Recent evidence demonstrated that cancer stem cells reside in close proximity to blood vessels in human head and neck squamous cell carcinomas (HNSCC). These findings suggest the existence of a supporting perivascular niche for cancer stem cells.

Objective

The purpose of this study was to evaluate the effect of endothelial cell-secreted factors on the behavior of head and neck cancer stem-like cells (HNCSC).

Materials and methods

HNCSC were identified by sorting UM-SCC-22A (cell line derived from a primary squamous cell carcinoma of the oropharynx) and UM-SCC-22B (derived from the metastatic lymph node of the same patient) for CD44 expression and ALDH (aldehyde dehydrogenase) activity. HNCSC (ALDH+CD44+) and control (ALDH−CD44−) cells were cultured in ultra-low attachment plates in presence of conditioned medium from primary human endothelial cells.

Results

ALDH+CD44+ generated more orospheres than control cells when cultured in suspension. The growth factor milieu secreted by endothelial cells protected HNCSC against anoikis. Mechanistic studies revealed that endothelial cell-secreted vascular endothelial growth factor (VEGF) induces proliferation of HNCSC derived from primary UM-SCC-22A, but not from the metastatic UM-SCC-22B. Likewise, blockade of VEGF abrogated endothelial cell-induced Akt phosphorylation in HNCSC derived from UM-SCC-22A while it had a modest effect in Akt phosphorylation in HNCSC from UM-SCC-22B.

Conclusion

This study revealed that endothelial cells initiate a crosstalk that protect head and neck cancer stem cells against anoikis, and suggest that therapeutic interference with this crosstalk might be beneficial for patients with head and neck cancer.

Emerging evidence indicates that a small population of cancer cells is highly tumorigenic, endowed with self-renewal, and has the ability to differentiate into cells that constitute the bulk of tumors. These cells are considered the “drivers” of the tumorigenic process in some tumor types, and have been named cancer stem cells. Epithelial-mesenchymal transition (EMT) appears to be involved in the process leading to the acquisition of stemness by epithelial tumor cells. Through this process, cells acquire an invasive phenotype that may contribute to tumor recurrence and metastasis. Cancer stem cells have been identified in human head and neck squamous cell carcinomas (HNSCC) using markers such as CD133 and CD44 expression, and aldehyde dehydrogenase (ALDH) activity. The head and neck cancer stem cells reside primarily in perivascular niches in the invasive front where endothelial-cell initiated events contribute to their survival and function. In this review, we discuss the state-of-the-knowledge on the pathobiology of cancer stem cells, with a focus on the impact of these cells to head and neck tumor progression.

Background

Loco-regional spread of disease causes high morbidity and is associated with the poor prognosis of malignant oral tumors. Better understanding of mechanisms underlying the establishment of lymph node metastasis is necessary for the development of more effective therapies for patients with oral cancer. The aims of this work were to evaluate a possible correlation between endothelial cell Bcl-2 and lymph node metastasis in patients with oral squamous cell carcinoma (OSCC), and to study signaling pathways that regulate Bcl-2 expression in lymphatic endothelial cells.

Methods

Endothelial cells were selectively retrieved from paraffin-embedded tissue sections of primary human OSCC from patients with or without lymph node metastasis by laser capture microdissection (LCM). RT-PCR was used to evaluate Bcl-2 expression in tumor-associated endothelial cells and in tumor cells. In vitro, mechanistic studies were performed to examine the effect of vascular endothelial growth factor (VEGF)-C on the expression of Bcl-2 in primary human lymphatic endothelial cells.

Results

We observed that Bcl-2 expression is upregulated in the endothelial cells of human oral tumors with lymph node metastasis as compared to endothelial cells from stage-matched tumors without metastasis. VEGF-C induced Bcl-2 expression in lymphatic endothelial cells via VEGFR-3 and PI3k/Akt signaling. Notably, OSCC cells express VEGF-C and induce Bcl-2 in lymphatic endothelial cells.

Conclusions

Collectively, this work unveiled a mechanism for the induction of Bcl-2 in lymphatic endothelial cells, and suggested that endothelial cell Bcl-2 contributes to lymph node metastasis in patients with oral squamous cell carcinoma.

