Intratumor IL-17-Positive Mast Cells Are the Major Source of the IL-17 That Is Predictive of Survival in Gastric Cancer Patients

Interleukin-17 (IL-17) is prevalent in tumor tissue and suppresses effective anti-tumor immune responses. However, the source of the increased tumor-infiltrating IL-17 and its contribution to tumor progression in human gastric cancer remain poorly understood. In this study, we enrolled 112 gastric cancer patients, immunofluorescence was used to evaluate the colocalization of CD3, CD4, CD56, CD20, CD68, and mast cell tryptase (MCT) with IL-17. Immunohistochemistry was used to evaluate the distribution of microvessel density (CD34), CD66b⁺, CD68⁺, and FoxP3⁺ cells in different microanatomical areas. Prognostic value was determined by Kaplan-Meier analysis and a Cox regression model. The results showed that mast cells, but not T cells or macrophages, were the predominant cell type producing IL-17 in gastric cancer. Significant positive correlations were detected between densities of mast cell-derived IL-17 and microvessels, neutrophils, and regulatory T cells (Tregs). Futhermore, we found that the majority of vascular endothelial cells expressing Interleukin-17 receptor (IL-17R). Kaplan-Meier analysis revealed that increasing intratumor infiltrated mast cells and IL-17⁺ cells, as well as MCT⁺ IL-17⁺ cells, were significantly associated with worse overall survival. These findings indicated that mast cells were the major source of IL-17 in gastric cancer, and intratumor IL-17 infiltration may have promoted tumor progression by enhancing angiogenesis in the tumor microenvironment through the axis of IL-17/IL-17R. IL-17-positive mast cells showed a prognostic factor in gastric cancer, indicating that immunotherapy targeting mast cells might be an effective strategy to control intratumor IL-17 infiltration, and consequently reverse immunosuppression in the tumor microenvironment, facilitating cancer immunotherapy.     

A protective role by interleukin-17F in colon tumorigenesis

Interleukin-17F (IL-17F), produced by Th17 cells and other immune cells, is a member of IL-17 cytokine family with highest homology to IL-17A. IL-17F has been shown to have multiple functions in inflammatory responses. While IL-17A plays important roles in cancer development, the function of IL-17F in tumorigenesis has not yet been elucidated. In the current study, we found that IL-17F is expressed in normal human colonic epithelial cells, but this expression is greatly decreased in colon cancer tissues. To examine the roles of IL-17F in colon cancer, we have used IL-17F over-expressing colon cancer cell lines and IL-17F-deficient mice. Our data showed decreased tumor growth of IL-17F-transfected HCT116 cells comparing to mock transfectants when transplanted in nude mice. Conversely, there were increased colonic tumor numbers and tumor areas in Il-17f(-/-) mice than those from wild-type controls after colon cancer induction. These results indicate that IL-17F plays an inhibitory role in colon tumorigenesis in vivo. In IL-17F over-expressing tumors, there was no significant change in leukocyte infiltration; instead, we found decreased VEGF levels and CD31(+) cells. While the VEGF levels were increased in the colon tissues of Il-17f(-/-) mice with colon cancer. Together, our findings demonstrate a protective role for IL-17F in colon cancer development, possibly via inhibiting tumor angiogenesis.     

Expression and Biological Significance of Leptin,Leptin Receptor,VEGF, and CD34 in Colorectal Carcinoma

To investigate the expression and biological significance of Leptin, Leptin receptor, Vascular Endothelial Growth Factor (VEGF), and CD34 protein in colorectal carcinoma tissues. The expression of Leptin, Leptin receptor, VEGF, and CD34 was detected in 68 cases of colorectal carcinoma tissues, paired para-carcinoma tissues and normal colorectal tissues by Immunohistochemical SP Method. The results and related clinicopathological data were analyzed. The positive rate of Leptin, Leptin receptor, and VEGF was significantly higher in colorectal carcinoma tissues than that in paired para-carcinoma tissues and normal colorectal tissues. The expression of Leptin, Leptin receptor, and VEGF was correlated with grade of tumor differentiation, depth of bowel wall invasion, lymph node metastasis, Dukes stage, distant metastasis, and lympho/vascular tumor embolization. Microvessel density (MVD) value in colorectal carcinoma was significantly higher than that in para-carcinoma tissues and normal colorectal tissues, and the density in para-carcinoma tissues was higher than that in normal colorectal tissues. The expression of Leptin, Leptin receptor, VEGF, and MVD value in colorectal carcinoma was positively correlated. In conclusion, microvessel density value is an important index of the growth, invasion, and metastasis of colorectal carcinoma. The binding of Leptin and Leptin receptor promotes the proliferation of colorectal carcinoma cells. The synergy between Leptin and VEGF accelerates the angiogenesis in colorectal carcinoma and accelerates the invasion and metastasis of the tumor cells.     

