Vasculogenic mimicry structures in melanoma support the recruitment of monocytes.

The progression of cancer is facilitated by infiltrating leukocytes which can either actively kill cancer cells or promote their survival. Our current understanding of leukocyte recruitment into tumors is largely limited to the adhesion molecules and chemokines expressed by conventional blood vessels that are lined by endothelial cells (ECs). However, cancer cells themselves can form their own vascular structures (a process known as vasculogenic mimicry (VM)); but whether they actively participate in the recruitment of leukocytes remains to be elucidated. Herein, we demonstrate that VM-competent human melanoma cell lines express multiple adhesion molecules (e.g. CD44, intercellular adhesion molecule (ICAM)-1 and junction adhesion molecules (JAMs)) and chemokines (e.g. CXCL8 and CXCL12) relevant for leukocyte recruitment. Microfluidic-based adhesion assays revealed that similar to ECs, VM-competent melanoma cells facilitate the rolling and adhesion of leukocytes, particularly monocytes, under conditions of shear flow. Moreover, we identified ICAM-1 to be a key participant in this process. Transwell assays showed that, similar to ECs, VM-competent melanoma cells facilitate monocyte transmigration toward a chemotactic gradient. Gene expression profiling of human melanoma patient samples confirmed the expression of numerous leukocyte capture adhesion molecules and chemokines. Finally, immunostaining of patient tissue microarrays revealed that tumors with high VM content also contained higher numbers of leukocytes (including macrophages). Taken together, this study suggests an underappreciated role of VM vessels in solid tumors via their active participation in leukocyte recruitment and begins to identify key adhesion molecules and chemokines that underpin this process.

Desmoglein-2 expression is an independent predictor of poor prognosis patients with multiple myeloma.

Multiple myeloma (MM) is the second most common haematological malignancy and is an incurable disease of neoplastic plasma cells (PC). Newly diagnosed MM patients currently undergo lengthy genetic testing to match chromosomal mutations with the most potent drug/s to decelerate disease progression. With only 17% of MM patients surviving 10-years postdiagnosis, faster detection and earlier intervention would unequivocally improve outcomes. Here, we show that the cell surface protein desmoglein-2 (DSG2) is overexpressed in ~ 20% of bone marrow biopsies from newly diagnosed MM patients. Importantly, DSG2 expression was strongly predictive of poor clinical outcome, with patients expressing DSG2 above the 70th percentile exhibiting an almost 3-fold increased risk of death. As a prognostic factor, DSG2 is independent of genetic subtype as well as the routinely measured biomarkers of MM activity (e.g. paraprotein). Functional studies revealed a nonredundant role for DSG2 in adhesion of MM PC to endothelial cells. Together, our studies suggest DSG2 to be a potential cell surface biomarker that can be readily detected by flow cytometry to rapidly predict disease trajectory at the time of diagnosis.

CD36 promotes vasculogenic mimicry in melanoma by mediating adhesion to the extracellular matrix.

BACKGROUND: The formation of blood vessels within solid tumors directly contributes to cancer growth and metastasis. Until recently, tumor vasculature was thought to occur exclusively via endothelial cell (EC) lined structures (i.e. angiogenesis), but a second source of tumor vasculature arises from the cancer cells themselves, a process known as vasculogenic mimicry (VM). While it is generally understood that the function of VM vessels is the same as that of EC-lined vessels (i.e. to supply oxygen and nutrients to the proliferating cancer cells), the molecular mechanisms underpinning VM are yet to be fully elucidated. METHODS: Human VM-competent melanoma cell lines were examined for their VM potential using the in vitro angiogenesis assays (Matrigel), together with inhibition studies using small interfering RNA and blocking monoclonal antibodies. Invasion assays and adhesion assays were used to examine cancer cell function. RESULTS: Herein we demonstrate that CD36, a cell surface glycoprotein known to promote angiogenesis by ECs, also supports VM formation by human melanoma cancer cells. In silico analysis of CD36 expression within the melanoma cohort of The Cancer Genome Atlas suggests that melanoma patients with high expression of CD36 have a poorer clinical outcome. Using in vitro 'angiogenesis' assays and CD36-knockdown approaches, we reveal that CD36 supports VM formation by human melanoma cells as well as adhesion to, and invasion through, a cancer derived extracellular matrix substrate. Interestingly, thrombospondin-1 (TSP-1), a ligand for CD36 on ECs that inhibits angiogenesis, has no effect on VM formation. Further investigation revealed a role for laminin, but not collagen or fibronectin, as ligands for CD36 expressing melanoma cells. CONCLUSIONS: Taken together, this study suggests that CD36 is a novel regulator of VM by melanoma cancer cells that is facilitated, at least in part, via integrin-α3 and laminin. Unlike angiogenesis, VM is not perturbed by the presence of TSP-1, thus providing new information on differences between these two processes of tumor vascularization which may be exploited to combat cancer progression.

