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Simple Summary: Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic tumours, representing greater than 90% of all diagnosed cases of pancreatic cancer. PDAC has the lowest 5-year survival rate of all tumour malignancies with less than 9% patient survival. A unique feature of PDAC tumours is the presence of a dense fibrotic fortress that creates a physical barrier around the cancer cells, thus resulting in a reduced penetrability of drugs and a ‘sanctuary’ in which cancer cells thrive, termed desmoplasia. Extensive desmoplasia in the PDAC tumour microenvironment (TME) is a crucial factor that influences PDAC development, progression, metastasis, and resistance to treatment. This review will focus on the role of the TME in PDAC, current treatments for PDAC, and a reflection on past and current clinical trials targeting components of the TME in PDAC. An overabundance of desmoplasia in the tumour microenvironment (TME) is one of the defining features that influences pancreatic ductal adenocarcinoma (PDAC) development, progression, metastasis, and treatment resistance. Desmoplasia is characterised by the recruitment and activation of fibroblasts, heightened extracellular matrix deposition (ECM) and reduced blood supply, as well as increased inflammation through an influx of inflammatory cells and cytokines, creating an intrinsically immunosuppressive TME with low immunogenic potential. Herein, we review the development of PDAC, the drivers that initiate and/or sustain the progression of the disease and the complex and interwoven nature of the cellular and acellular components that come together to make PDAC one of the most aggressive and difficult to treat cancers. We review the challenges in delivering drugs into the fortress of PDAC tumours in concentrations that are therapeutic due to the presence of a highly fibrotic and immunosuppressive TME. Taken together, we present further support for continued/renewed efforts focusing on aspects of the extremely dense and complex TME of PDAC to improve the efficacy of therapy for better patient outcomes. Refereed/Peer-reviewed

Leucine-rich repeat containing G-protein coupled receptor (LGR5) is stem cell marker that potentiates canonical signaling in the Wnt/β-catenin signaling pathway (Barker et al. 2007). LGR5 is known to be expressed on several tissues; particularly the stem cells within intestinal crypts and the stem cell niche at the base of hair follicles (Barker et al. 2007 and Jaks et al. 2008). The plasticity and stem phenotype of LGR5+ cells has led to their identification as a cancer stem cell marker for some solid tumors including colorectal cancer (CRC) (Barker et al. 2009). This presents LGR5 as a potential target molecule for CAR-T cell directed specific tumor cell killing. For treatment of CRC with LGR5-directed CAR-T cells, it is critical to gain an understanding of the level of LGR5 expression on normal tissue compared with cancerous tissue. The aim of this study was to develop a screening technique for patient selection, and importantly identify potential responders to LGR5-targeting CAR-T cell therapy.Utilizing commercially purchased tissue microarrays a cohort of patient primary tumor biopsies (n=472) were assessed for LGR5 expression; ~60% displayed moderate to high level LGR5 positivity, with a trend toward higher grade, later stage tumors having higher levels of LGR5. LGR5 expression levels on lymph node metastases showed a positive correlation to LGR5 expression on patient-matched primary tumor tissue. Seven out of ten liver biopsies from metastatic colon cancer lesions showed a medium to high level LGR5 staining, indicating this may be used as a screening tool for metastatic CRC patients. We also observed restricted expression of LGR5 across the many normal tissues, suggesting limited off-target effects of LGR5-targeting CAR-T cells.The data obtained in this study provides evidence that LGR5 IHC of biopsies could be used as a diagnostic screening tool of CRC patients for inclusion into a Phase1a/2 clinical trial. Barker N, et al., 2007, Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449: 1003–1007 Barker N. et al. 2009, Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457: 608–611Jaks V. et al. 2008, Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet. Nov;40(11):1291-9. Citation Format: Emma J. Thompson, Veronika Bandara, Batjargal Gundsambuu, Silvana Napoli, Stuart Mills, Jade Foeng, Dylan McPeake, Timona Tyllis, Caitlin Abbott, Allison Cowin, Timothy Sadlon, Shaun McColl, Carmela Ricciardelli, Simon C. Barry, Claudine S. Bonder. Assessing LGR5 expression levels on colorectal cancer tissue samples for use in LGR5-targeting CAR-T cell therapy clinical trial. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5545.

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. Refereed/Peer-reviewed

Chimeric antigen receptor (CAR)-T immunotherapy is a novel treatment that genetically modifies the patient’s own T cells to target and kill malignant cells. CAR-T cells demonstrated robust clinical activity against certain B-cell malignancies. However, identification of tumour-specific antigens expressed on multiple cancer types, especially on solid cancers, remains a major challenge. P2X purinoceptor 7 (P2X7) is an ATP gated cation channel that forms homotrimers and heterotrimers at the cell surface. When functioning normally, it controls ion transport in response to ATP. A dysfunctional version of P2X7, named nfP2X7, has been identified on cancer cells from a range of tissues, while being undetectable on healthy cells. We generated prototype nfP2X7-targeting human CAR-T cells, which demonstrated effective antigen-specific cytotoxicity against twelve solid cancer types including breast, prostate, lung, colorectal, brain and skin in vitro. In preclinical xenograft mouse models of aggressive breast and prostate cancer, CAR-T cells targeting nfP2X7 exhibited robust anti-tumour efficacy. These data indicate CAR-T cells targeting nfP2X7 have potential as a novel broad-spectrum cancer immunotherapy for solid tumours in humans.

