Abstract To facilitate survival, migration and evasion of immune surveillance, cancer cells tightly coordinate the synthesis and trafficking of a diverse repertoire of proteins to their cell surface and the surrounding tumor microenvironment. A key mechanism underlying this process is the intracellular membrane trafficking pathways, including vesicular transport systems. There remains a paucity of mechanistic insight into the regulatory components that mediate nascent protein trafficking and their dysregulation in cancer. Herein, we investigate Tumor Protein D54 (TPD54) as a central regulator of intracellular protein transport that is exploited by melanoma cells to promote disease progression. Integrative analyses of patient-derived tumor tissue specimens show that the expression of TPD52L2 (the gene encoding TPD54) is frequently overexpressed in melanoma and correlates with adverse clinical outcomes, including reduced responses to immune checkpoint blockade. Mechanistic investigations further revealed that TPD54 maintains Golgi integrity and orchestrates trafficking of early endosomes, anterograde vesicles and extracellular vesicles. Functionally, TPD54 augments the secretion of pro-cancerous cytokines, increases the cell surface expression of adhesion-signaling receptors (e.g. integrin-β1 and desmoglein-2), promotes melanoma cell migration and elevates their capability to undergo vasculogenic mimicry. Targeting TPD52L2 in two mouse models of melanoma (B16-F10 and HCmel12) showed significant attenuation of tumor growth, disrupted tumor vasculature, enhanced anti-tumor immunity with infiltration of CD8 + T cells and reduced metastatic disease. Collectively, these findings establish TPD54 as a critical and previously underappreciated regulator of protein trafficking in cancer cells that directly contributes to disease progression and highlights its potential as a novel therapeutic target to combat melanoma.

Solid tumours routinely access the blood supply by promoting endothelium‐dependent angiogenesis; but tumour vasculature can also be formed by cancer cells themselves via vasculogenic mimicry (VM). Investigation of the gene expression profile during the early stages of VM formation by MDA‐MB‐231‐LM2 breast cancer cells identified the transcriptional regulator inhibitor of DNA binding 1 (ID1) to be elevated ~ 10‐fold within the first 2 hours. This role for ID1 in promoting VM was supported by ID1 genetic knockdown or chemical inhibition interrupting VM formation by MDA‐MB‐231‐LM2 (breast) and BxPC‐3 (pancreatic) cancer cells. More specifically, reducing ID1 lowered cancer cell expression of endothelial cell genes (e.g. CDH5, TIE2) and production of pro‐angiogenic proteins (e.g. VEGF, CD31, MMP9 and IL‐8). In silico analysis of MDA‐MB‐231 cells engrafted into mice identified elevated ID1 expression in cancer cells that had metastasised to the lungs or liver, and an enrichment of pro‐angiogenic genes. Additionally, Id1 knockdown in 4T1.13 murine breast cancer cells demonstrated reduced tumour growth and metastasis in vivo. Taken together, this study further implicates ID1 in a vascular program within cancer cells that supports disease progression.

Small interfering RNA (siRNA) is a growing treatment avenue for cancer patients. However, formulating siRNA to achieve high tumoral delivery efficiency is challenging. Among various formulation strategies, porous silicon nanoparticles (pSiNPs) are unique due to the versatile nanoengineering options. This study builds on and aims to further refine two successful strategies previously developed for pSiNPs-based siRNA delivery systems: calcium silicate sealing and lipid coating. The application of alternative lipid composition based on ionisable lipids (dilinoleyl-methyl-4-dimethylaminobutyrate (MC3)) is investigated and head-to-head compared with previously developed coatings based on quaternary ammonium lipids (1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)) associated with potential in vivo safety concerns. While the MC3 formulation underperforms compared to the DOTAP-based formulation in terms of siRNA loading and in vitro cellular association, comparable lysosomal escape rate and acceptable levels of siRNA transfection are observed. In vivo biodistribution of formulations is compared head-to-head, with higher tumor accumulation observed for the MC3 formulation in an orthotopic breast cancer murine model. Formulations are well-tolerated at a 20 mg/kg dose. As another approach for refinement of the formulation for siRNA delivery, the lipid-coated pSiNPs are functionalized using an anti-human desmoglein-2 monoclonal antibody (aDSG2) to achieve better transfection efficacy.

