- Email: [email protected]
879: Cryotherapy enhances the antigen-specific T cell immune responses and therapeutic antitumor effects generated by therapeutic HPV DNA vaccine
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EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
875 Macrophage migration and polarization is triggered by oxygen gradient in glioblastoma 1 ´ , A. Corroyer-Dulmont1 , E. Petit1 , M. Bernaudin1 , M.M. Leblond1 , A.N. Gerault S. Valable1 . 1 UMR 6301 ISTCT-CNRS-CEA-Universite´ de Caen, CERVOxy Team, Caen, France
Background: Hypoxia is a common feature of tumors, highly pronounced in glioblastoma (GBM) and known to induce resistances to conventional therapies which may explains the poor life expectancy of GBM patients (Stupp et al., 2005). GBM growth is also associated with a marked inflammation of which a strong macrophage accumulation. It has been shown that macrophages found in the tumor can be polarized into a M1 (anti-tumoral) or a M2 (protumoral) phenotype (Murdoch et al., 2004). At present, it seems that tumor associated macrophages, found in patients with GBM, are M2 macrophages (Lu-Emerson et al., 2013). However, factors responsible for their migration and polarization are not yet identified. The aim of this study was, first, to evaluate the macrophage tropism toward tumor on two models of human GBM, well characterized in term of hypoxia degree (Corroyer-Dulmont et al., 2013), and secondly, to evaluate, in vitro and in vivo, the effect of hypoxia on macrophage polarization. Materials and Methods: Animals models consisted of orthotropic injection of U87 and U251 cells (5×104 /3 ml) into athymic nude rats. Hypoxia has been evaluated ex-vivo by pimonidazol immunostaining. Macrophages have been detected on free floating coronal slices thanks to a CD68 immunostaining and polarization toward M1 or M2 phenotype with iNOS and Arginase1 immunostainings, respectively. For in vitro experiments, primary cultures of macrophages have been prepared from mice bone marrow and cells have been exposed to 1% of oxygen and their polarization has been characterized by RT-qPCR and functional markers. Results: In both models, macrophages are localized inside the tumor, with a preferential migration toward U251 model which is the most hypoxic model (Corroyer-Dulmont et al., 2013). For this model, macrophages are localised preferentially in hypoxic areas of the tumor and express M2 markers, unlike macrophages found in the U87 model which exhibit M1 markers. In vitro, macrophages cultured in 1% of oxygen present a phenotype closed to M2 macrophages. Conclusion: In this study, using GBM models with various degree of oxygenation, we have shown that the number of macrophages found in the tumor is in closed relationship with the degree of hypoxia. We have also shown that hypoxia may trigger the acquisition of an M2 phenotype, suspected to contribute to tumor development. These results highlight a new function of hypoxia in the aggressiveness of GBM and may help to increase therapies for these tumors. No conflict of interest.
876 Allergen induced pulmonary inflammation enhances mammary tumor growth and metastasis: Role of CHI3L1 S. Libreros1 , R. Garcia-Areas1 , P. Robinson2 , A. Humbles3 , V. IragavarapuCharyulu1 . 1 Florida Atlantic University, Basic Science, Boca Raton, USA, 2 Florida Atlantic University, Clinical Science, Boca Raton, USA, 3 MedImmune LLC, Gaithersburg, USA Introduction: Metastasis is responsible for majority of cancer deaths and the incidence of metastasis to the lungs is higher in breast cancer patients with chronic pulmonary inflammatory illnesses. Lungs are a frequent site of inflammation. Pulmonary inflammatory illnesses have been increasing throughout the world with women more susceptible to pulmonary inflammatory diseases such as asthma. Patients with asthma demonstrate higher circulating levels of Chitinase-3 like 1 protein (CHI3L1). Recently, elevated serum levels of this glycoprotein have been correlated with poor prognosis and shorter survival of patients with metastatic breast cancer. Although a wealth of clinical evidence shows an association between CHI3L1 expression with tumor growth and short survival, little is known regarding the mechanisms leading to tumor metastasis. In this study we tested the hypothesis that pre-existing inflammation and CHI3L1 plays a role in enhancing growth of the primary tumor and metastasis to the lungs. Material and Methods: We use the combination of mouse models of allergic pulmonary inflammation and a well-established models breast cancer metastasis in wild type and CHI3L1 knockout mice. Results: Using 4T1 ragweed allergen sensitized mice we show accelerated tumor growth and metastasis, and overall decrease in survival. Allergen induced mice exhibit higher levels of proinflammatory/protumorigenic molecules CHI3L1, MMP-9, CXCL2/IL-8 and CCL2/MCP-1 in circulation and in pulmonary microenvironment. Accelerated tumor growth and the expression of CHI3L1 was also shown in two other mouse mammary tumor models 67NR and 4T07 induced for pulmonary inflammation. More significantly, ragweed allergen sensitized CHI3L1 knockout mice bearing mammary tumors had <50 fold decreased in tumor growth and metastasis with increased survival.
