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.

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.

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 70 th 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.

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.

Bone defects arising from fractures or disease represent a significant problem for surgeons to manage and are a substantial economic burden on the healthcare economy. Recent advances in the development of biomaterial substitutes provides an attractive alternative to the current "gold standard" autologous bone grafting. Despite ongoing research, we are yet to identify cost effective biocompatible, osteo-inductive factors that stimulate controlled, accelerated bone regeneration.We have recently reported that enzymes with peroxidase activity possess previously unrecognised roles in extracellular matrix biosynthesis, angiogenesis and osteoclastogenesis, which are essential processes in bone remodelling and repair. Here, we report for the first time, that plant-derived soybean peroxidase (SBP) possesses pro-osteogenic ability by promoting collagen I biosynthesis and matrix mineralization of human osteoblasts in vitro. Mechanistically, SBP regulates osteogenic genes responsible for inflammation, extracellular matrix remodelling and ossification, which are necessary for normal bone healing. Furthermore, SBP was shown to have osteo-inductive properties, that when combined with commercially available biphasic calcium phosphate (BCP) granules can accelerate bone repair in a critical size long bone defect ovine model. Micro-CT analysis showed that SBP when combined with commercially available biphasic calcium phosphate (BCP) granules significantly increased bone formation within the defects as early as 4 weeks compared to BCP alone. Histomorphometric assessment demonstrated accelerated bone formation prominent at the defect margins and surrounding individual BCP granules, with evidence of intramembranous ossification. These results highlight the capacity of SBP to be an effective regulator of osteoblastic function and may be beneficial as a new and cost effective osteo-inductive agent to accelerate repair of large bone defects.

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.

Objective: Osteosarcoma is the most common primary tumor of the bone, predominantly affecting children and adolescents. While localized osteosarcoma can be readily treated with the use of pre-operative chemotherapy in combination with surgery, patients who develop metastatic disease and tumor-induced osteolysis continue to have a poor prognosis. Many cancer cells express tumor-specific antigens, rendering them vulnerable to immune effector T cell killing. There is increasing evidence that highly cytotoxic gamma delta (Vγ9Vδ2) T cells together with the bone anti-resorptive drug zoledronate may hold significant clinical benefit in the treatment of a variety of tumor types. Methods: Ex vivo expanded Vγ9Vδ2 T cells were used to assess effector-mediated killing of osteosarcoma cells (BTK-143 and K-HOS) in response to zoledronate pre-treatment. An orthotopic mouse model of osteolytic osteosarcoma was used to verify Vγ9Vδ2 T cell cytotoxicity in combination with zoledronate on tumor growth, osteolysis and metastasis. Results: Pre-treatment of osteosarcoma cells with zoledronate enhanced Vγ9Vδ2 T cell rapid killing compared to untreated cells in vitro via blockade of the mevalonate pathway. When adoptively transferred into osteosarcoma bearing NOD/SCID mice in vivo, Vγ9Vδ2 T cells in combination with zoledronate potentiated the anti-cancer efficacy of Vγ9Vδ2T cells and inhibited tumor induced osteolysis. Importantly, Vγ9Vδ2 T cells alone reduced both the incidence and burden of lung metastases. Conclusion: This study demonstrated the dual-action of zoledronate to enhance the immunogenicity of osteosarcoma cells to Vγ9Vδ2 T cell cytotoxicity and provide protection against tumor-induced osteolysis.

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, well known for their antimicrobial activity, are released in high quantities by infiltrating immune cells in breast cancer. However, the functional importance of their presence within the tumour microenvironment is unclear. We have recently described a new role for peroxidases as key regulators of fibroblast and endothelial cell functionality. In the present study, we investigate for the first time, the ability of peroxidases to promote breast cancer development and progression. Using the 4T1 syngeneic murine orthotopic breast cancer model, we examined whether increased levels of peroxidases in developing mammary tumours influences primary tumour growth and metastasis. We showed that MPO and EPO stimulation increased mammary tumour growth and enhanced lung metastases, effects that were associated with reduced tumour necrosis, increased collagen deposition and neo-vascularisation within the primary tumour. In vitro, peroxidase treatment, robustly stimulated human mammary fibroblast migration and collagen type I and type VI secretion. Mechanistically, peroxidases induced the transcription of pro-tumorigenic and metastatic MMP1, MMP3 and COX-2 genes. Taken together, these findings identify peroxidases as key contributors to cancer progression by augmenting pro-tumorigenic collagen production and angiogenesis. Importantly, this identifies inflammatory peroxidases as therapeutic targets in breast cancer therapy.

