rWTC-MBTA Vaccine Induces Potent Adaptive Immune Responses Against Glioblastomas via Dynamic Activation of Dendritic Cells.

Despite strides in immunotherapy, glioblastoma multiforme (GBM) remains challenging due to low inherent immunogenicity and suppressive tumor microenvironment. Converting "cold" GBMs to "hot" is crucial for immune activation and improved outcomes. This study comprehensively characterized a therapeutic vaccination strategy for preclinical GBM models. The vaccine consists of Mannan-BAM-anchored irradiated whole tumor cells, Toll-like receptor ligands [lipoteichoic acid (LTA), polyinosinic-polycytidylic acid (Poly (I:C)), and resiquimod (R-848)], and anti-CD40 agonistic antibody (rWTC-MBTA). Intracranial GBM models (GL261, SB28 cells) are used to evaluate the vaccine efficacy. A substantial number of vaccinated mice exhibited complete regression of GBM tumors in a T-cell-dependent manner, with no significant toxicity. Long-term tumor-specific immune memory is confirmed upon tumor rechallenge. In the vaccine-draining lymph nodes of the SB28 model, rWTC-MBTA vaccination triggered a major rise in conventional dendritic cell type 1 (cDC1) 12 h post-treatment, followed by an increase in conventional dendritic cell type 2 (cDC2), monocyte-derived dendritic cell (moDC), and plasmacytoid dendritic cell (pDC) on Day 5 and Day 13. Enhanced cytotoxicity of CD4+ and CD8+ T cells in vaccinated mice is verified in co-culture with tumor cells. Analyses of immunosuppressive signals (T-cell exhaustion, myeloid-derived suppressor cells (MDSC), M2 macrophages) in the GBM microenvironment suggest potential combinations with other immunotherapies for enhanced efficacy. In conclusion, the authors findings demonstrate that rWTC-MBTA induces potent and long-term adaptive immune responses against GBM.

Application of toll-like receptors (TLRs) and their agonists in cancer vaccines and immunotherapy.

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) expressed in various immune cell types and perform multiple purposes and duties involved in the induction of innate and adaptive immunity. Their capability to propagate immunity makes them attractive targets for the expansion of numerous immunotherapeutic approaches targeting cancer. These immunotherapeutic strategies include using TLR ligands/agonists as monotherapy or combined therapeutic strategies. Several TLR agonists have demonstrated significant efficacy in advanced clinical trials. In recent years, multiple reports established the applicability of TLR agonists as adjuvants to chemotherapeutic drugs, radiation, and immunotherapies, including cancer vaccines. Cancer vaccines are a relatively novel approach in the field of cancer immunotherapy and are currently under extensive evaluation for treating different cancers. In the present review, we tried to deliver an inclusive discussion of the significant TLR agonists and discussed their application and challenges to their incorporation into cancer immunotherapy approaches, particularly highlighting the usage of TLR agonists as functional adjuvants to cancer vaccines. Finally, we present the translational potential of rWTC-MBTA vaccination [irradiated whole tumor cells (rWTC) pulsed with phagocytic agonists Mannan-BAM, TLR ligands, and anti-CD40 agonisticAntibody], an autologous cancer vaccine leveraging membrane-bound Mannan-BAM, and the immune-inducing prowess of TLR agonists as a probable immunotherapy in multiple cancer types.

End binding-3 inhibitor activates regenerative program in age-related macular degeneration.

Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) biologics, there is a need for the development of less invasive treatments. Here, we designed an allosteric inhibitor of end binding-3 (EB3) protein, termed EBIN, which reduces the effects of environmental stresses on endothelial cells by limiting pathological calcium signaling. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of wet AMD prevents both neovascular leakage and choroidal neovascularization. EBIN reverses the epigenetic changes induced by environmental stresses, allowing an activation of a regenerative program within metabolic-active endothelial cells comprising choroidal neovascularization (CNV) lesions. These results suggest the therapeutic potential of EBIN in preventing the degenerative processes underlying wet AMD.

rWTC-MBTA: autologous vaccine prevents metastases via antitumor immune responses.

