Establishment of gastric signet ring cell carcinoma organoid for the therapeutic drug testing

Signet ring cell carcinoma (SRCC) has specific oncogenesis and phenotypic and treatment resistance heterogeneity. Systemic therapies are often ineffective, and predictive biomarkers to guide treatment are urgently needed. Tumor organoids have recently emerged as an ideal model for drug testing and screening. Here, we report gastric organoids established from tumor tissues comprising four SRCCs and eight non-SRCCs. Tumor organoids demonstrated different growth characteristics and morphologies. Changes in the original tumor genome were maintained during long-term culture from whole-exome sequencing (WES) analysis. Immunohistochemistry and H&E staining showed that the tissue characteristics of the primary tumor could be recapitulated. In addition, organoid lines successfully formed tumors in immunodeficient mice and maintained tumorigenic character. Different responses to 5-fluorouracil, oxaliplatin, docetaxel and irinotecan treatment were observed in SRCC and non-SRCC organoids. These results demonstrate that gastric organoid drug models, including SRCC, were highly similar to the original tumors in phenotypic and genotypic profiling and could be as living biomarkers for drug response testing.

Paxillin Promotes Breast Tumor Collective Cell Invasion through Maintenance of Adherens Junction Integrity

Distant organ metastasis is linked to poor prognosis during cancer progression. The expression level of the focal adhesion adapter protein paxillin varies among different human cancers, but its role in tumor progression is unclear. Herein, we utilize a newly generated PyMT mammary tumor mouse model with conditional paxillin ablation in breast tumor epithelial cells, combined with in vitro 3D tumor organoids invasion analysis and 2D calcium switch assays, to assess the roles for paxillin in breast tumor cell invasion. Paxillin had little effect on primary tumor initiation and growth but is critical for the formation of distant lung metastasis. In paxillin-depleted 3D tumor organoids, collective cell invasion was substantially perturbed. Two-dimensional cell culture revealed paxillin-dependent stabilization of adherens junctions (AJ). Mechanistically, paxillin is required for AJ assembly through facilitating E-cadherin endocytosis and recycling and HDAC6-mediated microtubule acetylation. Furthermore, Rho GTPase activity analysis and rescue experiments with a RhoA activator or Rac1 inhibitor suggest paxillin is potentially regulating the E-cadherin-dependent junction integrity and contractility through control of the balance of RhoA and Rac1 activities. Together, these data highlight new roles for paxillin in the regulation of cell-cell adhesion and collective tumor cell migration to promote the formation of distance organ metastases. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

TMOD-29. STANDARDIZED GENERATION OF TUMOR-ORGANOIDS AS NOVEL DRUG SCREENING MODEL IN MENINGIOMA

Tumor-organoids (TOs) are novel, complex three-dimensional ex vivo tissue cultures that under optimal conditions accurately reflect genotype and phenotype of the original tissue with preserved cellular heterogeneity and morphology. They may serve as a new and exciting model for studying cancer biology and directing personalized therapies. The aim of our study was to establish TOs from meningioma (MGM) and to test their usability for large-scale drug screenings. We were capable of forming several hundred TO equal in size by controlled reaggregation of freshly prepared single cell suspension of MGM tissue samples. In total, standardized TOs from 60 patients were formed, including eight grade II and three grade III MGMs. TOs reaggregated within 3 days resulting in a reducted diameter by 50%. Thereafter, TO size remained stable throughout a 14 days observation period. TOs consisted of largely viable cells, whereas dead cells were predominantly found outside of the organoid. H&E stainings confirmed the successful establishment of dense tissue-like structures. Next, we assessed the suitability and reliability of TOs for a robust large-scale drug testing by employing nine highly potent compounds, derived from a drug screening performed on several MGM cell lines. First, we tested if drug responses depend on TO size. Interestingly, drug responses to these drugs remained identical independent of their sizes. Based on a sufficient representation of low abundance cell types such as T-cells and macrophages an overall number of 25.000 cells/TO was selected for further experiments revealing FDA-approved HDAC inhibitors as highly effective drugs in most of the TOs with a mean z-AUC score of -1.33. Taken together, we developed a protocol to generate standardized TO from MGM containing low abundant cell types of the tumor microenvironment in a representative manner. Robust and reliable drug responses suggest patient-derived TOs as a novel drug testing model in meningioma research.

