Development of a multicellular pancreatic tumor microenvironment system using patient-derived tumor cells.

In vitro culture for patient-derived cells offers many advances in the development of novel therapies for colorectal cancer. Although various culture systems have been developed, experimental systems in which the long-term expansion of patient-derived epithelial cells still remained difficult. Here, we demonstrated that epithelial cells isolated from colorectal cancer patient-derived xenograft can be efficiently immortalized in conditioned medium from irradiated feeder cells containing a Rho kinase inhibitor Y-27632. Patient-derived tumor cells were rapidly proliferated to reach confluence in approximately 6 days. Short tandem repeat (STR) analysis of tumor tissue and cultured cells demonstrate that they have 13 identical STR loci and Amelogenin, Penta E, Penta D, D2S1338 and D19S433. The epithelial character was confirmed by staining for EpCAM, cytokeratin 20 and vimentin as a mesenchymal marker. Cells were continued to proliferate and formed well-defined tumor spheroids when transferred to 3D culture. To study the mechanism for promoting unrestricted cell proliferation, we investigated the expression of several cellular genes that fall into two designated pathways, induction of telomerase and cytoskeletal remodeling/p16 pathway. Moreover, patient-derived tumor cells were utilized for high-throughput screening of FDA approved drugs allowing detection of gene-drug associations. Collectively, we demonstrate that the long-term expansion of patient-derived tumor epithelial cells and these cells could be a useful tool in order to investigate a personalized treatment approach. Citation Format: Hyekyung Hong, Nakhyeon Yun, Taewon Kim, Yeosong Lee, Sujeong Song, Jaemoon Bae, Wooyong Lee, Yongbeom Cho. Efficient primary culture model of patient-derived epithelial cells from colorectal cancer using a ROCK inhibitor and feeder cells [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 4086.

The potential of novobiocin to augment cancer chemotherapy was explored in the late 1980s and early 1990s in tumor cells and tumor-bearing mice and in Phase 1 clinical trials with cyclophosphamide or cisplatin. The molecular target of novobiocin was identified to be DNA POLθ which has a role in repair of DNA double strand breaks (DSB) during mitosis. Genetic alterations which may increase or decrease POLθ inhibitor effects have been elucidated. Thirty patient-derived tumor cell lines with known BRCA, ATM, ATR, POLQ, XRCC1, PALB2, PARP1, and LIG3 alterations were screened in a complex spheroid assay (tumor cells, endothelial cells, mesenchymal stem cells) with a POLθ inhibitor (novobiocin, ART-558, or RP6685), alone or in simultaneous combination with a FDA approved or investigational anticancer small molecule, using a 7-day exposure and a CellTiter-Glo 3D luminescence endpoint. As single agents, the POLθ inhibitors had little or no cytotoxicity. Potentiation of the topoisomerase II inhibitors doxorubicin (DOX) and etoposide by the POLθ inhibitors ART-558 or RP6685 was observed in spheroids grown from the serous endometrial adenocarcinoma 922993-354-T-J3, which has ATM and BRCA2 variants. The 379773-124-R-J2 endometrioid endometrial adenocarcinoma which harbors mutant POLθ demonstrated antagonism with ART-558 in combination with PARP inhibitors and less than additive to additive cytotoxicity in combination with DOX. Activity of the Chk1/2 inhibitor prexasertib was potentiated by either ART-558 or RP6685 in the 922993-354-T-J3 complex spheroids. The combination of POLθ inhibitor ART-558 and the Chk1/2 inhibitor prexasertib produced up to 1 log increase in cytotoxicity in the 922993-354-T-J3 complex spheroids. KRAS G12D inhibitor MRTX-133 cytotoxicity against 186277-243-T-J2 colon adenocarcinoma with the KRAS G12D variant was potentiated by either ART-558 or RP6685. Potential combinations to advance into in vivo studies will be presented. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Beverly A. Teicher, Thomas S. Dexheimer, Thomas Silvers, Nathan P. Coussens, J Paul Eder, James H. Doroshow. Polymerase θ (POLθ) inhibitors (novobiocin, ART-558, RP6685) were tested with 22 approved and investigational agents in multi-cell type spheroids of genetically selected patient-derived human tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB215.

