Losartan controls immune checkpoint blocker-induced edema and improves survival in glioblastoma mouse models.

Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma trials. Here, we found that ICBs induce cerebral edema in some patients and mice with glioblastoma. Through single-cell RNA sequencing, intravital imaging, and CD8+ T cell blocking studies in mice, we demonstrated that this edema results from an inflammatory response following antiprogrammed death 1 (PD1) antibody treatment that disrupts the blood-tumor barrier. Used in lieu of immunosuppressive corticosteroids, the angiotensin receptor blocker losartan prevented this ICB-induced edema and reprogrammed the tumor microenvironment, curing 20% of mice which increased to 40% in combination with standard of care treatment. Using a bihemispheric tumor model, we identified a "hot" tumor immune signature prior to losartan+anti-PD1 therapy that predicted long-term survival. Our findings provide the rationale and associated biomarkers to test losartan with ICBs in glioblastoma patients.

Combined therapy with thrombospondin-1 type I repeats (3TSR) and chemotherapy induces regression and significantly improves survival in a preclinical model of advanced stage epithelial ovarian cancer.

Most women are diagnosed with epithelial ovarian cancer (EOC) at advanced stage, where therapies have limited effectiveness and the long-term survival rate is low. We evaluated the effects of combined antiangiogenic and chemotherapy treatments on advanced stage EOC. Treatment of EOC cells with a recombinant version of the thrombospondin-1 type I repeats (3TSR) induced more apoptotic cell death (36.5 ± 9.6%) in vitro compared to untreated controls (4.1 ± 1.4). In vivo, tumors were induced in an orthotopic, syngeneic mouse model of advanced stage EOC. Mice were treated with 3TSR (4 mg/kg per day) alone or in combination with chemotherapy drugs delivered with maximum tolerated dose or metronomic scheduling. Pretreatment with 3TSR induced tumor regression, normalized tumor vasculature, and improved uptake of chemotherapy drugs. Combination 3TSR and metronomic chemotherapy induced the greatest tumor regression (6.2-fold reduction in size compared to PBS-treated controls) and highest survival when treatment was initiated at advanced stage. 3TSR binding to its receptor, CD36 (cluster of differentiation 36), increased binding of CD36 and SHP-1, which significantly inhibited phosphorylation of the VEGF receptor. In this study, we describe a novel treatment approach and mechanism of action with 3TSR and chemotherapy that induces regression of advanced stage EOC and significantly improves survival.-Russell, S., Duquette, M., Liu, J., Drapkin, R., Lawler, J., Petrik, J. Combined therapy with thrombospondin-1 type I repeats (3TSR) and chemotherapy induces regression and significantly improves survival in a preclinical model of advanced stage epithelial ovarian cancer.

Priming of the vascular endothelial growth factor signaling pathway by thrombospondin-1, CD36, and spleen tyrosine kinase.

CD36 plays a critical role in the inhibition of angiogenesis through binding to the type 1 repeats of thrombospondin-1 (TSP-1) and activating Fyn tyrosine kinase and MAPK pathways. Here, we reveal a novel association of CD36 with VEGFR-2 and spleen tyrosine kinase (Syk). We also address the correlation between the expression of CD36 and Syk by demonstrating that overexpression of CD36 in HUVECs up-regulates endogenous Syk expression. We also define a new role for TSP-1 and CD36 in the activation of the VEGFR-2 signaling pathway that requires Syk. Our findings also identify a role for Syk as a stimulator of VEGF-A-induced angiogenesis by increasing phosphorylation of Y1175 in VEGFR-2, which is a major tyrosine for promoting VEGF-A-induced endothelial cell migration. Together, these studies introduce a new signaling pathway for TSP-1, CD36, and Syk, and address the role of these proteins in regulating the angiogenic switch.

Anti-VEGF therapy improves EGFR-vIII-CAR-T cell delivery and efficacy in syngeneic glioblastoma models in mice.

