Normal Tissue Vasculature Research Articles

Tumor growth and vascular redistribution contributes to the dosimetric preferential effect of microbeam radiotherapy: a Monte Carlo study

The radiobiological mechanisms behind the favorable response of tissues to microbeam radiation therapy (MRT) are not fully described yet. Among other factors, the differential action to tumor and normal tissue vasculature is considered to contribute to MRT efficacy. This computational study evaluates the relevance of tumor growth stage and associated vascular redistribution to this effect. A multiscale approach was employed with two simulation softwares: TOPAS and CompuCell3D. Segmentation images of the angioarchitecture of a non-bearing tumor mouse brain were used. The tumor vasculature at different tumor growth stages was obtained by simulating the tumor proliferation and spatial vascular redistribution. The radiation-induced damage to vascular cells and consequent change in oxygen perfusion were simulated for normal and tumor tissues. The multiscale model showed that oxygen perfusion to tissues and vessels decreased as a function of the tumor proliferation stage, and with the decrease in uniformity of the vasculature spatial distribution in the tumor tissue. This led to an increase in the fraction of hypoxic (up to 60%) and necrotic (10%) tumor cells at advanced tumor stages, whereas normal tissues remained normoxic. These results showed that tumor stage and spatial vascular distribution contribute to the preferential effect of MRT in tumors.

Enhancement of immune surveillance in breast cancer by targeting hypoxic tumor endothelium: Can it be an immunological switch point?

Breast cancer ranks second among the causes of cancer-related deaths in women. In spite of the recent advances achieved in the diagnosis and treatment of breast cancer, further study is required to overcome the risk of cancer resistance to treatment and thereby improve the prognosis of individuals with advanced-stage breast cancer. The existence of a hypoxic microenvironment is a well-known event in the development of mutagenesis and rapid proliferation of cancer cells. Tumor cells, purposefully cause local hypoxia in order to induce angiogenesis and growth factors that promote tumor growth and metastatic characteristics, while healthy tissue surrounding the tumor suffers damage or mutate. It has been found that these settings with low oxygen levels cause immunosuppression and a lack of immune surveillance by reducing the activation and recruitment of tumor infiltrating leukocytes (TILs). The immune system is further suppressed by hypoxic tumor endothelium through a variety of ways, which creates an immunosuppressive milieu in the tumor microenvironment. Non responsiveness of tumor endothelium to inflammatory signals or endothelial anergy exclude effector T cells from the tumor milieu. Expression of endothelial specific antigens and immunoinhibitory molecules like Programmed death ligand 1,2 (PDL-1, 2) and T cell immunoglobulin and mucin-domain containing-3 (TIM-3) by tumor endothelium adds fuel to the fire by inhibiting T lymphocytes while promoting regulatory T cells. The hypoxic microenvironment in turn recruits Myeloid Derived Suppressor Cells (MDSCs), Tumor Associated Macrophages (TAMs) and T regulatory cells (Treg). The structure and function of newly generated blood vessels within tumors, on the other hand, are aberrant, lacking the specific organization of normal tissue vasculature. Vascular normalisation may work for a variety of tumour types and show to be an advantageous complement to immunotherapy for improving tumour access. By enhancing immune response in the hypoxic tumor microenvironment, via immune-herbal therapeutic and immune-nutraceuticals based approaches that leverage immunological evasion of tumor, will be briefly reviewed in this article. Whether these tactics may be the game changer for emerging immunological switch point to attenuate the breast cancer growth and prevent metastatic cell division, is the key concern of the current study.

PREPARATION AND CHARACTERIZATION OF ALGINATE CHITOSAN CROSSLINKED NANOPARTICLES BEARING DRUG FOR THE EFFECTIVE MANAGEMENT OF ULCERATIVE COLITIS