Recurrent metastatic breast cancer is difficult to treat and patients often exhaust standard-of-care regimens. P.A.R.I.S. is a CLIA certified high throughput chemosensitivity test developed by SEngine Precision Medicine. (Seattle, WA USA) Tumor cells derived from patient breast tissue are expanded as mammospheres, and challenged with a library of 123 clinically actionable, targeted drugs. These results are integrated with patient genomic data and reported to the oncologist to prioritize treatment options. To date, we have performed over 100 such drug screens using low passage patient-derived tumor cells in 70 independent replicate pairs. Of 18 viable primary breast tumor samples received to date, we have successfully derived organotypic cultures in 13 cases. (72%) Of these, 7 were challenged to the full 123 agent library. Three of the seven cases were HER2 positive and demonstrated clear sensitivity to HER2 kinase inhibitors. Overall, patients can be separated into two groups, characterized by families of drug sensitivities. Patients in group one respond preferentially to inhibitors of signaling pathways such as IGF1R, ALK, FGFR and EGFR. Patients in the second group responded to therapies targeting epigenetic modifiers, cell cycle control and apoptosis. Presented here is a premenopausal female, diagnosed in 2010 as pT2N0M0 (stage IIA), ER+ PR+ HER2+. This patient underwent surgery and received adjuvant chemotherapy, followed by tamoxifen. Upon progression to metastatic disease, biopsy of her recurrent lesion showed loss of ER and PR expression, but remained positive for HER-2 amplification. Sequencing of this biopsy exposed a PIK3CA G106R mutation and amplification of unknown significance. Tumor cells were isolated from ascites, cultured and the P.A.R.I.S. test was performed. Patient derived tumor cells were sensitive to HER2 targeting agents, concordant with the patient's HER2 amplification. Of these, the non-reversible inhibitor neratinib exhibited a significantly enhanced effect and achieved maximum growth inhibition of 80% over the duration of the assay. Two different AKT inhibitors, afuresertib and AZD5363, demonstrated activity in the patient cells consistent with the activating PIK3CA mutation. Also consistent with the loss of ER/PR expression, the patient derived tumor cells no longer responded to anti-estrogen therapies nor to chemotherapies the patient had been refractory to prior: cyclophosphamide, docetaxel, doxorubicin, gemcitabine, and paclitaxel.The PARIS assay is a robust ex vivo screening platform to objectively quantify patient specific sensitivities to a panel of 123 clinically actionable oncology drugs. We show here concordance with both genomic data and prior treatments the patient received in the course of disease however the same approach may be used to successfully identify unique sensitivities in the absence of established biomarkers. Citation Format: Michael J. Churchill, Franz X. Schaub, Hallie A. Swan, Rachele Rosati, Fengting Yan, Reid C. Shaw, Kay E. Gurley, Robert L. Diaz, Shalini C. Pereira, Carla Grandori, Christopher J. Kemp, Vijayakrishna K. Gadi. Patient derived tumor organoids identify actionable targets in heavily pretreated metastatic breast cancer patients [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 1603.

