Investigating the role of macrophages in the suppression of NK cell functions in mouse models of metastatic breast cancer
View/ Open
Date
27/06/2020Author
Brownlie, Demi Betty Beth
Metadata
Abstract
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.