CCR6 promotes tumor angiogenesis via the AKT/NF-κB/VEGF pathway in colorectal cancer

Chemokines and chemokine receptors play an important role in tumorigenesis. Angiogenesis is a vital part of the occurrence, development and metastasis of cancer. CCR6 is an important factor during tumor progression; however, its function in tumor angiogenesis is not fully understood. In our study, we found that CCR6 was significantly overexpressed in colorectal cancer (CRC) tissues and predicted a poor prognosis in CRC patients. We then verified the function of CCR6 on tumor angiogenesis in vivo and in vitro. We observed that silencing CCR6 could decrease angiogenesis by inhibiting the proliferation and migration of human umbilical vein endothelial cells (HUVECs), whereas overexpression of CCR6 can promote angiogenesis. Additionally, we investigated the molecular mechanisms and demonstrated that activation of the AKT/NF-κB pathway maybe involved in CCR6-mediated tumor angiogenesis, which was able to promote the secretion of vascular endothelial growth factor A (VEGF-A). In conclusion, CCR6 facilitates tumor angiogenesis via the AKT/NF-κB/VEGF pathway in colorectal cancer. CCR6 inhibition may be a novel option for anti-vascular treatment in CRC.     

The accumulation and prognosis value of tumor infiltrating IL-17 producing cells in esophageal squamous cell carcinoma

Background

The role of IL-17 producing cells in tumors is controversial. In the present study, we investigated the prognostic value of measuring tumor-infiltrating IL-17 producing cell levels in human esophageal squamous cell carcinoma (ESCC).

Methodology/Principal Findings

Immunohistochemical staining was performed to investigate the levels of IL-17+ tumor infiltrating lymphocytes (TILs), as well as CD8+ cytotoxic T lymphocytes (CTLs) and CD57+ natural killer (NK) cells from 181 ESCC patients. The prognostic value of measuring the densities of IL-17+TILs and the correlation with CTLs and NK was evaluated. IL-17 producing cells were detected in esophageal squamous cell carcinoma tissues. The IL-17 producing cells were major CD4 positive, but Foxp3 negative. The median level of IL-17+TILs was 3.90 cells/high power microscopic field (HPF). The density of IL-17 producing cells correlated negatively with T stage (P = 0.042). The higher densities of tumor infiltrating IL-17+ lymphocytes were associated with better overall survival (P = 0.031). Furthermore, we found that there were positive correlations between levels of IL-17 producing cells and the densities of CD8+cells, as well as CD57+cells (r = 0.198, P = 0.008 for CD8+ cells and r = 0.261, P<0.001 for CD57+ cells, respectively). The prognosis analysis also showed that the higher levels of CD8+ CTLs and CD57+ NK cells correlated with better overall survival of ESCC patients.

Conclusions

Our study suggests that tumor infiltrating IL-17 producing cells in ESCC patients may have protective roles in the tumor microenvironment and may be treated as a prognostic marker for ESCC patients.     

