Cells Of Tumor Blood Vessels Research Articles

The way forward in treating pancreatic cancer

The way forward in treating pancreatic cancer

Abstract A129: Targeted delivery of therapeutics to tumor cells and the tumor microenvironment

Abstract Human cancer is a complex disease caused by genetic instability and accumulation of multiple molecular alterations. Tumor growth and metastasis depend on the development of a neovasculature by a process called angiogenesis, which is controlled by numerous pro- and antiangiogenic factors. Therefore, vascular targeting emerges as an attractive therapeutic approach, since angiogenic blood vessels express distinctive molecular markers with direct access by the therapeutic agent. Moreover, ablation of immature angiogenic vessels causes reduction of the interstitial pressure gradient in the tumor periphery, improving drug delivery through the highly permeabilized vessel wall. Hence, additional therapeutic gains will be expected by “starving” tumor cells to death and by increasing the concentration of the therapeutic agent in the target cell. The present work, is aimed at investigating the ability of a system consisting of a ligand coupled to the extremity of poly(ethylene glycol)-grafted liposomes, containing doxorubicin (DXR), to simultaneously target human tumor cells and endothelial cells of tumor blood vessels. Cellular association studies were conducted by flow cytometry, fluorimetry and confocal microscopy with rhodamine-labeled liposomes. Competitive inhibition and mechanistic assays were also performed by fluorimetry. Cytotoxicity was evaluated with the MTT assay and biodistribution performed on Balb/c nude female mice bearing orthotopic implanted tumors was analyzed by radioactivity counts of a lipid and a drug tracer ([3H]CHE and [14C]DXR, respectively). Our results show a substantial increase in the levels of association for ligand-targeted liposomes, in vitro as well as ex vivo, in tumor cells removed from patients, after mastectomy or tumorectomy. These results suggest that internalization occurs, most likely through the clathrin-mediated endocytic pathway. Cytotoxicity studies show that ligand-targeted liposomes are more cytotoxic than the control formulation (4 to 180-fold), against tumor and endothelial cells, indicating that binding and internalization of the system are contributing to a more efficient delivery of the payload to the target cells. In vivo experiments report a higher tumor accumulation of the radiolabeled targeted system over non-targeted liposomes (approximately 18-fold). The observed improved tumor targeting is likely due to a strong vascular targeting component that is taking place in vivo. The data generated so far indicate that the same technological platform is able to target two distinct cell populations within the tumor niche. Overall, these results represent a novel and a valuable contribution for delivery strategies to the tumor and its microenvironment. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A129.

Intravascular Presence of Tumor Cells as Prognostic Parameter in Uveal Melanoma: A 35-Year Survey

Invasion of tumor cells into blood vessels is essential for metastasis of uveal melanoma. The occurrence of ingrowth of tumor cells in blood vessels in uveal melanoma was analyzed, and this parameter was compared with the survival of the patients. Between 1972 and 2007, 643 eyes primarily enucleated for uveal melanoma were evaluated histopathologically. Survival data were obtained from charts and from the Integral Cancer Center patient registry. No vascular ingrowth of tumor cells occurred in 59% of the eyes, whereas 18% had tumor cell ingrowth in vessels inside the tumor, 10% in vessels outside the tumor, and 8% in vessels inside as well as outside the tumor. The presence of any intravascular ingrowth of tumor cells correlated significantly with the diameter (P < 0.01) and prominence of the tumor (P < 0.01), as well as with non-spindle-cell type (P = 0.03) and intrascleral ingrowth (P < 0.01), and was associated with a worse survival. When extravascular matrix patterns were not included in the multivariate analysis, intravascular ingrowth came out as an independent prognostic factor, but this was not the case when extravascular matrix patterns were included in the multivariate model. Intravascular ingrowth of tumor cells in uveal melanoma occurs frequently in combination with well-known histopathologic factors such as large tumor size, epithelioid cell type, and intrascleral ingrowth.

