Vessels In Normal Tissue Research Articles

From Localized Mild Hyperthermia to Improved Tumor Oxygenation: Physiological Mechanisms Critically Involved in Oncologic Thermo-Radio-Immunotherapy.

(1) Background: Mild hyperthermia (mHT, 39-42 °C) is a potent cancer treatment modality when delivered in conjunction with radiotherapy. mHT triggers a series of therapeutically relevant biological mechanisms, e.g., it can act as a radiosensitizer by improving tumor oxygenation, the latter generally believed to be the commensurate result of increased blood flow, and it can positively modulate protective anticancer immune responses. However, the extent and kinetics of tumor blood flow (TBF) changes and tumor oxygenation are variable during and after the application of mHT. The interpretation of these spatiotemporal heterogeneities is currently not yet fully clarified. (2) Aim and methods: We have undertaken a systematic literature review and herein provide a comprehensive insight into the potential impact of mHT on the clinical benefits of therapeutic modalities such as radio- and immuno-therapy. (3) Results: mHT-induced increases in TBF are multifactorial and differ both spatially and with time. In the short term, changes are preferentially caused by vasodilation of co-opted vessels and of upstream normal tissue vessels as well as by improved hemorheology. Sustained TBF increases are thought to result from a drastic reduction of interstitial pressure, thus restoring adequate perfusion pressures and/or HIF-1α- and VEGF-mediated activation of angiogenesis. The enhanced oxygenation is not only the result of mHT-increased TBF and, thus, oxygen availability but also of heat-induced higher O2 diffusivities, acidosis- and heat-related enhanced O2 unloading from red blood cells. (4) Conclusions: Enhancement of tumor oxygenation achieved by mHT cannot be fully explained by TBF changes alone. Instead, a series of additional, complexly linked physiological mechanisms are crucial for enhancing tumor oxygenation, almost doubling the initial O2 tensions in tumors.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Immunohistochemical Staining for Lymphatic and Blood Vessels in Normal Tissues: Comparison between Routinely Paraffin-Embedded Tissues and Frozen Sections.

In the current study, we compared the distribution of blood and lymphatic vessels from paraffin-embedded tissues with those of frozen tissues of normal human and rhesus monkey. We performed immunocytochemical staining for lymphatic and blood vessels using LYVE-1 for lymphatic vessels and von Willebrand factor (F-8) for blood vessels. Normal tissues included spleen, lymph node, liver, pancreas, salivary gland, colon, diaphragm, heart, lung, thyroid, adrenal gland, kidney, ovary, endometrium, and prostate. Splenic sinusoids were stained for LYVE-1 and F-8 in the frozen sections, supporting that the sinusoid is a lymphoreticular system and blood vessel in structure and function. In frozen sections, the lymphatic sinusoids were consistently positive for LYVE-1, while hepatic sinusoids were positive for LYVE-1, but not for F-8. Thus, lymphatic and blood vessels were more readily detected in frozen tissue sections than in the paraffin-embedded sections. In the endometrium, lymphatic vessels were not diffusely immunostained in paraffin-embedded sections. However, frozen sections detected cyclic changes of lymphatic vessels, growing from basalis to functionalis in the menstrual cycle. Lymphatic vessels were immunostained in many organs using frozen sections. Small pulmonary blood vessels were not immunostained by F-8 in the periphery of the bronchial vessel tree most likely these smallest blood vessels were not immunostained due to less F-8 attached to their endothelia. The present findings illustrate the differences in the immunostaining of blood vessels in sections obtained from paraffin-embedded tissues and those from frozen tissue. These new findings may be relevant for the basic histology and histopathology of lymphatic and blood vessels.

Angiogenesis as a hallmark of solid tumors - clinical perspectives.