While blood-contacting materials are widely deployed in medicine in vascular stents, catheters, and cannulas, devices fail in situ because of thrombosis and restenosis. Furthermore, microbial attachment and biofilm formation is not an uncommon problem for medical devices. Even incremental improvements in hemocompatible materials can provide significant benefits for patients in terms of safety and patency as well as substantial cost savings. Herein, a novel but simple strategy is described for coating a range of medical materials, that can be applied to objects of complex geometry, involving plasma-grafting of an ultrathin hyperbranched polyglycerol coating (HPG). Plasma activation creates highly reactive surface oxygen moieties that readily react with glycidol. Irrespective of the substrate, coatings are uniform and pinhole free, comprising O─C─O repeats, with HPG chains packing in a fashion that holds reversibly binding proteins at the coating surface. In vitro assays with planar test samples show that HPG prevents platelet adhesion and activation, as well as reducing (>3 log) bacterial attachment and preventing biofilm formation. Ex vivo and preclinical studies show that HPG-coated nitinol stents do not elicit thrombosis or restenosis, nor complement or neutrophil activation. Subcutaneous implantation of HPG coated disks under the skin of mice shows no evidence of toxicity nor inflammation.

AbstractObjectivesCAR‐T cells are being investigated as a novel immunotherapy for glioblastoma, but clinical success has been limited. We recently described fibroblast activation protein (FAP) as an ideal target antigen for glioblastoma immunotherapy, with expression on both tumor cells and tumor blood vessels. However, CAR‐T cells targeting FAP have never been investigated as a therapy for glioblastoma.MethodsWe generated a novel FAP targeting CAR with CD3ζ and CD28 signalling domains and tested the resulting CAR‐T cells for their lytic activity and cytokine secretion function in vitro (using real‐time impedance, flow cytometry, imaging and bead‐based cytokine assays), and in vivo (using a xenograft mimicking the natural heterogeneity of human glioblastoma).ResultsFAP‐CAR‐T cells exhibited target specificity against model cell lines and potent cytotoxicity against patient‐derived glioma neural stem cells, even when only a subpopulation expressed FAP, indicating a bystander killing mechanism. Using co‐culture assays, we confirmed FAP‐CAR‐T cells mediate bystander killing of antigen‐negative tumor cells, but only after activation by FAP‐positive target cells. This bystander killing was at least partially mediated by soluble factors and amplified by IL‐2 which activated the non‐transduced fraction of the CAR‐T product. Finally, a low dose of intravenously administered FAP‐CAR‐T cells controlled, without overt toxicity, the growth of subcutaneous tumors created using a mixture of antigen‐negative and antigen‐positive glioblastoma cells.ConclusionsOur findings advance FAP as a leading candidate for clinical CAR‐T therapy of glioblastoma and highlight under‐recognised antigen nonspecific mechanisms that may contribute meaningfully to the antitumor activity of CAR‐T cells.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with a dismal 5-year survival rate of <13%. We have discovered that desmoglein-2 (DSG2), a cell surface protein, is highly expressed on PDAC cells. In silico data indicate that elevated levels of DSG2 is associated with PDAC progression and poor patient survival. Immunohistochemistry (IHC) analysis of a tissue microarray of 302 patient samples showed that DSG2 was expressed by >90% of PDAC patients. We hypothesized that DSG2 is an under-appreciated contributor to PDAC progression and that DSG2 can be targeted. The main aims of this study were to: 1) investigate the role of DSG2 in PDAC cell function in vitro, 2) investigate the molecular biology underpinning the role of DSG2 in PDAC, and 3) to use murine PDAC tumor models to evaluate the contribution of DSG2 to cancer progression in vivo. Our in vitro data showed that reducing the expression of DSG2 (via small interfering RNA (siRNA)) significantly decreased PDAC cell proliferation, migration, and invasion. Moreover, signaling analysis of PDAC cells with or without DSG2 (via siRNA) using a reverse-phase protein array and cytokine array revealed that DSG2 modulates pro-tumorigenic and pro-metastatic cellular pathways, through reducing the effects of certain proteins. Particular alterations were observed in pathways involving gene regulation (e.g., beta-catenin), migration (e.g., epidermal growth factor receptor and integrin-beta1), and cytokine release (e.g., serpine-1). Next, in an orthotopic xenograft mouse model, BxPC-3 PDAC cells with a stable knockdown of DSG2 (via short-hairpin RNA) were injected into NSG (NOD scid gamma) mice, where we observed a significant reduction in tumor burden and liver metastasis when compared to control mice. IHC staining of tumor sections revealed that the DSG2 knockdown tumors contained reduced levels of collagen, tumor vasculature and cancer associated fibroblasts. Thus, suggesting that DSG2 also has a modulatory effect on the tumor microenvironment (TME). A similar reduction in tumor burden, cancer progression and changes to the TME were observed when a neutralizing anti-DSG2 monoclonal antibody was administered intraperitoneally twice a week at 5mg/kg to BxPC-3 tumor bearing mice. Finally, to investigate the immune cell landscape, we used an orthotopic syngeneic mouse model, whereby Dsg2 was knocked out of KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) mouse PDAC cells via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. This mouse model also showed that loss of Dsg2 resulted in a significant reduction in tumor burden, cancer cell metastasis and changes to the TME. In summary, we have identified a potential new target for the treatment of PDAC, namely DSG2, and future experiments are pursuing clinical opportunities to improve PDAC patient survival. Citation Format: Charlie B Ffrench, Kay K Myo Min, Mark DeNichilo, Michaelia P Cockshell, Emma L Dorward, Emma J Thompson, Michael Ortiz, Michael S Samuel, Savio George Barreto, Claudine S Bonder. Desmoglein-2 is a regulator of pancreatic ductal adenocarcinoma progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A073.