Cationic liposomal paclitaxel in combination with gemcitabine in patients with advanced pancreatic cancer: A phase II trial

4526 Background: EndoTAG-1 is a novel cationic liposomal formulation of paclitaxel being developed for the treatment of solid malignancies. It acts by targeting activated negatively charged endothelial cells of tumor blood vessels. We present safety and efficacy data of a randomized, controlled phase II trial in pancreatic cancer (PC). Methods: 200 patients with advanced PC were randomized to 1st line treatment with weekly gemcitabine (GEM: 1000 mg/m2) and twice weekly infusions of EndoTAG-1 (E) at 3 different dose levels (Elow: 11 mg/m2, Emed: 22 mg/m2, Ehigh: 44 mg/m2) or GEM monotherapy. Patients were treated for 7 weeks and followed up for overall survival (OS) for at least 1 year. After finishing study treatment, any anti-tumor therapy was allowed. A subgroup of patients had the option to receive repeated cycles of combination therapy in case of at least stable disease according to RECIST until disease progression. Results: Median OS was substantially higher in the GEM+Emed and GEM+ Ehigh groups than the GEM monotherapy group. Adjusted hazard ratios for OS were 0.72 (95% CI 0.46–1.13) and 0.67 (0.43–1.07). In patients receiving &gt;1 treatment cycle, median OS was 11.5 months (GEM+Ehigh); in the GEM+Emed group 75% of patients were alive at 1 year. Treatment with EndoTAG-1 and gemcitabine was generally well tolerated. A trend for increasing adverse event frequency with EndoTAG-1 dose was observed for infusion-related reactions associated with chills and pyrexia, and thrombocytopenia. The overall frequency of serious adverse events in the GEM+E groups was low, the most frequent SAE being pyrexia in 4 (8%) patients in the GEM+Ehigh group. There was no indication for significant organ toxicity associated with EndoTAG-1, even in patients receiving multiple treatment cycles. Conclusions: This phase II trial indicates a considerable survival benefit for patients with advanced PC receiving EndoTAG-1 in combination with gemcitabine and a favourable safety profile warranting further development of EndoTAG-1 in this indication. [Table: see text]

58 POSTER Novel approaches to breast cancer therapy – simultaneous targeting to tumor and endothelial cells of tumor blood vessels

Metastasis of prostate cancer (PCa) is largely affected by natural killer (NK) cells. This study aimed to clarify the mechanisms underlying tumor cells escaping from NK cells mediated by HIF-1α.MiR-224 expression in lymphocytes and HIF-1α protein level in NK cells were determined by qRT-PCR and western blot, respectively. The amount of NKp46+ NK cells was detected with flow cytometry. The IFN-γ level was examined by enzyme linked immunosorbent assay (ELISA). NK cells were tested for cytolytic activity with a Non-Radioactive Cytotoxicity Assay, and treated with oxygenglucose deprivation (OGD) for hypoxia simulation. Interaction between miR-224 and NCR1 was evaluated with dual luciferase reporter assay.MiR-224 was down-regulated in lymphocytes isolated from prostate cancer tissues (n = 10). Overexpression of miR-224 protected prostate cancer from NK cells. HIF-1α increased miR-224 to inhibit the killing capability of NK cells on prostate cancer. MiR-224 controlled the expression of NCR1. Overexpression of miR-224 protected prostate cancer from NK cells through NCR1/NKp46 signaling. Suppression of HIF-1α enhanced the cytotoxicity of NK cells on prostate cancer via miR-224/NCR1 pathway.HIF-1α inhibits NCR1/NKp46 pathway through up-regulating miR-224, which affects the killing capability of NK cells on prostate cancer, thus inducing immune escape of tumor cells.

Anti-angiogenic effect of 5-Fluorouracil-based drugs against human colon cancer xenografts