Angiogenesis is a key and early step in tumorigenesis, and is known as a hallmark of solidtumors and a key promoter of tumor recurrence. Unlike normal tissue vessels, the architecture of the tumor vasculature is abnormal, being leaky, tortuous, fragile and blind-ended. Perivascular cells are either detached or absent, causing reduction of vascular integrity, an increase in vessel immaturity, incoherent perfusion, defective functionality and enhanced tumor dissemination and metastasis. The abnormal tumor vasculature along with the defective tumor vessel functionality finally causes bouts of hypoxia and acidity in the tumor microenvironment (TME), further reinvigorating tumor aggression. Interstitial hypertension or high interstitial fluid pressure (IFP) is an outcome of tumor hyper-permeability. High IFP can be a barrier for either effective delivery of anti-cancer drugs toward the TME or accumulation of drugs within the tumor area, thus promoting tumor resistance to therapy. Some tumors do, however, not undergo angiogenesis but instead undergo vessel co-option or vascular mimicry, thereby adding another layer of complexity to cancerdevelopment and therapy. Combination of anti-angiogenesis therapy with chemotherapy and particularly with immune checkpoint inhibitors (ICIs) is a promising strategy for a number of advanced cancers. Among the various approaches for targeting tumor angiogenesis, vascular normalization is considered as the most desired method, which allows effective penetration of chemotherapeutics into the tumor area, thus being an appropriate adjuvantto other cancer modalities.

Prostate-Specific Membrane Antigen (PSMA) Promotes Angiogenesis of Glioblastoma Through Interacting With ITGB4 and Regulating NF-κB Signaling Pathway.

BackgroundGlioblastoma multiforme (GBM) is the most common primary malignant tumor in the central nervous system (CNS), causing the extremely poor prognosis. Combining the role of angiogenesis in tumor progression and the role of prostate-specific membrane antigen (PSMA) in angiogenesis, this study aims to explore the functions of PSMA in GBM.MethodsClinical GBM specimens were collected from 60 patients who accepted surgical treatment in Fudan University Shanghai Cancer Center between January 2018 and June 2019. Immunohistochemical staining was used to detect PSMA and CD31 expression in GBM tissues. Prognostic significance of PSMA was evaluated by bioinformatics. Human umbilical vein endothelial cells (HUVECs) transfected with PSMA overexpression plasmids or cultured with conditioned medium collected based on GBM cells, were used for CCK8, Transwell and tube formation assays. High-throughput sequencing and immunoprecipitation were used to explore the underlying mechanism. Furthermore, the in vivo experiment had been also conducted.ResultsWe demonstrated that PSMA was abundantly expressed in endothelium of vessels of GBM tissues but not in vessels of normal tissues, which was significantly correlated with poor prognosis. Overexpression of PSMA could promotes proliferation, invasion and tube formation ability of human umbilical vein endothelial cells (HUVECs). Moreover, U87 or U251 conditioned medium could upregulated PSMA expression and induce similar effects on phenotypes of HUVECs, all of which could be partially attenuated by 2-PMPA treatment. The mechanistic study revealed that PSMA might promote angiogenesis of GBM through interacting with Integrin β4 (ITGB4) and activating NF-κB signaling pathway. The in vivo growth of GBM could be alleviated by the treatment of 2-PMPA.ConclusionThis study identified PSMA as a critical regulator in angiogenesis and progression of GBM, which might be a promising therapeutic target for GBM treatment.

A novel anti-CD146 monoclonal antibody, AA98, inhibits angiogenesis and tumor growth

AbstractThe goal of our study was to raise monoclonal antibodies (mAbs) against endothelial cell—surface proteins specific for tumor vasculature. Here, we describe the generation and intensive characterization of mAb AA98, including its functional properties and its antigen identification. In our study, an enhanced mAb AA98 immunoreactivity was observed on stimulated human umbilical vein endothelial cells (HUVECs). In addition, mAb AA98 showed remarkably restricted immunoreactivity against intratumoral neovasculature compared with blood vessels of normal tissues. We identified the AA98 antigen as human CD146, an adhesion molecule belonging to the immunoglobulin superfamily. Data from in vitro experiments imply structural and signaling functions for endothelial CD146; however, the role of CD146 in vivo is largely unknown. Here, we show that mAb AA98 displays antiangiogenic properties in vitro and in vivo. Proliferation and migration of HUVECs were inhibited by mAb AA98 as was angiogenesis in chicken chorioallantoic membrane (CAM) assays and tumor growth in 3 xenografted human tumor models in mice. Our data provide new insights into the function of CD146 on endothelial cells, validate CD146 as a novel target for antiangiogenic agents, and demonstrate that mAb AA98 has potential as a diagnostic and therapeutic agent in vascular and cancer biology. (Blood. 2003;102:184-191)

