Harvard University



Andrew Portell - AACR 2019 Abstract Submission Authors: Portell A., Greene, J., Taus, L., Lizotte, P., Ivanova, E., Menezes, K., Aref, A., Kirschmeier, P., Barbie, D., Paweletz, C.P.Title: Ex vivo single cell RNA-sequencing of tumor derived organotypic spheroids identifies a unique mesenchymal resistance program to PD-1 blockade Introduction: Immune checkpoint blockade, including ?PD-1 and ?PD-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 tumor spheroids from MC38s were grown and treated with ?PD-1 and isotype matched IgG in a microfluidic device as previously described1. At day 6, libraries were prepared from viable cells using the 10X Genomics platform and sequenced at DFCI. ?Sequence data were 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 ?PD-1 treated cells fell into 2 unique clusters with downregulated genes associated with apoptosis and interferon-γ response, such as 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 higher 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. ................
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