Abstract Introduction: Immune checkpoint blockade, including αPD-1 and αPD-L1 form the backbone of personalized medicine for lung cancer and other malignancies. Yet the underlying mechanisms of resistance to therapy are not fully characterized partly because functional models to perform mechanistic studies are lacking. Here we report on single cell RNA sequencing from murine (or patient) derived organotypic tumor spheroids (DOTS-seq) that enables analysis of tumor and immune cell intrinsic changes that occur during αPD-1 treatment ex vivo. Methods: Murine-derived organotypic spheroids from syngeneic MC38 tumors were grown and treated with αPD-1 and isotype matched IgG in a microfluidic device as previously described1. At day 6, libraries were prepared from individual viable cells using the 10X Genomics platform and sequenced at DFCI. Sequencing data was processed using the Seurat package and corrected for UMI, ribosomal, mitochondrial and cell cycle transcripts. Dimensionality reduction, clustering, and identification of differentially expressed genes were performed on log normalized data. Gene set enrichment analysis was performed using the SetRank package. Results: Transcripts were obtained for 2,543 IgG treated and 2,626 αPD-1 treated cells that were resistant to ex vivo killing. 60% of αPD-1 treated cells fell into 2 unique clusters which each had downregulated genes associated with apoptosis and interferon-γ response, such as Dap, Cxcl10 and B2m. Within these two, one cluster contained highly upregulated genes known to be E2F targets or important for G2M transition while the other did not. Interestingly, the quiescent subpopulation exhibited a unique epithelial to mesenchymal transition-like state characterized by expression of Snai1, Mmp2, Mmp14, and Vegfa. This subpopulation also upregulated transcripts of immuno-modulatory cytokines from the IL6 family, including Il11, Lif, and Osm. IL-6 extracellular levels are also elevated in treated cultures, suggesting a mesenchymal-like cell subpopulation responsible for this cytokine modulation of the tumor microenvironment. Conclusion: Here we show that profiling the interplay between tumor and immune cells at the single cell level is possible ex vivo. We identify a previously uncharacterized subpopulation of EMT-like cells that regulate the tumor microenvironment and promote resistance to αPD-1 in MC38 tumors. Reference: 1. Jenkins RW, et al. Cancer Discov DOI:10.1158/2159-8290 Citation Format: Andrew J. Portell, Jonathan Greene, Luke J. Taus, Patrick Lizotte, Elena Ivanova, Kalil O. Menezes, Amir R. Aref, Paul Kirschmeier, Russell W. Jenkins, David Barbie, Cloud P. Paweletz. Ex vivo single cell RNA-sequencing of tumor derived organotypic spheroids identifies a unique mesenchymal resistance program to PD-1 blockade [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 1483.