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IL-17C promotes tumor-associated inflammation and lung tumor growth
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
Sunday 10 July 2016 Poster Session
Tumour Immunology I 900 A combination of T regulatory cell-specific markers to determine the expanded subsets in cancer patients E. Elkord1,2,3 , M. Abd Al Samid2 , B. Chaudhary2 , K. Yazan3 , B. Ammori3 . 1 United Arab Emirates University, Medical Microbiology & Immunology, Al Ain, U.A.E., 2 University of Salford, Biomedical Research Institute, Manchester, United Kingdom, 3 University of Manchester, Institute of Cancer Sciences, Manchester, United Kingdom Background: Regulatory T cells (Tregs) comprise numerous heterogeneous subsets with distinct phenotypic and functional features. Identifying Treg markers is critical to investigate the role and clinical impact of various Treg subsets in pathological settings, and also for developing more effective immunotherapies. Material and Methods: We investigated different Treg subsets as defined by expression of FoxP3 and Helios transcription factors and GARP and LAP immunosuppressive markers in peripheral blood samples isolated from patients with pancreatic cancer (PC) and liver metastases from colorectal cancer (LICRC), and compared their levels to control groups. Results: We have recently shown that non-activated FoxP3−Helios+ and activated FoxP3+/−Helios+ CD4+ T cells express GARP/LAP immunosuppressive markers in healthy donors. In this study, we report similar observations in the peripheral blood of patients with pancreatic cancer (PC) and liver metastases from colorectal cancer (LICRC). Comparing levels of different Treg subpopulations in cancer patients and controls, we report that in PC patients, and unlike LICRC patients, there was no increase in Treg levels as defined by FoxP3 and Helios. However, defining Tregs based on GARP/LAP expression showed that FoxP3−LAP+ Tregs in non-activated and activated settings, and FoxP3+Helios+GARP+LAP+ activated Tregs were significantly increased in both groups of patients, compared with controls. Additionally, GARP−/+LAP+ CD4+ T cells made IL-10, and not IFN-g, and levels of IL-10-secreting CD4+ T cells were elevated in LICRC patients, especially with higher tumor staging. Conclusions: This work implies that a combination of Treg-specific markers could be used to more accurately determine expanded Treg subsets and to understand their contribution in cancer settings, and investigations of Treg levels in different cancers should consider diverse Treg-related markers such as GARP, LAP, Helios, and others and not only FoxP3 as a sole Treg-specific marker. No conflict of interest. 901 Development of an autoimmune-mediated strategy for bladder cancer vaccination in mice K. Izgi1 , B. Iskender2 , H.B. Ulusoy3 , H. Canatan2 . 1 Erciyes University School of Medicine, Medical Biochemistry, Kayseri, Turkey, 2 Erciyes University School of Medicine, Medical Biology, Kayseri, Turkey, 3 Erciyes University School of Medicine, Medical Pharmacology, Kayseri, Turkey Introduction: The more specific and effective therapies are intended to develop for bladder cancer due to reasons of superficial bladder cancer treated with BCG immunotherapy inducing non-specific immune response to tumor, having a high rate of recurrence and high toxicity. Organ or tissue-specific antigens produced by normal tissue or by cancer cells can be selected for cancer immunotherapy to target the tumor. In our previous study we have induced T-cell mediated bladder specific autoimmunity by targeting the bladder-specific Uroplakin 3A (UPK3A) protein. UPK3A is a well-chosen target to develop autoimmune response against bladder cancer since the antigen is also expressed in bladder tumor. In this study, we developed robust immune response in BALB/c mice using the well-characterized antigenic UPK3A 65−84 peptide in conjugation with an immunogenic carrier protein. We immunized the mice bearing bladder cancer to analyze the tumor reduction. Material and Method: In combination with the peptide, we utilised either CFA (Complete Freund’s Adjuvant) or CpG (Cytosine-phosphate-guanine) as effective adjuvants in order to break the tumor tolerance in OH-BBN induced bladder cancer model. The immune response evoked by UPK3A 65−84 peptide using two different adjuvants was compared profoundly with the detection of immune cells’ responses change in cytokine profile and immune cell populations in mice with bladder cancer. Results and Discussion: We demonstrated that CpG combined with KLH (Keyhole Limpet Hemocyanin) conjugated peptide antigen (UPK3A 65−84) promote robust immune response via induction of higher IL-2, IFN-g, TNFa, IL-17 production and activation of more immune cells (CD4+ T cell, CD8+ T cells, NK cells CD11b, CD45) than the KLH conjugated peptide antigen (UPK3A 65−84) with CFA. We found that immunoreactivity against Uroplakin 3A derived peptide provides substantial protection and therapy
