Blood Vessels In Tumor Tissue Research Articles

Delivery of aPD-L1 antibody to i.p. tumors via direct penetration by i.p. route: Beyond EPR effect.

Chemotherapy for peritoneal dissemination is poorly effective owing to limited drug transfer from the blood to the intraperitoneal (i.p.) compartment after intravenous (i.v.) administration. i.p. chemotherapy has been investigated to improve drug delivery to tumors; however, the efficacy continues to be debated. As anticancer drugs have low molecular weight and are rapidly excreted through the peritoneal blood vessels, maintaining the i.p. concentration as high as expected is a challenge. In this study, we examined whether i.p. administration is an efficient route of administration of high-molecular-weight immune checkpoint inhibitors (ICIs) for the treatment of peritoneal dissemination using a model of peritoneal disseminated carcinoma. After i.p. administration, the amount of anti-PD-L1 antibody transferred into i.p. tumors increased by approximately eight folds compared to that after i.v. administration. Intratumoral distribution analysis revealed that anti-PD-L1 antibodies were delivered directly from the i.p. space to the surface of tumor tissue, and that they deeply penetrated the tumor tissues after i.p. administration; in contrast, after i.v. administration, anti-PD-L1 antibodies were only distributed around blood vessels in tumor tissues via the enhanced permeability and retention (EPR) effect. Owing to the enhanced delivery, the therapeutic efficacy of anti-PD-L1 antibody in the peritoneal dissemination models was also improved after i.p. administration compared to that after i.v. administration. This is the first study to clearly demonstrate an EPR-independent delivery of ICIs to i.p. tumors by which ICIs were delivered in a massive amount to the tumor tissue via direct penetration after i.p. administration.

Novel Soloxolone Amides as Potent Anti-Glioblastoma Candidates: Design, Synthesis, In Silico Analysis and Biological Activities In Vitro and In Vivo.

The modification of natural or semisynthetic triterpenoids with amines can be explored as a promising strategy for improving their pharmacological properties. Here, we report the design and synthesis of 11 novel amide derivatives of soloxolone methyl (SM), a cyano enone-bearing derivative of 18βH-glycyrrhetinic acid. Analysis of their bioactivities in vitro and in silico revealed their high toxicity against a panel of tumor cells (average IC50(24h) = 3.7 µM) and showed that the formation of amide moieties at the C-30 position of soloxolone did not enhance the cytotoxicity of derivatives toward tumor cells compared to SM, though it can impart an ability to pass across the blood–brain barrier. Further HPLC–MS/MS and mechanistic studies verified significant brain accumulation of hit compound 12 (soloxolone tryptamide) in a murine model and showed its high anti-glioblastoma potential. It was found that 12 induced ROS-dependent and autophagy-independent death of U87 and U118 glioblastoma cells via mitochondrial apoptosis and effectively blocked their clonogenicity, motility and capacity to form vessel-like structures. Further in vivo study demonstrated that intraperitoneal injection of 12 at a dosage of 20 mg/kg effectively inhibited the growth of U87 glioblastoma in a mouse xenograft model, reducing the proliferative potential of the tumor and leading to a depletion of collagen content and normalization of blood vessels in tumor tissue. The obtained results clearly demonstrate that 12 can be considered as a promising leading compound for drug development in glioblastoma treatment.

Multimodal obstruction of tumorigenic energy supply via bionic nanocarriers for effective tumor therapy

