Fig 1: Quantitative proteomics shows a higher abundance of fibromodulin under the control of TGF-ß signaling in the differentiated glioma cell (DGC) secretome.(A) Volcano plot depicting relative protein abundance in glioma stem-like cell (GSC) (MGG4, MGG6, and MGG8) vs. their corresponding DGC conditioned media (CM). The black dots represent the nonsignificant proteins (p>0.05), while the red (higher abundance in GSC CM) and green (lower abundance in GSC CM) dots represent the significant ones (p<0.05) with a log2 fold change cutoff of >0.58 or <-0.58. (B) Venn diagram showing the relationship between proteins upregulated in DGC CM and annotated extracellular matrix (ECM) proteoglycans. Of the common proteins shown below, fibromodulin (FMOD) exhibits the highest DGC/GSC ratio (indicated by the more intense red color). (C) Label-free quantification (LFQ) of FMOD, expressed as log2 fold change in GSCs vs. DGCs CM. (D) RT-qPCR analysis shows upregulation of FMOD transcript in DGCs (red bars) compared to GSCs (blue bars). (E) Western blotting shows the presence of higher amounts of intracellular FMOD in DGCs compared with corresponding GSCs. (F) Western blotting shows the presence of higher amounts of FMOD in the DGC CM compared to GSC CM (top panel). Equal loading of the proteins assessed by Ponceau Red staining (bottom panel). (G) RT-qPCR analysis shows a reduction of FMOD transcript level in DGCs, but not in GSCs, upon treatment with SB431542 (10 µM), a TGF-ß inhibitor. Red bars indicate FMOD expression, and blue bars represent TGM2 (a bonafide TGF-ß pathway target gene) expression. (H) Western blotting shows the reduction of FMOD protein level in DGCs, but not in GSCs, upon treatment with SB431542 (10 µM) (intracellular, top, and secreted, bottom). Equal loading of the secreted proteins assessed by Ponceau Red staining. (I) Western blotting shows higher expression of pSAMD2 in DGCs than in GSCs, which is reduced by SB431542 treatment. (J) RT-qPCR shows significantly higher fold enrichment of pSMAD2 in the FMOD promoter, which is inhibited upon SB431542 treatment (10 µM). For panels (C), (D), (G), and (J), n=3, and p-value is calculated by unpaired t-test with Welch’s correction. p-Value <0.05 is considered significant with *, **, and *** representing p-values <0.05, 0.01, and 0.001, respectively. Figure 1—source data 1.Source data used to generate Figure 1A. Figure 1—source data 2.Source data used to generate Figure 1B. Figure 1—source data 3.Source data used to generate Figure 1C. Figure 1—source data 4.Source data used to generate Figure 1D. Figure 1—source data 5.Source data used to generate Figure 1E, F, H, I. Figure 1—source data 6.Source data used to generate Figure 1G. Figure 1—source data 7.Source data used to generate Figure 1J. Figure 1—source data 8.TGF-ß is activated in glioblastoma (GBM) over normal samples in multiple datasets.The table shows the gene set enrichment analysisGene Set Enrichment Analysis (GSEA) output, indicating significant positive enrichment of multiple TGF-ß-related gene sets in GBM over normal in multiple publicly available datasets. Darker to lighter red indicates the highest to lowest normalized enrichment score (NES), while the symbol '%' and green color indicate the significant gene sets p-Value <0.05 is considered significant. NA, not available. Figure 1—source data 9.Source data used to generate Figure 1—source data 8. Figure 1—source data 10.Mesenchymal gene expression signature and TGF-ß signaling pathway are enriched in differentiated glioma cells (DGCs).Gene Set Variance Analysis (GSVA) output indicates the molecular subtypes of the glioma stem-like cells (GSCs) and DGCs of MGG4, MGG6, and MGG8 (each in triplicates). The darkest red indicates the highest GSVA value for a subtype indicating the highest enrichment in that particular sample, with decreasing color intensity indicating a lower enrichment of the subtypes. The table also shows the GSVA score for the TGF-ß hallmark gene set from MSigDb across the multiple data sets. The darkest red depicts the highest enrichment, and gradual lighter colors indicate gradually decreasing enrichment. Figure 1—source data 11.Source data used to generate Figure 1—source data 10.
Fig 2: Growth of human glioma stem-like cell (GSC)-initiated tumors requires secreted fibromodulin (FMOD).(A) Flow cytometry analysis shows the relative levels of CD133-positive cells in MGG8-GSCs compared to MGG8-DGCs. (B) Kaplan–Meier graph shows the survival of mice (n = 10 per group) injected intracranially with MGG8-GSC/shNT or MGG8-GSC/shFMOD cells (1 × 105 cells per animal). (C) Hematoxylin and eosin staining shows a larger tumor (depicted by dark blue color due to high cell density) in mice brain injected with MGG8-GSC/shNT cells (top) compared to that of MGG8-GSC/shFMOD cells (bottom). Magnification = ×0.8. (D) Confocal microscopy analysis shows FMOD expression in brains of mice injected with MGG8-GSC/shNT (top panel) and MGG8-GSC/shFMOD (bottom panel) cells. Red indicates FMOD, and blue indicates H33342 (stains nuclei). Magnification = ×20, scale bar = 50 µm. Statistical significance for panel (B) was calculated using the log-rank test. p-Value <0.05 is considered significant. Figure 6—source data 1.Source data used to generate Figure 6A. Figure 6—source data 2.Source data used to generate Figure 6B. Figure 6—source data 3.Source data used to generate Figure 6C. Figure 6—source data 4.Source data used to generate Figure 6D.