BACKGROUND: Autologous tumor cell-based vaccines (ATVs) aim to prevent and treat tumor metastasis by activating patient-specific tumor antigens to induce immune memory. However, their clinical efficacy is limited. Mannan-BAM (MB), a pathogen-associated molecular pattern (PAMP), can coordinate an innate immune response that recognizes and eliminates mannan-BAM-labeled tumor cells. TLR agonists and anti-CD40 antibodies (TA) can enhance the immune response by activating antigen-presenting cells (APCs) to present tumor antigens to the adaptive immune system. In this study, we investigated the efficacy and mechanism of action of rWTC-MBTA, an autologous whole tumor cell vaccine consisting of irradiated tumor cells (rWTC) pulsed with mannan-BAM, TLR agonists, and anti-CD40 antibody (MBTA), in preventing tumor metastasis in multiple animal models. METHODS: The efficacy of the rWTC-MBTA vaccine was evaluated in mice using breast (4T1) and melanoma (B16-F10) tumor models via subcutaneous and intravenous injection of tumor cells to induce metastasis. The vaccine's effect was also assessed in a postoperative breast tumor model (4T1) and tested in autologous and allogeneic syngeneic breast tumor models (4T1 and EMT6). Mechanistic investigations included immunohistochemistry, immunophenotyping analysis, ELISA, tumor-specific cytotoxicity testing, and T-cell depletion experiments. Biochemistry testing and histopathology of major tissues in vaccinated mice were also evaluated for potential systemic toxicity of the vaccine. RESULTS: The rWTC-MBTA vaccine effectively prevented metastasis and inhibited tumor growth in breast tumor and melanoma metastatic animal models. It also prevented tumor metastasis and prolonged survival in the postoperative breast tumor animal model. Cross-vaccination experiments revealed that the rWTC-MBTA vaccine prevented autologous tumor growth, but not allogeneic tumor growth. Mechanistic data demonstrated that the vaccine increased the percentage of antigen-presenting cells, induced effector and central memory cells, and enhanced CD4+ and CD8+ T-cell responses. T-cells obtained from mice that were vaccinated displayed tumor-specific cytotoxicity, as shown by enhanced tumor cell killing in co-culture experiments, accompanied by increased levels of Granzyme B, TNF-α, IFN-γ, and CD107a in T-cells. T-cell depletion experiments showed that the vaccine's antitumor efficacy depended on T-cells, especially CD4+ T-cells. Biochemistry testing and histopathology of major tissues in vaccinated mice revealed negligible systemic toxicity of the vaccine. CONCLUSION: The rWTC-MBTA vaccine demonstrated efficacy in multiple animal models through T-cell mediated cytotoxicity and has potential as a therapeutic option for preventing and treating tumor metastasis with minimal systemic toxicity.

Inhibition of PP2A with LB-100 Enhances Efficacy of CAR-T Cell Therapy Against Glioblastoma.

Chimeric antigen receptor (CAR)-engineered T cells represent a promising modality for treating glioblastoma. Recently, we demonstrated that CAR-T cells targeting carbonic anhydrase IX (CAIX), a protein involved in HIF-1a hypoxic signaling, is a promising CAR-T cell target in an intracranial murine glioblastoma model. Anti-CAIX CAR-T cell therapy is limited by its suboptimal activation within the tumor microenvironment. LB-100, a small molecular inhibitor of protein phosphatase 2A (PP2A), has been shown to enhance T cell anti-tumor activity through activation of the mTOR signaling pathway. Herein, we investigated if a treatment strategy consisting of a combination of LB-100 and anti-CAIX CAR-T cell therapy produced a synergistic anti-tumor effect. Our studies demonstrate that LB-100 enhanced anti-CAIX CAR-T cell treatment efficacy in vitro and in vivo. Our findings demonstrate the role of LB-100 in augmenting the cytotoxic activity of anti-CAIX CAR-T cells and underscore the synergistic therapeutic potential of applying combination LB-100 and CAR-T Cell therapy to other solid tumors.