Malignant glioma stands as a formidable cancer with limited therapeutic options due to many factors including pronounced genetic heterogeneity, the blood-brain barrier, among others. Precision medicine, tailoring treatment based on a patient’s unique molecular profiles, is a fitting approach for addressing intra- and inter-tumor heterogeneity. Previously, we have shown the advantage of using patient-derived tumor cells (PDCs) to evaluate drug efficacy across various cancer types. Unlike cancer cell lines, which significantly differ from primary tumors, PDCs capture the molecular features of primary tumors, showing predictive clinical responses in a retrospective study. Building on these encouraging results, we have expanded PDC-based drug screening to specifically target malignant gliomas. We have established an initial cohort of over 500 malignant glioma PDCs (~ 370 patients) and screened more than 100 drugs. We have complemented our cohort with molecular profiles, including genomic and transcriptomic data to infer the potential pharmacogenomic associations of malignant gliomas. We constructed a multi-modal deep-learning model that seamlessly integrates molecular features and drug structure to predict the drug sensitivity. By employing an explainable machine learning method, we retrospectively analyzed the model to investigate the potential molecular biomarkers and their contribution to drug sensitivity. Collectively, our study demonstrates the efficacy of using patient-derived tumor cells (PDCs) combined with deep-learning models to predict drug sensitivity in malignant gliomas. This integrative approach can potentially refine personalized treatment strategies and improve clinical outcomes for glioma patients.

BACKGROUND:The MAP2K1 K57T mutation is known to be a potential mechanism of primary and secondary resistance to EGFR inhibitors in metastatic colorectal cancer (CRC) and has also been reported to promote resistance to BRAF and MEK inhibitors. It is important to overcome therapeutic resistance to EGFR inhibitors to improve the treatment outcomes of metastatic CRC.METHODS: We established patient-derived tumor cells (PDCs) from metastatic lesions that newly appeared during treatment with a BRAF inhibitor (LGX-818) plus an EGFR inhibitor (cetuximab) in a patient with BRAF-mutant CRC. To investigate therapeutic options to overcome acquired resistance due to MAP2K1 mutation in BRAF-mutant CRC, we performed cell viability assays using the PDCs.RESULTS:We tested whether the PDCs were resistant to an EGFR inhibitor (cetuximab) and a BRAF inhibitor (sorafenib) as these cells were established at the time of resistance to the EGFR plus BRAF inhibitors. Moreover, the anti-tumor effect of AZD6244 (MEK inhibitor) was evaluated because PDCs harbored a MAP2K1 mutation at the time of resistance to the EGFR plus BRAF inhibitors. MTT proliferation assays showed that monotherapy with cetuximab, sorafenib, or AZD6244 did not suppress cell viability. We next tested viability of the PDCs to combination treatment with cetuximab plus AZD6244 and sorafenib plus AZD6244. Proliferation of PDCs was significantly inhibited by sorafenib and AZD6244, but not by cetuximab plus AZD6244. Investigation of the combined effect of sorafenib and AZD6244 using the calculated combination index (CI) showed synergistic effects of sorafenib and AZD6244 in combination therapy applied to PDCs with the MAP2K1 K57T mutation.CONCLUSION:Our results suggest that combination treatment with BRAF and MEK inhibitors might be a novel treatment strategy for MAP2K1 K57T-mutant CRC. This finding will be helpful to guide treatment of patients with CRC that is resistant to EGFR inhibitors.

BackgroundNatural killer (NK) cells play a critical role in cancer immunosurveillance. Recent developments in NK cell ex-vivo expansion makes it possible to generate millions of activated NK cells from a small volume of peripheral blood. We tested the functionality of ex vivo expanded NK cells in vitro against breast cancer cell lines and in vivo using a xenograft mouse model. The study aim was to assess functionality and phenotype of expanded NK cells from breast cancer patients against breast cancer cell lines and autologous primary tumours.MethodsWe used a well-established NK cell co-culture system to expand NK cells ex vivo from healthy donors and breast cancer patients and examined their surface marker expression. Moreover, we tested the ability of expanded NK cells to lyse the triple negative breast cancer and HER2-positive breast cancer cell lines MDA-MB-231 and MDA-MB-453, respectively. We also tested their ability to prevent tumour growth in vivo using a xenograft mouse model. Finally, we tested the cytotoxicity of expanded NK cells against autologous and allogeneic primary breast cancer tumours in vitro.ResultsAfter 3 weeks of culture we observed over 1000-fold expansion of NK cells isolated from either breast cancer patients or healthy donors. We also showed that the phenotype of expanded NK cells is comparable between those from healthy donors and cancer patients. Moreover, our results confirm the ability of ex vivo expanded NK cells to lyse tumour cell lines in vitro. While the cell lines examined had differential sensitivity to NK cell killing we found this was correlated with level of major histocompatibility complex (MHC) class I expression. In our in vivo model, NK cells prevented tumour establishment and growth in immunocompromised mice. Finally, we showed that NK cells expanded from the peripheral blood of breast cancer patients show high cytotoxicity against allogeneic and autologous patient-derived tumour cells in vitro.ConclusionNK cells from breast cancer patients can be expanded similarly to those from healthy donors, have a high cytotoxic ability against breast cancer cell lines and patient-derived tumour cells, and can be compatible with current cancer treatments to restore NK cell function in cancer patients.

