Endothelial Cells In Tumor Tissues Research Articles

Combination of bulk RNA sequencing and scRNA sequencing uncover the molecular characteristics of MAPK signaling in kidney renal clear cell carcinoma.

The MAPK signaling pathway significantly impacts cancer progression and resistance; however, its functions remain incompletely assessed across various cancers, particularly in kidney renal clear cell carcinoma (KIRC). Therefore, there is an urgent need for comprehensive pan-cancer investigations of MAPK signaling, particularly within the context of KIRC. In this research, we obtained TCGA pan-cancer multi-omics data and conducted a comprehensive analysis of the genomic and transcriptomic characteristics of the MAPK signaling pathway. For in-depth investigation in KIRC, status of MAPK pathway was quantitatively estimated by ssGSEA and Ward algorithm was utilized for cluster analysis. Molecular characteristics and clinical prognoses of KIRC patients with distinct MAPK activities were comprehensively explored using a series of bioinformatics algorithms. Subsequently, a combination of LASSO and COX regression analyses were utilized sequentially to construct a MAPK-related signature to help identify the risk level of each sample. Patients in the C1 subtype exhibited relatively higher levels of MAPK signaling activity, which were associated with abundant immune cell infiltration and favorable clinical outcomes. Single-cell RNA sequencing (scRNA-seq) analysis of KIRC samples identified seven distinct cell types, and endothelial cells in tumor tissues had obviously higher MAPK scores than normal tissues. The immunohistochemistry results indicated the reduced expression levels of PAPSS1, MAP3K11, and SPRED1 in KIRC samples. In conclusion, our study represents the first integration of bulk RNA sequencing and single-cell RNA sequencing to elucidate the molecular characteristics of MAPK signaling in KIRC, providing a solid foundation for precision oncology.

Genomic Correlates of Outcome in Tumor-Infiltrating Lymphocyte Therapy for Metastatic Melanoma.

Adoptive cell therapy (ACT) of tumor-infiltrating lymphocytes (TIL) historically yields a 40%-50% response rate in metastatic melanoma. However, the determinants of outcome are largely unknown. We investigated tumor-based genomic correlates of overall survival (OS), progression-free survival (PFS), and response to therapy by interrogating tumor samples initially collected to generate TIL infusion products. Whole-exome sequencing (WES) data from 64 samples indicated a positive correlation between neoantigen load and OS, but not PFS or response to therapy. RNA sequencing analysis of 34 samples showed that expression of PDE1C, RTKN2, and NGFR was enriched in responders who had improved PFS and OS. In contrast, the expression of ELFN1 was enriched in patients with unfavorable response, poor PFS and OS, whereas enhanced methylation of ELFN1 was observed in patients with favorable outcomes. Expression of ELFN1, NGFR, and PDE1C was mainly found in cancer-associated fibroblasts and endothelial cells in tumor tissues across different cancer types in publicly available single-cell RNA sequencing datasets, suggesting a role for elements of the tumor microenvironment in defining the outcome of TIL therapy. Our findings suggest that transcriptional features of melanomas correlate with outcomes after TIL therapy and may provide candidates to guide patient selection.