Objective: Delivery of anticancer molecule to the liver remains a “holy grail” in molecular medicine and nanobiotechnology with conventional therapy, as conventional cancer chemotherapy does not prove effective as drug molecule does not reach to the target site at therapeutic concentration. Tumor vasculature differs from the vasculature of normal tissue both in morphology and biochemistry. Most of these differences appear too related to angiogenesis (formation of new blood vessels from pre-existing ones). For the present study nanoparticles (NPs) were chosen as a delivery system, because they have many advantages, e. g. they can pass through the smallest capillary vessels because of their ultra-tiny volume, can penetrate cells and tissue gap to arrive at, pH, ion and/or temperature sensitivity of materials, can improve the utility of drugs and reduce toxic side effects. Methods: PLGA (poly lactide co glycolic acid) was used for the preparation of NPs because of its biodegradability and biocompatibility. It degrades by hydrolysis of ester linkages in the presence of water in to two monomers lactic acid and glycolic acid. There are a number of ligands available for hepatic delivery, among them lactobionic acid (containing galactose moiety) was selected for present work. Preparation of plain nanoparticles was carried out using emulsification–diffusion method. Optimization of the polymer concentration is the first step during the study and it was performed by varying the polymer concentration where as keeping other variables constant. The prepared formulation was optimized on the basis of particle size and polydispersity index. Amount of drug was optimized on the basis of particle size and percentage entrapment efficiency. Results: Particle size and zeta potential of the nanoparticle were determined by zetasizer showed that particles are in nano range (blow 200 nm) and have acceptable range of zeta potential. Shape and surface morphology were determined by TEM and SEM analysis. The conjugation of lactobionic acid with PLGA polymer was proved by FTIR. The in vitro release profiles of entrapped drug from formulations were determined using dialysis membrane. For stability studies, the LDNPs (conjugated NPs) are stored at the temperatures 4±1 °C and room temperature. Human hepatoma cell line HepG2 by SRB assay was selected and it clearly suggests a dose dependent cytotoxicity response i.e. decrease in cell survival fraction with increasing concentration of drug. The in vivo study are important in evaluating the targeting efficacy of designed dosage form and also helps in establishing the correlation between the results obtained from in vitro experimentation to that from in vivo studies. The formulations were administered by tail vein to mice of four groups Group I: PBS 7.4 (control); Group II: 5-FU solution; Group III: DNPs; Group IV: LDNPs. Conclusion: The proposed targeting strategy is expected to enhance the therapeutic index of conventional anticancer drug as well as reduce its cytotoxic effects to normal cells.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Enhancing Anti-PD-1/PD-L1 Immune Checkpoint Inhibitory Cancer Therapy by CD276-Targeted Photodynamic Ablation of Tumor Cells and Tumor Vasculature.

Antiangiogenic therapies have been demonstrated to improve the efficacy of immune checkpoint inhibition by overcoming the immunosuppressive status of the tumor microenvironment. However, most of the current antiangiogenic agents cannot discriminate tumor angiogenesis from physiological angiogenesis. The aim of this study was to investigate whether a photodynamic therapy (PDT) agent that targets CD276, a receptor overexpressed in various tumor cells and tumor vasculature but with limited expression in normal tissue vasculature, could improve the tumor inhibitory efficacy of a PD-1/PD-L1 blockade. A CD276-targeting agent (IRD-αCD276/Fab) was synthesized by conjugating the Fab fragment of an anti-CD276 antibody with a photosensitizer IRDye700. The in vivo tumor-targeting efficacy and therapeutic effects of IRD-αCD276/Fab with or without an anti-PD-1/PD-L1 blockade were tested in subcutaneous and lung metastatic tumor models. PDT using IRD-αCD276/Fab significantly suppressed the growth of subcutaneous 4T1 tumor and inhibited its lung metastasis. Moreover, it triggered in vivo antitumor immunity by increasing the activation and maturation of dendritic cells. Tumor PD-L1 levels were also markedly increased after PDT using IRD-αCD276/Fab, as evidenced by noninvasive PD-L1-targeted small-animal PET imaging. In combination with an anti-PD-1/PD-L1 blockade, IRD-αCD276/Fab PDT markedly suppressed the growth of tumors and prevented their metastasis to the lung by recruiting the tumor infiltration of CD8+ T cells. Our data provide evidence for the role of CD276-targeted PDT for local immune modulation, and its combination with PD-L1/PD-1 axis inhibition is a promising strategy for eliminating primary tumors as well as disseminated metastases, by generating local and systemic antitumor responses.

Prostate-specific Membrane Antigen (PSMA) Expression in the Neovasculature of Gynecologic Malignancies: Implications for PSMA-targeted Therapy.

The goal of the study was to examine expression of prostate-specific membrane antigen (PSMA) in neovasculature of gynecologic cancers, as PSMA-targeted therapy has showed a promise in treatment of advanced carcinomas. The study included cervical carcinoma (n=28), vulvar carcinoma (n=20), endometrial carcinoma (n=23), primary ovarian carcinoma (n=21), metastatic ovarian carcinoma (n=25), and normal cervix (n=12) as negative control. All cases were immunostained using anti-CD31 antibody to delineate capillary endothelial cells. In parallel, all cases were immunostained using anti-PSMA antibody. The PSMA staining was assessed in tumor capillaries and in normal tissues and scored as a percentage of CD31 staining. PSMA expression was found in the tumor neovasculature, and no significant expression was identified in vasculature of normal tissues. The extent of PSMA staining in tumor capillaries varied from high expression in ovarian and endometrial cancers, to medium expression in cervical squamous cell carcinomas, and low expression in cervical adenocarcinomas and vulvar cancers. All (100%) cases of primary ovarian carcinoma, ovarian carcinoma metastases, and primary endometrial carcinoma showed PSMA expression in tumor vasculature, which was diffuse in majority of cases. The expression of PSMA in ovarian cancer metastases was similar among different metastatic foci of the same tumor. Fifteen percent of cervical squamous cell carcinoma, 50% of cervical adenocarcinoma, and 75% of vulvar carcinomas showed no capillary expression of PSMA. In conclusion, PSMA is highly and specifically expressed in the neovasculature of ovarian, endometrial, and cervical squamous carcinoma, rendering it a potential therapeutic vascular target.