In addition to the direct cytotoxic effects of chemotherapy agents on tumor cells, the anti-angiogenic activities attained by these agents by targeting proliferating endothelial cells in tumor blood vessels has attracted much research interest. In this study, we examined the antitumor activity of 5-Fluorouracil (5-FU)-based drugs (S-1 [1 M tegafur, 0.4 M 5-chloro-2,4-dihydroxypyridine and 1 M potassium oxonate] and capecitabine) on human colorectal cancer xenografts and evaluated their anti-angiogenic effects. Both drugs showed significant antitumor activities against COL-1 xenografts at a sub-maximum tolerated dose (sub-MTD), which was lower than the maximum tolerated dose (MTD). At the sub-MTD, a significant reduction in the microvessel number and the enhancement of tumor-associated microvessel endothelial cell apoptosis was seen in xenografts treated with S-1. In addition, we found that thrombospondin-1 (TSP-1) expression, known to be a mediator of the anti-angiogenic effects of metronomic chemotherapy, was significantly up-regulated in xenograft tumor tissues and plasma in animals treated with S-1 at a sub-MTD. Capecitabine also showed a trend toward the induction of TSP-1. These results suggest that 5-FU-based drugs inhibit tumor progression through different modes of action, including cytotoxic activity derived from 5-FU and the inhibition of angiogenesis through the induction of TSP-1.

US Imaging of Tumor Angiogenesis with Microbubbles Targeted to Vascular Endothelial Growth Factor Receptor Type 2 in Mice

To prospectively evaluate contrast material-enhanced ultrasonography (US) with microbubbles targeted to vascular endothelial growth factor receptor type 2 (VEGFR2) for imaging tumor angiogenesis in two murine tumor models. Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. A US contrast agent, consisting of encapsulated gaseous microbubbles, was developed specifically to bind to VEGFR2 (by using anti-VEGFR2 antibodies and biotin-streptavidin interaction) which is up-regulated on endothelial cells of tumor blood vessels. VEGFR2-targeted microbubbles (MB(V)), control microbubbles (MB(C)), and nonlabeled microbubbles (MB(N)) were tested for binding specificity on cells expressing VEGFR2 (mouse angiosarcoma SVR cells) and control cells (mouse skeletal myoblast C2C12 cells). Expression of mouse VEGFR2 in culture cells was tested with immunocytochemical and Western blot analysis. Contrast-enhanced US imaging with MB(V) and MB(C) was performed in 28 tumor-bearing nude mice (mouse angiosarcoma, n = 18; rat malignant glioma, n = 10). Differences were calculated by using analysis of variance. In cell culture, adherence of MB(V) on SVR cells (2.1 microbubbles per SVR cell) was significantly higher than adherence of control microbubbles (0.01-0.10 microbubble per SVR cell; P < .001) and significantly more MB(V) attached to SVR cells than to C2C12 cells (0.15 microbubble per C2C12 cell; P < .001). In vivo, contrast-enhanced US imaging showed significantly higher average video intensity when using MB(V) compared with MB(C) for angiosarcoma and malignant glioma tumors (P < .001). Results of immunohistochemical analysis confirmed VEGFR2 expression on vascular endothelial cells of both tumor types. US imaging with contrast microbubbles targeted to VEGFR2 allows noninvasive visualization of VEGFR2 expression in tumor vessels in mice.

Opening the gateway to tumors

T cells can turn back cancer, but they don't always reach tumor cells in sufficient numbers to do so. A close look at the cells of tumor blood vessels suggests a way to boost T cell migration in individuals with cancer (pages 28–36 ).

A case of vulvar superficial angiomyxoma with necrotizing angiitis-like lesions and expression of granulocyte-colony stimulating factor

A vulvar tumor of 3-year-old girl was resected. The tumor had a pedunculated polypoid appearance with a multinodular surface and was covered by normal colored skin. Histologically, the tumor was lobulated and consisted of sparse stellate- or spindle-shaped tumor cells with a large amount of edematous stroma admixed with myxomatous areas. The tumor was rich in blood vessels of various sizes. Several blood vessels showed fibrinoid necrosis. There was a diffuse neutrophilic infiltration in the stroma. The tumor was diagnosed as a superficial angiomyxoma. Immunohistochemically, the tumor cells diffusely expressed vimentin, focally α-smooth muscle actin and desmin. Both estrogen and progesterone receptors were negative. Occasionally, they expressed CD34. Most of the tumor cells expressed granulocyte-colony stimulating factor (G-CSF). Endothelial cells of tumor blood vessels occasionally expressed intercellular adhesion molecule-1 or E-selectin. Some endothelial cells in the tumor were immunolabeled by anti-vascular endothelial growth factor (VEGF) antibody along their luminal surfaces. In the present case, G-CSF and adhesion molecules to neutrophils may have played some roles in neutrophilic infiltration into the tumor and in fibrinoid necrosis of the blood vessels. In addition to these molecules, VEGF may have contributed to vascular growth, leading to edematous stroma.