Abstract 1315: Antibody-drug conjugate targeting glypican-1 inhibits tumor growth and tumor angiogenesis for glypican-1 positive cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) is one of highly malignant cancers. Systemic chemotherapy is used in many patients, however, there are few chemotherapy options, and their prognosis are poor. Therefore, it is necessary to develop new treatments. We have identified glypican-1 (GPC1) as a novel cancer antigen and reported that enhanced expression of GPC1 in esophageal squamous cell carcinoma and pancreatic cancer is significantly associated with poorer prognosis (Hara, Naka, et al. Br J Cancer. 2016, Nishigaki, Naka, et al. Br J Cancer. 2020). GPC1 is a heparan sulfate proteoglycan that is linked to the cell surface by a glycosylphosphatidylinositol anchor and promotes tumor growth, metastasis, and invasion by acting as a coreceptor, enhancing various signaling pathways. We recently identified that the expression of GPC1 was increased in cholangiocarcinoma. The present study aimed to develop a new therapy for cholangiocarcinoma using an antibody-drug conjugate (ADC) targeting glypican-1 (GPC1). Expression of GPC1 was evaluated in resected cholangiocarcinoma specimens and cell lines. GPC1 knockout cell was established from GPC1-positive cholangiocarcinoma cell line. The antitumor effect of monomethyl auristatin F conjugated GPC1-ADC was investigated in vitro and in vivo. GPC1 was highly expressed in cholangiocarcinoma cells and tissues. Immunohistochemical analysis of 49 extrahepatic cholangiocarcinoma patients revealed that 23 patients (47%) were high expression of GPC1, 24 patients (49%) were low expression of GPC1 and 2 patients (4%) were GPC1 negative. High-expression group demonstrated significantly poorer prognosis compared with the low-expression group in terms of disease-free survival and overall survival (p < 0.05). GPC1 was also expressed in tumor vessels of cholangiocarcinoma, but not on the vessels of nontumor tissues. Monomethyl auristatin F-conjugated GPC1-ADC significantly inhibited tumor growth against GPC1-positive cholangiocarcinoma cells in vitro and in vivo. In a GPC1 knockout xenograft model, GPC1-ADC partially inhibited tumor growth. Vascular endothelial cells in tumor tissues of GPC1-negative xenograft mice expressed GPC1 and were arrested in the G2/M phase of cell cycle by GPC1-ADC. In the present study, GPC1-ADC inhibits tumor growth and tumor angiogenesis for glypican-1 positive cholangiocarcinoma. Our preclinical data demonstrated GPC1-ADC as a promising strategy for GPC1-positive cholangiocarcinoma. Citation Format: Keiichiro Yokota, Satoshi Serada, Shigehiro Tsujii, Ichiro Murakami, Kazuhiro Hanazaki, Tetsuji Naka. Antibody-drug conjugate targeting glypican-1 inhibits tumor growth and tumor angiogenesis for glypican-1 positive cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1315.

Anti-Glypican-1 Antibody-drug Conjugate as Potential Therapy Against Tumor Cells and Tumor Vasculature for Glypican-1-Positive Cholangiocarcinoma.

Cholangiocarcinoma is a highly malignant cancer. Many patients need systemic chemotherapy to prevent tumor development and recurrence; however, their prognosis is poor due to the lack of effective therapy. Therefore, a new treatment option is urgently required. We recently identified glypican-1 (GPC1) as a novel cancer antigen of esophageal squamous cell carcinoma. We also demonstrated the efficacy and safety of GPC1-targeted ADC (GPC1-ADC) conjugating anti-GPC1 mAb possessing high internalization activity with monomethyl auristatin F (MMAF), which is a potent tubulin polymerizing inhibitor. In this study, we confirmed that GPC1 was highly expressed in cholangiocarcinoma cells and tissues. IHC analysis of 49 extrahepatic cholangiocarcinoma patient tumor specimens revealed high expression of GPC1 in 47% of patients. These patients demonstrated significantly poorer prognosis compared with the low-expression group in terms of disease-free survival and overall survival (P < 0.05). GPC1 was also expressed in tumor vessels of cholangiocarcinoma, but not on the vessels of nontumor tissues. MMAF-conjugated GPC1-ADC showed potent tumor growth inhibition against GPC1-positive cholangiocarcinoma cells in vitro and in vivo In a GPC1 knockout xenograft model, GPC1-ADC partially inhibited tumor growth. Vascular endothelial cells in tumor tissues of GPC1-negative xenograft mice expressed GPC1 and were arrested in the G2-M phase of cell cycle by GPC1-ADC. GPC1-ADC exhibits direct as well as indirect antitumor effects via inhibition of tumor angiogenesis. Our preclinical data highlight GPC1-ADC as a promising therapy for GPC1-positive cholangiocarcinoma.

A NIR light triggered disintegratable nanoplatform for enhanced penetration and chemotherapy in deep tumor tissues

Poor penetration and resultant low accumulation of nanomedicines in deep tumor tissues greatly reduce the chemotherapeutic efficiency. How to maximize the tumor accumulation is still a great challenge in the development of nanocarriers. Here, we developed a cyclic Arg-Gly-Asp-Phe-Lys peptide (cRGD) modified and near-infrared (NIR) light triggered disintegratable liposomal nanoplatform (PAM/Pt@IcLipo), where photosensitizer indocyanine green (ICG) was loaded in the out layer and polyamindoamine (PAMAM) dendrimers grafting cisplatin prodrug (PAM/Pt) were encapsulated inside. The cRGD ligands render the liposomes to target αvβ3 integrin receptors overexpressed by vascular endothelial cells in tumor tissues. Long blood circulation can be achieved owing to the relative large size (~162 nm) of the liposomes. When irradiated by NIR light locally at tumor site, ICG heating detonated the thermosensitive liposomes to release the small sized PAM/Pt nanoparticles (~8.6 nm), which were capable of penetrating into the deep tumor tissue. The in vivo results also showed that the PAM/Pt@IcLipo could significantly improve the penetration of cisplatin drug in deep tumor tissues under NIR light irradiation, resulting in an excellent antitumor activity. This nanoplatform solved the dilemma of long blood circulation of large sized nanoparticles and deep penetration of small sized nanoparticles, opening up a new strategy in the development of nanomedicines for cancer therapy.