Background: Preclinical in vitro and in vivo tumor models lack human stromal cells and are inadequate for the assessment of novel tumor microenvironment-targeted therapies. Here we report on a 3D-tumor growth assay (3D-TGA) in which patient-derived tumor cells are admixed with a basement membrane extract and bone marrow-derived mesenchymal stem cells (bmMSCs) in a 96-well format for screening. Response to standard-of-care (SoC) agents such as paclitaxel, targeted agents such as erlotinib were compared to subcutaneous xenograft responses in vivo. Methods: Tumor cells or xenograft materialthat is disaggregated to yield a pure epithelial population are admixed and suspended in basement membrane extract (Cultrex, Trevigen) with and without bmMSCs. Cells were maintained for three days before they were treated with a panel of test agents, such as paclitaxel, AZD2014, erlotinib, and carboplatin, and final cell viability was determined on day 7 by alamarBlue assay. IC50 curves were determined using GraphPad Prism 6.0. Subcutaneous tumor growth was evaluated three times a week by measuring the tumor in two dimensions using electronic calipers for the duration of the study, and tumor volumes were estimated. Treatment was initiated when tumor growth was established. Results: A panel of squamous NSCLC PDX models that contain various mutation sites (e.g., EGFR, KRAS) was assessed with a range of SoC (e.g., carboplatin, pemetrexed, and paclitaxel) or targeted agents (e.g., erlotinib, AZ2014). A squamous NSCLC model LU6422,that harbors a heterozygous mutation in the EGF-R intracellular kinase domain (c.2573T>G (L858R)) showed exquisite sensitivity to erlotinib in both 3D TGA and xenograft studies. Similarly, a squamous NSCLC model LU6402 was assessed with AZD2014 and paclitaxel in 3D-TGA in the presence of bmMSCs, which showed an additive effect of AZD2014 in combination of paclitaxel on tumor cell growth. A similar efficacy was observed in subcutaneous in vivo xenograft; however, a combination finding was not evident when the stromal cells bmMSCs were absent. The data illustrate that the comparable sensitivities to various test agents between 3D-TGA and in vivo xenograft studies were observed. Conclusions: The 3D tumor growth assay restores the interaction between the tumor and stroma in a 96-well format enabling screening to multiple dug concentrations and combinations in a 7-day assay. This assay provides a unique opportunity for interrogating therapies that target the tumor microenvironment. Citation Format: Simon Jiang, Jane Wrigley, Sumanjeet Malhi, Jamie Wood, Stephanie Morton, Louise Wainwright, Lucy Harris, Jason King, Yinfei Yin, Rajendra Kumari. Three-dimensional tumor growth assay for patient-derived tissue and cell line models incorporating stromal elements for screening [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A213.