SUMMARYConverging evidence indicates that extra-embryonic yolk sac is the source of both macrophages and endothelial cells in adult mouse tissues. Prevailing views are that these yolk sac-derived cells are maintained after birth by proliferative self-renewal in their differentiated states. Here we identify clonogenic, self-renewing endothelial-macrophage (EndoMac) progenitor cells in postnatal mouse aorta, heart and lung, that are independent of definitive hematopoiesis and derive from a CX3CR1+ and CSF1R+ yolk sac source. These bipotent progenitors are highly proliferative and vasculogenic, contributing to adventitial neovascularization in the aortic wall and forming perfused blood vessels after adoptive transfer into ischemic tissue. We establish a regulatory role for angiotensin II, which enhances their clonogenic, self-renewal and differentiation properties. Our findings demonstrate that tissue-resident EndoMac progenitors drive local inflammatory and vasculogenic responses by contributing to the renewal and expansion of yolk sac-derived macrophages and endothelial cells postnatally.

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.

Current challenges for CAR-T therapies in solid tumors relate to target specificity and T cell penetration of the tumor. A key determinant in CAR-T selection for clinical trials is the potency and persistence of the selected CAR-T to kill cancerous cells. This is often difficult to determine across different donors and batches. Additionally, these characteristics can be difficult to determine in vitro in a rapid, high-throughput manner. In this study we have utilized real-time impedance cytotoxicity assays to screen LGR5-targeting CAR-T cells. LGR5 is a well described cancer stem cell antigen implicated in tumor initiation and metastasis, and prognosis of colon, ovarian and neuroblastoma cancers. Impedance measurements are taken continuously, providing comprehensive real-time information about CAR-T cell efficiency over time, which is not achievable with single timepoint cytotoxicity assays. Through this validation process we have been able to concurrently test different Effector to Target (E:T) ratios and different CAR constructs to accurately and directly compare CAR-T killing efficiency. This readout is a key indicator of CAR-T potency. Similar to inhibitory concentration 50 (IC50) or lethal dose 50 (LD50) data, CAR-T potency can be expressed in a measure as kill time 50 (KT50), and represents the time required for the CAR-T to stimulate cytolysis in 50% of the target cancer cells. KT50 data was key to the selection of our lead clinical candidate; CNA3103. In addition to selection of our lead candidate, real-time impedance technology has allowed us to screen a broad range of LGR5-expressing cancer types. Our data indicates that CNA3103 can effectively target colon, neuroblastoma, and ovarian cancer cells in vitro. Real-time impedance assay has allowed us to analyze cancer cells with different levels of LGR5 expression to confirm the specificity of our CAR-T cells and to develop an understanding of how LGR5 expression levels might impact cancer cell killing. The data also indicates that our LGR5 targeting CAR-T cells have the potential to repeatedly kill, i.e. the same CAR-T cell is able to move on from one cancer cell to another and remain cytotoxic. Taken together, real-time cytotoxicity assays provide valuable pre-clinical data, enabling the selection of CNA3103 as our LRG5-CAR-T clinical candidate. This technology may also assist in patient and dose selection within a clinical trial. Citation Format: Emma J. Thompson, Veronika Bandara, Timothy Sadlon, Batjargal Gundsambuu, Lih Yin Tan, Carmela Ricciardelli, Simon C. Barry, Claudine S. Bonder. Real-time cytotoxicity assays as a pre-clinical screening tool for LGR5-targeting CAR-T cells for treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5184.

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. Refereed/Peer-reviewed

Cutaneous squamous cell carcinoma (cSCC) accounts for 25% of cutaneous malignancies diagnosed in Caucasian populations. Surgical removal in combination with radiation and chemother-apy are effective treatments for cSCC. Nevertheless, the aggressive metastatic forms of cSCC still have a relatively poor patient outcome. Studies have linked actin cytoskeletal dynamics and the Wnt/β-catenin signaling pathway as important modulators of cSCC pathogenesis. Previous studies have also shown that the actin-remodeling protein Flightless (Flii) is a negative regulator of cSCC. The aim of this study was to investigate if the functional effects of Flii on cSCC involve the Wnt/β-catenin signaling pathway. Flii knockdown was performed using siRNA in a human late stage aggressive metastatic cSCC cell line (MET-1) alongside analysis of Flii genetic murine models of 3-methylcholanthrene induced cSCC. Flii was increased in a MET-1 cSCC cell line and reducing Flii expression led to fewer PCNA positive cells and a concomitant reduction in cellular proliferation and symmetrical division. Knockdown of Flii led to decreased β-catenin and a decrease in the expression of the downstream effector of β-catenin signaling protein SOX9. 3-Methylcholanthrene (MCA)-induced cSCC in Flii overexpressing mice showed increased markers of cancer metastasis in-cluding talin and keratin-14 and a significant increase in SOX9 alongside a reduction in Flii associated protein (Flap-1). Taken together, this study demonstrates a role for Flii in regulating proteins involved in cSCC proliferation and tumor progression and suggests a potential role for Flii in aggressive metastatic cSCC. Refereed/Peer-reviewed

