Tumor-associated Blood Vessels Research Articles

Design and construction of a magnetic targeting pro-coagulant protein for embolic therapy of solid tumors

In this study, we have designed a magnetic targeting pro-coagulant protein (MTPCP) for the embolic therapy of solid tumours. The MTPCP consists of a magnetic carrier and a pro-coagulant protein. The pro-coagulant protein used in this study is the fusion protein tTF-EG3287 which is not pro-coagulant when free in the blood circulation, but presents strong pro-coagulant ability once bound to the Neuropilin-1(NRP-1) that is highly expressed on tumour-associated vascular endothelial cells. And the magnetic carrier is O-Carboxymethyl chitosan-coated iron oxide nanoparticles (OCMC/Fe3O4). In vitro, we assessed the NRP-1 targeting ability of the MTPCP using confocal microscopy and flow cytometry, and evaluated the potential pro-coagulant activity of the MTPCP using the Spectozyme FXa assay. In vivo, the magnetic targeting ability of the MTPCP was detected using a living imaging system. At last, we assessed the anticancer activity of the MTPCP on HepG2 tumour bearing BALB/c nude mice models including subcutaneous transplantation and orthotopic transplantation. HepG2 tumour bearing mice models revealed that after intravenous administration of the MTPCP, thrombosis specifically occurs on tumour-associated blood vessels, and resulting in tumour growth retardation. No apparent side effects, such as thrombosis in other organs or other treatment-related toxicity, were observed during the treatment. Our data showed that the MTPCP may be a promising embolic agent for the embolic therapy of solid tumours.

YAP1 Inhibition in Vascular Endothelial Cells Is Associated with Enhanced Release of lncRNA MALAT1-Containing Exosomes and Increased Hepatocarcinoma Invasion and Metastasis

Background: Liver cancer is one of the most common gastrointestinal malignancies. Anti-angiogenesis therapies have recently demonstrated promise in the treatment of liver cancer and other malignancies, although early treatment benefits may be accompanied by metastasis over time. Additional and more effective anti-angiogenic treatment modalities are therefore needed. We previously found that YAP1 expression is increased in hepatocellular carcinoma, particularly around tumor-associated blood vessels, suggesting a role in angiogenesis. The YAP1 inhibitor verteporfin (VP) is presently in anti-angiogenic clinical trials for the treatment of various cancers. Further elucidating the role of YAP1 in tumor angiogenesis may offer novel therapeutic targets. Methods and Results: We depleted YAP1 from vascular endothelial cells effectively reduces proliferation and tube formation, validating its utility as an anti-angiogenesis target. We also show that YAP1 depletion or inhibition in vascular endothelial cells leads to increased release of exosomes containing the lncRNA MALAT1 into the tumor microenvironment. Direct exosomal transfer of MALAT1 to hepatic cells leads to increased hepatic cell invasion and migration via activation of ERK1/2 signaling. Depletion of endothelial MALAT1 or inhibition of hepatocyte ERK1/2 signaling can suppress VP treatment-associated hepatocyte invasion and migration. Furthermore, we find that MALAT1 expression in patients with hepatocellular carcinoma is associated with decreased disease-free survival and increased lymph node metastasis. Conclusions: These observations may explain the occurrence of distant tumor metastasis with YAP1-associated anti-angiogenic therapy over time, and provide insight into new pathways and treatment paradigms that may be targeted to increase the long-term success of anti-angiogenic therapies. Funding Statement: This work was supported by grants from National Natural Science Foundation of China (No.81472302/No.81572425/No.81871983/No.81572831). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: The Institutional Review Board of the Fourth Affiliated Hospital of China Medical University approved the use of human tissue samples for this project. All patients gave their informed written consent for the use of clinical specimens for research.

Patient-specific organotypic blood vessels as an in vitro model for anti-angiogenic drug response testing in renal cell carcinoma.