Paneling human thyroid cancer cell lines for candidate proteins for targeted anti-angiogenic therapy

Tumor angiogenesis is believed to result from an imbalance of pro- and anti-angiogenic factors, some of which are candidates for targeted therapy. Such therapy has raised hopes for patients with undifferentiated thyroid carcinomas, who are facing a grave prognosis with a survival of only months. In this study, in vivo growth of xenografted human thyroid carcinomas unexpectedly responded quite differently to neutralizing anti-vascular endothelial growth factor (VEGF) antibody. In particular, lasting inhibition as well as accelerated growth occurred after treatment. Consequently, a panel of anti-angiogenic factors was addressed in a representative sample of thyroid carcinoma lines. VEGF, fibroblast growth factor (FGF-2), and endostatin were demonstrated by Western blotting and EIA, whereas PDGF-A, PDGF-B, and IL-6 were negative. Quantification of VEGF, FGF-2, and endostatin revealed a wide range of concentrations from 500 to 4,200 pg/ml VEGF, 5 to 60 pg/ml FGF-2, and 50 to 300 pg/ml endostatin, not related to a particular histologic thyroid carcinoma background. Angiostatin (kringles 1-3) was detected in all, but one of the cell lines. Finally, aaATIII was confirmed in FTC133 cells. These data highlight the complex regulation of angiogenesis in thyroid carcinoma cell lines and suggest that the array of angiogenic factors differs markedly between individual cell lines. For the first time, angiostatin, endostatin, and possibly also aaATIII are identified as novel candidate regulators of angiogenesis in thyroid carcinoma cells.     

Abnormally enhanced blood concentrations of vascular endothelial growth factor (VEGF) in metastatic cancer patients and their relation to circulating dendritic cells, IL-12 and endothelin-1

Elevated VEGF blood concentrations have been proven to be associated with poor prognosis in human neoplasms. This finding is generally explained as a consequence of the potential angiogenic properties of VEGF itself. However, preliminary experimental studies suggest that VEGF, in addition to its angiogenic activity, may also play an immunosuppressant role by inhibiting dendritic cell (DC) maturation. The present study was performed to analyze blood levels of VEGF in cancer patients in relation to those of another potentially angiogenic tumor growth factor, endothelin-1 (ET-1), and to the absolute number of circulating immature and mature DC, and serum levels of the best known antitumor cytokine, IL-12. The study was performed in 100 healthy controls and in 80 solid tumor patients (colorectal cancer: 24; gastric cancer: 17; cancer of pancreas: 4; lung cancer: 13; breast cancer: 11; renal cell cancer: 6; gynecologic tumors: 5), 48 of whom showed distant organ metastases. In each patient, we have evaluated serum concentrations of VEGF-165, total VEGF, ET-1, IL-12 and the circulating number of immature (CD123+) and mature (CD11c+) DC. Mean serum levels of VEGF-165 were significantly higher in metastatic patients than in controls or in non-metastatic patients, whereas the total amounts of VEGF were not significantly higher. Moreover, it has been observed that patients with abnormally elevated blood concentrations of VEGF-165 showed significantly lower mean values of immature DC, mature DC and IL-12 and significantly higher mean levels of ET-1 than those with normal concentrations. This study, by confirming that advanced neoplastic disease may be associated with increased endogenous secretion of VEGF, seems to suggest that the association between high blood levels of VEGF and poor prognosis in cancer does not depend only on VEGF-induced stimulation of the neovascularization, but also on VEGF-related immunosuppression.     

Up-regulation of miR-210 by vascular endothelial growth factor in ex vivo expanded CD34+ cells enhances cell-mediated angiogenesis

Ex vivo culture has been proposed as a means to augment and repair autologous cells in patients with chronic diseases, but the mechanisms governing improvement in cell function are not well understood. Although microRNAs (miRs) are increasingly appreciated as key regulators of cellular function, a role for these factors in CD34+ cell-mediated angiogenesis has not been elucidated. Vascular endothelial growth factor (VEGF) was previously shown to induce expression of certain miRs associated with angiogenesis in endothelial cells and promote survival and number of vascular colony forming units of haematopoietic stem cells (HSCs). We sought to evaluate the role of VEGF in expansion and angiogenic function of CD34+ cells and to identify specific miRs associated with angiogenic properties of expanded cells. Umbilical cord blood CD34+ cells were effectively expanded (18- to 22-fold) in culture medium containing stem cell factor (SCF), Flt-3 ligand (Flt-3), thrombopoietin (TPO) and interleukin-6 (IL-6) with (postEX/+VEGF) and without VEGF (postEX/noVEGF). Tube formation in matrigel assay and tissue perfusion/capillary density in mice ischaemic hindlimb were significantly improved by postEX/+VEGF cells compared with fresh CD34+ and postEX/noVEGF cells. MiR-210 expression was significantly up-regulated in postEX/+VEGF cells. MiR-210 inhibitor abrogated and 210 mimic recapitulated the pro-angiogenic effects by treatment of postEX/+VEGF and postEX/noVEGF cells respectively. Collectively, these observations highlight a critical role for VEGF in enhancing the angiogenic property of expanded cells, and identify miR-210 as a potential therapeutic target to enhance CD34+ stem cell function for the treatment of ischaemic vascular disease.     

