Innervation of cervical carcinoma is mediated by cancer-derived exosomes

Abstract

Objective: Recently, our laboratory identified sensory innervation within head and neck squamous cell carcinomas (HNSCCs) and subsequently defined a mechanism whereby HNSCCs promote their own innervation via the release of exosomes that stimulate neurite outgrowth. Interestingly, we noted that exosomes from human papillomavirus (HPV)-positive cell lines were more effective at promoting neurite outgrowth than those from HPV-negative cell lines. As nearly all cervical tumors are HPV-positive, we hypothesized that these findings would extend to cervical cancer.

Methods: We use an in vitro assay with PC12 cells to quantify the axonogenic potential of cervical cancer exosomes. PC12 cells are treated with cancer-derived exosomes, stained with the pan-neuronal marker (β-III tubulin) and the number of neurites quantified. To assess innervation in cervical cancer, we immunohistochemically stained cervical cancer patient samples for β-III tubulin and TRPV1 (sensory marker) and compared the staining to normal cervix.

Results: Here, we show the presence of sensory nerves within human cervical tumors. Additionally, we show that exosomes derived from HPV-positive cervical cancer cell lines effectively stimulate neurite outgrowth.

Conclusions: These data identify sensory nerves as components of the cervical cancer microenvironment and suggest that tumor- derived exosomes promote their recruitment.

Keywords: Cervical cancer; Exosomes; HPV; Innervation.

Figure 1:. Cervical cancer is innervated.

Representative bright field images of normal cervix (A-E) and cervical cancer (F-L) stained with H&E (A,F) and immunohistochemically stained for β-III tubulin (B,G), TRPV1 (C,H), VIP (D,I) and TH (E,J.). Panels K and L are examples of robust β-III tubulin and TRPV1 staining, respectively. Scale bars, 50 μm. Black boarding the cervical cancer is ink from the surgical procedure.

Figure 2.. Quantification of innervation.

Average IHC score of normal cervix (n= 10 cases) and cervical cancer (n=30 cases) for β-III tubulin and TRPV1. Statistical by unpaired student’s t-test, *, p< 0.05.

Figure 3:. TRPV1 and β- III tubulin fibers co-localize in cervical cancer.

Representative en face confocal images of cervical cancer sections from different patient samples (samples 1 and 2) that were processed for double immunofluorescence to localize TRPV1 (red) and β-III tubulin (green) positive nerve twigs. The merged (yellow) images demonstrate the co-localization of the two markers. Sample 1 scale bar, 20 μm; Sample 2 scale bar, 10 μm. Nuclie counterstained with DaPi (blue).

Figure 4:. Cervical cancer-derived exosomes induce neurite outgrowth.

(A) Representative fluorescent images of β-III tubulin staining (green) of PC12 cells treated with cervical cancer cell line derived exosomes. Scale bar, 20μm. (B) Quantitative PC12 neurite outgrowth assay results, showing both total neurites/well. Bars represent mean +/− SEM of n = 3 (exosome conditions), 6 (NGF), or 9 (PBS); *p < 0.05 vs. PBS alone; **p ≤ 0.005 vs. PBS alone; #p < 0.05 vs. all other cervical cancer exosomes. P-values reflect t-test results. (C) Western blot analysis of exosomes purified from the indicated cell lines for CD9 and CD81.