Endothelial Cells Of Tumor Blood Vessels Research Articles

Abstract 4413: Thrombospondin-1 is a master regulator of glioblastoma vascularization and infiltration

Abstract Glioblastoma are heterogeneous tumours composed of different subpopulations of cells with different behavioral characteristics. Areas of highly angiogenic cells co-exist with populations of non-angiogenic but highly invasive and infiltrative cells. This heterogeneity is a key clinical challenge in glioma treatment. We used a large scale RNA sequencing approach to investigate the molecular components which differentiate infiltrative from angiogenic cells, in a Patient Derived Xenograft (PDX) mouse model. Our bioinformatic analysis revealed TGFβ1 to be a master regulator of tumor development, and Thrombospondin-1 (Tsp1) to be up regulated in infiltrative areas as compared to angiogenic area. Thrombospondins are known as anti-angiogenic factors, however the full roles of these multi domain proteins in tumor development remain to be elucidated. We found Tsp1 expression to be upregulated in grade IV-GBM and in silico in the GBM mesenchymal subclass. It is expressed not only in tumor cells, but also in tumor blood vessel endothelial cells (ECs). TGFβ1 transcriptionally regulated Tsp1 via Smad1 and Smad3. However, contrary to previous results, we found that Tsp1 was not involved in TGFβ1-activation in tumor cells. We showed that Tsp1 regulates cell migration and invasion both in vitro and in vivo. Inhibition of Tsp1 expression in vivo correlated with increased tumor vascularization in both the chick CAM assay and the PDX mouse model. Anti-angiogenic treatments in the PDX mouse model leads to increased tumor hypoxia and invasive tumor cell behavior, as described in our previous work. In this context, both TGFβ1 and Tsp1 were upregulated in tumor cells. Downregulation of tumor-derived Tsp1 by shRNA in the presence of anti-angiogenic therapy led to reduced tumor growth and invasion in vivo. Finally, peptide-mediated inhibition of Tsp1 activity demonstrated that Tsp1/CD47 interaction is involved in the invasive capacity of GBM cells. Taken together, our data suggest that Tsp1 inhibition may be a promising therapeutic approach to limit tumor infiltration induced by treatment with anti-angiogenic agents. Citation Format: Thomas Daubon, Celine Leon, Kim Clarke, Mathilde Poulet, Hrvoje Miletic, Francesco Falciani, Rolf Bjerkvig, Andreas Bikfalvi. Thrombospondin-1 is a master regulator of glioblastoma vascularization and infiltration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4413.

Proapoptotic Peptide-Mediated Cancer Therapy Targeted to Cell Surface p32

Antiangiogenic therapy is a promising new treatment modality for cancer, but it generally produces only transient tumor regression. We have previously devised a tumor-targeted nanosystem, in which a pentapeptide, CGKRK, delivers a proapoptotic peptide into the mitochondria of tumor blood vessel endothelial cells and tumor cells. The treatment was highly effective in glioblastoma mouse models completely refractory to other antiangiogenic treatments. Here, we identify p32/gC1qR/HABP, a mitochondrial protein that is also expressed at the cell surface of activated (angiogenic) endothelial cells and tumor cells, as a receptor for the CGKRK peptide. The results demonstrate the ability of p32 to cause internalization of a payload bound to p32 into the cytoplasm. We also show that nardilysin, a protease capable of cleaving CGKRK, plays a role in the internalization of a p32-bound payload. As p32 is overexpressed and surface displayed in breast cancers, we studied the efficacy of the nanosystem in this cancer. We show highly significant treatment results in an orthotopic model of breast cancer. The specificity of cell surface p32 for tumor-associated cells, its ability to carry payloads to mitochondria, and the efficacy of the system in important types of cancer make the nanosystem a promising candidate for further development.

