Role of microRNAs in tumor-associated macrophages

Tumor-associated macrophages (TAMs) infiltrate both mouse and human tumors, typically in high numbers, and display complex interactions with cancer cells, endothelial cells (ECs) and other immune cells. TAMs comprise molecularly distinct subsets. Two such subsets, reminiscent of Th1 and Th2-activated macrophages, are frequently denoted M1 and M2-like TAMs, respectively. Whereas M2-like TAMs are immunosuppressive, proangiogenic and largely tumor-promoting; M1-like TAMs display a more proinflammatory profile. microRNAs (miRNAs), a large class of small noncoding RNAs that modulate gene expression post-transcriptionally, are expressed at high levels in macrophages and are involved in macrophage differentiation and activation. However, beyond specific miRNAs, the significance of general miRNA activity for TAM's immunological and tumor-regulatory functions remains unclear. Hence, the aim of this thesis is to characterize the role of miRNAs in TAMs, both endogenously and as secreted molecules. In the first part of this thesis, I used a genetic mouse model to conditionally delete the miRNA-processing enzyme Dicer in TAMs, and found that it greatly amplified their M1-like programming and inhibited tumor growth. TAM skewing fostered the recruitment and activation of cytotoxic T cells (CTLs), which exacerbated M1-like TAM polarization through interferon-gamma. Remarkably, TAM's DICER status determined pro versus anti-tumoral outcome of TAM or CTL depletion, and controlled response to immunotherapy. Finally, I found that the genetic rescue of the miRNA Let-7 in Dicer-deficient TAMs was sufficient to restore the M2-like TAM phenotype and decrease CTLs. These findings indicate that DICER/Let-7 activity opposes immunostimulatory TAM activation, with potential therapeutic implications. In the second part, I investigated the role of secreted, macrophage-derived miRNAs and their transfer to other cells via exosomes. Exosomes are small vesicles that have emerged as mediators of intercellular communication. They are released in the extracellular space where they can fuse with, and deliver their cargo to, other cells. Interestingly, specific miRNAs are enriched in exosomes in a cell-type-dependent fashion. However, the mechanisms by which miRNAs are sorted to exosomes and the significance of miRNA transfer to other cells are unclear. RNA profiling of macrophages and their exosomes showed that miRNA sorting to exosomes is modulated by cell-activation-dependent changes of miRNA target levels in the producer cells. Genetic perturbation of the expression of individual miRNAs, or their targeted transcripts, promoted bidirectional miRNA relocation from the cell cytoplasm (site of miRNA activity) to multivesicular-bodies (sites of exosome biogenesis) and controlled miRNA sorting to exosomes. The use of Dicer-deficient cells and reporter lentiviral vectors for miRNA activity further showed that exosomal miRNAs are transferred from macrophages to ECs to detectably repress tar