Discussion: Suppression of pulmonary inflammation and targeting CHI3L1 may be a valuable therapeutic strategy for breast cancer patients with pulmonary inflammatory illnesses diseases. No conflict of interest. 877 Changes of the white blood cells subsets in HER2 positive breast cancer patients treated with trastuzumab I. Matic1 , M. Grujic2 , B. Kolundzija1 , A. Damjanovic Velickovic1 , Z. Tomasevic3 , I. Bozovic Spasojevic3 , R. Dzodic4 , Z. Zdrale3 , Z. Juranic1 . 1 Institute of Oncology and Radiology of Serbia, Department of Experimental Oncology, Belgrade, Serbia, 2 Institute of Rheumatology, Third Hospital Department, Belgrade, Serbia, 3 Institute of Oncology and Radiology of Serbia, Department of Medical Oncology, Belgrade, Serbia, 4 Institute of Oncology and Radiology of Serbia, Department of Surgical Oncology, Belgrade, Serbia Background: The aim of research was to determine are there any changes in the percentages of lymphocytes, granulocytes, CD16+ and/or CD56+ lymphocytes in HER2+ breast cancer patients before or after treatment with trastuzumab in relation to response to therapy. Patients and Methods: This study involved 41 HER2+ breast cancer patients whose blood was analyzed before and two months after therapy with trastuzumab, applied alone or in combination with chemotherapeutics. Control group consisted of 41 healthy persons. The subsets of white blood cells were analyzed by flow cytometry. Results and Discussion: Our study demonstrates significantly lower percentages of the CD16+, CD56+ lymphocytes and CD16+CD56+ NK cells in breast cancer patients before therapy in comparison to healthy persons (p < 0.00001, p < 0.0004, p < 0.0001, respectively). Significantly lower percentage of lymphocytes (p < 0.000008), higher percentage of granulocytes (p < 0.007) and higher ratio of the percentage of granulocytes and percentage of lymphocytes (p < 0.00009) was observed in patients before treatment in relation to healthy controls. Clinical assessment showed progression of disease (PD) in 6 out of 41 patients. Surprisingly, patients who had PD after trastuzumab treatment had significantly higher percentage of CD16+ lymphocytes (p < 0.02), higher percentage of NK cells (p < 0.05) and also higher ratio of the percentage of granulocytes and percentage of lymphocytes (p < 0.04), before therapy, than patients without PD. While a significant decrease in the percentage of CD56+ lymphocytes in comparison to values before therapy was determined in patients without PD (p < 0.05), in patients with PD, after therapy, this phenomenon was more profound; more significant decrease in the percentage of CD56+ lymphocytes and NK cells (p < 0.004 and p < 0.007) was found. Conclusions: Results of our study indicate that tumor destroying action of trastuzumab is not occurring only through the known ADCC mechanism mediated by NK cells. This action could be accomplished through the killing of tumor cells by ROS released from trastuzumab activated CD64 receptors not only on monocytes but also on neutrophils, which could nonspecifically destroy neighboring CD56+ and CD16+ immune cells. This ROS-mediated trastuzumab toxicity seems to be more pronounced in patients with PD and could be the reason why pronounced decrease in the percentage of NK cells (and possibility for their antitumor action) in these patients was found. No conflict of interest. 879 Cryotherapy enhances the antigen-specific T cell immune responses and therapeutic antitumor effects generated by therapeutic HPV DNA vaccine S.Y. Lee1 , J.K. Sim1 , K.H. Min1 , G.Y. Hur1 , J.J. Shim1 , K.H. In1 , K.H. Kang1 , C.F. Hung2 , T.C. Wu2 . 1 Korea University Medical Center, Department of Internal Medicine, Seoul, Korea, 2 Johns Hopkins Medical Institutions, Department of Pathology, Baltimore, USA Background: Antigen-specific immunotherapy is an attractive approach for the treatment of cancers since it has the potency to specifically eradicate systemic tumors and control metastases without damaging normal cells. A favorable approach for antigen-specific immunotherapy is the use of DNA vaccines due to their safety, stability and ease of preparation. The therapeutic human papillomavirus (HPV) DNA vaccines encoding calreticulin (CRT) linked to HPV16 E7 antigen (CRT/E7) has showed the effects for enhancing the therapeutic HPV DNA vaccine potency. However, DNA vaccine suffers from insufficient immune response for antigen presenting cells and showed minimal clinical efficacies. Cryotherapy is an attractive therapeutic option for tumors due to its minimally invasive nature. And also cryotherapy is potentially immunogenic. Therefore, we explored the employment of cryotherapy in combination with CRT/E7 DNA vaccine in a preclinical model. Material and Methods: For in vivo tumor treatment, TC-1 tumor cells were subcutaneously injected into the left flank area of C57BL/6 mice. After 5 days, the mice were divided into four groups: group 1 received no treatment after the TC-1 tumor challenge, group 2 was treated with cryotherapy (20 seconds, two times), group 3 was immunized with the DNA vaccine, and group 4 was both immunized and treated with cryotherapy. Mice were monitored for E7-specific