Abstract 3375: Activation of the acid sphingomyelinase pathway as a potential mechanism of chemotherapy-induced normal tissue vasculature damage

Abstract Background: Several classes of chemotherapy confer substantial risk for late-term vascular morbidity. We aimed to investigate the mechanisms of chemotherapy-induced vascular toxicity in normal tissues. Methods: The effect of Doxorubicin and Cisplatin has been studied in-vitro using bovine aortic endothelial cells (BAEC) as a model of normal tissue vascular endothelium. Specifically, we investigated activation of the acid sphingomyelinase (ASMase) pathway, which may lead to endothelial dysfunction (vasoconstriction) and apoptosis following incubation with different concentrations of Doxorubicin and Cisplatin. ASMase activity was measured using radioenzymatic assay with [N-methyl-14C] sphingomyelin. Cellular Reactive Oxygen Species (ROS) production was assessed using the 2’,7’-dichlorofluorescin diacetate (DCFDA) detection assay kit (Abcam®). Endothelial apoptosis was quantified using the 3,5-bis-benzamide assay (Sigma-Aldrich®). For the in-vivo studies, mouse femoral arterial blood flow was measured in real-time during and after Doxorubicin (8 mg/kg intravenously) or Cisplatin (8 mg/kg) administration. Visualization of ovarian and femoral microvasculature was obtained by fluorescence optical imaging system, equipped with a confocal fiber microscope (Cell-viZio). Results: Incubation of BAEC with Doxorubicin (0.25 μM or 0.50 μM) resulted in increased ASMase activity, production of ROS and induction of apoptosis in a dose-dependent fashion. Cisplatin (25 μM or 50 μM) generated significantly reduced effects in BAEC yet apoptosis and ROS were notable. In-vivo endoscopic fibered confocal microscopy following chemotherapy administration revealed a differential pattern between the two agents. While post Doxorubicin injection, there was a marked reduction in fluorescent signal in small vessels (<15 μM) indicating vessel constriction and disintegration of vessel's wall in larger vessels, Cisplatin treatment resulted in very minor vascular changes. Conclusions: These findings suggest that chemotherapy-induced vascular damage in normal tissues may represent different modes of toxicity in vivo and in vitro, while is mediated via activation of the ASMase pathway in response to Doxorubicin and to a lesser extent in response to Cisplatin. Citation Format: Aviram Mizrachi. Activation of the acid sphingomyelinase pathway as a potential mechanism of chemotherapy-induced normal tissue vasculature damage. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3375.

The clinical and therapeutic uses of MDM2 and PSMA and their potential interaction in aggressive cancers.

Prostate-specific membrane antigen (PSMA) overexpression is observed in the neovasculature of solid tumors, but not in the vasculature of normal tissues. Increased PSMA expression is positively associated with tumor stage and grade, although its function in cancer remains unclear. Mouse double minute 2 (MDM2) is a negative regulator of the p53 tumor suppressor and is reported to regulate VEGF expression and angiogenesis. Both proteins have been considered as biomarkers and therapeutic targets for advanced solid tumors. Our work and a recent microarray-based gene profiling study suggest there could be signaling interplay between MDM2 and PSMA. We herein review the mechanisms underlining the outgrowth of tumors associated with PSMA and MDM2, their potential interaction and how this may be applied to anticancer therapeutics.

Clinical activity and correlative DCE-MRI imaging of G-202, a thapsigargin-based prostate-specific membrane antigen-activated prodrug, in progressive hepatocellular cancer.