The effect of flattening filter free delivery on endothelial dose enhancement with gold nanoparticles

Purpose The aim of this study is to quantify and to compare the dose enhancement factor from gold nanoparticles (AuNP) to tumor endothelial cells for different concentrations of AuNP, and clinical MV beam configurations. Methods Tumor endothelial cells are modeled as slabs measuring 10 × 10 × 2 lm. A spherical AuNP is simulated on the surface of the endothelial cell, within the blood vessel. 6 MV photon beams with and without the flattening filter are investigated for different field sizes, depths in material and beam modulation. The incident photon energy spectra for each configuration is generated using EGSnrc. The dose enhancement in the tumor endothelial cell is found using an analytical calculation. The endothelial dose enhancement factor is defined to be the ratio of the dose deposited with and without AuNPs. Results It is found that clinical beam parameters may be chosen to maximize the effect of gold nanoparticles during radiotherapy. This effect is further amplified ∼20% by the removal of the flattening filter. Modulation of the clinical beam with the multileaf collimator tends to decrease the proportion of low energy photons, therefore providing less enhancement than the corresponding open field. Conclusions The results of this work predict a dose enhancement to tumor blood vessel endothelial cells using conventional therapeutic (MV) X-rays and quantify the relative change in enhancement with treatment depth and field size.

Endoglin as a target of antitumor therapy

Blood vascular supply significantly affects progression of tumor growth. Inhibition of endothelial cell proliferation by antiangiogenic drugs should lead to growth arrest of both primary tumors and metastases. During the course of lengthy therapy, endothelial cells may, however, become refractory to the action of antiangiogenic agents. Novel approaches to anticancer treatment should explore the issue of drug resistance shown by endothelial cells. One possible therapeutic solution might be tumor immunotherapy directed against antigens expressed on the surface of endothelial cells which co-form tumor blood vasculature. Such therapy is supposed to break immune tolerance to own antigens and to eliminate tumor blood vessel endothelial cells by activating cytotoxic T lymphocytes. This kind of response can be obtained against endoglin (CD105). Endoglin is overexpressed in proliferating endothelial cells which line tumor blood vessels. Presence of endoglin in solid tumor blood vessels has prognostic value in cancer treatment. CD105 is also expressed by certain cancer cells (prostate, melanoma and Ewing sarcoma). It appears that therapeutic strategies directed against endoglin allow several mechanisms of resistance to antiangiogenic drugs to be omitted. The therapeutic approach that we propose, i.e. a tumor blood vessel-destroying strategy combined with immunotherapy, may become an effective therapeutic tool.

The effect of flattening filter free delivery on endothelial dose enhancement with gold nanoparticles

The aim of this study is to quantify and to compare the dose enhancement factor from gold nanoparticles (AuNP) to tumor endothelial cells for different concentrations of AuNP, and clinical MV beam configurations. Tumor endothelial cells are modeled as slabs measuring 10 × 10 × 2 μm. A spherical AuNP is simulated on the surface of the endothelial cell, within the blood vessel. 6 MV photon beams with and without the flattening filter are investigated for different field sizes, depths in material and beam modulation. The incident photon energy spectra for each configuration is generated using EGSnrc. The dose enhancement in the tumor endothelial cell is found using an analytical calculation. The endothelial dose enhancement factor is defined to be the ratio of the dose deposited with and without AuNPs. It is found that clinical beam parameters may be chosen to maximize the effect of gold nanoparticles during radiotherapy. This effect is further amplified ~20% by the removal of the flattening filter. Modulation of the clinical beam with the multileaf collimator tends to decrease the proportion of low energy photons, therefore providing less enhancement than the corresponding open field. The results of this work predict a dose enhancement to tumor blood vessel endothelial cells using conventional therapeutic (MV) x-rays and quantify the relative change in enhancement with treatment depth and field size.

Abstract 3042: Glypican-1 (GPC1) promotes S-phase entry and DNA replication in human glioma cells

