Differential gene expression in murine lung cancer in vitro vs. in vivo

The tumor microenvironment (TME) is incredibly important to the growth of tumors. To better understand the effect that the TME has on cancer cells, we assessed genes that were differentially expressed in murine lung tumors versus cells that were cultured in an incubator. We employed our orthotopic mouse model where murine lung cancer cells were implanted into the left lung of immunocompetent, syngeneic GFP-expressing mice of the C57BL/6 strain. After 3-4 weeks, tumors were harvested and a single cell suspension was prepared from the tumors. Using fluorescent-activated cell sorting, live (DAPI-negative), GFP-negative cells were isolated from the single cell suspensions. These GFP-negative cells constitute a pure population of tumor cells, excluding cells from the TME. RNA was isolated from these tumor cells, and submitted to RNA-seq. These are our “in vivo” samples. Concurrently, the cells from the same batch of cells that were initially injected into mice were cultured in vitro, and RNA was isolated from them and submitted to RNA-seq. These samples are our “in vitro” samples. Our study specifically utilizes 2 newly developed EML4-ALK fusion-driven murine lung cancer cell lines (EA1 and EA2 cells). Overall design: The following conditions were analyzed: (1) EA1 in vitro. RNA was extracted from EA1 cells cultured in vitro. This condition has 5 independent replicates; (2) EA1 in vivo. EA1 cells were injected into the left lung of GFP-expressing mice C57BL/6 mice. Tumors grew for 3-4 weeks, were isolated, and prepared into a single cell suspension. Tumor cells were isolated by flow sorting for GFP-negative cells, and RNA was isolated. This condition has 5 independent replicates. Each replicate is a pool of tumors from 3-5 mice; (3) EA2 in vitro. RNA was extracted from EA2 cells cultured in vitro. This condition has 3 independent replicates; and (4) EA2 in vivo. EA2 cells were injected into the left lung of GFP-expressing mice C57BL/6 mice. Tumors grew for 3-4 weeks, were isolated, and prepared into a single cell suspension. Tumor cells were isolated by flow sorting for GFP-negative cells, and RNA was isolated. This condition has 5 independent replicates. Each replicate is a pool of tumors from 3-5 mice.