BackgroundAnti-angiogenic treatment failure is often attributed to drug resistance, unsuccessful drug delivery, and tumor heterogeneity. Recent studies have speculated that anti-angiogenic treatments may fail due to characteristics inherent to tumor-associated blood vessels. Tumor-associated blood vessels are phenotypically different from their normal counterparts, having defective or permeable endothelial monolayers, abnormal sprouts, and abnormal vessel hierarchy. Therefore, to predict the efficacy of anti-angiogenic therapies in an individual patient, in vitro models that mirror individual patient's tumor vascular biology and response to anti-angiogenic treatment are needed.MethodsWe used a microfluidic in vitro organotypic model to create patient-specific biomimetic blood vessels from primary patient-specific tumor endothelial cells (TEnCs) and normal endothelial cells (NEnC). We assessed number of sprouts and vessel organization via microscopy imaging and image analysis. We characterized NEnC and TEnC vessel secretions via multiplex bead-based ELISA.FindingsUsing this model, we found that TEnC vessels exhibited more angiogenic sprouts than NEnC vessels. We also found a more disorganized and gap-filled endothelial monolayer. NEnCs and TEnC vessels exhibited heterogeneous functional drug responses across the five patients screened, as described in the clinic.InterpretationOur model recapitulated hallmarks of TEnCs and NEnCs found in vivo and captured the functional and structural differences between TEnC and NEnC vessels. This model enables a platform for therapeutic drug screening and assessing patient-specific responses with great potential to inform personalized medicine approaches.FundingNIH grants R01 EB010039, R33 CA225281, R01CA186134 University of Wisconsin Carbone Cancer Center (CA014520), and University of Wisconsin Hematology training grant T32 HL07899.

MCAM abnormal expression and clinical outcome associations are highly cancer dependent as revealed through pan-cancer analysis.

MCAM (CD146) is a cell surface adhesion molecule that has been reported to promote cancer development, progression and metastasis and is considered as a potential tumor biomarker and therapeutic target. However, inconsistent reports exist, and its clinical value is yet to be confirmed. Here we took advantage of several large genomic data collections (Genotype-Tissue Expression, The Cancer Genome Atlas and Cancer Cell Line Encyclopedia) and comprehensively analyzed MCAM expression in thousands of normal and cancer samples and cell lines along with their clinical phenotypes and drug response information. Our results show that MCAM is very highly expressed in large vessel tissues while majority of tissues have low or minimal expression. Its expression is dramatically increased in a few tumors but significantly decreased in most other tumors relative to their pairing normal tissues. Increased MCAM expression is associated with a higher tumor stage and worse patient survival for some less common tumors but not for major ones. Higher MCAM expression in primary tumors may be complicated by tumor-associated or normal stromal blood vessels yet its significance may differ from the one from cancer cells. MCAM expression is weakly associated with the response to a few small molecular drugs and the association with targeted anti-BRAF agents suggests its involvement in that pathway which warrants further investigation.

Loss of Multimerin-2 and EMILIN-2 Expression in Gastric Cancer Associate with Altered Angiogenesis.

Gastric cancer is a deadly tumor and a relatively common disease worldwide. Surgical resection and chemotherapy are the main clinical options to treat this type of disease, however the median overall survival rate is limited to one year. Thus, the development of new therapies is a highly necessary clinical need. Angiogenesis is a promising target for this tumor type, however clinical trials with the use of anti-angiogenic drugs have so far not met expectations. Therefore, it is important to better characterize the expression of molecules whose expression levels may impact on the efficacy of the treatments. In this study the characteristics of the gastric tumor associated blood vessels were first assessed by endomicroscopy. Next, we analyzed the expression of Multimerin-2, EMILIN-2 and EMILIN-1, three molecules of the EMI Domain ENdowed (EDEN) protein family. These molecules play important functions in the tumor microenvironment, affecting cancer progression both directly and indirectly impinging on angiogenesis and lymphangiogenesis. All the molecules were highly expressed in the normal mucosa whereas in a number of patients their expression was altered. We consider that better characterizing the gastric tumor microenvironment and the quality of the vasculature may achieve effective patient tailored therapies.

Correction: Targeting Vascular Endothelial-Cadherin in Tumor-Associated Blood Vessels Promotes T-cell-Mediated Immunotherapy.

Correction: Targeting Vascular Endothelial-Cadherin in Tumor-Associated Blood Vessels Promotes T-cell-Mediated Immunotherapy.

Oncolytic Viruses on Drugs: Achieving Higher Therapeutic Efficacy.