Chimeric antigen receptor (CAR)-T cells have revolutionized the treatment of multiple types of hematological malignancies, but have shown limited efficacy in patients with glioblastoma (GBM) or other solid tumors. This may be largely due to the immunosuppressive tumor microenvironment (TME) that compromises CAR-T cells' delivery and antitumor activity. We previously showed that blocking vascular endothelial growth factor (VEGF) signaling can normalize tumor vessels in murine and human tumors, including GBM, breast, liver, and rectal carcinomas. Moreover, we demonstrated that vascular normalization can improve the delivery of CD8+ T cells and the efficacy of immunotherapy in breast cancer models in mice. In fact, the US FDA (Food and drug administration) has approved seven different combinations of anti-VEGF drugs and immune checkpoint blockers for liver, kidney, lung and endometrial cancers in the past 3 years. Here, we tested the hypothesis that anti-VEGF therapy can improve the delivery and efficacy of CAR-T cells in immunocompetent mice bearing orthotopic GBM tumors. We engineered two syngeneic mouse GBM cell lines (CT2A and GSC005) to express EGFRvIII-one of the most common neoantigens in human GBM-and CAR T cells to recognize EGFRvIII. We found that treatment with the anti-mouse VEGF antibody (B20) improved CAR-T cell infiltration and distribution throughout the GBM TME, delayed tumor growth, and prolonged survival of GBM-bearing mice compared with EGFRvIII-CAR-T cell therapy alone. Our findings provide compelling data and a rationale for clinical evaluation of anti-VEGF agents with CAR T cells for GBM patients.

Wnt inhibition alleviates resistance to immune checkpoint blockade in glioblastoma.

Wnt signaling plays a critical role in the progression and treatment outcome of glioblastoma (GBM). Here, we identified WNT7b as a heretofore unknown mechanism of resistance to immune checkpoint inhibition (αPD1) in GBM patients and murine models. Acquired resistance to αPD1 was found to be associated with the upregulation of Wnt7b and ß-catenin protein levels in GBM in patients and in a clinically relevant, stem-rich GBM model. Combining the porcupine inhibitor WNT974 with αPD1 prolonged the survival of GBM-bearing mice. However, this combination had a dichotomous response, with a subset of tumors showing refractoriness. WNT974 and αPD1 expanded a subset of DC3-like dendritic cells (DCs) and decreased the granulocytic myeloid-derived suppressor cells (gMDSCs) in the tumor microenvironment (TME). By contrast, monocytic MDSCs (mMDSCs) increased, while T-cell infiltration remained unchanged, suggesting potential TME-mediated resistance. Our preclinical findings warrant the testing of Wnt7b/ß-catenin combined with αPD1 in GBM patients with elevated Wnt7b/ß-catenin signaling.

Lymphatic muscle cells are unique cells that undergo aging induced changes.

Lymphatic muscle cells (LMCs) within the wall of collecting lymphatic vessels exhibit tonic and autonomous phasic contractions, which drive active lymph transport to maintain tissue-fluid homeostasis and support immune surveillance. Damage to LMCs disrupts lymphatic function and is related to various diseases. Despite their importance, knowledge of the transcriptional signatures in LMCs and how they relate to lymphatic function in normal and disease contexts is largely missing. We have generated a comprehensive transcriptional single-cell atlas-including LMCs-of collecting lymphatic vessels in mouse dermis at various ages. We identified genes that distinguish LMCs from other types of muscle cells, characterized the phenotypical and transcriptomic changes in LMCs in aged vessels, and uncovered a pro-inflammatory microenvironment that suppresses the contractile apparatus in advanced-aged LMCs. Our findings provide a valuable resource to accelerate future research for the identification of potential drug targets on LMCs to preserve lymphatic vessel function as well as supporting studies to identify genetic causes of primary lymphedema currently with unknown molecular explanation.

FATTY ACID SYNTHESIS IS REQUIRED FOR BREAST CANCER BRAIN METASTASIS.