Fig 3: Reduced angiogenesis is characteristic of tumors initiated by fibromodulin (FMOD)-silenced glioma cells.(A) Confocal microscopy analysis shows CD31 expression in brain tumor sections of mice injected with AGR53-GSC/miRNT or AGR53-GSC/miRFMOD cells after doxycycline administration to the mice. Red indicates CD31, and blue indicates H33342 (stains nuclei). Magnification = ×20, scale bar = 50 µm.(B) Quantification of the mean fluorescence intensity of CD31 in brain tumor sections of mice injected with AGR53-GSC/miRNT or AGR53-GSC/miRFMOD cells after doxycycline administration to the mice. (C) Quantification of the mean area of blood vessels in brain tumor sections of mice injected with AGR53-GSC/miRNT or AGR53-GSC/miRFMOD cells after doxycycline administration to the mice. (D) Confocal microscopy analysis showing CD31 expression in brain tumor sections of mice injected with MGG8-GSC/shNT or MGG8-GSC/shFMOD cells. Red indicates CD31, and blue indicates H33342 (stains nuclei). Magnification = ×20, scale bar = 50 µm. (E) Quantification of the mean fluorescence intensity of CD31 in brain tumor sections of mice injected with MGG8-GSC/shNT or MGG8-GSC/shFMOD cells. (F) Quantification of the mean area of blood vessels in brain tumor sections of mice injected with MGG8-GSC/shNT or MGG8-GSC/shFMOD cells. (G) Confocal microscopy analysis showing CD31 and GFP expression in brain tumor sections of mice injected with AGR53-GSC/miRNT or AGR53-GSC/miRFMOD cells after doxycycline administration to the mice. Red indicates CD31, green indicates GFP, and blue indicates H33342 (stains nuclei). The yellow arrow indicates the region exhibiting colocalization of CD31 and GFP. Magnification = ×20, scale bar = 50 µm. (H) Quantification of the colocalization coefficient of CD31 and GFP staining in the brain tumor sections of mice injected with AGR53-GSC/miRNT and AGR53-GSC/miRFMOD cells after doxycycline injection to the mice. (I) The number of blood vessels with co-staining of CD31 and GFP was quantified in brain tumor sections of mice injected with AGR53-GSC/miRNT and AGR53-GSC/miRFMOD cells after doxycycline injection to the mice and plotted. (J) Confocal microscopy analysis showing CD31 and GFAP expression in brain tumor sections of mice injected with MGG8-GSC/shNT or MGG8-GSC/shFMOD cells. Red indicates CD31, green indicates GFAP, and blue indicates H33342 (stains nuclei). The yellow arrow indicates the region exhibiting colocalization of CD31 and GFAP. Magnification = ×20, scale bar = 50 µm. (K) Quantification of the colocalization coefficient of CD31 and GFAP staining in the brain tumor sections of mice injected with MGG8-GSC/shNT and MGG8-GSC/shFMOD cells. (L) The number of blood vessels with co-staining of CD31 and GFAP was quantified in brain tumor sections of mice injected with MGG8-GSC/shNT and MGG8-GSC/shFMOD cells and plotted. (M) A model depicting the functional interactions between different cell types in glioblastoma (GBM) tumors, which comprise a small proportion of glioma stem-like cells (GSCs), a massive number of differentiated glioma cells (DGCs), and stromal cells. FMOD, primarily secreted by DGCs, upregulates JAG1 through the activation of integrin signaling in endothelial cells. The higher expression of JAG1 causes the activation of the Notch signaling pathway, which results in the transcriptional upregulation of HES1 in endothelial cells. The integrin-dependent Notch pathway activation promotes angiogenesis and vascular mimicry, leading to glioma tumor growth. For panels (B), (C), (E), (F), (H), (I), (K), and (L), n=5, and p-value was calculated using an unpaired t-test with Welch’s correction. p-Value <0.05 was considered significant, with *, **, and *** representing p-values <0.05, 0.01, and 0.001, respectively. Figure 7—source data 1.Source data used to generate Figure 7A. Figure 7—source data 2.Source data used to generate Figure 7B, C, H, I. Figure 7—source data 3.Source data used to generate Figure 7D. Figure 7—source data 4.Source data used to generate Figure 7E, F, K, L. Figure 7—source data 5.Source data used to generate Figure 7G. Figure 7—source data 6.Source data used to generate Figure 7J. Figure 7—source data 7.Source data used to generate Figure 7M.