IAP inhibitor, Embelin increases VCAM-1 levels on the endothelium, producing lymphocytic infiltration and antitumor immunity

ABSTRACT There is an increasing unmet need for successful immunotherapeutic interventions. Lymphocyte extravasation via tumor tissue endothelial cells (TECs) is required for lymphocyte infiltration into tumor sites. This study aimed to investigate the clinical significance of dysfunctional TECs in pancreatic ductal adenocarcinoma (PDAC) and identify chemical compounds that boost tumor-infiltrating lymphocyte (TIL) numbers. We performed immunohistochemical detection and clinicopathological analysis of VCAM-1 on TECs, which is essential for lymphocyte trafficking. We characterized the gene expression profiles of TECs from fresh PDAC tissues. We isolated compounds that upregulated VCAM-1 and E-selectin expression in TECs and examined their biological activities. Compared to endothelial cells from chronic pancreatitis tissue, TECs showed significantly lower VCAM-1 and E-selectin expression and significant weaknesses in lymphocyte adhesion and transmigration, resulting in decreased T cell infiltration around vessels. Patients with a relatively high percentage of VCAM-1+ vessels among all vessels in PDAC tissue had an improved prognosis. A bioinformatics survey demonstrated that TNFR1 signaling was involved in abnormal VCAM-1 and E-selectin expression in TECs. We screened compounds affecting TNFR1 signaling, and the IAP inhibitor, Embelin, induced these molecules on TECs and enhanced T cell adhesion to TECs and transmigration. Furthermore, in vivo, Embelin enhanced tumor-infiltrating T cell numbers, leading to an antitumor immune response. Embelin accelerates TIL infiltration and the antitumor immune response by recovering VCAM-1 expression in TECs. Our strategy may be a therapeutic approach for accelerating the immunotherapeutic response in immune-quiescent tumors, leading to clinical trials’ success.

Suppression of LAT1 in endothelial cells of tumor tissues exhibits an anti-angiogenesis effect

Suppression of LAT1 in endothelial cells of tumor tissues exhibits an anti-angiogenesis effect

Abstract 3709: Deciphering the impact of tumor genetics on immune cell infiltration in major solid cancer types