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, hypoxia also leads to treatment opportunities as demonstrated by the development of compounds that target regions of hypoxia within tumors. Evofosfamide is a hypoxia‐activated prodrug that is created by linking the hypoxia‐seeking 2‐nitroimidazole moiety to the cytotoxic bromo‐isophosphoramide mustard (Br‐IPM). When evofosfamide is delivered to hypoxic regions of tumors, the DNA cross‐linking toxin, Br‐IPM, is released leading to cell death. This study assessed the anticancer efficacy of evofosfamide in combination with the Proapoptotic Receptor Agonists (PARAs) dulanermin and drozitumab against human osteosarcoma in vitro and in an intratibial murine model of osteosarcoma. Under hypoxic conditions in vitro, evofosfamide cooperated with dulanermin and drozitumab, resulting in the potentiation of cytotoxicity to osteosarcoma cells. In contrast, under the same conditions, primary human osteoblasts were resistant to treatment. Animals transplanted with osteosarcoma cells directly into their tibiae developed mixed osteosclerotic/osteolytic bone lesions and consequently developed lung metastases 3 weeks post cancer cell transplantation. Tumor burden in the bone was reduced by evofosfamide treatment alone and in combination with drozitumab and prevented osteosarcoma‐induced bone destruction while also reducing the growth of pulmonary metastases. These results suggest that evofosfamide may be an attractive therapeutic agent, with strong anticancer activity alone or in combination with either drozitumab or dulanermin against osteosarcoma.

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, hypoxia offers treatment opportunities, exemplified by the development of compounds that target hypoxic regions within tumors. Evofosfamide (TH-302) is a prodrug created by the conjugation of 2-nitroimidazole to bromo-isophosphoramide mustard (Br-IPM). When evofosfamide is delivered to hypoxic regions, the DNA cross-linking effector, Br-IPM, is released. This study assessed the cytotoxic activity of evofosfamide in vitro and its antitumor activity against osteolytic breast cancer either alone or in combination with paclitaxel in vivo. A panel of human breast cancer cell lines were treated with evofosfamide under hypoxia and assessed for cell viability. Osteolytic MDA-MB-231-TXSA cells were transplanted into the mammary fat pad, or into tibiae of mice, allowed to establish and treated with evofosfamide, paclitaxel, or both. Tumor burden was monitored using bioluminescence, and cancer-induced bone destruction was measured using micro-CT. In vitro, evofosfamide was selectively cytotoxic under hypoxic conditions. In vivo evofosfamide was tumor suppressive as a single agent and cooperated with paclitaxel to reduce mammary tumor growth. Breast cancer cells transplanted into the tibiae of mice developed osteolytic lesions. In contrast, treatment with evofosfamide or paclitaxel resulted in a significant delay in tumor growth and an overall reduction in tumor burden in bone, whereas combined treatment resulted in a significantly greater reduction in tumor burden in the tibia of mice. Evofosfamide cooperates with paclitaxel and exhibits potent tumor suppressive activity against breast cancer growth in the mammary gland and in bone.

Bone metastases occur in over 75% of patients with advanced breast cancer and are responsible for high levels of morbidity and mortality. In this study, ex vivo expanded cytotoxic Vγ9Vδ2 T cells isolated from human peripheral blood were tested for their anti-cancer efficacy in combination with zoledronic acid (ZOL), using a mouse model of osteolytic breast cancer. In vitro, expanded Vγ9Vδ2 T cells were cytotoxic against a panel of human breast cancer cell lines, and ZOL pre-treatment further sensitised breast cancer cells to killing by Vγ9Vδ2 T cells. Vγ9Vδ2 T cells adoptively transferred into NOD/SCID mice localised to osteolytic breast cancer lesions in the bone, and multiple infusions of Vγ9Vδ2 T cells reduced tumour growth in the bone. ZOL pre-treatment potentiated the anti-cancer efficacy of Vγ9Vδ2 T cells, with mice showing further reductions in tumour burden. Mice treated with the combination also had reduced tumour burden of secondary pulmonary metastases, and decreased bone degradation. Our data suggests that adoptive transfer of Vγ9Vδ2 T cell in combination with ZOL may prove an effective immunotherapeutic approach for the treatment of breast cancer bone metastases.