Human CCR4+ CCR6+ Th17 cells suppress autologous CD8+ T cell responses

The role of Th17 cells in cancer patients remains unclear and controversial. In this study, we have analyzed the phenotype of in vitro primed Th17 cells and further characterized their function on the basis of CCR4 and CCR6 expression. We show a novel function for a subset of IL-17-secreting CD4(+) T cells, namely, CCR4(+)CCR6(+)Th17 cells. When cultured together, CCR4(+)CCR6(+)Th17 cells suppressed the lytic function, proliferation, and cytokine secretion of both Ag-specific and CD3/CD28/CD2-stimulated autologous CD8(+) T cells. In contrast, CCR4(-)CCR6(+) CD4(+) T cells, which also secrete IL-17, did not affect the CD8(+) T cells. Suppression of CD8(+) T cells by CCR4(+)CCR6(+)Th17 cells was partially dependent on TGF-β, because neutralization of TGF-β in cocultures reversed their suppressor function. In addition, we also found an increase in the frequency of CCR4(+)CCR6(+), but not CCR4(-)CCR6(+) Th17 cells in peripheral blood of hepatocellular carcinoma patients. Our study not only underlies the importance of analysis of subsets within Th17 cells to understand their function, but also suggests Th17 cells as yet another immune evasion mechanism in hepatocellular carcinoma. This has important implications when studying the mechanisms of carcinogenesis, as well as designing effective immunotherapy protocols for patients with cancer.     

Human Tumor Cells Induce Angiogenesis through Positive Feedback between CD147 and Insulin-Like Growth Factor-I

Tumor angiogenesis is a complex process based upon a sequence of interactions between tumor cells and endothelial cells. Previous studies have shown that CD147 was correlated with tumor angiogenesis through increasing tumor cell secretion of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). In this study, we made a three-dimensional (3D) tumor angiogenesis model using a co-culture system of human hepatocellular carcinoma cells SMMC-7721 and humanumbilical vein endothelial cells (HUVECs) in vitro. We found that CD147-expressing cancer cells could promote HUVECs to form net-like structures resembling the neo-vasculature, whereas the ability of proliferation, migration and tube formation of HUVECs was significantly decreased in tumor conditioned medium (TCM) of SMMC-7721 cells transfected with specific CD147-siRNA. Furthermore, by assaying the change of pro-angiogenic factors in TCM, we found that the inhibition of CD147 expression led to significant decrease of VEGF and insulin-like growth factor-I (IGF-I) secretion. Interestingly, we also found that IGF-I up-regulated the expression of CD147 in both tumor cells and HUVECs. These findings suggest that there is a positive feedback between CD147 and IGF-I at the tumor-endothelial interface and CD147 initiates the formation of an angiogenesis niche.     

Vascular endothelial growth factor-dependent and -independent regulation of angiogenesis

