Fig 2: GBM PDXs with high CCL5 expression benefit from combined treatment of TMZ and MVC.a Development of adjuvant MVC treatment together with TMZ chemo-treatment according to CCL5 expression in GBM PDXs. PDXs were cultured and passaged in NCG mice in vivo. Representative immunofluorescence staining of CCL5 (green) and CD146 (red) in two human GBMs (CCL5high or CCL5low) with PDX model constructed. CCL5high PDXs were treated with two cycles of TMZ (5 mg/kg, i.p., 3 consecutive days per cycle starting from Day 7 and Day 13) and one cycle of MVC (100 mg/kg, i.p., 3 consecutive days starting from Day 13), while CCL5low PDXs were treated with two cycles of TMZ (5 mg/kg, i.p., 3 consecutive days per cycle starting from Day 7 and Day 13) alone. Scale bars, 50 μm. b, c Relative tumor volume of CCL5high PDXs (b) and CCL5low PDXs (c) with indicated treatments. n = 5 for each group. *P < 0.05; **P < 0.01. d Relative inhibition rate of CCL5high PDXs treated with TMZ (red), TMZ + MVC (green) and CCL5low PDXs treated with TMZ (blue). **P < 0.01. e Representative images of immunohistochemistry staining of phosphorylated AKT (Ser473), phosphorylated DNA-PKcs (Ser2056) and γ-H2AX (Ser139) in CCL5high PDXs treated with TMZ together with or without MVC as an adjuvant agent. Scale bars, 50 μm. f Schematic diagram of the mechanism underlying pericyte-mediated DDR and TMZ resistance.

Fig 3: CCL5-CCR5 paracrine signaling activates DDR signaling in GBM cells to potentiate TMZ resistance.a Apoptosis analysis of GBM-1 cells with indicated treatments. GBM cells were stimulated with CCL5 (10 ng/mL) or vehicle (PBS) followed by the administration of TMZ (500 μmol/L) or DMSO. ns, not significant. **P < 0.01. b Cell survival analysis of GBM-1 cells with indicated treatments. CM from pericytes expressing shNT or shCCL5 (sh-1 or sh-2) was collected and added to GBM cells. GBM cells were treated with TMZ (500 μmol/L) after CM addition. **P < 0.01. c Cell survival analysis of GBM-1 cells with indicated treatments. Pericyte CM was pretreated with neutralizing anti-CCL5 antibody or IgG before being added to GBM cells. GBM cells were treated with TMZ (500 μmol/L) after pericyte CM addition. **P < 0.01. d Apoptosis analysis of GBM-1 cells with indicated treatments. GBM cells expressing shNT or shCCR5 (sh-1 or sh-2) were pretreated with pericyte CM or control GBM cell CM followed by TMZ (500 μmol/L) treatment. *P < 0.05; **P < 0.01. e Schematic diagram of MVC-mediated inhibition of CCL5-CCR5 signaling. The binding of MVC to CCR5 leads to a conformational change of its extracellular domain to prevent ligand-stimulated CCR5 activation. f Apoptosis analysis of GBM-1 cells with indicated treatments. GBM-1 cells were pretreated with MVC (500 nmol/L) or DMSO for 1 h, followed by pericyte CM stimulation. Apoptosis and cell survival analyses were performed 48 h after TMZ treatment. ns, not significant. **P < 0.01. g DNA damage after the indicated treatments was assessed by comet assay. Scale bars, 50 μm. h Quantification of percentage of cells with comet tails with indicated treatments. ns, not significant. *P < 0.05; **P < 0.01. i, j Immunoblot analysis of phosphorylated AKT (Ser473) and AKT (i) or phosphorylated DNA-PKcs (Ser2056) and DNA-PKcs (j) in GBM-1 cells with indicated treatments. k Immunoblot analysis of phosphorylated AKT (Ser473), AKT, phosphorylated DNA-PKcs (Ser2056), DNA-PKcs, γ-H2AX in GBM-1 cells with indicated treatments. DNA-PKi (DNA-PKcs inhibitor) represents KU-57788; AKTi (AKT inhibitor) represents MK-2206. Experiments in a–k were independently performed three times.

Fig 4: The combined use of ZSTK474 and TMZ reduced HR repair efficiency. (a)–(d) SF295 and U87 cells were treated with ZSTK474 and TMZ as single agents or in combination for 48 h. (a) The formation of RAD51 foci was assessed using immunofluorescence. Cell nuclei were stained with DAPI. Scale bar, 20 μm. Representative images are shown. (b) Quantification of the number of RAD51-positive SF295 and U87 cells. (c) The cells were collected for western blotting detection of NBS1, p-ATM, BRCA1, BRCA2, DNA-PKcs, and Ku80. (d) Western blotting analysis of PI3K-p110β, p-Akt, p-mTOR, and c-Myc. All data are presented as the mean ± SD (n = 3). ∗P < 0.05; ∗∗P < 0.01.

Fig 5: Identification of HOXB7 interacting with Ku70, Ku80 and DNA‐PKcs. (a) Coimmunoprecipitation of HOXB7 with Ku70, Ku80 and DNA‐PKcs in kYSE150 and KYSE450 cells. Protein was extracted and precipitated using anti‐HOXB7 antibody. Precipitates were subjected to western blotting using anti‐Ku70, anti‐Ku80 and anti‐DNA‐PKcs. (b) GST‐HOXB7 fusion protein pulldown assay in KYSE150 and KYSE450 cells. (c) GST‐HOXB7 fusion constructs were bacterially expressed. KYSE150 and KYSE450 cell lysates were mixed with glutathione agarose containing GST, GST‐HOXB7 construct and probed by western blot with an antibody against Ku70, Ku80 and DNA‐PKcs. (d) The colocalization of HOXB7 with Ku70, Ku80, DNA‐PKcs was analyzed by immunofluorescence microscopy. HOXB7 colocalized with Ku70, Ku80, DNA‐PKcs in nuclear, which was more intensive upon exposure to cisplatin (2 μg/mL for KYSE150 and 1 μg/mL for KYSE450).