Histological characteristics of tumor in vessels and lymph nodes are significant predictors of progression of invasive ductal carcinoma of the breast: a prospective study

Invasive ductal carcinomas (IDCs) of the breast are composed of primary invasive tumors as well as tumor cells in blood vessels and lymph nodes. The purpose of this study was to determine whether the histological characteristics of tumor in the vessels and nodes are significantly associated with outcome. In a series of 393 patients, multivariate analyses showed that in IDCs without nodal metastasis and with fibrotic focus dimension, lymph vessel tumor emboli with >6 apoptotic figures and those invading >3 mm from the tumor margin had significantly higher hazard rates (HRs) for recurrence ( P <0.05). In IDCs with 1 to 3 nodal metastases, >2 apoptotic figures in tumor emboli in blood vessels and >5 invaded lymph vessels were associated with significantly higher HRs for tumor recurrence and death ( P <0.005). In IDCs with 4 or more nodal metastases, nodal tumors with >5 mitotic figures and >5 nodes with extranodal extension were associated with significantly higher HRs for tumor recurrence or death ( P <0.05). We conclude that several histological characteristics of tumors in vessels and nodes have significant implications for the progression of IDCs.

Nucleolin expressed at the cell surface is a marker of endothelial cells in angiogenic blood vessels.

A tumor-homing peptide, F3, selectively binds to endothelial cells in tumor blood vessels and to tumor cells. Here, we show that the cell surface molecule recognized by F3 is nucleolin. Nucleolin specifically bound to an F3 peptide affinity matrix from extracts of cultured breast carcinoma cells. Antibodies and cell surface biotin labeling revealed nucleolin at the surface of actively growing cells, and these cells bound and internalized fluorescein-conjugated F3 peptide, transporting it into the nucleus. In contrast, nucleolin was exclusively nuclear in serum-starved cells, and F3 did not bind to these cells. The binding and subsequent internalization of F3 were blocked by an antinucleolin antibody. Like the F3 peptide, intravenously injected antinucleolin antibodies selectively accumulated in tumor vessels and in angiogenic vessels of implanted “matrigel” plugs. These results show that cell surface nucleolin is a specific marker of angiogenic endothelial cells within the vasculature. It may be a useful target molecule for diagnostic tests and drug delivery applications.

Histological characteristics of tumors in blood vessels play an important role in tumor progression of invasive ductal carcinoma of the breast.

Whether the characteristics of tumor cells in blood vessels play an important role in the tumor progression of invasive ductal carcinoma (IDC) of the breast is not known. The purpose of this study was to investigate the significance of the characteristics of tumor cells in blood vessels in relation to tumor progression in 247 IDC patients with blood vessel invasion, in comparison with well-known histological parameters. Blood vessel tumor embolus dimensions were measured. Nuclear atypia, numbers of mitotic and apoptotic figures, and fibrosis grade of tumor cells in blood vessels were assessed. Cox proportional hazard multivariate analyses showed that >2 mitotic figures in blood vessel tumors significantly increased the hazard rates (HRs) of disease-free survival (P=0.002) and initial distant organ metastasis-free survival (IDOMS) in node-negative IDCs (P=0.005), and the HRs of disease-free survival (DFS, P=0.007) and IDOMS (P=0.015) in node-positive IDCs. Apoptotic figures >2 in blood vessel tumor emboli significantly increased the HR of overall survival (P=0.007) in node-positive IDCs. The present study showed the number of mitotic and apoptotic figures in tumor cells in the blood vessels to play a very important role in the tumor progression of IDCs.

The angiogenic factor midkine is aberrantly expressed in NF1-deficient Schwann cells and is a mitogen for neurofibroma-derived cells.

