e14178 Background: Colorectal Cancer (CRC) is the third most commonly diagnosed cancer in the world. Yet, CRC has been difficult to treat with immunotherapy, as majority (85%) of CRC are Microsatellite stable (MSS), lowly immunogenic and do not respond well. To investigate immuno-cytotoxicity in MSS cancers, we set up an ex vivo co-culture model using patient-derived tumor epithelial cells and autologous tumor-infiltrating lymphocytes (TILs). Aims: To perform phenotypic and functional characterisation of the model, and show we are able to modulate immuno-cytotoxicity. Methods: Fresh tumor, adjacent normal and/or liver metastasis samples were collected and freshly dissociated from 29 colorectal cancer patients undergoing surgery. TILs and tumor epithelial cells were grown in culture. Weekly immunophenotyping of TILs was performed via flow cytometry and data was analysed to identify temporal changes in proportions of cell types. To modulate cytotoxicity, epithelial cell lines were co-cultured with autologous and allogeneic TILs at various effector:target ratios with and without anti-PD1 (Pembrolizumab). Caspase dye was used to detect apoptosis and measure cell death. Results: TILs from most patients were successfully expanded, with a theoretical potential of up to 10,000 times. CD4+ T cells were selectively expanded, while CD8+ T cells decreased in proportion over time. A select group of patients showed an opposite trend. Cell type proportions in metastases mirrored those of the primary tumour while NK cells expanded more in tumour samples than normal samples. Selection proceeded up to 3 weeks then decreased. In the cytotoxicity experiment, we show that cell death is increased at higher effector:target ratios in the presence of autologous TILs. Addition of Pembrolizumab further modulates cytotoxicity in vitro. Conclusions: We have developed and characterised an autologous T cell and epithelial cell co-culture system to evaluate immuno-cytotoxicity in colorectal cancer. This will allow screening of perturbations for improved immuno-cytotoxicity in colorectal cancer.

Background: Immunotherapy has so far had limited success in colorectal cancer (CRC), with its efficacy restricted to a subset of microsatellite instability high (MSI-H) tumors. A comprehensive interrogation of the CRC tumor immune microenvironment (TME) is urgently needed. We present here an ongoing multi-platform study on early stage colon and rectal cancers, where immuno-genomic profiling of tumors and patient-derived cell models of tumor epithelia, cancer-associated fibroblasts and tumor-infiltrating lymphocytes (TIL) complement each other, with opportunities for mutual cross-validation between experimental and bioprofiling data. Methods: At the time of writing, 21 of a planned 50 early stage CRC patients have been recruited. Surgically resected tumors are processed for 4 broad classes of analyses: 1) Bulk tissue profiling by RNA and whole exome sequencing; 2) High resolution protein and transcriptome analysis comprising scRNA-seq and flow cytometry/CyToF; 3) H&E analysis and multiplex immunohistochemistry for TME-specific proteins; 4) Culture of epithelial, fibroblast, and TIL lines, and generation of patient-derived xenografts for functional studies. Results: Four tumors were MSI-H and 17 were microsatellite stable (MSS), with 1 POLE-mutant MSS patient harboring over 6000 mutations. Bulk genomics analysis revealed the most common mutations to be in TP53, APC, MUC17, and TGFBR2. The most frequently altered pathways were WNT, followed in order by p53, TGFβ, PI3K, and RAS-MAPK. scRNA-seq and flow cytometry/CyToF analyses are being performed to examine immune phenotypes, mediators of cell migration, and immune suppressive populations, which complement data on transcriptomic profile, histopathology, and spatial localization of TME cellular components. Of the 3 cultured cell types, 16 patients have lines of at least one cell compartment established in vitro. Characteristics of individual models will be reported at the meeting. Establishing the 3 cultured cell types from the same patient will enable us to develop an autologous patient-derived co-culture system to evaluate all 3 pairwise interactions, including TIL cytotoxicity toward epithelial cells, mutual modulation by fibroblast and epithelial cells in co-culture and their phenotypic alterations, and fibroblast regulation of TIL cytotoxic function. Autologous co-cultures with all 3 cell types are also planned. Conclusions: The generation of well-annotated multi-platform profiling data from CRC tumors, complemented by matched tri-compartment patient-derived cell cultures, enables mutual cross-validation between experimental models of the TME and bioprofiling data. Citation Format: Lindsay H. Kua, Fiona Y. Lee, Christine L. Eng, Harini Srinivasan, Rahul Nahar, Janice H. Oh, Nicole Ann L. Gunn, Kai Xian Thng, Ashley S. Yong, Adrian C. Sim, Rebecca Lim, Nicole Boo, Simeen Malik, Michael T. Wong, Tze Guan Tan, Shu Wen S. Ho, Shirleen Soh, Wan Jun Lim, Macalinao Dominique Camat, Joe P. Yeong, Clarinda W. Chua, Wei Qiang Leow, Ramanuj DasGupta, Si-Lin Koo, Lewis Hong, Brian Henry, Tony K. Lim, Iain B. Tan. Tri-compartment (epithelial, immune, fibroblast) patient-derived models of the tumor microenvironment from an immuno-genomic profiled cohort of early stage colorectal cancers [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 3990.