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144 CDx2 Promotes Tumor Pericyte Content and Vascular Function in Human Colon Cancer Xenografts
anticancer drug-induced apoptosis, while an anti-VEGF-neutralizing antibody did not completely block the resistance. To directly reveal function of VEGF mRNA itself, cells were transiently transfected with plasmids encoding full-length VEGF mRNA with mutation of signal sequence, VEGF mRNAs lacking UTRs, or mutated 5'UTR. We demonstrated that VEGF mRNA possessed the anti-apoptotic activity in the 5'UTR, which was independent of VEGF protein. We then established HCT116 clones stably expressing chimeric mRNA of either the VEGF 5'UTR or the mutated 5'UTR fused to the yellow fluorescent protein-coding gene. The clones expressing the 5'UTR, but not the mutated one, showed increased anchorageindependent growth In Vitro and progressive tumor formation when implanted in athymic nude mice. Microarray and real-time PCR analyses indicated that the VEGF 5'UTR-expressing tumors up-regulated anti-apoptotic genes and down-regulated pro-apoptotic genes. Notably, expression of STAT1 was markedly repressed in the 5'UTR-expressing tumors, resulting in down-regulation of a STAT1-responsive cluster of genes (43 genes). As a result, the tumors did not respond to interferon therapy at all. Our results indicated that cancer cells have a unique survival system that is regulated by VEGF mRNA and imply that both VEGF mRNA and its protein may synergistically promote the malignancy of tumor cells. Therefore, combination of anti-VEGF transcript strategies, such as siRNA-based gene silencing, with antiVEGF antibody therapy may provide effective and multiple anti-tumor effects.
A 20 Hours ELISA Method Chemosusceptibility Rapid Test for Helicobacter pylori Dino Vaira, Federico Perna BACKGROUND: Antimicrobial resistant strains of Helicobacter pylori (H. pylori) have been increasing in the United States and Europe, which may account for constant decreasing eradication rates reported in the literature (most recent trials reported eradication rate as low as 57-73% for 7-day triple therapy and 67-79% for 10 day triple therapy (Gastroenterology 2007;113:985-1001). The common methods to test chemosusceptibility are agar dilution, disk diffusion and the E-Test, that consist of performing the test on culture agar-based media with different types of supplements. All these methods require from 7-14 days obtaining results. Recently a 20 hours Elisa chemosusceptibility rapid test for H. pylori has been patented by Alma Mater Studio rum, University of Bologna (PCT/IT2007/000270) not based on DNA amplification, that reduce the time required from the 7-14 days to 20-24 hours. AIM: To assess the accuracy of the new “20 hours Elisa chemosusceptibility rapid test for H. pylori” compared to a gold standard (agar dilution method). METHODS: One hundred-five out of 115 H. pylori positive biopsies were tested for chemosusceptibility to Clarithromycin (≥ 1 micro/ml) Metronidazole (≥ 8 micro/ml) by the gold standard and by the new 20 hours Elisa chemosusceptibility rapid test for H. pylori (PCT/IT2007/000270). RESULTS: According to chemosusceptibility to clarithromycin, 75 sensitive cultures and 30 resistant cultures have been identified with the gold standard. Taking the new method into consideration, all 75 have been sensitive (sensitivity of the method - 100%), while 28 out of 30 have been identified as resistant, and two have been falsely sensitive (method specificity - 93.3%). According to chemosusceptibility to metronidazole, 68 sensitive cultures and 37 resistant cultures have been identified with gold standard. Taking the new method into consideration, 63 have been sensitive and 5 falsely resistant (method sensitivity - 92.6%), while 32 out of 37 have been identified as resistant, and 5 have been falsely sensitive (method specificity - 86.5%). CONSLUSION: A new simple rapid test for susceptibility of antibiotics against H pylori has been validated which is simple to perform, reduce the incubation time from 14 days to 20 hours and does not need molecular biology techniques.