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 CD8 (+) T cell immune responses, myeloid-derived suppressor cells (MDSC) and antitumor effects. In vitro assay, the secretion of HMGB1 protein in TC-1 cells after incubation in deep freezer was determined by Western blot. Results: We found that the combination of cryotherapy and CRT/E7 DNA vaccination significantly inhibited tumor growth and increased E7-specific CD8+ T cells in blood compared to CRT/E7 DNA vaccinated mice or mice treated with cryotherapy alone. Furthermore, combination treatment with cryotherapy and CRT/E7 vaccination reduces immunosuppressive MDSC. In vitro assay, we found that treatment with deep freezing and thawing led to up-regulation of HMGB1 in E7-expressing TC-1 tumor cells, rendering the tumor cells more susceptible to APCs and E7-specific CD8+ T cell-mediated killing. Conclusion: Our data indicate that cryotherapy can enhance therapeutic HPV DNA vaccine potency in generating improved antigen-specific immune responses and antitumor effects. These findings have important implications for future clinical translation. No conflict of interest. 880 Interactions of immunoglobulin-like cell adhesion molecule hepaCAM with CD137 in cell–matrix interactions, cell motility and escape from immune surveillance in Hodgkin Lymphoma J. Seah1 . 1 National University Of Singapore, Physiology, Singapore, Singapore Background: The study aims to determine how the interactions between hepaCAM (a cell adhesion molecule) and CD137 (a member of TNFR superfamily 9) contributes to cell adhesion, cell–extracellular matrix interactions and recruitment of leukocytes aiding in escape from immune surveillance in Hodgkin Lymphoma. Materials and Methods: Western blot analysis, confocal microscopy and flow cytometry were used to determine the expression and localisation of hepaCAM and CD137 in human Reed Sternberg (HRS) cell lines of Hodgkin Lymphoma. Co-immunoprecipitation was used to shed light on the physical interactions between hepaCAM and CD137 in HRS cells, and to determine the regions of hepaCAM responsible for interactions with CD137. Adhesion and cell spreading assays, coupled with neutralizing antibodies targeting hepaCAM, were used to determine the role of hepaCAM in cell migration and cell–extracellular matrix interactions in HRS cells. Purified hepaCAM proteins were used to determine the possible role of hepaCAM in binding to activated peripheral blood mononuclear cells (PBMCs) resulting in subsequent inhibition of activation of immune cells involving CD137. It may contribute to the escape of HRS cells from immune surveillance. Results: As CD137 is ectopically expressed on most classical Hodgkin Lymphoma cases and appeared to play a role in escape from immune surveillance in HRS cells, the interacting partners of CD137 may play a role in the escape mechanism as well. In addition, immunoglobulin-like cell adhesion molecules have been known to play roles in cancer progression and work in concert with members of TNFR superfamily. Firstly, the expression and localisation of hepaCAM on cell surfaces was found to be CD137 dependent in HRS cell lines. This indicates the possible interactions of hepaCAM and CD137 in HRS cells. Secondly, co-immunoprecipitation confirmed that hepaCAM and CD137 interacted with each other using HRS cell lines. The cytoplasmic tail of hepaCAM proved to be important in maintaining the interactions with CD137. Thirdly, neutralising hepaCAM with antibodies appeared to result in reduced cell adhesion and cell spreading on fibronectin which may indicate the importance of hepaCAM interactions with extracellular matrix in HRS cells. Lastly, hepaCAM was found to bind preferentially to activated PBMCs. Taken together with the expression, localisation and interactions results obtained, this data indicated the possibility of hepaCAM working in concert with CD137 in recruitment and binding of leukocytes resulting in inhibition of activation of PBMCs and escape from immune surveillance. Conclusion: The expression and localisation of hepaCAM on cell surfaces of HRS cells were CD137 dependent. The interactions between hepaCAM and CD137 may work in concert to affect cell adhesion, cell–extracellular matrix interactions and binding of leukocytes aiding in escape from immune surveillance. No conflict of interest. 881 Depletion of tumor-associated macrophages enhances peptide-based cancer immunotherapy S. Liu1 , K. Shen1 , Y. Song1 , I. Chen1 , P. Chong1 . 1 National Health Research Institutes, National Institute of Infectious Diseases and Vaccinology, Miaoli County, Taiwan Background: Cervical cancer is the life-threatened human papillomavirus (HPV) associated cancer. Although current prophylactic vaccines effectively prevent HPV infection and decrease incidence of cervical cancer, there
