Fig 2: Pharmacological inhibition of TGF-ß signaling blocks LAMC2-induced metastasis. a Volume of tumors formed following subcutaneously injection of EGFP+ and EGFP- cells in nude athymic mice. n = 10. b Representative H&E-stained sections of xenografts derived from EGFP+ or EGFP- cells. c qPCR analysis for LAMC2, EMT, MMP2 and MMP10 gene expression in EGFP+ or EGFP- cells, isolated from tumors. Data are normalized to GAPDH and are presented as fold change relative to EGFP-. d qPCR analysis or CD44 and CD133 expression in EGFP+ or EGFP- cells isolated from tumors. Data are normalized to GAPDH and are presented as fold change in gene expression relative to EGFP-. e Number of tumors generated by subcutaneous injection of EGFP+ or EGFP- cells. f Enrichment plot for EGFP+ versus EGFP- cells isolated by FACS from subcutaneous tumors. g Representative immunofluorescence images for pSMAD2 (violet), LAMC2 (green) and nuclei (blue, DAPI) in tumor sections derived from EGFP- or EGFP+ cells subcutaneously xenografted in nude athymic mice. h qPCR analysis for LAMC2 expression in PDAC cells treated with 10 ng/ml of rTGF-ß1 in the presence or absence of 80 µM Vactosertib. Data are normalized to GAPDH and are presented as fold change in gene expression relative to control. i Representative H&E-stained sections of lungs following tail vein injection of EGFP+ or EGFP- tumor cells. Mice were treated with Vactosertib (40 mg/kg mice) or vehicle. *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}$$\ge 5.$$\end{document}=5. Statistical significance was assessed by Student's t-test

Fig 3: DOX-resistant breast cancer cells spread resistance capacity by transferring resistance-associated proteins, especially CD44, to sensitive cells. (A) The exosomes extracted from MCF-7/ADR cells with three biological replications were labeled as A1, A2, and A3. The exosomes extracted from MCF-7 cells with three biological replications were labeled as M1, M2, and M3. A/M stands for the log2 (fold change) results, which was the average protein signal ratio of the A and M groups. Then we performed the heat map analysis based on the log2 (fold change) results. In the heat map analysis, blue indicates the downregulated proteins, and red indicates the upregulated. (B) We further verified these differently expressed proteins by flow cytometry analysis, which was also consistent with the proteomic analysis. (C) The immunogold staining showed that more immunogold particles were bound to the exosomes, thus? indicating that CD44 was highly stored inside A/Exo. (D,E) Western blot results indicated that the protein level of CD44 was significantly upregulated in A/Exo compared with that in S/Exo (p < 0.05), and the increase level of CD44 in A/Exo (14.76-fold) was much higher than that in MCF-7/ADR cells (6.56-fold). *p < 0.05.

Fig 4: 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 5: OSM-induced EMT and acquisition of CSC properties requires SMAD3 transcriptional activity in the nucleus. (a) CD24+/CD44- cells were exposed to OSM alone or in combination with RUX (10 µM) or SB431542 (SB; 10 µM) for 2 h, and then protein levels and gene expression were assessed by western blot analysis (top) and qRT-PCR analysis (bottom), respectively. (b) CD24+/CD44- cells were treated with SB431542 (SB) or TGF-ß for 2 h and subjected to nuclear protein fractionation, and then western blot analysis of nuclear lysates for P-SMAD3 and Lamin B2 (as a loading control) was performed. (c) CD24+/CD44- epithelial/non-CSC expressing shGFP, shSMAD3, shSMAD4, or DNSTAT3 were exposed to OSM for 2 h, and then western blot analysis (top) assessed P-STAT3, STAT3, and Actin (as a loading control) protein levels; and qRT-PCR analysis (bottom) assessed SNAI1 and SOCS3 gene expression. (d) Chromatin immunoprecipitation (ChIP) and qRT-PCR analysis for SMAD3 enrichment on the SNAI1 and HAS2 gene promoters in CD24+/CD44- epithelial/non-CSC exposed to SB431542 for 24 h, and then treated with OSM alone, SB431542 alone or in combination for 1 h. (e) CD24+/CD44- epithelial/non-CSCs were exposed to OSM or TGF-ß for 30 min, and then nuclear localization of SMAD3 (left) and STAT3 (right) was assessed by confocal microscopy (left panel) and quantified (right panel). Red triangles: cells with elevated nuclear localization of both SMAD3 and STAT3. Blue triangles: cells with low levels of SMAD3 and STAT3 nuclear localization (n=53 for the untreated and TGFß-treated cells and n=74 for OSM-treated cells). (f, g) HPAC cells expressing SMAD7 or empty control Vector were treated with OSM for 7 days, and then (f) CD24/44 surface expression was determined via flow cytometry; in addition (g) protein levels of phosphorylated-STAT3 Y705 (P-STAT3), Snail and Actin (as a loading control) were determined by western blot analysis. (h) HPAC cells expressing shRNA targeting SMAD2 (S2), SMAD3 (S3) or GFP as a control were exposed to OSM for 7 days. Single-gene qRT-PCR analysis (top) assessed SNAI1 gene expression, and western blot analysis (bottom) confirmed SMAD2 and SMAD3 knockdown with Actin used as a loading control.