Claire Lugassy

Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma

Intermittent intense ultraviolet (UV) exposure represents an important aetiological factor in the development of malignant melanoma. The ability of UV radiation to cause tumour-initiating DNA mutations in melanocytes is now firmly established, but how the microenvironmental effects of UV radiation influence melanoma pathogenesis is not fully understood. Here we report that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression, independent of its tumour-initiating effects. UV irradiation enhanced the expansion of tumour cells along abluminal blood vessel surfaces and increased the number of lung metastases. This effect depended on the recruitment and activation of neutrophils, initiated by the release of high mobility group box 1 (HMGB1) from UV-damaged epidermal keratinocytes and driven by Toll-like receptor 4 (TLR4). The UV-induced neutrophilic inflammatory response stimulated angiogenesis and promoted the ability of melanoma cells to migrate towards endothelial cells and use selective motility cues on their surfaces. Our results not only reveal how UV irradiation of epidermal keratinocytes is sensed by the innate immune system, but also show that the resulting inflammatory response catalyses reciprocal melanoma–endothelial cell interactions leading to perivascular invasion, a phenomenon originally described as angiotropism in human melanomas by histopathologists. Angiotropism represents a hitherto underappreciated mechanism of metastasis that also increases the likelihood of intravasation and haematogenous dissemination. Consistent with our findings, ulcerated primary human melanomas with abundant neutrophils and reactive angiogenesis frequently show angiotropism and a high risk for metastases. Our work indicates that targeting the inflammation-induced phenotypic plasticity of melanoma cells and their association with endothelial cells represent rational strategies to specifically interfere with metastatic progression.

Angiotropic melanoma and extravascular migratory metastasis: a review.

How metastases develop is poorly understood. The concept of intravascular dissemination of cancer cells has been widely accepted as a central paradigm. In addition to this explanation, however, other mechanisms may be operable. Ultrastructural studies have identified in malignant melanoma an angio-tumoral complex, in which tumor cells are linked to endothelium by a matrix containing laminin without evidence of intravasation. This observation has suggested that melanoma cells may migrate along the external surface of vessels and other anatomic structures, a mechanism termed “extravascular migratory metastasis” (EVMM). Angiotropism (melanoma cells cuffing the external surface of vessels) is the histopathologic counterpart of the angio-tumoral complex. The authors have recently drawn attention to the importance of angiotropism as a biologic phenomenom and prognostic factor in melanoma and as a likely correlate of EVMM. In addition, recent experimental studies strongly suggest a correlation of angiotropism of melanoma cells with EVMM. These studies, including cocultures of melanoma cells with capillarylike structures in vitro and the growth of green fluorescent protein-labeled melanoma cells in the shell-less chick chorioallantoic membrane model, have demonstrated the migration of angiotropic melanoma cells along the vascular channels, supporting the concept of EVMM. The new field of EVMM reviewed in this paper may prove useful in elucidating the molecular interactions involved in melanoma metastasis.

Pericytic-like angiotropism of glioma and melanoma cells

We have identified in malignant melanoma an angiotumoral complex in which tumor cells occupy a pericytic location along the endothelium of microvessels without evidence of intravasation. We have suggested that this pericytic-like angiotropism could be a marker of an extravascular migration of tumor cells along the abluminal surface of vessels. The extravascular migratory metastasis proposed for melanoma has close analogies with glioma migration. To compare our hypothesis of extravascular migration by melanoma with the migration of glioma cells, we have used the B16 murine melanoma cell line and the GL26 murine glioma cell line in an in vivo murine brain tumor model and in vitro using endothelial cells that have formed capillary-like structures and have been cocultivated with tumor cells. In the brain tumors, a clear progression of glioma and melanoma cells was observed along the abluminal surface of vessels, where they occupied a pericytic location along the periendothelial laminin. In vitro, time-lapse videomicroscopy recorded the migration of tumor cells toward endothelial tubules. After 24 hours, both the melanoma cells and the glioma cells were localized along the external surfaces of the vascular tubules, occupying a pericytic-like location. These similarities between glioma and melanoma support the hypothesis of an extravascular migration of melanoma cells, particularly along the abluminal surface of vessels.