Abstract Glypican-1 (GPC1), the most ubiquitously expressed member of the glypican family of heparan sulfate proteoglycans (HSPGs), is highly expressed in the developing brain and gliomas. In gliomas, GPC1 is overexpressed in both tumor blood vessel endothelial cells (ECs) and the neoplastic cells. Prior work by us in ECs indicates that GPC1 is overall mitogenic and essential for cell cycle progression and proliferation, implying a role in glioma angiogenesis. The present study focused on the role of GPC1 in glioma cells to explore potential roles in tumorigenesis and growth. The expression of GPC1 in U87 cells, a human glioma cell line with a relatively low basal level of GPC1, was titrated by adenoviral transduction. BrdU pulse-labeling showed a robust induction of S-phase entry and DNA replication by expression of GPC1 in a dose-dependent manner. GPC1 overexpression ultimately led to DNA re-replication and aneuploidy as well as enlarged nuclei and cell rounding. Consistent with these cell cycle effects, ectopic expression of GPC1 significantly induced downregulation of pRb, p21Cip1 and p27Kip1 and upregulation of cyclin E, CDK2 and E2F transactivation activity. These cellular and molecular activities of GPC1 were independent of serum concentrations in the growth medium, suggesting that they do not require exogenous growth factors. This activity required intact GPC1 as neither unglycanated GPC1 core protein nor isolated GPC1 HS chains induced the cell cycle and molecular alterations seen after intact GPC1 was added. The more general relevance of our observation to other cell types and the HS-dependency of the GPC1 activity were confirmed by GPC1 transduction of wild-type and HS synthesis-defective CHO cells. Overexpression of GPC1 induced dramatic S-phase entry in wild-type but not in HS-deficient mutant CHO cells. In summary, this work reveals a potent S-phase promoting activity of GPC1 in glioma cells, which may lead to accelerated cell growth and/or increased genomic instability and, therefore, implies a role for GPC1 in glioma tumorigenesis and growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3042. doi:1538-7445.AM2012-3042

Endothelial cell activation and proliferation in ovarian tumors: Two distinct steps as potential markers for antiangiogenic therapy response

Ovarian cancer is the second leading cause of cancer-related death in women worldwide. Since most patients are diagnosed in advanced disease stages, the starting point, early steps and molecular mechanisms of ovarian cancer angiogenesis are still incompletely characterized. Most immunohistochemical studies for assessment of microvessel density (MVD) in ovarian tumors are based on CD31, CD34 and CD105 immunostaining of tumor blood vessels. Yet, the proliferative status of tumor blood vessel endothelial cells has not as yet been used in the assessment of tumor blood vessels. The present study investigated the Ki67 proliferative index of tumor blood vessel endothelial cells highlighted with the CD34 panendothelial marker and the CD105 endothelial marker in ovarian cancers by antigen co-localization with a doublestaining immunohistochemical method. Lack of co-localization of CD105 and Ki67 in all types of ovarian tumors together with the presence of CD34+/Ki67-positive endothelial cells suggest that endothelial cell activation and proliferation are distinct steps in ovarian tumors. Differences in the proliferation index were observed in endothelial cells from blood vessels of the tumor core and those of the tumor peripheral zones. Potential specific targeting of activated and proliferating tumor blood vessels may provide clues for improving antiangiogenic therapy efficiency.

Localized Dose Enhancement to Tumor Blood Vessel Endothelial Cells via Megavoltage X-rays and Targeted Gold Nanoparticles: New Potential for External Beam Radiotherapy

Tumor endothelial cell damage during radiation therapy may contribute significantly to tumor eradication and treatment efficacy. Gold nanoparticles (AuNPs) delivered preferentially to the walls of tumor blood vessels produce low-energy, short-range photoelectrons during external beam radiotherapy, boosting dose to the tumor microvasculature. In this study dosimetry at the single-cell level is used to estimate the anticipated AuNP-mediated dose enhancement to tumor endothelial cells during 6-MV X-ray irradiation. Endothelial cells are modeled as thin slabs with 100-nm-diameter AuNPs attached within the blood vessel. The number of photoelectrons emitted per AuNP per gray of X-rays is computed at multiple points along the external beam central axis by use of a Monte Carlo-generated energy fluence spectrum. The energy deposited from AuNP emissions to the endothelium is calculated based on an analytic method incorporating the energy-loss formula of Cole. The endothelial dose enhancement factor (EDEF) is the ratio of the overall (externally plus internally generated) dose to endothelial cells in the presence of AuNPs to the dose without AuNPs (from the external beam only). At 20-cm depth, the EDEF is 1.7 (70% dose increase) for an intravascular AuNP concentration of 30 mg/g. Most of this dose enhancement arises from the low-energy (approximately 100 keV) portion of the linear accelerator X-ray spectrum. Furthermore, for AuNP concentrations ranging from 7 to 140 mg/g, EDEF values of 1.2 to 4.4 (20-340% dose increase) are calculated. In contrast to calculations assuming that AuNPs distributed homogeneously throughout the target volume (macrodosimetry), our cellular microdosimetry calculations predict a major dose enhancement to tumor microvasculature from conventional linear accelerator X-rays. This effect may enable the delivery of ablative therapeutic doses to these sensitive microstructures while maintaining established dose constraints for the organs at risk.