144 CDx2 Promotes Tumor Pericyte Content and Vascular Function in Human Colon Cancer Xenografts Kyunghee Burkitt, Sang Chun, John P. Lynch, Long H. Dang, Duyen Dang INTRODUCTION: CDX2 is a homeobox transcription factor that is important for the establishment and maintenance of intestinal epithelial cells. CDX2 has direct effects on tumor cell proliferation and survival. The effects of CDX2 expression on the tumor vasculature are not known. The development of a mature vascular network depends on crosstalk among multiple cell types, including epithelial cells, endothelial cells, and pericytes. Endothelial cells are central to microvessel formation. Pericytes are mural cells that surround endothelial tubes. Pericytes regulate the function, permeability, stability, and maturation of the vascular network. AIM: To determine the effects of CDX2 on tumor vasculature. METHODS: Three sets of human colon cancer cell lines were evaluated. LOVO cells express high CDX2 and LOVO CDX2-/- cells do not express CDX2 due to somatic disruption. COLO205 do not express CDX2 and COLO205 CDX2+ cells overexpress CDX2. HCT116 cells express low levels of CDX2 and HCT116 CDX2+ cells overexpress CDX2. Xenografts were grown and harvested from the flanks of athymic mice. Tumor microvessel density (MVD) was determined by CD31 endothelial cell staining. Tumor pericyte content was determined by staining for mature pericytes with alpha smooth muscle actin (SMA). Tumor vessel pericyte coverage was determined by the overlap in merged serial sections of CD31 endothelial and SMA pericytes stains. Tumor perfusion was visualized by Hoescht 33342 injection. RESULTS: CDX2 expression did not alter tumor MVD, but significantly altered microvessel morphology. Microvessels in CDX2 expressing xenografts were larger in diameter and length. While there were more microvessels in non-CDX2 expressing xenografts, they were short and truncated. There was ~4-fold increase in tumor pericyte content and vessel coverage in CDX2 expressing tumor xenografts. This was associated with ~2-fold increase in tumor perfusion. CONCLUSIONS: These data show, for the first time, that CDX2 expression in tumor epithelial cells regulates tumor vasculature. CDX2 increases tumor pericyte content, which in turn increases pericyte coverage of vessels, and a more mature vascular network. These changes are associated with increased tumor blood flow. While tumor vessel pericytes promote tumor blood flow and growth, they also limit tumor cell metastasis by stabilizing the microvessel wall. This might help explain the dichotomy in the CDX2 literature, in which CDX2 can be associated with increased tumor xenograft growth, but decreased metastasis. We conclude that CDX2 regulates epithelial cell interactions with pericytes and endothelial cells in colon cancer xenografts.
142 PLGF Expression By Tumor Stromal Cells Is Induced Via Tumor Cell-Tumor Stromal Crosstalk and Substantially Contributes to Tumor Growth Christian Fischer, Bart Jonckx, Massimilano Mazzone, Serena Zacchigna, Sonja Loges, Sabine Wyns, Jean Marie Stassen, Mieke Dewerchin, Desire Collen, Peter Carmeliet By releasing angiogenic factors, the tumor microenvironment contributes to tumor growth. Placental growth factor (PlGF), a VEGF homologue, that selectively binds and acts via VEGFR-1, is an angiogenic factor, which is expressed by tumor cells and leukocytes. To explore the role of stromal derived PlGF in tumor growth we used several In Vivo and In Vitro approaches. First, murine CT26 colon tumor cells in culture do not express PlGF under normoxia or hypoxia. When implanting these cells in PlGF-/- mice and their respective PlGF+/+ littermates, ELISA on tumor extracts revealed that PlGF was undetectable in CT26 tumors grown in PlGF-/- mice. In contrast, high PlGF levels were measured in CT26 tumors, grown in PlGF+/+ mice, demonstrating