Over the past 20 years there has been a dramatic expansion in the testing of oncolytic viruses (OVs) for the treatment of cancer. OVs are unique biotherapeutics that induce multimodal responses toward tumors, from direct cytopathic effects on cancer cells, to tumor associated blood vessel disruption, and ultimately potent stimulation of anti-tumor immune activation. These agents are highly targeted and can be efficacious as cancer treatments resulting in some patients experiencing complete tumor regression and even cures from OV monotherapy. However, most patients have limited responses with viral replication in tumors often found to be modest and transient. To augment OV replication, increase bystander killing of cancer cells, and/or stimulate stronger targeted anti-cancer immune responses, drug combination approaches have taken center stage for translation to the clinic. Here we comprehensively review drugs that have been combined with OVs to increase therapeutic efficacy, examining the proposed mechanisms of action, and we discuss trends in pharmaco-viral immunotherapeutic approaches currently being investigated.

Abstract 5771: Targeting EphA2 in bladder cancer using a novel antibody-directed nanotherapeutic

Abstract Ephrin receptor A2 (EphA2) is a member of the Ephrin-Ephrin receptors cell to cell signaling family of molecules and plays a key role in proliferation, differentiation, and migration. EphA2 is overexpressed in a broad range of cancers, including bladder cancer, and is associated with increased metastasis and poor prognosis. Several potential EphA2-targeted therapies were developed and showed promising preclinical activity which failed to translate clinically due to narrow therapeutic window. we propose to use a novel antibody-directed nanotherapeutic (ADN) approach to target EphA2 for the treatment of bladder cancer. This work characterizes the expression of EphA2 in bladder cancer patients, evaluates its prognostic power, and tests an EphA2-targeted ADN, MM-310, in patient-derived xenograft (PDX) models. EphA2 expression in tumors was investigated using a validated immunohistochemistry assay performed in 147 bladder cancer samples, enabling analysis of prevalence and prognostic power. Whole sections of primary and metastatic tumor resections were used to assess EphA2 expression in tumor-associated vessels and tumor cells. Four EphA2+ PDX models were used to evaluate the activity of MM-310 compared to free docetaxel, alone or in combination with gemcitabine. EphA2 was expressed in tumor cells and tumor-associated blood vessels in primary and metastatic lesions. EphA2 overexpression was seen in 80%-100% of tumors and correlated with shorter survival. In the PDX models, MM-310 controlled tumor growth, mediated greater regression, and was more active than free docetaxel at equitoxic dosing (Table). Additionally, the combination of MM-310 and gemcitabine controlled tumor growth better than each drug alone, and outperformed the combination of free docetaxel and gemcitabine in the single PDX model where it was compared. Thus, in bladder cancer models, a docetaxel-based ADN as a monotherapy and in combination with gemcitabine targeted EphA2 and led to significant tumor regression. Maximum tumor regression (%) & Time to regrowth (days)Model NameControlDocetaxelMM-310p valueBL0293-2% & 3 days-58% & 55 days-100% & 120 days<.05 & <.001BL03820% & 3 days-16% & 34 days-100% & 106 days<.001 & <.001BL04400% & 7 days-43% & 41 days-84% & 67 days<.01 & <.01BL4173620% & 5 days-47% & 69 days-100% & 121 days<.05 & <.05 Citation Format: Walid Kamoun, Elden Swindell, Christine Pien, Lia Luus, Jason Cain, Irawati Kandela, Richard Huang, Suresh Tipparaju, Dmitri Kirpotin, Wiam Bshara, Vasileios Askoxylakis, Carl Morrison, Daryl Drummond. Targeting EphA2 in bladder cancer using a novel antibody-directed nanotherapeutic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5771.

Effect of multiple cyclic RGD peptides on tumor accumulation and intratumoral distribution of IRDye 700DX-conjugated polymers

Strategic delivery of IRDye 700DX (photosensitizer) is a key for improving its effect in photodynamic therapy. In this study, we have synthesized IRDye 700DX-conjugated polymers containing multiple cyclic RGD peptides to deliver IRDye 700DX selectively to tumor cells and tumor-associated blood vessels overexpressing αvβ3 integrin. Our polymer has a backbone of hydrophilic poly(ethylene glycol)-poly(L-glutamic acid) block copolymer, and cyclic RGD peptides are conjugated to side chains of the poly(L-glutamic acid) while IRDye 700DX is conjugated to the terminal of poly(ethylene glycol). The polymers exhibited selective accumulation to the target sites in a subcutaneous solid tumor, and the accumulation was augmented with the increased number of cyclic RGD peptides. More importantly, the polymer containing 15 cyclic RGD peptides in one construct revealed preferential accumulation on the tumor-associated blood vessels without compromising penetration to deep portions of the tumor, thereby drastically inhibiting tumor growth upon photoirradiation, while the polymer containing 5 cyclic RGD peptides showed moderate antitumor activity despite efficient accumulation in the tumor with almost homogenous intratumoral distribution. These results suggest that controlling the intratumoral distribution of IRDye 700DX is critical for successful PDT, and our polymer containing multiple cyclic RGD peptides may be a promising carrier for this spatial control.

