Endothelial Cells In Normal Tissues Research Articles

Chloride Intracellular Channel Proteins (CLICs) and Malignant Tumor Progression: A Focus on the Preventive Role of CLIC2 in Invasion and Metastasis.

Simple SummaryAlthough chloride intracellular channel proteins (CLICs) have been identified as ion channel proteins, their true functions are still elusive. Recent in silico analyses show that CLICs may be prognostic markers in cancer. This review focuses on CLIC2 that plays preventive roles in malignant cell invasion and metastasis. CLIC2 is secreted extracellularly and binds to matrix metalloproteinase 14 (MMP14), while inhibiting its activity. As a result, CLIC2 may contribute to the development/maintenance of junctions between blood vessel endothelial cells and the inhibition of invasion and metastasis of tumor cells. CLIC2 may be a novel therapeutic target for malignancies.CLICs are the dimorphic protein present in both soluble and membrane fractions. As an integral membrane protein, CLICs potentially possess ion channel activity. However, it is not fully clarified what kinds of roles CLICs play in physiological and pathological conditions. In vertebrates, CLICs are classified into six classes: CLIC1, 2, 3, 4, 5, and 6. Recently, in silico analyses have revealed that the expression level of CLICs may have prognostic significance in cancer. In this review, we focus on CLIC2, which has received less attention than other CLICs, and discuss its role in the metastasis and invasion of malignant tumor cells. CLIC2 is expressed at higher levels in benign tumors than in malignant ones, most likely preventing tumor cell invasion into surrounding tissues. CLIC2 is also expressed in the vascular endothelial cells of normal tissues and maintains their intercellular adhesive junctions, presumably suppressing the hematogenous metastasis of malignant tumor cells. Surprisingly, CLIC2 is localized in secretory granules and secreted into the extracellular milieu. Secreted CLIC2 binds to MMP14 and inhibits its activity, leading to suppressed MMP2 activity. CLIC4, on the other hand, promotes MMP14 activity. These findings challenge the assumption that CLICs are ion channels, implying that they could be potential new targets for the treatment of malignant tumors.

Abstract 247: Targeting Notch4 with a novel neutralizing antibody inhibits tumor growth and alters immune cell populations within the tumor microenvironment

Abstract Endothelial Notch signaling is critical for tumor angiogenesis and growth. Inhibition of Notch1 or its ligand, Dll4, can induce tumor vessel hypersprouting, which interferes with perfusion and reduces tumor development. Unfortunately, blocking Notch1 in endothelial cells of normal tissue can lead to tissue hypoxia and vascular neoplasms. Notch4, another member of the Notch family, is upregulated and increased in the endothelium of certain tumor types compared to normal vasculature. Reducing Notch4, unlike Notch1, causes reduction in developmental and tumor angiogenesis. Using a newly developed anti-Notch4 antibody, E7011, we assessed the effects on Notch4 inhibition on developmental retinal angiogenesis. E7011 treatment did not alter capillary density within the vascular plexus or endothelial tip cell number, but did decrease radial outgrowth and vein diameter, supporting the hypothesis that Notch4 functions as a pro-angiogenic signal. These findings suggest that the targeting of Notch4 could interfere with tumor angiogenesis and offer a novel therapeutic avenue. To establish the role of Notch4 within the tumor microenvironment, we analyzed its expression in both human and murine tumor samples. Immunofluorescent staining revealed a range of Notch4 expression in both tumor cells and endothelium in human triple negative breast cancers. Several murine syngeneic tumors examined expressed Notch4 primarily in the endothelium with only rare tumor expression. We treated orthotopically implanted Py8119 murine breast carcinomas and B16F10 melanomas with E7011, which significantly reduced tumor size compared to IgG treated controls, indicating that suppression of Notch4 is anti-tumorigenic. To determine mechanistically how E7011 delays tumor growth, we performed single cell RNA sequencing on E7011 and control treated Py8119 tumors. Surprisingly, we found that while Notch4 transcripts were present only in endothelial cells, E7011 administration dramatically altered macrophage and monocyte populations within the tumor. Taken together, these findings indicate that inhibition of tumor endothelial Notch4 by E7011 can regulate the presence of immune cells within the tumor microenvironment and improve tumor control. Citation Format: Jason Eng, L.A. Naiche, Bhairavi Swaminathan, Yu Kato, Krishna Thakkar, Rahul Vadakath, James Herts, Rajyasree Emmadi, Junji Matsui, Jan Kitajewski. Targeting Notch4 with a novel neutralizing antibody inhibits tumor growth and alters immune cell populations within the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 247.