Whilst blood‐contacting materials are widely deployed in medicine in vascular stents, catheters and cannulas, devices fail in‐situ because of thrombosis and restenosis. Furthermore, microbial attachment and biofilm formation is not an uncommon problem for medical devices. Even incremental improvements in hemocompatible materials could provide significant benefits for patients in terms of safety and patency as well as substantial cost savings.Herein, we describe a novel but simple strategy for coating a range of medical materials, that can be applied to objects of complex geometry, involving plasma‐grafting of an ultra‐thin hyperbranched polyglycerol coating (HPG). Plasma activation creates highly reactive surface oxygen moieties that readily react with glycidol. Irrespective of the substrate, coatings are uniform and pinhole free, comprising O‐C‐O repeats, with HPG chains packing in a fashion that holds reversibly binding proteins at the coating surface.In vitro assays with planar test samples show that HPG prevents platelet adhesion and activation, as well as reducing (>3log) bacterial attachment and preventing biofilm formation. Ex vivo and preclinical studies show that HPG‐coated nitinol stents do not elicit thrombosis or restenosis, nor complement or neutrophil activation. Subcutaneous implantation of HPG coated disks under the skin of mice showed no evidence of toxicity nor inflammation. This article is protected by copyright. All rights reserved

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.

Cardiovascular disease is a leading cause of death worldwide; however, despite substantial advances in medical device surface modifications, no synthetic coatings have so far matched the native endothelium as the optimal hemocompatible surface for blood-contacting implants. A promising strategy for rapid restoration of the endothelium on blood-contacting biomedical devices entails attracting circulating endothelial cells or their progenitors, via immobilized cell-capture molecules; for example, anti-CD34 antibody to attract CD34+ endothelial colony-forming cells (ECFCs). Inherent is the assumption that the cells attracted to the biomaterial surface are bound exclusively via a specific CD34 binding. However, serum proteins might adsorb in-between or on the top of antibody molecules and attract ECFCs via other binding mechanisms. Here, we studied whether a surface with immobilized anti-CD34 antibodies attracts ECFCs via a specific CD34 binding or a nonspecific (non-CD34) binding. To minimize serum protein adsorption, a fouling-resistant layer of hyperbranched polyglycerol (HPG) was used as a "blank slate," onto which anti-CD34 antibodies were immobilized via aldehyde-amine coupling reaction after oxidation of terminal diols to aldehydes. An isotype antibody, mIgG1, was surface-immobilized analogously and was used as the control for antigen-binding specificity. Cell binding was also measured on the HPG hydrogel layer before and after oxidation. The surface analysis methods, x-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry, were used to verify the intended surface chemistries and revealed that the surface coverage of antibodies was sparse, yet the anti-CD34 antibody grafted surface-bound ECFCs very effectively. Moreover, it still captured the ECFCs after BSA passivation. However, cells also attached to oxidized HPG and immobilized mIgG1, though in much lower amounts. While our results confirm the effectiveness of attracting ECFCs via surface-bound anti-CD34 antibodies, our observation of a nonspecific binding component highlights the importance of considering its consequences in future studies.