Brain metastases are refractory to therapies that control systemic disease in patients with human epidermal growth factor receptor 2 (HER2+) breast cancer, and the brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for brain metastatic breast cancer growth may introduce liabilities that can be exploited for therapy. Here, we assessed how metabolism differs between breast tumors in brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in brain. We determine that this phenotype is an adaptation to decreased lipid availability in brain relative to other tissues, resulting in a site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reduces HER2+ breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies.

The crystal structure of the signature domain of cartilage oligomeric matrix protein: implications for collagen, glycosaminoglycan and integrin binding.

Cartilage oligomeric matrix protein (COMP), or thrombospondin-5 (TSP-5), is a secreted glycoprotein that is important for growth plate organization and function. Mutations in COMP cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). In this study, we determined the structure of a recombinant protein that contains the last epidermal growth factor repeat, the type 3 repeats and the C-terminal domain (CTD) of COMP to 3.15-A resolution limit by X-ray crystallography. The CTD is a beta-sandwich that is composed of 15 antiparallel beta-strands, and the type 3 repeats are a contiguous series of calcium binding sites that associate with the CTD at multiple points. The crystal packing reveals an exposed potential metal-ion-dependent adhesion site (MIDAS) on one edge of the beta-sandwich that is common to all TSPs and may serve as a binding site for collagens and other ligands. Disease-causing mutations in COMP disrupt calcium binding, disulfide bond formation, intramolecular interactions, or sites for potential ligand binding. The structure presented here and its unique molecular packing in the crystal identify potential interactive sites for glycosaminoglycans, integrins, and collagens, which are key to cartilage structure and function.

Thrombospondin-1 modulates vascular endothelial growth factor activity at the receptor level.

Vascular endothelial growth factor (VEGF) is a well-established stimulator of vascular permeability and angiogenesis, whereas thrombospondin-1 (TSP-1) is a potent angiogenic inhibitor. In this study, we have found that the TSP-1 receptors CD36 and beta1 integrin associate with the VEGF receptor 2 (VEGFR2). The coclustering of receptors that regulate angiogenesis may provide the endothelial cell with a platform for integration of positive and negative signals in the plane of the membrane. Thus, this complex may represent a molecular switch that regulates angiogenesis and determines endothelial cell behavior. In this context, physiological levels of TSP-1 appear to support VEGFR2 function on both the cellular and tissue level, because phosphorylation of VEGFR2 and vascular permeability in response to VEGF are decreased in TSP-1-null mice and isolated endothelial cells. A therapeutic agent based on the antiangiogenic domain of TSP-1, designated 3TSR (for three TSP-1 type 1 repeats), has significant antiangiogenic and antitumor efficacy. Systemic treatment of wild-type mice with 3TSR significantly decreased VEGF-induced permeability. Consistent with this result, VEGF-stimulated phosphorylation of VEGFR2 was also significantly decreased in lung extracts from 3TSR-treated mice. Moreover, 3TSR significantly decreased VEGF-stimulated VEGFR2 phosphorylation in human dermal microvascular endothelial cells in culture. Taken together, the results indicate that TSP-1 and 3TSR modulate the function of VEGFR2.

Corrigendum: Thrombospondin-1 Silencing Down-Regulates Integrin Expression Levels in Human Anaplastic Thyroid Cancer Cells With BRAFV600E: New Insights in the Host Tissue Adaptation and Homeostasis of Tumor Microenvironment.

[This corrects the article DOI: 10.3389/fendo.2013.00189.].

The effect of thrombospondin-1 on breast cancer metastasis.