Loss of the tumor suppressor gene NF1 in neurofibromatosis type 1 (NF1) contributes to the development of a variety of tumors, including malignant peripheral nerve sheath tumors (MPNST) and benign neurofibromas. Of the different cell types found in neurofibromas, Schwann cells usually provide between 40 and 80%, and are thought to be critical for tumor growth. Here we describe the identification of growth factors that are upregulated in NF1-/- mouse Schwann cells and are potential regulators of angiogenesis and cell growth. Basic fibroblast growth factor (FGF-2), platelet-derived growth factor (PDGF) and midkine (MK) were found to be induced by loss of neurofibromin and MK was further characterized. MK was induced in human neurofibromas, schwannomas, and various nervous system tumors associated with NF1 or NF2; midkine showed an expression pattern overlapping but distinct from its homolog pleiotrophin (PTN). Immunohistochemistry revealed expression of MK in S-100 positive Schwann cells of dermal and plexiform neurofibromas, and in endothelial cells of tumor blood vessels, but not in normal blood vessels. Furthermore, MK demonstrated potent mitogenic activity for human systemic and brain endothelial cells in vitro and stimulated proliferation and soft agar colony formation of human MPNST derived S100 positive cells and fibroblastoid cells derived from an NF1 neurofibroma. The data support a possible central role for MK as a mediator of angiogenesis and neurofibroma growth in NF1. Oncogene (2001) 20, 97 - 105.

Suppression of solid tumor growth by a monoclonal antibody against tumor vasculature in rats: involvement of intravascular thrombosis and fibrinogenesis.

We have reported that immunization of rat tumor-derived endothelial cells (TEC) isolated from KMT-17 solid tumors results in the generation of several monoclonal antibodies (MAbs). TES-23, one of these MAbs, recognizes a naturally occurring 80-kDa antigen expressed on endothelial cells of tumor blood vessels. To determine whether such MAbs can suppress solid tumor growth in vivo by impairment of endothelial cells in tumors following direct binding, we tested the biodistribution of (125)I-labeled TES-23 in rats bearing KMT-17 solid tumors. We also examined the effect of treatment using unconjugated TES-23 on tumor growth and histo-pathological changes in tumor tissues. Biodistribution studies showed localization of TES-23 into tumor tissues 60 min after intravenous injection. TES-23 suppressed significantly the growth of KMT-17 solid tumors following administration for 5 days. Histo-pathological examination showed that TES-23 caused degeneration, apoptosis and/or necrosis and denudation of endothelial cells in viable tumor areas following local aggregation and adhesion of lymphocytes, with subsequent intravascular thrombus formation by platelets and fibrin. Our results indicate that TES-23, which recognizes TEC, can target endothelial cells of solid tumor vasculature directly, resulting in growth suppression in vivo by reduction of blood flow due to intravascular thrombosis. Our results also suggest that targeting tumor vasculature is a potentially attractive approach for the treatment of solid tumors.

Identification of tumor vascular antigens by monoclonal antibodies prepared from rat-tumor-derived endothelial cells.

We have reported the isolation and specific in vitro properties of tumor-derived endothelial cells (TEC) from rat KMT-17 fibrosarcomas transplanted into rats. To develop antibody-based tumor vascular targeting therapy for solid tumors, we have generated monoclonal antibodies (MAbs) using passive immunization of outside-out membrane vesicles of rat epididymal-fat-pad-derived capillary endothelial cells (FCEC) followed by active immunization of those of rat TEC. The MAbs produced were screened against TEC and FCEC. Of all cultured hybridomas, 75 (3.3%) of the secreted MAbs preferentially recognized TEC. We selected a total of 7 MAbs which detected antigens highly abundant in TEC, although 5 of the 7 MAbs were weakly positive for FCEC in cell-ELISA and FACS analyses. The antigens recognized by these MAbs, with the exception of MAb TES-7, were present on endothelial cells of tumor blood vessels in KMT-17 fibrosarcoma tissues, as shown by immunohistochemical analysis. Antigens of 40- and 80-kDa were recognized by MAbs TES-1, 7, 17, 21 and 26 and by MAbs TES-23 and 27 respectively. Although the function of these antigens, which are preferentially expressed on rat tumor-derived endothelial cells, is still unknown, we believe that future studies of such antigens will help elucidate the role of endothelial cells in tumor vasculature. Our results indicate that MAbs may provide a novel tool for the development of antibody-based therapy targeting tumor vasculature.