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

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

Vasohibin-1 Is a Poor Prognostic Factor of Ovarian Carcinoma.

Vasohibin-1 (VASH1) is an identified negative feedback inhibitor of angiogenesis induced by vascular endothelial growth factor (VEGF) in vascular endothelial cells (ECs). Expression of VASH1 has been reported not only in ECs of normal tissue, but also in ECs surrounding malignant tumors. In malignant tumors, VASH1 is also gaining attention as a prognosis prediction marker. The aim of this study is to investigate the correlation between VASH1 expression and vascular-related factors and various clinicopathological outcomes in clinical cases of ovarian carcinoma. We retrospectively analyzed clinical records of 58 patients with ovarian carcinoma. The expression patterns of VASH1 and other vascular-related factors (CD31 as markers of microvessel density (MVD), VEGF receptor type 2 (VEGFR2), D2-40 as markers of lymphovessel density), and Ki67 (as proliferation markers of cancer cells) were examined immunohistochemically. We studied the correlation between immunohistochemical expression and overall survival. VASH1 expression pattern significantly differed between Federation of Obstetrics and Gynecology (FIGO) Stages. Numbers of VASH1-positive vessels had a significant positive correlation with MVD (Speaman's correlation coefficient (ρ) was 0.51, p < 0.001), VEGFR2-positive vessels (ρ = 0.61, p < 0.001), and percentage of Ki67 (ρ = 0.28, p = 0.034). The Cox univariable analyses revealed that the group of high VASH1 expression (> 14.6 vessels per mm2) at Stages I-III is a prognostic factor (HR = 3.3, 95%CI = 0.4-8.4; p = 0.013). Our results indicate that VASH1 expression in ovarian carcinoma is significantly associated with vascular-related factors and Ki67 expression. We propose that VASH1 is a prognostic marker in ovarian carcinoma.

Podoplanin Expression in Canine Melanoma.

A type I transmembrane protein, podoplanin (PDPN), is expressed in several normal cells such as lymphatic endothelial cells or pulmonary type I alveolar cells. We recently demonstrated that anticanine PDPN monoclonal antibody (mAb), PMab-38, recognizes canine PDPN of squamous cell carcinomas, but does not react with lymphatic endothelial cells. Herein, we investigated whether PMab-38 reacts with canine melanoma. PMab-38 reacted with 90% of melanoma cells (9/10 cases) using immunohistochemistry. Of interest, PMab-38 stained the lymphatic endothelial cells and cancer-associated fibroblasts in melanoma tissues, although it did not stain any lymphatic endothelial cells in normal tissues. PMab-38 could be useful for uncovering the function of PDPN in canine melanomas.

TNFSF15 suppresses VEGF production in endothelial cells by stimulating miR-29b expression via activation of JNK-GATA3 signals.

Vascular endothelial cell growth factor (VEGF) plays a pivotal role in promoting neovascularization. VEGF gene expression in vascular endothelial cells in normal tissues is maintained at low levels but becomes highly up-regulated in a variety of disease settings including cancers. Tumor necrosis factor superfamily 15 (TNFSF15; VEGI; TL1A) is an anti-angiogenic cytokine prominently produced by endothelial cells in a normal vasculature. We report here that VEGF production in mouse endothelial cell line bEnd.3 can be inhibited by TNFSF15 via microRNA-29b (miR-29b) that targets the 3'-UTR of VEGF transcript. Blocking TNFSF15 activity by using either siRNA against the TNFSF15 receptor known as death domain-containing receptor-3 (DR3; TNFRSF25), or a neutralizing antibody 4-3H against TNFSF15, led to inhibition of miR-29b expression and reinvigoration of VEGF production. In addition, we found that TNFSF15 activated the JNK signaling pathway as well as the transcription factor GATA3, resulting in enhanced miR-29b production. Treatment of the cells either with SP600125, an inhibitor of JNK, or with JNK siRNA, led to eradication of TNFSF15-induced GATA3 expression. Moreover, GATA3 siRNA suppressed TNFSF15-induced miR-29b expression. These findings suggest that VEGF gene expression can be suppressed by TNFSF15-stimulated activation of the JNK-GATA3 signaling pathway which gives rise to up-regulation of miR-29b.