Thrombospondin-1 (TSP-1) has been proposed to have both pro-metastatic and anti-metastatic properties. To elucidate its role in breast cancer metastasis, we compared tumor progression in the polyomavirus middle T antigen (Pyt) transgenic mouse and the TSP-1-null Pyt transgenic mouse. We characterized the tumors in these mice at 45, 60 and 90 days of age. Tumor size, areas of necrosis, macrophage infiltration, levels of active and total TGF-beta, vessel morphology, and lung and blood metastasis were measured in these mice. Mammary tumors were larger in the TSP-1-null mouse, and vessels were larger, but fewer in number in these tumors. The level of total TGF-beta was significantly higher in the Pyt tumors at 90 days of age. Importantly, significantly fewer metastases were observed in the lungs of the TSP-1-null/Pyt mouse. Primary Pyt tumor cells were more migratory than TSP-1-null Pyt tumor cells on collagen. Treatment of Pyt mice with recombinant proteins that contain the type-1 repeats of TSP-1 resulted in decreased primary tumor growth and metastasis. Sequences that are involved in CD36 binding and those required for TGF-beta activation mediated the inhibition of primary tumor growth. Thus, TSP-1 in the mammary tumor microenvironment inhibits angiogenesis and tumor growth, but promotes metastasis to the lung in the Pyt transgenic mouse. The ability of TSP-1 to support metastasis correlates with its ability to promote tumor cell migration.

Crystal structure of the TSP-1 type 1 repeats: a novel layered fold and its biological implication.

Thrombospondin-1 (TSP-1) contains three type 1 repeats (TSRs), which mediate cell attachment, glycosaminoglycan binding, inhibition of angiogenesis, activation of TGFbeta, and inhibition of matrix metalloproteinases. The crystal structure of the TSRs reported in this article reveals a novel, antiparallel, three-stranded fold that consists of alternating stacked layers of tryptophan and arginine residues from respective strands, capped by disulfide bonds on each end. The front face of the TSR contains a right-handed spiral, positively charged groove that might be the "recognition" face, mediating interactions with various ligands. This is the first high-resolution crystal structure of a TSR domain that provides a prototypic architecture for structural and functional exploration of the diverse members of the TSR superfamily.

The structures of the thrombospondin-1 N-terminal domain and its complex with a synthetic pentameric heparin.

The N-terminal domain of thrombospondin-1 (TSPN-1) mediates the protein's interaction with (1) glycosaminoglycans, calreticulin, and integrins during cellular adhesion, (2) low-density lipoprotein receptor-related protein during uptake and clearance, and (3) fibrinogen during platelet aggregation. The crystal structure of TSPN-1 to 1.8 A resolution is a beta sandwich with 13 antiparallel beta strands and 1 irregular strand-like segment. Unique structural features of the N- and C-terminal regions, and the disulfide bond location, distinguish TSPN-1 from the laminin G domain and other concanavalin A-like lectins/glucanases superfamily members. The crystal structure of the complex of TSPN-1 with heparin indicates that residues R29, R42, and R77 in an extensive positively charged patch at the bottom of the domain specifically associate with the sulfate groups of heparin. The TSPN-1 structure and identified adjacent linker region provide a structural framework for the analysis of the TSPN domain of various molecules, including TSPs, NELLs, many collagens, TSPEAR, and kielin.

Continuous administration of the three thrombospondin-1 type 1 repeats recombinant protein improves the potency of therapy in an orthotopic human pancreatic cancer model.

Thrombospondin-1 is one of most important natural angiogenic inhibitors. The three thrombospondin-1 type 1 repeats (3TSR), an anti-angiogenic domain of thrombospondin-1, is a promising novel agent for anti-angiogenic treatment. In the present study, we showed 3TSR was biologically stable at least for 7 days in mini-osmotic pumps in vivo, and continuous administration of 3TSR decreased the dosage and improved the potency of therapy in an orthotopic pancreatic cancer model. By using different dosage and delivery routes, we proved that the anti-tumor efficacy of 3TSR was correlated with its anti-angiogenic efficacy. 3TSR treatment also decreased tumor vessel patency and blood flow. The results indicate the advantage of continuous administration of angiogenic inhibitors and provide rationale for using such delivery methods for cancer treatment.

Antiangiogenic treatment with three thrombospondin-1 type 1 repeats versus gemcitabine in an orthotopic human pancreatic cancer model.

