Fig 2: Identification of smooth muscle-derived factor PERIOSTIN as an in vivo and in vitro substrate for MMP17.a List of growth factors and extracellular proteins found in muscle-SN. n = 3 biological replicates. b TPM values for Postn comparing crypts with smooth muscle tissue. n = 3 biological replicates. c In vitro digestion experiment with human recombinant proteins showing POSTN fragments when in contact with MMP17 catalytic domain. P, POSTN, M, MMP17, FL, full length. n = 2 experiments with increasing concentrations of POSTN or MMP17. d WB of mouse intestinal muscle showing decreased POSTN fragments in the absence of MMP17. n = 2 biological replicates. e Scheme of POSTN molecule showing putative sites of MMP17 cleavage based on digestion assays followed by MS. Sites that are noted all had 5 or more peptide-spectrum matches (PSMs). In blue and red are available sites after in silico modeling (see Fig. 8f for a model of the best fitted (red) site). f In silico model of MMP17 (magenta)- POSTN (green) docking showing close proximity of 664-665 POSTN cleavage site (cyan) to MMP17 catalytic site (yellow). g, h Representative brightfield images showing SI organoids morphology in the presence of POSTN and area quantification in (h) (Day 3). Scale 1250 and 200 µm in inset. n = 8 wells/genotype pooled from three independently performed experiments with 2–3 wells/experiment. i, j Representative confocal maximum intensity projection images showing YAP or Ki67 (green) staining in POSTN-treated SI organoids. Scale 100 µm; 25 µm in inset (YAP pictures) and 200 µm and 100 µm in Ki67 pictures. Numerical data are means ± SD. Data in (b) represents p-adjusted value from RNAseq analysis (padj value of 1.02e-06), and data in (h) was analyzed by Mann–Whitney test (two-sided, p = 0.0260). Source data are provided as a Source Data file.

Fig 3: POSTN in GSCs is essential for angiogenesis and tumor progression(A and B) Representative images (A) and quantification (B) of relative migration of iHUVECs following stimulation with CMs from GSC272 cells expressing shC and shPOSTN. Scale bar, 200 µm; n = 3 biological replicates.(C and D) Representative images (C) and quantification (D) of relative tube formation of iHUVECs following stimulation with CMs from GSC272 cells expressing shC and shPOSTN. Scale bar, 400 µm; n = 3 biological replicates.(E and F) Quantification of relative Transwell migration (E) and tube formation (F) of iHUVECs following stimulation with CMs from QPP7 GSCs expressing shC and shPostn. n = 3 biological replicates.(G) Survival curves of C57BL/6 mice implanted with QPP7 GSCs (2 × 104 cells) expressing shC and shPostn (n = 6–7 mice per group).(H) Survival curves of C57BL/6 mice implanted with CT2A cells (2 × 104 cells) expressing shC and shPostn (n = 5 mice for shC group and 10 mice for shPostn group).(I and J) Immunofluorescence (I) and quantification (J) of CD31 in tumors from C57BL/6J implanted with shC and shPostn QPP7 GSCs. Scale bar, 50 µm; n = 3 biological replicates.(K–M) High-resolution uniform manifold approximation and projection (UMAP) dimensional reduction of glioma cells from GBM patient tumors, partitioned into nine distinct clusters (K). The expression of CD44 (L) and POSTN (M) in glioma cells/GSCs are shown. Intensity of the blue color indicates the expression of individual cells. The analysis was performed on single-cell RNA sequencing data of glioma cells from samples of 16 patients with GBM.(N) The correlation between glioma cell/GSC POSTN level and the frequency of tumor microenvironment components (including immune cells, endothelial cells, and pericytes as indicated) based on single-cell RNA sequencing data from 16 GBM patient tumors. Red signal indicates positive correlation, and blue signal denotes negative correlation.(O) Endothelial cell frequency in patients with glioma with glioma cell/GSC POSTN high versus POSTN low. The analysis is based on single-cell RNA sequencing data from 16 GBM patient tumors.Data from multiple replicates are presented as mean. Error bars indicate mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA test (B and D–F), Student’s t test (O), and log rank test (G and H).See also Figures S4–S6.