Sufficient energy generation based on effective transport of nutrient via abundant blood vessels in tumor tissue and subsequent oxidative metabolism in mitochondria is critical for growth, proliferation and migration of tumor. Thus the strategy to cut off this transport pathway (blood vessels) and simultaneously close the power house (mitochondria) is highly desired for tumor treatment. Herein, we fabricated a bionic nanocarrier with core-shell-corona structure to give selective and effective tumor therapy via stepwise destruction of existed tumor vessel, inhibition of tumor angiogenesis and dysfunction of tumor mitochondria. The core of this bionic nanocarrier consists of combretastatin A4 phosphate (CA4P) and vitamin K2 (VK2) co-loaded mesoporous silica nanoparticle (MSNs), which is in charge of the vasculature destruction and mitochondrial dysfunction after cargos release. The N-tert-butylacrylamide (TBAM) and tri-sulfated N-acetylglucosamine (TSAG) shell served as artificial affinity reagent against vascular endothelial growth factor (VEGF) for angiogenesis inhibition. As to guarantee that these actions only happened in tumor, the hyaluronic acid (HA) corona was introduced to endow the nanocarrier with tumor targeting property and stimuli-responsiveness for accurate therapy. Both in vitro and in vivo results indicated that the CA4P/VK2-MSNs-TBAM/TSAG-HA (CVMMGH for short) nanocarrier combined well-controllable manipulation of tumor vasculature and tumor mitochondria to effectivly cut off the tumorigenic energy supply, which performed significant inhibition of tumor growth, demonstrating the great candidate of our strategy for effective tumor therapy.

Smart Polymeric Nanoparticles with pH-Responsive and PEG-Detachable Properties (II): Co-Delivery of Paclitaxel and VEGF siRNA for Synergistic Breast Cancer Therapy in Mice

BackgroundThe dual-loaded nano-delivery system can realize chemotherapeutic drug and small interfering RNA (siRNA) co-loading as well as enhance the therapeutic effect of drugs on tumors through a synergistic effect, while reducing their toxic and side effects on normal tissues.MethodsPreviously, we developed layered smart nanoparticles (NPs) to co-deliver survivin siRNA as well as small molecule drugs for lung cancer. In this study, we used such smart NPs to co-deliver paclitaxel (PTX) and siRNA against vascular endothelial growth factor (VEGF) gene for breast cancer therapy in mice models. For the prepared NPs, characterizations such as particle size, zeta potential, gel electrophoresis imaging and in vitro stability were investigated. Then, 4T1 cells were used to evaluate the in vitro VEGF silencing capacity, tumor cell inhibitory and anti-apoptotic abilities. Finally, an orthotopic model of mouse breast cancer was established to evaluate the in vivo antitumor effects and safety properties of PTX-siRNAVEGF-NPs.ResultsWe prepared PTX-siRNAVEGF-NPs with particle size of 85.25 nm, PDI of 0.261, and zeta potential of 5.25 mV. The NPs with VEGF siRNA effectively knocked down the expression of VEGF mRNA. Cell counting kit-8 (CCK-8) and apoptosis assays revealed that the PTX-siRNAVEGF-NPs exhibited antiproliferation effect of PTX on 4T1 cells. The in vivo anti-tumor study indicated that PTX-siRNAVEGF-NPs could exert an antitumor effect by inhibiting the formation and development of new blood vessels in tumor tissues, thereby cutting off nutrient and blood supplies required for tumor tissue growth. Both the anti-tumor efficacy and in vivo safety of the PTX-siRNAVEGF-NPs group were better than that of the PTX-NPs and siRNAVEGF-NPs groups.ConclusionThe combination of PTX and VEGF siRNA exerts good antitumor effect on 4T1 tumor cells. This study provides a theoretical and practical basis for breast cancer therapy.

Co-administration of combretastatin A4 nanoparticles and anti-PD-L1 for synergistic therapy of hepatocellular carcinoma

BackgroundAccording to data estimated by the WHO, primary liver cancer is currently the fourth most common malignant tumor and the second leading cause of death around the world. Hepatocellular carcinoma (HCC) is one of the most common primary liver malignancies, so effective therapy is highly desired for HCC.ResultsIn this study, the use of poly(l-Aspartic acid)-poly(ethylene glycol)/combretastatin A4 (CA4-NPs) was aimed to significantly disrupt new blood vessels in tumor tissues for targeted hepatic tumor therapy. Here, PEG-b-PAsp-g-CA4 showed significantly prolonged retention in plasma and tumor tissue. Most importantly, CA4-NPs were mainly distributed at the tumor site because of the triple target effects—enhanced permeability and retention (EPR) effect, acid-sensitive (pH = 5.5) effect to the tumor microenvironment (TME), and good selectivity of CA4 for central tumor blood vessel. Considering that CA4-NPs might induce severe hypoxic conditions resulting in high expression of HIF-1α in tumor tissues, which could induce the overexpression of PD-L1, herein we also used a programmed death-ligand 1 antibody (aPD-L1) to prevent immunosuppression. This way of complementary combination is able to achieve an ideal treatment effect in tumor site where CA4-NPs and aPD-L1 could respond to the inner area and peripheral area, respectively. As a result, a significant decrease in tumor volume and weight was observed in the combination group of CA4-NPs plus aPD-L1 compared with CA4-NPs or aPD-L1 monotherapy in subcutaneous Hepa1-6 hepatic tumor models.ConclusionsWe presented a new idea that co-administration of CA4-NPs and aPD-L1 possessed notable anti-tumor efficacy for HCC treatment.Graphic abstract

