Fig 2: Purification of pericytes from normal adjacent tissues and tumors derived from NSCLC and HCC patients using CD146+--FITC-MACS enrichment method.a–f Representative triple immunostaining images of CD34 (white), CD146 (green), and a-SMA (red) from 2 different microscopic fields in normal adjacent tissues and tumors derived from NSCLC and HCC patients are given. Bar charts represent the percentage of CD146 and a-SMA-double positive (+ve) blood vessels per field in each group (n = 10 NSCLC/HCC patients). Statistical tests were two-sided. g–j Representative FACS plots showing the gating strategy for the purification of pericytes from normal adjacent tissues and tumors derived from NSCLC patients with (+) or without (-) CD146+-FITC-microbead cell soring enrichment (MACS) method. Debris, doublets (TOF), and dead cells (FVD) were excluded, then CD45-CD31-CD146+PDGFRß+ pericytes were collected. Percentage refers to the proportion of cells in the previous parent gate. k, l Bar charts show the relative fold change in percentage of pericytes and endothelial cells isolated by the MACS enrichment coupled with FACS protocol compared to that isolated by FACS alone. Statistical tests were two-sided. Percentages of pericyte refer to cell population in the R1 gate. NSCLC non-small cell lung cancer, HCC hepatocellular carcinomas, FSC forward-scatter; SSC side-scatter, TOF time of fly, FVD fixable viability dye, EC endothelial cells, PC pericytes, NEC normal adjacent tissue derived endothelial cells, TEC tumor-derived endothelial cells, NPC normal adjacent tissue derived pericytes, TPC tumor-derived pericytes. FACS plots/bar charts are representative of three individual patient data. Means ± SEM are given. c, f, k, l Student’s t test. Scale bars in a, b, d, e represent 50 µm, a, b, d, e (magnified pictures) represent 25 µm.

Fig 3: Characterization of normal adjacent tissue derived pericytes and tumor-derived pericytes.a Flow cytometry analysis of pericytes isolated from normal adjacent tissue and tumor-derived from NSCLC patients showed that the purity of pericyte preparation was high. Pericyte preparations displayed good expression of pericyte markers PDGFRß, CD146, and CD13 and undetected or low level of endothelial cell markers CD31 and CD34 as well as immune cell marker CD45. b Immunofluorescent staining analysis of the endothelial cell, fibroblast, pericyte marker expression in NPC, TPC, HUVEC, and fibroblast (n = 3 independent samples). c RT-PCR analysis of the endothelial cell, fibroblast, smooth muscle cells, pericyte marker expression in NPC, TPC, HUVEC, fibroblasts, peripheral blood mononuclear cell (PBMC), smooth muscle cells (SMC) (for CD31, CD34, CD45, CNN1, MYOCD, and ACTA2 expression analysis: n = 4 independent NPC and TPC samples and n = 3 independent SMC, PBMC and HUVEC samples; for FAP, PDGFRA, PDGFRB, and CD146 expression analysis: n = 3 independent samples for each cell type). d Doubling time of NPC and TPC derived from NSCLC/HCC patients (n = 3 independent experiments). e ß-galactosidase staining assay showed the percentage of senescent cells at different passages of NSCLC/HCC-derived NPC and TPC (n = 3 independent experiments). Statistical tests were two-sided. f, g Wound healing assay showed no difference in the migration ability of NSCLC/HCC-derived NPC and TPC (n = 3 independent experiments). Statistical tests were two-sided. Means ± SEM are given. c One-way ANOVA. d Two-way ANOVA. e–g Student’s t test. Scale bars in b represent 50 µm, e–g 100 µm.

Fig 4: Tumor-derived pericytes display defective blood vessel supporting functions in vitro and in vivo.a CCK8 proliferation assay showed that exposure with conditioned medium from NSCLC/HCC-derived NPC or TPC significantly enhanced the proliferation of HUVEC as compared to untreated HUVEC (n = 5 experimental repeats). b Wound healing assay of the HUVEC after exposed with condition medium from NSCLC/HCC-derived NPC or TPC. Representative pictures of wound healing assay in each group are given (n = 3 independent experiments). c HUVEC were either cultured alone or co-cultured with CFSE (green fluorescent dye) labeled NSCLC-derived-NPC/-TPC, fibroblast or smooth muscle cells (SMC) in a tube formation assay. d–f Bar charts show the relative total tube length, branch points and number in each group (n = 3 independent experiments). g Representative images of the serial sections of matrix plugs after immunostained with CD34, PDGFRß, or collagen IV antibody in each group are given. h–k Violin plots show the quantification of the total number of blood vessels, lumen diameter, collagen IV level and percentage of coverage in each group (n = 3 mice per group). Results are given as means ± SEM. a Two-way ANOVA. b, d–f, h–k One-way ANOVA. Scale bars in b represents 100 µm, c 200 µm, g 50 µm.

Fig 5: Impaired hematopoietic supportive capacity of BT-MSCs.(A) Transwell migration assay of cord blood (CB) CD34+ toward HD-MSCs (n = 5; blue squares), BT-MSCs (n = 5; orange circles), and BT-MSCs (purple squares) treated with 100 µM DFO for 24 hours. Migration capacity is represented as absolute number of CD45+ cells migrated into the bottom chamber. Positive control (pos ctrl): SDF-1 (100 ng/ml). Negative control (neg ctrl): basal medium. Each error bar shows mean ± SEM. (B) Total number of live cord blood (CB) CD34+ cells after 3 days of coculture with HD-MSCs (red) and BT-MSCs (blue) in the presence or absence of proper cytokines. CB CD34+ cells cultured for 3 days in the presence or absence of proper cytokines were used as control (black). Data are mean ± SEM (HD: n = 3; BT: n = 3). (C) Absolute number of primitive HSPCs identified as Lin–, CD34hi, CD90+, CD45RA– on CD45+ cells after 3 days of coculture with HD-MSCs (red) and BT-MSCs (blue) in the presence or absence of proper cytokines. CB CD34+ cells cultured alone in the presence or absence of proper cytokines were used as control (black) (HD: n = 3; BT: n = 3). (D) Percentage (left panel) and absolute number (right panel) of human CD45+ cells detected in the peripheral blood of NSG mice 6 weeks after intra tail vein coinfusion of 2.5 × 105 human CB CD34 with 1 × 106 HD- or BT-MSCs. (E) Percentage (left panel) and absolute number (right panel) of human CD45+ cells detected in the peripheral blood of NSG mice at a later time point (12 weeks). (F) Percentage (left panel) and absolute number (right panel) of human T lymphocytes (CD3+) detected in the peripheral blood of NSG mice 12 weeks after transplantation. Mice transplanted with CB CD34+ cells alone were used as controls. Data are mean ± SEM. In all panels, each dot represents an irradiated mouse transplanted with CB CD34+ + HD-MSCs (light blue), CB CD34+ + BT-MSCs (orange), or CB CD34+ (black). P values were determined by Student’s t test (*P < 0.05; **P < 0.001).