Fig. 6

Blocking melanoma metastasis through overexpression of CypD. a Primary tumor growth. Control or CypD OE B16F10 cells were injected subcutaneously into the footpads of mice (tumor n = 8, 4 mice per group), and tumor volume was measured using a caliper. b Invasion into the dermis. The primary tumor tissues collected in (a) were stained with H&E (left). The infiltrating melanoma cells (black arrow) from primary tumors were counted (right, n = 12). Black scale bar, 40 µm; white scale bar, 50 µm. c Lymph node (LN) metastasis. Popliteal lymph nodes (pLNs) were collected at the end of the experiment in (a) (left, lymph node n = 8 per group), and the pigmented metastatic area was calculated using ImageJ (right). Scale bar, 1 mm. d Gene expression analysis. Total RNA from tumor tissues collected from (a) was analyzed by RT-qPCR. Relative mRNA levels of CypD OE tumors were compared with those in control tumors (n = 5). e Expression of HIF1α, CypD and EMT markers in primary tumors. Primary tumors collected from the mice in (a) were analyzed by immunohistochemistry (left). Quantification of fluorescence intensity (right, n = 6). Scale bar, 10 µm. Inhibition of lymph node metastasis by Ad-CypD. B16F10 (f) and A375P cells (g) were injected subcutaneously into the footpads of mice. After one week, either a control (Ad-Conn) or a CypD-overexpressing adenovirus (Ad-CypD) was administered intratumorally. pLNs were isolated and analyzed as in (c), 24 days (f), and 40 days (g) after virus injection (f, n = 7 per group; g, n = 10 per group). Scale bar, 1 mm. h, i RNA and protein expression. Tissues isolated from the A375P tumors shown in (g) were analyzed by RT-qPCR (h, n = 5) and western blotting (i, n = 5). j Reciprocal regulatory loop between HIF1α stabilization and CypD suppression to establish pseudohypoxic conditions. Data are presented as the mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns not significant