DDRE-42. TOWARDS PRECISION MEDICINE: AUTOMATED DRUG SCREENING PLATFORM UTILIZING TUMOR-ORGANOIDS TO IDENTIFY PATIENT-SPECIFIC DRUG-RESPONSES IN MENINGIOMA

Tumor-organoids (TO) are mini-tumors generated from tumor tissue preserving its genotype and phenotype by maintaining the cellular heterogeneity and important components of the tumor microenvironment. We recently developed a protocol to reliably establish TOs from meningioma (MGM) in large quantities. The use of TOs in combination with lab automation holds great promise for drug discovery and screening of comprehensive drug libraries. This might help to tailor patient-specific therapy in the future. The aim of our study was to establish an automated drug screening platform utilizing TOs. For this purpose, we established TOs by controlled reaggregation of freshly prepared single cell suspension of MGM tissue samples in the high-throughput format of 384-well plates. The drug screening was performed fully automated by utilizing the robotic liquid handler Hamilton Microlab STAR and a drug library containing 166 FDA-approved oncology agents. Viability was assessed with CellTiterGlo3D. In total, we performed the drug screening with 166 drugs on TOs from 11 patients suffering from MGM (n=8 WHO°I, n=2 WHO°II, n=1 WHO°III). The top five most effective drugs resulted in a decrease of TO viability ranging from 84.6–63.3%. K-means clustering analysis resulted in groupings of drugs with similar modes of action. One cluster consisted of epigenetic drugs while another cluster consisted of several proteasome inhibitors. However, when looking at a patient-individual level, in 11 patients 44 of 166 drugs, were among the top 10 most effective drugs, providing strong evidence for heterogeneous drug-responses in MGM patients. Taken together, we successfully developed an automated drug screening platform pipeline utilizing TOs from MGM to identify patient-specific drug-responses. The observed intra-individual differences of drug responses mandate for a personalized testing of comprehensive drug libraries in TOs to tailor more effective therapies in MGM patients.