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., Fouladzadeh, Dorraki and colleagues investigate the development of angiogenic networks for in vitro cancer cell lines. They demonstrate that during the growth stages of vasculogenic mimicry, the number of edges and vertices decreases but the edge length increases resulting in improved network efficiency.

BackgroundNovel targeted therapies for children diagnosed with medulloblastoma (MB), the most common malignant pediatric brain tumor, are urgently required. A major hurdle in the development of effective therapies is the impaired delivery of systemic therapies to tumor cells due to a specialized endothelial blood-brain barrier (BBB). Accordingly, the integrity of the BBB is an essential consideration in any preclinical model used for assessing novel therapeutics. This study sought to assess the functional integrity of the BBB in several preclinical mouse models of MB.MethodsDynamic contrast enhancement magnetic resonance imaging (MRI) was used to evaluate blood-brain-tumor barrier (BBTB) permeability in a murine genetically engineered mouse model (GEMM) of Sonic Hedgehog (SHH) MB, patient-derived orthotopic xenograft models of MB (SHH and Gp3), and orthotopic transplantation of GEMM tumor cells, enabling a comparison of the direct effects of transplantation on the integrity of the BBTB. Immunofluorescence analysis was performed to compare the structural and subcellular features of tumor-associated vasculature in all models.ResultsContrast enhancement was observed in all transplantation models of MB. No contrast enhancement was observed in the GEMM despite significant tumor burden. Cellular analysis of BBTB integrity revealed aberrancies in all transplantation models, correlating to the varying levels of BBTB permeability observed by MRI in these models.ConclusionsThese results highlight functional differences in the integrity of the BBTB and tumor vessel phenotype between commonly utilized preclinical models of MB, with important implications for the preclinical evaluation of novel therapeutic agents for MB.

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Aims: Cardiovascular disease (CVD) is the leading cause of mortality in patients with end‐stage renal disease (ESRD) receiving maintenance hemodialysis treatment. This study investigated the effect of a 12‐week intradialytic progressive resistance training (PRT) intervention on pulse wave velocity (PWV) and associated hemodynamic, anthropometric, and hematologic outcomes in patients with ESRD. Method: Twenty‐two patients with ESRD (59% men, 71.3 ± 11.0 years) were recruited. Supervised PRT (3 sets of 11 exercises) was prescribed three times per week during routine dialysis. The primary outcome was brachial‐ankle PWV via applanation tonometry. Secondary outcomes included augmentation index, brachial and aortic blood pressures, endothelial progenitor cells, C‐reactive protein, blood lipids and anthropometrics. Results: The intradialytic PRT regimen resulted in no significant change in PWV between control and intervention periods [mean difference = 0 (95% CI = ‐0.1 to 0.1); P=0.58]. Similarly, no significant change was noted in any secondary outcome measures between the control and intervention periods. Post‐hoc analyses limited to high adherers (≥75% attendance; n=11) did not differ from the primary analysis, indicating no dose‐response effect of our intervention Conclusion: Our 12‐week PRT intervention did not change PWV or any secondary outcomes. Future studies should determine if higher dosages of intradialytic PRT (i.e. longer duration and/or higher intensity) can be applied as a method improve arterial stiffness to potentially reduce cardiovascular disease and associated mortality this cohort Refereed/Peer-reviewed

Biologically compatible fluorescent ion sensors, particularly those that are reversible, represent a key tool for answering a range of fundamental biological questions. We report a rationally designed probe with a 6′-fluoro spiropyran scaffold (5) for the reversible sensing of zinc (Zn 2+ ) in cells. The 6′-fluoro substituent overcomes several limitations normally associated with spiropyran-based sensors to provide an improved signal-To-background ratio and faster photoswitching times in aqueous solution. In vitro studies were performed with 5 and the 6′-nitro analogues (6) in HEK 293 and endothelial cells. The new spiropyran (5) can detect exogenous Zn 2+ inside both cell types and without affecting the proliferation of endothelial cells. Studies were also performed on dying HEK 293 cells, with results demonstrating the ability of the key compound to detect endogenous Zn 2+ efflux from cells undergoing apoptosis. Biocompatibility and photoswitching of 5 were demonstrated within endothelial cells but not with 6, suggesting the future applicability of sensor 5 to study intracellular Zn 2+ efflux in these systems. Refereed/Peer-reviewed