It is well known that angiogenesis plays a critical role in the pathobiology of tumors. Recent clinical trials have shown that inhibition of angiogenesis can be an effective therapeutic strategy for patients with cancer. However, one of the outstanding issues in anti-angiogenic treatment for cancer is the development of toxicities related to off-target effects of drugs. Transcriptional targeting of tumor endothelial cells involves the use of specific promoters for selective expression of therapeutic genes in the endothelial cells lining the blood vessels of tumors. Recently, several genes that are expressed specifically in tumor-associated endothelial cells have been identified and characterized. These discoveries have enhanced the prospectus of transcriptionaly targeting tumor endothelial cells for cancer gene therapy. In this manuscript, we review the promoters, vectors, and therapeutic genes that have been used for transcriptional targeting of tumor endothelial cells, and discuss the prospects of such approaches for cancer gene therapy.

The specific targeting of diseases, particularly cancer, is a primary aim in drug development, as specificity reduces unwelcome effects on healthy tissue and increases drug efficacy at the target site. Drug specificity can be increased by improving the delivery system or by selecting drugs with affinity for a molecular ligand specific to the disease state. The role of the prosurvival Bcl-2 protein in maintaining the normal balance between apoptosis and cellular survival has been recognized for more than a decade. Bcl-2 is vital during development, much less so in adults. It has also been noted that some cancers evade apoptosis and obtain a survival advantage through aberrant expression of Bcl-2. The new and remarkably diverse class of drugs, small-molecule inhibitors of Bcl-2 (molecular weight approximately 400 to 800 Daltons), is examined herein. We present the activities of these compounds along with clinical observations, where available. The effects of Bcl-2 inhibition on attenuation of tumor cell growth are discussed, as are studies revealing the potential for Bcl-2 inhibitors as antiangiogenic agents. Despite an enormous body of work published for the Bcl-2 family of proteins, we are still learning exactly how this group of molecules interacts and indeed what they do. The small-molecule inhibitors of Bcl-2, in addition to their therapeutic potential, are proving to be an important investigative tool for understanding the function of Bcl-2.

The specific targeting of diseases, particularly cancer, is a primary aim in drug development, as specificity reduces unwelcome effects on healthy tissue and increases drug efficacy at the target site. Drug specificity can be increased by improving the delivery system or by selecting drugs with affinity for a molecular ligand specific to the disease state. The role of the pro-survival Bcl-2 protein in maintaining the normal balance between apoptosis and cellular survival has been recognized for over a decade. Bcl-2 is vital during development, much less so in adults. It has also been noted that some cancers evade apoptosis and obtain a survival advantage through aberrant expression of Bcl-2. The new and remarkably diverse class of drugs, small molecule inhibitors of Bcl-2 (mw ~400–800), is examined herein. We present the activities of these compounds along with clinical observations, where available. The effects of Bcl-2 inhibition on attenuation of tumor cell growth are discussed, as are studies revealing the potential for Bcl-2 inhibitors as anti-angiogenic agents. Despite an enormous body of work published for the Bcl-2 family of proteins, we are still learning exactly how this group of molecules interacts and indeed what they do. The small molecule inhibitors of Bcl-2, in addition to their therapeutic potential, are proving to be an important investigative tool for understanding the function of Bcl-2.

The success of tissue engineering depends on the rapid and efficient formation of a functional blood vasculature. Adult blood vessels comprise endothelial cells and peri-vascular mural cells that assemble into patent tubules ensheathed by a basement membrane during angiogenesis. Using individual vessel components, we characterized intra-scaffold microvessel self-assembly efficiency in a physiological in vivo tissue engineering implant context. Primary human microvascular endothelial- and vascular smooth muscle cells were seeded at different ratios in poly-L lactic acid (PLLA) scaffolds enriched with basement membrane proteins (Matrigel) and implanted subcutaneously into immunocompromised mice. Temporal intra-scaffold microvessel formation, anastomosis and perfusion were monitored by immunohistochemical, flow cytometric and in vivo multiphoton fluorescence microscopy analysis. Vascularization in the tissue engineering context was strongly enhanced in the implants seeded with a complete complement of blood vessel components: Human microvascular endothelial and vascular smooth muscle cells in vivo assembled a patent microvasculature within Matrigel-enriched PLLA scaffolds that anastomosed with the host circulation during the first week of implantation. Multiphoton fluorescence angiographic analysis of the intra-scaffold microcirculation showed a uniform, branched microvascular network. 3-D image reconstruction analysis of hPASMC distribution within vascularized implants was non-random and displayed a preferential peri-vascular localization. Hence, efficient microvessel self-assembly, anastomosis and establishment of a functional microvasculture in the native hypoxic in vivo tissue engineering context is promoted by providing a complete set of vascular components.

