Fig 2: Spinal cord stimulation reduces CSF1 levels and proinflammatory cytokine expression in the DH in CCI rats. A, Western blot analysis of CSF1 levels in the DH; *P = .0099, CCI + sham SCS rats versus naive rats; **P = .0367, CCI + SCS rats versus CCI + sham SCS rats. B, Real-time PCR analysis of mRNA expression for proinflammatory cytokines IL-1ß, IL-6, and TNF-a in the DH. n = 5 rats/per group; *P = .0003 for IL-1ß, P = .2671 for IL-6, and P = .031 for TNF-a, CCI + sham SCS rats versus naive rats; **P = .0494 for IL-1ß, P = .3623 for IL-6, and P = .0018 for TNF-a, CCI + SCS rats versus CCI + sham SCS rats. CCI indicates chronic constriction injury; CSF1, colony-stimulating factor 1; DH, spinal dorsal horn; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; mRNA, messenger ribonucleic acid; PCR, polymerase chain reaction; SCS, spinal cord stimulation; TNF-a, tumor necrosis factor-a.

Fig 3: Senescent tumor cells inhibit CD8+ T cell activation via monocytes to M2 type macrophage differentiation. A) CD8+ T cells activation in p16INK4A positive and negative CRC. p16INK4A negative and positive CRC were dissected serially and immunostained with p16INK4A/CD8, Ki67/CD8 and Tim3, and the percentage of Ki67+/CD8+ cells was analyzed. Three randomly selected areas of the tumor tissue were photographed (200×) and then analyzed for Ki67+/CD8+ cell infiltration and Tim3+ cell number; the results were averaged and then presented as a dot graph. The p value was calculated by the Mann–Whitney U test. B) CD68 and SA-ß-Gal staining analysis in CRC. C) Macrophage distribution analysis with CD206 in p16INK4A positive or negative CRC. The number of CD206 positive macrophages in CRC according to the grades of p16INK4A immunostaining was presented as a dot graph. D) T cell proliferation assay. Isolated primary monocytes were incubated with CM from SW480 or SW480/ROS for 7 days and then monocyte differentiation was analyzed (left lower panel). CFSE labeled CD8+ T activation by anti-CD3/CD28 beads was performed under coculture with differentiated macrophages for 96 h, and suppression of T cell proliferation was measured by flow cytometry. The p value was calculated by one-way ANOVA and post hoc analysis C,D). E) Microdissection analysis. The expression in RNA sequencing indicates the relative values of the p16INK4A positive region compared with the p16INK4A negative region. F) CRC tissues were serially sectioned for p16INK4A and CSF1 immunostaining. The p value was calculated using the ? 2 test. G) CSF1 polarized monocytes to M2 macrophages. Isolated primary monocytes were cocultured with SW480 (control, H2O2 treated, CSF1 overexpressing, and CXCL12 overexpressing) for 6 days and then analyzed for cell morphology (upper panel), and CD206 expression by FACS (lower panel), respectively. N and n indicated the number of cases and independent experiments, respectively.

Fig 4: Stimulation mode and the experimental timeline. A, Stimulation mode: frequency, 60 Hz; pulse width, 240 µs; intensity, 80% MoT; duration, 6 h‚ for 5 consecutive days. B, Representative X-ray images showing epidural placement of the SCS lead. C, Pattern diagram of extracting the DRG and DR. D, Pattern diagram of the immunofluorescence image area of a DRG with the DR. E, Experimental timeline showing CCI, electrode implantation, and SCS. F, Experimental timeline showing CCI, electrode implantation, SCS, and intrathecal injection of CSF1 or trehalose. CCI indicates chronic constriction injury; CSF1, colony-stimulating factor 1; DR, dorsal root fiber; DRG, dorsal root ganglion; MoT, motor threshold; SCS, spinal cord stimulation.

Fig 5: Differentially expressed proteins are mostly upregulated. (A) Heat-map depiction of upregulated continuously expressed proteins between 4D and 8D time points. Color scale and relative dot size represent Z-scored LFQ values. Blank spaces represent missing values. (B) Heat-map depiction of downregulated continuously proteins between 4D and 8D time points. Color scale and relative dot size represents Z-scored LFQ values. Blank spaces represent missing values (C) Western blot analysis of Csf1 and Mif expression levels in 4D and 8D TNFa treated adipocytes and corresponding LFQ fold change. Full-length blots are shown in Supplementary Fig. S4. N.D. no difference.