874 Development of BCA101, a bifunctional antibody capable of simultaneously disabling EGFR and TGFβ signaling, as novel single-agent immunotherapy

e14004 Background: Monoclonal antibodies are established treatment options for B cell-derived malignancies, but relapse is still the major challenge. Novel target structures may open alternative avenues to develop effective antibody therapies. Here, we characterized the novel tetravalent antibody ‘EBU-141 Tetra’ and identified the glycoantigen CD75s (α-2,6-sialylated lactosamines) as suitable target structure for antibody-based therapy. CD75s was detected on most B cell lymphomas, including Burkitt’s lymphoma, FL, DLBCL, MCL, CLL, and plasma cell tumors. Classical Hodgkin lymphomas were consistently negative while reactivity on individual cases of peripheral T cell lymphoma was seen. To evaluate CD75s as a target for antibody therapy, we generated a tetravalent, Fc-engineered chEBU-141 IgG1 antibody with enhanced avidity for CD75s and potent effector functions. Methods: ‘EBU-141 Tetra’ was produced by transient transfection and purified by affinity chromatography. Direct anti-tumor effects and Fc-mediated effector functions were investigated in cell proliferation assays, by fluorescence microscopy and in 51Cr release experiments using lymphoma and myeloma cell lines and patient-derived tumor cells. Peripheral blood mononuclear cells and monocyte-derived macrophages of healthy donors were used as human effector cells in the experiments. Results: ‘EBU-141 Tetra’ showed improved binding to CD75s on cell surface of mature B cell lymphoma as well as myeloma plasma cells compared to the conventional chimeric antibody chEBU-141 IgG1. The higher avidity for CD75s resulted in markedly improved ADCC activity of ‘EBU-141 Tetra’ against Daudi Burkitt’s lymphoma, U266 plasma cells and CLL patient-derived tumor cells with EC50 values in the low nanomolar range. In addition, ‘EBU-141 Tetra’ demonstrated efficient phagocytosis of Burkitt’s lymphoma and myeloma cell lines. Thus, the novel tetravalent, chimeric, Fc-engineered antibody ‘EBU-141 Tetra’ efficiently recruits immune effector cells for tumor cell lysis. Conclusions: Our findings further demonstrate that highly potent IgG-like antibodies against glycan-structures can be generated from mouse IgM antibodies and may open a new therapeutic window for therapy of patients with mature B cell lymphomas and multiple myeloma.

5-Fluorouracil (5-FU) remains the first-line treatment for colorectal cancer (CRC). Although 5-FU initially de-bulks the tumor mass, recurrence after chemotherapy is the barrier to effective clinical outcomes for CRC patients. Here, we demonstrate that p53 promotes WNT3 transcription, leading to activation of the WNT/β-catenin pathway in ApcMin/+/Lgr5EGFP mice, CRC patient-derived tumor organoids (PDTOs) and patient-derived tumor cells (PDCs). Through this regulation, 5-FU induces activation and enrichment of cancer stem cells (CSCs) in the residual tumors, contributing to recurrence after treatment. Combinatorial treatment of a WNT inhibitor and 5-FU effectively suppresses the CSCs and reduces tumor regrowth after discontinuation of treatment. These findings indicate p53 as a critical mediator of 5-FU-induced CSC activation via the WNT/β-catenin signaling pathway and highlight the significance of combinatorial treatment of WNT inhibitor and 5-FU as a compelling therapeutic strategy to improve the poor outcomes of current 5-FU-based therapies for CRC patients.

A glioma produces some of the most rapidly growing, angiogenic, and invasive primary brain tumor cells known. A lack of understanding about the intricately coupled molecular mechanisms that result in cell transformation is responsible, in part, for the minimal progress made in treating this disease over the past century. To begin dissecting molecular interrelationships in time and space within living normal and transformed cells, a morphological assay is being developed wherein patient-derived tumor cells are allowed to attach to a substrate, spread, change shape, and locomote. During this process (approximately 6.5 h), low magnification phase contrast images of the cells are recorded at 1 min intervals. Quantitative image analyses of these time-lapse images are used to measure dynamic parameter such as projected cell area, shape, and displacement for each cell. Although there is considerable cellular heterogeneity, patterns of patient-specific tumor cell behavior are beginning to emerge. In addition, the assay is being used to test the effects of drugs that alter specific intracellular processes (e.g., cytochalasin, 2-deoxyglucose, and chemotherapeutic drugs). To dissect tumor cell physiology into its molecular components, I have focused on the actin-cytoskeleton because it is involved in the temporal and spatial orchestration of ions, metabolites, macromolecules, and organelles that underlies the interconnected processes of tumor cell growth, motility, and differentiation. I have used fluorescent analogs of actin and its associated proteins in conjunction with multimode-based light microscopy of living cells to measure the complex interrelationships responsible for motility in patient-derived normal and transformed glia. I have simultaneously measured the dynamics of actin assembly and focal contact formation using new fluorescent analogs of actin and vinculin in single migrating human glioblastoma cells and used this information to begin developing a molecular model of tumor cell migration. By engineering new protein-based reagents and fluorescence spectroscopic methodologies we will be able to measure and manipulate a greater number of molecular processes and therefore further refine the model. The ultimate goal of this work is to use living cells to diagnose and design treatment for primary brain tumors in a more patient-specific manner.