Angiogenesis, the formation of blood vessels, is essential for preparing a closed circulatory system in the body, and for supplying oxygen and nutrition to tissues. Major diseases such as cancer, rheumatoid arthritis, and atherosclerosis include pathological angiogenesis in their malignant processes, suggesting anti-angiogenic therapy to be a new strategy for suppression of diseases. However, until the 1970s, the molecular basis of angiogenesis was largely unknown. In recent decades, extensive studies have revealed a variety of angiogenic factors and their receptors, including vascular endothelial growth factor (VEGF)-VEGFRs, Angiopoietin-Tie, Ephrin-EphRs and Delta-Notch to be the major regulators of angiogenesis in vertebrates. VEGF and its receptors play a central role in physiological as well as pathological angiogenesis, and functional inhibitors of VEGF and VEGFRs such as anti-VEGF neutralizing antibody and small molecules that block the tyrosine kinase activity of VEGFRs have recently been approved for use to treat patients with colorectal, lung, renal and liver cancers. These drugs have opened a novel field of cancer therapy, i.e. anti-angiogenesis therapy. However, as yet they cannot completely cure patients, and cancer cells could become resistant to these drugs. Thus, it is important to understand further the molecular mechanisms underlying not only VEGF-VEGFR signaling but also the VEGF-independent regulation of angiogenesis, and to learn how to improve anti-angiogenesis therapy.     

(−)-Epigallocatechin gallate inhibits growth and activation of the VEGF/VEGFR axis in human colorectal cancer cells

(?)-Epigallocatechin gallate (EGCG), the major constituent of green tea, inhibits the growth of colorectal cancer cells by inhibiting the activation of various types of receptor tyrosine kinases (RTKs). The RTK vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) axis induces tumor angiogenesis in colorectal cancer. This study examined the effects of EGCG on the activity of the VEGF/VEGFR axis and the expression of hypoxia-inducible factor (HIF)-1α, which promotes angiogenesis by elevating VEGF levels, in human colorectal cancer cells. Total and phosphorylated (i.e., activated) form (p-VEGFR-2) of VEGFR-2 proteins were overexpressed in a series of human colorectal cancer cell lines. Within 3 h, EGCG caused a decrease in the expression of HIF-1α protein and VEGF, HIF-1α, insulin-like growth factor (IGF)-1, IGF-2, epidermal growth factor (EGF), and heregulin mRNAs in SW837 colorectal cancer cells, which express a constitutively activated VEGF/VEGFR axis. A decrease was also observed in the expression of VEGFR-2, p-VEGFR-2, p-IGF-1 receptor, p-ERK, and p-Akt proteins within 6 h after EGCG treatment. Drinking EGCG significantly inhibited the growth of SW837 xenografts in nude mice, and this was associated with the inhibition of the expression and activation of VEGFR-2. The consumption of EGCG also inhibited activation of ERK and Akt, both of which are downstream signaling molecules of the VEGF/VEGFR axis, and reduced the expression of VEGF mRNA in xenografts. These findings suggest that EGCG may exert, at least in part, growth-inhibitory effects on colorectal cancer cells by inhibiting the activation of the VEGF/VEGFR axis through suppressing the expression of HIF-1α and several major growth factors. EGCG may therefore be useful in the chemoprevention and/or treatment of colorectal cancer.     

Interleukin-6 increases vascular endothelial growth factor and angiogenesis in gastric carcinoma

Interleukin-6 (IL-6) is a proinflammatory cytokine associated with the disease status of gastric carcinoma (GC). Vascular endothelial growth factor (VEGF) is a potent tumor angiogenic factor in GC. In this study, we attempted to clarify whether IL-6 can regulate VEGF and angiogenesis in GC. GC samples from 54 surgical specimens were subjected to immunohistochemical examination of IL-6, VEGF, and tumor microvessels, and results showed that IL-6 was positively correlated with VEGF expression and tumor vasculature. We determined VEGF expression in four GC cell lines by ELISA, revealing that GC cells can produce significant amount of VEGF with increasing dose and duration of IL-6 stimulation. Next, a luciferase reporter gene assay was employed to determine the signaling pathway driving the VEGF promoter by IL-6, which showed that the JAK/STAT pathway is involved in the stimulation of VEGF gene expression. The effects of IL-6 on angiogenesis in vitro and in vivo were evaluated by HUVEC studies and the Matrigel plug assay, respectively. Results showed that IL-6 effectively promoted HUVEC proliferation and tube formation in vitro and Matrigel plug vascularization in vivo, primarily by inducing VEGF in GC. This study provides evidence that the multifunctional cytokine, IL-6, may induce VEGF expression which increases angiogenesis in gastric carcinogenesis.     

