Desmogleins (DSG) are a family of cadherin adhesion proteins that were first identified in desmosomes and provide cardiomyocytes and epithelial cells with the junctional stability to tolerate mechanical stress. However, one member of this family, DSG2, is emerging as a protein with additional biological functions on a broader range of cells. Here we reveal that DSG2 is expressed by non-desmosome-forming human endothelial progenitor cells as well as their mature counterparts [endothelial cells (ECs)] in human tissue from healthy individuals and cancer patients. Analysis of normal blood and bone marrow showed that DSG2 is also expressed by CD34+CD45dim hematopoietic progenitor cells. An inability to detect other desmosomal components, i.e., DSG1, DSG3 and desmocollin (DSC)2/3, on these cells supports a solitary role for DSG2 outside of desmosomes. Functionally, we show that CD34+CD45dimDSG2+ progenitor cells are multi-potent and pro-angiogenic in vitro. Using a ‘knockout-first’ approach, we generated a Dsg2 loss-of-function strain of mice (Dsg2lo/lo) and observed that, in response to reduced levels of Dsg2: (i) CD31+ ECs in the pancreas are hypertrophic and exhibit altered morphology, (ii) bone marrow-derived endothelial colony formation is impaired, (iii) ex vivo vascular sprouting from aortic rings is reduced, and (iv) vessel formation in vitro and in vivo is attenuated. Finally, knockdown of DSG2 in a human bone marrow EC line reveals a reduction in an in vitro angiogenesis assay as well as relocalisation of actin and VE-cadherin away from the cell junctions, reduced cell–cell adhesion and increased invasive properties by these cells. In summary, we have identified DSG2 expression in distinct progenitor cell subpopulations and show that, independent from its classical function as a component of desmosomes, this cadherin also plays a critical role in the vasculature.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-016-9520-y) contains supplementary material, which is available to authorized users.
Although melanoma is initiated by acquisition of point mutations and limited focal copy number alterations in melanocytes-of-origin, the nature of genetic changes that characterise lethal metastatic disease is poorly understood. Here, we analyze the evolution of human melanoma progressing from early to late disease in 13 patients by sampling their tumours at multiple sites and times. Whole exome and genome sequencing data from 88 tumour samples reveals only limited gain of point mutations generally, with net mutational loss in some metastases. In contrast, melanoma evolution is dominated by whole genome doubling and large-scale aneuploidy, in which widespread loss of heterozygosity sculpts the burden of point mutations, neoantigens and structural variants even in treatment-naïve and primary cutaneous melanomas in some patients. These results imply that dysregulation of genomic integrity is a key driver of selective clonal advantage during melanoma progression.
Targeting the right cancer-specific peptides presented by Human Leukocyte antigen (HLA) class I and II molecules on the tumor cell surface is a crucial step in cancer immunotherapy. Numerous approaches have been proposed to predict the presentation of potential neoepitopes that may be targeted through immune-based therapies. Often founded on patient specific somatic mutations, the routine validation of their actual appearance on the tumor cell surface is a significant barrier to realising personalized cancer immunotherapy. This can be attributed to the lack of robust and adaptable assays for antigen presentation that offer the required sensitivity to deal with the limited amounts of patient tumor tissue available. Rather than personalize individual assays we propose the use mass spectrometry to identify tumor neoepitopes from HLA-bound peptides directly isolated form the surface of tumor biopsies. We have developed a microscale HLA-peptide complex immunoprecipitation protocol combined with tandem mass tagging (TMT) to directly sequence HLA-bound peptides using mass spectrometry. Using this strategy, we identified HLA-bound peptides from as few as ~1000 cultured cells and from a small piece (~1 mg) of whole melanoma tumour tissue, encompassing epitopes derived from Melanoma-associated antigens and potential neoantigens.
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