AbstractTumour-associated macrophages (TAMs) are abundant infiltrating immune cells in solid tumours and have been associated with poor clinical outcomes. A subset of TAMs identified by their expression of the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) reside proximal to blood vasculature and are therefore termed perivascular (PV) TAMs and contribute to disease progression. Previous work in the lab has shown that depleting LYVE-1+ TAMs in the spontaneous mouse mammary tumour virus-polyoma middle T antigen (MMTV-PyMT) model of breast cancer using liposome-based approaches resulted in tumour control, highlighting the importance of the TAM subset in cancer progression. To further investigate this subset and its role in tumour development we utilised a combination of genetically modified murine models, advanced immunofluorescence imaging, flow cytometry and RNA sequencing approaches. We demonstrated that LYVE-1+ TAMs co-express the immunosuppressive enzyme heme oxygenase-1 (HO-1) and form coordinated and dynamic multicellular ‘nest’ structures in the PV niche. As LYVE-1 is also expressed at high levels by the lymphatic endothelium, and HO-1 expression is selectively expressed by this TAM subset, we generated a knock-in reporter mouse for the Hmox1 gene which expresses click beetle luciferase and enhanced green fluorescence protein under the control of the Hmox1 promotor, to facilitate the study of these cells. Using this model, we show that LYVE-1+ macrophages expressing HO-1 can also be found populating healthy tissues but are not phenotypically identical to the LYVE-1+ TAM subset. We also reveal that the LYVE-1+ TAM population can be identified and tracked as F4/80+CD206hiMHCIIlo. Using IL-6 knockout (KO) MMTV-PyMT murine models alongside the HO-1 reporter, we show that LYVE-1+ TAM polarisation is dependent on IL-6 secreted by the endothelium in vivo. We also demonstrate that IL-6 induces the expression of the chemokine receptor CCR5. Using CCR5 KO MMTV-PyMT mice we reveal that LYVE-1+ TAM nest formation is dependent on a communication axis involving CCR5 and its cognate ligands CCL3/4. In relation to this, we also revealed that CCR5 plays an ongoing active role in maintaining the LYVE-1+ TAM nests post-formation. When CCR5 is therapeutically inhibited using the selective CCR5 inhibitor, maraviroc, post-tumour and nest formation, there was an observable dispersion of the LYVE-1+ TAM nests away from the PV space.
Through investigating the changes in the tumour microenvironment in the absence of LYVE-1+ TAMs or their nest structures, we observed a selective increase in CD8+ T-cell infiltration, suggesting that LYVE-1+ TAMs and their nests could be associated with CD8+ T-cell exclusion. Due to the clear link between the presence of tumour-infiltrating lymphocytes and the response to chemotherapy, we also investigated whether the disruption of the LYVE-1+ TAM nests could have a therapeutic implication. This investigation revealed that LYVE-1+ TAM nests were associated with resistance to immune-stimulating therapeutics, and both IL-6 and CCR5 KO MMTV-PyMT models were significantly more sensitive to the immune-stimulating clinical chemotherapy 5-Fluorouracil than their wild-type counterparts. This study highlights an unappreciated collaboration between TAMs and uncovers a spatially-driven therapeutic resistance mechanism of these cells in cancer which can be therapeutically targeted.
|Date of Award||1 Sept 2022|
|Supervisor||James Arnold (Supervisor) & Joy Burchell (Supervisor)|