Abstract A44: Clinical value of immunohistochemical expression of EGFL7 within high-grade serous ovarian cancer tissue.

Abstract Epidermal growth factor like domain 7 (Egfl7) is expressed by endothelial cells in normal tissues and by malignant cells in various human tumors. Aim: to analyze patterns of immunohistochemical (IHC) staining of Egfl7 within high grade serous ovarian cancer (HGSOC) tissue and to assess clinical value of this biomarker. Material and methods: The IHC staining and pathological analyses were performed on paraffin-embedded primary tumours collected from 73 HGSOC patients with verified clinical history. Mouse anti-human monoclonal antibody against Egfl7, was used. Egfl7 expressing tumors were classified qualitatively in accordance to intense of staining (HSCORE). The appropriate statistical analyses were performed. P-values of &amp;lt;0.05 were regarded as significant. Results: Egfl7 was expressed by the cancer cells within 63 of 73 primary HGSOC tumors (68.3%). Endothelial expression of this biomarker was sporadic. Cancer cells presented two patterns of staining: cytoplasmic only in 50/72 cases (69.4%) and nuclear/cytoplasmic in 13/72 cases (18%). Median duration of the follow-up was 47.25 months (range 17–79 months) Cytoplasmic expression of Egfl7 was not correlated with any of clinico-pathological features as well as with prognosis. The presence of nuclear/cytoplasmic staining within HGSOC predicts worse response for chemotherapy and correlates with increased preoperative levels of CA125. Conclusion: Cancer endogenous expression of Egfl7 is frequent within HGSOC tissue. Predictive value of nuclear/cytoplasmic staining of Egfl7 for chemoresistance should be further evaluated. Citation Format: Jacek Jan Sznurkowski, Anton Zawrocki, Maciej Pawlowski. Clinical value of immunohistochemical expression of EGFL7 within high-grade serous ovarian cancer tissue. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A44.

Expression of BCR/ABL fusion gene in circulating endothelial cells from chronic myelogenous leukemia patients and its clinical significance

Several studies have shown that the tumor endothelial cells are different from the normal tissue endothelial cells. These tumor endothelial cells may contribute to tumor neo-vasculogenesis. This study was purposed to analyze the biologic features and determine the expression level of CD133 and BCR/ABL fusion gene in circulating endothelial cells (CEC) isolated from peripheral blood of CML patients, as well as to investigate the role of CEC in disease progression. Mononuclear cells were isolated from peripheral blood by density gradient centrifugation; CEC were sorted by MACS and harvested in the endothelial growth medium. The morphologic features of CEC were observed by microscopy, the cell growth rate was calculated by cell counting, and the cells were identified by immunofluorescence staining for the expression of CD31,CD34,VWF and CD133. The expression of BCR/ABL fusion gene was examined by FISH in 12 CML patients. The results indicated that the isolated CEC displayed the typical cobble-stone morphology. These cells could be identified by the positive immunofluorescence staining for CD31, CD34 and VWF, and showed more increased proliferative potential as compared to that of healthy donors. It was found that the positive rate of CD133 was 31.29% in CML patients, which was significantly different from that of healthy donors (P < 0.05). In 12 CML patients, CEC carried the same chromosome aberration as the leukemia cells (10.77%). Higher expression level of CD133 and BCR/ABL fusion gene positively correlated with progression of disease. It is concluded that the CEC may participate in invasion and angiogenesis in patients with CML and possibly correlate to the spreading and progression of the disease.