Background Precision oncology is a clinical approach aimed towards tailoring treatment strategies for patients based on the genetic profile of each patient's malignancy. Available cell line models are greatly limited in recapitulating the genetic profile of the tumor and tumor microenvironment of patients and therefore limit preclinical evaluation of new targeted agents. Furthermore, a high failure rate of drug candidates can be attributed in part to use of two dimensional (2D) monolayer cultures as the initial screening method that frequently produces inaccurate results and does not predict chemoresistance. The ability of three dimensional (3D) cultured tumor cells derived directly from patients to recapitulate tumor-like microenvironment suggests that engineered tumor models may provide more accurate and reproducible information for drug testing. At the Institute for Precision Medicine, we grow patient-derived 2D cell strains and engineer 3D tumor spheroids for patient`s tumor specific drug testing. Design Fresh tissue samples from 30 different solid tumors were digested in Type II or IV Collagenase (Gibco) and then re-suspended in growth factor reduced Matrigel (BD), plated on plastic, and overlaid with cell type specific cell growth media tsCM, which consists of either serum free Advanced DMEM/F12 (Gibco) with multiple growth factors optimized to propagate benign primary tumor cells, or FBS enriched DMEM/F12 media. Cultures were maintained at 37°C in 5% CO2. Once established, early cell strain culture passage and tumor spheroids size < 500 μm were cryopreserved. Specific drug testing was performed following 3 models: 2D monolayer culture, 2D monolayer culture converted in a 3D system using the hanging drop system from Biomatrix 3D®, and engineered 3D tumor spheroids. Results Our success rate in generating 2D- cell strains and 3D tumor spheroids is about 30%-40%. In the initial 30 samples, we were able to establish 11 continuous 2D and 3D tumor spheroid cultures derived from distinct primary and metastatic cancers including prostate, kidney, bladder and endometrium, as well as sarcomas and a neuroendocrine tumor. Tumor content in biopsy represents a major factor for successful growth. Histology and cytology examination demonstrates that 3D tumor spheroids recapitulate harvested tumors. Next generation sequencing shows the presence of same genomic alterations compared with original tumor without generating additional mutations. Conclusion We have developed individual patient-derived 2D tumor cell strains and 3D engineered tumor spheroids as well as models for patient`s tumor specific drug testing. This effort has been implemented for Precision Care and Clinical Trials at our Institution. Citation Format: Chantal Pauli, Jonathan Pauwels, Myriam Kossai, Nikolai Steklov, Andrea Sboner, Rema Rao, Kenneth Hennrick, Brian Robinson, Juan Miguel Mosquera, Himisha Beltran, Mark A. Rubin. Development of 2D - cell strain and 3D - tumor spheroid models for Precision Medicine. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4672. doi:10.1158/1538-7445.AM2015-4672