Administration of bolus intravenous fluid is associated with respiratory dysfunction and increased mortality, findings with no clear mechanistic explanation. The objective of this study was to examine whether bolus intravenous (i.v.) fluid administration results in acute lung injury in a rat model and further, to examine whether this injury is associated with transient receptor potential vallinoid (TRPV)4 channel function and endothelial inflammatory response. Healthy male Sprague-Dawley rats were administered 60 ml/kg 0.9% saline i.v. over 30 min. Manifestation of acute lung injury was assessed by lung physiology, morphology, and markers of inflammation. The role of TRPV4 channels in fluid-induced lung injury was subsequently examined by the administration of ruthenium red (RR) in this established rat model and again in TRPV4 KO mice. In endothelial cell culture, permeability and P-selectin expression were measured following TRPV4 agonist with and without antagonist; 0.9% saline resulted in an increase in lung water, lavage protein and phospholipase A2, and plasma angiopoietin-2, with worsening in arterial blood oxygen (PaO2), lung elastance, surfactant activity, and lung histological injury score. These effects were ameliorated following i.v. fluid in rats receiving RR. TRPV4 KO mice did not develop lung edema. Expression of P-selectin increased in endothelial cells following administration of a TRPV4 agonist, which was ameliorated by simultaneous addition of RR. Bolus i.v. 0.9% saline resulted in permeability pulmonary edema. Data from ruthenium red, TRPV4 KO mice, and endothelial cell culture suggest activation of TRPV4 and release of angiopoietin 2 and P-selectin as the central mechanism. Refereed/Peer-reviewed

Skin cancer is associated with abnormal cellular metabolism which if identified early introduces the possibility of intervention to prevent its progress to a deadly metastatic stage. This study combines multiphoton microscopy with fluorescence lifetime imaging (FLIM) using an syngeneic melanoma mouse model, to detect changes in metabolic state of single epidermal cells as a metabolic marker to monitor the progress of tumor growth. This method utilizes imaging of the ratio of the amounts of the free and protein-bound forms of the intracellular autofluorescent metabolic co-enzyme nicotinamide adenine dinucleotide (NADH). Here, we investigate the impact of the primary tumor lesion on the epidermal layers at three different growth stages of melanoma lesion compared to normal skin as a control. We show a significant increase in the free-to-bound NADH ratio with the growth of the solid melanoma tumor, while concurrently the short and the long lifetime components of NADH remained constant. These results demonstrate the potential of FLIM for rapid, non-invasive and sensitive assessment of melanoma progression revealing its potential as a diagnostic tool for melanoma detection and as an aid for melanoma staging. Refereed/Peer-reviewed

Pancreatic islet transplantation is a promising clinical treatment for type 1 diabetes, but success is limited by extensive -cell death in the immediate posttransplant period and impaired islet function in the longer term. Following transplantation, appropriate vascular remodeling is crucial to ensure the survival and function of engrafted islets. The sphingosine kinase (SK) pathway is an important regulator of vascular beds, but its role in the survival and function of transplanted islets is unknown. We observed that donor islets from mice deficient in SK1 (Sphk1 knockout) contain a reduced number of resident intraislet vascular endothelial cells. Furthermore, we demonstrate that the main product of SK1, sphingosine-1-phosphate, controls the migration of intraislet endothelial cells in vitro. We reveal in vivo that Sphk1 knockout islets have an impaired ability to cure diabetes compared with wild-type controls. Thus, SK1-deficient islets not only contain fewer resident vascular cells that participate in revascularization, but likely also a reduced ability to recruit new vessels into the transplanted islet. Together, our data suggest that SK1 is important for islet revascularization following transplantation and represents a novel clinical target for improving transplant outcomes. Refereed/Peer-reviewed

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 fora 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 hyphalextensions 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. Refereed/Peer-reviewed

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.