Abstract 1139: CD206 positive M2-macrophage targeting engineered exosomes as a potential diagnostic and therapeutic tool

Abstract Tumor initiation and evolution is driven by the reciprocal actions between stromal and immune cells within the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) specifically M2-macrophages, a critical component of TME; participate in immune suppression, epithelial to mesenchymal transition, invasion, angiogenesis, tumor progression and subsequent metastasis foci formation. Given their prevalence in most of the human tumor tissue and correlation of their higher infiltration with lower survival, M2-macrophages represent promising targets for diagnosis, prognosis and anticancer therapy. Hence, early in vivo detection and intervention of M2-macrophages in the TME may boost the clinical outcome. Exosomes are biological nanoparticles sizing 30-150 nm, recently drawing huge attention for their potential application as therapeutic and diagnostic tool because they are more biocompatible and biodegradable with lesser toxicity than other synthetic nanoparticles and can easily percolate through the body’s barrier systems or through abnormally formed blood vessels in tumor tissue. We have generated engineered exosomes from HEK293 cells by lentiviral transfection system that carry specific peptide sequence on their external surface for targeting CD206 positive M2-macrophages. To determine the in vivo distribution of M2-macrophages, we adopted 111In-oxine based radio-labeling of the targeted exosomes and single-photon emission computed tomography (SPECT). Up to the time, among the used techniques, nuclear imaging is the foremost method to trace exosomes in vivo for better tissue penetration, sensitivity and quantitative analysis. After the labeling with 111In-oxine, we analyzed the binding efficacy and serum stability of the targeted exosome by thin layer paper chromatography (TLPC) using 100% methanol and 2M Sodium acetate solution (1:1) as eluent. More than 98% of 111In-oxine was bound to exosomes and only 5% of free 111In-oxine was dissociated from the exosomes even 24 hours after the incubation with 20% serum. When we injected these radio-labeled targeted exosomes in to 4T1 breast tumor-bearing mice, they went mostly to the periphery of the primary tumor, metastatic area in the lungs, spleen and to the liver. We performed ex vivo quantification of radioactivity from individual organ by gamma counter after final SPECT scan and observed similar distribution of the radio-labeled targeted exosomes. We also labeled the exosomes with DiI dye and injected into the same mice followed by euthanasia after 3 hours and organ collection. Fluorescent imaging from the tumor and spleen showed the adherence of exosomes to the CD206 positive macrophages confirming the targeting efficacy of the exosomes. Henceforth, we intend to utilize these exosomes as a therapeutic probe for carrying chemotherapeutic or antibody to intervene the actions of M2-macrophages in primary and distal TME. Citation Format: Mohammad H. Rashid, Thaiz F. Borin, Roxan Ara, Kartik Angara, Achyut Bhagelu, Jingwen Cai, Yutao Liu, Ali S. Arbab. CD206 positive M2-macrophage targeting engineered exosomes as a potential diagnostic and therapeutic tool [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1139.

Endothelium originated from colorectal cancer stem cells constitute cancer blood vessels.

