Growth Of Tumor Blood Vessels Research Articles

Targeted anti-cancer therapy: Co-delivery of VEGF siRNA and Phenethyl isothiocyanate (PEITC) via cRGD-modified lipid nanoparticles for enhanced anti-angiogenic efficacy

Anti-tumor angiogenesis therapy, targeting the suppression of blood vessel growth in tumors, presents a potent approach in the battle against cancer. Traditional therapies have primarily concentrated on single-target techniques, with a specific emphasis on targeting the vascular endothelial growth factor, but have not reached ideal therapeutic efficacy. In response to this issue, our study introduced a novel nanoparticle system known as CS-siRNA/PEITC&L-cRGD NPs. These chitosan-based nanoparticles have been recognized for their excellent biocompatibility and ability to deliver genes. To enhance their targeted delivery capability, they were combined with a cyclic RGD peptide (cRGD). Targeted co-delivery of gene and chemotherapeutic agents was achieved through the use of a negatively charged lipid shell and cRGD, which possesses high affinity for integrin αvβ3 overexpressed in tumor cells and neovasculature. In this multifaceted approach, co-delivery of VEGF siRNA and phenethyl isothiocyanate (PEITC) was employed to target both tumor vascular endothelial cells and tumor cells simultaneously. The co-delivery of VEGF siRNA and PEITC could achieve precise silencing of VEGF, inhibit the accumulation of HIF-1α under hypoxic conditions, and induce apoptosis in tumor cells. In summary, we have successfully developed a nanoparticle delivery platform that utilizes a dual mechanism of action of anti-tumor angiogenesis and pro-tumor apoptosis, which provides a robust and potent strategy for the delivery of anti-cancer therapeutics.

Anti‐angiogenic therapy enhances cancer immunotherapy: Mechanism and clinical application

AbstractImmunotherapy, specifically immune checkpoint inhibitors, is revolutionizing cancer treatment, achieving durable control of previously incurable or advanced tumors. However, only a certain group of patients exhibit effective responses to immunotherapy. Anti‐angiogenic therapy aims to block blood vessel growth in tumors by depriving them of essential nutrients and effectively impeding their growth. Emerging evidence shows that tumor vessels exhibit structural and functional abnormalities, resulting in an immunosuppressive microenvironment and poor response to immunotherapy. Both preclinical and clinical studies have used anti‐angiogenic agents to enhance the effectiveness of immunotherapy against cancer. In this review, we concentrate on the synergistic effect of anti‐angiogenic and immune therapies in cancer management, dissect the direct effects and underlying mechanisms of tumor vessels on recruiting and activating immune cells, and discuss the potential of anti‐angiogenic agents to improve the effectiveness of immunotherapy. Lastly, we outline challenges and opportunities for the anti‐angiogenic strategy to enhance immunotherapy. Considering the increasing approval of the combination of anti‐angiogenic and immune therapies in treating cancers, this comprehensive review would be timely and important.

Talin-dependent integrin activation is required for endothelial proliferation and postnatal angiogenesis.

Integrin activation contributes to key blood cell functions including adhesion, proliferation and migration. An essential step in the cell signaling pathway that activates integrin requires the binding of talin to the β-integrin cytoplasmic tail. Whereas this pathway is understood in platelets in detail, considerably less is known regarding how integrin-mediated adhesion in endothelium contributes to postnatal angiogenesis. We utilized an inducible EC-specific talin1 knock-out mouse (Tln1 EC-KO) and talin1 L325R knock-in mutant (Tln1 L325R) mouse, in which talin selectively lacks the capacity to activate integrins, to assess the role of integrin activation during angiogenesis. Deletion of talin1 during postnatal days 1-3 (P1-P3) caused lethality by P8 with extensive defects in retinal angiogenesis and widespread hemorrhaging. Tln1 EC-KO mice displayed reduced retinal vascular area, impaired EC sprouting and proliferation relative to Tln1 CTRLs. In contrast, induction of talin1 L325R in neonatal mice resulted in modest defects in retinal angiogenesis and mice survived to adulthood. Interestingly, deletion of talin1 or expression of talin1 L325R in ECs increased MAPK/ERK signaling. Strikingly, B16-F0 tumors grown in Tln1 L325R adult mice were 55% smaller and significantly less vascularized than tumors grown in littermate controls. EC talin1 is indispensable for postnatal development angiogenesis. The role of EC integrin activation appears context-dependent as its inhibition is compatible with postnatal development with mild defects in retinal angiogenesis but results in marked defects in tumor growth and angiogenesis. Inhibiting EC pan-integrin activation may be an effective approach to selectively target tumor blood vessel growth.

