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900: Changes in NK cell effector function, subset distribution and receptor repertoire following in vitro NK cell and K562 tumor cell contact in melanoma patients
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EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
898 Impact of inflammation in a spontaneous model of melanoma in zebrafish 1 E. Gomez-Abenza ´ , C. Gabellini1 , D. Garc´ıa-Moreno1 , M. Mione2 , V. Mulero1 . 1 University of Murcia, Department of Cell Biology and Histology Faculty of Biology, Murcia, Spain, 2 Karlsruhe Institute of Technology (KIT), Institute of Toxicology and Genetics (ITG), Eggenstein-Leopoldshafen, Germany Background: Inflammation and cancer have a profound yet ambiguous relationship. Inflammation, especially chronic inflammation, exerts protumorigenic effects but inflammatory cells may also kill tumor cells and besides immunosuppression is known to increase the risk for certain cancers. Using a spontaneous model of melanoma in zebrafish, we evaluated the impact of inflammation, in particular of the proinflammatory cytokine TNFa, in promoting melanoma cells proliferation in vivo. Materials and Methods: We used the zebrafish transgenic kita-GFP-RAS expressing oncogenic HRASV12 under the control of kita promoter, driving oncogene expression in developing melanocytes and goblet cells. To establish chronic inflammation, we used the hai-deficient zebrafish that exhibited skin inflammation and areas of epidermal hyperproliferation due to the lacking of a serine protease inhibitor of Matriptase1. To study the specific involvement of TNFa signaling pathways, we modulated the expression of TNF receptors (TNFR) using two approaches: (i) morpholino gene-mediated inactivation of TNFR1 and TNFR2 and (ii) TNFR2 overexpression in transformed melanocytes using the combinatorial Gal4-UAS system. Results: We observed that hai1 knock-down significantly increased the number of oncogenically transformed cells expressing HRASV12 larvae at 3 dpf compared to their wild type siblings. Similarly, TNFR2 deficiency resulted in the inflammation of the skin and a concomitant increased number of oncogenically transformed cells at 3 dpf, while the inhibition of TNFR1 signaling did not affect skin homeostasis and weakly increased the proliferation of transformed cells. Interestingly, restoration of TNFR2 signaling in transformed cells did not reverse their enhanced proliferation, indicating that TNFR2 signaling in stromal cells was responsible for the increased proliferation of oncogenically transformed cells. Conclusions: Our data suggest that inflammation may promotes tumor cell proliferation, identifying a key role of TNFR2 signaling in the cross-talk between oncogenically transformed cells and the tumor microenvironment. No conflict of interest. 899 Identifying genes involved in the colon cancer initiating cells (CICs) survivability against montelukast treatment in xenograft model K. Bellamkonda1 , S. Savari1 , N. Chandrashekar1 , A. Sjolander1 . 1 Clinical ¨ Sweden Research Centre, Department of Laboratory Medicine, Malmo, Background: Colorectal Cancer (CRC) is the third leading form of cancer and significant cause of mortality in western populations. Inflammation is believed as one of the major risk factor for CRC. In this context, we have reported that lipid inflammatory mediator such as LTD4 stimulated the proliferation, survival and migration of colon cancer cells. Moreover pharmological inhibition of CysLT (1) R prevented the growth of colon cancer xenograft. Materials and Methods: Human colon cancer xenograft was induced in in female 5−6 week old nude mice by injecting 2.5×106 SW480 or HT29 cells. The tumors appeared after 7 days. The mice then recieved daily i.p. injections of either DMSO or Montelukast (5 mg/kg) for consecutive 14 days. Tumors thus obtained were processed for qPCR and immunohistochemistry (IHC) for the identification of altered gene expression and protein levels. Furthermore, we performed colony formation assay with SW480 and HT29 cell lines treated with LTD4 , PGE2 and their receptor antagonists. Results: The tumors obtained following Montelukast and DMSO treatment differ markedly in their size. To identify the basis of this difference we classified the tumors as resistant and susceptible. It was observed that resistant tumors have upregulated gene expression of ALDH1B1 and GLI1 which represents the increased CICs percentage. Moreover, increased BCL2 and COX-2 expression in resistant xenografts suggested the improved survivability and proliferation of colon cancer cells. IHC data replicated the gene findings with overexpressed COX-2, b-catenin and BCL2 proteins. Under in-vitro circumstances, LTD4 and PGE2 increased markedly the colony formation in both cell lines. The antagonists of LTD4 and PGE2 receptors significantly reduced the number of colonies. Conclusions: In conclusion our data showed that tumor responsiveness to antagonist of inflammatory mediators depends upon the percentage population of CICs present. Thus targeting the CICs along with inflammatory pathway could be of better therapeutic importance. No conflict of interest.