Cytokine regulation of angiogenesis in breast cancer: the role of tumor-associated macrophages.

Studies over the past 20 years have established that the development of new capillaries from an existing vascular network (a process called angiogenesis) is an essential component of tumor growth. Malignant tumors do not grow beyond 2-3 mm3 in size unless they stimulate the formation of new blood vessels and thus provide a route for the increased inflow of nutrients and oxygen and outflow of waste products. Tumor angiogenesis also provides an essential exit route for metastasizing tumor cells from the tumor to the bloodstream. Indeed, extensive neovascularization is a poor prognostic factor in several forms of human cancer. Angiogenesis is a complex, multistep process driven by many local signals within the tumor. This involves the degradation of the extracellular matrix around a local venule after the release of collagenases and proteases, the proliferation and migration of capillary endothelial cells, and their differentiation into functioning capillaries. Cytokines produced by various cell types present within the microenvironment of solid tumors form a complex, dynamic network in which they have multiple effects on tumor progression. Herein we review our work on the presence, and possible regulatory influence on tumor angiogenesis, of a number of these cytokines within invasive breast carcinomas. We have combined immunocytochemistry with a single cell cytokine release assay called the reverse hemolytic plaque assay to investigate the cellular sources of the key angiogenic cytokines, vascular endothelial growth factor, basic fibroblast growth factor, and tumor necrosis factor-alpha. Tumor-associated macrophages in the stromal compartment of these tumors and/or malignant epithelial cells were seen to be a major producer cell for these cytokines, whereas tumor necrosis factor-alpha receptors were expressed by leukocytes, malignant cells, and endothelial cells in tumor blood vessels.

Cellular Localization of Endothelin Receptor mRNAs (ETA and ETB) in Brain Tumors and Normal Human Brain

We performed in situ hybridization (ISH), using radioactively labeled deoxyoligonucleotides, to localize endothelin (ET) receptor subtype mRNAs (ETA and ETB) in a series of meningiomas in comparison to normal human meninges. Our examination also addressed the cerebellum adjacent to the meninges investigated. Tumor cells of all meningiomas examined showed strong hybridization signals for ETA receptor mRNA, whereas for ETB receptor mRNA only weak hybridization signals could be detected in endothelial cells of tumor blood vessels and in some cell clusters. mRNAs for neither ET receptor were detectable in normal leptomeninges. In human cerebellum we found ETA receptor mRNA only in large blood vessels; ETB receptor mRNA was intensely expressed in Bergmann glial cells. Neither subtype was detectable in neurons.

Experimental gliomas: an autoradiographic study of the endothelial component.

Autochthonous gliomas were induced in rats by intracerebral inoculation of avian of avian sarcoma virus and studied by 3H-thymidine autoradiography. Parenchymal glial tumor cells had a 3H-labeling index (LI) of 3.0 to 13.6%. Endothelial cells in tumor blood vessels had an LI of 2.6 to 34.3%, independent of and in most instances higher than the LI of the glial tumor. Endothelial cells of normal blood vessels had an average LI of 0.3%. This study documents the high proliferative rate of the endothelial cells in anaplastic experimental gliomas, and emphasizes the necessity for seeking direct, incontrovertible evidence to determine whether or not the rapidly proliferating endothelial cells are malignant.

Relationship between transcerebral passage of tumor cells and brain metastasis.

Quantitative study was carried out on transcerebral passage of tumor cells using Yoshida sarcoma and 6 strains of rat ascites hepatoma. The counting was done every second for 40 sec. In relation to the results obtained, histological study was made on the cerebral metastasis of these tumor cells. Metastatic foci in the brain parenchyma, except the meninges and choroid plexus, were formed by island-forming strains (AH-130, AH-272, and AH-7971) which showed comparatively low passage rate and not be single-cell strains (Yoshida sarcoma, AH-7974F, AH-66F, and AH-13) which showed comparatively high rates. Accordingly, it is obvious that there is a correlation between transcerebral passage rates and brain metastasis formation. The mechanisms of metastasis formation were discussed with special reference to arrest of tumor cells in blood vessels.