SummaryRAMA1 is a novel kinetochore protein involved in kinetochore-microtubule attachment

In order to improve the efficacy of conventional radiotherapy, attention has been paid to immune cells, which not only modulate cancer cell response to therapy but are also highly recruited to tumours after irradiation. Particularly, the effect of ionizing radiation on macrophages, using therapeutically relevant doses, is not well understood. To evaluate how radiotherapy affects macrophage behaviour and macrophage-mediated cancer cell activity, human monocyte derived-macrophages were subjected, for a week, to cumulative ionizing radiation doses, as used during cancer treatment (2 Gy/fraction/day). Irradiated macrophages remained viable and metabolically active, despite DNA damage. NF-kappaB transcription activation and increased Bcl-xL expression evidenced the promotion of pro-survival activity. A significant increase of pro-inflammatory macrophage markers CD80, CD86 and HLA-DR, but not CCR7, TNF and IL1B was observed after 10 Gy cumulative doses, while anti-inflammatory markers CD163, MRC1, VCAN and IL-10 expression decreased, suggesting the modulation towards a more pro-inflammatory phenotype. Moreover, ionizing radiation induced macrophage morphological alterations and increased their phagocytic rate, without affecting matrix metalloproteases (MMP)2 and MMP9 activity. Importantly, irradiated macrophages promoted cancer cell-invasion and cancer cell-induced angiogenesis. Our work highlights macrophage ability to sustain cancer cell activities as a major concern that needs to be addressed to improve radiotherapy efficacy.

Orchestration of bone repair processes requires crosstalk between different cell populations, including immune cells and mesenchymal stem/stromal cells (MSC). Extracellular vesicles (EV) as mediators of these interactions remain vastly unexplored. Here, we aimed to determine the mechanism of MSC recruitment by Dendritic Cells (DC), hypothesising that it would be mediated by EV. Primary human DC-secreted EV (DC-EV), isolated by ultracentrifugation, were characterized for their size, morphology and protein markers, indicating an enrichment in exosomes. DC-EV were readily internalized by human bone marrow-derived MSC, without impacting significantly their proliferation or influencing their osteogenic/chondrogenic differentiation. Importantly, DC-EV significantly and dose-dependently promoted MSC recruitment across a transwell system and enhanced MSC migration in a microfluidic chemotaxis assay. DC-EV content was analysed by chemokine array, indicating the presence of chemotactic mediators. Osteopontin and matrix metalloproteinase-9 were confirmed inside EV. In summary, DC-EV are naturally loaded with chemoattractants and can contribute to cell recruitment, thus inspiring the development of new tissue regeneration strategies.