Abstract With the advent of immune therapies including inhibition of the PD-1/PD-L1 axis there is an urgent need to identify cancer patients that benefit from these therapies and to overcome mechanisms of resistance. Here, we analyzed the impact of tumor genetics on the composition of tumor microenvironment across 21 solid cancer types from the TCGA project. Using specific mRNA markers of 10 cell populations (MCPcounter), we estimated the content of T cells, CD8+ T cells, cytotoxic lymphocytes, B lineage cells, NK cells, monocytic lineage cells, myeloid dendritic cells, neutrophils, fibroblasts and endothelial cells in tumor tissues. Immune cell compositions were correlated with mutational load, the activity of 27 mutational signatures (MutSigs) and PD-L1 mRNA expression. Correlation strength was assessed using Spearman’s rho statistics. Multiple testing was addressed using the Benjamini-Hochberg method and FDR control at 5%. MutSig 1 (clock-like, age-related) showed significant positive correlations with immune cell infiltrations in low-grade gliomas, negative correlations in breast cancer, melanoma, stomach cancer, lung and prostate adenocarcinoma, but no significant correlations in the remaining 15 cancer types. Of the two APOBEC-related signatures, MutSig 2 showed positive significant correlations with immune cell infiltrates in cervical cancer, bladder cancer, lung adenocarcinoma, head and neck cancer and thyroid cancer, MutSig 13 in breast and cervical cancer. In particular, correlation of MutSig 2 with cytotoxic lymphocytes was significant in cervical cancer, bladder cancer and lung adenocarcinoma (R = 0.29, R= 0.17 and R = 0.17), the corresponding correlation of MutSig 13 in breast cancer (R = 0.17). MutSig 4 (tobacco smoke-related) showed a positive significant correlation with CD8+ T cells and NK cells in lung adenocarcinoma (R = 0.15 and R = 0.14). In contrast, correlation of MutSig 4 with immune cell infiltrates was non-significant or negative in head and neck cancer. In colorectal cancer, MutSig 6 (defective mismatch repair) showed significant positive correlations with NK cells, cytotoxic lymphocytes, monocytic lineage cells and neutrophils infiltration (R = 0.45, R = 0.37, R = 0.23 and R = 0.19). PD-L1 mRNA expression and immune cell infiltrates correlated positively in all 21 cancer types. In particular, correlation with monocytic lineage cells was positive and significant in all cancer types and strong (R &amp;gt; 0.50) in 12 cancer types. Correlation with T cells was positive and significant in 18 cancer types and strong in 10 cancer types. In summary, pan-cancer analysis of DNA and RNAseq data showed that specific mutational signatures contribute to cancer cell immunogenicity, while the ubiquitous clock-like mutational process did not positively correlate with immune cell infiltrates in 20 of 21 cancer types. Moreover, PD-L1 mRNA expression strongly correlated with infiltrates of immune cells in the majority of cancer types. Citation Format: Jan Budczies, Michael Bockmayr, Frederick Klauschen, Abrecht Stenzinger, Carsten Denkert. Deciphering the impact of tumor genetics on immune cell infiltration in major solid cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3709. doi:10.1158/1538-7445.AM2017-3709

β-Tubulin-III Is an Immunohistochemical Marker for the Early Invasive Foci of Nonmucinous Lung Adenocarcinoma

Objective: The present study is to investigate the expression of CD34, β-Tubulin-III and Collagen IV-Laminin in adenocarcinoma in situ (AIS), the AIS component of minimally invasive adenocarcinoma (MIA), and early invasive foci, in order to find a valuable immunohistochemical marker for discriminating AIS and its early invasive foci. Methods: A total of 51 AIS patients and 88 MIA patients were included in the present study. In addition, 40 atypical adenomatous hyperplasia (AAH) patients and 54 invasive adenocarcinoma (IA) patients were included as control. Immunohisto-chemical staining of β-Tubulin-III, CD34, CD31, F8 and Collagen IV-Laminin was performed by serial sectioning. β-Tubulin-III was used to show invasive adenocarcinoma foci, CD34 was used to indicate interstitial cells in AIS, CD31 and F8 were used to identify capillary endothelial cells in tumor tissues, and Collagen IV-Laminin was used to visualize the basement membrane component of AIS. Results: The basement membranes and interstitial cells of AAH, AIS and the AIS component of MIA had positive expression of CD34, while mucinous AIS and various invasive adenocarcinomas had no CD34-positive basement membranes or interstitial cells. Invasive cancers such as alveolar adenocarcinoma, papillary adenocarcinoma, micropapillary adenocarcinoma and solid adenocarcinoma had strong positive expression of β-Tubulin-III, while AAH, AIS and the AIS component of MIA, and invasive mucinous adenocarcinoma had negative expression of β-Tubulin-III. AAH, AIS and the AIS component of MIA were surrounded by basement membranes with positive expression of Collagen IV-Laminin, AIS and the AIS component of MIA had significantly thickened basement membranes, and none of invasive adenocarcinomas was surrounded by basement membranes. Conclusions: The present study demonstrates that immunohistochemical staining of CD34, β-Tubulin-III, and Collagen IV-Laminin discriminates AIS component of lung adenocarcinoma from early invasive foci, with the efficacy of β-Tubulin-III being the best. Staining of β-Tubulin-III precisely identifies the early invasive foci of MIA, and can be used as a marker for the identification of the early invasive foci of nonmucinous lung adenocarcinoma.