PURPOSE: In this study, we investigated the antitumor efficacy of thrombospondin-1 three type 1 repeats (3TSR), the antiangiogenic domain of thrombospondin-1, in comparison and in combination with gemcitabine, in an orthotopic pancreatic cancer model. EXPERIMENTAL DESIGN: Human pancreatic cancer cells were injected into the pancreas of severe combined immunodeficient mice. The animals were treated with 3TSR, gemcitabine, 3TSR plus gemcitabine, or vehicle for 3 weeks. Subsequently, the effects of 3TSR and/or gemcitabine on tumor growth, tumor necrosis, microvessel density, cancer cell proliferation, apoptosis, and endothelial cell apoptosis were analyzed. RESULTS: After 3 weeks of treatment, 3TSR reduced tumor volume by 65%, and gemcitabine by 84%. Tumor volume was not statistically different between gemcitabine group and combinatorial treatment group. Extensive necrotic areas were observed in tumors from 3TSR-treated mice, whereas tumors from gemcitabine and combinatorially treated mice were less necrotic than control tumors. 3TSR reduced tumor microvessel density and increased tumor blood vessel endothelial cell apoptosis. In contrast, gemcitabine induced apoptosis and inhibited proliferation of cancer cells. CONCLUSION: 3TSR, the antiangiogenic domain of thrombospondin-1, showed comparable antitumor efficacy to gemcitabine in a human pancreatic cancer orthotopic mouse model. No synergistic effect was found when the two drugs were combined and possible reasons are discussed in detail. A delicate balance between normalization and excessive regression of tumor vasculature is important when initiating alternative combinatorial regimens for treatment of patients with pancreatic cancer.

Antiangiogenic treatment with the three thrombospondin-1 type 1 repeats recombinant protein in an orthotopic human pancreatic cancer model.

PURPOSE: This study investigates the antiangiogenesis and antitumor efficacy of a recombinant protein composed of the three type 1 repeats (3TSR) of thrombospondin-1 in an orthotopic human pancreatic cancer model and provides useful preclinical data for pancreatic cancer treatment. EXPERIMENTAL DESIGN: Human pancreatic cancer cells (AsPC-1) were injected into the pancreas of severe combined immunodeficient mice. The animals were treated with 3TSR (3 mg per kg per day) or PBS for 3 weeks. Subsequently, the effects of 3TSR on tumor growth, microvessel density, cancer cell proliferation, apoptosis, and endothelial cell apoptosis were analyzed. The in vitro effects of 3TSR on human pancreatic cancer cells were also studied. RESULTS: 3TSR treatment significantly reduced angiogenesis and tumor growth of orthotopic pancreatic cancer. 3TSR-treated mice had a 69% reduction in tumor volume (316.6 +/- 79.3 versus 1,012.2 +/- 364.5 mm(3); P = 0.0001), and a significant increase in tumor necrotic area. After 3TSR treatment, both the vessel number and average microvessel size were significantly decreased, and microvessel density was decreased from 8.0% to 3.7% (P < 0.0001). The apoptotic rate of tumoral endothelial cells in 3TSR-treated tumors increased to 14.7% comparing to 4.2% in control tumors (P < 0.0001). 3TSR showed no direct effects on pancreatic cancer cell proliferation or apoptosis either in vivo or in vitro. CONCLUSION: 3TSR, a domain of a natural occurring angiogenesis inhibitor, showed potent therapeutic effect in pancreatic cancer by inhibiting tumor angiogenesis and may prove to be a promising agent for clinical pancreatic cancer treatment.

Metastasis-associated MCL1 and P16 copy number alterations dictate resistance to vemurafenib in a BRAFV600E patient-derived papillary thyroid carcinoma preclinical model.

