Tumor-endothelium interactions are critical for tumor survival and metastasis. Melanomas can rapidly metastasize early in tumor progression, but the dependence of this aggressive behavior on tumor-stromal interaction is poorly understood. To probe the mechanisms involved, we developed a heterotypic coculture methodology, allowing simultaneous tracking of genomic and phenotypic changes in interacting tumor and endothelial cells in vitro. We found a dramatic rearrangement of endothelial cell networks into patterns reminiscent of vascular beds, even on plastic and glass. Multiple genes were upregulated in the process, many coding for cell surface and secreted proteins, including Neuropilin-2 (NRP2). A critical role of NRP2 in coordinated cell patterning and growth was confirmed using the coculture system. We conclude that NRP2 represents an important mediator of melanoma-endothelial interactions. Furthermore, the described methodology represents a powerful yet simple system to elucidate heterotypic intercellular interactions mediating diverse physiological and pathological processes. Cancer Res; 71(7); 2433-44. Ó2011 AACR.

Neuropilin-2 (NRP2), a cell surface receptor involved in angiogenesis and axonal guidance, has recently been shown to be a critical mediator of tumor-associated lymphangiogenesis. Given that lymphangiogenesis is a major conduit of metastasis in melanomas and that blocking NRP2 function in vivo is effective in inhibiting tumor cell metastasis, we sought to determine the clinical relevance of NRP2 expression in cutaneous melanoma. Immunohistochemical analysis of NRP2 expression was evaluated in nevomelanocytic proliferations that included a tissue microarray (TMA) and histologic sections (HS) from samples of primary melanomas (n=42; 40 TMA, 2 HS), metastatic melanomas (n=30; 22TMA, 8 HS) and nevi (n=30; 5 TMA, 25HS), as well as a panel of normal human tissues and select non-melanocytic tumors. Staining for grading and intensity of NRP2 expression was estimated semi-quantitatively as follows for the former: <20%, 20-60% and >60% of tissue present and, for the latter from 0-3 with 3 being the highest and 0 the lowest intensity. In nevomelanocytic proliferations, >20% staining for NRP2 was noted in 36/42 cases (86%) of primary melanoma, in 27/30 cases (90%) of metastatic melanoma and in 9/30 cases (30%) of nevi with differences achieving statistical significance between melanoma (primary and metastatic) and nevi (p<0.0001). For staining intensity, an intensity of 2 or more was noted in 36/42 cases (86%) of primary melanoma, in 17/30 cases (57%) of metastatic melanoma and in 7/23 (30%) of nevi with differences achieving statistical significance between melanoma (primary and metastatic) and nevi (p<0.0001). In normal human tissue, consistently strong NRP2 staining was noted in kidney (glomerular endothelial cells, collecting tubules and collecting ducts), skin (epidermal keratinocytes) and testes (epithelium of the seminiferous tubules), while in tumoral tissue, consistently strong staining was noted only in renal cell carcinoma but not in any of the other tumors studied. More recently, using a heterotypic co-culture methodology with melanoma and endothelial cells, we have demonstrated successful up-regulation of NRP2 and confirmed the critical role of NRP2 in melanoma-endothelial interactions. Since these co-culture methods were developed to model melanoma metastasis, the significantly increased and enhanced expression of NRP2 staining in primary and metastatic melanoma versus nevi in the current study suggests that it is also relevant in vivo.

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