Fig 2: The AMPK inhibitor, WZ4003, and the p38 MAPK inhibitor, SB203580, could inhibit the APN-induced overexpression of CXCL1 and CXCL8 and h-JBMMSCs chemotaxis. After treated with WZ4003 and SB203580, the expression of CXCL1 (A and B) and CXCL8 (C and D) in the cells and supernatants of h-JBMMSCs treated with or without APN was evaluated by the ELISA assay (n = 3; *P < 0.05; **P < 0.01). (E) After treated with WZ4003 and SB203580, the chmotaxis of h-JBMMSCs treated with or without APN was evaluated by the transwell assay (n = 3; *P < 0.05).

Fig 3: APN promoted h-JBMMSCs chemotaxis through CXCL1/8 and activated the phosphorylation of AMPK and p38 MAPK in a time-dependent manner. (A) SB225002 could inhibited h-JBMMSCs chemotaxis in both the co-culture and the monoculture transwell systems (n = 3; *P < 0.05). (B) The h-JBMMSCs were cultured in 60-mm dishes, then starved for 12 hrs after reaching 90% confluence and treated with 1 µg/ml APN at the indicated time-points. Then, phospho-p38 MAPK, total p38 MAPK, phospho-AMPK, total AMPK and actin were examined by Western blotting. The protein concentration was also analysed using Image J software (n = 3; **P < 0.01).

Fig 4: APN promoted h-JBMMSCs up-regulating CXCL1 and CXCL8. (A) h-JBMMSCs were cultured in osteoblast-inducing conditional media with or without APN (1 µg/ml) for 1 week, and the total RNA was analysed using RNA-seq. A total of 198 genes showing differential expression were identified, including 185 up-regulated and 13 down-regulated genes in the APN-treatment group (P < 0.005). The CXCL1 and CXCL8 were selected as the target genes. To validate the results of RNA-seq, real-time PCR (B) and (C) ELISA were used to measure the expression of CXCL1 and CXCL8 in the control and APN-treated groups (n = 3; *P < 0.05; **P < 0.01). (D) The exogenous CXCL1 or CXCL8 proteins were added in the lower compartment to verify its chemotaxis effect. The cell number was displayed as mean ± standard deviation (n = 3; *P < 0.05). (E) The Western blot was used to evaluate the expression of CXCR1 and CXCR2, the specific receptor of CXCL1 and CXCL8, after different treatments (n = 3; *P < 0.05; **P < 0.01).

Fig 5: SLC25A22 knockout abrogates recruitment and activation of MDSC in KRAS-mutant CRC via a CXCL1-CXCR2 axis.a Flow cytometry validation of isolated mouse MDSC (CD11b+ Gr-1+) and human MDSC (CD11b+CD33+) from the spleens of tumor-implanted syngeneic mice and PBMC humanized mice, respectively. b Conditioned medium from sgControl cells induced MDSC migration, which was impaired by SLC25A22 knockout (n = 4). Each dot represents an independent sample. c siCXCL1, but not siCXCL3, abrogated induction of MDSC migration in sgControl cell conditioned medium (n = 4). Each dot represents an independent sample. d Anti-Cxcl1 neutralizing antibody (0.25 µg/mL) suppressed MDSC migration in sgControl cell conditioned medium; but had no effect on SLC25A22 knockout cells (CT26: n = 3; Colo26: n = 4). Each dot represents an independent sample. e Recombinant Cxcl1 (1 ng/ml) rescued MDSC migration in CT26-Slc-KO and Colo26-Slc-KO conditioned medium (n = 4). Each dot represents an independent sample. f CXCR2 inhibitors SX682 (2 µM) or SB265610 (10 µM) abolished MDSC migration in sgControl cell conditioned medium (n = 4). Each dot represents an independent sample. g Tumoral CXCL1 positively correlated with MDSC infiltration in ApcMin/+KrasG12D/+ organoids (n = 10) in C57BL/6 mice, CT26 allografts (n = 9) in BALB/c mice, and DLD1 xenografts (sgControl, n = 15; SLC-KO1, n = 10) in PBMC humanized mice. Each dot represents an independent mouse. h qPCR of isolated MDSC from CT26 allografts showed that activation markers PD-L1, iNOS, and ARG1 were enhanced in intratumoral MDSC compared to that of splenic MDSC, and was abrogated in SLC25A22 knockout tumors (n = 3). Each dot represents an independent mouse. i Flow cytometry of MDSC from CT26 allografts mice showed that surface PD-L1 protein on MDSC were induced in tumors compared with spleen, which was impaired in SLC25A22 knockout tumors (spleen: n = 3; tumors: n = 10). Each dot represents an independent sample. j SB265610 suppressed the growth of CT26-sgControl allografts (n = 10) and k downregulated MDSC (n = 9), but had no effect on CT26-Slc-KO allografts in BALB/c mice. MDSC positively correlated with tumor weight (n = 36). Each dot represents an independent tumor. Data are shown as mean ± SD. Two-tailed one-way ANOVA (b—d, f, h–k). Two-tailed Student’s t test analysis for two-group comparison e. Spearman correlation test (g, k). Source data are provided as a Source Data file.