Angiotropism of Human Melanoma: Studies Involving In Transit and Other Cutaneous Metastases and the Chicken Chorioallantoic Membrane: Implications for Extravascular Melanoma Invasion and Metastasis

Melanoma cell migration along the outside of vessels has been termed “extravascular migratory metastasis” (EVMM), as distinct from intravascular dissemination. Previous studies in both human and experimental melanoma models have shown angiotropism of melanoma cells, suggesting EVMM. Our objectives are to study the mechanism of dissemination of human melanoma cells in the chick chorioallantoic membrane (CAM) and to compare the histopathology in the CAM with that of patients with in transit and other cutaneous melanoma metastases.Human and murine melanoma cells were inoculated onto the CAM and observed over a 10-day period for tumor dissemination. Both human melanoma specimens from 26 patients and melanoma cells growing on the CAM showed the presence of tumor cell angiotropism at the invasive front of the tumor and at some distance from the tumor mass. In addition, a clear progression of melanoma cells spreading on the CAM was observed along the abluminal surface of vessels, where they occupied a perivascular location. By day 10 after injection, small micrometastases had developed along vessels, in a pattern similar to that in transit and other cutaneous melanoma metastases. In addition, the results suggested that the number of micrometastases directly correlated with increasing tumor volume. Taken together, these data suggest that the CAM is a relevant model for studying tumor cell dissemination, and that EVMM may be a mechanism by which some melanoma cells spread to nearby and even distant sites.

Pilot Study on “Pericytic Mimicry” and Potential Embryonic/Stem Cell Properties of Angiotropic Melanoma Cells Interacting with the Abluminal Vascular Surface

The interaction of tumor cells with the tumor vasculature is mainly studied for its role in tumor angiogenesis and intravascular metastasis of circulating tumor cells. In addition, a specific interaction of tumor cells with the abluminal surfaces of vessels, or angiotropism, may promote the migration of angiotropic tumor cells along the abluminal vascular surfaces in a pericytic location. This process has been termed extravascular migratory metastasis. The abluminal vascular surface may also provide a vascular niche inducing or sustaining stemness to angiotropic tumor cells. This pilot study investigated if angiotropic melanoma cells might represent a subset population with pericytic and embryonic or stem cell properties. Through microarray analysis, we showed that the interaction between melanoma cells and the abluminal surface of endothelial cells triggers significant differential expression of several genes. The most significantly differentially expressed genes have demonstrated properties linked to cancer cell migration (CCL2, ICAM1 and IL6), cancer progression (CCL2, ICAM1, SELE, TRAF1, IL6, SERPINB2 and CXCL6), epithelial to mesenchymal transition (CCL2 and IL6), embryonic/stem cell properties (CCL2, PDGFB, EVX1 and CFDP1) and pericytic recruitment (PDGFB). In addition, bioinformatics-based analysis of the differentially expressed genes has shown that the most significantly enriched functional groups included development, cell movement, cancer, and embryonic development. Finally, the investigation of pericyte/mesenchymal stem cells markers via immunostaining of human melanoma samples revealed expression of PDGFRB, NG2 and CD146 by angiotropic melanoma cells. Taken together, these preliminary data are supportive of the “pericytic mimicry” by angiotropic melanoma cells, and suggest that the interaction between melanoma cells and the abluminal vascular surface induce differential expression of genes linked to cancer migration and embryonic/stem cell properties.

Angiotropic Neonatal Congenital Melanocytic Nevus: How Extravascular Migration of Melanocytes may Explain the Development of Congenital Nevi

In the following report we describe a medium-sized congenital melanocytic nevus (CMN) on the upper back of a female patient biopsied at 9 days of age. This case is a unique variant of CMN occurring in the neonatal period that mimics malignant melanoma. This is not only because of histologic features such as a large round or ovoid cellular phenotype of melanocytes mimicking melanoma cells but also because of conspicuous angiotropism, a finding not previously reported in such CMN. Immunostaining for blood and lymphatic vessels demonstrated angiotropism of melanocytes about blood vessels but not lymphatics. We have already emphasized the significance of angiotropism as a marker of extravascular migratory metastasis (EVMM) of melanoma. EVMM, a process by which tumor cells migrate along vessels and other tracks, has striking parallels with the migration of embryonic stem cells from the neural crest. Thus we propose, because angiotropism is a common finding in CMN and metastatic melanoma, that (1) such pathways of cellular migration may result in the genesis of CMN and other melanocytic neoplasms; and (2) the dysregulation of such embryonic pathways may result in the retrograde migratory phenomena of melanoma as already described. In summary, extravascular cellular migration of melanocytes seems to be fundamental for melanoma (perhaps other cancer) metastasis but also hypothetically may be important for the development of other melanocytic lesions such as CMN and requires further investigation.