The early recruitment of inflammatory cells to sites of bone fracture and trauma is a critical determinant in successful fracture healing. Released by infiltrating inflammatory cells, myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, whose functional involvement in bone repair has mainly been studied in the context of providing a mechanism for oxidative defense against invading microorganisms. We report here novel findings that show peroxidase enzymes have the capacity to stimulate osteoblastic cells to secrete collagen I protein and generate a mineralized extracellular matrix in vitro. Mechanistic studies conducted using cultured osteoblasts show that peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl hydroxylase-dependent manner, which does not require ascorbic acid. Our studies demonstrate that osteoblasts rapidly bind and internalize both MPO and EPO, and the catalytic activity of these peroxidase enzymes is essential to support collagen I biosynthesis and subsequent release of collagen by osteoblasts. We show that EPO is capable of regulating osteogenic gene expression and matrix mineralization in culture, suggesting that peroxidase enzymes may play an important role not only in normal bone repair, but also in the progression of pathological states where infiltrating inflammatory cells are known to deposit peroxidases.

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, well known for their antimicrobial activity, are released in abundance by innate immune infiltrates at sites of inflammation and injury. We have discovered new and previously unrecognised roles for heme peroxidases in extracellular matrix biosynthesis, angiogenesis, and bone mineralisation, all of which play an essential role in skeletal integrity. In this study we used in vitro models of osteoclastogenesis to investigate the effects of heme peroxidase enzymes on osteoclast differentiation and bone resorbing activity, pertinent to skeletal development and remodelling. Receptor activator of nuclear factor kappa B-ligand (RANKL) stimulates the formation of tartate-resistant acid phosphatase (TRAP) positive multinucleated cells and increases bone resorption when cultured with human peripheral blood mononuclear cells (PBMCs) or the RAW264.7 murine monocytic cell line. When RANKL was added in combination with either MPO or EPO, a dose-dependent inhibition of osteoclast differentiation and bone resorption was observed. Notably, peroxidases had no effect on the bone resorbing activity of mature osteoclasts, suggesting that the inhibitory effect of the peroxidases was limited to osteoclast precursor cells. Mechanistically, we observed that osteoclast precursor cells readily internalize peroxidases, and inhibited the phosphorylation of JNK, p38 MAPK and ERK1/2, important signalling molecules central to osteoclastogenesis. Our findings suggest that peroxidase enzymes, like MPO and EPO, may play a fundamental role in inhibiting RANKL-induced osteoclast differentiation at inflammatory sites of bone fracture and injury. Therefore, peroxidase enzymes could be considered as potential therapeutic agents to treat osteolytic bone disease and aberrant bone resorption.

Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases.

Peroxidases are heme-containing enzymes released by activated immune cells at sites of inflammation. To-date their functional role in human health has mainly been limited to providing a mechanism for oxidative defence against invading bacteria and other pathogenic microorganisms. Our laboratory has recently identified a new functional role for peroxidase enzymes in stimulating fibroblast migration and collagen biosynthesis, offering a new insight into the causative association between inflammation and the pro-fibrogenic events that mediate tissue repair and regeneration. Peroxidases are found at elevated levels within and near blood vessels however, their direct involvement in angiogenesis has never been reported. Here we report for the first time that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are readily internalised by human umbilical vein endothelial cells (HUVEC) where they promote cellular proliferation, migration, invasion, and stimulate angiogenesis both in vitro and in vivo. These pro-angiogenic effects were attenuated using the specific peroxidase inhibitor 4-ABAH, indicating the enzyme's catalytic activity is essential in mediating this response. Mechanistically, we provide evidence that MPO and EPO regulate endothelial FAK, Akt, p38 MAPK, ERK1/2 phosphorylation and stabilisation of HIF-2α, culminating in transcriptional regulation of key angiogenesis pathways. These findings uncover for the first time an important and previously unsuspected role for peroxidases as drivers of angiogenesis, and suggest that peroxidase inhibitors may have therapeutic potential for the treatment of angiogenesis related diseases driven by inflammation.