Translating transcriptome of cancer cells in situ in mesenchymal-rich tumor microenvironment

A mesenchymal rich stroma such as cancer-associated fibroblasts (CAFs) in breast tumors favors the selection of cancer clones with enhanced bone metastatic ability. To determine the cancer cell transcriptomic response to the mesenchymal stroma, we supplemented experimental mammary tumours with or without exogenous mesenchymal cells. We used bone marrow-derived human mesenchymal stem cells (MSCs) as a source of mesenchymal stroma, as MSCs have been shown to undergo CAF-like differentiation. We engineered the cancer cells to express an EGFP-tagged version of ribosomal protein L10a (EGFP-L10a). This allows the retrieval of cancer cell specific transcripts rapidly from whole tumor lysates by translating ribosome affinity purification (TRAP) and direct profiling of cancer cell gene expression patterns when they are in situ. EGFP-10a+ MDA-MB-231 cells were orthotopically injected into the mammary fat pad with or without 1:1 ratio of MSCs. The mammary tumors were retrieved for TRAP-RNAseq profiling after 3 weeks.