Abstract 1488: A novel Notch4 neutralizing antibody inhibits angiogenesis and tumor growth via a distinct mechanism from endothelial Notch1 inhibition

Abstract Endothelial Notch signaling is critical for tumor angiogenesis and growth. Inhibitors of Notch1 or its ligand Dll4 cause tumor vessel hypersprouting but interfere with vessel perfusion causing reduced tumor growth. Unfortunately, normal tissue vessels respond to these inhibitors causing tissue hypoxia or vascular neoplasms. Mice null for Notch4, unlike Notch1 mutants, exhibit reduced developmental and tumor angiogenesis. Notch4 expression is increased in the endothelium of some tumor types. These results suggest that specifically targeting Notch4 could reduce tumor angiogenesis with fewer effects on normal tissue and represents an unexplored therapeutic opportunity. We use a newly developed anti-Notch4 antibody, E7011, and show that in endothelial cells, inhibition of Notch4 signaling suppresses expression of a subset of canonical Notch target genes. Treatment with E7011 also suppresses expression of a unique set of genes not regulated by canonical Dll4-Notch1 signaling, which appear to be distinct Notch4 targets. We administered E7011 to neonatal mice to examine the effects on developmental retinal angiogenesis. A single dose of E7011 significantly reduced retinal vascular outgrowth, demonstrating that E7011 has anti-angiogenic activity in vivo. E7011 treatment did not alter capillary density within the vascular plexus or endothelial tip cell number, both of which are typically increased by loss of Notch1 signaling. Because the E7011 phenotype was markedly distinct from the Notch1 loss of function, we combined E7011 treatment with endothelial-specific loss of Notch1 (Notch1ECKO) to compare Notch1 to Notch4 function. E7011-treated Notch1ECKO retina showed a compound phenotype, exhibiting both the loss of vascular outgrowth characteristic of E7011/Notch4 and the hypersprouting characteristic of Notch1 loss of function. These results suggest that Notch1 and Notch4 have distinct roles in developmental angiogenesis, with Notch4 being a pro-angiogenic signaling pathway. We evaluated Notch4 expression in the breast cancer tumor microenvironment to determine if Notch4 functions in tumor vessels. Human triple negative breast cancer (TNBC) shows a range of expression of Notch4 in both tumor cells and tumor endothelium. The anti-angiogenic effects that we observe in the retina lead us to hypothesize that E7011 will show anti-tumor activity against tumors that induce Notch4 expression in neighboring endothelium, and that targeting endothelial Notch4 is a novel anti-angiogenic approach. Citation Format: L. A. Naiche, Bhairavi Swaminathan, Yu Kato, Sarita Das, Jason W. Eng, Qanber Raza, Krishna Thakkar, James H. Herts, Rajyasree Emmadi, Junji Matsui, Jan K. Kitajewski. A novel Notch4 neutralizing antibody inhibits angiogenesis and tumor growth via a distinct mechanism from endothelial Notch1 inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1488.

Endothelial, pericyte and tumor cell expression in glioblastoma identifies fibroblast activation protein (FAP) as an excellent target for immunotherapy.

ObjectivesTargeted 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.MethodsGlioblastoma 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).ResultsCompared 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.ConclusionFibroblast 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.

Acute cellular and vascular responses to photodynamic therapy using EGFR-targeted nanobody-photosensitizer conjugates studied with intravital optical imaging and magnetic resonance imaging.