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are no therapeutic effects against pre-existing HPV infection and the tumor progression. Lipopeptides have been recognized that could be used to elicit cytotoxic T lymphocytes (CTLs) responses against viral diseases and cancer. In our previous study, we found that mono-palmitoylated peptide could enhance anti-tumor responses in the absence of adjuvant activity. Here, we are interested to investigate whether di-palmitoylate peptide with TLR2 agonist activity is able to induce better anti-tumor immunity and the reduction of tumor associate immunosuppressive factors facilitate anti-tumor effects of lipopeptide. Material and Methods: We synthesized a di-palmitic acid conjugated long peptide that contains a murine CTL epitope of HPV E749−57 (Pam2IDG). Bone marrow-derived dendritic cells (BMDCs) of TLRs knockout mice were stimulated with Pam2IDG to identify the co-receptor usage. The HPV16 E7expressed cell line, TC-1, was used to establish tumor model. To investigate whether depletion of immunosuppressive factors enhances therapeutic effects of lipopeptide, chrodonate, anti-IL10R or cyclooxygenase 2 (COX-2) inhibitor were used in this study. Results: Pam2IDG stimulated bone marrow-derived dendritic cells (BMDCs) maturation through TLR2/6. After immunization, Pam2IDG induced higher level of T cell responses than non-lipidated counterpart (IDG) immunization. In prophylactic model, Pam2IDG immunization completely inhibits tumor growth but IDG immunization. However, Pam2IDG immunization could not effectively inhibit established large tumor growth. However, we found that depletion of immunosuppressive factors is able to improve therapeutic effects of Pam2IDG. Conclusions: In conclusion, our data showed that therapeutic effects of Pam2IDG were improved by diminishing TAMs function, IL10 receptor blocking antibody or COX-2 inhibitor. No conflict of interest. 882 Early development of CD49d-high Th1 memory phenotype CD4+ T cells in the murine peritoneal cavity Y. Bae1 , H. Moon2 , J.G. Lee2 , S.H. Shin2 , C.H. Park2 , J.H. Lee2 , K.J. Kang3 , T.J. Kim2 . 1 Seoul National University College of Medicine, Parasitology, Seoul, South Korea, 2 Sungkyunkwan University School of Medicine, Immunobiology, Suwon, South Korea, 3 Sungkyunkwan University School of Medicine, Anatomy Cell Biology, Suwon, South Korea The peritoneal CD4+ T cells that may regulate the functions of natural antibodyproducing innate-like B-1 cells are not well characterized. In the pursuit of CD4+ T helper cells that interact B-1 cells, we identified a population of CD49d (integrin a4)high CD4+ T cells that constituted about half of CD4+ T cells in the peritoneal cavity, but not in other lymphoid compartments. The peritoneal CD49dhigh CD4+ T cells were CD1d-independent CD44high CD62Llow pro-inflammatory Th1 cells, which expressed CXCR3 and integrin a4b1, but not a4b7, and rapidly secreted IFNg, TNFa, and IL-2 upon stimulation with PMA and ionomycin or IL-12. They developed spontaneously as early as in 12 days of age, were maintained as a self-renewing population after the neonatal thymectomy, but proliferated to a lesser extent upon stimulation than the CD49dlow CD4+ T cells. Noticeably, the peritoneal CD49dhigh CD4+ T cells also showed partial features of follicular helper T cells such as the expression of PD-1, ICOS, IL-21, and CXCR5 and migration towards CXCL13. They supported IgM and IgG antibody production by B-1a cells as well as splenic B-2 cells more efficiently than peritoneal CD49dlow CD4+ T cells and splenic CD4+ T cells, demonstrating their potential functional role as follicular helper T cells. These data suggest that peritoneal CD49dlow CD4+ T cells are memory phenotype CD4+ T cells having bi-potential properties of Th1 and follicular helper T cells for both cellular and humoral immune responses. These innatelike helper T cells may have a role against peritoneal seeding of tumors and infections. No conflict of interest. 883 Serum-dependent processing of late apoptotic cells for enhanced efferocytosis Y. Liang1 , T. Arnold2 , A. Michlmayr2 , D. Rainprecht2 , B. Perticevic2 , A. Spittler2 , R. Oehler2 . 1 Medical University of Vienna, Wien, Austria, 2 Medical University of Vienna, Surgery, Wien, Austria Background: Binding of complement component C1q to the surface of dying cells facilitates their clearance by phagocytes in a process termed efferocytosis. Here we investigate during which phase of the apoptotic cell death progression the C1q binding takes place and wheater this is influenced by other serum factors. Material and Methods: Four different chemotherapeutic drugs (oxaliplatin, irinotecan, etoposide, and 5-FU) and UV-C irradiation were applied to induce apoptosis in human leukemic Jurkat T-cells. Apoptosis was confirmed by Western blotting and cell volume measurements. C1q binding and phagocytosis was assessed by flow cytometry. Results: Incubating apoptotic cells in normal human serum (NHS) resulted in the formation of a subpopulation of late apoptotic/secondary necrotic cells