Nanoparticle Formulation Derived from Carboxymethyl Cellulose, Polyethylene Glycol, and Cabazitaxel for Chemotherapy Delivery to the Brain.

Nanoparticles provide a unique opportunity to explore the benefits of selective distribution and release of cancer therapeutics at sites of disease through varying particle sizes and compositions that exploit the enhanced permeability of tumor-associated blood vessels. Though delivery of larger as opposed to smaller and/or actively transported molecules to the brain is prima facie a challenging endeavor, we wondered whether nanoparticles could improve the therapeutic index of existing drugs for use in treating brain tumors via these vascular effects. We therefore selected a family of nanoparticles composed of cabazitaxel-carboxymethyl cellulose amphiphilic polymers to investigate the potential for delivering a brain-penetrant taxane to intracranial brain tumors in mice. Among a small set of nanoparticle formulations, we found evidence for nanoparticle accumulation in the brain, and one such formulation demonstrated activity in an orthotopic model of glioma, suggesting that such nanoparticles could be useful for the treatment of glioma and brain metastases of other tumor types.

Selective Inhibition of Histone Deacetylation in Melanoma Increases Targeted Gene Delivery by a Bacteriophage Viral Vector.

The previously developed adeno-associated virus/phage (AAVP) vector, a hybrid between M13 bacteriophage (phage) viruses that infect bacteria only and human Adeno-Associated Virus (AAV), is a promising tool in targeted gene therapy against cancer. AAVP can be administered systemically and made tissue specific through the use of ligand-directed targeting. Cancer cells and tumor-associated blood vessels overexpress the αν integrin receptors, which are involved in tumor angiogenesis and tumor invasion. AAVP is targeted to these integrins via a double cyclic RGD4C ligand displayed on the phage capsid. Nevertheless, there remain significant host-defense hurdles to the use of AAVP in targeted gene delivery and subsequently in gene therapy. We previously reported that histone deacetylation in cancer constitutes a barrier to AAVP. Herein, to improve AAVP-mediated gene delivery to cancer cells, we combined the vector with selective adjuvant chemicals that inhibit specific histone deacetylases (HDAC). We examined the effects of the HDAC inhibitor C1A that mainly targets HDAC6 and compared this to sodium butyrate, a pan-HDAC inhibitor with broad spectrum HDAC inhibition. We tested the effects on melanoma, known for HDAC6 up-regulation, and compared this side by side with a normal human kidney HEK293 cell line. Varying concentrations were tested to determine cytotoxic levels as well as effects on AAVP gene delivery. We report that the HDAC inhibitor C1A increased AAVP-mediated transgene expression by up to ~9-fold. These findings indicate that selective HDAC inhibition is a promising adjuvant treatment for increasing the therapeutic value of AAVP.

Meeting the needs of breast cancer: A nucleolin's perspective.

A major challenge in the management of breast cancer disease has been the development of metastases. Finding new molecular targets and the design of targeted therapeutic approaches to improve the overall survival and quality of life of these patients is, therefore, of great importance. Nucleolin, which is overexpressed in cancer cells and tumor-associated blood vessels, have been implicated in various processes supporting tumorigenesis and angiogenesis. Additionally, its overexpression has been demonstrated in a variety of human neoplasias as an unfavorable prognostic factor, associated with a high risk of relapse and low overall survival. Hence, nucleolin has emerged as a relevant target for therapeutic intervention in cancer malignancy, including breast cancer. This review focus on the contribution of nucleolin for cancer disease and on the development of therapeutic strategies targeting this protein. In this respect, it also provides a critical analysis about the potential and pitfalls of nanomedicine for cancer therapy.

A Chimeric Antibody against ACKR3/CXCR7 in Combination with TMZ Activates Immune Responses and Extends Survival in Mouse GBM Models.

