GSE131271
Cancer-cell transcriptional signatures associated with Socs1 knockdown in an immunocompetent and orthotopic mouse model of non-small lung cancer
How cancer cells respond to signals coming from the tumor microenvironment cannot be fully elucidated in vitro. Additionally, RNA-Sequencing data from bulk human tumors does not assess the contribution of cancer cells alone, but from a complex mixture of both host and cancer cells. Using an immunocompetent and orthotopic mouse model of human Non-small Cell Lung Cancer, we sought to determine cancer cell specific transcriptomes from bulk tumors and compared these to cancer cells grown in vitro. We hypothesized that knockdown of Socs1, or suppressor of cytokine signaling 1 in Lewis Lung Carcinoma (LLC) cells would not only impact their response to single-agent immunotherapy, but also lead to complex changes in the tumor microenvironment. First, we harvested RNA from either LLC-NT (LLC cells transduced with a non-targeting control vector) or LLC-sh21 (LLC cells transduced with a Socs1 knockdown vector) cell lines grown in vitro. To determine the transcriptome of these cells grown in vivo, we injected LLC-NT or LLC-sh21 cells into the left lung of transgenic GFP-expressing C57BL/6J mice. After three weeks, tumor-bearing lung lobes were isolated from mice and were made into single cell suspensions. The GFP-negative cancer cell population was sorted from GFP-positive host cells by fluorescence activated cell sorting. RNA was extracted from freshly isolated cancer cells grown in vitro or from cancer cells isolated from tumors. Our RNA-seq analysis shows that LLC-sh21 cells respond to IFNg coming from the microenvironment relative to LLC-NT cells which have a blunted response due to high basal expression of SOCS1. These data suggest that specific genes in the IFNg response pathway are important for response to immunotherapy. Overall design: The following conditions were analyzed: (1) LLC-NT In Vitro. RNA was extracted from LLC-NT cells cultured in vitro. This condition has 3 independently cultured and harvested replicates.(2) LLC-NT In Vivo. RNA was extracted from LLC cells that were injected into transgenic GFP-expressing C57BL/6J mice, formed a primary tumor and after three weeks, the GFP-negative cancer cell population was isolated by fluorescence activated cell sorting. This condition has 3 independently injected and harvested replicates. Each replicate is a pool of 5 tumor-bearing lung lobes. (3) LLC-sh21 In Vitro. RNA was extracted from LLC-sh21 cells cultured in vitro. This condition has 3 independently cultured and harvested replicates.(4) LLC-sh21 In Vivo. RNA was extracted from LLC-sh21 cells that were injected into transgenic GFP-expressing C57BL/6J mice, formed a primary tumor and after three weeks, the GFP-negative cancer cell population was isolated by fluorescence activated cell sorting. This condition has 3 independently injected and harvested replicates. Each replicate is a pool of 5 tumor-bearing lung lobes.