Defining the pro-tumour impact of the evolving stromal microenvironment
Much like normal tissues, tumours require a supporting microenvironment for growth and survival, known as the tumour stroma. However, tumours represent a dynamic and turbulent environment, in which factors such as hypoxia, fibrosis, nutrient deprivation and the local cytokine milieu continually fluctuate as the tumour grows and develops. These factors influence stromal phenotypes and create heterogeneity, which can confound our understanding of their role within the microenvironment. In particular, cancer associated fibroblasts (CAFs) represent a diverse population of cells, which cannot be identified by one universal marker. CAFs promote tumour growth and dissemination by secreting growth factors, stimulating angiogenesis, aiding the development of tumour-promoting inflammation and remodelling the extra-cellular matrix (ECM). However, recent investigations have shown these functions belong to defined populations that differ between tumour types. This project aimed to investigate stromal heterogeneity across melanoma development, with a specific focus on the CAF compartment. Whilst conventional techniques such as IF and flow cytometry showed varied expression of fibroblast markers, they lacked the resolution to discern functional subsets. Thus, we employed single cell RNA sequencing (scRNAseq) to profile CAF populations at different stages of tumour development. To avoid bias, CAFs were isolated from the B16-F10 melanoma model using a ‘negative selection’ approach. Our data revealed the presence of 3 functionally distinct fibroblast populations, termed ‘immune’, ‘desmoplastic’ and ‘contractile’, which expressed genes involved in immune cross talk, matrix remodelling and stress fibre contraction respectively. Furthermore, these populations are dynamic, changing in prevalence as the tumour grows. While ‘immune’ and ‘desmoplastic’ populations were present from early stages, ‘contractile’ CAFs were more abundant at later time points. Owing to their unique marker profiles, we were able to identify these populations within the tumour stoma and validate their temporal nature. Subsequent investigation into the contribution of these subsets to tumour growth, revealed that ‘immune’ CAFs promoted accumulation of suppressive macrophages by production of C3 and its cleavage product C3a. Significantly, inhibition of the C3a/C3aR axis reduced the number of macrophages and decreased tumour volume. This reduction in tumour growth was accompanied by increased CD8 T-cell infiltration, implying that ‘immune’ CAFs may inhibit adaptive anti-tumour immunity through controlling the myeloid compartment. The interaction between C3 producing CAFs and C3aR expressing macrophages was conserved in different murine tumours and human cancer. Thus, ‘immune’ CAFs and C3a signalling may represent therapeutic targets in multiple cancer types. Overall, our data highlights the complexity of stromal phenotypes and microenvironment interactions, which likely reflects the convoluted climate of the developing tumour.
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