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«Myeloid Derived Suppressor Cells in Dogs with Cancer: Phenotype, Function and Clinical Implications A DISSERTATION SUBMITTED TO THE FACULTY OF THE ...»

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Myeloid Derived Suppressor Cells in Dogs with Cancer:

Phenotype, Function and Clinical Implications

A DISSERTATION

SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL

OF THE UNIVERSITY OF MINNESOTA

BY

Michelle Rodrigues Goulart

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

Elizabeth Pluhar, D.V.M., Ph.D Advisor June 2014 @ Michelle R. Goulart 2014 Acknowledgements First and foremost I want to thank God for blessing me with amazing opportunities in life and my mother for giving me all of the support and encouragement to move out and pursue my professional achievements. Life could have been easier and joyful in Brazil, but thank you for understanding why I took the road less traveled.

Dr. Pluhar my advisor, thank for seeing the potential in me. I am extremely grateful for the opportunity you gave me and for your contributions to my education and further professional development.

Dr. Ohlfest my co-advisor, thank you for the amazing mentorship and for inspiring me to build my skills as an investigator. I will never forget the day that you gave me the Gabrilovich paper “Myeloid-derived suppressor cells as regulators of the immune system” and said: “Michelle I want you to read this”.

Dr. Olin, my co-advisor, thank you for sharing your knowledge with me in the first years of my lab training, especially with the flow cytometry that we both very much like, and for all the supporting and guidance at your new lab in this past year. Lastly thank you for the laughs we shared during all of these years.

Next time I go to a conference, I promise I will never miss my poster presentation.

Drs. Pennell and Modiano my committee members, thank you. I am fortunate and deeply grateful to have you as members of my committee and helping me with your scientific knowledge.

I also want to thank Dr. Gerry O’Sullivan for the histopathological analysis of canine gliomas, my friend Amber Winter from the Canine Investigation Center and members of the Veterinary Oncology Services at the UMN Veterinary Medical Center who helped me collect the blood samples analyzed in the study. I also need to thank all the members of the Ohlfest and Olin lab with whom I spent most of my time, especially Brian Andersen, Flavia Popescu, Zoe Zhang, Charlie Seiler, Zhengming Xiong, Adam Litterman, David Zellmer and Nate Waldron.

Thank you for the support, friendship and encouragement.

i I want to thank my lovely and caring family that though far away is always present, Rita and David Wilson - the family I lived with during my first year in the U.S and all my friends for their friendship, patience, understanding, motivation and joy.

Lastly but not least, I want to thank my dogs Mini and Gizmo. Thank you for making my life complete.

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This thesis is dedicated to all of man's best friends. For the true love and friendship that only they can give to us. Dogs are pure and true and fill our hearts with joy. They know how to deeply touch our souls through simple but unforgettable demonstrations of love, friendship, affection and companionship… and all we wanted is that they be immortal.

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Acknowledgements..………………………………………………………………….…i Dedication……………………………………………………………………………….iii Table of contents.….……………………………..……………………………….……iv Abstract…………………………………………………………………………….…….vi List of Tables …………………...………………………………………………...…...viii List of Figures…………………...………………………………………………………ix List of Abbreviations………………………………………………………………........xi Chapter I: Introduction…………………………………………………………….……1 Overview on cancer in dogs and the canine cancer model……………2 Cancer immunosurveillance and tumor-immune cell interactions..….. 3 Tumor-immunosuppression……….…………………………………..…..5 Origins of myeloid-derived suppressor cells (MDSC)

Accumulation of myeloid-derived suppressor cells in cancer..………...9 Characteristics of MDSC – phenotype and subsets…………………..14 Mechanisms of myeloid-derived suppressor cell immunosuppression………………………………………………………17 MDSC as therapeutic targets….………………………………………...19 Chapter II: Identification of Myeloid Derived Suppressor Cells in Dogs with

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Summary…………..………………………………………..……………….....24 Introduction……………………………………………………………………..26 Materials and Methods………………………………………………….....….28 Results……………...……………………………………………………….…..35 Discussion……………………………………………………………………….38 Chapter III: Myeloid-derived suppressor cells accumulation in the peripheral blood of dogs with glioma……………………………………………………………..58 Summary…..……………………………………………………………………59 Introduction………………………………………………………………….….60 Materials and methods………………………………………………………..62 Results





Discussion……………………………….……………………………………..70 Chapter IV: Conclusions and Future Directions……………………….………..…84 Bibliography…………………………………………………………………….….…..99

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Myeloid-derived suppressor cells comprise phenotypically heterogeneous population of myeloid cells at different stages of differentiation endowed with potent immunosuppressive activity. Abnormal accumulation of MDSC in tumor models and cancer patients produce profound immune suppression, severely impairing T cell antitumor immunity, contributing to angiogenesis, cell invasion and metastasis, and constitute a major hurdle in achieving successful immunebased therapies.

Understanding the mechanism that drives MDSC expansion and enhances function in humans and dogs is crucial for the development of efficacious immunotherapy.

Studies in dogs with several tumor types, including sarcoma, carcinomas, mast cell tumors and gliomas confirmed MDSC expansion in the peripheral blood of dog cancer patients. MDSC have been identified in dogs using the combination of three-marker phenotype CD11b+CD14-MHCII-cells for granulocytic and CD11b+CD14+MHCII-cells for monocytic subsets. Granulocytic MDSC accumulated in the peripheral blood of dogs with advanced sarcoma, carcinomas and mast cell tumors, co-purified with peripheral blood mononuclear cell (PBMC) fraction and expressed polymorphic mononuclear morphology. This subset of cells showed the ability to efficiently inhibit T cell proliferation and IFN-γ secretion of autologous T cells, as well as allogenic T cells from healthy dogs, and expressed ARG1, iNOS2, TGF-β and IL-10. Monocytic MDSC also

–  –  –

accumulated in the peripheral blood of dogs with glioma. Elevated levels of arginase activity found in the serum of dogs with glioma could potentially be due to the presence of elevated numbers of MDSC. Evaluation of the anti-mouse Gr1 antibody for MDSC staining and identification revealed that does not cross react and therefore is not suitable for canine cells.