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
875 Macrophage migration and polarization is triggered by oxygen gradient in glioblastoma 1 ´ , A. Corroyer-Dulmont1 , E. Petit1 , M. Bernaudin1 , M.M. Leblond1 , A.N. Gerault S. Valable1 . 1 UMR 6301 ISTCT-CNRS-CEA-Universite´ de Caen, CERVOxy Team, Caen, France
Background: Hypoxia is a common feature of tumors, highly pronounced in glioblastoma (GBM) and known to induce resistances to conventional therapies which may explains the poor life expectancy of GBM patients (Stupp et al., 2005). GBM growth is also associated with a marked inflammation of which a strong macrophage accumulation. It has been shown that macrophages found in the tumor can be polarized into a M1 (anti-tumoral) or a M2 (protumoral) phenotype (Murdoch et al., 2004). At present, it seems that tumor associated macrophages, found in patients with GBM, are M2 macrophages (Lu-Emerson et al., 2013). However, factors responsible for their migration and polarization are not yet identified. The aim of this study was, first, to evaluate the macrophage tropism toward tumor on two models of human GBM, well characterized in term of hypoxia degree (Corroyer-Dulmont et al., 2013), and secondly, to evaluate, in vitro and in vivo, the effect of hypoxia on macrophage polarization. Materials and Methods: Animals models consisted of orthotropic injection of U87 and U251 cells (5×104 /3 ml) into athymic nude rats. Hypoxia has been evaluated ex-vivo by pimonidazol immunostaining. Macrophages have been detected on free floating coronal slices thanks to a CD68 immunostaining and polarization toward M1 or M2 phenotype with iNOS and Arginase1 immunostainings, respectively. For in vitro experiments, primary cultures of macrophages have been prepared from mice bone marrow and cells have been exposed to 1% of oxygen and their polarization has been characterized by RT-qPCR and functional markers. Results: In both models, macrophages are localized inside the tumor, with a preferential migration toward U251 model which is the most hypoxic model (Corroyer-Dulmont et al., 2013). For this model, macrophages are localised preferentially in hypoxic areas of the tumor and express M2 markers, unlike macrophages found in the U87 model which exhibit M1 markers. In vitro, macrophages cultured in 1% of oxygen present a phenotype closed to M2 macrophages. Conclusion: In this study, using GBM models with various degree of oxygenation, we have shown that the number of macrophages found in the tumor is in closed relationship with the degree of hypoxia. We have also shown that hypoxia may trigger the acquisition of an M2 phenotype, suspected to contribute to tumor development. These results highlight a new function of hypoxia in the aggressiveness of GBM and may help to increase therapies for these tumors. No conflict of interest.
876 Allergen induced pulmonary inflammation enhances mammary tumor growth and metastasis: Role of CHI3L1 S. Libreros1 , R. Garcia-Areas1 , P. Robinson2 , A. Humbles3 , V. IragavarapuCharyulu1 . 1 Florida Atlantic University, Basic Science, Boca Raton, USA, 2 Florida Atlantic University, Clinical Science, Boca Raton, USA, 3 MedImmune LLC, Gaithersburg, USA Introduction: Metastasis is responsible for majority of cancer deaths and the incidence of metastasis to the lungs is higher in breast cancer patients with chronic pulmonary inflammatory illnesses. Lungs are a frequent site of inflammation. Pulmonary inflammatory illnesses have been increasing throughout the world with women more susceptible to pulmonary inflammatory diseases such as asthma. Patients with asthma demonstrate higher circulating levels of Chitinase-3 like 1 protein (CHI3L1). Recently, elevated serum levels of this glycoprotein have been correlated with poor prognosis and shorter survival of patients with metastatic breast cancer. Although a wealth of clinical evidence shows an association between CHI3L1 expression with tumor growth and short survival, little is known regarding the mechanisms leading to tumor metastasis. In this study we tested the hypothesis that pre-existing inflammation and CHI3L1 plays a role in enhancing growth of the primary tumor and metastasis to the lungs. Material and Methods: We use the combination of mouse models of allergic pulmonary inflammation and a well-established models breast cancer metastasis in wild type and CHI3L1 knockout mice. Results: Using 4T1 ragweed allergen sensitized mice we show accelerated tumor growth and metastasis, and overall decrease in survival. Allergen induced mice exhibit higher levels of proinflammatory/protumorigenic molecules CHI3L1, MMP-9, CXCL2/IL-8 and CCL2/MCP-1 in circulation and in pulmonary microenvironment. Accelerated tumor growth and the expression of CHI3L1 was also shown in two other mouse mammary tumor models 67NR and 4T07 induced for pulmonary inflammation. More significantly, ragweed allergen sensitized CHI3L1 knockout mice bearing mammary tumors had <50 fold decreased in tumor growth and metastasis with increased survival.