Photodynamic therapy involves an antiangiogenic mechanism and is enhanced by ferrochelatase inhibitor in urothelial carcinoma

The purpose of the present study was to investigate the mechanism of photodynamic therapy (PDT) supplemented with exogenously added 5-aminolevulinic acid (ALA) on human urothelial cancer (UC). Moreover, we aimed to determine whether the therapeutic effects of ALA-based PDT (ALA-PDT) for UC could be enhanced by deferoxamine (DFX), an inhibitor of ferrochelatase. The efficiency of ALA-PDT on these cells was analyzed using flow cytometry and the type of cell death was also assessed. The ALA-PDT promoting effect of DFX was examined on both UC cells and human umbilical vein endothelial cells (HUVEC). The ALA-PDT decreased levels of mitochondrial membrane potential and induced cell death mainly via apoptosis in these cells. Moreover, inhibition of ferrochelatase by DFX led to an increase of protoporphyrin IX (PpIX) accumulation and enhanced the effect of ALA-PDT on UC cells. We further investigated the effect of DFX on in vivo PDT with a tumor-bearing animal model and found that DFX efficiently enhanced tumor cell apoptosis. ALA-PDT induced death of neovascular endothelial cells in tumors but did not affect small vessel endothelial cells in normal tissues surrounding the tumor. Furthermore, DFX enhanced inhibition of neovascularization. These results demonstrated ALA-PDT dominantly induced apoptosis over necrosis by direct action on UC as well as via antiangiogenic action on neovacular endothelial cells, suggesting that the therapeutic damage by ALA-PDT could be kept to a minimum in the surrounding normal tissues. In addition, increased accumulation of PpIX by DFX could enhance this effectiveness of ALA-PDT.

Expression of Egfl7 correlates with low-grade invasive lesions in human breast cancer

Egfl7 (VE-statin) is specifically expressed by endothelial cells of normal tissues but its expression is deregulated in human cancers. Analysis of expression of Egfl7 protein and transcripts in 211 human breast cancer samples shows that Egfl7 is strongly expressed by breast tumor cells. Egfl7 expression is significantly higher in invasive ductal than in invasive lobular carcinoma. Expression of Egfl7 transcripts is also higher in lower SBR grade lesions and in lesions which are not associated with lymph node invasion. Within the invasive ductal carcinoma sub-population, expression of Egfl7 transcripts is correlated with the SBR score and with the ER+ status. High transcript and Egfl7 protein levels significantly correlate with the absence of axillary lymph node invasion. In lymph nodes, the levels of Egfl7 are correlated with the histological type of the primary lesions; they are higher in ductal than in lobular carcinoma. Egfl7 expression is thus associated with better prognosis factors and with the absence of lymph node invasion in human breast cancer lesions.

Egfl7 Promotes Tumor Escape from Immunity by Repressing Endothelial Cell Activation

Downregulating the leukocyte adhesion molecules expressed by endothelial cells that line tumor blood vessels can limit the entry of immune effector cells into the tumor mass, thereby contributing to tumoral immune escape. Egfl7 (also known as VE-statin) is a secreted protein specifically expressed by endothelial cells in normal tissues and by cancer cells in various human tumors. High levels of Egfl7 correlate with higher tumor grade and poorer prognosis. Here we show that expression of Egfl7 in breast and lung carcinoma cells accelerates tumor growth and metastasis in immunocompetent mice but not in immunodeficient mice. Tumors expressing Egfl7 were infiltrated relatively poorly by immune cells and were characterized by reduced levels of immunostimulatory cytokines [IFN-γ, interleukin-12 (IL-12)] and fewer endothelial adhesion molecules [intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1)]. In vitro studies revealed that Egfl7 inhibited the expression of leukocyte adhesion molecules by endothelial cells, preventing lymphocyte adhesion. In contrast, Egfl7 did not exert any effects on immune cell activation. Human breast cancer lesions expressing high levels of Egfl7 also expressed less ICAM-1 and VCAM-1 in their blood vessels, also indicating an inverse correlation between expression levels of Egfl7 and IFN-γ. Thus, Egfl7 expression in tumors promotes tumor progression by reducing the expression of endothelial molecules that mediate immune cell infiltration. Our findings highlight a novel mechanism through which tumors escape immune control.

