Figure 5. Epithelial–mesenchymal transition (EMT) controls tumor axonogenesis and metastasis in vivo.

(A) Characterization of dominant negative TGFβ type II receptor strategy in NMuMG model. Micrographs of NMuMG cell cultures expressing WT (NMuMG WT) or dominant negative TGFβ type II receptor (NMuMG DN RII) and treated with TGFβ to induce EMT. (B) Quantitative PCR analysis of Sema4F, fibronectin (FN1; mesenchymal marker), and E-cadherin (CDH1; epithelial marker) transcripts induction by TGFβ in the dominant negative TGFβ type II receptor model. Data are mean ± SD (n = 3). (C) Schematic of the dominant negative TGFβ type II receptor (RII) expression strategy to block TGFβ signaling. (D) Western blot analysis of Smad2 phosphorylation 30 m after TGFβ exposure (0.5 ng.mL⁻¹) in the mouse 4T1 breast cancer cells expressing either the wild-type (WT) or dominant negative TGFβ type II receptor (DN RII) in vitro. (E) (Top) Lung metastases quantification are reported in the table. LM+ = number of mice positive for lung metastases; five mice per group. (Bottom) Representative picture of mice lungs 3 wk after mammary fat pad injection of 4T1 cells expressing WT (WT RII) or truncated TGFβ type II receptor (DN RII) & obtained tumors weight (g). Data are mean ± SD (n = 5 mice per group). (F) Tumor-related axonogenesis of tumors generated by mammary fat pad injection of NOD/SCID mice with 4T1 cells enabled (WT) or blocked (DN RII) for TGFβ signaling. Tubulin β3 immunostaining visualizes intra-tumoral innervation. (G) Western blot analysis of axonogenesis in tumors abolished for TGFβ signaling. Tubulin β3 and TH neuronal markers were used to quantify axonogenesis. Densitometry quantification of Tubulin β3 is provided in scattered dot plots on the right of the blots. Data are mean ± SD; ***P < 0.001. HSP90 is used as a loading control. (n = 5 mice per group). (H) Characterization of primary tumors innervation in the 4T1 orthotopic model of tumor progression. Co-immunofluorescence analysis of neuron-specific marker Tubulin β3 (Tubβ3 – green) with sympathetic-specific marker tyrosine hydroxylase (TH), sensory-specific marker capsaicin receptor (TRPV1), or parasympathetic marker choline acetyltransferase (ChAT). For each marker, a representative nerve twig or a more organized fiber is displayed. 120× magnification. (I) Cross-correlation between EMT and tumor innervation in human breast cancer samples. (Top) Analysis of human breast cancer primary tumor axonogenesis (TUBB3 gene expression) and EMT-related mesenchymal (TGFβ1, FN1, CDH2, and SNAI1) and epithelial (TJP1 and CTNNB1) genes expression in 1,218 human breast tumor samples from the Cancer Genome Atlas BRCA RNA-sequencing database. Tumor innervation is ranked through tubulin-β3 expression analysis. Red color indicates tumors with the highest innervation. (J) Correlation analysis between primary tumor axonogenesis and EMT related factors. Genes expression is expressed in Log₂ (normalized count + 1) and Pearson’s rho (r=) and P-value (p=) is provided for each correlation. HSP90 serves as a loading control. (K) Kaplan–Meier survival curve comparing breast cancer innervation to overall survival. Data obtained from the KM plotter breast dataset in overall breast cancer samples (n = 4,929). HSP90 serves as a loading control. Experiments have been repeated three times or as specified in the legend. Scale bars: 50 μm.