Glioblastoma (GBM) is the least treatable type of brain tumor, afflicting over 15,000 people per year in the United States. Patients have a median survival of 16 months, and over 95% die within 5 years. The chemokine receptor ACKR3 is selectively expressed on both GBM cells and tumor-associated blood vessels. High tumor expression of ACKR3 correlates with poor prognosis and potential treatment resistance, making it an attractive therapeutic target. We engineered a single chain FV-human FC-immunoglobulin G1 (IgG1) antibody, X7Ab, to target ACKR3 in human and mouse GBM cells. We used hydrodynamic gene transfer to overexpress the antibody, with efficacy in vivo. X7Ab kills GBM tumor cells and ACKR3-expressing vascular endothelial cells by engaging the cytotoxic activity of natural killer (NK) cells and complement and the phagocytic activity of macrophages. Combining X7Ab with TMZ allows the TMZ dosage to be lowered, without compromising therapeutic efficacy. Mice treated with X7Ab and in combination with TMZ showed significant tumor reduction by MRI and longer survival overall. Brain-tumor-infiltrating leukocyte analysis revealed that X7Ab enhances the activation of M1 macrophages to support anti-tumor immune response in vivo. Targeting ACKR3 with immunotherapeutic monoclonal antibodies (mAbs) in combination with standard of care therapies may prove effective in treating GBM.

A DNA nanorobot functions as a cancer therapeutic in response to a molecular trigger in vivo.

Nanoscale robots have potential as intelligent drug delivery systems that respond to molecular triggers. Using DNA origami we constructed an autonomous DNA robot programmed to transport payloads and present them specifically in tumors. Our nanorobot is functionalized on the outside with a DNA aptamer that binds nucleolin, a protein specifically expressed on tumor-associated endothelial cells, and the blood coagulation protease thrombin within its inner cavity. The nucleolin-targeting aptamer serves both as a targeting domain and as a molecular trigger for the mechanical opening of the DNA nanorobot. The thrombin inside is thus exposed and activates coagulation at the tumor site. Using tumor-bearing mouse models, we demonstrate that intravenously injected DNA nanorobots deliver thrombin specifically to tumor-associated blood vessels and induce intravascular thrombosis, resulting in tumor necrosis and inhibition of tumor growth. The nanorobot proved safe and immunologically inert in mice and Bama miniature pigs. Our data show that DNA nanorobots represent a promising strategy for precise drug delivery in cancer therapy.

Antibody-based delivery of tumor necrosis factor (L19-TNFα) and interleukin-2 (L19-IL2) to tumor-associated blood vessels has potent immunological and anticancer activity in the syngeneic J558L BALB/c myeloma model.

To analyze the impact of TNFα or IL2 on human lymphocytes in vitro and the anti-tumor and immune-modifying effects of L19-IL2 and L19-TNFα on subcutaneously growing J558L myeloma in immunocompetent mice. PBMCs from three healthy volunteers were incubated with IL2, TNFα, or with IL2 plus addition of TNFα (final 20h). BALB/c J558L mice with subcutaneous tumors were treated with intravenous L19-TNFα plus L19-IL2, or controls. Tumor growth and intra- and peri-tumoral tissues were analyzed for micro-vessel density, necrosis, immune cell composition, and PD1 or PD-L1 expressing cells. Exposure of PBMC in vitro to IL2, TNFα, or to IL2 over 3 and 5 days plus TNFα for the final 20h resulted in an approximately 50 and 75% reduction of the CD25low effector cell/CD25high Treg cell ratio, respectively, compared to medium control. IL2 or TNFα increased the proportion of CD4- CD25low effector lymphocytes while reducing the proportion of CD4+ CD25low Teff cells. In the J558L myeloma model, tumor eradication was observed in 58, 42, 25, and 0% of mice treated with L19-TNFα plus L19-IL2, L19-TNFα, L19-IL2, and PBS, respectively. L19-TNFα/L19-IL2 combination caused tumor necrosis, capillary density doubling, peri-tumoral T cell and PD1+ T cell reduction (- 50%), and an increase in PD-L1+ myeloma cells. IL2, TNFα, or IL2 plus TNFα (final 20h) increased the proportion of CD4- CD25low effector lymphocytes possibly indicating immune activation. L19-TNFα/L19-IL2 combination therapy eradicated tumors in J558L myeloma BALB/c mice likely via TNFα-induced tumor necrosis and L19-TNFα/L19-IL2-mediated local cellular immune reactions.

Targeting Vascular Endothelial-Cadherin in Tumor-Associated Blood Vessels Promotes T-cell-Mediated Immunotherapy.