301 Background: G-202 is a thapsigargin-based prodrug whose cytotoxic activity is blocked by a masking peptide that is cleaved by prostate-specific membrane antigen (PSMA), a membrane-bound protease expressed in prostate cancer and the endothelium of tumor vasculature but not in most other tissues or normal tissue vasculature. In a Phase I study of G-202, prolonged disease stabilization was observed in the subset of patients with hepatocellular carcinoma (HCC) and prompted development of a Phase II study to further evaluate activity of G-202 in patients with HCC who have progressed on sorafenib. Methods: G-202 is administered by iv infusion on Days 1, 2 and 3 of a 28-day cycle; prophylactic hydration is administered with infusion to ameliorate incidence of creatinine elevations. HCC is typically highly-vascularized, and DCE-MRI is performed in consenting patients at baseline and after treatment on approximately Day 6 of Cycle 2 to evaluate possible effects of G-202 on blood flow metrics in hepatic lesions. DCE-MRI is a non-invasive method of investigating vascular structure and function and is sensitive to alterations in vascular permeability and blood flow. DCE-MRI measurements were made on 1.5 Tesla MRI and the volume transfer coefficients, Ktrans, of HCC tumor lesions were calculated using arterial input function derived from signal in abdominal aorta. Results: Among the 22 patients treated to date, G-202 related SAEs in this patient population have been creatinine increase/acute renal failure/acute kidney injury (3 pts) and congestive heart failure (1 pt). While objective responses (CR, PR) have not been observed in these patients with advanced disease, the rate of disease stabilization has been remarkable, with >70% of patients exhibiting SD. In patients undergoing DCE-MRI, an average 56% decrease in Ktranshas been observed after administration of G-202. Conclusions: G-202 is generally well-tolerated and promotes disease stabilization in patients with advanced HCC who have progressed on sorafenib. DCE-MRI findings of decreased tumor Ktrans values suggests that G-202 reduces early arterial blood flow in hepatic lesions. Clinical trial information: NCT01777594.

Abstract B244: A first-in-human phase 1 clinical study of G-202, a thapsigargin-based Prostate-Specific Membrane Antigen (PSMA) activated prodrug, in patients with advanced solid tumors.

Abstract Background: Thapsigargin induces apoptosis through disruption of calcium homeostasis. G-202 is a thapsigargin-based prodrug whose cytotoxic activity is blocked by a masking peptide that is cleaved by PSMA, a membrane-bound protease expressed in prostate cancer cells and the endothelium of tumor vasculature but not in most other tissues or the vasculature of normal tissue. An open-label, single-arm, dose-escalation Phase I study was performed in patients with advanced solid tumors to evaluate the safety and pharmacokinetics of G-202 and establish a Phase II dosing regimen. Methods: 3+3 dose escalation of G-202 starting at 1.2 mg/m2 administered by intravenous infusion on Days 1, 2 and 3 of a 28-day cycle. Dose escalated in 100% increments until occurrence of at least two G-202 related equivalent Grade 2 adverse events (AEs) in the same cohort, at which time 33% dose increments were implemented. A Phase II dosing regimen was established and evaluated in an expansion cohort. PK parameters were measured in Cycle 1. Results: In the dose escalation, 28 patients (pts) were treated at 8 dose levels (1.2 to 88 mg/m2). A protocol-defined MTD was not reached, but infusion-related reactions and creatinine elevation at 88 mg/m2 led to declaration of 66.8 mg/m2 as MTD. A modified regimen to reduce infusion-related reactions of G-202 at 40 mg/m2 on Day 1 and 66.8 mg/m2 on Days 2 and 3 was evaluated in an expansion cohort of 16 pts with a total of 48 cycles administered (avg 3/pt, range 1-12). Among the 44 pts treated on the study, there were 132 drug-related AEs, 60% of which were Grade 1. Related AEs of any grade reported in at least 4 (10%) patients were fatigue (16 pts), rash (16 pts), increased creatinine/acute kidney injury/acute renal failure (12 pts), nausea (12 pts), fever (8 pts), infusion-related reaction (8 pts), anorexia (7 pts), chills (7 pts), pruritis (7 pts), AST elevation (5 pts), heartburn (5 pts), vomiting (5 pts), and thrombocytopenia(4 pts). Prophylactic intravenous hydration on the day of infusion appeared to ameliorate creatinine elevations. G-202 has a mean half-life of 21 hours and steady state distribution volume of 4995 mL/m2, suggesting confinement to plasma. Because PSMA is highly expressed in hepatocellular carcinoma (HCC), the expansion cohort was enriched with HCC pts. Five HCC pts were enrolled and received a total of 29 cycles (avg 6/pt, range 2-12). One pt completed 12 cycles before exhibiting disease progression and one pt completed 9 cycles before being withdrawn due to an unrelated treatment delay. Conclusions: The MTD of G-202 administered intravenously for 3 consecutive days of a 28-day cycle is 66.8 mg/m2. A dosing regimen of 40 mg/m2 on Day 1 and 66.8 mg/m2 on Days 2 and 3 appears to be well-tolerated and may have activity in HCC. G-202 is being evaluated in a Phase 2 study in patients with HCC who have progressed on sorafenib. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B244. Citation Format: Devalingam Mahalingam, Jeremy Cetnar, George Wilding, Samuel Denmeade, John Sarantopoulos, Michael Kurman, Michael Carducci. A first-in-human phase 1 clinical study of G-202, a thapsigargin-based Prostate-Specific Membrane Antigen (PSMA) activated prodrug, in patients with advanced solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B244.

