Abstract Immunotherapy has proven to be a powerful anti-tumor therapy, harnessing the body’s own immune system to target and kill tumor cells. However, immunotherapy is not successful in all cancer patients due to both intrinsic non-responsiveness and adaptive resistance. Developing predictive biomarkers and understanding mechanisms of resistance are major goals of the immuno-oncology community. The extracellular matrix (ECM), an important factor for promoting tumor growth, survival, and migration of tumor cells, can also act as a physical barrier to prevent immune cell infiltration and promote tumor immune escape. Components of the ECM such as COL11A1, COL4A1, and LOXL2 have been shown to be associated with cancer progression. Furthermore, new data suggests that TGFβ activation leads to up-regulation of ECM genes in cancer-associated fibroblasts and immune suppression. However, it remains poorly understood which cells in the tumor microenvironment (TME) are the sources of ECM gene expression and how they are related to tumor infiltrating cytotoxic T lymphocytes (CTLs). In this study, we employed a highly sensitive and specific RNAscope in situ hybridization (ISH) duplex assay to directly visualize the tissue distribution of cells expressing COL4A1, COL11A1, LOXL2, and TGFB1 in relation to tumor infiltrating CTLs in non-small cell lung carcinoma (NSCLC). NSCLC tissue microarrays (TMAs) consisting of 63 independent patient FFPE tumor samples were analyzed using this ISH assay with the following probe combinations: Hs-CD8/Hs-IFNG, Hs-CD4/Hs-FOXP3, Hs-LOXL2/Hs-COL4A1, and Hs-TGFB1/Hs-COL11A1. We observed COL4A1 expression in both tumor and tumor-associated stromal cells in different samples. In contrast, COL11A1 was only expressed in tumor-associated stromal cells. Interestingly, high COL4A1 expression was associated with high CD8+ T cell infiltration, whereas high COL11A1 expression was associated with poor CD8+ T cell infiltration. In addition, tumor expression of TGFB1 was positively correlated with COL11A1 expression. These data depict a complex landscape of ECM gene expression and their relationship to T cell infiltration in the tumor and TME. Taken together, these results demonstrate that the RNAscope assay provides a powerful approach to directly examine the interactions between tumor, ECM, and T cell immune infiltration, and offers advantages over immunohistochemistry (IHC) for identifying the cellular sources of secreted proteins such as ECM components in the TME. Citation Format: NA LI, Hongzhe Sun, Xin Wang, Zhifu Zhang, Ying Zhou, Courtney Anderson, Xiao-Jun Ma. Extracellular matrix gene expression and cytotoxic T lymphocyte infiltration in the tumor microenvironment in non-small cell lung cancer [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 4965.