BRAF(V600E) mutation exerts an essential oncogenic function in many tumors, including papillary thyroid carcinoma (PTC). Although BRAF(V600E) inhibitors are available, lack of response has been frequently observed. To study the mechanism underlying intrinsic resistance to the mutant BRAF(V600E) selective inhibitor vemurafenib, we established short-term primary cell cultures of human metastatic/recurrent BRAF(V600E)-PTC, intrathyroidal BRAF(V600E)-PTC, and normal thyroid (NT). We also generated an early intervention model of human BRAF(V600E)-PTC orthotopic mouse. We find that metastatic BRAF(V600E)-PTC cells elicit paracrine-signaling which trigger migration of pericytes, blood endothelial cells and lymphatic endothelial cells as compared to BRAF(WT)-PTC cells, and show a higher rate of invasion. We further show that vemurafenib therapy significantly suppresses these aberrant functions in non-metastatic BRAF(V600E)-PTC cells but lesser in metastatic BRAF(V600E)-PTC cells as compared to vehicle treatment. These results concur with similar folds of down-regulation of tumor microenvironment-associated pro-metastatic molecules, with no effects in BRAF(WT)-PTC and NT cells. Our early intervention preclinical trial shows that vemurafenib delays tumor growth in the orthotopic BRAF(WT/V600E)-PTC mice. Importantly, we identify high copy number gain of MCL1 (chromosome 1q) and loss of CDKN2A (P16, chromosome 9p) in metastatic BRAF(V600E)-PTC cells which are associated with resistance to vemurafenib treatment. Critically, we demonstrate that combined vemurafenib therapy with BCL2/MCL1 inhibitor increases metastatic BRAF(V600E)-PTC cell death and ameliorates response to vemurafenib treatment as compared to single agent treatment. In conclusion, short-term PTC and NT cultures offer a predictive model for evaluating therapeutic response in patients with PTC. Our PTC pre-clinical model suggests that combined targeted therapy might be an important therapeutic strategy for metastatic and refractory BRAF(V600E)-positive PTC.

Members of the thrombospondin gene family bind stromal interaction molecule 1 and regulate calcium channel activity.

The thrombospondins (TSPs) are a family of matricellular proteins that regulate cellular phenotype through interactions with a myriad of other proteins and proteoglycans. We have identified a novel interaction of the members of the TSP gene family with stromal interaction molecule 1 (STIM1). This association is robust since it is preserved in Triton X-100, can be detected with multiple anti-TSP-1 and anti-STIM1 antibodies, and is detected in a wide range of cell types. We have also found that STIM1 co-immunoprecipitates with TSP-4 and cartilage oligomeric matrix protein (COMP), and that a recombinant version of the N-terminal domain of STIM1 binds to the signature domain of TSP-1 and COMP. The association of the TSPs with STIM1 is observed in both the presence and absence of calcium indicating that the calcium-dependent conformation of the signature domain of TSPs is not required for binding. Thus, this interaction could occur in the ER under conditions of normal or low calcium concentration. Furthermore, we observed that the expression of COMP in HEK 293 cells decreases STIM1-mediated calcium release activated calcium (CRAC) channel currents and increases arachidonic acid calcium (ARC) channel currents. These data indicate that the TSPs regulate STIM1 function and participate in the reciprocal regulation of two channels that mediate calcium entry into the cell.

Thrombospondin-1 Modulates Actin Filament Remodeling and Cell Motility in Mouse Mammary Tumor cells in Vitro.

It is well established that the secretion of thrombospondin-1 (TSP-1) by activated stromal cells and its accumulation in the tumor microenvironment during dysplasia inhibits primary tumor growth through inhibition of angiogenesis. This inhibitory function of TSP-1 is actuated either by inhibiting MMP9 activation and the release of VEGF from extracellular matrix or by an interaction with CD36 on the surface of endothelial cells resulting in an increase in apoptosis. In contrast, several published articles have also shown that as tumor cells become more invasive and enter the early stage of carcinoma, they up-regulate TSP-1 expression, which may promote invasion and migration. In our in vivo studies using the polyoma middle T antigen (PyT) transgenic mouse model of breast cancer, we observed that the absence of TSP-1 significantly increased the growth of primary tumors, but delayed metastasis to the lungs. In this study, we propose a mechanism for the promigratory function of TSP-1 in mouse mammary tumor cells in vitro. We demonstrate the correlations between expression of TSP-1 and its receptor integrin α3ß1, which is considered a promigratory protein in cancer cells. In addition we propose that binding of TSP-1 to integrin α3ß1 is important for mediating actin filament polymerization and therefore, cell motility. These findings can help explain the dual functionality of TSP-1 in cancer progression.