부산대학교 도서관

Tumour-associated macrophages (TAMs) are key immune-suppressive constituents of the breast tumour microenvironment (TME) and correlate with poor prognosis. Tenascin-C (TNC) is an immunomodulatory extracellular matrix (ECM) protein which is upregulated in breast tumours, and is associated with increased metastasis and worsened prognosis. Our previous work showed that perturbation of the breast TME by tumour-derived TNC skews macrophages towards an immune-suppressive phenotype featuring downregulated antigen-presentation. Using TNC as an exemplar perturbant, this thesis sought to understand how macrophage effector functions such as antigen presentation become enhanced or suppressed within the TME. To achieve this, single-cell RNA sequencing (scRNAseq) was performed on infiltrating myeloid cells derived from syngeneic, orthotopically-engrafted, murine breast tumours that were engineered to express high or low levels of TNC. scRNAseq revealed marked heterogeneity in the intra-tumoural myeloid compartment with 7 distinct subsets observed, 5 of which were macrophages (Mac1-5). Notably, macrophages delegated key effector functions including antigen presentation (Mac2) and chemokine production (Mac3) to discrete subsets that were phenotypically, spatially and temporally distinct at the protein level by flow cytometry and confocal immunofluorescence imaging. The role of TNC was predominantly exerted at the level of subset abundance (rather than gene transcription), with 4 out of 7 myeloid subsets exhibiting TNC-dependent changes in abundance, including two macrophage subsets (Mac1 & Mac2). Over time, TNC modestly weakened the segregation of Mac2 and Mac3 phenotypes, suggesting it may drive dysfunctional delegation of effector functions. Pseudotemporal analysis of scRNAseq data implicated the epigenetic regulator, Kdm6b, in governing the acquisition of Mac3 effector function, and pharmacological inhibition of KDM6B in vivo shifted the proportion of Mac3 via TAM reprogramming. Finally, mouse-derived myeloid subsets were well-conserved in the most comprehensive scRNAseq atlas of human breast cancer, and gene signature analyses in bulk clinical datasets demonstrated significant differences in prognosis for patients with distinct myeloid 'ecotype' profiles. Together, these data demonstrate functionally divergent roles for TAM subsets in breast tumour progression and metastasis, while providing mechanistic insights into the impact of ECM-derived cues such as TNC on shaping the myeloid compartment, with potential therapeutic and prognostic implications for breast cancer patients.