Fig. 2
Melanoma liver metastases share a pushing type histopathological growth pattern, but differ in metastatic size, the number of necrosis and proliferation rate. A. Images of H&E stainings of hepatic metastases of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma. Scale bars = 200 µm, n = 5. B. The size of liver metastases was measured and area in mm2 is presented. A Dunn’s test was applied (p = 0.0338 for B16F10 vs. HCmel12; p < 0.0001 for B16F10 vs. D4M; p = 0.0004 for RET vs. D4M; p < 0.0001 for WT31 vs. D4M; p < 0.0001 for HCmel12 vs. D4M). Second, H&E stainings of liver metastases were analyzed for necrosis. The percentages of necrotic metastases in comparison to total number of metastases analyzed are presented. A Dunn’s test was applied (p = 0.0009 for B16F10 vs. WT31; p < 0.0001 for B16F10 vs. D4M; p = 0.0128 for RET vs. WT31; p = 0.0006 for RET vs. D4M; p = 0.0361 for WT31 vs. HCmel12; p = 0.0027 for HCmel12 vs. D4M). Third, the percentage of necrotic areas in comparison to total size of the metastases was quantified. A Dunn’s test was applied (p = 0.0107 for B16F10 vs. RET; p < 0.0001 for B16F10 vs. WT31; p = 0.0026 for B16F10 vs. HCmel12; p < 0.0001 for B16F10 vs. D4M). Number of animals analyzed = 5 (B16F10 luc2), 3 (RET), 5 (WT31), 6 (HCmel12), 4 (D4M). Number of metastases analyzed = 15 (B16F10 luc2), 7 (RET), 11 (WT31), 12 (HCmel12), 15 (D4M). C. Immunofluorescence stainings for cleaved Caspase3 (cCasp3) and DAPI. Representative images of the center of metastases are shown. Necrotic cores (NC) are marked. Scale bar = 100 µm, n = 5. Number of animals analyzed = 3 (B16F10 luc2), 3 (RET), 4 (WT31), 4 (HCmel12), 4 (D4M). Number of metastases analyzed = 10 (B16F10 luc2), 3 (RET), 9 (WT31), 8 (HCmel12), 9 (D4M). D. Immunofluorescence stainings for Ki-67 and DAPI. Representative images of the center of metastases are shown. Necrotic cores (NC) are marked. Scale bar = 100 µm, n = 5. Insert shows a representative picture of a smaller metastasis of HCmel12 melanoma. Scale bar = 100 µm. Number of animals analyzed = 3 (B16F10 luc2), 3 (RET), 3 (WT31), 4 (HCmel12), 5 (D4M). Number of metastases analyzed = 10 (B16F10 luc2), 3 (RET), 5 (WT31), 9 (HCmel12), 8 (D4M). E. The Percentages of cCasp3+ and DAPI+ (p = 0,0225 for B16F10 vs. WT31) or Ki-67+ and DAPI+ (p = 0,0360 for RET vs. HCmel12) as compared to total DAPI+ melanoma cells are shown. Dunn’s tests were performed. Besides, the number of DAPI+ tumor cells related to tumor area is presented (p = 0.0007 for B16F10 vs. D4M; p = 0.0002 for WT31 vs. D4M). A Dunn’s test was applied. Number of animals analyzed = 3 (B16F10 luc2), 3 (RET), 4 (WT31), 4 (HCmel12), 4 (D4M). Number of metastases analyzed = 10 (B16F10 luc2), 3 (RET), 9 (WT31), 8 (HCmel12), 9 (D4M). Data information: *P < 0.05, **P < 0.01, *** P < 0.0001, n.s. = not significant.