Fig. 8.

Silencing PRRX1 in melanoma cells increases cell proliferation in primary tumors and spontaneous metastasis in NSG orthotopic xenografts. (A, B) Detection of cells undergoing mitosis by phospho‐ Histone 3 (pH3) immunostaining in 793's (A) and A375MM (B) primary tumors. Left bar plots show the mean and SEM of pH3 positive cells in either controls or shPRRX, detected in xenografts (40×) (6 fields per tumor and n = 5 independent tumors per group). ****P < 0.0001 determined by unpaired t‐tests. Bars, 50 μm. (C, D) Silencing PRRX1 in melanoma cells increases spontaneous metastasis in NSG mice. Immunohistochemical images of PRRX1 expression in primary tumors and spontaneous liver and lung metastasis from human melanoma xenografts. Representative images of the primary tumor, liver, and lung sections from NSG mice after orthotopic injection of 793 mock or 793 shPRRX1 (C) cells and A375MM mock or A375shPRRX1 (D) cells, respectively. One out of three independent experiments are presented. The positive immunostaining for PRRX1 or Tyrosinase is indicated at the bottom. Both stainings were made from consecutive sections of the same sample. PRRX1 positive staining was visualized as an intense reddish‐brown stain in the nuclei of cells of the primary tumor and metastatic foci of mock controls (upper panels). Positive immunoreactivity was also detected in collagen fibers, fibroblasts, and the dermis surrounding the primary tumor of both groups. Remarkably, the nuclei of the primary tumors or metastatic foci from shPRRX1 were negative for PRRX1 immunostaining although a diffuse stain was observed in the cytoplasm. Scale bars 100 μm (primary tumors and liver metastasis) and 50 μm (enlarged areas of primaries and lung metastasis, except when indicated). All sections were counterstained with Hematoxylin. (E) Detection of p75 NTR in primary tumors of melanoma xenografts. One out of three independent experiments are presented. Representative images of primary tumor sections from NSG mice after orthotopic injection of 793 mock or 793 shPRRX1 cells and A375MM mock or A375 shPRRX1 cells. Immunostaining for p75NTR is indicated at the bottom and was visualized as an intense reddish‐brown stain in cell membranes and cytoplasm of the shPRRX1 primary tumors (bottom panels). In contrast, positive staining was only seen in isolated few cells of mock controls (upper panels). Scale bars 100 μm. All sections were counterstained with Hematoxylin.