Discussion: Suppression of pulmonary inflammation and targeting CHI3L1 may be a valuable therapeutic strategy for breast cancer patients with pulmonary inflammatory illnesses diseases. No conflict of interest. 877 Changes of the white blood cells subsets in HER2 positive breast cancer patients treated with trastuzumab I. Matic1 , M. Grujic2 , B. Kolundzija1 , A. Damjanovic Velickovic1 , Z. Tomasevic3 , I. Bozovic Spasojevic3 , R. Dzodic4 , Z. Zdrale3 , Z. Juranic1 . 1 Institute of Oncology and Radiology of Serbia, Department of Experimental Oncology, Belgrade, Serbia, 2 Institute of Rheumatology, Third Hospital Department, Belgrade, Serbia, 3 Institute of Oncology and Radiology of Serbia, Department of Medical Oncology, Belgrade, Serbia, 4 Institute of Oncology and Radiology of Serbia, Department of Surgical Oncology, Belgrade, Serbia Background: The aim of research was to determine are there any changes in the percentages of lymphocytes, granulocytes, CD16+ and/or CD56+ lymphocytes in HER2+ breast cancer patients before or after treatment with trastuzumab in relation to response to therapy. Patients and Methods: This study involved 41 HER2+ breast cancer patients whose blood was analyzed before and two months after therapy with trastuzumab, applied alone or in combination with chemotherapeutics. Control group consisted of 41 healthy persons. The subsets of white blood cells were analyzed by flow cytometry. Results and Discussion: Our study demonstrates significantly lower percentages of the CD16+, CD56+ lymphocytes and CD16+CD56+ NK cells in breast cancer patients before therapy in comparison to healthy persons (p < 0.00001, p < 0.0004, p < 0.0001, respectively). Significantly lower percentage of lymphocytes (p < 0.000008), higher percentage of granulocytes (p < 0.007) and higher ratio of the percentage of granulocytes and percentage of lymphocytes (p < 0.00009) was observed in patients before treatment in relation to healthy controls. Clinical assessment showed progression of disease (PD) in 6 out of 41 patients. Surprisingly, patients who had PD after trastuzumab treatment had significantly higher percentage of CD16+ lymphocytes (p < 0.02), higher percentage of NK cells (p < 0.05) and also higher ratio of the percentage of granulocytes and percentage of lymphocytes (p < 0.04), before therapy, than patients without PD. While a significant decrease in the percentage of CD56+ lymphocytes in comparison to values before therapy was determined in patients without PD (p < 0.05), in patients with PD, after therapy, this phenomenon was more profound; more significant decrease in the percentage of CD56+ lymphocytes and NK cells (p < 0.004 and p < 0.007) was found. Conclusions: Results of our study indicate that tumor destroying action of trastuzumab is not occurring only through the known ADCC mechanism mediated by NK cells. This action could be accomplished through the killing of tumor cells by ROS released from trastuzumab activated CD64 receptors not only on monocytes but also on neutrophils, which could nonspecifically destroy neighboring CD56+ and CD16+ immune cells. This ROS-mediated trastuzumab toxicity seems to be more pronounced in patients with PD and could be the reason why pronounced decrease in the percentage of NK cells (and possibility for their antitumor action) in these patients was found. No conflict of interest. 879 Cryotherapy enhances the antigen-specific T cell immune responses and therapeutic antitumor effects generated by therapeutic HPV DNA vaccine S.Y. Lee1 , J.K. Sim1 , K.H. Min1 , G.Y. Hur1 , J.J. Shim1 , K.H. In1 , K.H. Kang1 , C.F. Hung2 , T.C. Wu2 . 1 Korea University Medical Center, Department of Internal Medicine, Seoul, Korea, 2 Johns Hopkins Medical Institutions, Department of Pathology, Baltimore, USA Background: Antigen-specific immunotherapy is an attractive approach for the treatment of cancers since it has the potency to specifically eradicate systemic tumors and control metastases without damaging normal cells. A favorable approach for antigen-specific immunotherapy is the use of DNA vaccines due to their safety, stability and ease of preparation. The therapeutic human papillomavirus (HPV) DNA vaccines encoding calreticulin (CRT) linked to HPV16 E7 antigen (CRT/E7) has showed the effects for enhancing the therapeutic HPV DNA vaccine potency. However, DNA vaccine suffers from insufficient immune response for antigen presenting cells and showed minimal clinical efficacies. Cryotherapy is an attractive therapeutic option for tumors due to its minimally invasive nature. And also cryotherapy is potentially immunogenic. Therefore, we explored the employment of cryotherapy in combination with CRT/E7 DNA vaccine in a preclinical model. Material and Methods: For in vivo tumor treatment, TC-1 tumor cells were subcutaneously injected into the left flank area of C57BL/6 mice. After 5 days, the mice were divided into four groups: group 1 received no treatment after the TC-1 tumor challenge, group 2 was treated with cryotherapy (20 seconds, two times), group 3 was immunized with the DNA vaccine, and group 4 was both immunized and treated with cryotherapy. Mice were monitored for E7-specific
