Supplementary Materials1. xenograft and patient tumors. qPCR, Western blot, lentiviral-mediated gene knockdown, and human microRNA arrays were performed for mechanistic studies. Results By focusing on LAPC9 model, we show that this TM+ cells are CSCs with both tumor-initiating and tumor-propagating abilities for CRPC. Moreover, primary patient samples have TM+ cells, which possess CSC activities in castrated culture conditions. Mechanistically, we find that 1) the phenotypic markers are causally involved in CRPC development; 2) the TM+ cells preferentially express castration resistance and stem cell-associated molecules that regulate their CSC characteristics; and 3) the TM+ cells possess distinct microRNA expression profiles and miR-499-5p functions as an oncomir. Conclusions Our results define the TM+ PCa cells as a populace of pre-existent stem-like cancer cells that can both mediate and propagate CRPC and spotlight the TM+ cell populace as a therapeutic target. value 0.05 considered statistically significant. See also Supplementary Materials & Methods Results The TM+ (ALDHhi CD44+ 21+) PCa cell populace is usually enriched in experimental CRPC models In our earlier buy Tideglusib cDNA microarray analysis, we compared gene expression profiles between PSA?/lo versus PSA+ LAPC9 PCa cells and found that PSA?/lo PCa cells overexpressed several dozens of stem cell (SC) related genes, including and (16). ALDHhiCD44+21+ or TM+ LAPC9 cells regenerated much larger tumors when implanted in castrated mice than the corresponding ALDHloCD44?21? or TM? cells (16), suggesting that TM+ PCa cells may play an important role in CRPC development. To directly test this suggestion, buy Tideglusib we established serially passaged androgen-independent (AI, i.e., castration-resistant) xenograft models, including LAPC9, LAPC4, LNCaP and HPCa101 (25) from their respective androgen-dependent (AD) parental tumors (Fig. 1A). As illustrated in Fig. 1B, both LAPC9 and LAPC4 buy Tideglusib buy Tideglusib AI tumors showed a prominent upregulation of N-Cadherin, a molecule known to be involved in CRPC (24). In contrast, E-Cadherin levels did not significantly change in AI tumors in comparison to AD tumors (Fig. 1B). Interestingly, the AI LAPC4 tumors showed increased AR protein whereas the LAPC9 AI tumors gradually lost AR, similar to earlier reports by others (24, 26). However, both AI tumor models showed decreased amounts of PSA (Fig. 1B), consistent with our earlier observations that castration resistance is associated with decreasing tumor cell PSA levels and increasing PSA?/lo PCSCs (16, 19). Together, these results indicate that we have successfully established experimental CRPC models. Open in a separate windows Physique 1 TM+ cells in AD and AI PCa modelsA. Strategies in establishing AD and AI PCa lines. B. Western blot analysis of the molecules indicated in AD and AI LAPC9 and LAPC4 tumors. Du145 and LNCaP cells were used as controls. C. qRT-PCR analysis of mRNA levels for in Foxd1 LAPC9 AD and AI tumors. The relative transcript abundance was normalized to levels. Error bars represent the mean S.D. *in sorted TM+ and isogenic TM? cells purified from 6 and 15 LAPC9 AI tumors. Bars represent the mean S.D. *and (11) mRNAs than AD tumors (Fig. 1C). A pattern of increased mRNA in LAPC9 AI tumors was also observed (Fig. 1C). Importantly, the and mRNA levels in TM+ LAPC9 cells purified from serially passaged AI tumors were significantly higher than in the corresponding TM? cells (Fig. 1D). FACS analysis, using the gating strategies we developed, showed that this percentage of TM+ cells dramatically increased in serially passaged LAPC9 AI tumors (Supplementary Fig. S1). IHC and IF staining in formalin fixed paraffin embedded (FFPE) samples confirmed that CD44+ cells were highly enriched in LAPC9 AI tumors compared to AD tumor (Fig. 1E-F and Supplementary Fig. S2A). Recent studies have linked aldehyde dehydrogenase (ALDH) activity in PCSCs to PCa development (10, 11, 19, 27, 28) that might be conferred by several isoforms including ALDH1A1 (10, 27, 28) and ALDH7A1 (11). However, whether these ALDH isoforms are expressed in CRPC samples is usually unclear. We found that buy Tideglusib the cells with high ALDH activity were increased in LAPC9 AI tumors (Supplementary Fig. S1, panels e), and the abundance of ALDH1A1+ and ALDH7A1+ PCa cells was also higher in AI vs. AD tumors (Supplementary Fig. S2B-C). In addition, integrin 2+ cells were increased in both LAPC9 (Fig. 1G-H) and LAPC4 (Supplementary Fig. S2D) AI tumor models. We employed FACS analysis to further investigate TM+ cells in several different.