Endothelial, pericyte and tumor cell expression in glioblastoma identifies fibroblast activation protein (FAP) as an excellent target for immunotherapy.

OBJECTIVES: Targeted immunotherapies such as chimeric antigen receptor (CAR)-T cells are emerging as attractive treatment options for glioblastoma, but rely on identification of a suitable tumor antigen. We validated a new target antigen for glioblastoma, fibroblast activation protein (FAP), by undertaking a detailed expression study of human samples. METHODS: Glioblastoma and normal tissues were assessed using immunostaining, supported by analyses of published transcriptomic datasets. Short-term cultures of glioma neural stem (GNS) cells were compared to cultures of healthy astrocytes and neurons using flow cytometry. Glioblastoma tissues were dissociated and analysed by high-parameter flow cytometry and single-cell transcriptomics (scRNAseq). RESULTS: Compared to normal brain, FAP was overexpressed at the gene and protein level in a large percentage of glioblastoma tissues, with highest levels of expression associated with poorer prognosis. FAP was also overexpressed in several paediatric brain cancers. FAP was commonly expressed by cultured GNS cells but absent from normal neurons and astrocytes. Within glioblastoma tissues, the strongest expression of FAP was around blood vessels. In fact, almost every tumor vessel was highlighted by FAP expression, whereas normal tissue vessels and cultured endothelial cells (ECs) lacked expression. Single-cell analyses of dissociated tumors facilitated a detailed characterisation of the main cellular components of the glioblastoma microenvironment and revealed that vessel-localised FAP is because of expression on both ECs and pericytes. CONCLUSION: Fibroblast activation protein is expressed by multiple cell types within glioblastoma, highlighting it as an ideal immunotherapy antigen to target destruction of both tumor cells and their supporting vascular network.

Desmoglein 2 promotes vasculogenic mimicry in melanoma and is associated with poor clinical outcome.

Tumors can develop a blood supply not only by promoting angiogenesis but also by forming vessel-like structures directly from tumor cells, known as vasculogenic mimicry (VM). Understanding mechanisms that regulate VM is important, as these might be exploitable to inhibit tumor progression. Here, we reveal the adhesion molecule desmoglein 2 (DSG2) as a novel mediator of VM in melanoma. Analysis of patient-derived melanoma cell lines and tumor tissues, and interrogation of The Cancer Genome Atlas (TCGA) data, revealed that DSG2 is frequently overexpressed in primary and metastatic melanomas compared to normal melanocytes. Notably, this overexpression was associated with poor clinical outcome. DSG2+ melanoma cells self-organized into tube-like structures on Matrigel, indicative of VM activity, which was inhibited by DSG2 knockdown or treatment with a DSG2-blocking peptide. Mechanistic studies revealed that DSG2 regulates adhesion and cell-cell interactions during tube formation, but does not control melanoma cell viability, proliferation or motility. Finally, analysis of patient tumors revealed a correlation between DSG2 expression, VM network density and expression of VM-associated genes. These studies identify DSG2 as a key regulator of VM activity in human melanoma and suggest this molecule might be therapeutically targeted to reduce tumor blood supply and metastatic spread.