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against growth of tumors in OH-BBN induced bladder cancer mouse model. Thus, uroplakin 3A derived peptide vaccination may provide safe and effective protection against the development of bladder cancer. Conclusion: CFA or CpG as an adjuvant combined with KLH conjugated peptide antigen (UPK3A 65−84) reduced tumor growth more than 50% in preclinical models of bladder cancer. This peptide is a well-chosen target for the development of bladder cancer immunotherapy. Acknowledgement: This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK), Grant Number: 113S927. No conflict of interest. 902 IL-17C promotes tumor-associated inflammation and lung tumor growth C. Jungnickel1 , L. Bittigkoffer1 , A. Kamyschnikow1 , C. Herr1 , R. Bals1 , C. Beisswenger1 . 1 University of the Saarland, Internal Medicine V, Homburg/Saar, Germany Background: Smoking is the main risk factor for the development of lung cancer and chronic obstructive lung disease (COPD). Lungs of smokers and COPD patients are frequently colonized with bacterial pathogens. The epithelial cytokine IL-17C mediates the expression of proinflammatory cytokines and chemokines. Therefore, we characterized the function of IL-17C in a preclinical mouse model of tumor growth. Material and Methods: Lewis lung carcinoma (LLC) cells were injected to initiate the growth of tumors in the lungs of wild-type (WT) mice and mice deficient for IL-17C. Mice were exposed to air or cigarette smoke (CS) in combination with and without exposure to inactivated nontypeable Haemophilus influenzae (NTHi) 7 days after the LLC injection for additional 7 days. Results: Tumor proliferation and growth were significantly decreased in IL-17C deficient mice exposed to NTHi and CS/NTHi compared to WT mice. Numbers of neutrophils and TNF-a expression were significantly decreased in tumor tissue of IL-17C deficient mice. IL-17C significantly increased the expression of the neutrophil recruiting cytokines KC and MIP-2 in LLC cells in vitro. Conclusion: Our data indicate that IL-17C regulates the tumor microenvironment, including the recruitment of tumor-associated neutrophils, and tumor growth in inflamed airways. No conflict of interest. 903 Rewiring the cytokine network in melanoma R. Nagaraju1 , C. Dee1 , J. Barriuso1 , H. Young1 , M. Abdelmouti1 , A. Hurlstone1 . 1 University of Manchester, Faculty of Life sciences, Manchester, United Kingdom Malignant melanoma is one of the most aggressive types of malignancies in humans resulting in 50,000 deaths worldwide annually. Fortuitously, malignant melanoma is highly immunogenic and it is now well established that the host immune system can detect and kill melanoma. T helper type-1 lymphocytes (TH1) play a pivotal role in inducing and maintaining anti-tumour immune responses. In melanoma patients, it has been observed that higher number of TH1 cells in the tumour micro-environment leads to better prognosis. Here, using an autochthonous zebrafish melanoma model, we report that by forcing the expression of interferon gamma (IFNg) specifically in the tumour microenvironment, thereby potentially enhancing TH1 differentiation, we induced an inflammatory immune response leading to complete tumour regression. Using transposon mediated BAC transgenesis, we have generated a Tg(cd4:mcherry) reporter line labelling CD4+ cells in zebrafish. By driving Human NRASQ61L in zebrafish melanocytes we have modelled melanoma in zebrafish and later when a tumour is developed we forced the expression of IFNg in melanoma cells using a (Z)-4-hydroxytamoxifen (4-OHT) inducible LSL/CreERT2 system driven by mitfa minimal promoter. Experiments were performed using animals with and without CreERT2. Following 4-OHT administration, we observed loss of pigmentation in all the IFNg induced tumours by 2 weeks post induction which gradually increased over the study period whereas no loss of pigmentation was observed in control tumours (no IFNg induction) throughout the study period. At 9 weeks post induction of IFNg, the study was terminated and tumours were histologically analysed. In tumours were IFNg was induced, we observed that the tumour tissue was now replaced with fibrotic tissue accompanied by a marked lymphocyte infiltration and patches of necrosis, while we observed no signs of tumour regression in control tumours. However, new tumour nodules were also observed to develop in the vicinity of regressing nodules which are unpigmented and potentially hypo-immunogenic. Local induction of IFNg in a zebrafish melanoma micro-environment can induce an inflammatory immune response leading to complete tumour regression. Although preliminary, our data is very promising as it will now open new avenues for developing combinatorial therapies in melanoma. Using a high throughput in-vivo model system such as zebrafish, it is an exciting