900 Changes in NK cell effector function, subset distribution and receptor repertoire following in vitro NK cell and K562 tumor cell contact in melanoma patients G. Konjevic1 , A. Vuletic1 , K. Mirjacic Martinovic1 . 1 Insitute of Oncology and Radiology, Department of Immunology, Belgrade, Serbia Background: NK cells are divided into two functionally distinct subsets: cytotoxic CD16bright and regulatory CD16dim subset based on CD16 receptor expression. Expression of functionally opposing, activating and inhibitory NK cell receptors regulate NK cell cytotoxicity against malignant cells. Ligation of activating receptors to stress-induced ligands stimulates, while binding of inhibitory, KIR receptors to MHC class I molecules on tumor cells inhibits NK cell cytotoxicity, although KIR expression is inherent to more mature, cytotoxic CD16bright subset. K562 human erythromyeloid tumor cell line is MHC class I deficient and sensitive to NK cell lysis, and it expresses stress induced MICA ligand for NKG2D activating NK cell receptor. The aim of this study was to investigate the effect of co-culture of melanoma patients’ NK cells with K562 tumor cell line on effector functions and the expression of several receptors on CD3− CD16+ NK cells and their subsets. Material and Methods: PBMC obtained from 15 metastatic melanoma patients (MM) were co-cultured with K562 cell line in target to effector ratio 2:3. Samples were incubated for 4 h at 37ºC in a humidified atmosphere in CO2 incubator. The expression of NK cell receptors (CD16, NKG2D, CD161, CD158a, CD158b), degranulation marker (CD107a) and intracellular IFNg production were estimated on CD3− CD16+ NK cells and on CD3− CD16bright and CD3− CD16dim NK subsets by flow cytometry. Results: Conjugate formation between NK cells and NK-sensitive K562 target tumor cell line led to degranulation, a hallmark of NK cell cytotoxicity, with an increase in CD107a expression on NK cells and cytotoxic CD16bright NK cell subset, as well as activation-induced IFNg production also in NK cells and in both their subsets. Moreover, the up-regulation in IFNg production was preferentially found in cytotoxic CD16bright NK cell subset. We also found a decrease in the percent of CD3− CD16+ NK cells and a subsequent CD16bright to CD16dim NK cell subset transition, suggesting CD16 shedding. NK cell– target cell interaction led to lower post-contact NKG2D receptor expression on NK cells and CD16bright NK cell subset. Furthermore, we show post contact increase in KIR and CD161 inhibitory receptors on the regulatory CD16dim NK cell subset. Conclusions: In this study we show in MM patients that contact between NK and tumor cells leads to degranulation and affects NK cell subset distribution and receptor repertoire. The obtained results reflect NKG2D ligand-induced internalization and degradation, early and prompt post-contact IFNg up regulation in the cytotoxic CD3− CD16bright NK cell subset, recently described in healthy individuals, only, and potential induction of NK cell maturation into terminally differentiated cytotoxic effectors based on the found KIR up regulation on CD3− CD16dim subset. No conflict of interest.
Sunday 6 July 2014 Poster Session
Radiobiology/Radiation Oncology I 901 Psammaplin A (PsA) derivatives inhibit DNA methyltransferase (DNMT) and radiosensitize A549 lung cancer cells H.J. Kim1 , E.S. Ma2 , B.S. Shin2 , J.H. Kim1 , I.H. Kim1 . 1 Seoul National University Hospital, Radiation Oncology, Seoul, South Korea, 2 Catholic University of Daegu, College of Pharmacy, Daegu, South Korea Background: Psammaplin A (PsA), a novel DNA methyltransferase (DNMT) inhibitor and histone deacetylase (HDAC) inhibitor, was reported to induce radiosensitivity in lung cancer cell line. Concerning in vivo tissue distribution characteristics, PsA was found to be highly distributed to lung tissues. However, it appeared to be unstable in biological matrices. Here we report the DNMT inhibitory effect of PsA derivatives and their potential for radiation sensitization in lung cancer cell line. Material and Methods: A549 lung cancer cell line was used in verification of DNMT inhibitory effect with cultured cell DNA extraction kit. A549 cell line was exposed to radiation with or without a total of 9 PsA derivatives for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Results: These 9 PsA derivatives all showed DNMT inhibitory effect and inhibited cell proliferation at the ranges of IC50 30–120mM. Furthermore, radiation clonogenic assays revealed that these compound shows radiosensitizing properties in A549 lung cancer cell line. Conclusions: This preliminary data support the further investigation of these derivatives for use as radiation sensitizing agents with potential for clinical application. No conflict of interest.