Targeted photodynamic therapy (PDT) has the potential to selectively damage tumor tissue and to increase tumor vessel permeability. Here we characterize the tissue biodistribution of two EGFR-targeted nanobody-photosensitizer conjugates (NB-PS), the monovalent 7D12-PS and the biparatopic 7D12-9G8-PS. In addition, we report on the local and acute phototoxic effects triggered by illumination of these NB-PS which have previously shown to lead to extensive tumor damage.Methods: Intravital microscopy and the skin-fold chamber model, containing OSC-19-luc2-cGFP tumors, were used to investigate: a) the fluorescence kinetics and distribution, b) the vascular response and c) the induction of necrosis after illumination at 1 or 24 h post administration of 7D12-PS and 7D12-9G8-PS. In addition, dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) of a solid tumor model was used to investigate the microvascular status 2 h after 7D12-PS mediated PDT.Results: Image analysis showed significant tumor colocalization for both NB-PS which was higher for 7D12-9G8-PS. Intravital imaging showed clear tumor cell membrane localization 1 and 2 h after administration of 7D12-9G8-PS, and fluorescence in or close to endothelial cells in normal tissue for both NB-PS. PDT lead to vasoconstriction and leakage of tumor and normal tissue vessels in the skin-fold chamber model. DCE-MRI confirmed the reduction of tumor perfusion after 7D12-PS mediated PDT. PDT induced extensive tumor necrosis and moderate normal tissue damage, which was similar for both NB-PS conjugates. This was significantly reduced when illumination was performed at 24 h compared to 1 h after administration.Discussion: Although differences were observed in distribution of the two NB-PS conjugates, both led to similar necrosis. Clearly, the response to PDT using NB-PS conjugates is the result of a complex mixture of tumor cell responses and vascular effects, which is likely to be necessary for a maximally effective treatment.

Modulation of pericytes by a fusion protein comprising of a PDGFRβ-antagonistic affibody and TNFα induces tumor vessel normalization and improves chemotherapy

The delivery of anticancer drugs is hampered by tumor vessels with abnormal structure and function, which requires that vessel normalization be mediated by pharmaceutics. The current strategies for vessel normalization focus on direct modulation of endothelial cells (ECs), which frequently affect vessels in normal tissues. Modulating EC-supporting cells, such as pericytes (PCs), is a new direction. Here, we produced a fusion protein, Z-TNFα, by fusing the platelet-derived growth factor receptor β (PDGFRβ)- antagonistic affibody ZPDGFRβ to tumor necrosis factor α (TNFα). Owing to the affinity of fused ZPDGFRβ for PDGFRβ, Z-TNFα binds PDGFRβ+ PCs but not PDGFRβ− ECs. Low-dose (1 μg/mouse) Z-TNFα treatment remodeled the tumor vessels, thus reducing vessel permeability and increasing vessel perfusion. As a result, the Z-TNFα treatment improved the delivery of doxorubicin (DOX) and enhanced its antitumor effect, indicating that Z-TNFα induced normalization of tumor vessels. Mechanically, the tumor vessel normalization mediated by Z-TNFα might be attributed to the reduction of vascular endothelial growth factor (VEGF) secretion by PCs and the elevated expression of intercellular cell adhesion molecule-1 (ICAM-1) in PCs, which might suppress the proliferation and migration of ECs and simultaneously trigger interaction between perivascular macrophages and PCs. These results demonstrated that tumor-associated PCs could be considered novel target cells for vessel normalization, and Z-TNFα might be developed as a potential tool for antitumor combination therapy.

Rhodocetin-αβ selectively breaks the endothelial barrier of the tumor vasculature in HT1080 fibrosarcoma and A431 epidermoid carcinoma tumor models.

The tumor vasculature differs from normal blood vessels in morphology, composition and stability. Here, we describe a novel tumor vessel-disrupting mechanism. In an HT1080/mouse xenograft tumor model rhodocetin-αβ was highly effective in disrupting the tumor endothelial barrier. Mechanistically, rhodocetin-αβ triggered MET signaling via neuropilin-1. As both neuropilin-1 and MET were only lumen-exposed in a subset of abnormal tumor vessels, but not in normal vessels, the prime target of rhodocetin-αβ were these abnormal tumor vessels. Consequently, cells lining such tumor vessels became increasingly motile which compromised the vessel wall tightness. After this initial leakage, rhodocetin-αβ could leave the bloodstream and reach the as yet inaccessible neuropilin-1 on the basolateral side of endothelial cells and thus disrupt nearby vessels. Due to the specific neuropilin-1/MET co-distribution on cells lining such abnormal tumor vessels in contrast to normal endothelial cells, rhodocetin-αβ formed the necessary trimeric signaling complex of rhodocetin-αβ-MET-neuropilin-1 only in these abnormal tumor vessels. This selective attack of tumor vessels, sparing endothelial cell-lined vessels of normal tissues, suggests that the neuropilin-1-MET signaling axis may be a promising drugable target for anti-tumor therapy, and that rhodocetin-αβ may serve as a lead structure to develop novel anti-tumor drugs that target such vessels.

