Fig 2: CCR5 conformation frequencies in different cell lines. JC53 (A) and U87.CD4 (B) cell lines were transfected with CCR5-GFP, grown in culture for 24 h post-transfection, and stained with one of six antibodies: CTC5 (NT), CTC8 (NT), T 21/8 (NT), 2D7 (ECL2), 45531 (ECL2), or 45523 (ECL1/ECL2). All primary antibodies were labeled with a secondary antibody conjugated with AlexaFluor 647. Cells were imaged and colocalization coefficients normalized to total GFP were calculated using ZEN Blue 2.3 software at ×1.3 zoom using 10 different cells. Data was graphed using GraphPad Prism 9. **p < 0.01; ***p < 0.001

Fig 3: Increased CCL5 informs poor therapeutic efficacy of TMZ and worse outcome of GBM patients.a Therapeutic response of TMZ in two representative GBM patients with high or low CCL5 expression. Representative MRI, immunofluorescence staining of CD146 (red) and CCL5 (green) and PFS were shown. Tumor border was marked by red dotted lines in enhanced T1 image (T1c) of MRI. Patients with low CCL5/pericyte proportion benefit more from TMZ treatment relative to those with high CCL5/pericyte proportion. Scale bars, 50 µm. b, c Correlation analysis of CCL5 level (b) or CD146 level (c) and PFS of glioma patients (n = 28). High level of CCL5 or CD146 indicates rapid glioma recurrence. d Therapeutic response to TMZ in glioma patients with high or low CCL5 expression (n = 28). Therapeutic responses were evaluated by the Response Assessment in Neuro-Oncology (RANO) standard. CR, complete response; PR, partial response; PD, progressive disease; SD, stable disease. e Expression patterns of CCL5, CCR5 and pericyte markers ACTA2, MCAM, PDGFRB and DES in human gliomas with different molecular signatures using the TCGA pan-glioma (GBM-LGG) dataset (n = 669). Codel, co-deletion of chromosomes 1p and 19q; G-CIMP, glioma-CpG island methylator phenotype (CpG, C-phosphate-G); PA-like, pilocytic astrocytoma-like; LGm6-glioblastoma, a subgroup of glioma enriched for histologic low-grade gliomas but also contains a subset of tumors with GBM-defining histologic criteria; MGMT, O6-methylguanine-DNA methyltransferase; IDH, isocitrate dehydrogenase; WT, wild type; NA, not available.

Fig 4: CCL5-CCR5 paracrine axis represents a molecular link between pericytes and GBM cells.a Top 10 upregulated cytokines in human pericytes relative to GBM cells identified by RNA-Seq and qRT-PCR analyses. b GSEA of pericyte score in human GBMs from the TCGA database (n = 152). c ELISA of CCL5 level in T cell, macrophage, microglia (MG), GBM pericytes (GBM PC-1, -2), HBMECs, GBM cells (GBM cell-1, -2) and astrocyte. Expression of CCL5 in CD3+ T cells was used as a positive control. d, e Immunofluorescence staining (d) and correlation (e) of CCL5 (green) and CD146 (red) expressions in human GBMs (n = 28). Scale bars, 50 µm. **P < 0.01. f, g Immunofluorescence staining of CCR5 (red) and GFAP (green) (f) and proportion of CCR5+/GFAP+ GBM cells in CCR5+ cells (g) in human GBMs. Scale bars, 50 µm. h Immunofluorescence staining of CD146 (red) and CCL5 (green) in GBM tumor and peri-tumor area of GBM. Scale bars, 25 µm. i, j Expression of CCL5 (i) and CCR5 (j) in human GBMs (n = 528) and non-tumor tissues (n = 10) from the TCGA database. *P < 0.05; **P < 0.01. Experiments were performed three times independently (a, c).

Fig 5: Endocytosis and recycling rates of CCR5 surface populations in U87.CD4.CCR5 cells: temperature versus RANTES trigger. U87.CD4 cells were transfected with CCR5-GFP. 24 h post-transfection, cells were divided into groups and treated with or without 100 nM RANTES at 37 °C (A). After 0, 15, 30, 45, 60, 75, or 90 min, cells were fixed and then stained with CTC8 (blue lines), 2D7 (green lines), or 45523 (red lines). Colocalization percentages first were normalized to the 0-min timepoint and graphed. To determine recovery rates, U87.CD4 cells were transfected with CCR5-GFP. 24 h post-transfection, cells were divided into groups and treated with RANTES for 90 min at 37 °C. RANTES was removed, and cells were treated with 1 µM cytochalasin D, 0.45 M sucrose, or left as a volume equivalent control for 0, 30, 60, or 120 min at 37 °C (B). After treatment, cells were fixed and stained with CTC8 (blue lines), 2D7 (green lines), or 45523 (red lines). Colocalization coefficients were calculated using ZEN Blue 2.3 software at ×1.3 zoom using 10 different cells and graphed as a percentage of the control (without treatment)