Blockade of Toll-like Receptor 2 Expression and Membrane Translocation in Rat Corneal Epithelial Cells by Glucocorticoid (TobraDex) After Penetrating Keratoplasty

To evaluate the role of Toll-like receptor (TLR) 2 and the effect of glucocorticoid on immune rejection of penetrating keratoplasty. Allograft corneal transplantation was performed between host Sprague Dawley (SD) and Wistar donor rats. The expression of TLR2 messenger RNA (mRNA) and protein in corneas was determined by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and immunofluorescence on days 5, 7, and 9 after operation. Three groups were included: allograft, allograft treated with TobraDex (Alcon, Rijksweg, Belgium), and isograft. Normal rat corneas were included as an additional control. Various degrees of congregation of inflammatory cells and neovascularization of grafts were confirmed by histopathology. Immunohistochemistry revealed that TLR2 was expressed in epithelial, stromal, and endothelial cells of normal tissue, and in all of the grafts. Immunofluorescence analysis of TLR2 showed membrane staining of epithelial cells in the allografts on days 7 and 9. This was absent in the isografts and the allografts treated with TobraDex. TLR2 mRNA was detected in normal corneas, and levels were increased in all of the grafts, as determined by quantitative reverse transcription-polymerase chain reaction. By day 9 after transplantation, a 3.6-fold increase in TLR2 mRNA was observed in the allografts compared with the isografts or the allografts treated with TobraDex, which was statistically significant, at P < 0.005. Expression of TLR2 in the rat cornea was significantly increased and concurred with the allograft rejection, but was effectively blocked by treatment with TobraDex.

Abstract 4588: Tumor distribution of anti-neuropilin-1 is affected by antibody dose level in a tumor xenograft mouse model

Abstract Neuropilin-1 (NRP1) is a VEGF165 and semaphorin receptor widely expressed by vascular endothelial cells in normal tissues and on some tumor cells. Blocking NRP1 with human IgG1 against its b domain has been shown to inhibit tumor growth in several preclinical models. The objective of this study was to assess tumor distribution of anti-NRP1 at different dose levels with the aim of supporting the clinical development of this antibody. Nude mice bearing HM7 tumors were administered [111In]-DOTA labeled anti-NRP1 in the presence of increasing amounts of unlabeled anti-NRP1 via single IV injection. Following dosing, blood and tumors were collected at pre-selected time points. Tumor distribution was evaluated by measuring the radioactivity levels in the tumor and blood samples using a gamma counter. The mice that received only the radiolabeled antibody had relatively low plasma and tumor radioactivity levels at all time points. The plasma and tumor radioactivity levels for all other dose groups were higher than those of the group that received only radiolabeled antibody due to the saturation of the normal tissue sink previously identified. Comparing the tumor to blood radioactivity ratios demonstrated that the different dose groups maintained maximum exposure in the tumor for variable lengths of time, depending on the dose level. At 24 hours post-dose, all doses greater than the radiolabeled antibody alone were able to maintain maximum exposure. Conversely, at 48 hours post-dose, doses ≥ 10 mg/kg were able to maintain that level, while at 120 hours post-dose, only doses ≥ 20 mg/kg were able to maintain maximum exposure. Taken together, this suggests that tumor distribution is both dose and time dependent. This approach has been used to aid the clinical dose selection for anti-NRP1 and may be a valuable tool for the clinical development of other targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4588. doi:10.1158/1538-7445.AM2011-4588

Abstract 696: Anti-NRP1 tumor uptake is impacted by normal tissue sinks in a tumor bearing mouse model

Abstract Neuropilin-1 (NRP1) is a VEGF165 and semaphorin receptor widely expressed by vascular endothelial cells in normal tissues and on some tumor cells. Blocking NRP1 with a human IgG1 against the b domain of NRP1 has been shown to inhibit tumor growth in several preclinical models. However, due to the wide expression of NRP1 in normal tissues, the uptake of anti-NRP1 by tumor cells may be impacted by the uptake in normal tissues. The objective of this study was to determine the normal tissue uptake of anti-NRP1 and assess its impact on tumor tissue uptake in a tumor bearing mouse model. Nude mice bearing tumors with either low, HM7, or high, SKMES, NRP1 expression were dosed with [111In]-DOTA labeled anti-NRP1 with or without excess amounts of unlabeled anti-NRP1 via single IV injection. Following dosing, blood, tumor, and several normal tissues were collected at pre-selected time points for the determination of radioactivity levels using a gamma counter. In the absence of unlabeled anti-NRP1, 3% Injected Dose (ID)/ml of radioactivity was observed in the blood at 15 minutes post-dose and decreased quickly. There was minimal radioactivity seen in tumor tissue. In contrast, the majority of radioactivity was detected in the lungs and liver. With increasing amount of unlabeled anti-NRP1, the radioactivity in the lungs/liver dropped, while increasing in blood and tumor tissue. At ∼10 mg/kg unlabeled anti-NRP1, there was no additional decrease of radioactivity in these normal tissues whereas the tumor radioactivity appeared to reach its peak. Taken together, these data suggest that the lungs/liver are tissue sinks that sequester a majority of the anti-NRP1 when given at lower doses, thus hampering tumor uptake. It is important to optimize the dose and dose regimen in order to overcome this tissue sink binding and allow for a sufficient amount of antibody to be available for tumor uptake. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 696.