Protocol for bevacizumab purification using Ac-PHQGQHIGVSK-agarose

Bevacizumab is a monoclonal antibody, produced in CHO cells, used for the treatment of many human cancers. It is an anti-vascular endothelial growth factor (antsi-VEGF) that blocks the growth of tumor blood vessels. Nowadays its purification is achieved by affinity chromatography (AC) using protein A which is a very expensive ligand. On the other hand, the peptide Ac-PHQGQHIGVSK contained in the VEGF fragment binds bevacizumab with high affinity. This short peptide ligand has higher stability and lower cost than protein A and it can be prepared very easily by solid phase peptide synthesis. The present protocol describes the synthesis of Ac-PHQGQHIGVSK-agarose and its use for affinity chromatography purification of bevacizumab from a clarified CHO cell culture.•Ac-PHQGQHIGVSK-agarose capacity and selectivity are equivalent to those of protein A matrices.•The peptide ligand shows a greater stability and lower cost. The lack of Trp, Met or Cys in the peptide ligand prevents its oxidation and extends the useful life of the chromatographic matrix.•Mild conditions used during chromatography preserved the integrity of bevacizumab.

Low dose angiostatic treatment counteracts radiotherapy-induced tumor perfusion and enhances the anti-tumor effect.

The extent of tumor oxygenation is an important factor contributing to the efficacy of radiation therapy (RTx). Interestingly, several preclinical studies have shown benefit of combining RTx with drugs that inhibit tumor blood vessel growth, i.e. angiostatic therapy. Recent findings show that proper scheduling of both treatment modalities allows dose reduction of angiostatic drugs without affecting therapeutic efficacy. We found that whilst low dose sunitinib (20 mg/kg/day) did not affect the growth of xenograft HT29 colon carcinoma tumors in nude mice, the combination with either single dose RTx (1x 5Gy) or fractionated RTx (5x 2Gy/week, up to 3 weeks) substantially hampered tumor growth compared to either RTx treatment alone. To better understand the interaction between RTx and low dose angiostatic therapy, we explored the effects of RTx on tumor angiogenesis and tissue perfusion. DCE-MRI analyses revealed that fractionated RTx resulted in enhanced perfusion after two weeks of treatment. This mainly occurred in the center of the tumor and was accompanied by increased tissue viability and decreased hypoxia. These effects were accompanied by increased expression of the pro-angiogenic growth factors VEGF and PlGF. DCE-MRI and contrast enhanced ultrasonography showed that the increase in perfusion and tissue viability was counteracted by low-dose sunitinib. Overall, these data give insight in the dynamics of tumor perfusion during conventional 2 Gy fractionated RTx and provide a rationale to combine low dose angiostatic drugs with RTx both in the palliative as well as in the curative setting.

Automated computational framework of blood vessel quantification in chick chorioallantoic membrane angiogenesis.

Chick chorioallantoic membrane (CAM) angiogenesis assay has been widely used for finding drugs targeting new blood vessel development in cancer research. In addition to the setup materials and protocols, laboratory findings depend on the quantification and analysis of microscopic blood vessel images. However, it is still a challenging problem because of the high complexity of blood vessel branching structures. We applied preprocessing on CAM microscopic images by keeping the integrity of minor branches in the vessel structure. We then proposed an efficient way to automatically extract blood vessel centerlines based on vector tracing starting from detected seed points. Finally, all branches were coded to construct an abstract model of the branching structure, which enabled more accurate modeling for in-depth analysis. The framework was applied in quantifying Icaritin (ICT) inhibition effects on angiogenesis in a CAM model. Experimental results showed the high accuracy in blood vessel quantification and modeling compared with semimanual measurements. Meanwhile, a set of blood vessel growth indicators were extracted to provide fully automated analysis for angiogenesis assays. Further analysis proved that ICT took effect in a dose-dependent manner which could be applied in suppressing tumor blood vessel growth.

