Fig 2: Effects of GrA on the expression and distribution of pancreatic CSC markers. a Protein expression profiles of CD133 and CD44 in BxPC-3 and MIA PaCa-2 cells treated with 0.1, 0.01 and 0.001 µM of GrA for 48 h represented graphically. b Densitometry quantitation of CD44 band intensities in western blot. Cells were treated with GrA for 24 h and 48 h, respectively. c Immunofluorescence staining images showed CD47 distribution in BxPC-3 cells treated with 0.05 µM GrA for 24 h. d Immunofluorescence staining images showed CD47 distribution in MIA PaCa-2 cells treated with 0.05 µM GrA for 24 h. Cellular nucleus was stained by DAPI exhibiting blue fluorescence while CD47 on cell surface was marked by antibodies with green fluorescence. The white arrow indicates the CD47 cluster

Fig 3: Calreticulin’s action on tumor-invaded bone and its interaction with CD47.The single asterisk indicates p<0.05. CALR = calreticulin, pl = placebo, siNC = nonspecific siRNA, siCD47=CD47 siRNA, CW CM = CW008-treated MSC conditioned medium, and Cas = caspase 3. (A) X-ray images of the proximal tibia of NSG mice that received inoculation of TT2 OS cells. (B) MicroCT image-based increase in BMD (bone mineral density) of cortical bone in the proximal tibia (N=8) of NSG mice by daily injection of calreticulin (10 µg/kg). (C) MicroCT image-based increase in BV/TV (bone volume ratio) and BMD of trabecular bone of NSG mice in the proximal tibia (n=8). (D) Reciprocal co-immunoprecipitation of calreticulin and CD47 using TT2 osteosarcoma (OS) cell lysate. (E) Suppression of calreticulin-induced changes of p-Src, Snail, and cleaved caspase 3 (c-Cas) in U2OS cells by silencing CD47. (F) Favorable %survival with a high level of calreticulin and a low level of CD47 in all cancer patients. (G) Elevated expression level of CD47 in three OS cell lines (U2OS, TT2, and MG63), compared to non-OS cells (MSCs). (H) Tumor selectivity, larger than 1 for CW008-treated MSC CM and calreticulin, indicating the selective inhibition of OS cells (TT2, U2OS, and MG63) compared to MSCs. Of note, tumor selectivity for cisplatin, smaller than 1, indicates cisplatin’s non-selective inhibition of tumor and non-tumor cells. (Scale bar, 1 mm, error bars indicate standard deviation.) Figure 5—source data 1.Original files for the gels in Figure 5D, E and G.

Fig 4: Disturbing THBS1-CD47 interaction promoted mLVs restoration via inhibiting STAT3/BCL2-mediated apoptosis in mLECs. A) Flow cytometric analysis of mLECs percentage in sham, SAH + igG, SAH + anti-CD47, and SAH + anti-THBS1 group at 24 h post-SAH, n = 4 per group, ***p < 0.001 by paired two-tailed Student's t-test. B) Representative images and quantification of beads accumulation in dCLNs of Sham, SAH + igG, SAH + anti-CD47, and SAH + anti-THBS1 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 < 0.001 by paired two-tailed Student's t-test. Scale bar: 200 µm. C) Representative confocal images of mLVs region of Sham, SAH + igG, SAH + anti-CD47, and SAH + anti-THBS1 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. D) Modified Garcia test, E) time turn, F) time total, and G) wire hanging test at 24 h after SAH revealed anti-CD47 and anti-THBS1 therapy improved short-term neurological function compared with Sham or SAH + igG group. n = 10–12 per group. *p < 0.05; **p < 0.01, ***p < 0.001 by paired two-tailed Student's t-test. H) GSEA showed apoptosis pathway was activated in mLECs after SAH. I) Representative immunoblot images showing effects of rTHBS1 (100 ng mL-1) treatment on pSTAT3 and Bcl-2 inhibition in primary mLECs. J–M) Representative immunoblot images of STAT3, pSTAT3, Bax, and Bcl-2 in different groups.

Fig 5: 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.