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 CD8 (+) T cell immune responses, myeloid-derived suppressor cells (MDSC) and antitumor effects. In vitro assay, the secretion of HMGB1 protein in TC-1 cells after incubation in deep freezer was determined by Western blot. Results: We found that the combination of cryotherapy and CRT/E7 DNA vaccination significantly inhibited tumor growth and increased E7-specific CD8+ T cells in blood compared to CRT/E7 DNA vaccinated mice or mice treated with cryotherapy alone. Furthermore, combination treatment with cryotherapy and CRT/E7 vaccination reduces immunosuppressive MDSC. In vitro assay, we found that treatment with deep freezing and thawing led to up-regulation of HMGB1 in E7-expressing TC-1 tumor cells, rendering the tumor cells more susceptible to APCs and E7-specific CD8+ T cell-mediated killing. Conclusion: Our data indicate that cryotherapy can enhance therapeutic HPV DNA vaccine potency in generating improved antigen-specific immune responses and antitumor effects. These findings have important implications for future clinical translation. No conflict of interest. 880 Interactions of immunoglobulin-like cell adhesion molecule hepaCAM with CD137 in cell–matrix interactions, cell motility and escape from immune surveillance in Hodgkin Lymphoma J. Seah1 . 1 National University Of Singapore, Physiology, Singapore, Singapore Background: The study aims to determine how the interactions between hepaCAM (a cell adhesion molecule) and CD137 (a member of TNFR superfamily 9) contributes to cell adhesion, cell–extracellular matrix interactions and recruitment of leukocytes aiding in escape from immune surveillance in Hodgkin Lymphoma. Materials and Methods: Western blot analysis, confocal microscopy and flow cytometry were used to determine the expression and localisation of hepaCAM and CD137 in human Reed Sternberg (HRS) cell lines of Hodgkin Lymphoma. Co-immunoprecipitation was used to shed light on the physical interactions between hepaCAM and CD137 in HRS cells, and to determine the regions of hepaCAM responsible for interactions with CD137. Adhesion and cell spreading assays, coupled with neutralizing antibodies targeting hepaCAM, were used to determine the role of hepaCAM in cell migration and cell–extracellular matrix interactions in HRS cells. Purified hepaCAM proteins were used to determine the possible role of hepaCAM in binding to activated peripheral blood mononuclear cells (PBMCs) resulting in subsequent inhibition of activation of immune cells involving CD137. It may contribute to the escape of HRS cells from immune surveillance. Results: As CD137 is ectopically expressed on most classical Hodgkin Lymphoma cases and appeared to play a role in escape from immune surveillance in HRS cells, the interacting partners of CD137 may play a role in the escape mechanism as well. In addition, immunoglobulin-like cell adhesion molecules have been known to play roles in cancer progression and work in concert with members of TNFR superfamily. Firstly, the expression and localisation of hepaCAM on cell surfaces was found to be CD137 dependent in HRS cell lines. This indicates the possible interactions of hepaCAM and CD137 in HRS cells. Secondly, co-immunoprecipitation confirmed that hepaCAM and CD137 interacted with each other using HRS cell lines. The cytoplasmic tail of hepaCAM proved to be important in maintaining the interactions with CD137. Thirdly, neutralising hepaCAM with antibodies appeared to result in reduced cell adhesion and cell spreading on fibronectin which may indicate the importance of hepaCAM interactions with extracellular matrix in HRS cells. Lastly, hepaCAM was found to bind preferentially to activated PBMCs. Taken together with the expression, localisation and interactions results obtained, this data indicated the possibility of hepaCAM working in concert with CD137 in recruitment and binding of leukocytes resulting in inhibition of activation of PBMCs and escape from immune surveillance. Conclusion: The expression and localisation of hepaCAM on cell surfaces of HRS cells were CD137 dependent. The interactions between hepaCAM and CD137 may work in concert to affect cell adhesion, cell–extracellular matrix interactions and binding of leukocytes aiding in escape from immune surveillance. No conflict of interest. 881 Depletion of tumor-associated macrophages enhances peptide-based cancer immunotherapy S. Liu1 , K. Shen1 , Y. Song1 , I. Chen1 , P. Chong1 . 1 National Health Research Institutes, National Institute of Infectious Diseases and Vaccinology, Miaoli County, Taiwan Background: Cervical cancer is the life-threatened human papillomavirus (HPV) associated cancer. Although current prophylactic vaccines effectively prevent HPV infection and decrease incidence of cervical cancer, there