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Table 1. Characteristics of dogs with cancer in the study……………………….

.39 Table 2. Characteristics of healthy dogs in the study……………………………..40 Table S1. Summary data for dogs with advanced stage or metastatic tumors…50 Table S2. Summary data for dogs with early stage or non-metastatic tumors…51 Table S3. Table of canine patient samples and the experiments in which the PBMCs were used……………….…………………………………………………....52 Table S4. Primer sequences for genes evaluated by semi-quantitative PCR.…53 Chapter III Table 1. Summary data of dogs in the study………………………………...……..76

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Figure 1. Immunophenotyping gating strategy and morphological analysis for MDSC identification in peripheral blood of dogs……………………………………41 Figure 2.

Percentage of circulating CD11b+CD14-MHCII- cells in dogs with cancer correlates with clinical tumor stage…………..………………………….….42 Figure 3. CD11b+CD14-MHCII- cells suppress T cell proliferation and cytokine elaboration………………………...…………………………………………………..43 Figure 4. CD11b+CD14-MHCII- cells suppress T cell proliferation…………….…44 Figure 5. CD11b+CD14-MHCII- cells express MDSC-derived immunosuppressive factors……………...……………………………………………………………………45 Figure S1. Mouse anti-CD11b and Gr-1 antibodies cross-react with canine sample………………………………………………………………………………..…46 Figure S2. CD11b+CD14+MHCII- cells demonstrate ability to suppress T cell proliferation………………………………………………………..……………………47 Figure S3. Frequency of MDSCs measured was not significantly altered by cryopreservation……………………………………………..………………………...48 Figure S4. No significant effect of pretreatment on MDSC burden……………...49 Chapter III Figure 1. Flow cytometry analysis for identification of MDSC in dogs with glioma…………………………………………………………………………………...77 Figure 2. Dogs with glioma have significant increased percentage of CD11b+CD14+MHCII- cells……………………………………………………………78 ix Figure 3. Percentage of CD11b+CD14+MHCII- cells is significantly increased in all of the glioma subtypes………………………………………………….…….……79 Fig 4. Dogs with glioma have elevated levels of serum arginase activity…….…80 Fig 5. Cells isolated with Gr-1 magnetic-coated beads inhibit T cell proliferation in health PBMCs and autologous PBMCs.………………………………………….81 Fig 6. Gr-1 antibody binding of canine PBMCs is nonspecific……………………82 Fig 5. Anti-mouse Gr-1 antibody failed to cross-react with canine tissues……...83

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FoxP3 Forkhead box protein 3 G-CSF granulocyte colony stimulating factor GM-CSF granulocyte macrophage colony stimulating factor G-MDSC granulocytic myeloid-derived suppressor cells HLA human leukocyte antigen HPC hematopoietic progenitor cell HSCs hematopoietic stem cells IFNα interferon-alpha IFNγ interferon-gamma

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M-CSF macrophage colony stimulating factor MDSC myeloid-derived suppressor cells MHCI major histocompatibility complex I M-MDSC monocytic myeloid-derived suppressor cells MMPs matrix metalloproteinases NSAID non-steroidal anti-inflammatory drug

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TGF-β transforming growth factor beta TLR tool-like receptor TME tumor microenvironment Treg T regulatory cells TAA tumor-associated antigens TNF tumor necrosis factor

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Introduction Overview on cancer in dogs and the canine cancer model Approximately four million dogs are diagnosed with cancer each year in the United States, making cancer the leading cause of death in adult dogs and the major health care concern of pet owners in the United States, Australia, Japan and Europe (1).

Naturally occurring canine malignances often share a wide variety of biologic and clinical features often observed with human cancers. There are many similarities between human and canine malignances including spontaneous neoplasm development, tumor biology, genetics, incidence rates, histological appearance, and response to conventional treatments (1-5). In both humans and dogs, tumor initiation and progression can be influenced by similar factors such as age, nutrition, sex, reproductive status, and exposure to environmental risk factors. Spontaneous tumors in dogs evolve over long periods of time, interacting dynamically with the host immune system, which recapitulates the mechanisms of tolerance observed in human disease (3). These similarities, the ability to collect serum, urine, blood, biopsy tissue samples, and the use of advanced imaging diagnostic tests to monitor clinical parameters, tumor progression and efficacy of the therapy make dogs with spontaneous tumors a strong model for translational cancer research (2, 6, 7).

Although murine cancer models have proven to be extremely powerful to determine molecular pathways involved in cancer initiation, promotion, and progression, there are important limitations due to differences in size and physiology of this model. For instance, murine models do not reproduce some essential features of cancer in humans; such as tumor grow over long periods, immune system function, tumor microenvironment and stroma interactions. More importantly, they have not been predictive of toxicity or efficacy of treatments in humans (2).

Unquestionably, an advantage of the canine model of spontaneous cancer includes the possibility to develop an almost identical treatment scheme to those used in humans, including surgery, radiotherapy, chemotherapy, and immunotherapy that may better predict the response in humans to novel therapies. For this reason, in the past few years, translational studies using pet dogs have been developed to assess novel therapeutic approaches for a variety of cancers (1, 2, 4).



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