Tumor growth depends on the formation of blood vessels that provide the supply of nutrients and oxygen. Previous data have shown that glioblastoma stem cells are able to give rise to vascular cells to constitute the functional vessels in tumor tissues. However, which kinds of vascular cells are generated from glioblastoma stem cells is largely debated. In addition, there is little evidence showing that the stem cells from other kinds of tumors can produce vascular cells to constitute the functional blood vessels in tumor tissues. Here we show that cancer stem cells of human colorectal carcinomas (CoCSC) can give rise to vascular endothelial cells and compose the vasculatures in cancer tissues. The human‐cell‐specific nuclear antigen NuMA + vascular endothelial cells were detected in the blood vessels in xenografts derived from CoCSC. NuMA + endothelial cells incorporated into functional blood vessels. Our data indicate that the cancer stem cells derived from human colorectal carcinomas have the capacity to generate functional blood vessels and provide a new mechanism for tumor vasculogenesis in carcinoma.

Abstract 599: MS17-38 mAb targeting of PODXL-v2 inhibits gastric cancer growth and metastasis

Abstract Background: Gastric cancer is the primary cause of cancer-specific mortality worldwide. PODXL (podocalyxin-like protein, or podocalyxin) is a cell-surface glycoprotein that belongs to CD34 family of sialomucins. PODXL-v1 is widely expressed on vascular endothelium, mesothelial cells, and platelets; in contrast, PODXL-v2, a truncated short form of PODXL is specifically expressed or up-regulated in numerous cancer cell types as well as in small blood vessels in tumor tissues. There remains considerable interest in generating monoclonal antibodies (mAbs) directed against specific tumor targets for antigen (Ag) discovery, diagnosis and therapy. Methods: Mixed live cells from four human GC cell lines were used as the immunogen in A/J mice. MS17-38 mAb was generated by hybridoma technology and from high throughput screening (HTS), with GC cell lines versus normal human PBMC live cells. Purified MS17-38 mAb was used to confirm the target Ag through a combination of western blot (WB), immunoprecipitation (IP) and mass spectrometry (MS). Peptide microarrays of the identified Ag were layered on chips and double screening was performed to achieve epitope mapping of binding of the mAb its target. Dose-dependent treatment of GC cells with MS17-38 mAb and target siRNA-was followed by staining with CCK-8 and CyQuant GR dyes and read at 450nm and 480/520nm for cell proliferation and transwell migration assays. GC MKN-45 cells were injected subcutaneously (s.c.) into the flank of nu/nu mice treated with MS17-38 mAb or control mAb. In addition, tumor tissues from 42 human GC patients were stained by immunohistochemistry (IHC) and evaluated for PODXL expression using MS17-38, and the survival of the GC patients compared to target antigen expression levels. Results: MS17-38 mAb was generated by live cells HTS and was demonstrated to bind to a specific conformational epitope of PODXL-v2 on GC cells. PODXL-v2 expression inhibition by PODXL-v2 siRNA or the PODXL-v2-neutralizing antibody MS17-38 resulted in inhibition of GC cell growth and prevention of GC cell migration in vitro. Furthermore, in vivo studies demonstrated that MS17-38 mAb can potently inhibit tumor growth and prevent MKN-45 metastasis to the lungs in nu/nu mouse models. Finally, high expression of PODXL-v2 was associated with advanced GC stage and short survival time (P<0.01). Additional engineering of this mAb into chimeric or humanized forms could permit development of MS17-38 for diagnosis, staging or therapy of human malignancy. Conclusions: Our findings indicate that PODXL-v2 is specifically expressed in the extracellular matrix of GC, and MS17-38 mAb directed against a conformational epitope of PODXL-v2 identified through HTS can functionally inhibit GC cancer cell growth and migration/metastasis both in vitro and in vivo. MS17-38 is a promising functional antibody directed against GC that could potentially be developed into a therapeutic mAb for clinical application. Citation Format: Runhua Feng, Ming Li, Yuling Wang, Dongqing Zhang, Xiaolian Gao, Xuehua Chen, Joe X. Zhou, Victor G. Prieto, Jian H. Song, Shenying Fang, Binya Liu, Zhenggang Zhu, Xiangcang Ye, Lee M. Ellis, Mason Lu, Jeffery E. Lee. MS17-38 mAb targeting of PODXL-v2 inhibits gastric cancer growth and metastasis. [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 599.