Prostate Specific Membrane Antigen (PSMA) Regulates Angiogenesis Independently of VEGF during Ocular Neovascularization

BackgroundAberrant growth of blood vessels in the eye forms the basis of many incapacitating diseases and currently the majority of patients respond to anti-angiogenic therapies based on blocking the principal angiogenic growth factor, vascular endothelial growth factor (VEGF). While highly successful, new therapeutic targets are critical for the increasing number of individuals susceptible to retina-related pathologies in our increasingly aging population. Prostate specific membrane antigen (PSMA) is a cell surface peptidase that is absent on normal tissue vasculature but is highly expressed on the neovasculature of most solid tumors, where we have previously shown to regulate angiogenic endothelial cell invasion. Because pathologic angiogenic responses are often triggered by distinct signals, we sought to determine if PSMA also contributes to the pathologic angiogenesis provoked by hypoxia of the retina, which underlies many debilitating retinopathies.Methodology/Principal FindingsUsing a mouse model of oxygen-induced retinopathy, we found that while developmental angiogenesis is normal in PSMA null mice, hypoxic challenge resulted in decreased retinal vascular pathology when compared to wild type mice as assessed by avascular area and numbers of vascular tufts/glomeruli. The vessels formed in the PSMA null mice were more organized and highly perfused, suggesting a more ‘normal’ phenotype. Importantly, the decrease in angiogenesis was not due to an impaired hypoxic response as levels of pro-angiogenic factors are comparable; indicating that PSMA regulation of angiogenesis is independent of VEGF. Furthermore, both systemic and intravitreal administration of a PSMA inhibitor in wild type mice undergoing OIR mimicked the PSMA null phenotype resulting in improved retinal vasculature.Conclusions/SignificanceOur data indicate that PSMA plays a VEGF-independent role in retinal angiogenesis and that the lack of or inhibition of PSMA may represent a novel therapeutic strategy for treatment of angiogenesis-based ocular diseases.

Monitoring Sunitinib-Induced Vascular Effects to Optimize Radiotherapy Combined with Soy Isoflavones in Murine Xenograft Tumor

Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to monitor vascular changes induced by sunitinib within a murine xenograft kidney tumor, we previously determined a dose that caused only partial destruction of blood vessels leading to "normalization" of tumor vasculature and improved blood flow. In the current study, kidney tumors were treated with this dose of sunitinib to modify the tumor microenvironment and enhance the effect of kidney tumor irradiation. The addition of soy isoflavones to this combined antiangiogenic and radiotherapy approach was investigated based on our studies demonstrating that soy isoflavones can potentiate the radiation effect on the tumors and act as antioxidants to protect normal tissues from treatment-induced toxicity. DCE-MRI was used to monitor vascular changes induced by sunitinib and schedule radiation when the uptake and washout of the contrast agent indicated regularization of blood flow. The combination of sunitinib with tumor irradiation and soy isoflavones significantly inhibited the growth and invasion of established kidney tumors and caused marked aberrations in the morphology of residual tumor cells. DCE-MRI studies demonstrated that the three modalities, sunitinib, radiation, and soy isoflavones, also exerted antiangiogenic effects resulting in increased uptake and clearance of the contrast agent. Interestingly, DCE-MRI and histologic observations of the normal contralateral kidneys suggest that soy could protect the vasculature of normal tissue from the adverse effects of sunitinib. An antiangiogenic approach that only partially destroys inefficient vessels could potentially increase the efficacy and delivery of cytotoxic therapies and radiotherapy for unresectable primary renal cell carcinoma tumors and metastatic disease.

A Thapsigargin Prodrug Produces Sustained Growth Inhibition and Substantial Regression of Human Breast Cancers In Vivo with Minimal Host Toxicity.