Localized Dose Enhancement to Tumor Blood Vessel Endothelial Cells Via Targeted Gold Nanoparticles: New Potential for External Beam Radiotherapy

Tumor endothelial cell damage during radiation therapy is assumed to contribute significantly to tumor eradication and the success of treatment. For example, in ablative radiotherapy, large radiation dose fractions to tumor vasculature can hasten the destruction of the tumor infrastructure. However, the radiation doses needed to achieve large scale endothelial cell death are not always attainable due to patient-specific factors. Gold nanoparticles (AuNP) delivered preferentially to the walls of tumor blood vessels produce low energy, short range photoelectrons during external beam radiotherapy, thus boosting dose to the tumor microvasculature while minimally affecting normal tissues.

Heterogeneity of the Tumor Vasculature

The blood vessels supplying tumors are strikingly heterogeneous and differ from their normal counterparts with respect to organization, structure, and function. Six distinctly different tumor vessel types have been identified, and much has been learned about the steps and mechanisms by which they form. Four of the six vessel types (mother vessels, capillaries, glomeruloid microvascular proliferations, and vascular malformations) develop from preexisting normal venules and capillaries by angiogenesis. The two remaining vessel types (feeder arteries and draining veins) develop from arterio-venogenesis, a parallel, poorly understood process that involves the remodeling of preexisting arteries and veins. All six of these tumor vessel types can be induced to form sequentially in normal mouse tissues by an adenoviral vector expressing vascular endothelial growth factor (VEGF)-A164. Current antiangiogenic cancer therapies directed at VEGF-A or its receptors have been of only limited benefit to cancer patients, perhaps because they target only the endothelial cells of the tumor blood vessel subset that requires exogenous VEGF-A for maintenance. A goal of future work is to identify therapeutic targets on tumor blood vessel endothelial cells that have lost this requirement.

Phase I pharmacokinetic study of the vascular endothelial growth factor receptor tyrosine kinase inhibitor vatalanib (PTK787) plus imatinib and hydroxyurea for malignant glioma

This study determined the maximum tolerated dose (MTD) and dose-limiting toxicities (DLT) of the oral vascular endothelial growth factor receptor (VEGFR) inhibitor, vatalanib, when administered with imatinib and hydroxyurea on a continuous daily schedule among recurrent malignant glioma patients. All patients received 500 mg of hydroxyurea twice daily. Imatinib was dosed at 400 mg per day for patients not taking enzyme-inducing antiepileptic drugs (EIAEDs; stratum A) and at 500 mg twice-a-day for patients taking EIAEDs (stratum B). Vatalanib was escalated from 500 mg to 1250 mg twice daily in successive cohorts, independently for each stratum. Pharmacokinetics of each drug were assessed. A total of 37 recurrent patients, 34 (92%) with glioblastoma and 3 (8%) with grade 3 malignant glioma, were enrolled. Nineteen patients (51%) were taking EIAEDs. The MTD of vatalanib for all patients was 1000 mg twice-a-day. DLTs were hematologic, gastrointestinal, renal, and hepatic. No patients developed intracranial hemorrhage. Concurrent administration of imatinib and hydroxyurea did not affect vatalanib exposure, but EIAEDs decreased vatalanib and imatinib plasma exposures. Vatalanib doses up to 1000 mg twice-a-day combined with imatinib and hydroxyurea were well tolerated. Strategies to target tumor blood vessel endothelial cells and pericytes by inhibiting VEGFR and platelet-derived growth factor, respectively, were safe among recurrent malignant glioma patients and may enhance antiangiogenesis activity.