Y-Box Binding Protein-1 is Involved in Tumor Angiogenesis and a Promising Therapeutic Target

ABSTRACT Aim: Anti-angiogenesis is a promising strategy to control refractory solid cancers. A recent study has demonstrated that Y-box binding protein-1 (YB-1) is overexpressed in the angiogenic endothelial cells of tumor tissues as well as in cancer cells. The aim of this study was to elucidate the biological significance of YB-1 in vascular endothelial cells (EC) and to analyze whether YB-1 is a target molecule to control the angiogenic switch of tumor growth. Methods: YB-1 expression in EC (HUVEC and HPAEC) was inhibited by specific small interfering RNAs (YB-1 siRNA) or antisense oligonucleotide (YB-1 ASO). The effects of YB-1 knockdown on the proliferation, apoptosis, cell cycle transition and tube formation of EC cells were evaluated by MTT assay, Annexin V-fluorescein isothiocyanate (FITC)/ propidium iodide (PI) staining, flow cytometry and tube formation assay, respectively. The changes of gene expression in EC by YB-1 knockdown were analyzed with DNA microarray, quantative RT-PCR and Western blotting. To examine the effect of YB-1 knockdown on tumor angiogenesis, we administered YB-1 ASO into subcutaneous tumors in colon(HCT116) and lung(A549) cancer xenograft mouse models, and evaluated tumor growth by measurement of tumor volume and microvascular density (MVD) by CD31 immunohistochemistry. Results: Compared with the siRNA- or ASO-control, YB-1 knockdown by YB-1 siRNA or YB-1 ASO inhibited cell proliferation, induced G1 phase cell cycle arrest, increased the percentage of apoptotic cells in EC cells, and decreased the length of tubular network structure of EC cells significantly. The expressions of cell cycle- and apoptosis signaling-related factors (CCNB1, CCND, CDK1, CDK2, CDK4, p21, p27 and PARP1) were changed. In vivo experiment, local administration of YB-1 ASO inhibited tumor growth and induced significant decrease of MVD with down-regulation of YB-1 expression in tumor vessels. Conclusions: These results suggest that YB-1 is a key factor for angiogenesis and may be a promising target for the treatment of refractory malignancies. Disclosure: All authors have declared no conflicts of interest.

A novel approach to overcome multidrug resistance: Utilization of P-gp mediated efflux of paclitaxel to attack neighboring vascular endothelial cells in tumors

We tried to overcome the paclitaxel (PTX) resistance of cancer cells due to P-glycoprotein (P-gp) overexpression in the in vivo anti-tumor chemotherapy by utilizing polyethylene glycol-modified liposomal paclitaxel (PL-PTX). First of all, established were PTX-resistant Colon-26 cancer cells (C26/PTX) overexpressing P-gp, which provided IC50 value of PTX solution about 30 times larger than that obtained for control C26 (C26/control) in the in vitro MTT assay. Western blot analysis confirmed P-gp expression in C26/PTX 10 times higher than that in C26/control, indicating that the resistance acquisition of C26/PTX to PTX would be ascribed to the enhanced efflux of PTX by P-gp overexpressed in C26/PTX. However, the in vivo anti-tumor effect of PL-PTX in C26/PTX-bearing mice was similar to that in C26/control-bearing mice. Double immunohistochemical staining of vascular endothelial cells and apoptotic cells within tumor tissues demonstrated that the apoptotic cell death was preferentially observed in vascular endothelial cells in C26/PTX tumors after intravenous administration of PL-PTX, while that was in tumor cells in C26/control tumors. These results suggest that the in vivo anti-tumor effect of PL-PTX in C26/PTX-bearing mice would be ascribed to the cytotoxic action of PTX pumped out of tumor cells by overexpressed P-gp to vascular endothelial cells in tumor tissues.

Prognostic Significance of Annexin A1 Expression in Pancreatic Ductal Adenocarcinoma

Annexin A1 is a 37-kDa calcium- and phospholipid-binding protein of the annexin superfamily considered to play an important role in tumorigenesis. However, associations with clinicopathological features in pancreatic ductal adenocarcinoma (PDAC) cases have yet to be fully defined. We therefore investigated the prognostic value of annexin A1 protein as a PDAC biomarker in 83 tumor and matched non-cancerous tissues or normal pancreas tissues. Expression was analyzed using real-time RT-PCR, Western blotting and immunohistochemistry. In non-tumor tissue, myoepithelial cells showed no or weak expression of annexin A1 while expression was strong and sometimes even located in the nuclei of endothelial cells in tumor tissue. High expression was significantly associated with advanced stage (P <0.05) and a worse overall survival (P <0.05). These results provide new insights to better understand the role of annexin A1 in PDAC survival, and might be relevant to prediction of prognosis and development of more effective therapeutic strategies aimed at improving survival.

