NanoString nCounter GX Cancer Reference Kit and the custom made ITESM (14 genes associated with prostate cancer)

Cancer stem cells (CSC) drive prostate cancer tumor survival and metastasis. Nevertheless, the development of specific therapies against CSCs is hindered by the scarcity of these cells in prostate tissues. Suspension culture systems have been reported to enrich CSCs in primary cultures and cell lines. However, the molecular mechanisms underlying this phenomenon have not been fully explored. We describe a prostasphere assay for the enrichment of CD133+ CSCs in four commercial PCa cell lines: 22Rv1, DU145, LNCaP, and PC3. Overexpression of CD133, as determined by flow cytometric analysis, correlated with an increased clonogenic, chemoresistant, and invasive potential in vitro. This phenotype is concordant to that of CSCs in vivo. Gene expression profiling was then carried out using the Cancer Reference kit and the custom made ITESM CodeSets with the nCounter system from NanoString Technologies. This analysis revealed several upregulated transcripts that can be further explored as potential diagnostic markers or therapeutic targets. Furthermore, functional annotation analysis suggests that ΔNp63α modulates the activation of developmental pathways responsible for the increased stem identity of cells growing in suspension cultures. We conclude that profiling the genetic mechanisms involved in CSC enrichment will help us to better understand the molecular pathways that underlie CSC pathophysiology. This platform can be readily adapted to enrich and assay actual patient samples, in order to design patient-specific therapies that are aimed particularly against CSCs. PC3 prostasphere cultures were sampled on day 0 (Parental monolayers) (EPI_1), day 4 (EPI_2), day 8 (EPI_3), and day 12 (EPI_4) of culture. Day 12 prostaspheres were also enzymatically dissociated and sorted via MACS using the Anti-CD133/2 (293C3)-PE Antibody (Miltenyi) into CD133+ (EPI_P) and CD133- (EPI_N) fractions. Total RNA was extracted from these fractions and analyzed using the nCounter GX Cancer Reference kit and the custom made ITESM CodeSet from NanoString Technologies. Three independent experiments were carried out in parallel.