Recent studies have demonstrated that cancer stem cells play an important role in the pathobiology of head and neck squamous cell carcinomas (HNSCC). However, little is known about functional interactions between head and neck cancer stem-like cells (CSC) and surrounding stromal cells. Here, we used Aldehyde Dehydrogenase activity and CD44 expression to sort putative stem cells from primary human HNSCC. Implantation of 1,000 CSC (ALDH+CD44+Lin−) led to tumors in 13 (out of 15) mice, while 10,000 non-cancer stem cells (NCSC; ALDH−CD44−Lin−) resulted in 2 tumors in 15 mice. These data demonstrated that ALDH and CD44 select a sub-population of cells that are highly tumorigenic. The ability to self-renew was confirmed by the observation that ALDH+CD44+Lin− cells sorted from human HNSCC formed more spheroids (orospheres) in 3-D agarose matrices or ultra-low attachment plates than controls and were serially passaged in vivo. We observed that approximately 80% of the CSC were located in close proximity (within 100-µm radius) of blood vessels in human tumors, suggesting the existence of perivascular niches in HNSCC. In vitro studies demonstrated that endothelial cell-secreted factors promoted self-renewal of CSC, as demonstrated by the upregulation of Bmi-1 expression and the increase in the number of orospheres as compared to controls. Notably, selective ablation of tumor-associated endothelial cells stably transduced with a caspase-based artificial death switch (iCaspase-9) caused a marked reduction in the fraction of CSC in xenograft tumors. Collectively, these findings indicate that endothelial cell-initiated signaling can enhance the survival and self-renewal of head and neck cancer stem cells.

Purpose

To investigate the effect of a metronomic (low dose, high frequency) small molecule inhibitor of Bcl-2 (TW-37) in combination with radiotherapy on microvascular endothelial cells in vitro and in tumor angiogenesis in vivo.

Methods and materials

Primary human dermal microvascular endothelial cells (HDMEC) were exposed to ionizing radiation and/or TW-37, and colony formation as well as capillary sprouting in 3-D collagen matrices, was evaluated. Xenografts vascularized with human blood vessels were engineered by co-transplantation of human squamous cell carcinoma cells (OSCC3) and HDMEC seeded in highly porous biodegradable scaffolds into the subcutaneous space of immunodeficient mice. Mice were treated with metronomic TW-37 and/or radiation, and tumor growth was evaluated.

Results

Low dose TW-37 sensitized primary endothelial cells to radiation-induced inhibition of colony formation. Low dose TW-37 or radiation partially inhibited endothelial cell sprout formation, while in combination these therapies abrogated new sprouting. Combination of metronomic TW-37 and low dose radiation inhibited tumor growth and resulted in significant increase in time to failure as compared to controls, whereas single agents did not. Notably, histopathological analysis revealed that tumors treated with TW-37 (with or without radiation) are more differentiated and showed more cohesive invasive fronts, which is consistent with less aggressive phenotype.

Conclusions

These results demonstrate that metronomic TW-37 potentiates the anti-tumor effects of radiotherapy, and suggest that patients with head and neck cancer might benefit from the combination of small molecule inhibitor of Bcl-2 and radiation therapy.

There is increasing demand for biomedical implants to correct skeletal defects caused by trauma, disease, or genetic disorder. In this study, the MG-63 cells were grown on metals coated with ordered and disordered fluorapatite (FA) crystal surfaces to study the biocompatibility, initial cellular response, and the underlying mechanisms during this process. The long-term growth and mineralization of the cells were also investigated. After 3 days, the cell numbers on etched metal surface are significantly higher than those on the ordered and disordered FA surfaces, but the initial adherence of a greater number of cells did not lead to earlier mineral formation at the cell–implant interface. Of the 84 cell adhesion and matrix-focused pathway genes, an up- or down-regulation of a total of 15 genes such as integrin molecules, integrin alpha M and integrin alpha 7 and 8 was noted, suggesting a modulating effect on these adhesion molecules by the ordered FA surface compared with the disordered. Osteocalcin expression and the mineral nodule formation are most evident on the FA surfaces after osteogenic induction (OI) for 7 weeks. The binding of the ordered FA surfaces to the metal, with and without OI, was significantly higher than that of the disordered FA surfaces with OI. Most significantly, even without the OI supplement, the MG-63 cells grown on FA crystal surfaces start to differentiate and mineralize, suggesting that the FA crystal could be a simple and bioactive implant coating material.