Identification of immature granulocytes in cancer chemotherapy patients by cell counting vs. microscopic examination of blood smears

Tumor cell formation occurs through various mechanisms that may result in the growth of tumor blood vessels. Thus, novel methods are required to provide tumor therapy. The aim of the present study was to investigate the reliability of the abnormality-indicating alarm information provided by the automatic hematology analyzer in the measurement of immature granulocytes (IG) and assess the factors involved. The quality control groups at three concentration levels were repeatedly determined within 20 days to observe the within-run precision. The quality control groups at two concentration levels were repeatedly determined within 20 days to observe the between-run precision. The results obtained by the two methods were compared and the reliability of IG measurement using the hematology analyzer was analyzed. Additionally, IG parameters of 120 venous blood specimens collected from cancer patients were measured by cell counting using a hematology analyzer and stained blood smears were observed. The within-run precision experiment showed that the coefficient of variation (CV) values of the IG percentage in the high, middle and low concentration levels were 3.62, 6.75 and 13.69%, respectively, while the CV values of the IG percentage in the two-level between-run precision experiment were 5.6 and 7.1%, respectively. All of the CV values were <15% and within the permitted range. The true-positive rates obtained using the hematology analyzer in the IG≤1%, 1<IG≤10% and IG>10% groups were 11.5, 65.4 and 95.0%, respectively, and the false-negative rates were 0. The IG percentages obtained through the hematology analyzer measurement and from the microscopic observation were (6.98±11.18) and (9.36±20.71)%, respectively. Results of the correlation analysis revelaed that there was an excellent correlation between the two methods (r=0.1364). A significant difference between the two methods was observed using the signed-rank test (P=0.001). Manual microscopic observation is essential for the IG specimen that has been measured using an automatic hematology analyzer and has received an abnormality alarm. For cancer patients who receive chemotherapy, this application helps to provide laboratory data for clinical disease diagnosis and treatment monitoring.

Nanosecond pulsed electric fields as a novel drug free therapy for breast cancer: An in vivo study

Nanosecond pulsed electric fields (nsPEFs) is a novel non-thermal approach to induce cell apoptosis. NsPEFs has been proven effective in treating several murine tumors, but few studies focus on its efficacy in treating human tumors. To determine if nsPEFs is equally effective in treatment of human breast cancer, 30 human breast cancer tumors across 30Balb/c (nu/nu) mice were exposed to 720 pulses of 100ns duration, at 4pulsespersecond and 30kV/cm. Two weeks after treatment, the growth of treated tumors was inhibited by 79%. Morphological changes of tumors were observed via a 3.0T clinical magnetic resonance imaging (MRI) system with a self-made surface coil. Pulsed tumors exhibited apoptosis evaluated by TUNEL staining, inhibition in Bcl-2 expression and decreased blood vessel density. Notably, CD34, vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) expression in treated tumors were strongly suppressed. To evaluate the might-be adverse effects of nsPEFs in healthy tissues, normal skin was treated exactly the same way as tumors, and pulsed skin showed no permanent damages. The results suggest nsPEFs is able to inhibit human breast cancer development and suppress tumor blood vessel growth, indicating nsPEFs may serve as a novel therapy for breast cancer in the future.

Oral administration of monogalactosyl diacylglycerol from spinach inhibits colon tumor growth in mice

Previously, we observed that purified monogalactosyl diacylglycerol (MGDG), a major glycoglycerolipid from spinach, selectively inhibits the activities of mammalian replicative DNA polymerases (α, δ and ε). However, the function of MGDG following ingestion is not well-known. In the present study, spinach MGDG suppressed the proliferation of Colon26 mouse colon cancer cells with an LD50 of 24 μg/ml in vitro. γ-cyclodextrin (CD)-MGDG complex was prepared and administered orally following Colon26 mouse tumor adhesion for 26 days. It was observed that 20 mg/kg equivalent (eq.) of the CD-MGDG complex reduced tumor volume by ∼60% compared with that of the vehicle-treated controls. In immunohistochemical analysis, the CD-MGDG complex group showed a decreased number of proliferating cell nuclear antigen (PCNA)-positive cells and reduction of mitosis in the tumor cells compared with the control group. In addition, the CD-MGDG complex increased the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive apoptotic cells and inhibited CD31-positive tumor blood vessel growth significantly. These results suggest that MGDG has the potential for cancer prevention and health promotion.

On the Hunt for Vascular Endothelial Stem Cells

On the Hunt for Vascular Endothelial Stem Cells

Grape seed proanthocyanidins inhibit angiogenesis via the downregulation of both vascular endothelial growth factor and angiopoietin signaling