The Multicompartmental p32/gClqR as a New Target for Antibody-based Tumor Targeting Strategies

Tumor-associated cell surface antigens and tumor-associated vascular markers have been used as a target for cancer intervention strategies. However, both types of targets have limitations due to accessibility, low and/or heterogeneous expression, and presence of tumor-associated serum antigen. It has been previously reported that a mitochondrial/cell surface protein, p32/gC1qR, is the receptor for a tumor-homing peptide, LyP-1, which specifically recognizes an epitope in tumor cells, tumor lymphatics, and tumor-associated macrophages/myeloid cells. Using antibody phage technology, we have generated an anti-p32 human monoclonal antibody (2.15). The 2.15 antibody, expressed in single-chain fragment variable and in trimerbody format, was then characterized in vivo using mice grafted subcutaneously with MDA-MB-231 human breast cancers cells, revealing a highly selective tumor uptake. The intratumoral distribution of the antibody was consistent with the expression pattern of p32 in the surface of some clusters of cells. These results demonstrate the potential of p32 for antibody-based tumor targeting strategies and the utility of the 2.15 antibody as targeting moiety for the selective delivery of imaging and therapeutic agents to tumors.

Expression of PC cell-derived growth factor and vascular endothelial growth factor in esophageal squamous cell carcinoma and their clinicopathologic significance

Esophageal carcinoma is a common kind of malignant tumor and about 90% of which is esophageal squamous cell carcinoma (ESCC), and it has a high incidence in China. In recent years it has been argued that angiogenesis plays a key role in tumor growth and metastasis and it is a complex process influenced by many factors. This study was aimed to investigate the expression of PC cell-derived growth factor (PCDGF or progranulin) and vascular endothelial growth factor (VEGF) in ESCC tissue and their relationship with clinical pathological parameters of ESCC, clarify the role of PCDGF and VEGF in the angiogenesis of ESCC. The expression of PCDGF and VEGF in 50 surgical specimens from patients with ESCC and 20 with normal esophageal mucosa were detected by immunohistochemistry. The vascular endothelial cells in tumor tissues were labeled by antibody to CD105 for counting microvessel density (MVD). The expression of PCDGF and VEGF in ESCC was significantly higher than that in normal mucosa. Expression correlated with the depth of tumor invasion, lymph node metastasis, and TNM (classification tumor, nodes, metastasis). In ESCC, both the expression level of PCDGF and VEGF had a positive correlation with MVD and the expression of PCDGF had a significant correlation with the expression of VEGF. These results show that both of PCDGF and VEGE have higher expression in ESCC, which indicate that they have a close relationship with angiogenesis. They may be involved in tumor growth, infiltration and metastasis through promoting tumor angiogenesis and may be an important index reflecting biological behavior and prognosis of ESCC.

Immunotherapy of Tumors with Protein Vaccine Based on Chicken Homologous Tie-2

Tie-2 is an endothelium-specific receptor tyrosine kinase known to play a key role in tumor angiogenesis. The present study explores the feasibility of immunotherapy of tumors by using a protein vaccine based on chicken Tie-2 as a model antigen to break the immune tolerance against Tie-2 in a cross-reaction between the xenogeneic homologous and self-Tie-2. In this study, a chicken homologous Tie-2 protein vaccine (chTie-2) and a corresponding mouse Tie-2 vaccine as a control were prepared and the antitumor effect of these vaccines was tested in two tumor models (murine B16F10 melanoma and murine H22 hepatoma). Immunotherapy with chTie-2 was found effective in two tumor models. Autoantibodies against mouse Tie-2 were detected in sera of mice immunized with chTie-2 through Western blot analysis and ELISA assay. Anti-Tie-2 antibody-producing B cells were detectable by ELISPOT. Histologic examination revealed that autoantibodies were deposited on the endothelial cells of tumor tissues. Purified immunoglobulins from chTie-2-immunized mice could induce the apoptosis of human umbilical vein endothelial cells in vitro. Importantly, adoptive transfer of purified immunoglobulins led to antitumor effect in vivo; apparently, angiogenesis was significantly inhibited in these tumors. Furthermore, the antitumor activity and production of autoantibodies could be abrogated by depletion of CD4+ T lymphocytes. Our findings may provide a vaccine strategy for cancer therapy and show the potential utilization of interference with Tie-2 pathway.

Molecular imaging of tumor angiogenesis.