A138: In vitro oncolytic effects of wild Newcastle disease virus strains and results in vivo virotherapy

Oncolytic virus therapy is an area in experimental cancer therapy that is under active investigation. A wide variety of viruses have shown huge potent oncolytic activity against human tumors in cell and animal models without major side effects. Several strains have already being tested in clinical trials in patients. Virotherapy is making promising progress for the treatment of malignancies. Nonetheless, when used in patients, the response rate to oncolytic virus is low. One of the main ways to increase oncolytic capacity is to refuse of excessive attenuation and to use the wild type of oncolytic strains. Naturally occurring oncolytic viruses (OVs) have shown their high cancer-targeted antitumor effects with selectively replication and destroying tumor cells while sparing normal cells, because of defects in IFN-signaling pathways in tumor cell lines. Newcastle disease virus (NDV) is one of the promising therapeutic agent for virotherapy. It belongs to avian paramyxovirus (APMV-1) that is able to infect over 240 species of birds and induce severe disease. However, NDV is not a human pathogen. Newcastle disease virus exhibits effectively oncolytic activity in a range of tumors in pre-clinical and clinical studies. We have previously reported in vitro oncolytic effects of three wild NDV strains. Here we demonstrate data that describe oncolytic capacity NDV collection gathered in 2008–2014, Russia. Antitumor effect of NDV strains on a panel of tumor human cell lines (HCT116, MCF7, A549, HeLa, Skbr, H1299) were investigated. Cell viability was examined over 96 hours by MTT assay. Dose-dependent cell cytotoxicity was demonstrated. In vitro study shows the capacity of several wild NDV strains to lyse tumor cell lines. Two strains of them have significant strong oncolytic activity on human tumor cells of various histogenesis. Recent work in our research also describes the results of in vivo experiments. One of them consists of comparing the efficacy of NDV strain to exhibit specific oncolytic activity in C57Bl/6 mice on subcutaneous murine tumors (LLC and B16) . Our results show that both intravenous and intratumoral injections with multiple doses of wild NDV strain lead to survival prolongation in B16- and LLC-bearing mice and prevent tumor development. We demonstrate antitumor activity of NDV/Altai/pigeon/770/2011 against murine KREBS-2 solid tumor in preclinical model on immunocompetent BALB/c mice. Recently we have demonstrated that NDV/Altai/pigeon/770/2011 can destroy murine KREBS-2 cells in vitro. Moreover, in pilot study we have shown that intratumoral NDV injections decrease of tumor volume and result in complete regression of KREBS-2 tumor in MRI images. Here we test intratumoral administration of NDV and show suppression of Krebs-2 tumor growth in intramuscularly allograft model (2.6-fold less size that in untreated group). Importantly, spread of the virus causes significant tumor necrosis in Krebs-2 tumors. By 20 days after virotherapy, widespread necrosis of NDV-treated tumor is observed. We describe that the vast fields of necrosis in NDV-treated group are the results of formation of ischemic foci in tumor tissue with the rapid development of tumor node and slow neoangiogenesis. Histological examinations and morphometric analysis of tumors in NDV-treated and control groups show that the number of blood vessels including the newly formed in the untreated group is significantly higher than in the experimental group. Immunohistochemical staining (CD34, VEGFR) shows that blood vessels in tumor tissue is strongly reduced to 20 days post-treatment and neoangiogenesis progresses in untreated tumor tissue. Thus, results suggest that vascular disruption in the NDV-treated group indicates the virus ability to directly or indirectly affect tumor angiogenesis and regulate tissue trophism in that way. Understanding how and in which step this effect occurs may provide capacity to use oncolytic NDV strains for therapeutic benefit.

Hybrid PET/optical imaging of integrin αVβ3 receptor expression using a (64)Cu-labeled streptavidin/biotin-based dimeric RGD peptide.

