways in the presence of targeted inhibition. The derivation and characterization of these cell lines have facilitated the study of cell signaling in oncogene-driven cancer, have helped identify new resistance mechanisms, and have facilitated drug development for rare oncogenes such as NTRK1 gene fusions and EGFR exon 20 insertions. We expect that this growing library of cell lines will continue to further our understanding of oncogenic-driven tumor biology and provide mechanistic insight towards the development of novel therapeutics and drug combinations. Citation Format: Anh Tuan Le, Adriana Estrada-Bernal, Laura Schubert, Andrea Doak, Nan Chen, Kurtis Davies, Aria Vaishnavi, Mary Jackson, Vignesh Narayana, Kimi Kondo, John Mitchell, Michael Weyant, Tom Purcell, Paul Bunn, Ross Camidge, Janet Freeman-Daly, Robert Doebele. The CUTO panel of patient-derived NSCLC cell lines reveals unique molecular characteristics and responses to targeted therapies [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A29.

Establishment of a nasopharyngeal carcinoma cell line capable of undergoing lytic Epstein–Barr virus reactivation

Epstein–Barr virus (EBV) infects more than 90% of the adult human population. Undifferentiated nasopharyngeal carcinoma (NPC) is common in Southeast Asia, with a particularly high incidence among southern Chinese. The EBV genome can be detected in practically all cancer cells in undifferentiated NPC. The role of EBV in pathogenesis of undifferentiated NPC remains elusive. NPC cell lines are known to be difficult to establish in culture. The EBV+ve NPC cell lines, even if established in culture, rapidly lost their EBV episomes upon prolonged propagation. At present, the C666-1 NPC cell line, which is defective in lytic EBV reactivation, is the only EBV+ve NPC cell line available for NPC and EBV research. The need to establish new and representative NPC cell lines is eminent for NPC and EBV research. In this study, we report the use of the Rho-associated kinase inhibitor (Y-27632) has facilitated the establishment of a new EBV+ve NPC cell line from an earlier established NPC xenograft, C17. The C17 cell line was tumorigenic in immune-deficient mice (NOD/SCID). It retained the EBV episomes and could be induced to undergo productive lytic reactivation of EBV to generate infectious virus particles. The C17 cell line represents a new investigative tool for NPC and EBV studies. The ability of C17 to undergo lytic reactivation is unique and opens up the opportunity to examine regulation of latent and lytic infection of EBV and their contributions to NPC pathogenesis.EBV-positive NPC cell lines are difficult to establish in culture which seriously hampered research progress in NPC and EBV. The ROCK inhibitor (Y-27632) facilitated the establishment of a new and unique EBV-positive NPC cell line from the C17 NPC xenograft capable of undergoing productive lytic EBV reactivation.

3100 - A Novel G Protein Coupled Receptor Circuit Regulates Hematopoiesis

Hematopoietic stem cell (HSC) generation is a complex process that is tightly regulated by a variety of integrated molecular signals. Gpr56, a G-coupled receptor protein, is most highly expressed in HSCs as they are generated. Despite the demonstration that Gpr56 is required for HSC generation in zebrafish, its role in mammalian haematopoiesis remains unclear and controversial. In this study, we examine the function of Gpr56 by taking advantage of our unique mouse Gata2Venus embryonic stem cell line (G2V ESC). G2V ESCs are an excellent tool to examine the role of Gpr56 in hematopoiesis in vitro. In vitro hematopoietic differentiation of mouse G2V ESC showed that Gpr56 is mainly expressed in the Gata2-expressing cell fraction, which is enriched in hematopoietic progenitor cells (HPC). When Gpr56 knockout (G56-/-) G2V ESCs are differentiated, a surprising increase in Gata2 expressing cells and HPCs was found. Preliminary data demonstrate that another G-coupled receptor protein, Gpr97 (located at same locus as Gpr56) is highly expressed when Gpr56 is deleted. A Gpr56 gain-of-function approach is also being taken to explore the mechanism by which this receptor acts in HSCs. To this end, we are using an innovative approach to target mouse and human pluripotent stem cells to ensure the Gpr56 overexpression during the different stages of in vitro differentiation. The data so far collected demonstrate that Gpr56 is required for the maintenance of HSC physiology, and suggest that Gpr97 is a novel molecule that could support a compensatory, and possibly an additional, function in HSCs.

Abstract 3356: The EWS/FLI1 transcriptome is characterized by marked heterogeneity across Ewing sarcoma cell lines

Abstract BACKGROUND: Ewing sarcoma (ES) is the second most common pediatric bone cancer. ES cells are dependent on the activity of the EWS/FLI1 transcription factor for cell survival. EWS/FLI1 drives proliferation, dysregulates the cell cycle and establishes a block in differentiation. Despite the known dependence of Ewing sarcoma on EWS/FLI1, the exact identity of all the downstream targets of EWS/FLI1 is not known. While gene signatures have been reported for EWS/FLI1, these fail to capture the marked heterogeneity in the EWS/FLI1 transcriptome or correlate these expression changes with cellular phenotype. METHODS: Next generation RNA sequencing with deep coverage was used to generate a comprehensive analysis of the EWS/FLI1 transcriptome by silencing EWS/FLI1 in 5 different ES cell lines at two different time points. These results were organized into gene expression networks and signaling nodes were identified by weighted correlation network analysis (WGCNA). The networks were validated by a 500-gene signature of EWS/FLI1 targets using a next generation sequencing capture assay and the Illumina platform. The cellular endpoint of EWS/FLI1 silencing was evaluated by caspase 3,7 cleavage to measure apoptosis, BGAL staining to measure senescence and scratch assays to capture migration. RESULTS: Next generation RNA sequencing with siRNA silencing of EWS/FLI1 revealed marked heterogeneity in the EWS/FLI1 transcriptome among cell lines. Targets of EWS/FLI1 that were common across cell lines exhibited small magnitude gene expression changes (log fold <2), while large magnitude gene expression changes were unique to individual cell lines (log fold>2). When the log fold change in expression was restricted to greater than 2, only 13 common repressed targets and 4 common induced targets were identified in all 5 cell lines. In addition, silencing of EWS/FLI1 induced dramatically different phenotypes across cell lines, where p53 wild-type cell lines underwent senescence and the p53 mutant cell lines did not. Most cell lines did not continue to proliferate with EWS/FLI1 silencing with the exception being the widely used A673 cell line. Cellular migration did not change with EWS/FLI1 silencing in many of the cell lines in contrast to A673 cells. CONCLUSIONS: EWS/FLI1 drives different gene expression signatures and different phenotypes in unique cell lines. This diversity needs to be considered as EWS/FLI1 directed therapies are developed. Citation Format: Susan M. Kitchen-Goosen, Megan J. Bowman, Marie Adams, Penny Berger, Patrick J. Grohar. The EWS/FLI1 transcriptome is characterized by marked heterogeneity across Ewing sarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3356.