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Desmoglein-2 (DSG2) is a calcium-binding single pass transmembrane glycoprotein and a member of the large cadherin family. Until recently, DSG2 was thought to only function as a cell adhesion protein embedded within desmosome junctions designed to enable cells to better tolerate mechanical stress. However, additional roles for DSG2 outside of desmosomes are continuing to emerge, particularly in cancer. Herein, we review the current literature on DSG2 in cancer and detail its impact on biological functions such as cell adhesion, proliferation, migration, invasion, intracellular signaling, extracellular vesicle release and vasculogenic mimicry. An increased understanding of the diverse repertoire of the biological functions of DSG2 holds promise to exploit this cell surface protein as a potential prognostic biomarker and/or target for better patient outcomes. This review explores the canonical and non-canonical functions of DSG2, as well as the context-dependent impacts of DSG2 in the realm of cancer.

Glioblastoma is an aggressive primary brain tumor that has seen few advances in treatments for over 20 years. In response to this desperate clinical need, multiple immunotherapy strategies are under development, including CAR-T cells, immune checkpoint inhibitors, oncolytic viruses and dendritic cell vaccines, although these approaches are yet to yield significant clinical benefit. Potential reasons for the lack of success so far include the immunosuppressive tumor microenvironment, the blood-brain barrier, and systemic changes to the immune system driven by both the tumor and its treatment. Furthermore, while T cells are essential effector cells for tumor control, dendritic cells play an equally important role in T cell activation, and emerging evidence suggests the dendritic cell compartment may be deeply compromised in glioblastoma patients. In this review, we describe the immunotherapy approaches currently under development for glioblastoma and the challenges faced, with a particular emphasis on the critical role of the dendritic cell-T cell axis. We suggest a number of strategies that could be used to boost dendritic cell number and function and propose that the use of these in combination with T cell-targeting strategies could lead to successful tumor control.

The clinical success of immune-checkpoint inhibitors (ICI) in both resected and metastatic melanoma has confirmed the validity of therapeutic strategies that boost the immune system to counteract cancer. However, half of patients with metastatic disease treated with even the most aggressive regimen do not derive durable clinical benefit. Thus, there is a critical need for predictive biomarkers that can identify individuals who are unlikely to benefit with high accuracy so that these patients may be spared the toxicity of treatment without the likely benefit of response. Ideally, such an assay would have a fast turnaround time and minimal invasiveness. Here, we utilize a novel platform that combines mass spectrometry with an artificial intelligence-based data processing engine to interrogate the blood glycoproteome in melanoma patients before receiving ICI therapy. We identify 143 biomarkers that demonstrate a difference in expression between the patients who died within six months of starting ICI treatment and those who remained progression-free for three years. We then develop a glycoproteomic classifier that predicts benefit of immunotherapy (HR=2.7; p=0.026) and achieves a significant separation of patients in an independent cohort (HR=5.6; p=0.027). To understand how circulating glycoproteins may affect efficacy of treatment, we analyze the differences in glycosylation structure and discover a fucosylation signature in patients with shorter overall survival (OS). We then develop a fucosylation-based model that effectively stratifies patients (HR=3.5; p=0.0066). Together, our data demonstrate the utility of plasma glycoproteomics for biomarker discovery and prediction of ICI benefit in patients with metastatic melanoma and suggest that protein fucosylation may be a determinant of anti-tumor immunity.

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.

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.

Abstract 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.

Abstract Background Organoids are a reliable model used in the study of human brain development and under pathological conditions. However, current methods for brain organoid culture generate tissues that range from 0.5 to 2 mm of size, which need to be constantly agitated to allow proper oxygenation. The culture conditions are, therefore, not suitable for whole-brain organoid live imaging, required to study developmental processes and disease progression within physiologically relevant time frames (i.e. days, weeks, months). Results Here we designed 3D-printed microplate inserts adaptable to standard 24 multi-well plates, which allow the growth of multiple organoids in pre-defined and fixed XYZ coordinates. This innovation facilitates high-resolution imaging of whole-cerebral organoids, allowing precise assessment of organoid growth and morphology, as well as cell tracking within the organoids, over long periods. We applied this technology to track neocortex development through neuronal progenitors in brain organoids, as well as the movement of patient-derived glioblastoma stem cells within healthy brain organoids. Conclusions This new bioengineering platform constitutes a significant advance that permits long term detailed analysis of whole-brain organoids using multimodal inverted fluorescence microscopy.

