Fig 1: Validation of THBS1 impacting mLVs function after SAH. SAH modeling and behavioral tests were performed 4 weeks after the AAV injection. A) Flow cytometric analysis of mLECs percentage in Sham, AAV‐control, and AAV‐THBS1 group at 72 h post‐SAH, n = 4 per group, **p < 0.01, ***p < 0.001 by paired two‐tailed Student's t‐test. B) Representative images and quantification of beads accumulation in dCLNs of Sham, AAV‐control, and AAV‐THBS1 group at 72 h post‐SAH, each data point represents an average of the 2 dCLNs from one individual mouse, n = 8 per group, **p < 0.01, ***p < 0.001 by paired two‐tailed Student's t‐test. Scale bar: 200 µm. C) Representative confocal images of mLVs region of Sham, AAV‐control, and AAV‐THBS1 group at 72 h post‐SAH. An enlarged view of TS and SSS is shown on the right in each group. Lyve1 (blue), Beads (green), and CD31 (red). Scale bar: 1000 µm. D) Modified Garcia test, E) time turn, F) time total, and G) wire hanging test at 72 h after SAH revealed AAV‐THBS1 delivery aggravated short‐term neurological function compared with Sham or AAV‐control group, n = 10–12 per group. *p < 0.05; **p < 0.01, ***p < 0.001 by paired two‐tailed Student's t‐test. H) Flow cytometric analysis of mLECs percentage in Sham, SAH‐WT, and THBS1‐KO group at 24 h post‐SAH, n = 4 per group, **p < 0.01, ***p < 0.001 by paired two‐tailed Student's t‐test. I) Representative images and quantification of beads accumulation in dCLNs in sham, SAH‐WT, and THBS‐KO group at 24 h post‐SAH, each data point represents an average of the 2 dCLNs from one individual mouse. n = 8 per group, **p < 0.01 by paired two‐tailed Student's t‐test. Scale bar: 200 µm. J) Representative confocal images of mLVs region of Sham, SAH‐WT, and THBS1‐KO group at 24 h post‐SAH. An enlarged view of TS and SSS is shown on the right in each group. Lyve1 (blue), Beads (green), and CD31 (red). Scale bar: 1000 µm. K) Modified Garcia test, L) time turn, M) time total, and N) wire hanging test at 24 h after SAH revealed THBS1 knockout improved short‐term neurological function compared with Sham or WT‐SAH group. n = 10–12 per group. *p < 0.05; **p < 0.01, ***p < 0.001 by paired two‐tailed Student's t‐test.
Fig 2: The THBS1-YAP pathway operates in human low-grade adenomas.(A) Correlation matrix between the expression levels of THBS1 and the YAP targets CTGF, CYR61, and LGR5 in human colon tumours from the TCGA colon cancer bulk datasets. R indicates Spearman’s coefficient. (B–E) Representative sections of low-grade human adenomas (B, D) or advanced human carcinomas (C, E) processed by single-molecule RNA fluorescence in situ hybridisation (smRNA FISH) for Thbs1 (pThbs1, red dots) and Lgr5 (pLgr5, green dots in B, C) or immunostained with anti-YAP1 antibodies (D, E). White arrows highlight tumour cells presenting high nuclear YAP in (D, E). n = 5 human low-grade adenomas in (B, D) and n = 5 advanced human adenocarcinomas in (C, E). (F) Graphical summary of paracrine interactions between wildtype (WT) organoids and tumoroids along the THBS1-YAP axis. Mutant tumoroids ‘corrupt’ genetically WT organoids by secreting THBS-1 (orange arrows). This results in YAP1 nuclear translocation (black nuclei in organoids or tumoroids) and ectopic proliferation as well as cystic morphology in a subset of organoids.
Fig 3: Intercellular communication networks in and around mLVs. A) Spatial visualization of immune cell infiltration based on mLECs distribution. B) Outgoing and incoming signal patterns among all meningeal cells. C) THBS signaling pathway network among all cell types. D) Spatial visualization of THBS1‐CD47 L‐R pair distribution by stLearn. E) Volcano plots showing that THBS1 is one of the upregulated DEGs in the SAH group compared to the NPH group identified by mass spectrometry. F) Quantification of THBS1, THBS2, and THBS4 expression in CSF samples from SAH patients compared to those from NPH patients by ELISA assay, *** p < 0.001 versus NPH by paired two‐tailed Student's t‐test. G) Representative confocal images of mLVs region of the negative control (NC) group and recombinant THBS1 protein treatment (rTHBS1) group. An enlarged view of TS and SSS is shown on the right in each group. Lyve1 (blue), Beads (green), and CD31 (red). Scale bar: 1000 µm. H) Flow cytometric analysis of mLECs percentage in NC group and rTHBS1 group, n = 4 per group, *** p < 0.001 versus NC by paired two‐tailed Student's t‐test. I) Representative images of beads accumulation in dCLNs of the NC group and rTHBS1 group. Scale bar: 200 µm.