that PlGF is exclusively tumor stroma derived in this tumor model. PlGF is expressed by endothelial cells. However, to further unravel which other tumor stromal cells express PlGF, we isolated F4/80+ tumor associated macrophages (TAM) from CT26 In Vivo growing tumors. RT-PCR revealed substantial amounts of PlGF mRNA transcripts in TAM extracts as compared to CT26 tumor cell cultures. To analyze whether the tumor cells might crosstalk to stromal fibroblasts and induce the production of PlGF by these stromal cells, we co-cultured CT26 tumor cells with murine embryonic fibroblasts (MEFs) isolated from PlGF+/+ and PlGF-/- mice. MEFs of either genotype did not produce any detectable PlGF. When PlGF-/- MEFs were co-cultured with CT26 tumor cells, no PlGF was detectable in the conditioned medium. In contrast, when PlGF+/+ MEFs were co-cultured with CT26 tumor cells, abundant amounts of PlGF were detected, indicating that the CT26 tumor cells induced MEFs to release PlGF. To finally delineate the role of tumor stromal derived PlGF in tumor growth and angiogenesis, we used an anti-mPlGF antibody (αPlGF) that only blocks mouse but not human PlGF. When CT26 tumor cells were implanted into syngeneic mice, αPlGF inhibited tumor growth and angiogenesis. Furthermore, we transplanted human pancreatic DANG xenograft tumor cells into NMRInu/ nu mice. These tumors expressed human and murine PlGF. Even though human PlGF, released by the malignant cells, was not neutralized by αPlGF, treatment of αPlGF inhibited the growth of DANG tumors by 55%, reduced plasma levels of the tumor markers CA199 and CEA, and prolonged survival of tumor-bearing mice. Thus, PlGF is not only produced by various stromal cell types, including endothelial cells, leukocytes and fibroblasts, but also induced upon tumor cell-tumor stromal crosstalk and substantially contributes to tumor growth.
145 Tumor Derived Colonic Myofibroblasts Promote Enhanced Expansion of CD4+ Cd25High FOXP3+ Regulatory T Cells Via PGE2 Dependent Mechanism Iryna V. Pinchuk, Jamal I. Saada, Ellen J. Beswick, Gushyalatha Boya, Sahil Mittal, John F. Di Mari, Victor E. Reyes, Don W. Powell Background & Aims: Colorectal cancer (CRC) affects hundreds of thousands of people annually worldwide and conveys a significant mortality risk among the malignant disease in both men and women. Recent studies demonstrated that immunosupression by CD4+ CD25high FoxP3+ regulatory T cells (Treg) in peripheral tissue (adaptive Treg) is a crucial tumor immune-evasion mechanisms and likely represents an important obstacle to successful immunotherapy. While the contribution of the adaptive Treg to CRC progression has been recently studied, mechanisms underlying Treg induction in the colonic mucosa remain unknown. Antigen presenting cells (APCs) are likely to be involved in this process since they are key regulators of T cell responses. We recently reported that human colonic myofibroblasts (CMFs) are novel non professional APCs and are the most abundant MHC class II expressing cell phenotype in the normal human colonic mucosa. Our recent studies also showed that normal CMFs stimulate expansion of T regulatory cells (Treg) with a CD4+ CD25high FoxP3+ phenotype via a PGE2-dependent mechanism. Because cancer-associated CMFs are avid producers of prostaglandins, we hypothesize that PGE2 producing colonic CMFs can contribute to the immunosuppressive, tumor-evasion mechanism during CRC via enhanced expansion of the adaptive CD4+ CD25high FoxP3+ Treg cells. Methods: Naive or resting T helper cells were cocultured with CMFs. T cell phenotype was determined by immunostaining followed by FACS analysis. Proliferation assays, real-time RT-PCR and cytokine ELISAs were used to evaluate activation of Treg cells co-cultured with CMFs. Results: Our data demonstrate that CMFs induce de novo generation and proliferation of allogeneic CD4+ CD25high FoxP3+ Treg cells. The newly generated Treg were negative for CD127 marker, expressed CTLA-4, produced TGF-β and had suppressive function, since they inhibited the proliferation of activated autologous CD4+ effector T cells. CRC-derived CMFs significantly enhanced the de novo generation of the adaptive CD4+ CD4+ CD25high FoxP3+ Treg cells from naïve CD4+ CD25- FoxP3- T helper cells when compare to the