Purpose

Thrombospondin 1 (TSP1) is a matrix glycoprotein that regulates cell adhesion, migration, and proliferation, and is a natural inhibitor of angiogenesis. Recent evidence suggests that TSP1 is a major physiologic activator of latent transforming growth factor-β1 (TGF-β1), and that TGF-β1 is important for wound healing. The purpose of this study was to examine whether excisional wound healing in TSP1-deficient mice is compromised as a result of deficient TGF-β1 activation.

Materials and Methods

Punch wounds were made on the dorsum of TSP1 deficient and wild-type mice and the area of granulation tissue, number of microvessels, and inflammatory cell infiltration was evaluated over a period of 28 days.

Results

TSP1 deficient mice showed impaired wound healing with persistent granulation tissue, decreased collagen content over time, and delayed arrival of macrophages compared to wild-type littermates. The number of microvessels in wounds of TSP1-deficient mice was approximately two-fold greater than in wild-type littermates 10 days after injury. Topical application of TSP1, or KRFK (a peptide derived from TSP1 that activates latent TGF-β1), to wounds of TSP1-deficient mice rescued wild-type patterns of wound repair and partially recovered local levels of TGF-β1 expression. Topical application of anti-TGF-β neutralizing antibody impaired the ability of KRFK to rescue normal patterns of wound neovascularization in TSP1-deficient mice.

Conclusions

These results demonstrate that TSP1 plays a key role in the orchestration of wound healing, and that TSP1-mediated activation of local TGF-β1 is an important step in this process.

Laser capture microdissection (LCM) allows for the microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. According to the recent development of the LCM technologies and methodologies, the LCM has been used in various kinds of tissue specimens in dental research. For example, the real-time polymerase-chain reaction (PCR) can be performed from the formaldehyde-fixed, paraffin-embedded, and immunostained sections. Thus, the advance of immuno-LCM method allows us to improve the validity of molecular biological analysis and to get more accurate diagnosis in pathological field in contrast to conventional LCM. This paper is focused on the presentation and discussion of the existing literature that covers the fields of RNA analysis following LCM in dentistry.

Summary

Two genes are considered synthetic lethal if mutation of either alone allows cell viability, while simultaneous mutation leads to cell death. A synthetic lethal screen unveiled the dependency of Wnt/B-catenin-addicted colorectal cancer cells on vascular endothelial growth factor receptor-1 kinase activity and suggested a novel therapeutic approach for this malignancy.

Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by RT-PCR and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4°C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. GAPDH and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM.

Vascular endothelial growth factor (VEGF) signaling is critical for tumor angiogenesis. However, therapies based on the inhibition of VEGF receptors have shown modest results in patients with cancer. Surprisingly little is known about mechanisms underlying the regulation of VEGFR1 and VEGFR2 expression, the main targets of these drugs. Here, analysis of tissue microarrays revealed an inversely reciprocal pattern of VEGF receptor regulation in the endothelium of human squamous cell carcinomas (high VEGFR1, low VEGFR2), as compared to the endothelium of control tissues (low VEGFR1, high VEGFR2). Mechanistic studies demonstrated that VEGF signals through the Akt/ERK pathway to inhibit constitutive ubiquitination and induce rapid VEGFR1 accumulation in endothelial cells. Surprisingly, VEGFR1 is primarily localized in the nucleus of endothelial cells. In contrast, VEGF signals through the JNK/c-Jun pathway to induce endocytosis, nuclear translocation, and downregulation of VEGFR2 via ubiquitination. VEGFR1 signaling is required for endothelial cell survival, while VEGFR2 regulates capillary tube formation. Notably, the antiangiogenic effect of Bevacizumab (anti-VEGF antibody) requires the normalization of VEGFR1 and VEGFR2 levels in human squamous cell carcinomas vascularized with human blood vessels in immunodeficient mice. Collectively, this work demonstrate that VEGF-induced angiogenesis requires the inverse regulation of VEGFR1 and VEGFR2 in tumor-associated endothelial cells.