Background Macrophages and endothelial cells share an intimate relationship during neovessel formation in different pathophysiological conditions. Recent studies have determined that in some tissues, both cell types are derived embryonically from yolk sac (YS) progenitor cells and are maintained postnatally without contribution from circulating sources. The mechanism by which this local “self-maintenance” occurs is unknown. Purpose We previously identified that mouse arteries contain macrophage and endothelial progenitor cells in their adventitial Sca-1+CD45+ compartment. Here we investigated at a clonal level for the existence of postnatal adventitial haemangioblasts and studied their developmental origins. Methods and results Single cell digests were prepared from murine aortas to perform colony-forming unit (CFU) assays in methylcellulose. Aortic cells from C57BL/6J mice selectively generated macrophage colonies (CFU-M) which contained progenitor cells that displayed >95% positive for expression of CD45, Sca-1, c-Kit, CX3CR1 and CSF1R, but negative for Lineage markers, as well as mature monocyte/macrophage (CD11b, F4/80) and endothelial (CD144) markers. Secondary replating of CFU-M progenitors from adult aortas revealed their self-renewal capacity, with 1 in 10 cells forming new CFU-M. Lineage mapping using Flt3CrexRosamT/mG mice demonstrated that aortic CFU-M progenitors were FLT3-ve, indicating that they were not derived from definitive bone marrow haematopoiesis. CFU-M prevalence in C57BL/6J aortas was highest in neonatal mice and diminished progressively with increasing age (∼100 per 105 cells at P1, ∼15 at 12w, ∼5 at 52w, P4/gp), consistent with prenatal seeding. Embryonic profiling determined that CFU-M progenitors first appeared in extra-embryonic yolk sac around E9.5 and in aorta-gonad-mesonephros at E10.5, before the emergence of definitive haematopoietic stem cells. Inducible fate-mapping then confirmed that aortic CFU-M progenitors originated from CX3CR1+ and CSF1R+ cells in E9.5 yolk sac. Both yolk sac and postnatal aortic CFU-M progenitors generated vascular-like networks when cultured in Matrigel in vitro, containing M2-like macrophages (CD11b+F4/80+CD206+) and endothelial cells (CD31+CD144+). They produced similar progeny and rescued adventitial vascular sprouting when seeded around aortic rings whose adventitia had been stripped. Finally, adoptive transfer of CFU-M progenitors into a mouse model of hindlimb ischaemia resulted in 80% augmentation in hindlimb perfusion compared to cell-free control, with de novo transformation of donor cells into macrophages, endothelial cells and perfused neovessels (n=6). Conclusion To the best of our knowledge, this is the first ever definitive proof at a clonal level for the existence of haemangioblasts in postnatal tissue. Adventitial haemangioblasts originate from extra-embryonic YS and are a source of vasculogenesis in the arterial wall, relevant to vasa vasorum formation. Acknowledgement/Funding NHMRC of Australia (GNT1086796, CDF1161506), NHFA (FLF100412, FLF102056) Royal Australasian College of Physicians

The cellular origins of vasa vasorum are ill-defined and may involve circulating or local progenitor cells. We previously discovered that murine aortic adventitia contains Sca-1+CD45+ progenitors that produce macrophages. Here we investigated whether they are also vasculogenic. In aortas of C57BL/6 mice, Sca-1+CD45+ cells were localised to adventitia and lacked surface expression of endothelial markers (ApoE−/− aortas. Although Sca-1+CD45+ cells from C57BL/6 aorta did not express CD31, they formed CD31+ colonies in endothelial differentiation media and produced interconnecting vascular-like cords in Matrigel that contained both endothelial cells and a small population of macrophages, which were located at branch points. Transfer of aortic Sca-1+CD45+ cells generated endothelial cells and neovessels de novo in a hindlimb model of ischaemia and resulted in a 50% increase in perfusion compared to cell-free control. Similarly, their injection into the carotid adventitia of ApoE−/− mice produced donor-derived adventitial and peri-adventitial microvessels after atherogenic diet, suggestive of newly formed vasa vasorum. These findings show that beyond its content of macrophage progenitors, adventitial Sca-1+CD45+ cells are also vasculogenic and may be a source of vasa vasorum during atherogenesis.

Nitric oxide (NO) is a key regulator of endothelial cell and vascular function. The direct measurement of NO is challenging due to its short half-life, and as such surrogate measurements are typically used to approximate its relative concentrations. Here we demonstrate that ruthenium-based [Ru(bpy)2(dabpy)]2+ is a potent sensor for NO in its irreversible, NO-bound active form, [Ru(bpy)2(T-bpy)]2+. Using spectrophotometry we established the sensor’s ability to detect and measure soluble NO in a concentration-dependent manner in cell-free media. Endothelial cells cultured with acetylcholine or hydrogen peroxide to induce endogenous NO production showed modest increases of 7.3 ± 7.1% and 36.3 ± 25.0% respectively in fluorescence signal from baseline state, while addition of exogenous NO increased their fluorescence by 5.2-fold. The changes in fluorescence signal were proportionate and comparable against conventional NO assays. Rabbit blood samples immediately exposed to [Ru(bpy)2(dabpy)]2+ displayed 8-fold higher mean fluorescence, relative to blood without sensor. Approximately 14% of the observed signal was NO/NO adduct-specific. Optimal readings were obtained when sensor was added to freshly collected blood, remaining stable during subsequent freeze-thaw cycles. Clinical studies are now required to test the utility of [Ru(bpy)2(dabpy)]2+ as a sensor to detect changes in NO from human blood samples in cardiovascular health and disease.

Objective A key mediator of vascular EC barrier integrity, S1P, is derived from phosphorylation of sphingosine by the SK-1 and SK-2. While previous work indicates that SK-1 can regulate EC barrier integrity, whether SK-2 has a similar role remains to be determined. Methods A cell impedance assay was used to assess human umbilical vein EC and bone marrow EC barrier integrity in vitro, with application of the SK inhibitors ABC294640, PF543, SKi, and MP-A08. In vivo studies were conducted using intravital microscopy to assess EC barrier integrity in SK-1 (Sphk1−/−) and SK-2 (Sphk2−/−) knock-out mice. Results Only ABC294640 and MP-A08, which can both inhibit SK-2, caused a decrease in EC barrier integrity in vitro in both cell types. Intravital microscopy revealed that Sphk1−/− mice had reduced EC barrier integrity compared to WT mice, whereas no change was evident in Sphk2−/− mice. Conclusions Our data suggest that in vitro inhibition of SK-2, can compromise the integrity of the EC monolayer, while SK-1 exerts a more dominant control in vivo. These data may have clinical implications and could aid in the development of new treatments for disorders of vascular barrier function.

