Fig 2: Extracellular matrix (ECM) deposition in fetal bovine serum (FBS) or human platelet lysate (HPL)-cultured adipose-derived stem cell (ASC) sheets. (A) Gene expression of collagen 1a1 (COL1A1), fibronectin (FN1), and laminin (LAMA1) in ASCs under different culture condition (n = 3). (B) Sircol assay of ASCs cultured under different culture condition (n = 3). (C) Western blot analysis of the expression of the ECM proteins in FBS or HPL-cultured ASC sheets. (D) Venn diagram of the number of proteins detected on LC/MS in the ECM of ASC sheets cultured with FBS and HPL, respectively (*p < 0.05; **p < 0.01; ***p < 0.005 from control, #p < 0.05; ##p < 0.01; ###p < 0.005 between the indicated groups).

Fig 3: BMP1 promotes collagen I deposition.a, b Cultured BMP1 oe cells deposit more ColI compared with control BxPC3 cells (a) although COL1A1 mRNA levels are comparable (b). a Shows images representative of three independent experiments with ß-actin as a sample processing control. The quantified WB signal is represented as mean ± SD. Note that, while a1(I) protein is elevated, mRNA is not. c Representative IHC on xenograft tumor sections showed increased intensities of both human cancer-cell- and murine stroma-derived ColI staining upon BMP1 overexpression. The intensities were quantified in d. N numbers are 5, 6, 6 (left to right). e, f Western blotting (WB) of enriched ECM samples from xenograft tumors showed increased cancer-cell-derived (human) ColI deposition in oe-BMP1 tumors. In f, each column represents the quantification of the band intensities for four tumors (two were shown in e and two came from an independent repeat, see Methods section). g The intensity of protein hydroxyproline (Hypro) normalized to either the intensity of protein proline or aspartic acid in xenograft tumors suggested a slight increase in overall collagen upon BMP1 overexpression for one of the guides but not the other. h, i Cultured shBMP1 cells deposit less a1(I) compared to control BxPC3 cells (h) although COL1A1 mRNA level is comparable (i, N = 3). h is an image representative of three independent experiments with ß-actin as a sample processing control. j IHC on xenograft shBMP1 tumor sections showed somewhat reduced ColI staining for human cancer-cell-derived ColI but no change in stroma-derived ColI staining upon knocking down COL1A1 in cancer cells. The intensities were quantified in k (N = 7 for both columns). l WB of enriched ECM samples from xenograft shBMP1 tumors showed reduced cancer-cell-derived (human) ColI deposition. The WB band intensities were quantified in m (N = 4). n The intensity of protein Hypro normalized to either the intensity of protein proline or aspartic acid in xenograft tumors suggested no change in overall collagen quantity upon BMP1 knockdown (N = 3). All p-values come from two-tailed Student’s t tests. All columns are represented by mean ± SD.

Fig 4: COL1A1 mutated to remove the BMP1 cleavage site promotes tumor growth and metastasis.a Generation of cell clones that were knocked down for COL1A1 by shRNA targeting the 5' UTR region and expressing either WT or mutated COL1A1. qPCR showed comparable expression levels for WT and mut COL1A1 (N = 4). b Genotyping for the COL1A1 mutated transcript showed that 67% of all COL1A1 transcripts in the cells expressing COL1A1 mut were mutated (see Methods section). c Cultured cells expressing WT COL1A1 deposit more a1(I) compared to those expressing mut COL1A1 (A) although COL1A1 RNA levels are comparable (see panel a). c Shows images representative of three independent experiments with ß-actin as a sample processing control. The quantified WB signal is represented as mean ± SD. d Cultured cells expressing COL1A1 mut have slightly faster growth rate than do cells expressing COL1A1, represented here by 1/doubling time in hours. e–i Orthotopic injection of BxPC3 cells expressing COL1A1 mut compared to cells expressing COL1A1 WT resulted in slightly increased primary tumor weight (f), and increased metastasis load in lung (g) and liver (h). Representative lung and liver metastasis images are shown in I. Mouse numbers are 6 for both groups. j, k IHC on xenograft tumor sections showed reduced staining intensities for cancer-cell-derived ColI but not for stroma-derived ColI staining upon BMP1 mutation. k is the quantification of the band intensities of four tumors from two independent experiments. l, m WB on enriched ECM samples from xenograft tumors showed decreased cancer-cell-derived (human) ColI deposition in COL1A1 mut tumors but a slight increase in mouse ColI. The WB band intensities were quantified in m (N = 4). n COL1A1 expression was similar in the COL1A1 WT and mut xenograft tumors in species-specific COL1A1 qPCR experiment using human-specific COL1A1 and ß-actin primers (N = 3). o Working hypothesis summarizing the findings: in cancer cells, BMP1 promotes increased ColI deposition, which can be enhanced by PCOLCE and disrupted by mutating the BMP1 cleavage site in a1(I), and that, in turn, leads to suppression of tumor growth and metastasis. All p-values come from two-tailed Student’s t tests. All columns are represented by mean ± SD.

Fig 5: WNT10A overexpression in hTCEpi cells. Both WNT10A transcript (A) and protein levels (C, D) are elevated in the WNT10A overexpression plasmid wells (band size for WNT10A, 46 kDA; COL1A1, ~120 kDA). COL1A1 transcript (B) and protein (C, D) are also increased in wells overexpressing WNT10A.