Type 1 diabetes is a complex disease characterized by the lack of endogenous insulin secreted from the pancreatic β-cells. Although β-cell targeted autoimmune processes and β-cell dysfunction are known to occur in type 1 diabetes, a complete understanding of the cell-to-cell interactions that support pancreatic function is still lacking. To characterize the pancreatic endocrine compartment, we studied pancreata from healthy adult donors and investigated a single cell surface adhesion molecule, desmoglein-2 (DSG2). Genetically-modified mice lacking Dsg2 were examined for islet cell mass, insulin production, responses to glucose, susceptibility to a streptozotocin-induced mouse model of hyperglycaemia, and ability to cure diabetes in a syngeneic transplantation model. Herein, we have identified DSG2 as a previously unrecognized adhesion molecule that supports β-cells. Furthermore, we reveal that DSG2 is within the top 10 percent of all genes expressed by human pancreatic islets and is expressed by the insulin-producing β-cells but not the somatostatin-producing δ-cells. In a Dsg2 loss-of-function mice (Dsg2lo/lo), we observed a significant reduction in the number of pancreatic islets and islet size, and consequently, there was less total insulin content per islet cluster. Dsg2lo/lo mice also exhibited a reduction in blood vessel barrier integrity, an increased incidence of streptozotocin-induced diabetes, and islets isolated from Dsg2lo/lo mice were more susceptible to cytokine-induced β-cell apoptosis. Following transplantation into diabetic mice, islets isolated from Dsg2lo/lo mice were less effective than their wildtype counterparts at curing diabetes. In vitro assays using the Beta-TC-6 murine β-cell line suggest that DSG2 supports the actin cytoskeleton as well as the release of cytokines and chemokines. Taken together, our study suggests that DSG2 is an under-appreciated regulator of β-cell function in pancreatic islets and that a better understanding of this adhesion molecule may provide new opportunities to combat type 1 diabetes.

Abstract Synthetic materials with an innate ability to avoid the foreign-body-response remain an unrealized goal that would transform the medical device industry. The balance of bulk material properties, that enable a device to perform as intended, with surface properties, that provide bio- and hemocompatibility, has always required the former to be prioritized. While all materials used in modern devices have an acceptable level of biocompatibility, imperfection remains, ultimately leading to device failure, or requiring pharmacological intervention for it to be tolerated. Where such devices cause damage or place strain on the normal architecture of the surrounding tissue, these impacts may initiate inflammatory responses that can also led to failure. This is most evident in the treatment of vessels in the lower extremity in patients with peripheral arterial disease (PAD), where in-stent restenosis (ISR) remains a significant challenge for vascular surgeons. Blood-contacting devices, such as stents and artificial grafts, are considered particularly difficult to shield from the foreign-body-response due to the immediate and direct exposure to blood and therefore, the full gamut of the body’s immune responses. Pharmacological treatment is currently paramount to successful percutaneous vascular intervention (PVI) with antiplatelet therapies being prescribed to manage the risk of thrombosis and cytotoxic drug-eluting coatings to reduce restenosis. Here, we present data that indicate a nano-thin coating of hyperbranched polyglycerol (HPG) can greatly improve the safety and durability of endovascular metal stents. The HPG coating successfully prevents the binding and activation of platelets and greatly reduces the thrombogenicity of nitinol stents when studied ex vivo, using fresh human blood. In vivo, HPG-coated stainless-steel stents remained patent after 28 days in apolipoprotein E (ApoE) knockout mice while control stents all became completely occluded, highlighting the HPG coating’s ability to reduce restenosis. Together, these properties could help alleviate the industry’s dependence on blood-thinning and antiproliferative drugs to resolve device compatibility issues; thereby greatly improve patients’ quality of life through faster recovery, fewer complications and fewer repeat interventions. Furthermore, this coating technology is compatible with a range of materials commonly used in the production of implantable medical devices, such as stainless steel, nitinol, silicone, and polytetrafluoroethylene, while being highly scalable, cost effective and stable. Taken together, HPG presents itself as an alternative coating suitable for a broad range of vascular devices including stents and grafts.

