Stem cells play a critical role in development and in tissue regeneration. The dental pulp contains a small sub-population of stem cells that are involved in the response of the pulp to caries progression. Specifically, stem cells replace odontoblasts that have undergone cell death as a consequence of the cariogenic challenge. Stem cells also secrete factors that have the potential to enhance pulp vascularization and provide the oxygen and nutrients required for the dentinogenic response that is typically observed in teeth with deep caries. However, the same angiogenic factors that are required for dentin regeneration may ultimately contribute to the demise of the pulp by enhancing vascular permeability and interstitial pressure. Recent studies focused on the biology of dental pulp stem cells revealed that the multipotency and angiogenic capacity of these cells could be exploited therapeutically in dental pulp tissue engineering. Collectively, these findings suggest new treatment paradigms in the field of Endodontics. The goal of this review is to discuss the potential impact of dental pulp stem cells to Regenerative Endodontics.
Tissue Engineering; Dental pulp; Odontoblasts; Angiogenesis; Differentiation
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.
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.
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.
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.
Head and neck squamous cell carcinoma; Perivascular niche; Angiogenesis; Tumor microenvironment; Metastasis
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.
Oral cancer; Tumorigenesis; Epithelial-mesenchymal transition; EMT; Self-renewal; Stemness; Perivascular niche; Squamous cell carcinoma; Angiogenesis
Neovascularization is a limiting factor in tumor growth and progression. It is well known that changes in the tumor microenvironment, such as hypoxia and glucose deprivation (GD), can induce VEGF production. However, the mechanism linking GD to tumor growth and angiogenesis is unclear. We hypothesize that GD induces the angiogenic switch in tumors through activation of the unfolded protein response (UPR). We report that UPR activation in human tumors results in elevated expression of proangiogenic mediators and a concomitant decrease in angiogenesis inhibitors. cDNA microarray results showed that GD-induced UPR activation promoted upregulation of a number of proangiogenic mediators (VEGF, FGF2, IL6, etc.) and downregulation of several angiogenic inhibitors (THBS1, CXCL14 and CXCL10). In vitro studies revealed that partially blocking UPR signaling by silencing PERK or ATF4 significantly reduced the production of angiogenesis mediators induced by GD. However, suppressing the alpha subunit of hypoxia-inducible factors had no effect on this process. Chromatin immunoprecipitation confirmed binding of ATF4 to a regulatory site in the VEGF gene. In vivo results confirmed that knockdown of PERK in tumor cells slows down tumor growth and decreases tumor blood vessel density. Collectively, these results demonstrate that the PERK/ATF4 arm of UPR mediates the angiogenic switch and is a potential target for antiangiogenic cancer therapy.
Glucose deprivation; UPR; angiogenic switch; VEGF; THBS1
Recent studies have shown that Bcl-2 functions as a pro-angiogenic signaling molecule in addition to its well-known effect as an inhibitor of apoptosis. The discovery of AT101, a BH3-mimetic drug that is effective and well tolerated when administered orally, suggested the possibility of using a molecularly targeted drug in a metronomic regimen. Here, we generated xenograft squamous cell carcinomas (SCC) with humanized vasculature in immunodeficient mice. Mice received taxotere in combination with either daily 10 mg/kg AT101 (metronomic regimen) or weekly 70 mg/kg AT101 (bolus regimen). The effect of single drug AT101 on angiogenesis, and combination AT101/taxotere on the survival of endothelial cells and SCC cells, were also evaluated in vitro. Metronomic AT101 increased mouse survival (p=0.02), decreased tumor mitotic index (p=0.0009), and decreased tumor microvessel density (p=0.0052), as compared to bolus delivery of AT101. Notably, the substantial potentiation of the anti-tumor effect observed in the metronomic AT101 group was achieved using the same amount of drug and without significant changes in systemic toxicities. In vitro, combination of AT101 and taxotere showed additive toxicity for endothelial cells and synergistic or additive toxicity for tumor cells (SCC). Interestingly, low-dose (sub-apoptotic) concentrations of AT101 potently inhibited the angiogenic potential of endothelial cells. Taken together, these data unveiled the benefit of metronomic delivery of a molecularly targeted drug, and suggested that patients with squamous cell carcinomas might benefit from continuous administration of low dose BH3-mimetic drugs.