BackgroundHybrid PET/optical imaging provides quantitative and complementary information for diagnosis of tumors. Herein, we developed a 64Cu-labeled AlexaFluor 680-streptavidin ((AF)SAv)/biotin-based dimeric cyclic RGD peptide (RGD2) for hybrid PET/optical imaging of integrin αVβ3 expression.Methods64Cu-1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA)-(AF)SAv/biotin-PEG-RGD2 was prepared by formation of a complex comprising DOTA-(AF)SAv and biotin-PEG-RGD2, followed by radiolabeling with 64Cu. Receptor binding studies of DOTA-(AF)SAv/biotin-PEG-RGD2 were performed using U87MG cells and 125I-RGDyK as the radioligand, and cellular uptake studies of 64Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 were also performed. MicroPET imaging followed by optical imaging of U87MG tumor-bearing mice was acquired after injection of the hybrid probe, and region of interest (ROI) analysis of tumors was performed. Ex vivo PET/optical imaging and biodistribution studies of the major tissues were performed after the in vivo imaging, and immunofluorescence staining of the tumor tissue sections was carried out.Results64Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 was prepared in 52.1 ± 5.4 % radiochemical yield and with specific activity of 1.0 ± 0.1 GBq/mg. Receptor binding studies showed that DOTA-(AF)SAv/biotin-PEG-RGD2 had higher binding affinity for integrin αVβ3 than RGD2, reflecting a possible polyvalency effect. Moreover, the hybrid probe revealed time-dependent uptake by U87MG cells. In a microPET/optical imaging study, the hybrid probe demonstrated high accumulation in tumors; ROI analysis revealed 2.7 ± 0.2 % ID/g at 1 h and 4.7 ± 0.2 % ID/g at 21 h after injection, and subsequently acquired optical images showed tumors with strong fluorescence intensity. Ex vivo PET/optical images of the major tissues confirmed the in vivo imaging data, and biodistribution studies demonstrated high and specific uptake in tumors (4.8 ± 0.1 % ID/g). Immunofluorescence staining showed the formation of new blood vessels in tumor tissues, suggesting that the tumor uptake was due to specific binding of the hybrid probe to integrin αVβ3 expressed on tumor cells.ConclusionsThese results indicate that a 64Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 is able to provide quantitative information on hybrid PET/optical imaging of integrin αVβ3 expression.

Drug Targets for Cancer Treatment: An Overview

Cancer is one of the major causes of the death worldwide.Malignant cells display metabolic changes, when compared to normal cells, because of both genetic and epigenetic alterations. Number of drugs being used for the cancer treatment follows different mechanisms of action. Therapeutic strategies include targeting of drugs at specific genes or proteins/enzymes found in cancer cells or the internal tissueenvironment which contributes to growth and survival of these cells. Targeted therapy is often used along with chemotherapy and other treatments to restrict the growth and spread of cancer cells. During the past few decades, targeted therapy has emerged as a promising approach for the development of selective anticancer agents. There is a class of targeted therapy drugs called angiogenesis inhibitors which focus on blocking the development of new blood vesselsin tumor tissues. In addition, anticancer drugs also include DNA intercalators, DNA synthesis inhibitors, transcription regulators, enzyme inhibitors etc. This review focuses on major classes of anticancer drug targets and their therapeutic importance.

Hollow submicron spheres fabricated from cyanoacrylate instant adhesive

Hollow spherical particles of less than 1mm, consisting of either single or multi-voids have several advantages, including low effective density and high surface specificity. Moreover, particles with dimensions <100nm are known to selectively access cancer cells through enlarged porous surfaces of blood vessels in tumor tissues, and the drug delivery systems with these submicron spheres have been studied intensively in recent years.In this paper, we demonstrate a novel and cost-effective method to fabricate hollow submicron spheres with polymer shells by simply pumping the vapor of a commonly used instant adhesive into a citric acid solution to form microbubbles. The main component of the instant adhesive is a cyanoacrylate monomer, which is polymerized within seconds in the presence of water; however in the case of the citric acid solution, the polymerization occurs at a much slower rate. The polymer shell is composed of a stable biocompatible material, used for adhesion to human tissue, i.e., skin, blood vessels and organs. Moreover, the dimension of the fabricated submicron sphere is less than 1µm in diameter, (one-tenth the size of a blood capillary; approximately 10µm), making them suitable for use as a hollow nanocarrier in drug delivery applications, including cancer research.