This study outlines the synthetic and purification process used to obtain hyperbranched polyglycerol (HPG) within a molecular weight range not previously obtainable. Purified fractions with low polydispersity are achieved via successive precipitations from acetone. Furthermore, the use of the initiator molecule 5-hexyne-1-ol introduces alkyne functionality at the core of each HPG molecule. Alkyne-azide cycloaddition chemistry in combination with NMR and field flow fractionation analysis is used to verify the availability of the alkyne core for bioconjugation across the range of molecular weights. (Figure presented.). Refereed/Peer-reviewed

Background and objective:Pulmonary arterial hyper-tension (PAH) is characterized by increased resistance in the distal pulmonary arteries, ultimately leading to right heart failure and, despite the available therapeutics, survival remains poor. Reduced expression of bone morphogenetic protein receptor type 2 (BMPR2) is strongly associated with PAH. Cell therapies are of interest in PAH, but whether this approach can upregulate BMPR2 is not known. Our objective was to evaluate a preclinical cell therapy approach based on upregulation of BMPR2 Methods: We assessed the therapeutic effect of intravenously injected BMPR2-augmented rat bone marrow-derived endothelial-like progenitor cells (BMPR2-BM-ELPC) on PAH in the rat monocrotaline (MCT) model. Results: The cells accumulate in the lungs with negligible systemic distribution, but the vast majority are lost from the lungs by 24 h. Lungs from rats treated with BMPR2-BM-ELPC exhibited an immediate increase in BMPR2 and related intracellular signalling proteins. Treatment with BMPR2-BM-ELPC attenuated PAH as demonstrated by a reduction in right ventricular hypertrophy as well as right ventricular systolic and mean pulmonary arterial pressures. In addition, this treatment reversed PAH-induced vascular remodelling with a significant reduction in vessel thickness and muscularization. In view of the short retention time of injected cells in the lungs, the mechanism for the effects seen may be intracellular communication via exosomes. In support of this hypothesis, we demonstrate that BMPR2-transduced outgrowth endothelial progenitor cells (OECs) release BMPR2-expressing exosomes. Conclusion: BMPR2-augmented ELPC demonstrate therapeutic benefits in the rat model and may have clinical translation potential Refereed/Peer-reviewed

While the two mammalian sphingosine kinases, SK1 and SK2, both catalyze the generation of pro-survival sphingosine 1-phosphate (S1P), their roles vary dependent on their different subcellular localization. SK1 is generally found in the cytoplasm or at the plasma membrane where it can promote cell proliferation and survival. SK2 can be present at the plasma membrane where it appears to have a similar function to SK1, but can also be localized to the nucleus, endoplasmic reticulum or mitochondria where it mediates cell death. Although SK2 has been implicated in cancer initiation and progression, the mechanisms regulating SK2 subcellular localization are undefined. Here, we report that SK2 interacts with the intermediate chain subunits of the retrograde-directed transport motor complex, cytoplasmic dynein 1 (DYNC1I1 and -2), and we show that this interaction, particularly with DYNC1I1, facilitates the transport of SK2 away from the plasma membrane. DYNC1I1 is dramatically downregulated in patient samples of glioblastoma (GBM), where lower expression of DYNC1I1 correlates with poorer patient survival. Notably, low DYNC1I1 expression in GBM cells coincided with more SK2 localized to the plasma membrane, where it has been recently implicated in oncogenesis. Re-expression of DYNC1I1 reduced plasma membrane-localized SK2 and extracellular S1P formation, and decreased GBM tumor growth and tumor-associated angiogenesis in vivo. Consistent with this, chemical inhibition of SK2 reduced the viability of patient-derived GBM cells in vitro and decreased GBM tumor growth in vivo. Thus, these findings demonstrate a tumor-suppressive function of DYNC1I1, and uncover new mechanistic insights into SK2 regulation which may have implications in targeting this enzyme as a therapeutic strategy in GBM. Refereed/Peer-reviewed