The emergence of angiogenesis as an important target for cancer therapy has led to increased research aimed at understanding the mechanisms underlying the development, maintenance, and destruction of tumor vasculature. Concurrently, molecular imaging technologies have been developed and are being incorporated as integral components of biomedical research due to their ability to noninvasively monitor in vivo molecular events. With the evaluation of numerous anti-angiogenic agents in clinical trials, the adaptation and validation of molecular imaging modalities for monitoring angiogenesis is actively being pursued. The importance of selecting appropriate molecular targets in the study of angiogenesis has become increasingly complex due to the pleiotropy of vascular phenotypes. Furthermore, due to both the relatively low abundance of endothelial cells in tumor tissue and the inherent difficulties of detecting molecular events, molecular imaging of vasculature necessitates continued improvements in achieving higher sensitivity. While several studies have been published that set the groundwork for imaging angiogenesis, much has yet to be accomplished. Various tumor models and transgenic mice provide an excellent resource for developing molecular imaging technologies for the understanding of angiogenesis. This research may play a particularly crucial role in evaluating mechanism and efficacy during pre-clinical testing of anti-angiogenic drugs. Due to practical limitations, however, the implementation of angiogenesis-directed molecular imaging may not extend beyond highly specialized clinical trials. That is, imaging modalities that evaluate angiogenesis at a functional level may prove more appropriate. Despite future technical challenges, molecular imaging will become an important research and clinical tool in evaluating tumor angiogenesis.

Contortrostatin, a dimeric disintegrin from Agkistrodon contortrix contortrix, inhibits breast cancer progression.

We report the results of a multidisciplinary study on the inhibitory effect of a snake venom disintegrin, contortrostatin, a 13.5 kDa homodimeric protein isolated from Agkistrodon contortrix contortrix (southern copperhead) venom, on breast cancer progression. We demonstrate that contortrostatin binds to integrins and blocks the adhesion of human breast cancer cells (MDA-MB-435) to extracellular matrix (ECM) proteins including fibronectin and vitronectin, but it has no effect on adhesion of the cells to laminin and Matrigel. Contortrostatin also prevents invasion of MDA-MB-435 cells through an artificial Matrigel basement membrane. Daily local injection of contortrostatin (5 microg per mouse per day) into MDA-MB-435 tumor masses in an orthotopic xenograft nude mouse model inhibits growth of the tumor by 74% (p = 0.0164). More importantly, it reduces the number of pulmonary macro-metastasis of the breast cancer by 68% (p < 0.001), and micro-metastasis by 62.4% (p < 0.001). Contortrostatin is not cytotoxic to cancer cells, and does not inhibit proliferation of the breast cancer cells in vitro. However, contortrostatin inhibits angiogenesis induced by the breast cancer, as shown by immunohistochemical quantitation of the vascular endothelial cells in tumor tissue removed from the nude mice. We have identified alpha(v)beta3, an important integrin mediating cell motility and tumor invasion, as one of the binding sites of contortrostatin on MDA-MB-435 cells. We conclude that contortrostatin blocks alpha(v)beta3, and perhaps other integrins, and thus inhibits in vivo progression.

Correlation of basic fibroblast growth factor expression levels with the degree of malignancy and vascularity in human gliomas.

Basic fibroblast growth factor (FGF) is a mitogen, a differentiation factor for neuroectoderm-derived cells, and a potent angiogenic factor. The authors have previously demonstrated that the messenger ribonucleic acid of basic FGF is expressed in more than 90% of human gliomas. In the present study, they examined the expression of basic FGF in human glioma tissues using immunohistochemical techniques with a mouse monoclonal antibody against human basic FGF. They also correlated the basic FGF level with the histological grades of malignancy assessed by the number of nucleolar organizer regions (NOR's). Basic FGF was detected in 18 of 19 gliomas, whereas it was undetectable in two normal brains. The expression level of basic FGF peptide increased proportionally with the degree of malignancy. There was also a tendency for the number of NOR's in glioma cells to increase in glioma samples with a high level of basic FGF expression. Furthermore, most of the cases with increased vascularity demonstrated on cerebral angiograms showed a relatively high level of basic FGF expression of tumor cells and a large number of NOR's in endothelial cells in tumor tissues. These results suggest that basic FGF is actually produced in most gliomas and is involved in tumorigenesis and malignant progression as an autocrine growth factor. Moreover, basic FGF may play an important role in tumor neovascularization as a paracrine angiogenic factor.