Figure 8. Expression of Col XVII and laminin 5 by TICs in human lung cancer-related malignant pleural effusion and the requirement of the Col XVII/laminin 5 pathway in malignant lung pleural effusion induced by enriched TICs from A549 cells.

(a) Cells derived from human malignant pleural effusion natively formed spheres, which could be maintained in spheroid culture for weeks, while cells derived from benign pleural effusion of congestive heart failure did not form spheres even when cultured under spheroid conditions. (b,c) Immunofluorescence revealed that tumour cells in human malignant pleural effusion adopted spheroid morphology and expressed CD44, a surface marker for lung cancer stem cells, Col XVII and laminin 5. (d) Cluster fractions and single-cell fractions were separated by passing the pleural effusion fluids or ascites using the cell strainers (with 40-μm pores), followed by transmission electron microscopy (TEM) analysis. TEM images show the existence of hemidesmosomes in the cluster fractions but not in the single-cell fractions of lung cancer malignant plural effusion and colon cancer malignant ascites. Scale bar, 0.1 μm. (e) Immunofluorescence revealed the expression of Col XVII and laminin 5 by enriched TIC derived from A549 cells. Scale bar, 20 μm. (f) The ability of enriched TICs from A549 cells to form malignant pleural effusion is dependent on the Col XVII-laminin 5 pathway. Micro-CT was performed before killing the mice, to demonstrate intrapleural and extrapulmonary tumour growth. Left, gross images of the pleural cavities of mice receiving injections of indicated numbers of bulk or enriched TICs from A549 cells. Right, the ratio of injections with indicated numbers of cells from different conditions to form malignant pleural effusion. The stem cell frequencies for A549 bulk cancer cells (Bulk) and spheroid culture-enriched TICs (SPH) are 1:25,000 and 1:4,500, respectively. Asterisks indicate significant differences (*P<0.05 versus bulk or CTR) determined by Fisher's exact test.