T-cell infiltration of solid tumors is associated with improved prognosis and favorable responses to immunotherapy. Mechanisms that enable tumor infiltration of CD8+ T cells have not been defined, nor have drugs that assist this process been discovered. Here we address these issues with a focus on VE-cadherin, a major endothelial cell-specific junctional protein that controls vascular integrity. A decrease in VE-cadherin expression is associated with tumor pathology. We developed an oligonucleotide-based inhibitor (CD5-2), which disrupted the interaction of VE-cadherin with its regulator miR-27a, resulting in increased VE-cadherin expression. Administration of CD5-2 in tumor-bearing mice enhanced expression of VE-cadherin in tumor endothelium, activating TIE-2 and tight junction pathways and normalizing vessel structure and function. CD5-2 administration also enhanced tumor-specific T-cell infiltration and spatially redistributed CD8+ T cells within the tumor parenchyma. Finally, CD5-2 treatment enhanced the efficacy of anti-PD-1 blocking antibody. Our work establishes a role for VE-cadherin in T-cell infiltration in tumors and offers a preclinical proof of concept for CD5-2 as a therapeutic modifier of cancer immunotherapy via effects on the tumor vasculature. Cancer Res; 77(16); 4434-47. ©2017 AACR.

Abstract 793: Human endothelial progenitor cells: A new target for anti-vascular therapy

Abstract Breast cancer affects one in eight women in the USA. Early diagnosis and newer treatment modalities have rendered breast cancer manageable. However, triple negative breast cancer is still difficult to treat and warrantes a search for newer targets. One strategy that has emerged in cancer research involves targeting of tumor associated blood vessels which provide growing tumors with oxygenated blood and growth factors necessary for maintenance and metastasis. Antiangiogenic drug therapy is transient and has not been able to gain mainstream therapeutic modality. We discovered that endothelial progenitor cells (EPCs) are mobilized from the bone marrow to the tumor site and contribute to the development of breast tumor vessel formation in an estrogen dependent manner. Therefore, characterization of tumor associated endothelial progenitor cells in breast cancer may provide a more specific antivascular therapy. Using the highly proliferative human umbilical cord blood derived EPCs, having the phenotype (CD133+, CD34+, VEGFR-2+), the effect of growth factor and chemokine rich EPCs conditioned medium (CM) was assessed in luminal (MCF-7), and post-EMT (MDA-MB-231) breast carcinoma cell lines. We observed an initial halt in cellular proliferation in MCF-7 followed by a significant increase in proliferation after forty eight hours of treatment. On the other hand, MDA-MB-231 showed decreased proliferation even after forty eight hours of treatment. Treating the EPCs with breast cancer conditioned medium resulted in morphological and cellular growth changes in the EPCs. MDA-MB-231 CM resulted in an increase of the EPCs proliferation and differentiation by increasing the number of spindle shaped attaching cells, and MCF-7 CM resulted only in an increase in the differentiation rate by increasing the number of cell clusters. This increase in EPCs proliferation and differentiation associated with MDA-MB-231 CM treatment might explain the invasiveness of this breast cancer cells through the increase in the tumor associated neovascularization. The analysis of the paracrine interaction between breast cancer cells and EPCs along with the associated cellular changes will facilitate identification of the interactive mediators and subsequent development of effective antivascular therapy. Citation Format: Ghada Ben Rahoma, Neha Tuli, Rachana Maniyar, Sanjukta Chakraborty, Sarnath Singh, Abraham Mittelman, Raj K. Tiwari. Human endothelial progenitor cells: A new target for anti-vascular therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 793. doi:10.1158/1538-7445.AM2017-793

Abstract 1824: Endothelial mTORC2 deficiency inhibits tumor angiogenesis, tumor progression and metastasis in non-small cell lung cancer (NSCLC)