Drug Targets to Pro-Angiogenetic Factors with Special Reference to Primary Peritoneal Mesothelioma

Angiogenesis is necessary for growth and the spread of human tumors. Animal studies also suggest that angiogenesis is an important interspecies biological mechanism of tumor development. Angiogenesis is a complex multistep cascade modulated by both positive soluble factors, such as vascular endothelial growth factor, thymidine phosphorylase, basic-fibroblast growth factor and negative soluble factors such as angiostatin and endostatin. From the imbalance of the above angiogenesis regulators, tumor endothelial cells may divide up to 50 times more frequently than endothelial cells of normal tissue. Published studies have suggested that the assessment of microvessel density (MVD) or endothelial area (EA) can be considered as surrogate markers of angiogenesis with biological and prognostic relevance. Literature data on angiogenesis of mesothelioma are inconclusive, with only a few studies performed in primary peritoneal mesothelioma (PPM) due to the rarity of the disease. We assessed immunohistochemically MVD and EA and their biological and clinical significance in a consecutive series of 23 PPM cases. MVD and EA were detected in "hot spots" by a computerized image analyzer. The mean value of MVD and EA was 27 +/- 14 and 26.04 +/- 8.35 x10(-2) micro(2) per field (400x), respectively. Patients with a high MVD or EA tumors showed a more clinical aggressiveness due to the presence of ascites and a shorter overall survival. Our results suggest that PPM is an angiogenesis-dependent neoplasia. Therefore, antiangiogenic compounds should be tested particularly in those patients with highly vascularized PPM.

Vector Prime-Protein Boost Vaccine Induces Immune Response Against “Self-Antigens” Associated with Epithelial Neoplasms and Tumor Vascular Endothelial Cells.

In order to develop a method to overcome the immune tolerance of cancer, we have designed an Ad-sig-TAA/ecdCD40L adenoviral vector vaccine for the in vivo activation and tumor antigen loading of dendritic cells (DCs). Subcutaneous (sc) injection of the Ad-sig-TAA/ecdCD40L adenoviral vector results in the secretion for 10 days from the vector infected cells of a fusion protein composed of a fragment of a tumor associated antigen (TAA) fused to the extracellular domain (ecd) of the CD40 ligand (CD40L). CD40L is a homo-trimeric protein normally found on B cells and helper CD4+ T cell lymphocytes. All of the sequences necessary to stabilize this trimeric structure of the protein are contained within the ecd of the CD40L protein. The binding of the TAA/ecdCD40L protein to DCs induces migration of these DCs to the regional lymph nodes. These DCs carry fragments of TAA bound to surface MHC Class I molecules. We have shown that the Ad-sig-TAA/ecdCD40L vector strategy can induce a cellular and humoral immune that persists for over a year indicating that a durable memory response is generated. We showed that sc injections of the Ad-sig-rH2N/ecdCD40L vector in rH2N.Tg mice induces a cellular and humoral immune response against the rat Her-2-Neu (rH2N) antigen which is associated with breast cancer. We showed that the sc injection of the Ad-sig-rH2N/ecdCD40L adenoviral vector in a rH2N.Tg transgenic mouse induced resistance to the growth of rH2N positive cancer cells in mice previously anergic to the rH2N antigen. We demonstrated that the sc injection of the Ad-sig-hMUC-1/ecdCD40L vector suppressed the growth of tumor cells positive for the human MUC-1 (hMUC-1) antigen in hMUC-1.Tg mice which were previously anergic to the hMUC-1 antigen. The sc injection of the Ad-sig-hMUC-1/ecdCD40L vector followed by sc injection of two booster injections of the hMUC-1/ecdCD40L protein induced high levels of hMUC-1 specific tumor infiltrating effector CD8 positive T cells and hMUC-1 antibodies which bound to human breast and prostate cancer cells. In addition, we recently showed that the Ad-sig-TAA/ecdCD40L strategy could be used to activate a cellular and humoral immune response against Annexin A1 (AnxA1), which is a marker uniquely displayed on the luminal membrane of tumor vascular endothelial cells but not on the luminal membrane of vascular endothelial cells of normal tissue. The subcutaneous injection of the Ad-sig-AnxA1/ecdCD40L vector suppressed the growth of AnxA1 negative tumor cells in a syngeneic mouse tumor model. This vector prime/protein boost vaccination was found to induce increased levels of effector CD8 positive T cells in the target tumor. These effector T cells were shown express increased levels of the genes encoding the CCR5 chemokine receptor and the CCL3 chemokine ligand which promote the infiltration of antigen specific effector T cells in the target tumor tissues. The response to cancer vaccines is often reduced in older individuals in part due to an intrinsic functional defect in CD4 cells. The Ad-sig-TAA/ecdCD40L vaccine may circumvent this defect because we have shown that the induction of the immune response is CD4 independent. These data suggest that this vector prime-protein boost vaccination strategy will be useful in the reduction of the frequency of recurrence following initial therapy for a wide variety of neoplastic diseases.