Glioblastoma is the most common and aggressive form of primary brain cancer, with no improvements in the 5-year survival rate of 4.6% over the past three decades. T-cell-based immunotherapies such as immune-checkpoint inhibitors and chimeric antigen receptor T-cell therapy have prolonged the survival of patients with other cancers and have undergone early-phase clinical evaluation in glioblastoma patients. However, a major challenge for T-cell-based immunotherapy of glioblastoma and other solid cancers is T-cell infiltration into tumours. This process is mediated by chemokine-chemokine receptor and integrin-adhesion molecule interactions, yet the specific nature of the molecules that may facilitate T-cell homing into glioblastoma are unknown. Here, we have characterised chemokine receptor and integrin expression profiles of endogenous glioblastoma-infiltrating T cells, and the chemokine expression profile of glioblastoma-associated cells, by single-cell RNA-sequencing. Subsequently, chemokine receptors and integrins were validated at the protein level to reveal enrichment of receptors CCR2, CCR5, CXCR3, CXCR4, CXCR6, CD49a, and CD49d in glioblastoma-infiltrating T-cell populations relative to T cells in matched patient peripheral blood. Complementary chemokine ligand expression was then validated in glioblastoma biopsies and glioblastoma-derived primary cell cultures. Together, enriched expression of homing receptor-ligand pairs identified in this study implicate a potential role in mediating T-cell infiltration into glioblastoma. Importantly, our data characterising the migratory receptors on endogenous tumour-infiltrating T cells could be exploited to enhance the tumour-homing properties of future T-cell immunotherapies for glioblastoma.

Glioblastoma is the most common form of primary brain tumour in adults. For more than a decade, conventional treatment has produced a relatively modest improvement in the overall survival of glioblastoma patients. The immunosuppressive mechanisms employed by neoplastic and non-neoplastic cells within the tumour can limit treatment efficacy, and this can include the secretion of immunosuppressive cytokines and chemokines. These factors can play a significant role in immune modulation, thus disabling anti-tumour responses and contributing to tumour progression. Here, we review the complex interplay between populations of immune and tumour cells together with defined contributions by key cytokines and chemokines to these intercellular interactions. Understanding how these tumour-derived factors facilitate the crosstalk between cells may identify molecular candidates for potential immunotherapeutic targeting, which may enable better tumour control and improved patient survival.

Circulating endothelial progenitor cells (EPCs) provide revascularisation for cardiovascular disease and the expansion of these cells opens up the possibility of their use as a cell therapy. Herein we show that interleukin-3 (IL3) strongly expands a population of human non-adherent endothelial forming cells (EXnaEFCs) with low immunogenicity as well as pro-angiogenic capabilities in vivo, making their therapeutic utilisation a realistic option. Non-adherent CD133+ EFCs isolated from human umbilical cord blood and cultured under different conditions were maximally expanded by day 12 in the presence of IL3 at which time a 350-fold increase in cell number was obtained. Cell surface marker phenotyping confirmed expression of the hematopoietic progenitor cell markers CD133, CD117 and CD34, vascular cell markers VEGFR2 and CD31, dim expression of CD45 and absence of myeloid markers CD14 and CD11b. Functional experiments revealed that EXnaEFCs exhibited classical properties of endothelial cells (ECs), namely binding of Ulex europaeus lectin, up-take of acetylated-low density lipoprotein and contribution to EC tube formation in vitro. These EXnaEFCs demonstrated a pro-angiogenic phenotype within two independent in vivo rodent models. Firstly, a Matrigel plug assay showed increased vascularisation in mice. Secondly, a rat model of acute myocardial infarction demonstrated reduced heart damage as determined by lower levels of serum creatinine and a modest increase in heart functionality. Taken together, these studies show IL3 as a potent growth factor for human CD133+ cell expansion with clear pro-angiogenic properties (in vitro and in vivo) and thus may provide clinical utility for humans in the future.