Abstract Vascular endothelial cells (ECs) are specialized components of the tumor microenvironment that orchestrates tumor growth and invasion. They form tumor-associated blood vessels (angiogenic) that supply nutrients and oxygen, remove waste products, and also provide an entry site for tumorigenic cells to spread to secondary sites in various organs. One of the major signaling events in tumor vessels is PI3K/PDK-AKT-mTOR activation. mTOR is a serine/threonine kinase that functions in two distinct complexes, mTORC1 and mTORC2,which regulate a diverse array of cellular processes including cell growth, survival and metabolism. Although mTORC1, and to lesser extent, mTORC2, has been broadly studied in cancers and diseases, little is known regarding the relative contributions of mTORC1 versus mTORC2 in tumor endothelium. Using mouse model of endothelium-specific Rictor (a mTORC2 specific cofactor) gene targeting, we discovered that endothelial Rictor deletion decreased tumor neovascularization, suppressed tumor growth, and prevented metastasis in vivo. Direct co-culture of endothelial cells and NSCLC tumor cells in vitro showed that knockdown of endothelial Rictor inhibited tumor cell proliferation while Raptor (a mTORC1 specific cofactor) depletion had a modest effect. However, loss of endothelial Rictor or Raptor both markedly decreased tumor cell extravasation in response to endothelial cells, and also inhibited tumor spheroid formation in a direct co-culture system. Furthermore, a screen of phospho-kinase arrays revealed that Raptor or Rictor depletion in endothelial cells decreased various phosphor-RTKs in tumor cells that were co-cultured with ECs, including members of the Eph receptor family, Insulin receptor family and FGF receptor family. Additionally, endothelial Rictor knockdown suppressed phosphorylation levels of nerve growth factor receptors TrkB and TrkC in tumor cells. Collectively, these data suggest that endothelial mTORC1 and mTORC2 play critical roles in regulating tumor progression through distinct signaling pathways. Note: This abstract was not presented at the meeting. Citation Format: Shan Wang. Endothelial mTORC2 deficiency inhibits tumor angiogenesis, tumor progression and metastasis in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1824. doi:10.1158/1538-7445.AM2017-1824

High expression of insulin receptor on tumour-associated blood vessels in invasive bladder cancer predicts poor overall and progression-free survival.

Bladder cancer is a frequently recurring disease with a very poor prognosis once progressed to invasive stages, and tumour-associated blood vessels play a crucial role in this process. In order to identify novel biomarkers associated with progression, we isolated blood vascular endothelial cells (BECs) from human invasive bladder cancers and matched normal bladder tissue, and found that tumour-associated BECs greatly up-regulated the expression of insulin receptor (INSR). High expression of INSR on BECs of invasive bladder cancers was significantly associated with shorter progression-free and overall survival. Furthermore, increased expression of the INSR ligand IGF-2 in invasive bladder cancers was associated with reduced overall survival. INSR may therefore represent a novel biomarker to predict cancer progression. Mechanistically, we observed pronounced hypoxia in human bladder cancer tissue, and found a positive correlation between the expression of the hypoxia marker gene GLUT1 and vascular INSR expression, indicating that hypoxia drives INSR expression in tumour-associated blood vessels. In line with this, exposure of cultured BECs and human bladder cancer cell lines to hypoxia led to increased expression of INSR and IGF-2, respectively, and IGF-2 increased BEC migration through the activation of INSR in vitro. Taken together, we identified vascular INSR expression as a potential biomarker for progression in bladder cancer. Furthermore, our data suggest that IGF-2/INSR mediated paracrine crosstalk between bladder cancer cells and endothelial cells is functionally involved in tumour angiogenesis and may thus represent a new therapeutic target. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tumor-Associated Lymphatic Vessels Upregulate PDL1 to Inhibit T-Cell Activation.

Tumor-associated lymphatic vessels (LVs) play multiple roles during tumor progression, including promotion of metastasis and regulation of antitumor immune responses by delivering antigen from the tumor bed to draining lymph nodes (LNs). Under steady-state conditions, LN resident lymphatic endothelial cells (LECs) have been found to maintain peripheral tolerance by directly inhibiting autoreactive T-cells. Similarly, tumor-associated lymphatic endothelium has been suggested to reduce antitumor T-cell responses, but the mechanisms that mediate this effect have not been clarified. Using two distinct experimental tumor models, we found that tumor-associated LVs gain expression of the T-cell inhibitory molecule PDL1, similar to LN resident LECs, whereas tumor-associated blood vessels downregulate PDL1. The observed lymphatic upregulation of PDL1 was likely due to IFN-g released by stromal cells in the tumor microenvironment. Furthermore, we found that blocking PDL1 results in increased T-cell stimulation by antigen-presenting LECs in vitro. Taken together, our data suggest that peripheral, tumor-associated lymphatic endothelium contributes to T-cell inhibition, by a mechanism similar to peripheral tolerance maintenance described for LN resident LECs. These findings may have clinical implications for cancer therapy, as lymphatic expression of PDL1 could represent a new biomarker to select patients for immunotherapy with PD1 or PDL1 inhibitors.