AKAP12 knockdown suppresses primary tumor growth and lung metastasis in an orthotopic model of melanoma. (A) Serial sections of intradermal tumors stained for AKAP12 and hypoxia (Hypoxyprobe). Arrows indicate overlap. (B) K457 shScram or shAKAP12 stable cells were injected intradermally into mice and average tumor growth ± SEM is shown (*P ≤ 0.05, two-tailed t test). (C) Average Ki67+ cells per field ± SEM, with three fields per mouse; *P < 0.01, two-tailed t test. Representative images at 40×. (Scale bar, 50 µm.) (D) Average Meca32+ vessels per field ± SEM, with six fields per mouse, *P < 0.001, two-tailed t test. Images at 40×. (Scale bar, 100 µm.) (E) Metastasis was detected in the shScram mice through CT scan and confirmed on H&E sections of the lung (2 of 6; images at 5× in the orthotopic model). (Scale bar, 200 µm.) qRT-PCR on shScram vs. shAKAP12 injected mice to examine RNA expression of hGAPDH/18S within the lungs. (F) K457 shScram or shAKAP12 stable cells were injected into the tail vein of mice, and lung colonization, a measurement of metastatic potential, was assessed (*P = 0.01 and *P = 0.04, one-tailed t test). (G and H) Average tumor burden of tail vein injected mice with K457 (G) and K3291 stable cells (H) is graphed ± SEM (two-tailed t test). Representative H&E shown. [Scale bar, 100 (G) and 400 µm (H).]