Background High-grade gliomas including glioblastoma (GBM) and diffuse midline gliomas (DMG) represent the most lethal and aggressive brain cancers where current treatment modalities offer limited efficacy. Chimeric antigen receptor (CAR) T cell therapies have emerged as a promising strategy, boasting tumor-specific targeting and the unique ability to penetrate the blood-brain barrier. However, the effective clinical application hinges on the optimal choice of antigen, with a limited number, currently under investigation.Methods We employed cell surface proteomic analysis of primary human high-grade glioma samples from both adult and pediatric patients. This led to the identification of Ephrin type-A receptor 3 (EphA3) as a prevalently expressed target. We engineered a second-generation EphA3-targeted CAR T cell and assessed function using in vitro and in vivo models of GBM and DMG.Results EphA3-targeted CAR T cells demonstrated robust antigen-specific killing of human GBM and DMG cell lines in vitro. In an orthotopic xenograft NSG mouse model, EphA3-targeted CAR T cells not only effectively eradicated tumors but also established a functional T cell population protective on rechallenge. Remarkably, mice rechallenged with a second contralateral orthotopic tumor implantation achieved complete tumor clearance and maintained a sustained complete response 6 months following initial treatment.Conclusion Building on the proven safety profile of EphA3 antibodies in clinical settings, our study provides compelling preclinical evidence supporting the efficacy of EphA3-targeted CAR T cells against high-grade gliomas. These findings underscore the potential for transitioning this innovative therapy into clinical trials, aiming to revolutionize the treatment landscape for patients afflicted with these formidable brain cancers.

Abstract γδ T cells are a unique subset of T lymphocytes, exhibiting features of both innate and adaptive immune cells and are involved with cancer immunosurveillance. They present an attractive alternative to conventional T cell‐based immunotherapy due, in large part, to their lack of major histocompatibility (MHC) restriction and ability to secrete high levels of cytokines with well‐known anti‐tumour functions. To date, clinical trials using γδ T cell‐based immunotherapy for a range of haematological and solid cancers have yielded limited success compared with in vitro studies. This inability to translate the efficacy of γδ T‐cell therapies from preclinical to clinical trials is attributed to a combination of several factors, e.g. γδ T‐cell agonists that are commonly used to stimulate populations of these cells have limited cellular uptake yet rely on intracellular mechanisms; administered γδ T cells display low levels of tumour‐infiltration; and there is a gap in the understanding of γδ T‐cell inhibitory receptors. This review explores the discrepancy between γδ T‐cell clinical and preclinical performance and offers viable avenues to overcome these obstacles. Using more direct γδ T‐cell agonists, encapsulating these agonists into lipid nanocarriers to improve their pharmacokinetic and pharmacodynamic profiles and the use of combination therapies to overcome checkpoint inhibition and T‐cell exhaustion are ways to bridge the gap between preclinical and clinical success. Given the ability to overcome these limitations, the development of a more targeted γδ T‐cell agonist‐checkpoint blockade combination therapy has the potential for success in clinical trials which has to date remained elusive.

Abstract Objectives CAR‐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. Methods We 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). Results FAP‐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. Conclusions Our 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.