Enhanced expression of Programmed cell death 1 (PD-1) protein in benign vascular anomalies

Programmed cell death 1 (PD-1) and its ligands have been shown to play a significant role in evasion of malignant tumour cells from the immune system. Last year, the United States Food and Drug Administration (FDA) approved anti-PD-1 inhibitors for treatment of non-small cell lung carcinoma and recently has approved anti-PD-L1 blocker for treatment of metastatic urothelial cell carcinoma. However, the role that the immune system might have on benign tumours including vascular anomalies has received less attention. In this study, we evaluated PD-1 and PD-L1 expression on two benign vascular anomalies: infantile haemangiomas and venous malformations. Tissue microarrays (TMAs) from these two entities were stained for PD-1 and PD-L1 antibodies. Blood vessels from normal tissue were used as control. The endothelial cells in both infantile haemangioma and venous malformation showed high expression of PD-1 but were negative for PD-L1. Endothelial cells within the blood vessels in normal tissues were negative for both PD-1 and PD-L1. Our results showed over-expression of PD-1 in subsets of vascular anomalies, while PD-L1 was negative. This would raise the possibility of immunotherapy in benign vascular tumour when other options are exhausted.

Multiphoton imaging reveals that nanosecond pulsed electric fields collapse tumor and normal vascular perfusion in human glioblastoma xenografts

Despite the biomedical advances of the last century, many cancers including glioblastoma are still resistant to existing therapies leaving patients with poor prognoses. Nanosecond pulsed electric fields (nsPEF) are a promising technology for the treatment of cancer that have thus far been evaluated in vitro and in superficial malignancies. In this paper, we develop a tumor organoid model of glioblastoma and apply intravital multiphoton microscopy to assess their response to nsPEFs. We demonstrate for the first time that a single 10 ns, high voltage electric pulse (35–45 kV/cm), collapses the perfusion of neovasculature, and also alters the diameter of capillaries and larger vessels in normal tissue. These results contribute to the fundamental understanding of nsPEF effects in complex tissue environments, and confirm the potential of nsPEFs to disrupt the microenvironment of solid tumors such as glioblastoma.

Tumor vessel normalization after aerobic exercise enhances chemotherapeutic efficacy.

Targeted therapies aimed at tumor vasculature are utilized in combination with chemotherapy to improve drug delivery and efficacy after tumor vascular normalization. Tumor vessels are highly disorganized with disrupted blood flow impeding drug delivery to cancer cells. Although pharmacologic anti-angiogenic therapy can remodel and normalize tumor vessels, there is a limited window of efficacy and these drugs are associated with severe side effects necessitating alternatives for vascular normalization. Recently, moderate aerobic exercise has been shown to induce vascular normalization in mouse models. Here, we provide a mechanistic explanation for the tumor vascular normalization induced by exercise. Shear stress, the mechanical stimuli exerted on endothelial cells by blood flow, modulates vascular integrity. Increasing vascular shear stress through aerobic exercise can alter and remodel blood vessels in normal tissues. Our data in mouse models indicate that activation of calcineurin-NFAT-TSP1 signaling in endothelial cells plays a critical role in exercise-induced shear stress mediated tumor vessel remodeling. We show that moderate aerobic exercise with chemotherapy caused a significantly greater decrease in tumor growth than chemotherapy alone through improved chemotherapy delivery after tumor vascular normalization. Our work suggests that the vascular normalizing effects of aerobic exercise can be an effective chemotherapy adjuvant.

