Figure 6.

Metastatic EVs promote tumorsphere formation, a property diminished by triple chaperone knockdown. (a–d) In vitro tumorigenesis assay in the three-dimensional (3D) culture system. HSC-3 cells were treated with MEX (50 μg/mL), T-MEX (50 μg/mL), TGFβ (5 ng/mL) or PBS in nano-culture plates and then (a–c) tumorsphere formation and (d) protein expression levels were examined. (a) Representative photomicrographs of tumorspheres. Scale bars, 100 μm. (b) Column scatter plotting showing tumorsphere size. The size of the top 300 tumorspheres was measured in each group using ArrayScan HCS System. The horizontal lines indicate mean ± SD analysed by ANOVA Turkey’s multiple comparisons test. (c) The number of tumorspheres altered by MEX or T-MEX. Tumorspheres larger than 200 mm² were counted. n = 3, P = 0.0201. (d) Western blotting showing CD326/EpCAM, E-cadherin, β-catenin, claudin-1 and β-actin. The cell lysates were prepared from the tumorspheres. (e) Scatter plot showing co-expression correlation between CD326/EpCAM and CD9 in head and neck squamous cell carcinoma (HNSCC). A regression line was shown in red. Data were represented as log2. The data set of HNSCC with 523 patient-derived 523 samples (TCGA, Pan-Cancer Atlas) was analysed.