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
898 Impact of inflammation in a spontaneous model of melanoma in zebrafish 1 E. Gomez-Abenza ´ , C. Gabellini1 , D. Garc´ıa-Moreno1 , M. Mione2 , V. Mulero1 . 1 University of Murcia, Department of Cell Biology and Histology Faculty of Biology, Murcia, Spain, 2 Karlsruhe Institute of Technology (KIT), Institute of Toxicology and Genetics (ITG), Eggenstein-Leopoldshafen, Germany Background: Inflammation and cancer have a profound yet ambiguous relationship. Inflammation, especially chronic inflammation, exerts protumorigenic effects but inflammatory cells may also kill tumor cells and besides immunosuppression is known to increase the risk for certain cancers. Using a spontaneous model of melanoma in zebrafish, we evaluated the impact of inflammation, in particular of the proinflammatory cytokine TNFa, in promoting melanoma cells proliferation in vivo. Materials and Methods: We used the zebrafish transgenic kita-GFP-RAS expressing oncogenic HRASV12 under the control of kita promoter, driving oncogene expression in developing melanocytes and goblet cells. To establish chronic inflammation, we used the hai-deficient zebrafish that exhibited skin inflammation and areas of epidermal hyperproliferation due to the lacking of a serine protease inhibitor of Matriptase1. To study the specific involvement of TNFa signaling pathways, we modulated the expression of TNF receptors (TNFR) using two approaches: (i) morpholino gene-mediated inactivation of TNFR1 and TNFR2 and (ii) TNFR2 overexpression in transformed melanocytes using the combinatorial Gal4-UAS system. Results: We observed that hai1 knock-down significantly increased the number of oncogenically transformed cells expressing HRASV12 larvae at 3 dpf compared to their wild type siblings. Similarly, TNFR2 deficiency resulted in the inflammation of the skin and a concomitant increased number of oncogenically transformed cells at 3 dpf, while the inhibition of TNFR1 signaling did not affect skin homeostasis and weakly increased the proliferation of transformed cells. Interestingly, restoration of TNFR2 signaling in transformed cells did not reverse their enhanced proliferation, indicating that TNFR2 signaling in stromal cells was responsible for the increased proliferation of oncogenically transformed cells. Conclusions: Our data suggest that inflammation may promotes tumor cell proliferation, identifying a key role of TNFR2 signaling in the cross-talk between oncogenically transformed cells and the tumor microenvironment. No conflict of interest. 899 Identifying genes involved in the colon cancer initiating cells (CICs) survivability against montelukast treatment in xenograft model K. Bellamkonda1 , S. Savari1 , N. Chandrashekar1 , A. Sjolander1 . 1 Clinical ¨ Sweden Research Centre, Department of Laboratory Medicine, Malmo, Background: Colorectal Cancer (CRC) is the third leading form of cancer and significant cause of mortality in western populations. Inflammation is believed as one of the major risk factor for CRC. In this context, we have reported that lipid inflammatory mediator such as LTD4 stimulated the proliferation, survival and migration of colon cancer cells. Moreover pharmological inhibition of CysLT (1) R prevented the growth of colon cancer xenograft. Materials and Methods: Human colon cancer xenograft was induced in in female 5−6 week old nude mice by injecting 2.5×106 SW480 or HT29 cells. The tumors appeared after 7 days. The mice then recieved daily i.p. injections of either DMSO or Montelukast (5 mg/kg) for consecutive 14 days. Tumors thus obtained were processed for qPCR and immunohistochemistry (IHC) for the identification of altered gene expression and protein levels. Furthermore, we performed colony formation assay with SW480 and HT29 cell lines treated with LTD4 , PGE2 and their receptor antagonists. Results: The tumors obtained following Montelukast and DMSO treatment differ markedly in their size. To identify the basis of this difference we classified the tumors as resistant and susceptible. It was observed that resistant tumors have upregulated gene expression of ALDH1B1 and GLI1 which represents the increased CICs percentage. Moreover, increased BCL2 and COX-2 expression in resistant xenografts suggested the improved survivability and proliferation of colon cancer cells. IHC data replicated the gene findings with overexpressed COX-2, b-catenin and BCL2 proteins. Under in-vitro circumstances, LTD4 and PGE2 increased markedly the colony formation in both cell lines. The antagonists of LTD4 and PGE2 receptors significantly reduced the number of colonies. Conclusions: In conclusion our data showed that tumor responsiveness to antagonist of inflammatory mediators depends upon the percentage population of CICs present. Thus targeting the CICs along with inflammatory pathway could be of better therapeutic importance. No conflict of interest.