usc Purpose: Mechanical ventilation is a well-established therapy for patients with acute respiratory failure. However, up to 35% of mortality in acute respiratory distress syndrome may be attributed to ventilation-induced lung injury (VILI). We previously demonstrated the efficacy of the synthetic tripeptide feG for preventing and ameliorating acute pancreatitis-associated lung injury. However, as the mechanisms of induction of injury during mechanical ventilation may differ, we aimed to investigate the effect of feG in a rodent model of VILI, with or without secondary challenge, as a preventative treatment when administered before injury (prophylactic), or as a therapeutic treatment administered following initiation of injury (therapeutic). Methods: Lung injury was assessed following prophylactic or therapeutic intratracheal feG administration in a rodent model of ventilation-induced lung injury, with or without secondary intratracheal lipopolysaccharide challenge. Results: Prophylactic feG administration resulted in significant improvements in arterial blood oxygenation and respiratory mechanics, and decreased lung oedema, bronchoalveolar lavage protein concentration, histological tissue injury scores, blood vessel activation, bronchoalveolar lavage cell infiltration and lung myeloperoxidase activity in VILI, both with and without lipopolysaccharide. Therapeutic feG administration similarly ameliorated the severity of tissue damage and encouraged the resolution of injury. feG associated decreases in endothelial adhesion molecules may indicate a mechanism for these effects. Conclusions: This study supports the potential for feG as a pharmacological agent in the prevention or treatment of lung injury associated with mechanical ventilation. Refereed/Peer-reviewed

usc Objective: Activation of epidermal stem cells (EpSCs) from their quiescent niche is an integral component of wound reepithelialization and involves Wnt/β-catenin (β-Cat) signaling and remodeling of the actin cytoskeleton. The aim of this study was to investigate the effect of Flightless I (Flii), a cytoskeletal protein and inhibitor of wound healing, on EpSC activation during wound repair. Approach: Genetically modified Flii mice (Flii knockdown: Flii+/−, wild type: WT, Flii overexpressing: FliiTg/Tg) received two incisional wounds along the lateral axis of the dorsal skin. Indicators of EpSC activation (epidermal growth factor receptor 1 [EGFR1], leucine-rich repeats and immunoglobulin-like domains-1 [Lrig1], K14), Wnt/β-Cat signaling (Lgr6, Flap2, β-Cat, and axis inhibition protein 2 [Axin2]), and cell proliferation (proliferating cell nuclear antigen [PCNA]) were assessed using immunohistochemistry. β-Cat stabilization was examined using western blotting with cell cycling and differentiation of isolated CD34+ITGA6high EpSCs examined using real time-quantitative polymerase chain reaction after treatment with wound-conditioned media. Results:Flii+/− led to increased numbers of activated EpSCs expressing PCNA, elevated EGFR1, and decreased Lrig1. EpSCs in Flii+/− hair follicle niches adjacent to the wounds also showed expression of Wnt-activation markers including increased β-Cat and Lgr6, and decreased Axin2. EpSCs (CD34+ITGA6high) isolated from Flii+/− unwounded skin showed elevated expression of cell-cycling genes including ΔNp63, filaggrin (Fila), involucrin (Invo), cyclin D1 (Ccnd1), and cell-division cycle protein-20 (Cdc20); and elevated ΔNp63 and Invo after treatment with wound-conditioned media compared with WT and FliiTg/Tg counterparts. Innovation: Flii was identified as an inhibitor of EpSC activation that may explain its negative effects on wound reepithelialization. Conclusion: Flii may inhibit EpSC activation by interrupting Wnt/β-Cat signaling. Strategies that reduce Flii may increase activation of EpSCs and promote reepithelialization of wounds. Refereed/Peer-reviewed

Introduction/Aim: Previously we reported the therapeutic effect of BMPR2 augmented endothelial progenitor cell (EPC) therapy in a rat PH model. EPCs are have a role in vascular repair, angiogenesis and are known to mobilise in response to hypoxia. In this study, we have assessed the functional differences between PAH patient and control peripheral blood derived outgrowth EPCs (OECs) in response to hypoxia. Methods: OECs were isolated and cultured from both PAH and control patient’s peripheral blood (15mL). Following characterisation and expansion, cells were exposed to either normoxia (5%CO2/ atmosO2 and N2) or hypoxia (1%O2/5% CO2/94% N2). Proliferation and apoptosis were assessed using MTT colourimetric assays and flow cytometry. Immunofluorescence (IF) was used to assess the effect of hypoxia on cell matrix changes and reversal of these changes with BMP7 treatment. Protein analysis of cell lysates was conducted via western blots. Results: In hypoxia the proliferation rate of control OECs decreased significantly while the PAH OECs continued to proliferate. This proliferation could be due to apoptosis resistance in PAH OECs as apoptosis assays revealed a lower level of apoptosis after hypoxia exposure for 5 days in PAH lines than in control lines. IF showed significant cellular remodelling via fibronectin staining in all cells in hypoxia, as well as the PAH OECs in normoxia. This was reversed with BMP7 treatment. (Figure) Western blot analysis has shown significant decreses in VEGFR2 and pSmad1/5/8 with a significant increase in p-p38MAPK. Conclusion: PAH derived OECs display functional differences under hypoxic conditions when compared to control OECs.

