The effects of changes in energy balance on immune regulation during the early stage of 4T1.2 mammary tumor development
Open Access
- Author:
- Xu, Yitong
- Graduate Program:
- Integrative and Biomedical Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 01, 2020
- Committee Members:
- Connie Jo Rogers, Dissertation Advisor/Co-Advisor
Connie Jo Rogers, Committee Chair/Co-Chair
Margherita Teresa-Anna Cantorna, Committee Member
Joshua D Lambert, Committee Member
Todd Schell, Outside Member
Donna Hope Korzick, Program Head/Chair - Keywords:
- breast cancer
physical activity
energy restriction
tumor microenvironment
immunosuppression
4T1.2-HER2 mammary tumor model
antigen-specific immune response - Abstract:
- Energy balance-related host factors such as body weight and physical activity (PA) can significantly impact breast cancer risk and progression. Given the prevalence of breast cancer, it is of great importance to understand the biological mechanisms underlying the cancer prevention effect of changes in energy balance and develop novel therapeutic strategies to improve clinical outcomes. Emerging evidence suggests that the immune system may be an important mediator linking changes in energy balance to breast cancer prevention. Using the 4T1.2 murine breast cancer model, we have shown that weight maintenance achieved by the combination of PA and a mild (10%) energy restriction (ER) significantly reduces primary tumor growth, metastasis and improves survival. These changes occur concurrently with an increase in T cells and a reduction in myeloid-derived suppressor cells (MDSCs) and multiple immunosuppressive markers within the tumor microenvironment (TME) at the late stage of tumor progression. The overarching aim of this dissertation was to investigate how changes in energy balance, specifically by PA and/or ER, may modulate the TME and antigen-specific immune responses at the early stage of 4T1.2 mammary tumor development to prevent tumor progression. Because MDSC accumulation is positively correlated with mammary tumor burden, the first goal of Study 1 was to determine if PA and/or ER induced beneficial changes within the TME when controlling for tumor size. The combination of PA+ER, but not PA or ER alone, significantly reduced tumor-infiltrating MDSCs and increased tumor-infiltrating T cells when tumor sizes were equal. In addition, the combination of PA+ER induced a global reduction in chemokines associated with the recruitment of immunosuppressive cells, proinflammatory and immunosuppressive markers within the TME. Further, a downregulation of programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase (IDO) was evident within tumor-infiltrating immune cells. These results suggest that preventing weight gain by PA+ER may prevent an immunosuppressive TME, thus allowing for greater effector cell infiltration and function to prevent tumor progression. Because therapeutic cancer vaccines may benefit from combinatorial approaches that reduce immunosuppression within the TME, the second goal of Study 1 was to determine if weight maintenance (WM) achieved by the combination of PA+ER could enhance the efficacy of a 4T1.2 whole tumor cell vaccine (VAX). We did not observe a VAX effect in reducing primary tumor growth. However, there was a synergistic effect of WM and VAX in reducing the gene expression of various immunosuppressive markers within the TME, which provides an initial piece of evidence that energy balance interventions may be used in combination with immune-based therapies to modulate cancer immune responses and improve clinical outcomes. Antigen-specific immune response is a critical component of anti-tumor immunity. However, no studies to date have assessed its potential role in mediating the cancer prevention effect of changes in energy balance. Our findings from Study 1 suggest that weight maintenance achieved by the combination of PA+ER enhanced T cell cytotoxicity against allogeneic antigens. However, the 4T1.2 model is poorly immunogenic with no defined tumor antigens, which limits our ability to further study the effect of energy balance interventions on antigen-specific immune responses in this clinically relevant breast cancer model. Therefore, the goal of Study 2 was to characterize cancer immune responses in a modified 4T1.2 model that expresses a well-defined surrogate tumor antigen, human epidermal growth factor receptor-2 (HER2, rodent homolog neu), to provide a tool for future investigation of antigen-specific immune responses under therapeutic interventions. In contrast to the rapid, continuous tumor growth of the parental 4T1.2 model, the 4T1.2-HER2 model demonstrated an initial tumor growth followed by spontaneous tumor regression, and a second phase of tumor outgrowth or complete tumor rejection. Tumor regression was dependent on the adaptive immune system, and was associated with an increase in splenic and tumor-infiltrating T cells, the capacity of effector cells to produce cytokines (IFNγ, IL-2) and cytotoxic molecules (perforin), and importantly, HER2-specific immune response as indicated by IFNγ secretion. These results suggest that the integration of the HER2 antigen enhanced the immunogenicity of the 4T1.2 tumor and induced antigen-specific immune responses. Therefore, the orthotopic 4T1.2-HER2 breast cancer model could be a valuable tool to study the effect of lifestyle or other therapeutic interventions on antigen-specific immune responses in a clinically relevant setting. Using the 4T1.2-HER2 model, the goal of Study 3 was to determine if PA and/or ER had beneficial effects on tumor growth and HER2-specific immune responses during the early stage of tumor development. When immune outcomes were assessed at a same time point, there was no significant difference in the distribution of tumor-infiltrating immune cells or HER2-specific IFNγ secretion between treatment groups. However, the combination of PA+ER significantly delayed 4T1.2-HER2 tumor growth, resulting in a later onset but more rapid tumor regression period. To better understand the immune responses underlying the shifted tumor growth pattern by PA+ER, we conducted a time course experiment to assess the effects of PA and ER-induced weight maintenance on immune responses throughout 4T1.2-HER2 initial tumor growth and spontaneous regression. WM induced a modest increase in splenic CD8+ T cells during the initial tumor growth period, but did not change the distribution of tumor-infiltrating immune cells throughout the initial tumor growth and regression. Assessment of HER2-specific IFNγ response via different methods yielded contrasting results. During their respective tumor regression phase, WM mice demonstrated a higher percentage of HER2-specifc effector cells that were IFNγ+, whereas weight gain (WG) control mice demonstrated greater HER2-specific IFNγ secretion. Together, these results do not provide sufficient evidence to conclude if weight maintenance achieved by PA+ER enhanced HER2-specific immune responses. However, Study 3 presents the first investigation on the effect of PA and ER-induced weight maintenance on antigen-specific immune responses in a clinically relevant breast cancer model, and powers future studies to better understand energy balance-related changes in anti-tumor immune responses. Together, results from this dissertation provide a further understanding on the potential immune mechanisms by which changes in energy balance protect against breast cancer. First, weight maintenance achieved by the combination of PA+ER had multifaceted effects in reducing the immunosuppression within the TME. Specifically, weight maintenance downregulated a range of chemokines associated with immunosuppressive cells, proinflammatory and immunosuppressive markers within the TME. In addition, weight maintenance reduced tumor-infiltrating MDSCs and may reduce the immunosuppressive capacity of multiple tumor-infiltrating immune cells by downregulating PD-1, PD-L1 and IDO. A synergistic effect of weight maintenance and a whole tumor cell vaccine was observed in reducing immunosuppressive markers within the TME, which provides an initial piece of evidence that lifestyle interventions may be combined with immune-based therapies to improve clinical outcomes. Second, weight maintenance achieved by PA+ER may also enhance anti-tumor immune responses. Specifically, weight maintenance increased T cell infiltration and enhanced T cell cytotoxicity against allogeneic antigens. Further, we characterized a valuable breast cancer model to study antigen-specific immune responses in a clinically relevant setting, and evaluated the effect of weight maintenance on tumor antigen-specific immune responses. Despite the insufficient evidence to conclude if weight maintenance enhances antigen-specific responses, this presents an important initial exploration and powers future studies to better understand energy balance-related changes in anti-tumor immune responses.