Vascular Homing Peptides with Cell-Penetrating Properties

In vivo screening of phage-displayed peptide libraries has revealed extensive molecular heterogeneity in the blood vessels of individual normal tissues and shown that pathological lesions put their signature on the vasculature. In tumors, both blood and lymphatic vessels differ from normal vessels. Moreover, the molecular changes in the vasculature parallel progression in tumor development, hence making the vessels in premalignant lesions distinguishable from normal vessels and from the vessels in malignant tumors of the same tissue. Some of the tumor-homing peptides penetrate into tumor endothelial cells (and tumor cells), but not into endothelial cells in normal tissues or other normal cells. Thus, these cell-penetrating peptides are cell type-specific. Peptides that home to tumor vasculature have been shown to be useful in directing therapeutic agents to experimental tumors. The cell penetrating peptides may be particularly useful in drug delivery because they can take their payload inside the target cells and even into a specific subcellular organelle such as the nucleus.

Endoglin (CD105) expression in head and neck basaloid squamous cell carcinoma

Conclusions. This comparison of neo-angiogenesis performed by analysing CD105 expression seems to suggest that the biological behaviour of head and neck BSCCs is similar to that of site- and stage-matched conventional SCCs. Objective. Basaloid squamous cell carcinoma (BSCC) is an uncommon, high-grade biomorphic variant of SCC with a predilection for the head and neck region. It is a matter of controversy whether the biological and clinical behaviour of BSCC is more aggressive than that of SCC. Angiogenesis is essential for tumour growth. It has been established that endothelial cells of tumour-associated neovasculature proliferate more rapidly than endothelial cells of normal tissue. Endoglin (CD105) has been shown to be a useful marker for identifying proliferating endothelium involved in tumour angiogenesis. The aim of this study was to investigate the expression of CD105 in head and neck BSCCs and to compare it with that in SCCs. Material and methods. Nine head and neck BSCCs (five cases in the larynx, three in the tongue and one in the tonsil) were considered. A group of nine site- and stage-matched SCCs was examined simultaneously. For each sample, CD105 reactivity was evaluated immunohistochemically. The mean area fraction (percentage of fields occupied by vessels), the percentage of vessel-presenting fields and the vessel density were considered. Results. The median values of the mean area fraction were 1.01% and 0.9% in BSCCs and SCCs, respectively. The median values of the percentage of vessel-presenting fields were 57.5% and 46.67% in BSCCs and SCCs, respectively. The median values of vessel density were 2.07% and 1.58% in BSCCs and SCCs, respectively. Statistical analysis did not disclose any significant differences between BSCCs and SCCs for any of the above-mentioned parameters.

