Fig. 7: NMB-activated Schwann cells promote PNI by secreting CCL2.

Representative brightfield (a) and PGP9.5 staining (b) images were shown as indicated in PD168368 suppressed PNI in DRG model co-cultured with NMB knockout tumor cells. DRG was placed in the center of Matrigel. Scale bars: 200 μm. c Quantification of neurite length in (b) was shown (n = 3, one-way ANOVA and Tukey’s multiple comparisons test). d Representative brightfield images were shown as indicated in cancer cell-DRG co-culture model treated with NMB or/and PD168368. DRG was placed in the center of Matrigel. 50×magnification, scale bars: 200 μm. e Representative brightfield images were shown as indicated in DRG model co-cultured with NMB overexpressed CaSki and SiHa cells. DRG was placed in the center of Matrigel. ×50 magnification, scale bars: 200 μm. f–i CCL2 mRNA expression of RSC96 cells cocultured with HeLa and ME180 cells (f), NMB recombinant protein (g), NMB and PD168368 (h), and NMB knockout tumor cells were examined by qRT-PCR (n = 3, one-way ANOVA and Tukey’s multiple comparisons test). j Serum CCL2 was examined in healthy women (n = 8), cervical cancer patients with and without PNI (Non-PNI patients, n = 18; PNI positive patients, n = 20. One-way ANOVA and Tukey’s multiple comparisons test). k The correlation between CCL2 and NMB expression in cervical cancer patients’ serum was analyzed by Pearson’s correlation coefficient assay (n = 38). l ROC curve for the sensitivity and specificity of CCL2 combined with or without NMB in serum tested by ELISA in detecting cervical cancer PNI (n = 38). m Working model for cervical cancer-produced NMB triggers PNI. First, cervical cancer cells produce neuropeptide NMB, which is the initial signal to induce PNI. Second, NMB activates Schwann cells through its receptor NMBR-mediated calcium influx by PKA activation, thus triggering Schwann cell reprogramming and secreting CCL2. Third, the activated Schwann cells promote axon regeneration to form the direct contact with cervical cancer cells. Fourth, CCL2 attracts and facilitates the invasion of cervical cancer cells along the regenerated neurites. All these sequential steps lead to the initiation and progression of PNI in cervical cancer. Data are shown as photographs from one representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.