Developmental therapeutics; targeted therapy; angiogenesis; Bcl-2; squamous cell carcinoma
As tumors continue to grow and exceed their blood supply, nutrients become limited leading to deficiencies in amino acids (AAD), glucose (GD), and oxygen (hypoxia). These alterations result in significant changes in gene expression. While tumors have been shown to overcome the stress associated with GD or hypoxia by stimulating vascular endothelial growth factor (VEGF)-mediated angiogenesis, the role of AAD in tumor angiogenesis remains to be elucidated. We found that in human tumors, the expression of the general control non-derepressible 2 (GCN2, an AAD sensor) kinase is elevated at both protein and mRNA levels. In vitro studies revealed that VEGF expression is universally induced by AAD treatment in all five cell lines tested (five of five). This is in contrast to two other angiogenesis mediators interleukin-6 (two of five) and fibroblast growth factor 2 (two of five) that have a more restricted expression. Suppressing GCN2 expression significantly decreased AAD-induced VEGF expression. Silencing activating transcription factor 4 (ATF4), a downstream transcription factor of the GCN2 signaling pathway, is also associated with strong inhibition of AAD-induced VEGF expression. PKR-like kinase, the key player in GD-induced unfolded protein response is not involved in this process. In vivo xenograft tumor studies in nonobese diabetic/severe combined immunodeficient mice confirmed that knockdown of GCN2 in tumor cells retards tumor growth and decreases tumor blood vessel density. Our results reveal that the GCN2/ATF4 pathway promotes tumor growth and angiogenesis through AAD-mediated VEGF expression and, thus, is a potential target in cancer therapy.
Patients with mucoepidermoid carcinoma exhibit poor long-term prognosis due to the lack of therapeutic strategies that effectively block tumor progression. We have previously characterized the Ms cells as a highly metastatic mucoepidermoid carcinoma cell line that expresses high levels of transforming growth factor β1 (TGF-β1). Here, we studied the effect of suppressing TGF-β1 by RNA silencing on the invasive and metastatic potential of mucoepidermoid carcinoma.
Cell motility, substratum adhesion and transmembrane invasion were estimated by migration, matrigel adhesion and metrigel invasion assay. Matrix metalloproteinase (MMP)-2 and MMP-9 activity were determined using gelatin gel zymography. Mice lung metastatic model was used to test the matastatic ability. Lung metastatic tumors were experimentally induced by mice tail vein inoculation of cancer cells.
TGF-β1 silencing inhibits cell motility, substratum adhesion and transmembrane invasion. In vivo, a significant decrease in lung metastasis was observed when mice received tail vein injections of TGF-β1-silenced mucoepidermoid carcinoma cells, as compared to controls.
These results unveil a critical role for TGF-β1 in the progression of mucoepidermoid carcinomas, and suggest that patients with this malignancy may benefit from therapeutic inhibition of the effectors of the TGF-β1 pathway.
Tumor progression; Metastasis; Salivary gland cancer; Extracellular matrix; Matrix metalloproteinase
In this study, the effect of ordered rod-like FA coatings of metal discs on adipose-derived stem cell (ASC)’s growth, differentiation and mineralization was studied in vitro; and their mineral inductive effects in vivo. After 3 and 7 days, the cell number on the metal surfaces was significantly higher than those on the ordered and disordered FA surfaces. However, after 4 weeks much greater amounts of mineral formation was induced on the two FA surfaces with and even without osteogenesis induction. The osteogenic profiles showed the up regulation of a set of pro-osteogenic transcripts and bone mineralization phenotypic markers when the ASCs were grown on FA surfaces compared to metal surfaces at 7 and 21 days. In addition to BMP and TGFβ signaling pathways, EGF and FGF pathways also appeared to be involved in ASC differentiation and mineralization. In vivo studies showed accelerated and enhanced mineralized tissue formation integrated within ordered FA coatings. After 5 weeks, over 80 % of the ordered FA coating was integrated with a mineralized tissue layer covering the implants. Both the intrinsic properties of the FA crystals and the topography of the FA coating appeared to dominate the cell differentiation and mineralization process.
Fluorapatite; differentiation; mineralization; gene profile; stem cells
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.
Cancer; angiogenesis; promoter; tumor microenvironment; review
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.
Developmental therapeutics; Tumor; Neovascularization; Pharmacology
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.
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.
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.
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.
Oral cancer; Angiogenesis; Lymphangiogenesis; Biomarkers; VEGF; Head and Neck Cancer
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.
angiogenesis; scaffold; endothelial; mural cell; microcirculation; multiphoton
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.
Tumor microenvironment; perivascular niche; anti-angiogenic therapy; squamous cell carcinoma; stemness
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.
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.
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.
Developmental therapeutics; Radiotherapy; Head and Neck Cancer; Apoptosis; Neovascularization
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.
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.
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.
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.
angiogenesis; neovascularization; oral cavity; apoptosis
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.
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.
Synthetic lethality; Cell signaling; Receptor Tyrosine Kinase; Developmental Therapeutics; Gene Silencing
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.
laser capture microdissection; endothelial cells; gene expression; tumor angiogenesis
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.
Angiogenesis; Apoptosis; Receptor Tyrosine Kinase; Differentiation; Tumor Microenvironment
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.
apoptosis; neovascularization; cisplatin; cancer
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.
angiogenesis; neovascularization; apoptosis; suicide gene
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.