Abstract Background: Thapsigargin (TG) is a cytotoxic natural product isolated in high yields from the plant Thapsia garganica. In the NCI 60 Cancer Cell Line screen, TG has a GI50 of ∼10-10 M which compares favorably with agents such as paclitaxel (10-8 M) and doxorubicin (10-7 M) in this assay. TG is a non-cell type specific cytotoxin that kills cells in a proliferation independent manner via its potent inhibition of a critical intracellular protein, the Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase (SERCA) pump. Sustained inhibition of the SERCA pump by TG produces an elevation of intracellular calcium to micromolar levels which induces apoptosis via activation of the ER stress response and release of apoptotic factors from the mitochondria. TG has no therapeutic index in vivo with an LD100 of 0.2 mg/kg.Methods: In order to create a theraputic index that would allow for sytemic therapy of human breast cancer we generated prodrugs by coupling water soluble acidic amino acid containing peptides to a potent analog of thapsigargin, (12Aminododecanoyl)-8-O debutanoylthapsigargin (12ADT). In addition to solubilization, the peptide also serves to mask the cytotoxicity of the TG analog until it is released by specific active proteases present within tumor sites. One of these prodrugs (G202) was selected for further in vivo evaluation based on its solubility and ability to be hydrolyzed by the carboxypeptidase Prostate-Specific Membrane Antigen (PSMA). PSMA is membrane protein that is highly expressed by normal and malignant prostate epithelial cells. PSMA, however, is also expressed by the neovasculature within most solid tumors, but not by normal tissue vasculature. In this study we evaluated the toxicity and efficacy of this PSMA-activated thapsigargin prodrug against human MCF-7 breast cancer xenografts.Results: In preliminary studies with prostate cancer xenografts we determined that a single intravenous dose of 56 mg/kg G202 formulated in propylene glycol/Solutol could be administered safely without acute toxicity. Mice bearing MCF-7 xenografts were thus treated with one, two or three doses of 56 mg/kg G202. Three doses produced significant tumor regressions and prolonged growth inhibition but was associated with toxicity whereas a single dose of G202 had no discernible toxicity but produced only transient growth inhibition. In contrast, two consecutive doses of G202 produced only transient weight loss and resulted in significant tumor regression and sustained growth inhibition, with complete responses observed in a subset of animals. High levels (∼5 μM) of the active drug 12ADT-Asp were measurable in the tumor tissue 96 hrs post injection while negligible hydrolysis of G202 was observed in plasma. Discussion: The effectiveness of G202 against a panel of ER positive and negative breast cancer xenografts is currently being evaluated. On the basis of this encouraging preliminary data continued clinical development of G202 as therapy for metastatic breast cancer appears warranted. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5074.

A Novel Gene Expression Profile in Lymphatics Associated with Tumor Growth and Nodal Metastasis

Invasion of lymphatic vessels is a key step in the metastasis of primary tumors to draining lymph nodes. Although the process is enhanced by tumor lymphangiogenesis, it is unclear whether this is a consequence of increased lymphatic vessel number, altered lymphatic vessel properties, or both. Here we have addressed the question by comparing the RNA profiles of primary lymphatic endothelial cells (LEC) isolated from the vasculature of normal tissue and from highly metastatic T-241/vascular endothelial growth factor (VEGF)-C fibrosarcomas implanted in C57BL/6 mice. Our findings reveal significant differences in expression of some 792 genes (i.e., >or=2-fold up- or down-regulated, P <or= 0.05) that code for a variety of proteins including components of endothelial junctions, subendothelial matrix, and vessel growth/patterning. The tumor LEC profile, validated by immunohistochemical staining, is distinct from that of normal, inflammatory cytokine, or mitogen-activated LEC, characterized by elevated expression of such functionally significant molecules as the tight junction regulatory protein endothelial specific adhesion molecule (ESAM), the transforming growth factor-beta coreceptor Endoglin (CD105), the angiogenesis-associated leptin receptor, and the immunoinhibitory receptor CD200, and reduced expression of subendothelial matrix proteins including collagens, fibrillin, and biglycan. Moreover, we show similar induction of ESAM, Endoglin, and leptin receptor within tumor lymphatics in a series of human head and neck and colorectal carcinomas, and uncover a dramatic correlation between ESAM expression and nodal metastasis that identifies this marker as a possible prognostic indicator. These findings reveal a remarkable degree of phenotypic plasticity in cancer lymphatics and provide new insight into the processes of lymphatic invasion and lymph node metastasis.

Molecular heterogeneity of tumor endothelium

Tumors depend on a vascular supply for their growth. Tumor blood vessels, which are derived from normal tissue vasculature, display a markedly abnormal phenotype. Tumor endothelial properties are highly varied in space and time. An important goal is to delineate the range of phenotypes in tumor endothelium and to identify tumor endothelial-specific molecular signatures. This information should lead to a more complete understanding of the mechanisms of tumor growth, the discovery of new therapeutic targets, and the development of biomarkers for diagnosis and surveillance. The goals of this review are to outline recent advances in dissecting tumor endothelial-cell-specific gene expression, to address mechanisms of phenotypic heterogeneity in tumor vascular beds, and to discuss the therapeutic implications of these findings.