143 5'-Untranslated Region of the Human VEGF mRNA Has a Novel TumorPromoting Function in Human Colon Cancer Cells Kiyoshi Masuda, Shigetada Teshima-kondo, Yoshiko Nishikawa, Naoko Yamagishi, Kensei Nishida, Hideyuki Sasaki, Kumiko Tominaga, Tomoko Kawai, Keiya Nakamura, Kazuhito Rokutan Vascular endothelial growth factor-A (VEGF) is one of the key regulators of tumor development, hence it is considered to be an important therapeutic target for cancer treatment. However, clinical trials suggested that the anti-VEGF monotherapy was less effective than standard chemotherapy. Here we show a novel function of VEGF that the 5'untranslated region (UTR) of VEGF transcript could promote tumor malignancy of colon cancer HCT116 cells. Knockdown of VEGF with vegf-targeting siRNAs increased susceptibility of HCT116 to apoptosis caused with 5-fluorouracil. Recombinant human VEGF did not completely inhibit this apoptosis. Alternatively, overexpression of VEGF increased resistance to the
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A 20 Hours ELISA Method Chemosusceptibility Rapid Test for Helicobacter pylori Dino Vaira, Federico Perna BACKGROUND: Antimicrobial resistant strains of Helicobacter pylori (H. pylori) have been increasing in the United States and Europe, which may account for constant decreasing eradication rates reported in the literature (most recent trials reported eradication rate as low as 57-73% for 7-day triple therapy and 67-79% for 10 day triple therapy (Gastroenterology 2007;113:985-1001). The common methods to test chemosusceptibility are agar dilution, disk diffusion and the E-Test, that consist of performing the test on culture agar-based media with different types of supplements. All these methods require from 7-14 days obtaining results. Recently a 20 hours Elisa chemosusceptibility rapid test for H. pylori has been patented by Alma Mater Studio rum, University of Bologna (PCT/IT2007/000270) not based on DNA amplification, that reduce the time required from the 7-14 days to 20-24 hours. AIM: To assess the accuracy of the new “20 hours Elisa chemosusceptibility rapid test for H. pylori” compared to a gold standard (agar dilution method). METHODS: One hundred-five out of 115 H. pylori positive biopsies were tested for chemosusceptibility to Clarithromycin (≥ 1 micro/ml) Metronidazole (≥ 8 micro/ml) by the gold standard and by the new 20 hours Elisa chemosusceptibility rapid test for H. pylori (PCT/IT2007/000270). RESULTS: According to chemosusceptibility to clarithromycin, 75 sensitive cultures and 30 resistant cultures have been identified with the gold standard. Taking the new method into consideration, all 75 have been sensitive (sensitivity of the method - 100%), while 28 out of 30 have been identified as resistant, and two have been falsely sensitive (method specificity - 93.3%). According to chemosusceptibility to metronidazole, 68 sensitive cultures and 37 resistant cultures have been identified with gold standard. Taking the new method into consideration, 63 have been sensitive and 5 falsely resistant (method sensitivity - 92.6%), while 32 out of 37 have been identified as resistant, and 5 have been falsely sensitive (method specificity - 86.5%). CONSLUSION: A new simple rapid test for susceptibility of antibiotics against H pylori has been validated which is simple to perform, reduce the incubation time from 14 days to 20 hours and does not need molecular biology techniques.