R-Ras Inhibits VEGF-Induced p38MAPK Activation and HSP27 Phosphorylation in Endothelial Cells

R-Ras is a Ras family small GTPase that is highly expressed in mature functional blood vessels in normal tissues. It inhibits pathological angiogenesis and promotes vessel maturation and stabilization. Previous studies suggest that R-Ras affects cellular signaling in endothelial cells, pericytes and smooth-muscle cells to regulate vessel formation and remodeling in adult tissues. R-Ras suppresses VEGF-induced endothelial permeability and vessel sprouting while promoting normalization of pathologically developing vessels in mice. It attenuates VEGF receptor-2 (VEGFR2) activation by inhibiting internalization of the receptor upon VEGF ligand binding, leading to significant reduction of VEGFR2 autophosphorylation. Here, we show that R-Ras strongly suppresses the VEGF-dependent activation of stress-activated protein kinase-2/p38 mitogen-activated protein kinase (SAPK2/p38MAPK) and the phosphorylation of downstream heat-shock protein 27 (HSP27), a regulator of actin cytoskeleton organization, in endothelial cells. The suppression of p38MAPK activation and HSP27 phosphorylation by R-Ras concurred with altered actin cytoskeleton architecture, reduced membrane protrusion and inhibition of endothelial cell migration toward VEGF. Silencing of endogenous R-Ras by RNA interference increased membrane protrusion and cell migration stimulated by VEGF, and these effects were offset by p38MAPK inhibitor SB203580. These results suggest that R-Ras regulates angiogenic activities of endothelial cells in part via inhibition of the p38MAPK-HSP27 axis of VEGF signaling.

A Novel Vascular Homing Peptide Strategy to Selectively Enhance Pulmonary Drug Efficacy in Pulmonary Arterial Hypertension

A major limitation in the pharmacological treatment of pulmonary arterial hypertension (PAH) is the lack of pulmonary vascular selectivity. Recent studies have identified a tissue-penetrating homing peptide, CARSKNKDC (CAR), which specifically homes to hypertensive pulmonary arteries but not to normal pulmonary vessels or other tissues. Some tissue-penetrating vascular homing peptides have a unique ability to facilitate transport of co-administered drugs into the targeted cells/tissues without requiring physical conjugation of the drug to the peptide (bystander effect). We tested the hypothesis that co-administered CAR would selectively enhance the pulmonary vascular effects of i.v. vasodilators in Sugen5416/hypoxia/normoxia-exposed PAH rats. Systemically administered CAR was predominantly detected in cells of remodeled pulmonary arteries. Intravenously co-administered CAR enhanced pulmonary, but not systemic, effects of the vasodilators, fasudil and imatinib, in PAH rats. CAR increased lung tissue imatinib concentration in isolated PAH lungs without increasing pulmonary vascular permeability. Sublingual CAR was also effective in selectively enhancing the pulmonary vasodilation by imatinib and sildenafil. Our results suggest a new paradigm in the treatment of PAH, using an i.v./sublingual tissue-penetrating homing peptide to selectively augment pulmonary vascular effects of nonselective drugs without the potentially problematic conjugation process. CAR may be particularly useful as an add-on therapy to selectively enhance the pulmonary vascular efficacy of any ongoing drug treatment in patients with PAH.

Genetically modified T cells targeting neovasculature efficiently destroy tumor blood vessels, shrink established solid tumors and increase nanoparticle delivery

Converting T cells into tumor cell killers by grafting them with a chimeric antigen receptor (CAR) has shown promise as a cancer immunotherapeutic. However, the inability of these cells to actively migrate and extravasate into tumor parenchyma has limited their effectiveness in vivo. Here we report the construction of a CAR containing an echistatin as its targeting moiety (eCAR). As echistatin has high binding affinity to αvβ3 integrin that is highly expressed on the surface of endothelial cells of tumor neovasculature, T cells engrafted with eCAR (T-eCAR) can efficiently lyse human umbilical vein endothelial cells and tumor cells that express αvβ3 integrin when tested in vitro. Systemic administration of T-eCAR led to extensive bleeding in tumor tissues with no evidence of damage to blood vessels in normal tissues. Destruction of tumor blood vessels by T-eCAR significantly inhibited the growth of established bulky tumors. Moreover, when T-eCAR was codelivered with nanoparticles in a strategically designed temporal order, it dramatically increased nanoparticle deposition in tumor tissues, pointing to the possibility that it may be used together with nanocarriers to increase their capability to selectively deliver antineoplastic drugs to tumor tissues.