The growth of solid tumours relies on an ever-increasing supply of oxygen and nutrients that are delivered via vascular networks. Tumour vasculature includes endothelial cell lined angiogenesis and the less common cancer cell lined vasculogenic mimicry (VM). To study and compare the development of vascular networks formed during angiogenesis and VM (represented here by breast cancer and pancreatic cancer cell lines) a number of in vitro assays were utilised. From live cell imaging, we performed a large-scale automated extraction of network parameters and identified properties not previously reported. We show that for both angiogenesis and VM, the characteristic network path length reduces over time; however, only endothelial cells increase network clustering coefficients thus maintaining small-world network properties as they develop. When compared to angiogenesis, the VM network efficiency is improved by decreasing the number of edges and vertices, and also by increasing edge length. Furthermore, our results demonstrate that angiogenic and VM networks appear to display similar properties to road traffic networks and are also subject to the well-known Braess paradox. This quantitative measurement framework opens up new avenues to potentially evaluate the impact of anti-cancer drugs and anti-vascular therapies.

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-α 3 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.

Hyperbranched polyglycerol (HPG) was previously investigated as a nonfouling hydrophilic grafted layer on biomaterial surfaces, analogous to the well-known poly(ethylene oxide) (PEO), but the range of adsorbing cells and proteins tested was limited and at times the assays used were not the most sensitive. Thus, the questions arise whether HPG-grafted layers can indeed efficiently resist adsorption of a wider range of adsorbing biological entities, and how would different biological entities interact with such a coating. An HPG coating of 25 nm thickness was grafted onto a spin-coated and plasma-treated polystyrene (PS) layer on a silicon wafer substrate; this provided a well-suited system for surface analyses by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and atomic force microscopy (AFM), which verified the presence of a uniform, smooth grafted HPG layer. Adsorption of bovine serum albumin, lysozyme, fibrinogen, and endothelial cell growth medium 2 (EGM2) was reduced by >90%, with the adsorbed amounts close to the detection limit of XPS but still detectable by ToF-SIMS using principal component analysis. With human serum, however, the reduction in adsorption was slightly less pronounced. Smooth muscle cells (SMCs) and fibroblasts were virtually unable to attach onto the grafted HPG layer, with >99% reductions at 6 h compared with plasma-treated PS; the few attached cells remaining rounded and unable to spread. Their attachment might have resulted from coating defects. Testing with full blood showed that unlike for the control surface (plasma-treated PS), platelets did not adhere to the HPG surface, but there was attachment of some cells that stained CD11b positive and likely are neutrophils. Cells of the fungal organism Candida albicans were also able to attach onto the HPG surface to a limited extent, but in contrast to the control surface, the attached cells on HPG did not form hyphal extensions and thus seem to be compromised in their ability to invade and to form biofilms. Our data suggest that "low-fouling" is a better term than nonfouling for a grafted HPG layer as the resistance to adsorption is not uniform across a range of proteins and cells. It is also important in future work to study whether the cells that do attach can still exert their normal functions; our observation of the absence of hyphal extensions for C. albicans suggests that this may not be so. Hence, the potential utility of a grafted HPG layer may be not just a function of adsorbed amounts but also of the functionality of adsorbed proteins and cells.

Tumour vasculature supports the growth and progression of solid cancers with both angiogenesis (endothelial cell proliferation) and vasculogenic mimicry (VM, the formation of vascular structures by cancer cells themselves) predictors of poor patient outcomes. Increased circulating platelet counts also predict poor outcome for cancer patients but the influence of platelets on tumour vasculature is incompletely understood. Herein, we show with in vitro assays that platelets did not influence angiogenesis but did actively inhibit VM formation by cancer cell lines. Both platelet sized beads and the releasates from platelets were partially effective at inhibiting VM formation suggesting that direct contact maximises the effect. Platelets also promoted cancer cell invasion in vitro. B16F10 melanomas in Bcl-xPlt20/Plt20 thrombocytopenic mice showed a higher content of VM than their wildtype counterparts while angiogenesis did not differ. In a xenograft mouse model of breast cancer with low-dose aspirin to inactivate the platelets, the burden of MDA-MB-231-LM2 breast cancer cells was reduced and the gene expression profile of the cancer cells was altered; but no effect on tumour vasculature was observed. Taken together, this study provides new insights into the action of platelets on VM formation and their involvement in cancer progression.