900 Changes in NK cell effector function, subset distribution and receptor repertoire following in vitro NK cell and K562 tumor cell contact in melanoma patients G. Konjevic1 , A. Vuletic1 , K. Mirjacic Martinovic1 . 1 Insitute of Oncology and Radiology, Department of Immunology, Belgrade, Serbia Background: NK cells are divided into two functionally distinct subsets: cytotoxic CD16bright and regulatory CD16dim subset based on CD16 receptor expression. Expression of functionally opposing, activating and inhibitory NK cell receptors regulate NK cell cytotoxicity against malignant cells. Ligation of activating receptors to stress-induced ligands stimulates, while binding of inhibitory, KIR receptors to MHC class I molecules on tumor cells inhibits NK cell cytotoxicity, although KIR expression is inherent to more mature, cytotoxic CD16bright subset. K562 human erythromyeloid tumor cell line is MHC class I deficient and sensitive to NK cell lysis, and it expresses stress induced MICA ligand for NKG2D activating NK cell receptor. The aim of this study was to investigate the effect of co-culture of melanoma patients’ NK cells with K562 tumor cell line on effector functions and the expression of several receptors on CD3− CD16+ NK cells and their subsets. Material and Methods: PBMC obtained from 15 metastatic melanoma patients (MM) were co-cultured with K562 cell line in target to effector ratio 2:3. Samples were incubated for 4 h at 37ºC in a humidified atmosphere in CO2 incubator. The expression of NK cell receptors (CD16, NKG2D, CD161, CD158a, CD158b), degranulation marker (CD107a) and intracellular IFNg production were estimated on CD3− CD16+ NK cells and on CD3− CD16bright and CD3− CD16dim NK subsets by flow cytometry. Results: Conjugate formation between NK cells and NK-sensitive K562 target tumor cell line led to degranulation, a hallmark of NK cell cytotoxicity, with an increase in CD107a expression on NK cells and cytotoxic CD16bright NK cell subset, as well as activation-induced IFNg production also in NK cells and in both their subsets. Moreover, the up-regulation in IFNg production was preferentially found in cytotoxic CD16bright NK cell subset. We also found a decrease in the percent of CD3− CD16+ NK cells and a subsequent CD16bright to CD16dim NK cell subset transition, suggesting CD16 shedding. NK cell– target cell interaction led to lower post-contact NKG2D receptor expression on NK cells and CD16bright NK cell subset. Furthermore, we show post contact increase in KIR and CD161 inhibitory receptors on the regulatory CD16dim NK cell subset. Conclusions: In this study we show in MM patients that contact between NK and tumor cells leads to degranulation and affects NK cell subset distribution and receptor repertoire. The obtained results reflect NKG2D ligand-induced internalization and degradation, early and prompt post-contact IFNg up regulation in the cytotoxic CD3− CD16bright NK cell subset, recently described in healthy individuals, only, and potential induction of NK cell maturation into terminally differentiated cytotoxic effectors based on the found KIR up regulation on CD3− CD16dim subset. No conflict of interest.
Sunday 6 July 2014 Poster Session
Radiobiology/Radiation Oncology I 901 Psammaplin A (PsA) derivatives inhibit DNA methyltransferase (DNMT) and radiosensitize A549 lung cancer cells H.J. Kim1 , E.S. Ma2 , B.S. Shin2 , J.H. Kim1 , I.H. Kim1 . 1 Seoul National University Hospital, Radiation Oncology, Seoul, South Korea, 2 Catholic University of Daegu, College of Pharmacy, Daegu, South Korea Background: Psammaplin A (PsA), a novel DNA methyltransferase (DNMT) inhibitor and histone deacetylase (HDAC) inhibitor, was reported to induce radiosensitivity in lung cancer cell line. Concerning in vivo tissue distribution characteristics, PsA was found to be highly distributed to lung tissues. However, it appeared to be unstable in biological matrices. Here we report the DNMT inhibitory effect of PsA derivatives and their potential for radiation sensitization in lung cancer cell line. Material and Methods: A549 lung cancer cell line was used in verification of DNMT inhibitory effect with cultured cell DNA extraction kit. A549 cell line was exposed to radiation with or without a total of 9 PsA derivatives for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Results: These 9 PsA derivatives all showed DNMT inhibitory effect and inhibited cell proliferation at the ranges of IC50 30–120mM. Furthermore, radiation clonogenic assays revealed that these compound shows radiosensitizing properties in A549 lung cancer cell line. Conclusions: This preliminary data support the further investigation of these derivatives for use as radiation sensitizing agents with potential for clinical application. No conflict of interest.