Figure 5.

Tumor ascites is the major source of exosome and circSKA3

(A) B16 cells were incubated with a basal medium with 20% mouse ascites for 3 days followed by immunostaining and ImageJ analysis. Ascites from tumor-bearing mice injected with circSKA3-transfected B16 cells or MB-231 cells displayed increased levels of circSKA3 (n = 6). (B) MCF-7 cells were cultured in a basal medium with 20% ascites from mice injected with cells for 3 days and subjected to an invasion assay with or without ascites at the lower wells. Ascites from mice injected with circSKA3-transfected B16 or MB-231 cells enhanced cell invasion (n = 4). (C) MCF-7 cells were cultured with 30% ascites from mice injected with B16 or MB-231 cells. Treatment with ascites from tumor-bearing mice injected by circSKA3-transfected B16 or MB-231 cells increased cell motility in wound-healing (left) and chamber migration (right) assays (n = 4). (D) MCF-7 cells were cultured with 30% ascites from mice injected with B16 or MB-231 cells. Treatment with ascites of tumor-bearing mice injected by circSKA3-transfected B16 or MB-231 cells increased the circSKA3 uptake into MCF-7 cells, which was prevented by RNAse A, Cytochalasin D, Digoxin, and MβCD (n = 3). (E) Ascites from mice injected with circSKA3-transfected B16 or MB-231 cells enhanced MCF-7 cell migration in Transwell (left) and cell invasion in Matrigel (right), which could be blocked by RNAse A, Cytochalasin D, Digoxin, and MβCD treatment (n = 3). (F) Tumor sections were subjected to H&E staining. Tumors formed by MB-231 cells showed invasion into the connective stroma (green arrows) and muscles (blue arrows) in the oligo-treated samples that were blocked by treatment with MβCD and circSKA3 siRNA, ∗ p<0.05; ∗∗ p<0.01; Error bars, SD.