Members of the Bcl-2 family play a major role in the pathobiology of head and neck cancer. We have shown that Bcl-2 orchestrates a crosstalk between tumor cells and endothelial cells that have a direct impact on the progression of head and neck squamous cell carcinoma (HNSCC). Notably, Bcl-2 is significantly upregulated in the tumor associated endothelial cells as compared to the endothelial cells of normal oral mucosa in patients with HNSCC. Here, we evaluated the effect of TW-37, a small molecule inhibitor of Bcl-2, on the cell cycle and survival of endothelial cells and HNSCC and on the progression of xenografted tumors. TW-37 has an IC50 of 1.1 µM for primary human endothelial cells and averaged 0.3 µM for head and neck cancer cells (OSCC3, UM-SCC-1, UM-SCC-74A). Combination of TW-37 and cisplatin showed enhanced cytotoxic effects for endothelial cells and HNSCC in vitro, as compared with single drug treatment . Notably, while cisplatin led to an expected G2/M cell cycle arrest, TW-37 mediated an “S” phase cell cycle arrest in endothelial cells and in HNSCC. In vivo, TW-37 inhibited tumor angiogenesis and induced tumor apoptosis without significant systemic toxicities. Combination of TW-37 and cisplatin enhanced the time to tumor failure (i.e. 4-fold increase in tumor volume), as compared to either drug given separately. Collectively, these data reveal that therapeutic inhibition of Bcl-2 function with TW-37 is sufficient to arrest endothelial cells and HNSCC in the “S” phase of cell cycle, and to inhibit head and neck tumor angiogenesis.

Antiangiogenic therapies have shown varying results partly because each tumor type secretes a distinct panel of angiogenic factors to sustain its own microvascular network. In addition, recent evidence demonstrated that tumors develop resistance to antiangiogenic therapy by turning on alternate angiogenic pathways when one pathway is therapeutically inhibited. Here, we test the hypothesis that expression of a caspase-based artificial death switch in tumor-associated endothelial cells will disrupt tumor blood vessels and slowdown tumor progression irrespective of tumor type. Adenoviral vectors expressing inducible Caspase-9 (iCaspase-9) under transcriptional regulation with the endothelial cell specific VEGFR2 promoter (Ad-hVEGFR2-iCaspase-9) induced apoptosis of proliferating human dermal microvascular endothelial cells (HDMEC), but not human tumor cells (UM-SCC-17B, head and neck squamous cell carcinoma; HepG2, hepatocellular carcinoma; PC-3, prostate adenocarcinoma; SLK, Kaposi’s sarcoma; MCF-7, breast adenocarcinoma). Notably, apoptosis was dependent upon activation of iCaspase-9 with the dimerizer drug AP20187. Local delivery of Ad-hVEGFR2-iCaspase-9 followed by intraperitoneal injection of AP20187 ablated tumor microvessels and inhibited xenografted tumor growth in all tumor models evaluated here. We conclude that a cancer gene therapy strategy based on a transcriptionally targeted viral vector expressing an inducible caspase allows for selective and controlled ablation of microvessels of histopathologically diverse tumor types.

It is well known that cancer cells secrete angiogenic factors to recruit and sustain tumor vascular networks. However, little is known about the effect of endothelial cell-secreted factors on the phenotype and behavior of tumor cells. The hypothesis underlying this study is that endothelial cells initiate signaling pathways that enhance tumor cell survival and migration. Here, we observed that soluble mediators from primary human dermal microvascular endothelial cells induce phosphorylation of signal transducer and activator of transcription 3 (STAT3), Akt, and extracellular signal-regulated kinase (ERK) in a panel of head and neck squamous cell carcinoma (HNSCC) cells (OSCC-3, UM-SCC-1, UM-SCC-17B, UM-SCC-74A). Gene expression analysis demonstrated that interleukin-6 (IL- 6), interleukin-8 (CXCL8), and epidermal growth factor (EGF) are upregulated in endothelial cells cocultured with HNSCC. Blockade of endothelial cell-derived IL-6, CXCL8, or EGF by gene silencing or neutralizing antibodies inhibited phosphorylation of STAT3, Akt, and ERK in tumor cells, respectively. Notably, activation of STAT3, Akt, and ERK by endothelial cells enhanced migration and inhibited anoikis of tumor cells. We have previously demonstrated that Bcl-2 is upregulated in tumor microvessels in patients with HNSCC. Here, we observed that Bcl-2 signaling induces expression of IL-6, CXCL8, and EGF, providing a mechanism for the upregulation of these cytokines in tumor-associated endothelial cells. This study expands the contribution of endothelial cells to the pathobiology of tumor cells. It unveils a new mechanism in which endothelial cells function as initiators of molecular crosstalks that enhance survival and migration of tumor cells.