Fig 4: Differentiated glioma cell (DGC)-secreted fibromodulin (FMOD) is essential for tumor growth initiated by glioma stem-like cells (GSCs) in vivo in a co-implantation experiment.(A) Diagram of the inducible shFMOD lentiviral construct. (B) Schema depicts the GSC-DGC co-implantation experiment in C57BL/6 mice (n = 5 per group). Mice were injected subcutaneously with a combination of DBT-Luc-GSCs and DBT-Luc-DGCs transduced with either miRNT (nontargeting) or miRFMOD lentiviruses. To induce miRNT or miRFMOD (and mCherry), mice received doxycycline (100 µl of 1 mg/ml per animal) as intraperitoneal injections at indicated times. The control groups were only injected with DBT-Luc-GSCs or DBT-Luc-DGCs and did not receive doxycycline. (C) In vivo imaging of the injected mice shows tumor growth over time by bioluminescence and mCherry fluorescence, according to the timeline shown in (B). (D) Quantification of the total radiance. The different colors represent the different groups of animals. Significant differences between each of the groups were calculated using ANOVA. The p-values for days 28 and 32 are shown. A detailed comparison of the p-values between different groups is provided in Supplementary file 2. Figure 2—source data 1.Source data used to generate Figure 2C. Figure 2—source data 2.Source data used to generate Figure 2D.
Fig 5: Conditional silencing of fibromodulin (FMOD) in differentiated glioma cells (DGCs) formed de novo by glioma stem-like cell (GSC)-initiated tumors inhibits tumor growth.(A) Schema depicts the timeline of the intracranial orthotopic mouse glioma model using murine glioma stem cells (AGR53-GSC) in C57BL/6 mice (n = 10 per group). Mice were injected with AGR53-GSCs (1 × 105 cells per animal) transduced with either miRNT (nontargeting) or miRFMOD lentiviruses. Tumors were allowed to grow till day 13 and then miRNT or miRFMOD (and mCherry) were induced by doxycycline (100 µl of 1 mg/ml per animal) intraperitoneal injections at indicated times. Note that in vitro characterization shows that the highest knockdown of FMOD was obtained on the seventh day after doxycycline administration. First, in vivo imaging for mCherry expression depicting tumor size was done on day 21 post-injection, followed by imaging at regular intervals (as noted by the orange marks). (B) In vivo fluorescence (mCherry) imaging of mice injected with either AGR53-GSC/miRNT or AGR53-GSC/miRFMOD cells as per the timeline shown in (A). (C) The radiance efficiency for each time point in the two groups of animals as indicated was plotted. (D) Kaplan–Meier graph showing the survival of mice bearing tumors formed by AGR53-GSC/miRNT (Dox+) and AGR53-GSC/miRFMOD (Dox+) cells. (E) Hematoxylin and eosin staining shows a larger tumor (depicted by dark blue color due to extremely high cell density) in mice brain injected with AGR53-GSC/miRNT (Dox+) cells (top) compared to that of AGR53-GSC/miRFMOD (Dox+) cells (bottom). Magnification = ×0.8. (F) Confocal microscopy analysis showing FMOD expression in brains of mice injected with AGR53-GSC/miRNT (Dox+) and AGR53-GSC/miRFMOD (Dox+) cells. Red indicates FMOD, and blue indicates H33342 (stains nuclei). The merged images are shown for representation. Magnification = ×20, scale bar = 50 µm. (G) Brain sections showing areas of fluorescence (GFP, mCherry, and DAPI) for both AGR53-GSC/miRNT (Dox+) (left panel) and AGR53-GSC/miRFMOD (Dox+) (right panel) groups of animals. Note that the AGR53 cell line stably expresses GFP while mCherry expression is induced upon doxycycline addition. On day 13, prior to the administration of doxycycline, both AGR53-GSC/miRNT (left) and AGR53-GSC/miRFMOD (right) do not have any mCherry expression but have almost similar GFP expression. However, over time after the onset of doxycycline administration, both mCherry and GFP expression decreased in the miRFMOD group but not in the miRNT group. Merged images show an overlap of GFP and mCherry-positive tumor areas. Magnification = ×20, scale bar = 50 µm. For panel (C), the p-value was calculated by unpaired t-test with Welch’s correction, and for (D), the p-value was calculated by log-rank test. p-Value <0.05 was considered significant with *, **, and *** representing p-values <0.05, 0.01, and 0.001, respectively. Figure 5—source data 1.Source data used to generate Figure 5B. Figure 5—source data 2.Source data used to generate Figure 5C. Figure 5—source data 3.Source data used to generate Figure 5D. Figure 5—source data 4.Source data used to generate Figure 5E. Figure 5—source data 5.Source data used to generate Figure 5F. Figure 5—source data 6.Source data used to generate Figure 5G.
Supplier Page from OriGene Technologies for Fibromodulin (FMOD) (NM_002023) Human Recombinant Protein