Fig 2: BMP7 is confined to differentiated CRC cells. a Immunofluorescence analysis of BMP7 (green color) and LGR5 (red color) on peritumoral mucosa and colon cancer paraffin-embedded tissues performed on CSC#8. One representative tumor from twenty different tumors examined is shown. Nuclei were counterstained by Toto-3 (blue color). White arrowheads indicate LGR5+ cells at the base of colon crypt. The scale bar represents 100 µm. b Immunohistochemical analysis of BMP7 on CRC TMAs in lack, low, medium, and high staining intensity (red color). Nuclei were counterstained by aqueous hematoxylin (blue color). The scale bar represents 100 µm. c Association of BMP7 expression with score medium/high and the pathological grading in CRC TMAs provided by TRISTAR technology group. d Immunohistochemical analysis of BMP7 (red color) in paraffin-embedded sections of colon adenomas and adenocarcinoma (COAD). Nuclei were counterstained by aqueous hematoxylin (blue color). The scale bar represents 100 µm. e Immunofluorescence analysis of BMP7 (green color) in CRC sphere cells and their differentiated progeny SDACs. One representative of fifteen different CR-CSC lines (CSC#1–3, 5–7, 10,11, 14–16, 18, 25, 33, and 40) is shown. Nuclei were counterstained by Toto-3 (blue color). The scale bars represent 20 µm. f Representative flow cytometry analysis of CD133 in CRC sphere cells and its relative isotype-matched control (IMC) (upper panels) performed on CSC#4, 8, and 23–26. Immunofluorescence analysis of BMP7 (green color) in CD133+ and CD133- enriched CRC sphere cell subpopulations (lower panels). Nuclei were counterstained by Toto-3 (blue color). The scale bars represent 20 µm. g CD44v6 expression profiles of cells as described in f (upper panels). Expression of BMP7 (green color) in CD44v6+ and CD44v6- enriched CRC sphere cell subpopulations assessed by immunofluorescence analysis (lower panels). Nuclei were counterstained by Toto-3 (blue color). The scale bars represent 20 µm. h Flow cytometry analysis of BMP7 (green histograms) in enriched CD44v6-/CD133-, CD44v6-/CD133+, and CD44v6+/CD133+ CRC subpopulations performed as shown in f. Dotted line histograms indicate the relative IMC

Fig 3: BMP7v treatment promotes CR-CSC differentiation. a Phase-contrast microscopy analysis of CD44v6+ CRC sphere cells treated with BMP7v at the indicated time points. One representative of CSC#1, 2, 4, 5, 7, and 23–26 is shown. The scale bar represents 20 µm. b Percentage of CK20 positive cells in CD44v6+ CR-CSCs treated with vehicle or BMP7v up to 21 days evaluated by immunofluorescence analysis. Data are expressed as mean ± SD of experiments performed in 15 CRC sphere cell lines (CSC#1–3, 5–7, 10,11, 14–16, 18, 25, 33, and 40). c Flow cytometry analysis of CD133/CD44v6 on CRC sphere cells treated with vehicle or BMP7v for 14 days. Data reported are mean ± SD of 15 CRC sphere cell lines analyzed (CSC#1–8, 10,11, 14–16, 18, and 25). d (left panels) Immunofluorescence analysis of CDX2 on CR-CSCs upon 14 days of BMP7v treatment. One representative of CSC# 3, 9, and 21 is shown. Nuclei were stained with Toto-3 (blue color). The scale bars represent 20 µm. (right panel) Percentage of CDX2 positive cells in CD44v6+ CR-CSCs treated with vehicle or BMP7v up to 14 days evaluated by immunofluorescence analysis. Data are expressed as mean ± SD of experiments performed in CSC# 3, 9, and 21. e Flow cytometry analysis of TOP-dGFP or CD44v6 in enriched CD44v6+ sphere cells treated with BMP7v up to 14 days. One representative experiment of CSC#1, 2, 4, 7, and 10 is shown. f Phase-contrast microscopy analysis of TOP-dGFP CRC sphere cells grown in matrigel drops and treated with vehicle, BMP7v or FBS for 14 days. One representative of CSC# 8, 9, and 11 is shown. The scale bar represents 100 µm. g Immunofluorescence analysis of E-cadherin, vimentin, and ß-catenin (green color) in CD44v6+ CRC cells exposed to vehicle or BMP7v for 14 days. One representative experiment performed in cells as in e is shown. Nuclei were stained with Toto-3 (blue color). The scale bars represent 20 µm. h Migrating CD44v6+ and CD44v6- cells treated with vehicle or BMP7v up to 48 h. Data are shown as mean ± SD of three independent experiments performed in five CRC sphere cell lines (CSC#1, 5, 7, 10, and 12). i Cell viability percentage of enriched CD44v6+ and CD44v6- cells treated with vehicle or BMP7v up to 96 h. Data are shown as mean ± SD of different experiments performed in CSC#1, 2, 4, 7, and 10. j Cell cycle analysis in CD44v6+ CR-CSCs exposed to vehicle or BMP7v for 72 h. The data show percentage of cell number in sub-G0, G0/G1, S, and G2/M phases. Data are expressed as mean ± SD of three independent experiments performed in five different CRC sphere cell lines as in e. k Immunoblot analysis of PARP, cleaved PARP (cPARP), Caspase-3 (Casp-3), cleaved Caspase-3 (cCasp-3), Bcl-2, Bcl-xL in CD44v6+, and CD44v6- enriched cells treated as in e for 72 h. ß-actin was used as loading control. One representative experiment performed in three different CRC sphere cell lines (CSC#1, 4, and 7)

Fig 4: ADAM10 and ADAM17 are expressed in patient-derived GSCs and anti-ADAM17 inhibits protease function. a Representative confocal images of metalloproteinases ADAM10 (A10, green) and ADAM17 (A17, green) and stem cell markers SOX2 (red), CD133 (red) and nestin (Nes; red) staining of GSCs cultured as monolayers from four cell lines (G002, G049, G100 and G112). Nuclei stained with DAPI (blue), scale bar = 20 µm. N.B. G112 ADAM17 and nestin staining are from different wells, hence the absence of a merge picture. b The protease activity of purified rhADAM17 (left) or G002 cell lysates (right) pre-treated with either IgG isotype control (10 µg/ml), anti-ADAM17 (10 µg/ml) or TAPI-2 (10 µg/ml). Two separate G002 cell lysates were collected at passages 3 and 5, respectively, and tested in triplicate; the purified rhADAM17 was tested once in triplicate. **P < 0.01, ***P < 0.001 compared to IgG control using one-way ANOVA followed by Tukey’s posthoc test. c Tumourspheres from G112 after 11 days in vitro, scale bar = 100 µm

Fig 5: Evaluation of CD133/2 and CD24 expression in kidney progenitor cells by flow cytometry.Representative flow cytometry dot plot graphs showing the percentage of CD133 and CD24 positive cells in primary kidney progenitor cells at passage P1 (B). Staining of the same cells with isotype control antibodies is shown in (A).