Abstract 1680: Patient-derived <i>ex vivo</i> TME-models and single-cell sequencing reveal transcriptional responses to immunotherapy
Authors: Anuja Sathe, Jiamin Chen, Susan M. Grimes, Carlos I. Ayala, George Poultsides, Hanlee P. Ji
Journal: Cancer Research (2021)

Abstract The tumor microenvironment (TME) contains diverse cell phenotypes. This heterogeneity influences the cancer immunity cycle and response to immunotherapy. We examined the effects of immunotherapy in a unique patient-derived ex vivo system that maintains the TME in its near-native state. We leveraged single-cell RNA sequencing (scRNA-seq) to conduct an unbiased analysis of transcriptional responses in heterogenous TME cell types. We established slice cultures using a vibratome from five surgical resections of colorectal or gastric cancer. Ex vivo slice cultures (‘TME-models') were treated with isotype control antibody, GITR agonist, TIGIT inhibitor or PMA/Ionomycin for 24 hours. TME-models and original surgical resection (‘T0') were dissociated into single-cell suspensions and subjected to scRNA-seq. Following quality control, we performed dimensionality reduction and differential expression analysis. We sequenced 219000 single cells detecting an average of 841 median genes per cell. All cell lineages identified in the T0 resection including tumor epithelium, CD4, CD8, Treg, NK, B, plasma, mast, dendritic cells, macrophages, fibroblasts and endothelial cells were maintained in the corresponding ex vivo TME-model. No consistent differences in proportions or transcriptional profiles were observed, indicating that original TME is preserved in the near-native state. PMA/ionomycin led to an activation phenotype in CD8, CD4 and regulatory T cells including a significant increase in expression of effector cytokines (GZMB, IFNG, PRF1, CCL3, etc.) , activation markers (IL2RA, CD69), immune checkpoints (PDCD1, TIGIT, LAG3, etc.) and co-stimulatory molecules (TNFRSF18, TNFRSF9, etc.). PMA/Ionomycin also led to upregulation of chemokines (CXCL9, CXCL5, etc.) and interferon response genes (ISG20, IRF1, etc.) in fibroblasts and tumor epithelial cells potentially indicative of indirect T-cell mediated effects. GITR agonist and TIGIT inhibitor led to an increase in cytotoxic genes including IFNG in effector CD8 T cells but not in naïve cells. Moreover, the extent of transcriptional response varied across tumors. Experiments on additional tumors are ongoing. Ex vivo TME-models derived from surgical resections maintain all TME components in their near native state. In combination with scRNA-seq, this system can be utilized to test targets in the TME and provide insights into their mechanisms of action and resistance. Using this approach, we successfully identified heterogenous patient responses to GITR stimulation and TIGIT inhibition in gastrointestinal cancers. Citation Format: Anuja Sathe, Jiamin Chen, Susan M. Grimes, Carlos I. Ayala, George Poultsides, Hanlee P. Ji. Patient-derived ex vivo TME-models and single-cell sequencing reveal transcriptional responses to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1680.