Fig 2: Analysis of CD44+/CD22- stem cell population in theviable percentage of DU145 and PC3 cells.A, B. Mean fluorescence intensity of CD44+ and CD24+ cell populations in DU145 and PC3 comparing total and viable populations using Annexin V/PI assay. The values repres ent the mean ± SD (n = 6). *** p < 0.001, **** p ≤ 0.0001, significantly different from total vs viable. ## p ≤ 0.001, # p ≤ 0.0001, significantly different from the total population control. %% p ≤ 0.001, % p ≤ 0.0001, significantly different from the viable supopulation control.

Fig 3: Signals from the TME promote cellular plasticity. (a) Transformed HMEC consists of both epithelial/non-CSC (CD24+/CD44−) and mesenchymal/CSC (CD24−/CD44+) populations, that can be (b) separated by differential trypsinization or FACS. (c) Exogenous cytokine exposure drives cell-state changes of transformed HMEC (left) to either CD24−/CD44+ CSC (right, top) or CD24+/CD44− non-CSC (right, bottom). (d) Cytokine screen using a mixed population of transformed HMEC (as shown in a) that were exposed for 2 weeks to the indicated cytokines at the concentrations described in Table 1. Cytokine-treated populations were analyzed for CD24/CD44 using flow cytometry. Percent change in CSC was calculated as the difference in CSC population (identified as CD24−/CD44+, as shown in b) following exposure to each cytokine was calculated relative to untreated control.

Fig 4: LAMC2 expression correlates with stemness and chemoresistance. a qPCR analysis for LAMC2 gene expression in adherent cells versus spheres. Data are normalized to GAPDH and are presented as fold change in gene expression relative to adherent cells. b Western blot analysis for LAMC2 in adherent cells versus spheres. Parallel β-ACTIN immunoblotting was performed. c qPCR analysis for CD44 and LAMC2 gene expression in CD44+ sorted cells. Data are normalized to GAPDH and are presented as fold change in gene expression relative to CD44− cells. d qPCR analysis for CD133 and LAMC2 gene expression in CD133+ sorted cells. Data are normalized to GAPDH and are presented as fold change in gene expression relative to CD133− cells. e Western blot analysis for LAMC2 in sh empty and LAMC2 knockdown cells. Parallel β-ACTIN immunoblotting was performed. f qPCR analysis for LAMC2 and CD44 expression for sh empty versus LAMC2 knockdown cells. Data are normalized to GAPDH expression and are presented as fold change in gene expression relative to sh empty. g Flow cytometry quantification of CD44 in sh empty and LAMC2 knockdown cells. h Representative images of sh empty and LAMC2 knockdown cells grown as spheres. i Sphere formation capacity of sh empty and LAMC2 knockdown cells. P1 = 1st generation; P2 = 2nd generation. j Organoid formation capacity for sh empty versus LAMC2 knockdown cells. k Growth capacity of sh empty and LAMC2 knockdown cells in the presence of 100 μM Gemcitabine (GEM). l qPCR analysis for LAMC2, CNT1, CNT2 and CNT3 genes in sh empty versus LAMC2 knockdown cells. Data are normalized to GAPDH expression and are presented as fold change in gene expression relative to sh empty. *p < 0.05, **p < 0.005, ***p < 0.0005. n \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\ge 3.$$\end{document}≥3. Statistical significance was assessed by Student's t-test

Fig 5: Maintenance of EMT and CSC properties requires sustained exposure to OSM. (a) AIG of CD24High/CD44− or CD24Medium/CD44− epithelial/non-CSC in soft agar after exposure to OSM for 2 weeks. At the time of plating, OSM was removed from cells that were previously exposed (removed). (b) CD24+/CD44− epithelial/non-CSC exposed to OSM and that grew anchorage independently were recovered from soft agar, grown in two-dimensional (2D) culture with or without continuous OSM exposure for another 2 or 4 weeks, and then analyzed by flow cytometry. % CSC depicted by indicated numbers. (c) CD24+/CD44− epithelial/non-CSCs were continuously exposed to OSM for 3 weeks, and then exposed to OSM or had OSM removed continuously for another 3 weeks (6 weeks total) and were assessed by flow cytometry. (d, e) CD24+/CD44− epithelial/non-CSCs were continuously exposed to OSM for 6 weeks, and then had OSM removed for 4 weeks and were analyzed by (d) brightfield microscopy (× 40), or (e) confocal microscopy (× 100) for e-cadherin (red), vimentin (green) and nuclei (blue).