Targeting hypoxia downstream signaling protein, CAIX, for CAR T-cell therapy against glioblastoma.

BACKGROUND: Glioblastoma survival remains unchanged despite continuing therapeutic innovation. Herein, we aim to (i) develop chimeric antigen receptor (CAR) T cells with a specificity to a unique antigen, carbonic anhydrase IX (CAIX), which is expressed in the hypoxic microenvironment characteristic of glioblastoma, and (ii) demonstrate its efficacy with limited off-target effects. METHODS: First we demonstrated expression of CAIX in patient-derived glioblastoma samples and available databases. CAR T cells were generated against CAIX and efficacy was assessed in 4 glioblastoma cell lines and 2 glioblastoma stem cell lines. Cytotoxicity of anti-CAIX CAR T cells was assessed via interferon gamma, tumor necrosis factor alpha, and interleukin-2 levels when co-cultured with tumor cells. Finally, we assessed efficacy of direct intratumoral injection of the anti-CAIX CAR T cells on an in vivo xenograft mouse model using the U251 luciferase cell line. Tumor infiltrating lymphocyte analyses were performed. RESULTS: We confirm that CAIX is highly expressed in glioblastoma from patients. We demonstrate that CAIX is a suitable target for CAR T-cell therapy using anti-CAIX CAR T cells against glioblastoma in vitro and in vivo. In our mouse model, a 20% cure rate was observed without detectable systemic effects. CONCLUSIONS: By establishing the specificity of CAIX under hypoxic conditions in glioblastoma and highlighting its efficacy as a target for CAR T-cell therapy, our data suggest that anti-CAIX CAR T may be a promising strategy to treat glioblastoma. Direct intratumoral injection increases anti-CAIX CAR T-cell potency while limiting its off-target effects.

Analysis of biological networks in the endothelium with biomimetic microsystem platform.

Here we describe a novel method for studying the protein "interactome" in primary human cells and apply this method to investigate the effect of posttranslational protein modifications (PTMs) on the protein's functions. We created a novel "biomimetic microsystem platform" (Bio-MSP) to isolate the protein complexes in primary cells by covalently attaching purified His-tagged proteins to a solid microscale support. Using this Bio-MSP, we have analyzed the interactomes of unphosphorylated and phosphomimetic end-binding protein-3 (EB3) in endothelial cells. Pathway analysis of these interactomes demonstrated the novel role of EB3 phosphorylation at serine 162 in regulating the protein's function. We showed that phosphorylation "switches" the EB3 biological network to modulate cellular processes such as cell-to-cell adhesion whereas dephosphorylation of this site promotes cell proliferation. This novel technique provides a useful tool to study the role of PTMs or single point mutations in activating distinct signal transduction networks and thereby the biological function of the protein in health and disease.

A Transgenic Mouse Model of Pacak⁻Zhuang Syndrome with An Epas1 Gain-of-Function Mutation.

We previously identified a novel syndrome in patients characterized by paraganglioma, somatostatinoma, and polycythemia. In these patients, polycythemia occurs long before any tumor develops, and tumor removal only partially corrects polycythemia, with recurrence occurring shortly after surgery. Genetic mosaicism of gain-of-function mutations of the EPAS1 gene (encoding HIF2α) located in the oxygen degradation domain (ODD), typically p.530-532, was shown as the etiology of this syndrome. The aim of the present investigation was to demonstrate that these mutations are necessary and sufficient for the development of the symptoms. We developed transgenic mice with a gain-of-function Epas1A529V mutation (corresponding to human EPAS1A530V), which demonstrated elevated levels of erythropoietin and polycythemia, a decreased urinary metanephrine-to-normetanephrine ratio, and increased expression of somatostatin in the ampullary region of duodenum. Further, inhibition of HIF2α with its specific inhibitor PT2385 significantly reduced erythropoietin levels in the mutant mice. However, polycythemia persisted after PT2385 treatment, suggesting an alternative erythropoietin-independent mechanism of polycythemia. These findings demonstrate the vital roles of EPAS1 mutations in the syndrome development and the great potential of the Epas1A529V animal model for further pathogenesis and therapeutics studies.