Angiogenesis and vascular targeting: Relevance for hyperthermia

The creation of a functional blood supply from the normal tissue vasculature via the process of angiogenesis is critical for the continued growth and development of solid tumours. This importance has led to the concept of targeting the tumour vasculature as a therapeutic strategy, and two major types of vascular targeting agents (VTAs) have developed; those that inhibit the angiogenic process–angiogenesis inhibiting agents (AIAs)–and those that specifically damage the already established neovasculature–vascular disrupting agents (VDAs). The tumour vasculature also plays a critical role in influencing the response to hyperthermia. Generally, the poorer the blood supply the better the heat effect, thus combining VTAs with heat would appear to be a logical approach. Numerous pre-clinical studies have now demonstrated the benefits of combining AIAs or VDAs with heat to improve tumour response. VDAs have also been shown to enhance thermoradiosensitisation, especially at mild hyperthermia temperatures. Although some enhancement of normal tissue response has been observed when VDAs are given with heat, the effects are always less than those seen in tumours, and no increased normal tissue reactions are seen when radiation, VDAs and heat are combined, thus a therapeutic benefit exists. These pre-clinical studies clearly encourage clinical trials with these combinations.

Flow Correlated Percolation during Vascular Remodeling in Growing Tumors

A theoretical model based on the molecular interactions between a growing tumor and a dynamically evolving blood vessel network describes the transformation of the regular vasculature in normal tissues into a highly inhomogeneous tumor specific capillary network. The emerging morphology, characterized by the compartmentalization of the tumor into several regions differing in vessel density, diameter, and necrosis, is in accordance with experimental data for human melanoma. Vessel collapse due to a combination of severely reduced blood flow and solid stress exerted by the tumor leads to a correlated percolation process that is driven towards criticality by the mechanism of hydrodynamic vessel stabilization.

Targeting Coagulation to the Tumor Microvasculature: Perspectives and Therapeutic Implications From Preclinical Studies

Pioneering studies at the National Cancer Institute by Glenn Algire in 1945 led to the conclusion that “ the rapid growth of tumor explants is dependent on the development of a rich vascular supply ” ( 1 ) . There is now little doubt that most tumors are dependent on neovascularization for oxygen and nutrients to sustain progressive growth ( 2 ) . In 1971, Judah Folkman ( 3 ) proposed the innovative idea that angiogenesis inhibitors could be used to treat cancer. In 2004, the U.S. Food and Drug Administration approved the fi rst antiangiogenic drug for the treatment of cancer, a humanized monoclonal antibody against VEGF-A named Avastin (bevacizumab). Other drugs in this category are under development. There are at least two general approaches to depriving tumors of their blood supply. One approach is to inhibit tumor-induced neovascularization from both the sprouting neighboring vessels and the recruitment of endothelial cell precursors from the bone marrow ( 4 – 8 ) . The essential role of vascular endothelial growth factor (VEGF) in this process is well established, and several angiogenesis inhibitors have shown effi cacy at reducing tumor neovascularization and tumor growth, at least in preclinical models ( 9 – 11 ) . In some cases, rapidly growing lesions were converted into dormant lesions ( 12 ) . A second approach to reducing the tumor blood supply is to cause zonal tumor necrosis by targeting the preexisting vasculature ( 13 , 14 ) . This approach is used successfully in the clinic, where transarterial embolization is used to obstruct vessels, particularly in hepatocellular carcinoma ( 15 ) . Recently, randomized controlled trials have shown increased survival of patients treated in this way ( 16 , 17 ) . As elegantly illustrated by Dienst et al. in this issue ( 18 ), recent advances in the molecular characterization of tumor endothelium have permitted an evolution of this catheter-based approach to vessel occlusion. The targeting of vascular endothelial cells presents several advantages compared with tumor cell targeting. First, effective and uniform delivery of anticancer drugs to solid tumors has proved challenging: Drugs do not reach deep beyond the perivascular region due to physical barriers by fi brous tumor tissue and to elevated interstitial pressure, which reduces fl uid convection ( 19 ) . By contrast, endothelial cells lining the vessels are easily accessible to the bloodstream. Second, vascular endothelial cells are less heterogeneous than tumor cells and, as generally normal, nontransformed cells, are less likely than tumor cells to acquire mutations leading to drug resistance ( 7 ) . Third, complete cell death in all endothelial cells lining a vessel is not necessary; rather, interruption of the blood stream for a period of hours may suffi ce. At the same time, these advantages are the base of diffi culties to overcome. The most critical point is specifi city of tumor vascular targeting to avoid undesirable effects to normal tissue blood supply. Thus, selection of tumor-specifi c target molecules is the key to the success of this approach. Fortunately, tumor and normal tissue vasculature differ in morphology and function.