164 Potent tumor organoid infiltration and killing by PBMC-derived effector cells

BackgroundAlloplex Biotherapeutics has developed a novel autologous cellular therapy for cancer that uses ENgineered Leukocyte ImmunoSTimulatory cell lines called ENLIST cells to activate and expand a heterogeneous population of tumor killing effector cells from human peripheral blood mononuclear cells (PBMCs). The 2-week manufacturing process from PBMCs consistently results 300-fold expansion of NK cells, CD8+ T cells, gamma/delta T cells, NKT cells and some CD4+ T cells, collectively called SUPLEXA therapeutic cells. SUPLEXA cells will be delivered back to cancer patients via intravenous administrations on a weekly schedule as an autologous adoptive cellular immunotherapy for cancer. In this study, we tested SUPLEXA cells developed from normal healthy volunteer PBMCs for their ability to infiltrate and kill patient-derived tumor organoids (PDO) as a pre-clinical assessment for potency against 2 different types of tumor organoids.MethodsTumor organoids derived from colorectal cancer (CRC) or non-small cell lung carcinoma (NSCLC) patients were labeled with cell-trace red dye and plated at equal density in a 96-well plate. After 3 days culture, SUPLEXA cells were thawed (82.8% viable), labeled with cell-trace violet dye, and added to PDO at 1:2 serial diluted numbers ranging from 2 million to 7,800 cells per well. Fluorescent images were captured at 24 hours after adding SUPLEXA cells to PDO models to measure PDO size, tumor infiltration, and PDO killing.ResultsAdding SUPLEXA cells to PDO from CRC and NSCLC resulted in significant infiltration and killing of organoids by 24 hours as shown by the fluorescent images and the organoid size plot for the CRC PDO model (figure 1). Significant reduction in PDO size was observed by adding 31,240 SUPLEXA cells. Similar results were observed with the NSCLC PDO model with significant reduction in PDO size by adding 15,600 SUPLEXA cells. Obvious organoid infiltration was observed in both PDO models and organoid fluorescence was significantly reduced by addition of SUPLEXA cells in both PDO models to suggest that SUPLEXA cells were able to reduce tumor burden (figure 2).Abstract 164 Figure 1CRC organoid infiltration and killing by SUPLEXA. A representative fluorescent image of CRC organoid killing with addition of increasing SUPLEXA cell numbers and a plot showing statistical analysis of 6 replicate wells for changes in CRC organoid size in relation to SUPLEXA cell number additionsAbstract 164 Figure 2Dose-dependent killing in CRC and NSCLC PDO models. CRC and NSCLC organoids were detected by total red fluorescence at 24 hours after adding the indicated numbers of SUPLEXA cells. Loss of red fluorescence after adding SUPLEXA is a measure of overall tumor cell killing/burden in organoids. Data is plotted as mean ± SEM for n=6 replicates per group.ConclusionsSUPLEXA cells infiltrated and killed tumor cells in patient-derived organoids within 24 hours of culture at low cell concentrations indicating potent tumor killing activity. The observed activity in both colorectal and lung cancer organoid models support broad anti-tumor killing activity by SUPLEXA. These results provide further evidence that PBMCs from cancer patients can be activated and expanded by our approach as a novel autologous cellular immunotherapy for cancer.

Empirical identification and validation of tumor-targeting T cell receptors from circulation using autologous pancreatic tumor organoids

BackgroundTumor-specific cytotoxic T cells and T cell receptors are effective tools for cancer immunotherapy. Most efforts to identify them rely on known antigens or lymphocytes that have infiltrated into the tumor bed. Approaches to empirically identify tumor-targeting T cells and T cell receptors by exploiting all antigens expressed on tumor cell surfaces are not well developed for most carcinomas, including pancreatic cancer.MethodsAutologous tumor organoids were stimulated with T cells from the patients’ peripheral blood for 2 weeks to generate the organoid-primed T (opT) cells. opT cell phenotype was analyzed by monitoring changes in the expression levels of 28 cell surface and checkpoint proteins. Expression of ligands of the immune checkpoints was investigated by immunohistochemistry staining. T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and assayed by flow cytometry to monitor tumor-induced T cell proliferation changes. opT cell-mediated killing of three-dimensional organoids was measured using an M30 ELISA kit. T cell receptors (TCRs) were identified by deep sequencing of gDNA isolated from T cells, and the TCR specificity was confirmed by transferring TCRs to the T cell line SKW-3 or donor T cells.ResultsThe co-culture was effective in the generation of CD8 + or CD4+opT cells. The opT cells killed autologous tumors in a granzyme B or Fas-Fas ligand-dependent manner and expressed markers of tissue-resident memory phenotype. Each patient-derived opT cell culture displayed a unique complement of checkpoint proteins. Interestingly, only NKG2A blockade showed a potent increase in the interferon-γ production compared with blocking programmed cell death protein 1 (PD-1) or programmed cell death ligand 1 (PD-L1) or TIM3 or TIGIT or LAG3. Importantly, TCR sequencing demonstrated a dramatic clonal expansion of T cells with a restricted subset of TCRs. Cloning and transferring the TCRs to heterologous T cells was sufficient to confer tumor cell recognition and cytotoxic properties in a patient-specific manner.ConclusionWe report a platform for expanding tumor-targeting T cells from the peripheral blood of patients with pancreatic cancer. We identify the NKG2A-HLA-E axis as a potentially important checkpoint for CD8 +T cells for pancreatic cancer. Lastly, we demonstrate empirical identification of tumor-targeting TCRs that can be used for TCR-therapeutics.