N-cadherin signaling via Trio assembles adherens junctions to restrict endothelial permeability.

Vascular endothelial (VE)-cadherin forms homotypic adherens junctions (AJs) in the endothelium, whereas N-cadherin forms heterotypic adhesion between endothelial cells and surrounding vascular smooth muscle cells and pericytes. Here we addressed the question whether both cadherin adhesion complexes communicate through intracellular signaling and contribute to the integrity of the endothelial barrier. We demonstrated that deletion of N-cadherin (Cdh2) in either endothelial cells or pericytes increases junctional endothelial permeability in lung and brain secondary to reduced accumulation of VE-cadherin at AJs. N-cadherin functions by increasing the rate of VE-cadherin recruitment to AJs and induces the assembly of VE-cadherin junctions. We identified the dual Rac1/RhoA Rho guanine nucleotide exchange factor (GEF) Trio as a critical component of the N-cadherin adhesion complex, which activates both Rac1 and RhoA signaling pathways at AJs. Trio GEF1-mediated Rac1 activation induces the recruitment of VE-cadherin to AJs, whereas Trio GEF2-mediated RhoA activation increases intracellular tension and reinforces Rac1 activation to promote assembly of VE-cadherin junctions and thereby establish the characteristic restrictive endothelial barrier.

Molecular evaluation of a sporadic paraganglioma with concurrent IDH1 and ATRX mutations.

PURPOSE: Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors of neural crest origin. Germline or somatic mutations of numerous genes have been implicated in the pathogenesis of PPGLs, including the isocitrate dehydrogenase 1 (IDH1) gene and alpha thalassemia/mental retardation syndrome X-linked (ATRX) gene. Although concurrent IDH1 and ATRX mutations are frequently seen in gliomas, they have never been reported together in PPGLs. The aim of this study was to characterize one paraganglioma with concurrent IDH1 and ATRX mutations identified by whole exome sequencing. METHODS: Leukocyte and tumor DNA were used for whole exome sequencing and Sanger sequencing. 2-hydroxyglurarate level and the global DNA methylation status in the tumor were measured. ATRX's cDNA transcripts were analyzed. Tyrosine hydroxylase (TH), HIF1α and ATRX staining, as well as telomere-specific FISH was also performed. RESULTS: The presence of a somatic IDH1 (c.394C>T, p.R132C) mutation and a concurrent somatic ATRX splicing mutation (c.4318-2A>G) in the current case was confirmed. Dramatic accumulation of 2-hydroxyglutarate was detected in the paraganglioma without the global DNA hypermethylation, and pseudohypoxia was also activated. Importantly, immunohistochemistry revealed negative TH staining in the tumor and the first exon region of TH gene was hypermethylated resulting in normal plasma metanephrines. The splicing ATRX mutation resulted in two transcripts, causing frameshifts. Immunohistochemistry revealed scarce ATRX staining in the tumor. Alternative lengthening of telomeres (ALT) was detected by FISH. CONCLUSIONS: This case represents the first concurrence of IDH1 and ATRX mutations in PPGLs. Although relatively rare, a somatic R132C mutation of IDH1 might play a role in a small subset of sporadic PPGLs.

Enhancement of mitochondrial biogenesis and paradoxical inhibition of lactate dehydrogenase mediated by 14-3-3η in oncocytomas.