Vascular endothelial growth factor (VEGF)/VEGF receptor 2 and angiopoietin 1/tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 signaling pathways regulate different, but complementary, aspects of blood vessel growth in tumors. Simultaneous inhibition of both pathways not only exhibits additive antiangiogenic effects but also overcomes the resistance to anti-VEGF therapy. Grape seed proanthocyanidins (GSPs) are widely consumed dietary supplements with antiangiogenic activity. However, the molecular mechanisms underlying their antiangiogenic action have not been fully understood. We hypothesized that GSPs modulate multiple signaling pathways to exhibit antiangiogenic effects. In the present study, we aimed to test this hypothesis by examining the effects of GSPs on human microvascular endothelial cell-1 and chick chorioallantoic membrane. Our results showed that GSPs inhibited the migration, matrix metalloproteinase-2 and -9 secretion, and tube formation of human microvascular endothelial cell-1 in vitro in a dose-dependent manner. In addition, chick chorioallantoic membrane angiogenesis assay showed that GSPs inhibited neovascularization in a dose-dependent manner. Furthermore, we demonstrated that GSPs inhibited the phosphorylation of VEGF receptor 2 and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 as well as downstream signaling component extracellular signal-regulated kinase 1/2. In summary, these data suggest that GSPs inhibit both VEGF and angiopoietin 1 signaling to execute the antiangiogenic effects and indicate that GSPs could be developed as a pharmacologically safe chemopreventive agent against cancer.

Application of polychromatic flow cytometry to identify novel subsets of circulating cells with angiogenic potential

Defining whether human circulating proangiogenic cells represent a subset of the hematopoietic system and express CD45 or are hematopoietic derivatives that do not express CD45 (and are called endothelial progenitor cells) remains controversial. We have previously developed a polychromatic flow cytometry (PFC) protocol to isolate subsets of hematopoietic cells and we now identify the circulating pool of CD34(+)CD45(dim) cells representing functional circulating hematopoietic stem and progenitor cells (CHSPCs) that can be separated on the basis of AC133 expression and report that the AC133(+) subset of the CHSPCs enhances the growth of tumor blood vessels in vivo in immunodeficient mice. In addition, the ratio of AC133(+) proangiogenic CHSPCs to AC133(-) nonangiogenic CHSPCs unambiguously correlates with the severity of the clinical state of patients with peripheral arterial disease. In sum, a PFC protocol validated via in vitro and in vivo analyses, can be used to interrogate the roles of human hematopoietic elements in the growth and maintenance of the vasculature.

Non-invasive magnetic resonance imaging of blood vessel growth in tumors using nanosized contrast agents

Non-invasive magnetic resonance imaging of blood vessel growth in tumors using nanosized contrast agents

New foundation focused on the early detection of cancer

New foundation focused on the early detection of cancer

Micro research with big impact

Micro research with big impact

Abstract A14: sFLT01: An antiangiogenic protein that neutralizes placental growth factor

Abstract Introduction: Anti-angiogenic agents have shown clinical value in combination with chemotherapy by targeting the VEGF pathways. The development of tumor vasculature in human renal cell carcinomas (RCC) is stimulated not only by VEGF but by other angiogenic growth factors such as placental growth factor (PlGF) [1,2]. In the current study, we used a novel fusion protein, sFLT01, that includes human VEGFR1 domain 2 and an IgG1 Fc, which binds to both human VEGF and PlGF. The anti-angiogenic activity of sFLT01 was evaluated in several preclinical models of RCC. Methods: The binding affinity of sFLT01 for PlGF was evaluated in vitro in binding assays quantified by ELISA and Octet and in proliferation assays where endothelial cells are stimulated by PlGF. The ability of sFLT01 to slow tumor growth was evaluated in the 786-O human RCC xenograft model as well as in the mouse RENCA RCC orthotopic and subcutaneous tumor models. sFLT01 was delivered by intraperitoneal injection twice per week at doses ranging from 5–25 mg/kg. Microvessel density (MVD) and other vascular parameters were analyzed in the syngeneic and xenograft tumors by immunohistochemical methods with an antibody against mouse CD31 to identify endothelial cells. Results: sFLT01 bound to human PlGF with great affinity and inhibited HUVEC proliferation with an IC90 of approximately 2.3 nM. Subcutaneous 786-O RCC tumors were inhibited at a dose of 25 mg/kg and median survival time was increased by 33% in the orthotopic RENCA model. sFLT01 reduced MVD in subcutaneous RENCA tumors by approximately 50% and lumen area by approximately 66%. In the 786-O xenograft model, 5 or 25 mg/kg sFLT01 reduced the blood vessel area, lumen size, and vessel perimeter compared to control. Conclusions: sFLT01 is effective at inhibiting blood vessel growth in tumors by binding VEGF and PlGF thereby preventing the development of new vasculature and decreasing existing vasculature. sFLT01 reduces intratumoral blood vessel count, prolongs survival, and delays tumor progression in the RENCA and 786-O renal cell carcinoma models. As an anti-angiogenic agent, sFLT01 may be useful in treating human renal cell carcinomas. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A14.