144 CDx2 Promotes Tumor Pericyte Content and Vascular Function in Human Colon Cancer Xenografts Kyunghee Burkitt, Sang Chun, John P. Lynch, Long H. Dang, Duyen Dang INTRODUCTION: CDX2 is a homeobox transcription factor that is important for the establishment and maintenance of intestinal epithelial cells. CDX2 has direct effects on tumor cell proliferation and survival. The effects of CDX2 expression on the tumor vasculature are not known. The development of a mature vascular network depends on crosstalk among multiple cell types, including epithelial cells, endothelial cells, and pericytes. Endothelial cells are central to microvessel formation. Pericytes are mural cells that surround endothelial tubes. Pericytes regulate the function, permeability, stability, and maturation of the vascular network. AIM: To determine the effects of CDX2 on tumor vasculature. METHODS: Three sets of human colon cancer cell lines were evaluated. LOVO cells express high CDX2 and LOVO CDX2-/- cells do not express CDX2 due to somatic disruption. COLO205 do not express CDX2 and COLO205 CDX2+ cells overexpress CDX2. HCT116 cells express low levels of CDX2 and HCT116 CDX2+ cells overexpress CDX2. Xenografts were grown and harvested from the flanks of athymic mice. Tumor microvessel density (MVD) was determined by CD31 endothelial cell staining. Tumor pericyte content was determined by staining for mature pericytes with alpha smooth muscle actin (SMA). Tumor vessel pericyte coverage was determined by the overlap in merged serial sections of CD31 endothelial and SMA pericytes stains. Tumor perfusion was visualized by Hoescht 33342 injection. RESULTS: CDX2 expression did not alter tumor MVD, but significantly altered microvessel morphology. Microvessels in CDX2 expressing xenografts were larger in diameter and length. While there were more microvessels in non-CDX2 expressing xenografts, they were short and truncated. There was ~4-fold increase in tumor pericyte content and vessel coverage in CDX2 expressing tumor xenografts. This was associated with ~2-fold increase in tumor perfusion. CONCLUSIONS: These data show, for the first time, that CDX2 expression in tumor epithelial cells regulates tumor vasculature. CDX2 increases tumor pericyte content, which in turn increases pericyte coverage of vessels, and a more mature vascular network. These changes are associated with increased tumor blood flow. While tumor vessel pericytes promote tumor blood flow and growth, they also limit tumor cell metastasis by stabilizing the microvessel wall. This might help explain the dichotomy in the CDX2 literature, in which CDX2 can be associated with increased tumor xenograft growth, but decreased metastasis. We conclude that CDX2 regulates epithelial cell interactions with pericytes and endothelial cells in colon cancer xenografts.
142 PLGF Expression By Tumor Stromal Cells Is Induced Via Tumor Cell-Tumor Stromal Crosstalk and Substantially Contributes to Tumor Growth Christian Fischer, Bart Jonckx, Massimilano Mazzone, Serena Zacchigna, Sonja Loges, Sabine Wyns, Jean Marie Stassen, Mieke Dewerchin, Desire Collen, Peter Carmeliet By releasing angiogenic factors, the tumor microenvironment contributes to tumor growth. Placental growth factor (PlGF), a VEGF homologue, that selectively binds and acts via VEGFR-1, is an angiogenic factor, which is expressed by tumor cells and leukocytes. To explore the role of stromal derived PlGF in tumor growth we used several In Vivo and In Vitro approaches. First, murine CT26 colon tumor cells in culture do not express PlGF under normoxia or hypoxia. When implanting these cells in PlGF-/- mice and their respective PlGF+/+ littermates, ELISA on tumor extracts revealed that PlGF was undetectable in CT26 tumors grown in PlGF-/- mice. In contrast, high PlGF levels were measured in CT26 tumors, grown in PlGF+/+ mice, demonstrating that PlGF is exclusively tumor stroma derived in this tumor model. PlGF is expressed by endothelial cells. However, to further unravel which other tumor stromal cells express PlGF, we isolated F4/80+ tumor associated macrophages (TAM) from CT26 In Vivo growing tumors. RT-PCR revealed substantial amounts of PlGF mRNA transcripts in TAM extracts as compared to CT26 tumor cell cultures. To analyze whether the tumor cells might crosstalk to stromal fibroblasts and induce the production of PlGF by these stromal cells, we co-cultured CT26 tumor cells with murine embryonic fibroblasts (MEFs) isolated from PlGF+/+ and PlGF-/- mice. MEFs of either genotype did not produce any detectable PlGF. When PlGF-/- MEFs were co-cultured with CT26 tumor cells, no PlGF was detectable in the conditioned medium. In contrast, when PlGF+/+ MEFs were co-cultured with CT26 tumor cells, abundant amounts of PlGF were detected, indicating that the CT26 tumor cells induced MEFs to release PlGF. To finally delineate the role of tumor stromal derived PlGF in tumor growth and angiogenesis, we used an anti-mPlGF antibody (αPlGF) that only blocks mouse but not human PlGF. When CT26 tumor cells were implanted into syngeneic mice, αPlGF inhibited tumor growth and angiogenesis. Furthermore, we transplanted human pancreatic DANG xenograft tumor cells into NMRInu/ nu mice. These tumors expressed human and murine PlGF. Even though human PlGF, released by the malignant cells, was not neutralized by αPlGF, treatment of αPlGF inhibited the growth of DANG tumors by 55%, reduced plasma levels of the tumor markers CA199 and CEA, and prolonged survival of tumor-bearing mice. Thus, PlGF is not only produced by various stromal cell types, including endothelial cells, leukocytes and fibroblasts, but also induced upon tumor cell-tumor stromal crosstalk and substantially contributes to tumor growth.