Sunday 10 July 2016 Poster Session
Tumour Immunology I 900 A combination of T regulatory cell-specific markers to determine the expanded subsets in cancer patients E. Elkord1,2,3 , M. Abd Al Samid2 , B. Chaudhary2 , K. Yazan3 , B. Ammori3 . 1 United Arab Emirates University, Medical Microbiology & Immunology, Al Ain, U.A.E., 2 University of Salford, Biomedical Research Institute, Manchester, United Kingdom, 3 University of Manchester, Institute of Cancer Sciences, Manchester, United Kingdom Background: Regulatory T cells (Tregs) comprise numerous heterogeneous subsets with distinct phenotypic and functional features. Identifying Treg markers is critical to investigate the role and clinical impact of various Treg subsets in pathological settings, and also for developing more effective immunotherapies. Material and Methods: We investigated different Treg subsets as defined by expression of FoxP3 and Helios transcription factors and GARP and LAP immunosuppressive markers in peripheral blood samples isolated from patients with pancreatic cancer (PC) and liver metastases from colorectal cancer (LICRC), and compared their levels to control groups. Results: We have recently shown that non-activated FoxP3−Helios+ and activated FoxP3+/−Helios+ CD4+ T cells express GARP/LAP immunosuppressive markers in healthy donors. In this study, we report similar observations in the peripheral blood of patients with pancreatic cancer (PC) and liver metastases from colorectal cancer (LICRC). Comparing levels of different Treg subpopulations in cancer patients and controls, we report that in PC patients, and unlike LICRC patients, there was no increase in Treg levels as defined by FoxP3 and Helios. However, defining Tregs based on GARP/LAP expression showed that FoxP3−LAP+ Tregs in non-activated and activated settings, and FoxP3+Helios+GARP+LAP+ activated Tregs were significantly increased in both groups of patients, compared with controls. Additionally, GARP−/+LAP+ CD4+ T cells made IL-10, and not IFN-g, and levels of IL-10-secreting CD4+ T cells were elevated in LICRC patients, especially with higher tumor staging. Conclusions: This work implies that a combination of Treg-specific markers could be used to more accurately determine expanded Treg subsets and to understand their contribution in cancer settings, and investigations of Treg levels in different cancers should consider diverse Treg-related markers such as GARP, LAP, Helios, and others and not only FoxP3 as a sole Treg-specific marker. No conflict of interest. 901 Development of an autoimmune-mediated strategy for bladder cancer vaccination in mice K. Izgi1 , B. Iskender2 , H.B. Ulusoy3 , H. Canatan2 . 1 Erciyes University School of Medicine, Medical Biochemistry, Kayseri, Turkey, 2 Erciyes University School of Medicine, Medical Biology, Kayseri, Turkey, 3 Erciyes University School of Medicine, Medical Pharmacology, Kayseri, Turkey Introduction: The more specific and effective therapies are intended to develop for bladder cancer due to reasons of superficial bladder cancer treated with BCG immunotherapy inducing non-specific immune response to tumor, having a high rate of recurrence and high toxicity. Organ or tissue-specific antigens produced by normal tissue or by cancer cells can be selected for cancer immunotherapy to target the tumor. In our previous study we have induced T-cell mediated bladder specific autoimmunity by targeting the bladder-specific Uroplakin 3A (UPK3A) protein. UPK3A is a well-chosen target to develop autoimmune response against bladder cancer since the antigen is also expressed in bladder tumor. In this study, we developed robust immune response in BALB/c mice using the well-characterized antigenic UPK3A 65−84 peptide in conjugation with an immunogenic carrier protein. We immunized the mice bearing bladder cancer to analyze the tumor reduction. Material and Method: In combination with the peptide, we utilised either CFA (Complete Freund’s Adjuvant) or CpG (Cytosine-phosphate-guanine) as effective adjuvants in order to break the tumor tolerance in OH-BBN induced bladder cancer model. The immune response evoked by UPK3A 65−84 peptide using two different adjuvants was compared profoundly with the detection of immune cells’ responses change in cytokine profile and immune cell populations in mice with bladder cancer. Results and Discussion: We demonstrated that CpG combined with KLH (Keyhole Limpet Hemocyanin) conjugated peptide antigen (UPK3A 65−84) promote robust immune response via induction of higher IL-2, IFN-g, TNFa, IL-17 production and activation of more immune cells (CD4+ T cell, CD8+ T cells, NK cells CD11b, CD45) than the KLH conjugated peptide antigen (UPK3A 65−84) with CFA. We found that immunoreactivity against Uroplakin 3A derived peptide provides substantial protection and therapy
