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Title: Investigating the role of macrophages in the suppression of NK cell functions in mouse models of metastatic breast cancer
Author: Brownlie, Demi Betty Beth
ISNI:       0000 0004 8509 2978
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2020
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Breast cancer is the leading cause of cancer-related death in females worldwide. Although 5-year survival of breast cancer patients in early stages is 89-100%, that of patients with metastatic tumours is reduced to just 21%, suggesting the requirement of effective therapies for metastatic breast cancer (MBC). MBC is primarily treated with chemotherapeutics however the efficacy of such treatments is limited due to resistance. As an alternative approach, NK cell-based immunotherapy (i.e., adoptive transfer of NK cells to patients) has been focused on since it shows significant therapeutic effects on haematopoietic tumours. Nevertheless, its efficacy is limited in MBC probably due to an immune suppressive tumour microenvironment (TME). Tumour-associated macrophages (TAMs) are an abundant cell type within the TME of breast cancer and promote the metastatic process such as cancer cell egress from the primary tumour in mouse models. In mouse models of breast cancer, a distinct population of TAMs in the metastatic site called metastasis-associated macrophages (MAMs) can promote tumour cell seeding, survival and growth. Moreover, we have recently shown that MAMs and their progenitor cells can suppress cytotoxicity of CD8+ T cells in vitro. Interestingly, a recent study suggests that TAMs isolated from the 'primary' mammary tumour in mice can suppress tumour killing ability of NK cells in vitro, whereas the effects of MAMs on NK functions in the 'metastatic' tumour is largely unknown. We hypothesise that MAMs in the metastatic site suppress NK cell function, and that the depletion of these MAMs can improve NK cell immunotherapy efficacy for MBC. To investigate this hypothesis, we first established an in vitro NK cell cytotoxicity assay whereby mouse breast cancer cells were co-cultured with splenic NK cells, and the resultant tumour cell apoptosis was determined by quantitative fluorescence microscopy. Using this assay, we found that the NK cell-induced tumour cell apoptosis was significantly reduced in the presence of MAMs isolated from metastatic tumours in the lung of tumour cell injected mice. We also found that bone marrow-derived macrophages cultured with M-CSF (M- BMMs) that resemble MAMs also reduced NK cell cytotoxicity in a cell-to-cell contact dependent manner. In contrast, BMMs cultured with GM-CSF that represent pro-inflammatory macrophages did not suppress NK cell cytotoxicity. We further identified by flow cytometry that MAMs and M-BMMs expressed high levels of NK cell inhibitory ligands such as H2-Kb and H2-Db, and NK cells in the metastatic lung expressed high levels of their receptors. However, blockade of H2-Kb or H2-Db did not prevent macrophage mediated NK cell suppression in our assay. Alternatively, we found that M-BMMs expressed higher levels of membrane bound TGF-b than GM-BMMs and blocking TGF-b rescued the macrophage-mediated NK cell suppression, although these data must be confirmed. Using a mouse model of breast cancer metastasis, we further demonstrated that depletion of MAMs promoted maturation of NK cells in the metastatic lung as well as recruitment of NK cells towards the metastatic site. Importantly, the MAM depletion in this model significantly increased the efficacy of transferred NK cells in reducing metastatic tumour burden whereas NK cell transfer on its own did not suppress metastatic tumour growth. Collectively, our data suggest that MAMs in metastatic tumours can suppress NK cell cytotoxicity towards breast cancer cells by direct contact with NK cells that transmit suppressive signals via membrane bound TGF-b as well as by suppressing NK cell maturation and recruitment in the metastatic site. Our data also indicate that the depletion of MAMs can alter the immune suppressive TME and thereby improve the efficacy of NK cell infusion therapy efficacy. Further investigation of the mechanisms behind MAM-mediated NK suppression would lead to the increased success of NK cell-based immunotherapy for MBC.
Supervisor: Pollard, Jeffrey ; Kitamura, Takanori Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: cancer ; immunology ; NK cells ; macrophages ; breast cancer