Fig 4: Tumour conditioned medium (T-cM) induces YAP pathway activation and a foetal-like state in wildtype (WT) organoids.Gene Set Enrichment Analyses (GSEA) showing the correlation between differentially expressed genes in WT organoids cultured in T-cM (A–C) or tumoroids (D–F) and the indicated transcriptional signatures. NES: Normalised Enrichment Score; green NES: positive correlation; red NES: inverse correlation. (G) Representative image of WT organoids expressing Cas9-GFP (in green) transduced with an sgRNA targeting Yap1 (sgYap1 in red). Higher magnification of a budding Yap1KO organoid (in yellow in G') and a cystic Yap1WT organoid expressing only Cas9-GFP but no sgRNA (in green in G''). (H) Percentage of cystic organoids induced by exposure to T-cM in WT, Yap1KO or Tead4KO organoids, as indicated. (I–N) Max projections of immunostaining for YAP1 (in red) of WT organoids exposed to WT-cM (I), or T-cM (J–L) for 24 hr presenting cystic (J) or budding (K, L) morphologies. Organoids in (L) are treated by neutralising antibodies targeting THBS1 (A6.1), which rescues the budding morphology. Organoids in (M) overexpress THBS1 (LentiThbs1) and tumoroids are shown in (N). DAPI stains DNA in blue. White arrowheads pinpoint YAPHIGH cells in Z-section insets. (O) Quantification of the percentage of nuclear YAP (nYAPHIGH) cells/organoid based on the ratio of nuclear vs. cytoplasmic YAP1 in cystic and budding WT organoids grown in WT-cM or T-cM for 24 hr, in WT organoids overexpressing Thbs1 (lenti-Thbs1) or tumoroids, as indicated. (P) Quantification of the percentage of nYAPHIGH cells/organoid in WT organoids cultured with T-cM and control IgG1 or anti-THBS1 (A6.1) antibodies for 24 hr. Scale bars correspond to 100 µm in (G, I–N). Graphs indicate average values ± SD. Statistical analysis was performed with two-tailed unpaired Welch’s t-tests. For the lenti-Thbs1 sample, a Welch’s corrected t-test was applied to compare the percentage of nYAPHIGH cells/organoid between Thbs1-expressing organoids and WT organoids infected with an empty lentivirus. Figure 4—source data 1.Source data related to Figure 4H. Figure 4—source data 2.Source data related to Figure 4O. Figure 4—source data 3.Source data related to Figure 4P.
Fig 5: Thrombospondin-1 (THBS1) is essential for the growth of tumoroids.(A–D) Representative bright-field images of wildtype (WT) organoids (A, B) or tumoroids (C, D) incubated with IgG1 isotype control antibodies (A, C) or anti-THBS1 A6.1-neutralising antibody (B, D) (10 µg/ml). (E, F) Representative images of tumoroids infected with a lentivirus CRISPR-GFP without sgRNA (control in E) or with an sgRNA targeting Thbs1 (Thbs1-KO in F) 48 hr after replacement of single-cell seeding medium (ENRC) by tumoroid medium (EN). (G, H) Quantification of the number of tumoroids upon antibody neutralisation relative to their size: small tumoroids between 30 and 150 µm in (G); large tumoroids of more than 150 µm diameter in (H). (I) Quantification of the percentage of cystic tumoroids upon treatment by IgG1 isotype control antibodies or three different neutralising antibodies targeting THBS1 (as indicated) for 48 hr. (J) Paired quantification of the number of living tumoroids derived from four independent tumours (from four mice) upon treatment with three neutralising antibodies targeting THBS1 for 48 hr. Antibody concentration: 10 µg/ml. (K–N) Immunofluorescence staining for proliferative cells (EdU in red) and apoptosis (anti-cleaved caspase-3, CASP3 in green) in WT organoids (K, L) or tumoroids (M, N) exposed to IgG1 isotype control antibodies (K, M) or to anti-THBS1 A6.1-neutralising antibody (L, N). (O, P) Quantification of EdU+ cells per organoid (O) or tumoroid (P) in the presence of IgG1 control or anti-THBS1 (A6.1) antibodies. Scale bars = 100 µm. Graphs indicate average values ± SD. Statistical analysis was performed with paired Student’s t-test in (G–J) and two-tailed unpaired Welch’s t-tests in (O) and (P). Figure 3—source data 1.Source data related to Figure 3G. Figure 3—source data 2.Source data related to Figure 3H. Figure 3—source data 3.Source data related to Figure 3I. Figure 3—source data 4.Source data related to Figure 3J. Figure 3—source data 5.Source data related to Figure 3O. Figure 3—source data 6.Source data related to Figure 3P.
Supplier Page from R&D Systems, a Bio-Techne Brand for Thrombospondin-1 Protein
Available conjugates: Sizes Available: 50 ug