Interleukin-17 in acute myeloid leukemia

There are several reports that angiogenesis plays important roles in hematological malignancies including acute myeloid leukemia (AML). Human interleukin-17 (IL-17) is a proinflammatory cytokine produced by activated CD4 T cells. IL-17 plays a potential role in T cell mediated angiogenesis. The role of IL-17 in pathologic angiogenesis has not been evaluated yet. The aim of the study was to determine plasma level of IL-17 in patients with AML. IL-17 levels were measured by ELISA in plasma samples taken from 68 adult patients with AML before chemotherapy was administered. In addition 20 out of 68 patients were reanalysed after achieving complete remission (CR). Ten samples from healthy volunteers were evaluated as the control. In this study we have demonstrated that serum level of IL-17 is not elevated in AML patients. These results suggest that angiogenesis in AML is not mediated by CD4 T cells. To our knowledge this is the first report about IL-17 serum level in acute leukemias. We are currently evaluating IL-17 levels in others haematological malignancies.     

Changes in circulating dendritic cells and IL-12 in relation to the angiogenic factor VEGF during IL-2 immunotherapy of metastatic renal cell cancer

Angiogenesis and immunosuppression are the main biological mechanisms responsible for cancer progression. Moreover, recent observations suggesting a negative influence of angiogenesis on anticancer immunity have shown that some angiogenic factors, such as VEGF, may induce immunosuppression. In addition, the evidence of abnormally high blood levels of VEGF has been proven to be associated with resistance to IL-2 immunotherapy. The present study was performed to establish a possible relation ship between the efficacy of IL-2 cancer immunotherapy and changes in circulating levels of VEGF, IL-12, mature and immature dendritic cells (DC). The study included 25 metastatic renal cell cancer patients who underwent subcutaneous low-dose IL-2 immunotherapy (6 MIU/day for 6 days/week for 4 weeks). Immature and mature DCs were identified as CD123+ and CD11c+ cells, respectively. The clinical response consisted of partial response (PR) in five, stable disease (SD) in 11 and progressive disease (PD) in the remaining nine patients. The mean IL-12 levels observed during IL-2 immunotherapy were significantly higher in patients with PR or SD than in those with PD, whereas the mean VEGF concentrations were significantly higher in patients who had PD than in those with PR or SD. Finally, a significant increase in the mean number of circulating mature DCs occurred only in patients with PR or SD, whereas no significant change was seen in patients with PD. By contrast, no significant change was observed in the mean number of immature DCs. This study shows that the efficacy of IL-2 immunotherapy is associated with a significant increase in circulating mature DCs and IL-12, without any concomitant increase in VEGF concentrations. Further studies will be required to better define the relationship between activation of anticancer immunity and control of angiogenesis-related mechanisms.     

The effects of surgery, with or without rhGM-CSF, on the angiogenic profile of patients treated for colorectal carcinoma

Wound healing is a process with immunological and angiogenic aspects. rhGM-CSF is known to stimulate the immune system and angiogenesis via multiple pathways. In this study we investigated the combined effects of surgery, with or without rhGM-CSF, on angiogenic parameters in patients with a colorectal carcinoma. In this phase II randomized, placebo-controlled trial, 16 patients were assigned to perioperative rhGM-CSF (2.8 microg/kg body weight) treatment or saline. Patients received subcutaneous injections from three days before surgery until four days after. IL-6, VEGF, endostatin and angiostatin levels were measured perioperatively. rhGM-CSF enhanced the production of IL-6 and VEGF, but had no effect on the antiangiogenic agents endostatin and angiostatin. Surgery induced a transient decrease of endostatin. Two types of angiostatin (kringle 1-3 and kringle 1-4) became visible postoperatively. We conclude that this study demonstrated the immediate initiation of angiogenesis postoperatively, reflected by the increase of VEGF and a transient decrease of endostatin, followed by the appearance of two angiostatin bands, which confirms physiological wound healing in these cancer patients.     