A fusion protein containing murine vascular endothelial growth factor and tissue factor induces thrombogenesis and suppression of tumor growth in a colon carcinoma model

Induction of tumor vasculature occlusion by targeting a thrombogen to newly formed blood vessels in tumor tissues represents an intriguing approach to the eradication of primary solid tumors. In the current study, we construct and express a fusion protein containing vascular endothelial growth factor (VEGF) and tissue factor (TF) to explore whether this fusion protein has the capability of inhibiting tumor growth in a colon carcinoma model. The murine cDNA of VEGF A and TF were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned into prokaryotic expression plasmid pQE30 with a linker. The expression product recombinant VEGF-TF (rVEGF-TF) was purified and proved to have comparable enzyme activity to a commercial TF and the capability of specific binding to tumor vessels. Significant decrease of tumor growth was found in the mice administered with rVEGF-TF on Day 6 after initiated rVEGF-TF treatment (P<0.05), and the tumor masses in 2 of 10 mice were almost disappeared on Day 14 after the first treatment. In addition, valid thrombogenesis and tumor necrosis were observed in the tumor tissues injected with rVEGF-TF. Our results demonstrate that occlusion of tumor vasculature with rVEGF-TF is potentially an effective approach for cancer therapy.

Antiangiogenic Effect of Bile Acid Acylated Heparin Derivative

Chemically modified heparin-DOCA was prepared and found to have markedly lower anticoagulant activity than heparin. In the present study, we elucidated the antiangiogenic and antitumoral activities of heparin-DOCA derivative. To evaluate the antiangiogenic and antitumoral effects of heparin-DOCA, capillary-like tube formation assay, Matrigel plug assay in vivo, western blotting for FGFR phosphorylation, ERK and p38 MAPK activities, tumor growth of SCC in vivo and immunostaining of blood vessels in tumor tissues were performed. Heparin-DOCA inhibited capillary-like tubular structures of endothelial cells and bFGF-induced neovascularizations in Matrigel plug assays. Signaling experiments showed that heparin-DOCA significantly inhibited angiogenesis by suppressing the phosphorylation of FGFR and its downstream signal pathways (ERK and p38 MAPK activities). The antiangiogenic activity of this heparin derivative was found to be closely associated with antitumoral activity in a mouse model. In addition, histological evaluations supported the inhibitory effect of heparin-DOCA on blood vessel formation in tumor tissues. Heparin-DOCA derivative exerted a significant antitumoral effect by inhibiting angiogenesis resulting from the disruption of FGF/FGFR and its downstream signal pathways, and could be applied to treat various angiogenic diseases.

Matriptase activates stromelysin (MMP‐3) and promotes tumor growth and angiogenesis

Matriptase/MT-SP1, a type II membrane serine protease widely expressed in normal epithelial cells and human carcinoma cells, is thought to be involved in cancer progression. To clarify this possibility, we overexpressed exogenous matriptase in the human stomach cancer cell line AZ521. In vitro, the matriptase transfectant (Mat-AZ521) and the control transfectant (Mock-AZ521) showed a similar growth rate, although the saturation cell density was significantly higher with the Mat-AZ521. When implanted into nude mice subcutaneously or intraperitoneally, Mat-AZ521 cells grew faster and produced much larger solid tumors than Mock-AZ521 cells. The overexpression of matriptase in AZ521 cells shortened the survival time of tumor-bearing mice. Histological analysis showed that both the number and the size of blood vessels in tumor tissues were significantly higher in the Mat-AZ521 tumors than the Mock-AZ521 ones. Moreover, it was found that purified matriptase activated one of the important matrix metalloproteinases, stromelysin (MMP-3). These results suggest the possibility that the matriptase-dependent activation of MMP-3, as well as the direct activity of matriptase, promotes tumor growth and angiogenesis by enhancing extracellular matrix degradation in tumor cell microenvironments.

Procedure for quantitative determination of effectiveness of photoinduced destruction of malignant tumors

We have developed a procedure for analysis of the functional status of blood vessels in tumor tissues using computer-assisted color scanning of tumor slices and also for a quantitative assessment of the effectiveness of photoinduced destruction of tumor tissues in animal experiments. Its major advantage is direct determination of the size of the tumor necrosis zone. The procedure has been tested in an experiment on three strains of malignant tumors with different morphologies.