S209
against growth of tumors in OH-BBN induced bladder cancer mouse model. Thus, uroplakin 3A derived peptide vaccination may provide safe and effective protection against the development of bladder cancer. Conclusion: CFA or CpG as an adjuvant combined with KLH conjugated peptide antigen (UPK3A 65−84) reduced tumor growth more than 50% in preclinical models of bladder cancer. This peptide is a well-chosen target for the development of bladder cancer immunotherapy. Acknowledgement: This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK), Grant Number: 113S927. No conflict of interest. 902 IL-17C promotes tumor-associated inflammation and lung tumor growth C. Jungnickel1 , L. Bittigkoffer1 , A. Kamyschnikow1 , C. Herr1 , R. Bals1 , C. Beisswenger1 . 1 University of the Saarland, Internal Medicine V, Homburg/Saar, Germany Background: Smoking is the main risk factor for the development of lung cancer and chronic obstructive lung disease (COPD). Lungs of smokers and COPD patients are frequently colonized with bacterial pathogens. The epithelial cytokine IL-17C mediates the expression of proinflammatory cytokines and chemokines. Therefore, we characterized the function of IL-17C in a preclinical mouse model of tumor growth. Material and Methods: Lewis lung carcinoma (LLC) cells were injected to initiate the growth of tumors in the lungs of wild-type (WT) mice and mice deficient for IL-17C. Mice were exposed to air or cigarette smoke (CS) in combination with and without exposure to inactivated nontypeable Haemophilus influenzae (NTHi) 7 days after the LLC injection for additional 7 days. Results: Tumor proliferation and growth were significantly decreased in IL-17C deficient mice exposed to NTHi and CS/NTHi compared to WT mice. Numbers of neutrophils and TNF-a expression were significantly decreased in tumor tissue of IL-17C deficient mice. IL-17C significantly increased the expression of the neutrophil recruiting cytokines KC and MIP-2 in LLC cells in vitro. Conclusion: Our data indicate that IL-17C regulates the tumor microenvironment, including the recruitment of tumor-associated neutrophils, and tumor growth in inflamed airways. No conflict of interest. 903 Rewiring the cytokine network in melanoma R. Nagaraju1 , C. Dee1 , J. Barriuso1 , H. Young1 , M. Abdelmouti1 , A. Hurlstone1 . 1 University of Manchester, Faculty of Life sciences, Manchester, United Kingdom Malignant melanoma is one of the most aggressive types of malignancies in humans resulting in 50,000 deaths worldwide annually. Fortuitously, malignant melanoma is highly immunogenic and it is now well established that the host immune system can detect and kill melanoma. T helper type-1 lymphocytes (TH1) play a pivotal role in inducing and maintaining anti-tumour immune responses. In melanoma patients, it has been observed that higher number of TH1 cells in the tumour micro-environment leads to better prognosis. Here, using an autochthonous zebrafish melanoma model, we report that by forcing the expression of interferon gamma (IFNg) specifically in the tumour microenvironment, thereby potentially enhancing TH1 differentiation, we induced an inflammatory immune response leading to complete tumour regression. Using transposon mediated BAC transgenesis, we have generated a Tg(cd4:mcherry) reporter line labelling CD4+ cells in zebrafish. By driving Human NRASQ61L in zebrafish melanocytes we have modelled melanoma in zebrafish and later when a tumour is developed we forced the expression of IFNg in melanoma cells using a (Z)-4-hydroxytamoxifen (4-OHT) inducible LSL/CreERT2 system driven by mitfa minimal promoter. Experiments were performed using animals with and without CreERT2. Following 4-OHT administration, we observed loss of pigmentation in all the IFNg induced tumours by 2 weeks post induction which gradually increased over the study period whereas no loss of pigmentation was observed in control tumours (no IFNg induction) throughout the study period. At 9 weeks post induction of IFNg, the study was terminated and tumours were histologically analysed. In tumours were IFNg was induced, we observed that the tumour tissue was now replaced with fibrotic tissue accompanied by a marked lymphocyte infiltration and patches of necrosis, while we observed no signs of tumour regression in control tumours. However, new tumour nodules were also observed to develop in the vicinity of regressing nodules which are unpigmented and potentially hypo-immunogenic. Local induction of IFNg in a zebrafish melanoma micro-environment can induce an inflammatory immune response leading to complete tumour regression. Although preliminary, our data is very promising as it will now open new avenues for developing combinatorial therapies in melanoma. Using a high throughput in-vivo model system such as zebrafish, it is an exciting