Recombinant human angiostatin (rhAngiostatin) in combination with paclitaxel and carboplatin in patients (pts) with advanced NSCLC: Preliminary results of a phase II trial

7105 Background: Angiostatin, an endogenous protein derived from plasminogen, functions as a potent inhibitor of angiogenesis by inhibiting vascular endothelial cell growth. Because the replication rate of endothelial cells in tumor capillary vessels can be 100-fold greater than the endothelial cells of normal tissues, inhibition by rhAngiostatin may lead to involution of microvessels and cause tumor regression. We report the preliminary results of a single institution, randomized phase II study combining rhAngiostatin with paclitaxel and carboplatin in chemotherapy-naïve pts with stage IIIb (with pleural effusion) or IV NSCLC. Methods: Pts (n=24) received paclitaxel 175 mg/m2 i.v. + carboplatin AUC (5) i.v. day 1 + rhAngiostatin at 15mg (n=13) or 60 mg (n=11) subcutaneously twice daily. Chemotherapy cycles were repeated every 21 days for up to 6 cycles. Pts with non-PD after completing chemotherapy continued on maintenance rhAngiostatin until PD. The primary aims were to assess the safety and response rates of each dose combination. Pts with coagulopathies, on anti-coagulants, or with brain metastases were ineligible. Results: There were 16 males, 8 females, median age 66 years (range 45–78), ECOG PS 0 (46%) or 1 (54%), stage IIIb (16.6%) or IV (83.3%), and 62.5% had adenocarcinoma. A median of 4 cycles (range 1–6) of chemotherapy was given and the median duration of rhAngiostatin was 125 days (range, 1–437+). G3/4 toxicities included: neutropenia (41.6%), fatigue (41.6%) and dyspnea (25%). Also, 3 pts developed atrial fibrillation, 2 had a PE and 1 had a TIA. Grade 1–3 skin rash was observed in 91.6% of pts (75% grade 1). 23 of 24 were evaluable for response. PR rate was 39.1% with SD rate of 39.1%. 4 pts had PR's for approximately 8+ months, including 1 pt in a PR for 14.5+ months. Survival data are not yet mature. Conclusion: rhAngiostatin in combination with paclitaxel and carboplatin is well tolerated and active, including several patients who have had durable PR's. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Entremed Entremed Entremed

Recombinant human angiostatin (rhAngiostatin) in combination with paclitaxel and carboplatin in patients (pts) with advanced NSCLC: Preliminary results of a phase II trial

7105 Background: Angiostatin, an endogenous protein derived from plasminogen, functions as a potent inhibitor of angiogenesis by inhibiting vascular endothelial cell growth. Because the replication rate of endothelial cells in tumor capillary vessels can be 100-fold greater than the endothelial cells of normal tissues, inhibition by rhAngiostatin may lead to involution of microvessels and cause tumor regression. We report the preliminary results of a single institution, randomized phase II study combining rhAngiostatin with paclitaxel and carboplatin in chemotherapy-naïve pts with stage IIIb (with pleural effusion) or IV NSCLC. Methods: Pts (n=24) received paclitaxel 175 mg/m2 i.v. + carboplatin AUC (5) i.v. day 1 + rhAngiostatin at 15mg (n=13) or 60 mg (n=11) subcutaneously twice daily. Chemotherapy cycles were repeated every 21 days for up to 6 cycles. Pts with non-PD after completing chemotherapy continued on maintenance rhAngiostatin until PD. The primary aims were to assess the safety and response rates of each dose combination. Pts with coagulopathies, on anti-coagulants, or with brain metastases were ineligible. Results: There were 16 males, 8 females, median age 66 years (range 45–78), ECOG PS 0 (46%) or 1 (54%), stage IIIb (16.6%) or IV (83.3%), and 62.5% had adenocarcinoma. A median of 4 cycles (range 1–6) of chemotherapy was given and the median duration of rhAngiostatin was 125 days (range, 1–437+). G3/4 toxicities included: neutropenia (41.6%), fatigue (41.6%) and dyspnea (25%). Also, 3 pts developed atrial fibrillation, 2 had a PE and 1 had a TIA. Grade 1–3 skin rash was observed in 91.6% of pts (75% grade 1). 23 of 24 were evaluable for response. PR rate was 39.1% with SD rate of 39.1%. 4 pts had PR's for approximately 8+ months, including 1 pt in a PR for 14.5+ months. Survival data are not yet mature. Conclusion: rhAngiostatin in combination with paclitaxel and carboplatin is well tolerated and active, including several patients who have had durable PR's. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Entremed Entremed Entremed