Imaging of Angiotropism/Vascular Co-Option in a Murine Model of Brain Melanoma: Implications for Melanoma Progression along Extravascular Pathways.

Angiotropism/pericytic mimicry and vascular co-option involve tumor cell interactions with the abluminal vascular surface. These two phenomena may be closely related. However, investigations of the two processes have developed in an independent fashion and different explanations offered as to their biological nature. Angiotropism describes the propensity of tumor cells to spread distantly via continuous migration along abluminal vascular surfaces, or extravascular migratory metastasis (EVMM). Vascular co-option has been proposed as an alternative mechanism by which tumors cells may gain access to a blood supply. We have used a murine brain melanoma model to analyze the interactions of GFP human melanoma cells injected into the mouse brain with red fluorescent lectin-labeled microvascular channels. Results have shown a striking spread of melanoma cells along preexisting microvascular channels and features of both vascular co-option and angiotropism/pericytic mimicry. This study has also documented the perivascular expression of Serpin B2 by angiotropic melanoma cells in the murine brain and in human melanoma brain metastases. Our findings suggest that vascular co-option and angiotropism/pericytic mimicry are closely related if not identical processes. Further studies are needed in order to establish whether EVMM is an alternative form of cancer metastasis in addition to intravascular cancer dissemination.

Could pericytic mimicry represent another type of melanoma cell plasticity with embryonic properties

We hypothesize that the interaction between angiotropic melanoma cells and the abluminal vascular surface can induce or sustain embryonic and/or stem cell migratory properties in these tumor cells. As a result, such angiotropic melanoma cells may migrate along the abluminal vascular surface, demonstrating pericytic mimicry. Through these cellular interactions, melanoma cells may migrate toward secondary sites.

Angiotropism is an independent predictor of microscopic satellites in primary cutaneous melanoma

Wilmott J, Haydu L, Bagot M, Zhang Y, Jakrot V, McCarthy S, Lugassy C, Thompson J, Scolyer R & Barnhill R 
(2012) Histopathology 61, 889–898

Gene expression profiling of human angiotropic primary melanoma: Selection of 15 differentially expressed genes potentially involved in extravascular migratory metastasis

BACKGROUND AND AIM Angiotropism and extravascular migratory metastasis (EVMM) are an important alternative means of melanoma spread. In EVMM, melanoma cells migrate along the external surfaces of vascular channels to distant sites and demonstrate microscopic angiotropism, i.e. melanoma cells arrayed along the external surfaces of vascular endothelium. Pertinent to EVMM are the origin of melanocytes from the neural crest (NC) and strong analogies of EVMM with NC cell migration. Our aim is to elucidate the molecular mechanisms underlying angiotropism and EVMM. METHODS Frozen primary melanomas, previously utilised for gene expression profiling, were analysed for angiotropism as a differential marker. From the results of this new microarray analysis, we sought to identify genes which were directly relevant to the basic mechanisms underlying EVMM. RESULTS Among 66 melanomas from patients who developed metastases or remained disease-free at 4 years of follow-up, 26 melanomas were angiotropic while 35 were not, and five were equivocal. The new microarray analysis identified 128 genes differentially expressed in angiotropic versus non-angiotropic melanomas. Among these 128 genes, 15 genes were potentially directly involved in EVMM, based their respective expressions in the NC (seven genes), in other malignant tumours of NC origin (three genes), in cell motility and/or migration (four genes) and in neurotropism (one gene). CONCLUSION The detection of these 15 genes provides additional support for the importance of angiotropism and the mechanism of EVMM in melanoma. Ongoing studies on this new profile of 15 genes may potentially identify new targets for controlling melanoma metastasis.