Fig. 1: Cervical cancer promotes neurogenesis of axons through activating Schwann cells.

a HE staining was analyzed in PNI-positive tumor tissues of cervical cancer patients. 200×magnification, scale bars: 50 μm; N, nerves, T, tumors; white arrows indicate areas of perineural invasion by cervical cancer cells. b Neurogenesis was analyzed by PGP9.5 and Neurofilament L (NF-L) staining. 400×magnification, scale bars: 50 μm. Relative IHC score was analyzed using unpaired t-test. c Representative brightfield images were shown as indicated in HeLa and ME180 cells induced PNI in an in vitro model. DRG was placed in the center of Matrigel. ×50 magnification, scale bars: 200 μm. d Double immunofluorescence staining of neurites (PGP9.5, red) and cervical cancer cells (Pan-CK, green) in the PNI model was analyzed. 100×magnification, upper: HeLa, scale bars: 100 μm; lower: ME180, scale bars: 100 μm. e Schwann cells are the antecedent cells of PNI in cervical cancer. Living cell images show nerve innervation in the in vitro PNI models with HeLa cells (Zsgreen, green) and DRG. The left column shows Schwann cells demyelinating from DRG and moving toward the tumor cell clusters, while the right column shows the latter remains stationary. Times are shown as minutes (see Supplementary Movie 1, 2). f Immunofluorescent staining was analyzed for the movement of RSC96 cells in a 3D PNI model. Red: mScarlet labeled RSC96 cells. Green: Zsgreen labeled HcerEpic, HeLa and ME180 cells. Scale bars: 80,000 μm. g Relative invasion distance of RSC96 cells in (f) was analyzed (n = 3). h Immunofluorescent staining of CM-Dil was analyzed in Hela and ME180 cells (red) co-cultured with DRG for 1 or 4 days. Top row: day 1; bottom row: day 4. ×100 magnification, scale bars: 200 μm. i Live-cell imaging was analyzed in the crosstalk between Schwann cells and tumor cells (Zsgreen, green). Tumor cells formed membrane protrusion and moved toward DRGs at a contact point with Schwann cells. Times are shown as minutes (see Supplemental Movie 3). j Immunofluorescent staining of CK-17 was analyzed in HeLa and ME180 co-cultured with or without DRGs. ×1000 magnification, scale bars: 20 μm. Data are shown as photographs from one representative of three independent experiments. *P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001.