Endothelial cell apoptosis plays a critical role in the disruption of blood vessels mediated by natural inhibitors of angiogenesis and by anti-vascular drugs. However, the proportion of endothelial cells required to mediate a significant decrease in microvessel density is unknown. A system based on an inducible caspase (iCaspase-9) offers a unique opportunity to address this question. The dimerizer drug AP20187 induces apoptosis of human dermal microvascular endothelial cells stably transduced with iCaspase-9 (HDMEC-iCaspase-9), but not control cells (HDMEC-LXSN). Here, we generated blood vessels containing several HDMEC-iCaspase-9:HDMEC-LXSN ratios, and developed a mathematical modeling involving a system of differential equations to evaluate experimentally inaccessible ratios. A significant decrease in capillary sprouts was observed when at least 17% of the endothelial cells underwent apoptosis in vitro. Exposure to vascular endothelial growth factor (VEGF165) did not prevent apoptosis of HDMEC-iCaspase-9, but increased the apoptotic requirement for sprout disruption. In vivo experiments showed the requirement of at least 22% apoptotic endothelial cells for a significant decrease in microvascular density. The combined use of biological experimentation with mathematical modeling allowed us to conclude that apoptosis of a relatively small proportion of endothelial cells is sufficient to mediate a significant decrease in microvessel density.

Field cancerization involves the lateral spread of premalignant or malignant disease and contributes to the recurrence of head and neck tumors. The overall hypothesis underlying this work is that endothelial cells actively participate in tumor cell invasion by secreting chemokines and creating a chemotactic gradient for tumor cells. Here we demonstrate that conditioned medium from head and neck tumor cells enhance Bcl-2 expression in neovascular endothelial cells. Oral squamous cell carcinoma-3 (OSCC3) and Kaposi's sarcoma (SLK) show enhanced invasiveness when cocultured with pools of human dermal microvascular endothelial cells stably expressing Bcl-2 (HDMEC-Bcl-2), compared to cocultures with empty vector controls (HDMEC-LXSN). Xenografted OSCC3 tumors vascularized with HDMEC-Bcl-2 presented higher local invasion than OSCC3 tumors vascularized with control HDMEC-LXSN. CXCL1 and CXCL8 were upregulated in primary endothelial cells exposed to vascular endothelial growth factor (VEGF), as well as in HDMEC-Bcl-2. Notably, blockade of CXCR2 signaling, but not CXCR1, inhibited OSCC3 and SLK invasion toward endothelial cells. These data demonstrate that CXC chemokines secreted by endothelial cells induce tumor cell invasion and suggest that the process of lateral spread of tumor cells observed in field cancerization is guided by chemotactic signals that originated from endothelial cells.

Background

The microenvironment surrounding cells can exert multiple effects on their biological responses. In particular the extracellular matrix surrounding cells can profoundly influence their behavior. It has been shown that the extracellular matrix composition in tumors is vastly different than that found in normal tissue with increased amounts of certain matrices such as collagen I. It has been previously demonstrated that VEGF stimulation of endothelial cells growing on type I collagen results in the induction of bcl-2 expression and enhanced endothelial cell survival. We sought to investigate whether this increased endothelial cell survival resulted in the failure of angiostatic molecules to inhibit angiogenesis.

Results

We now demonstrate that VEGF-induced survival on collagen I impairs the ability of three known angiostatic molecules, TSP-1, IP-10 and endostatin to inhibit endothelial cell proliferation. Apoptosis of endothelial cells, growing on collagen I, induced by TSP-1 and IP-10 was also inhibited following VEGF stimulation. In contrast, endostatin induced apoptosis in these same cells. Further analysis determined that endostatin did not decrease the expression of bcl-2 nor did it increase activation of caspase-3 in the presence of VEGF. Alternatively, it appeared that in the presence of VEGF, endostatin induced the activation of caspase-8 in endothelial cells grown on collagen I. Furthermore, only endostatin had the ability to inhibit VEGF-induced sprout formation in collagen I gels.

Conclusion

These data suggest that TSP-1, IP-10 and endostatin inhibit endothelial cells via different mechanisms and that only endostatin is effective in inhibiting angiogenic activities in the presence of collagen I. Our results suggest that the efficacy of angiostatic treatments may be impaired depending on the context of the extracellular matrix within the tumor environment and thus could impede the efficacy of angiostatic therapies.