Fig 4: TBK1 mediates POSTN-induced angiogenesis in GBM(A) Identification of the overlapping signature pathways in TCGA GBM patient tumors (POSTN high versus POSTN low) and in endothelial cells from single-cell RNA sequencing (scRNA-seq) data of GBM patient tumors (glioma cell/GSC POSTN high versus POSTN low).(B) Co-immunofluorescence for P-TBK1 and CD31 in QPP7 and CT2A tumors established in C57BL/6J mice (top and middle panels) and in GBM patient tumors (bottom panels). Scale bar, 50 µm.(C) Immunoblots for TBK1 and P-TBK1 in lysates of primary mouse brain endothelial cells treated with or without CMs from CT2A cells expressing shC and shPostn.(D and E) Immunoblots for TBK1 and P-TBK1 in lysates of iHUVECs treated with POSTN recombinant protein at 500 ng/mL for different time points (D) and for 60 min at different concentrations (E).(F and G) Quantification of relative Transwell migration (F) and tube formation (G) of iHUVECs following stimulation with POSTN recombinant protein (500 ng/mL) in the presence or absence of TBK1 inhibitor BX795 (1 µM) and CMPD1 (1 µM). n = 3 biological replicates.(H) Schematic of generation of endothelial cell-specific TBK1 knockout (TBK1-eKO) mice by crossing TBK1fl/fl mice with Cdh5(PAC)-CreERT2 mice. TBK1 KO in endothelial cells is induced by injection of tamoxifen (TAM; 75 mg/kg i.p.) for 5 days.(I and J) Survival curves of TBK1-WT and TBK1-eKO mice implanted with 2 × 104 QPP7 GSCs (I, n = 8–9 mice/group) and CT2A cells (J, n = 5 mice/group).(K and L) Immunofluorescence (K) and quantification (L) of relative CD31 in tumors from TBK1-WT and TBK1-eKO mice implanted with CT2A cells. Scale bar, 50 µm n = 3 biological replicates.(M) Representative images show low, medium, and high expression of POSTN, P-TBK1, and CD31 in human GBM tumor samples based on immunohistochemistry staining. Scale bar, 100 µm.(N–P) Quantification of immunohistochemistry staining showing strong positive correlation between POSTN and P-TBK1 (N), POSTN and CD31 (O), and P-TBK1 and CD31 (P) in human GBM tumor samples (n = 23). R and p values are shown. Pearson test.Data from multiple replicates are presented as mean. Error bars indicate mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA test (F and G), Student’s t test (L), and log rank test (I and J).See also Figure S8.

Fig 5: CLOCK-directed OLFML3-HIF1a axis upregulates POSTN expression and angiogenesis(A) Volcano plots showing the fold change of genes (log2 scale) between OLFML3-high and OLFML3-low patient tumors (y axis, log10 scale). POSTN is the top one highly expressed in OLFML3-high tumors.(B) The correlation of POSTN and OLFML3 in TCGA GBM patient tumors. R and p values are shown. Pearson test.(C) Immunoblots for OLFML3 and POSTN in lysates of GSC272 cells expressing shC and shOLFML3.(D) Quantification of relative Transwell migration of iHUVECs following stimulation with CMs from GSC272 cells expressing shC and shOLFML3. n = 3 biological replicates.(E) Immunohistochemistry staining for CD31 (top panels) and POSTN (bottom panels) in tumors from SCID mice implanted with shOLFML3 and shC GSC272 cells. Scale bar, 100 µm.(F and G) Quantification of immunohistochemistry staining for CD31 (F) and POSTN (G) in tumors from SCID mice implanted with shOLFML3 and shC GSC272 cells. n = 3 biological replicates.(H and I) Representative images (H) and quantification (I) of HIF1A ChIP-PCR in the POSTN promoter of GSC272 cells. n = 3 biological replicates.(J) qRT-PCR for POSTN in lysates of GSC272 cells treated with HIF1a inhibitor acriflavine (ACF) at indicated concentrations. n = 3 biological replicates. Values are expressed as relative expression levels with respect to housekeeping gene ACTB.(K) Immunoblots for POSTN in lysates of GSC272 cells treated with ACF at indicated concentrations.(L and M) Quantification of relative Transwell migration (L) and tube formation (M) of iHUVECs following stimulation with CMs from GSC272 cells treated with or without ACF at indicated concentrations. n = 3 biological replicates.Data from multiple replicates are presented as mean. Error bars indicate mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA test (D, F, G, J, L, and M) and Student’s t test (I).See also Figure S7.