444. A New Vector Which Targets Breast Cancer and Its Vasculature

20% of women diagnosed with breast cancer will succumb to their disease due to resistance of their cancer cells to chemotherapy and hormonal therapy. One of the reasons for resistance to existing chemotherapy is that 70% of breast cancer cells are non-dividing and are therefore relatively resistant to the best chemotherapy for breast cancer. In order to overcome this problem of resistance of non-dividing breast cancer cells to chemotherapy, our laboratory designed the AdCDIRESE1A vector, which carries a chimeric transcription unit composed of the cytosine deaminase gene (CD) linked to the viral E1A proliferation gene. 5-Fluorocytosine (5FC) is converted into 5-Fluorouracil (5FU) by the bacterial gene cytosine deaminase (CD), which is produced in the vector infected cells. The levels of 5FU generated in vector infected cells are so high that even non dividing cells will die due to disruption of protein synthesis. Previous data from our laboratory had shown that the intratumoral injection of this AdCDIRESE1A vector followed by 10 days of ip 5FC induced regressions of existing sc breast cancer nodules. The E1A is an adenoviral gene necessary for replication. We then constructed a conditionally replication-competent adenoviral vector Ad.Lp-CD-IRES-E1A(control) in which the expression of both the prodrug activating cytosine deaminase gene and the viral replication E1A gene were driven by the L-plastin tumor specific promoter. As reported in this abstract this vector has been further modified by simultaneously ablating the native tropism of the vector to the primary CAR receptor and inserting a RGD-4C peptide into the HI loop of the fiber protein, which allows the vector to use the |[alpha]|v|[beta]|3 and |[alpha]|v|[beta]|5 integrins as alternative receptors. The |[alpha]|v|[beta]|3 integrin receptor is increased in breast cancer cells and in dividing endothelial cells. Dividing endothelial cells are increased (15%) in tumor vasculature as opposed to vasculature in normal tissues (0.5%). The resulting vector was named Ad.Lp-CD-IRES-E1A(MRGD). The transduction efficiency of the vector for breast cancer cell lines which have low expression level of CAR was increased both in vitro and in vivo. The Ad.Lp-CD-IRES-E1A(MRGD) vector produces a higher vector particle yield and a greater cytotoxic effect in tumor cells which have a low expression level of CAR, than did the Ad.Lp-CD-IRES-E1A(control) vector. Intratumoral injection of the Ad.Lp-CD-IRES-E1A(MRGD) vector following the intraperitoneal injection of 5FC into xenotransplanted human breast cancer cell lines led to tumor regression in vivo. The inactivation of the CAR receptor and the addition of the RGD will increase the safety of this vector in vivo, and will decrease the wasteful binding of the vector to non-neoplastic cells. In addition, the altered tropism will circumvent neutralizing antibodies, and will target this vector directly to luminal endothelial cells. Thus, since this vector does not need to cross over from the intravascular to the extravascular space, it is possible that it will be useful clinically.

Plasmalemmal vesicle-associated protein (PLVAP) is expressed by tumour endothelium and is upregulated by vascular endothelial growth factor-A (VEGF)

Vascular endothelial growth factor-A (VEGF) is an important regulator of vascular permeability. In preclinical studies, VEGF induces endothelial fenestrations in pre-existing and neo-vasculature, while inhibition of VEGF leads to a reduction in endothelial fenestrations. Recently, vascular regression in response to VEGF inhibition has been shown to correlate with the presence of endothelial fenestrations. Plasmalemmal vesicle-associated protein (PLVAP) is believed to be a component of diaphragmed endothelial fenestrations, but a direct relationship with VEGF signalling has not been established. The aim of this study was to characterize the expression pattern of PLVAP and investigate whether PLVAP is a transcriptional target of VEGF signal transduction. The expression pattern of PLVAP was characterized in normal and neoplastic human tissues by in situ hybridization and/or immunohistochemistry. The role of VEGF signal transduction in the regulation of PLVAP expression was investigated in vitro using receptor-selective engineered forms of VEGF, a neutralizing monoclonal antibody against VEGF, and inhibitors of downstream signalling pathways. PLVAP mRNA and protein were widely expressed in the endothelium of normal and neoplastic tissues. In cultured endothelial cells, VEGF signalling through receptor 2 stimulated expression of PLVAP total RNA and protein. This induction could be blocked with an anti-VEGF monoclonal antibody and by inhibitors of phosphatidylinositol 3-kinase (LY294002) or p38 mitogen-activated protein kinase (SB203580), but not by PD98059, a mitogen-activated protein/extracellular signal-regulated kinase 1 inhibitor. These data show that PLVAP is more widely expressed in the vasculature of normal tissues than previously thought and that it is expressed in the vasculature of most human tumours. We suggest that PLVAP is a downstream target of VEGF signalling. This work solidifies the association between VEGF and the appearance and maintenance of fenestrations by providing a potential mechanistic link.