Biological response modifiers in cancer

We have seen a surge in the use of immunotherapy for the treatment of cancer. Biological response modifiers can act passively by enhancing the immunologic response to tumor cells or actively by altering the differentiation/growth of tumor cells. Active immunotherapy with cytokines such as interferons (IFNs) and interleukins (IL-2) is a form of nonspecific active immune stimulation. The use of IL-2 has recently been approved by the United States Food and Drug Administration (FDA) for the treatment of renal cell carcinoma and metastatic colorectal cancer. Considerable success has been achieved with the use of immunotherapy, especially in the area of passive immunotherapy using monoclonal antibodies--in particular, radiolabeled monoclonal antibodies. In addition to the various monoclonal antibodies that have been used in clinical trials, other strategies such as the use of antiangiogenic agents and matrix metalloprotease inhibitors (MMPIs) have also met with some success. Recently, the FDA approved bevacizumab, an anti-vascular endothelial growth factor (VEGF) agent, for the treatment of metastatic melanoma. This review also sheds light on the various angiogenesis inhibitors in clinical trials, the increasing use of thalidomide in cancer, and the upcoming potential cancer vaccines designed to activate cell-mediated immune responses against tumor antigens.     

Model of angiogenesis in mice with severe combined immunodeficiency (SCID) and xenoengrafted with Epstein-Barr virus-transformed B cells

Xenoengraftment of human cells in mice with severe combined immunodeficiency (SCID) has been used as a model system to study the mechanisms of B-cell lymphomagenesis. In the study reported here, we determined that SCID mice can also be used as a model to study angiogenesis in B-cell lymphomas. The C.B-17 scid/scid mice were xenotransplanted with Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL), and we determined whether CD31, a marker found on endothelial cells, was detected in the human B-cell lymphomas that developed in these mice. Microvessel formation was identified by use of immunohistochemical staining for CD31. To assess possible mechanisms of angiogenic stimulus, we analyzed the expression of interleukin 8 (IL-8), a chemokine documented to promote angiogenesis, in non-small-cell lung cancer and bronchogenic carcinomas. We observed that a panel of LCL and LCL-lymphomas expressed IL-8 mRNA and protein. Neutralization of IL-8, however, did not inhibit lymphomagenesis, suggesting that IL-8 is not essential for angiogenesis in this model. To examine other parameters of angiogenesis, we identified expression of vascular endothelial growth factor in the lymphomas. These data suggest that angiogenesis accompanies EBV-associated B-cell lymphoma development, but IL-8 is not essential for this process. Thus, the SCID mouse model is amenable to testing of anti-angiogenic factors.     

Hypoxia-Induced Aggressiveness of Pancreatic Cancer Cells Is Due to Increased Expression of VEGF,IL-6 and miR-21, Which Can Be Attenuated by CDF Treatment

Hypoxia is known to play critical roles in cell survival, angiogenesis, tumor invasion, and metastasis. Hypoxia mediated over-expression of hypoxia-inducible factor (HIF) has been shown to be associated with therapeutic resistance, and contributes to poor prognosis of cancer patients. Emerging evidence suggest that hypoxia and HIF pathways contributes to the acquisition of epithelial-to-mesenchymal transition (EMT), maintenance of cancer stem cell (CSC) functions, and also maintains the vicious cycle of inflammation-all which lead to therapeutic resistance. However, the precise molecular mechanism(s) by which hypoxia/HIF drives these events are not fully understood. Here, we show, for the first time, that hypoxia leads to increased expression of VEGF, IL-6, and CSC signature genes Nanog, Oct4 and EZH2 consistent with increased cell migration/invasion and angiogenesis, and the formation of pancreatospheres, concomitant with increased expression of miR-21 and miR-210 in human pancreatic cancer (PC) cells. The treatment of PC cells with CDF, a novel synthetic compound inhibited the production of VEGF and IL-6, and down-regulated the expression of Nanog, Oct4, EZH2 mRNAs, as well as miR-21 and miR-210 under hypoxia. CDF also led to decreased cell migration/invasion, angiogenesis, and formation of pancreatospheres under hypoxia. Moreover, CDF decreased gene expression of miR-21, miR-210, IL-6, HIF-1α, VEGF, and CSC signatures in vivo in a mouse orthotopic model of human PC. Collectively, these results suggest that the anti-tumor activity of CDF is in part mediated through deregulation of tumor hypoxic pathways, and thus CDF could become a novel, and effective anti-tumor agent for PC therapy.