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Learn MoreCancer metastasis is the most deadly stage in cancer progression. Despite significant efforts over the last few decades, it remains elusive why only a very small fraction of tumorigenic cancer cells is able to generate metastatic colonization. Recently, we have shown that mechanically selected tumor-repopulating cells (TRCs), a highly tumorigenic subpopulation of mouse melanoma cells, can grow in 3D soft fibrin gels. However, the novel cell surface biomarkers and gene regulatory features of TRCs remain largely unknown. Here, we utilized RNA-sequencing method that uses next-generation sequencing (NGS) techniques for whole transcriptome profiling to elucidate novel cell surface biomarkers and active gene regulatory features. RNA-sequencing data was utilized in DAVID for identification of differentially expressed clusters of genes, including cell adhesion cluster. This allowed us to identify novel cell surface biomarkers such as Col2a1, NCAM1, F11R, and Negr1. We have validated the expression of these genes by real-time qPCR. Among the biomarkers, expression level of Col2a1 was found to be low in TRCs but 20 fold higher compared to parental B16F1 cells, thus making the marker very specific for TRCs. Our immunofluorescence microcopy study also shows high expression of Col2a1 in TRCs compared with parental B16F1 cells. KEGG pathway analysis showed JAK/STAT pathway, hypoxia, and Akt3 pathway to be highly active in TRCs. In addition, aerobic glycolysis pathway was found to be very active, indicating a typical Warburg effect of highly tumorigenic cells. Together, our study revealed novel and specific biomarkers of melanoma TRCs and their cryptic cell signaling pathways. Future studies will uncover if perturbation of these pathways can successfully neutralize TRCs. SOURCE: Michael,C.,Saul (michael.saul@jax.org) - Chesler Lab The Jackson Laboratory
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