Stem cells are unique in their ability to self-renew and differentiate into various cell types. Because of these features, stem cells are key to the formation of organisms and play fundamental roles in tissue regeneration and repair. Mechanisms controlling their fate are thus fundamental to the development and homeostasis of tissues and organs. Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are bioactive phospholipids that play a wide range of roles in multiple cell types, during developmental and pathophysiological events.Considerable evidence now demonstrates the potent roles of LPA and S1P in the biology of pluripotent and adult stem cells, from maintenance to repair. Here we review their roles for each main category of stem cells and explore how those effects impact development and physiopathology. Refereed/Peer-reviewed

Severe dengue virus (DENV) infection is associated with overactivity of the complement alternative pathway (AP) in patient studies. Here, the molecular changes in components of the AP during DENV infection in vitro were investigated. mRNA for factor H (FH), a major negative regulator of the AP, was significantly increased in DENV-infected endothelial cells (EC) and macrophages, but, in contrast, production of extracellular FH protein was not. This discord was not seen for the AP activator factor B (FB), with DENV induction of both FB mRNA and protein, nor was it seen with Toll-like receptor 3 or 4 stimulation of EC and macrophages, which induces both FH and FB mRNA and protein. Surface-bound and intracellular FH protein was, however, induced by DENV, but only in DENV antigen-positive cells, while in two other DENV-susceptible immortalized cell lines (ARPE-19 and human retinal endothelial cells), FH protein was induced both intracellularly and extracellularly by DENV infection. Regardless of the cell type, there was an imbalance in AP components and an increase in markers of complement AP activity associated with DENV-infected cells, with lower FH relative to FB protein, an increased ability to promote AP-mediated lytic activity, and increased deposition of complement component C3b on the surface of DENV-infected cells. For EC in particular, these changes are predicted to result in higher complement activity in the local cellular microenvironment, with the potential to induce functional changes that may result in increased vascular permeability, a hallmark of dengue disease. IMPORTANCE Dengue virus (DENV) is a significant human viral pathogen with a global medical and economic impact. DENV may cause serious and life-threatening disease, with increased vascular permeability and plasma leakage. The pathogenic mechanisms underlying these features remain unclear; however, overactivity of the complement alternative pathway has been suggested to play a role. In this study, we investigate the molecular events that may be responsible for this observed alternative pathway overactivity and provide novel findings of changes in the complement system in response to DENV infection in primary cell types that are a major target for DENV infection (macrophages) and pathogenesis (endothelial cells) in vivo . Our results suggest a new dimension of cellular events that may influence endothelial cell barrier function during DENV infection that could expand strategies for developing therapeutics to prevent or control DENV-mediated vascular disease.

Background and objective Pulmonary arterial hypertension (PAH) continues to be a fatal disease and is associated with downregulation of bone morphogenetic protein receptor type-2 (BMPR2). Our approach is to upregulate BMPR2 in the pulmonary vasculature allowing us to examine the changes in endothelial cell signalling and better understand what pathways are altered when disease is attenuated using this treatment approach. Methods We used gene delivery of BMPR2 to human pulmonary endothelial cells to investigate downstream signalling, then assessed the impact of this approach on downstream signalling in vivo in rats with PAH using the monocrotaline (MCT) model. Results Gene delivery of BMPR2 leads to an increase in BMPR2 protein expression, and this is associated with increased Smad1/5/8 and reduced Smad2/3 signalling. Additionally, we have found that BMPR2 modulation has effects on non-Smad signalling with increases found in phosphoinositide-3 kinase (PI3K) and a decrease in phosphorylated-p38-mitogen activated protein kinase (p38-MAPK) in vivo. These findings are associated with amelioration of PAH (reduced right ventricular, mean pulmonary artery pressures and Fulton Index). Conclusion These results indicate that the therapeutic effect of BMPR2 gene delivery on PAH is associated with a switch between TGF-β-Smad2/3 signalling to BMPR2-Smad1/5/8 signalling. This supports the further development of this treatment approach.

Although endothelial cell (EC) infection is not widespread during dengue virus (DENV) infection in vivo, the endothelium is the site of the pathogenic effects seen in severe DENV disease. In this study, we investigated DENV infection of primary EC and defined factors that influence infection in this cell type. Consistent with in vivo findings where EC infection is infrequent, only 3%–15% of EC became productively DENV-2-infected in vitro. This low level infection could not be attributed to inhibition by heparin, EC donor variation, heterogeneity, or biological source. DENV-infection of EC was associated with induction of innate immune responses, including increased STAT1 protein, STAT1- phosphorylation, interferon (IFN)-β, OAS-1, IFIT-1/ISG56, and viperin mRNA. Antibody blocking of IFN-β inhibited the induction of OAS1, IFIT1/ISG56, and viperin while shRNA knockdown of viperin enhanced DENV-infection in EC. DENV-infection of EC resulted in increased activity of sphingosine kinase 1, a factor important in maintaining vascular integrity, and altered basal and stimulated changes in barrier integrity of DENV-infected EC monolayers. Thus, DENV productively infects only a small percentage of primary EC but this has a major influence on induction of IFN-β driven innate immune responses that can restrict infection while the EC themselves are functionally altered. These changes may have important consequences for the endothelium and are reflective of pathogenic changes associated with vascular leakage, as seen in DENV disease. Refereed/Peer-reviewed