Anti-tumor effect of endostatin

Endostatin is another type of specific endogenous inhibitor after the discovery of angiosta-tin. It can block a variety of pathways which promote the growth of tumor blood vessels, and specifically inhibit the proliferation and migration of endothelial cells resulting in suppression of tumor growth and metastasis. En-dostatin has a broad-spectrum of antitumor effect with slight toxicity and is devoid of acquired drug-resistance, furthermore, it also has a synergistic effect when combining with other therapeutic approaches. Key words: Neoplasms; Angiogenesis inhibitors

Delphinidin, a dietary anthocyanidin, inhibits platelet-derived growth factor ligand/receptor (PDGF/PDGFR) signaling

Most cancers are dependent on the growth of tumor blood vessels and inhibition of tumor angiogenesis may thus provide an efficient strategy to retard or block tumor growth. Recently, tumor vascular targeting has expanded to include not only endothelial cells (ECs) but also smooth muscle cells (SMCs), which contribute to a mature and functional vasculature. We have reported previously that delphinidin, a major biologically active constituent of berries, inhibits the vascular endothelial growth factor-induced phosphorylation of vascular endothelial growth factor receptor-2 and blocks angiogenesis in vitro and in vivo. In the present study, we show that delphinidin also inhibits activation of the platelet-derived growth factor (PDGF)-BB receptor-beta [platelet-derived growth factor receptor-beta (PDGFR-beta)] in SMC and that this inhibition may contribute to its antitumor effect. The inhibitory effect of delphinidin on PDGFR-beta was very rapid and led to the inhibition of PDGF-BB-induced activation of extracellular signal-regulated kinase (ERK)-1/2 signaling and of the chemotactic motility of SMC, as well as the differentiation and stabilization of EC and SMC into capillary-like tubular structures in a three-dimensional coculture system. Using an anthocyan-rich extract of berries, we show that berry extracts were able to suppress the synergistic induction of vessel formation by basic fibroblast growth factor-2 and PDGF-BB in the mouse Matrigel plug assay. Oral administration of the berry extract also significantly retarded tumor growth in a lung carcinoma xenograft model. Taken together, these results provide new insight into the molecular mechanisms underlying the antiangiogenic activity of delphinidin that will be helpful for the development of dietary-based chemopreventive strategies.

Tumor-Induced Sentinel Lymph Node Lymphangiogenesis and Increased Lymph Flow Precede Melanoma Metastasis

Lymphangiogenesis is associated with human and murine cancer metastasis, suggesting that lymphatic vessels are important for tumor dissemination. Lymphatic vessel alterations were examined using B16-F10 melanoma cells implanted in syngeneic C57Bl/6 mice, which form tumors metastasizing to draining lymph nodes and subsequently to the lungs. Footpad tumors showed no lymphatic or blood vessel growth; however, the tumor-draining popliteal lymph node featured greatly increased lymphatic sinuses. Lymph node lymphangiogenesis began before melanoma cells reached draining lymph nodes, indicating that primary tumors induce these alterations at a distance. Lymph flow imaging revealed that nanoparticle transit was greatly increased through tumor-draining relative to nondraining lymph nodes. Lymph node lymphatic sinuses and lymph flow were increased in mice implanted with unmarked or with foreign antigen-expressing melanomas, indicating that these effects are not due to foreign antigen expression. However, tumor-derived immune signaling could promote lymph node alterations, as macrophages infiltrated footpad tumors, whereas lymphocytes accumulated in tumor-draining lymph nodes. B lymphocytes are required for lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes, as these alterations were not observed in mice deficient for B cells. Lymph node lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes may actively promote metastasis via the lymphatics.

Antiangiogenic and radiotherapy for cancer treatment.

Tumor growth and progression depends on tumor angiogenesis, the growth of tumor blood vessels, therefore, targeting tumor angiogenesis is a very promising approach for controlling tumor growth and/or causing regression. Tumor blood vessels have been recognized as a critical component of radiation response to the point of being independent of tumor oxygenation during radiation. An anti-angiogenic approach has been considered less likely to develop drug resistance. But recent findings suggest that anti-angiogenesis causes hypoxia that selects tumor cells (due to genetic instability) that are less dependent on blood supply and leads to drug resistance. The approach of combination of anti-angiogenesis with ionizing radiation by targeting both endothelial and tumor cells should minimize this possibility. The combination may produce a synergistic anti-tumor effect.