Oncocytomas represent a subset of benign pituitary adenomas that are characterized by significant mitochondrial hyperplasia. Mitochondria are key organelles for energy generation and metabolic intermediate production for biosynthesis in tumour cells, so understanding the mechanism underlying mitochondrial biogenesis and its impact on cellular metabolism in oncocytoma is vital. Here, we studied surgically resected pituitary oncocytomas by using multi-omic analyses. Whole-exome sequencing did not reveal any nuclear mutations, but identified several somatic mutations of mitochondrial DNA, and dysfunctional respiratory complex I. Metabolomic analysis suggested that oxidative phosphorylation was reduced within individual mitochondria, and that there was no reciprocal increase in glycolytic activity. Interestingly, we found a reduction in the cellular lactate level and reduced expression of lactate dehydrogenase A (LDHA), which contributed to mitochondrial biogenesis in an in vitro cell model. It is of note that the hypoxia-response signalling pathway was not upregulated in pituitary oncocytomas, thereby failing to enhance glycolysis. Proteomic analysis showed that 14-3-3η was exclusively overexpressed in oncocytomas, and that 14-3-3η was capable of inhibiting glycolysis, leading to mitochondrial biogenesis in the presence of rotenone. In particular, 14-3-3η inhibited LDHA by direct interaction in the setting of complex I dysfunction, highlighting the role of 14-3-3η overexpression and inefficient oxidative phosphorylation in oncocytoma mitochondrial biogenesis. These findings deepen our understanding of the metabolic changes that occur within oncocytomas, and shine a light on the mechanism of mitochondrial biogenesis, providing a novel perspective on metabolic adaptation in tumour cells. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Fatty acid oxidation contributes to IL-1ß secretion in M2 macrophages and promotes macrophage-mediated tumor cell migration.

Tumor-associated macrophages (TAMs) are predominantly M2 phenotype in solid cancers including hepatocellular carcinoma (HCC). Though differentiation of M2 macrophages has been recently linked to fatty acid oxidation (FAO), whether FAO plays a role in functional maintenance of M2 macrophages is still unclear. Here, we used an in vitro model to mimic TAM-HCC interaction in tumor microenvironment. We found that M2 monocyte-derived macrophages (MDMs) enhanced the proliferation, migration, and invasion of HCC cells through an FAO-dependent way. Further investigations identified that IL-1ß mediated the pro-migratory effect of M2 MDM. Using etomoxir and siRNA to inhibit FAO and palmitate to enhance FAO, we showed that FAO was responsible for the up-regulated secretion of IL-1ß and, thus, the pro-migratory effect in M2 MDMs. In addition, we proved that IL-1ß induction was reactive oxygen species and NLRP3-dependent. Our study demonstrates that FAO plays a key role in functional human M2 macrophages by enhancing IL-1ß secretion to promote HCC cell migration. These findings provide evidence for different dependency of energy sources in macrophages with distinct phenotypes and functions, and suggest a novel strategy to treat HCC by reprogramming cell metabolism or modulating tumor microenvironment.

IP3 receptor signaling and endothelial barrier function.

The endothelium, a monolayer of endothelial cells lining vessel walls, maintains tissue-fluid homeostasis by restricting the passage of the plasma proteins and blood cells into the interstitium. The ion Ca2+, a ubiquitous secondary messenger, initiates signal transduction events in endothelial cells that is critical to control of vascular tone and endothelial permeability. The ion Ca2+ is stored inside the intracellular organelles and released into the cytosol in response to environmental cues. The inositol 1,4,5-trisphosphate (IP3) messenger facilitates Ca2+ release through IP3 receptors which are Ca2+-selective intracellular channels located within the membrane of the endoplasmic reticulum. Binding of IP3 to the IP3Rs initiates assembly of IP3R clusters, a key event responsible for amplification of Ca2+ signals in endothelial cells. This review discusses emerging concepts related to architecture and dynamics of IP3R clusters, and their specific role in propagation of Ca2+ signals in endothelial cells.