First two authors contributed equally Last two authors contributed equally Recent studies have shown that ex vivo propagation of normal tissues or patient-derived tumor cells in presence of irradiated fibroblast feeder cells and ROCK inhibitor can rapidly establish conditionally reprogramed cells (CRCs). In case of normal tissues, the induction of CRCs was reversible when the ROCK inhibitor and the feeder cells were removed, resulting in CRCs differentiating to its tissue origin (Liu et al.2012). Previous publications suggested that the establishment of such cell models provides new strategies to understand acquired resistance during treatment (Crystal et al 2015) and to predict treatment response (Liu et al. 2014). However, gene expression modulations and genomic drifting during the establishment of CRC propagation have not been thoroughly studied. The primary goal of this study is to molecularly characterize alterations between the original tumor tissues and the derived models growing with or without ROCK inhibitor. Understanding in-depth molecular fluctuations in this patient-derived ex vivo system will facilitate its appropriate use for tumor biology experimentation. Herein, tumors from prostate cancer and breast cancer patients were surgical removed and cryopreserved at the clinical site then processed and cultured as previously described (Liu et al. 2012). Gene expression profiling and next-generation sequencing were carried out on the original tumor tissues and cellular models passaged during the CRC propagation in the presence or absence of ROCK inhibitor. Gene expression analysis of the prostate cancer cells and the breast cancer cells were carried out using a 93-gene prostate cancer-focused Fluidigm panel and a 800 gene NanoString breast cancer-focused panel, respectively. Cancer hotspot mutations were analyzed using the Ion Torrent Cancer Hotspot v2 NGS assay. Through aforementioned genomic and transcriptomic interrogations, we demonstrated the extent of indication-relevant gene expression modulation during establishment and propagation of these cells. We also characterize cancer hotspot mutations in the primary tumor cells and the stability of those mutations during ex vivo propagation. These results should begin to inform the appropriate use of the CRC model for tumor biology experimentation. Citation Format: Jessica Li, Bonnie Liu, Rin Nakamura, Heidi Savage, Shoji Ikeda, Timothy Wilson, Teiko Sumiyoshi, Garret Hampton, Lukas Amler, Mark Lackner, Shih-Min A. Huang, Walter C. Darbonne. Gene expression and genomic drift comparative analysis between patient-derived conditionally reprogrammed cells and original tumors. [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 4257.

Objective:Mutant KRAS, the principal isoform of RAS, plays a pivotal role in the oncogenesis of colorectal cancer by constitutively activating the RAF/MEK/ERK and PI3K/AKT pathways. Effective targeted therapies are urgently needed. We investigated whether rigosertib, a benzyl styryl sulfone RAS signaling disruptor, could selectively kill KRAS-mutant colorectal cancer cells.Methods:CCK-8 was used to determine the cell viability. Patient-derived tumor and cancer cell xenograft models were used to detect the inhibitory efficacy of rigosertib. Flow cytometry was used to evaluate the apoptosis and cell cycle progression. Apoptosis and cell cycle arrest markers were detected by Western blot. DCFH-DA was used to determine the reactive oxygen species. Immunohistochemistry staining and Western blot were performed to characterize RAS signaling markers in colorectal cancer tissues and cells.Results:Rigosertib (RGS) exhibited a cytotoxic effect against colorectal cancer cells, which was greater in KRAS-mutant cells. Furthermore, RGS induced mitotic arrest and oxidative stress-dependent apoptosis in KRAS-mutant DLD1 and HCT116 cells. Besides, RGS disrupted RAS signaling, and the inhibition of RAS/MEK/ERK was independent of cellular oxidative stress. Using patient-derived xenograft models, the response and tumor inhibition of RGS were significantly higher in the KRAS-mutant subgroup, while p-MEK, p-ERK, and p-AKT levels of RGS-treated tumors were significantly decreased. Finally, in a KRAS-mutant, chemotherapy-resistant patient-derived xenograft model, RGS showed a stronger therapeutic effect than the combination standard therapy involving fluoropyrimidine + oxaliplatin/irinotecan + bevacizumab.Conclusions:These data showed that targeting RAS signaling using RGS could be a therapeutic treatment for KRAS-mutant colorectal cancer patients.

When “No-Smoking” is not enough: Hypoxia and nicotine acetylcholine receptor signaling may drive lung adenocarcinoma progression in never-smokers