Identification and Functional Analysis of Phosphorylated Tyrosine Residues within EphA2 Receptor Tyrosine Kinase

EphA2 is a member of the Eph family of receptor tyrosine kinases. EphA2 mediates cell-cell communication and plays critical roles in a number of physiological and pathologic responses. We have previously shown that EphA2 is a key regulator of tumor angiogenesis and that tyrosine phosphorylation regulates EphA2 signaling. To understand the role of EphA2 phosphorylation, we have mapped phosphorylated tyrosines within the intracellular region of EphA2 by a combination of mass spectrometry analysis and phosphopeptide mapping using two-dimensional chromatography in conjunction with site-directed mutagenesis. The function of these phosphorylated tyrosine residues was assessed by mutational analysis using EphA2-null endothelial cells reconstituted with EphA2 tyrosine-to-phenylalanine or tyrosine-to-glutamic acid substitution mutants. Phosphorylated Tyr(587) and Tyr(593) bind to Vav2 and Vav3 guanine nucleotide exchange factors, whereas Tyr(P)(734) binds to the p85 regulatory subunit of phosphatidylinositol 3-kinase. Mutations that uncouple EphA2 with Vav guanine nucleotide exchange factors or p85 are defective in Rac1 activation and cell migration. Finally, EphA2 mutations in the juxtamembrane region (Y587F, Y593F, Y587E/Y593E), kinase domain (Y734F), or SAM domain (Y929F) inhibited ephrin-A1-induced vascular assembly. In addition, EphA2-null endothelial cells reconstituted with these mutants were unable to incorporate into tumor vasculature, suggesting a critical role of these phosphorylation tyrosine residues in transducing EphA2 signaling in vascular endothelial cells during tumor angiogenesis.

From Combinatorial Chemistry to Cancer-Targeting Peptides

Several monoclonal antibodies that target cell surface receptors have gained approval by the U.S. Food and Drug Administration and are widely used in the treatment of some cancers. These include but are not limited to the anti-CD20 antibody Rituximab, used in lymphoma treatment, as well as anti-HER-2 antibody for breast cancer therapy. The efficacy of this cancer immunotherapy modality is, however, limited by the large size of the antibody (160 kd) and its relatively nonspecific binding to the reticuloendothelial system. This latter property is particularly problematic if the antibody is used as a vehicle to deliver radionuclides, cytotoxic drugs, or toxins to the tumor site. Peptides, peptidomimetic, or small molecules are thus attractive as alternative cell surface targeting agents for cancer imaging and therapy. Cancer cell surface targeting peptides can be derived from known native peptide hormones such as somatostatin and bombesin, or they can be identified through screening combinatorial peptide libraries against unknown cell surface receptor targets. Phage-display peptide library and one-bead one-compound (OBOC) combinatorial library methods have been successfully used to discover peptides that target cancer cells or tumor blood vessel endothelial cells. The phage-display peptide library method, because of its biological nature, can only display l-amino acid peptides. In contrast, the OBOC combinatorial library method allows for bead-surface display of peptides that contain l-amino acids, d-amino acids, unnatural amino acids, or other organic moieties. We have successfully used the OBOC method to discover and optimize ligands against unique cell surface receptors of prostate cancer, T- and B-cell lymphoma, as well as ovarian and lung cancers, and we have used some of these peptides to image xenografts in nude mice with high specificity. Here, we (i) review the literature on the use of phage-display and OBOC combinatorial library methods to discover cancer and tumor blood vessel targeting ligands, and (ii) report on the use of an ovarian cancer targeting ligand, OA02, as an in vivo PET imaging probe in a xenograft model in nude mice.

Regulation of the expression of the VEGF/VPS and its receptors: role in tumor angiogenesis

Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPS) plays a crucial role for the vascularization of tumors including breast cancers. Tumors produce ample amounts of VEGF, which stimulates the proliferation and migration of endothelial cells (ECs), thereby inducing tumor vascularization by a paracrine mechanism. VEGF receptors (VEGF-Rs) are highly expressed by the ECs in tumor blood vessels. VEGF expression can be induced in various cell types by a number of stimuli including hypoxia, differentiation, growth factors and tumor promoters of the phorbol ester class, such as TPA. The VEGF inductive pathways comprise kinases, oncogenes, tumor suppressor genes, and steroid hormone transcription factors, many of which seem to converge on the activator protein (AP-1) transcription factor. Much less is known about the regulation of VEGF-R expression, which is restricted to ECs. This expression is greatly enhanced in diseased tissue such as solid tumors. So far, it appears that growth factors, cytokines, and tumor promoters are involved in the control of VEGF-R expression. Here we review current knowledge about the regulation of the expression of VEGF and its receptors.