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are no therapeutic effects against pre-existing HPV infection and the tumor progression. Lipopeptides have been recognized that could be used to elicit cytotoxic T lymphocytes (CTLs) responses against viral diseases and cancer. In our previous study, we found that mono-palmitoylated peptide could enhance anti-tumor responses in the absence of adjuvant activity. Here, we are interested to investigate whether di-palmitoylate peptide with TLR2 agonist activity is able to induce better anti-tumor immunity and the reduction of tumor associate immunosuppressive factors facilitate anti-tumor effects of lipopeptide. Material and Methods: We synthesized a di-palmitic acid conjugated long peptide that contains a murine CTL epitope of HPV E749−57 (Pam2IDG). Bone marrow-derived dendritic cells (BMDCs) of TLRs knockout mice were stimulated with Pam2IDG to identify the co-receptor usage. The HPV16 E7expressed cell line, TC-1, was used to establish tumor model. To investigate whether depletion of immunosuppressive factors enhances therapeutic effects of lipopeptide, chrodonate, anti-IL10R or cyclooxygenase 2 (COX-2) inhibitor were used in this study. Results: Pam2IDG stimulated bone marrow-derived dendritic cells (BMDCs) maturation through TLR2/6. After immunization, Pam2IDG induced higher level of T cell responses than non-lipidated counterpart (IDG) immunization. In prophylactic model, Pam2IDG immunization completely inhibits tumor growth but IDG immunization. However, Pam2IDG immunization could not effectively inhibit established large tumor growth. However, we found that depletion of immunosuppressive factors is able to improve therapeutic effects of Pam2IDG. Conclusions: In conclusion, our data showed that therapeutic effects of Pam2IDG were improved by diminishing TAMs function, IL10 receptor blocking antibody or COX-2 inhibitor. No conflict of interest. 882 Early development of CD49d-high Th1 memory phenotype CD4+ T cells in the murine peritoneal cavity Y. Bae1 , H. Moon2 , J.G. Lee2 , S.H. Shin2 , C.H. Park2 , J.H. Lee2 , K.J. Kang3 , T.J. Kim2 . 1 Seoul National University College of Medicine, Parasitology, Seoul, South Korea, 2 Sungkyunkwan University School of Medicine, Immunobiology, Suwon, South Korea, 3 Sungkyunkwan University School of Medicine, Anatomy Cell Biology, Suwon, South Korea The peritoneal CD4+ T cells that may regulate the functions of natural antibodyproducing innate-like B-1 cells are not well characterized. In the pursuit of CD4+ T helper cells that interact B-1 cells, we identified a population of CD49d (integrin a4)high CD4+ T cells that constituted about half of CD4+ T cells in the peritoneal cavity, but not in other lymphoid compartments. The peritoneal CD49dhigh CD4+ T cells were CD1d-independent CD44high CD62Llow pro-inflammatory Th1 cells, which expressed CXCR3 and integrin a4b1, but not a4b7, and rapidly secreted IFNg, TNFa, and IL-2 upon stimulation with PMA and ionomycin or IL-12. They developed spontaneously as early as in 12 days of age, were maintained as a self-renewing population after the neonatal thymectomy, but proliferated to a lesser extent upon stimulation than the CD49dlow CD4+ T cells. Noticeably, the peritoneal CD49dhigh CD4+ T cells also showed partial features of follicular helper T cells such as the expression of PD-1, ICOS, IL-21, and CXCR5 and migration towards CXCL13. They supported IgM and IgG antibody production by B-1a cells as well as splenic B-2 cells more efficiently than peritoneal CD49dlow CD4+ T cells and splenic CD4+ T cells, demonstrating their potential functional role as follicular helper T cells. These data suggest that peritoneal CD49dlow CD4+ T cells are memory phenotype CD4+ T cells having bi-potential properties of Th1 and follicular helper T cells for both cellular and humoral immune responses. These innatelike helper T cells may have a role against peritoneal seeding of tumors and infections. No conflict of interest. 883 Serum-dependent processing of late apoptotic cells for enhanced efferocytosis Y. Liang1 , T. Arnold2 , A. Michlmayr2 , D. Rainprecht2 , B. Perticevic2 , A. Spittler2 , R. Oehler2 . 1 Medical University of Vienna, Wien, Austria, 2 Medical University of Vienna, Surgery, Wien, Austria Background: Binding of complement component C1q to the surface of dying cells facilitates their clearance by phagocytes in a process termed efferocytosis. Here we investigate during which phase of the apoptotic cell death progression the C1q binding takes place and wheater this is influenced by other serum factors. Material and Methods: Four different chemotherapeutic drugs (oxaliplatin, irinotecan, etoposide, and 5-FU) and UV-C irradiation were applied to induce apoptosis in human leukemic Jurkat T-cells. Apoptosis was confirmed by Western blotting and cell volume measurements. C1q binding and phagocytosis was assessed by flow cytometry. Results: Incubating apoptotic cells in normal human serum (NHS) resulted in the formation of a subpopulation of late apoptotic/secondary necrotic cells