Background Aggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain tumors are limited, and 5-year survival rates remain poor. Immune checkpoint inhibitor therapy has transformed treatment of some other cancers but has yet to significantly benefit patients with glioblastoma. Early phase trials of chimeric antigen receptor (CAR) T-cell therapy in patients with glioblastoma have demonstrated that this approach is safe and feasible, but with limited evidence of its effectiveness. The choices of appropriate target antigens for CAR-T-cell therapy also remain limited.Methods We profiled an extensive biobank of patients’ biopsy tissues and patient-derived early passage glioma neural stem cell lines for GD2 expression using immunomicroscopy and flow cytometry. We then employed an approved clinical manufacturing process to make CAR- T cells from patients with peripheral blood of glioblastoma and diffuse midline glioma and characterized their phenotype and function in vitro. Finally, we tested intravenously administered CAR-T cells in an aggressive intracranial xenograft model of glioblastoma and used multicolor flow cytometry, multicolor whole-tissue immunofluorescence and next-generation RNA sequencing to uncover markers associated with effective tumor control.Results Here we show that the tumor-associated antigen GD2 is highly and consistently expressed in primary glioblastoma tissue removed at surgery. Moreover, despite patients with glioblastoma having perturbations in their immune system, highly functional GD2-specific CAR-T cells can be produced from their peripheral T cells using an approved clinical manufacturing process. Finally, after intravenous administration, GD2-CAR-T cells effectively infiltrated the brain and controlled tumor growth in an aggressive orthotopic xenograft model of glioblastoma. Tumor control was further improved using CAR-T cells manufactured with a clinical retroviral vector encoding an interleukin-15 transgene alongside the GD2-specific CAR. These CAR-T cells achieved a striking 50% complete response rate by bioluminescence imaging in established intracranial tumors.Conclusions Targeting GD2 using a clinically deployed CAR-T-cell therapy has a sound scientific and clinical rationale as a treatment for glioblastoma and other aggressive primary brain tumors.

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

Abstract Chimeric antigen receptor (CAR)‐T cell therapy is now approved in the United States and Europe as a standard treatment for relapsed/refractory B‐cell malignancies. It has also been approved recently by the Therapeutic Goods Administration in Australia and may soon be publicly reimbursed. This advance has accentuated scientific, clinical and commercial interest in adapting this exciting technology for the treatment of solid cancers where it is widely recognised that the challenges of overcoming a hostile tumor microenvironment are most acute. Indeed, CAR‐T cell technology may be of the greatest value for those cancers that lack pre‐existing immunity because they are immunologically ‘cold’, or have a low somatic tumor mutation load, or both. These cancers are generally not amenable to therapeutic immune checkpoint blockade, but CAR‐T cell therapy may be effective because it provides an abundant supply of autologous tumor‐specific T cells. This is achieved by using genetic engineering to re‐direct autologous T‐cell cytotoxicity towards a tumor‐associated antigen, bypassing endogenous T‐cell requirements for antigen processing, MHC‐dependent antigen presentation and co‐stimulation. One of the most challenging solid cancers is glioblastoma, which has among the least permissive immunological milieu of any cancer, and which is almost always fatal. Here, we argue that CAR‐T cell technology may counter some glioblastoma defences and provide a beachhead for furthering our eventual therapeutic aims of restoring effective antitumor immunity. Although clinical investigation of CAR‐T cell therapy for glioblastoma is at an early stage, we discuss three recently published studies, which feature significant differences in target antigen, CAR‐T cell phenotype, route of administration and tumor response. We discuss the lessons, which may be learned from these studies and which may guide further progress in the field.

Cancer vaccines are designed to expand tumor antigen-specific T cells with effector function. However, they may also inadvertently expand regulatory T cells (Treg), which could seriously hamper clinical efficacy. To address this possibility, we developed a novel assay to detect antigen-specific Treg based on down-regulation of surface CD3 following TCR engagement, and used this approach to screen for Treg specific to the NY-ESO-1 tumor antigen in melanoma patients treated with the NY-ESO-1/ISCOMATRIX™ cancer vaccine. All patients tested had Treg (CD25(bright) FoxP3(+) CD127(neg)) specific for at least one NY-ESO-1 epitope in the blood. Strikingly, comparison with pre-treatment samples revealed that many of these responses were induced or boosted by vaccination. The most frequently detected response was toward the HLA-DP4-restricted NY-ESO-1(157-170) epitope, which is also recognized by effector T cells. Notably, functional Treg specific for an HLA-DR-restricted epitope within the NY-ESO-1(115-132) peptide were also identified at high frequency in tumor tissue, suggesting that NY-ESO-1-specific Treg may suppress local anti-tumor immune responses. Together, our data provide compelling evidence for the ability of a cancer vaccine to expand tumor antigen-specific Treg in the setting of advanced cancer, a finding which should be given serious consideration in the design of future cancer vaccine clinical trials.