145 Tumor Derived Colonic Myofibroblasts Promote Enhanced Expansion of CD4+ Cd25High FOXP3+ Regulatory T Cells Via PGE2 Dependent Mechanism Iryna V. Pinchuk, Jamal I. Saada, Ellen J. Beswick, Gushyalatha Boya, Sahil Mittal, John F. Di Mari, Victor E. Reyes, Don W. Powell Background & Aims: Colorectal cancer (CRC) affects hundreds of thousands of people annually worldwide and conveys a significant mortality risk among the malignant disease in both men and women. Recent studies demonstrated that immunosupression by CD4+ CD25high FoxP3+ regulatory T cells (Treg) in peripheral tissue (adaptive Treg) is a crucial tumor immune-evasion mechanisms and likely represents an important obstacle to successful immunotherapy. While the contribution of the adaptive Treg to CRC progression has been recently studied, mechanisms underlying Treg induction in the colonic mucosa remain unknown. Antigen presenting cells (APCs) are likely to be involved in this process since they are key regulators of T cell responses. We recently reported that human colonic myofibroblasts (CMFs) are novel non professional APCs and are the most abundant MHC class II expressing cell phenotype in the normal human colonic mucosa. Our recent studies also showed that normal CMFs stimulate expansion of T regulatory cells (Treg) with a CD4+ CD25high FoxP3+ phenotype via a PGE2-dependent mechanism. Because cancer-associated CMFs are avid producers of prostaglandins, we hypothesize that PGE2 producing colonic CMFs can contribute to the immunosuppressive, tumor-evasion mechanism during CRC via enhanced expansion of the adaptive CD4+ CD25high FoxP3+ Treg cells. Methods: Naive or resting T helper cells were cocultured with CMFs. T cell phenotype was determined by immunostaining followed by FACS analysis. Proliferation assays, real-time RT-PCR and cytokine ELISAs were used to evaluate activation of Treg cells co-cultured with CMFs. Results: Our data demonstrate that CMFs induce de novo generation and proliferation of allogeneic CD4+ CD25high FoxP3+ Treg cells. The newly generated Treg were negative for CD127 marker, expressed CTLA-4, produced TGF-β and had suppressive function, since they inhibited the proliferation of activated autologous CD4+ effector T cells. CRC-derived CMFs significantly enhanced the de novo generation of the adaptive CD4+ CD4+ CD25high FoxP3+ Treg cells from naïve CD4+ CD25- FoxP3- T helper cells when compare to the
143 5'-Untranslated Region of the Human VEGF mRNA Has a Novel TumorPromoting Function in Human Colon Cancer Cells Kiyoshi Masuda, Shigetada Teshima-kondo, Yoshiko Nishikawa, Naoko Yamagishi, Kensei Nishida, Hideyuki Sasaki, Kumiko Tominaga, Tomoko Kawai, Keiya Nakamura, Kazuhito Rokutan Vascular endothelial growth factor-A (VEGF) is one of the key regulators of tumor development, hence it is considered to be an important therapeutic target for cancer treatment. However, clinical trials suggested that the anti-VEGF monotherapy was less effective than standard chemotherapy. Here we show a novel function of VEGF that the 5'untranslated region (UTR) of VEGF transcript could promote tumor malignancy of colon cancer HCT116 cells. Knockdown of VEGF with vegf-targeting siRNAs increased susceptibility of HCT116 to apoptosis caused with 5-fluorouracil. Recombinant human VEGF did not completely inhibit this apoptosis. Alternatively, overexpression of VEGF increased resistance to the
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