Semaphorin 3E expression correlates inversely with Plexin D1 during tumor progression

Abstract

Plexin D1 (PLXND1) is broadly expressed on tumor vessels and tumor cells in a number of different human tumor types. Little is known, however, about the potential functional contribution of PLXND1 expression to tumor development. Expression of semaphorin 3E (Sema3E), one of the ligands for PLXND1, has previously been correlated with invasive behavior and metastasis, suggesting that the PLXND1-Sema3E interaction may play a role in tumor progression. Here we investigated PLXND1 and Sema3E expression during tumor progression in cases of melanoma. PLXND1 was not expressed by melanocytic cells in either naevi or melanomas in situ, whereas expression increased with invasion level, according to Clark's criteria. Furthermore, 89% of the metastatic melanomas examined showed membranous PLXND1-staining of tumor cells. Surprisingly, expression of Sema3E was inversely correlated with tumor progression, with no detectable staining in melanoma metastasis. To functionally assess the effects of Sema3E expression on tumor development, we overexpressed Sema3E in a xenograft model of metastatic melanoma. Sema3E expression dramatically decreased metastatic potential. These results show that PLXND1 expression during tumor development is strongly correlated with both invasive behavior and metastasis, but exclude Sema3E as an activating ligand.

Figure 1

Immunohistochemical analysis of PLXND1 and Sema3E expression in naevi. PLXND1 is not detected in melanocytes in naevocellular (A) and dysplastic (B) naevi. Note the PLXND1-positivity of macrophage- (arrow in A) and fibroblast-like (arrowhead in A) cells. Melanocytes in naevocellular (C) and dysplastic (D) naevi abundantly express Sema3E. Note that vessels in a dysplastic naevus express Sema3E (arrows in D). Magnification = original ×200 (A, B) and ×100 (C, D).

Figure 2

PLXND1 expression in primary melanomas and a melanoma metastasis as revealed by immunostaining with single domain antibody A12. Shown are PLXND1 positive tumor cells in a Clark II melanoma (A), Clark IV melanomas (B and C) and a lymph node metastasis (D). Note in B that tumor vessels in a melanoma with a Breslow thickness of 3.2 mm express PLXND1 (arrows), while PLXND1 is absent in the vasculature of a Clark IV melanoma with a Breslow thickness of 0.65 mm (arrow in C). The insets in B, C and D show Sema3E staining in a Clark IV melanoma (B), a Clark II melanoma (C), and a lymph node metastasis (D). Magnification = original ×200 (A–D) and ×100 (insets).

Figure 3

Analysis of subcutaneous Mel57-VEGF-A xenografts co-expressing Sema3E or Sema3C. A: PLXND1 expression in subcutaneous Mel57-VEGF-A xenografts as revealed by immunohistochemistry using single domain antibody A12. Both Mel57-VEGF-A cells and endothelium express PLXND1 (the inset shows a negative control staining with anti-VSV-G antibody). B: Tumor growth curves of Sema3E (•) and Sema3C(▴)-expressing Mel57-VEGF-A xenografts and control Mel57-VEGF-A/EGFP lesions (♦). Tumor volumes are calculated as height × depth × width. Note that the xenografts co-expressing Sema3(C/E) show comparable growth rates. Histological analysis of subcutaneous Mel57-VEGF-A/Sema3E and -/Sema3C tumors by H&E staining (C and E) and anti-CD34 staining (D and F). Note the absence of micronodular transformation in Sema3E-expressing tumors (C and D), while Mel57-VEGF-A/Sema3C xenografts show the typically VEGF-A induced micronodular growth pattern (E and F). In contrast to Mel57-VEGF-A/Sema3C xenografts, Sema3E-expressing tumors only show a well vascularized tumor rim. The insets in C and E show Sema3E and Sema3C immunostainings on Mel57-VEGF-A/Sema3E (C) and -/Sema3C (E) xenografts respectively. Magnification = original ×100 (A, insets), ×25 (C, E) and ×50 (D, F).

Figure 4

Analysis of metastatic burden, localization and composition. A: Metastasic count of mice carrying different subcutaneous Mel57-VEGF-A xenografts. Lung lesions were counted after H&E staining. Note that in mice carrying subcutaneous Mel57-VEGF-A/Sema3E xenografts metastatic burden is significantly reduced compared to Mel57-VEGF-A/EGFP (P = 0.022) and -/Sema3C tumors (P = 0.006). In contrast, metastatic load in mice carrying Sema3C expressing tumors is not significantly different from Mel57-VEGF-A/EGFP xenografts (P = 0.289). Histochemical analysis of lung metastases by H&E staining (B and C) and anti-CD34 staining (insets) show that lung metastases derived from Mel57-VEGF-A/Sema3C xenografts (B) are predominantly located in the larger branches of the pulmonary vessels. Note in C that the small lesion in the tip of the lung derived from a Mel57-VEGF-A/Sema3E tumor is not surrounded by endothelial cells (inset in C). EGFP immunostaining on lung metastases derived from Mel57-VEGF-A/Sema3C (D) and -/Sema3E (E) xenografts tagged with EGFP-expressing tumor cells. Note that most lung metatases derived from Mel57-VEGF-A/Sema3C xenografts are of multicellular origin with both Mel57-VEGF-A/Sema3C and Mel57-EGFP cells (D), while the EGFP-negative lung lesion in mice carrying a subcutaneous Mel57-VEGF-A/Sema3E xenograft likely originates from clonal expansion of a single tumor cell (E). Magnification = original ×50 (B) and ×100 (C, D, E, insets).

Figure 5

Fold-changes in mRNA expression of extracellular matrix and adhesion molecules in Sema3E overexpressing tumors relative to the control subcutaneous Mel57-VEGF-A lesions as revealed by RT-PCR array analysis (A). Columns represent fold changes in expression in Sema3E-expressing tumors relative to subcutaneous Mel57-VEGF-A lesions. Upper columns (>1.00) represent molecules whose mRNA expression is up-regulated, lower columns (<1.00) molecules whose expression is down-regulated. Immunohistochemical analysis of THBS expression in subcutaneous Mel57-VEGF-A (B) and Mel57-VEGF-A/Sema3E xenografts (C). In contrast to Mel57-VEGF-A/Sema3E lesions THBS is abundantly expressed in Mel57-VEGF-A tumors (the insets show negative control staining with anti-mouse antibody). Note in B that THBS expression is clearly associated with the network of vessel wall elements and tumor cells. Magnification = original ×50 (B, C, insets) and ×400 (enlargements).