Fig. 4.

Airway smooth muscle cell (ASMC) is a metastatic niche in the lung. a H&E staining of lung sections revealing typical metastatic lung foci within alveolar spaces that were formed from vector control T24 cancer cells. (*) denotes alveolar spaces. Scale bar:100 μm. b H&E staining of lung sections revealing morphologically distinct metastatic foci residing adjacent to ASMCs (outlined by arrows), which formed from CD167a-overexpressing T24 cancer cells. Scale bar:100 μm. c Quantification of unique lung metastases with proximal ASMCs localization (control, n = 7; CD167a, n = 7). d Immunofluorescent co-staining of α-SMA (an ASMCs marker, red), and CD167a (to label metastatic cancer cells, green), in a serial section to b. Scale bar:100 μm. e–i Serial sections demonstrating H&E staining, IF and IHC staining of various markers illustrating the colonization of CD167a-expressing cancer cells within COL3-rich ASMCs. Anti-CD167a antibody was used to outline metastatic cancer cells, anti-α-SMA and anti-collagen III antibodies were used to label ASMCs and secreted collagens, respectively. Scale bar:100 μm. j Serial transplantation scheme to establish a highly-metastatic model (T24-LungMET) that efficiently colonized into lung tissues, by serially transplanting parental T24 cells into tail vein five consecutive times, monitored by bioluminescence imaging. k, l Representative serial sections of metastatic foci stained with anti-CD167a to mark metastatic cancer cells and its relative localization to COL3 in T24-LungMET model. m Immunofluorescence co-staining images of CD167a (green) and α-SMA (red) showing the relative localization of CD167a+ metastatic cancer cells and their proximity to α-SMA+ ASMCs. Scale bar:100 μm. n Experimental scheme to establish a spontaneous human bladder metastatic PDX model. o, p Representative serial sections of metastatic foci stained with anti-CD167a to mark metastatic cancer cells and their proximity to COL3. q Immunofluorescence co-staining images of CD167a (green) and α-SMA (red) showing the relative localization of CD167a+metastatic cancer cells and their proximity to α-SMA+ASMCs. Scale bar:100 μm. r Western blot analysis comparing CD167a protein expression in parental T24 cells, T24-LungMET model, CD167a-overexpressing T24 model and the human bladder metastatic PDX model. s Quantification of lung metastases proximal to ASMCs, in the metastatic bladder carcinoma models evaluated in r. Statistical analysis: a two-tailed, unpaired student’s t-test. Error bar: mean ± SEM. **p < 0.01, ***p < 0.005