Vascular Endothelial-Targeted Therapy Combined with Cytotoxic Chemotherapy Induces Inflammatory Intratumoral Infiltrates and Inhibits Tumor Relapses after Surgery

Surgery is the most effective therapy for cancer in the United States, but disease still recurs in more than 40% of patients within 5 years after resection. Chemotherapy is given postoperatively to prevent relapses; however, this approach has had marginal success. After surgery, recurrent tumors depend on rapid neovascular proliferation to deliver nutrients and oxygen. Phosphatidylserine (PS) is exposed on the vascular endothelial cells in the tumor microenvironment but is notably absent on blood vessels in normal tissues. Thus, PS is an attractive target for cancer therapy after surgery. Syngeneic mice bearing TC1 lung cancer tumors were treated with mch1N11 (a novel mouse chimeric monoclonal antibody that targets PS), cisplatin (cis), or combination after surgery. Tumor relapses and disease progression were decreased 90% by combination therapy compared with a 50% response rate for cis alone (P = .02). Mice receiving postoperative mch1N11 had no wound-related complications or added systemic toxicity in comparison to control animals. Mechanistic studies demonstrated that the effects of mch1N11 were associated with a dense infiltration of inflammatory cells, particularly granulocytes. This strategy was independent of the adaptive immune system. Together, these data suggest that vascular-targeted strategies directed against exposed PS may be a powerful adjunct to postoperative chemotherapy in preventing relapses after cancer surgery.

Identification and angiogenic role of the novel tumor endothelial marker CLEC14A

Tumor endothelial markers (TEMs) that are highly expressed in human tumor vasculature compared with vasculature in normal tissue hold clear therapeutic potential. We report that the C-type lectin CLEC14A is a novel TEM. Immunohistochemical and immunofluorescence staining of tissue arrays has shown that CLEC14A is strongly expressed in tumor vasculature when compared with vessels in normal tissue. CLEC14A overexpression in tumor vessels was seen in a wide range of solid tumor types. Functional studies showed that CLEC14A induces filopodia and facilitates endothelial migration, tube formation and vascular development in zebrafish that is, CLEC14A regulates pro-angiogenic phenotypes. CLEC14A antisera inhibited cell migration and tube formation, suggesting that anti-CLEC14A antibodies may have anti-angiogenic activity. Finally, in endothelial cultures, expression of CLEC14A increased at low shear stress, and we hypothesize that low shear stress due to poor blood flow in the disorganized tumor vasculature induces expression of CLEC14A on tumor vessels and pro-angiogenic phenotypes.

Epithin/PRSS14 proteolytically regulates angiopoietin receptor Tie2 during transendothelial migration

AbstractEpithin/PRSS14, a type II transmembrane serine protease, is involved in normal epithelial development and tumor progression. Here we report, as an interacting substrate of epithin, a receptor tyrosine kinase Tie2 that is well known for important roles in the vessel stability. Epithin interacts with and degrades the Tie2 extracellular portion that contains the ligand-binding domain. Epithin is located in the neighbor of Tie2-expressing vessels in normal tissue. Furthermore, epithin can cleave and degrade Tie2 not only in the same cell but also from neighboring cells nearby, resulting in the degradation of the Tie2 ectodomain. The remaining Tie2 fragment was highly phosphorylated and was able to recruit a downstream effector, phosphatidylinositol 3-kinase. Knocking down epithin expression using short hairpin RNA in thymoma cell severely impaired the migration through endothelial cells that show the actin rearrangement during the process. The diminution of epithin protein expression in 4T1 breast cancer cells caused the significant decrease in the number of transendothelial migrating cells in vitro as well as in those of metastasizing tumor nodules in vivo, Therefore, we propose that epithin, which regulates endothelial Tie2 functions, plays a critical role in the fine tuning of transendothelial migration for normal and cancer cells.