Collective directional migration drives the formation of heteroclonal cancer cell clusters.

Metastasisation occurs through the acquisition of invasive and survival capabilities that allow tumour cells to colonise distant sites. While the role of multicellular aggregates in cancer dissemination is acknowledged, the mechanisms that drive the formation of multiclonal cell aggregates are not fully elucidated. Here, we show that cancer cells of different tissue of origins can perform collective directional migration and can actively form heteroclonal aggregates in 3D, through a proliferation-independent mechanism. Coalescence of distant cell clusters is mediated by subcellular actin-rich protrusions and multicellular outgrowths that extend towards neighbouring aggregates. Coherently, perturbation of cytoskeletal dynamics impairs collective migration while myosin II activation is necessary for multicellular movements. We put forward the hypothesis that cluster attraction is mediated by secreted soluble factors. Such a hypothesis is consistent with the abrogation of aggregation by inhibition of PI3K/AKT/mTOR and MEK/ERK, the chemoattracting activity of conditioned culture media and with a wide screening of secreted proteins. Our results present a novel collective migration model and shed light on the mechanisms of formation of heteroclonal aggregates in cancer.

Activation of RSK by phosphomimetic substitution in the activation loop is prevented by structural constraints.

The activation of the majority of AGC kinases is regulated by two phosphorylation events on two conserved serine/threonine residues located on the activation loop and on the hydrophobic motif, respectively. In AGC kinase family, phosphomimetic substitutions with aspartate or glutamate, leading to constitutive activation, have frequently occurred at the hydrophobic motif site. On the contrary, phosphomimetic substitutions in the activation loop are absent across the evolution of AGC kinases. This observation is explained by the failure of aspartate and glutamate to mimic phosphorylatable serine/threonine in this regulatory site. By detailed 3D structural simulations of RSK2 and further biochemical evaluation in cells, we show that the phosphomimetic residue on the activation loop fails to form a critical salt bridge with R114, necessary to reorient the αC-helix and to activate the protein. By a phylogenetic analysis, we point at a possible coevolution of a phosphorylatable activation loop and the presence of a conserved positively charged amino acid on the αC-helix. In sum, our analysis leads to the unfeasibility of phosphomimetic substitution in the activation loop of RSK and, at the same time, highlights the peculiar structural role of activation loop phosphorylation.

Dynamic Interplay between Pericytes and Endothelial Cells during Sprouting Angiogenesis.

Vascular physiology relies on the concerted dynamics of several cell types, including pericytes, endothelial, and vascular smooth muscle cells. The interactions between such cell types are inherently dynamic and are not easily described with static, fixed, experimental approaches. Pericytes are mural cells that support vascular development, remodeling, and homeostasis, and are involved in a number of pathological situations including cancer. The dynamic interplay between pericytes and endothelial cells is at the basis of vascular physiology and few experimental tools exist to properly describe and study it. Here we employ a previously developed ex vivo murine aortic explant to study the formation of new blood capillary-like structures close to physiological situation. We develop several mouse models to culture, identify, characterize, and follow simultaneously single endothelial cells and pericytes during angiogenesis. We employ microscopy and image analysis to dissect the interactions between cell types and the process of cellular recruitment on the newly forming vessel. We find that pericytes are recruited on the developing sprout by proliferation, migrate independently from endothelial cells, and can proliferate on the growing capillary. Our results help elucidating several relevant mechanisms of interactions between endothelial cells and pericytes.

Plasticity of human adipose-derived stem cells - relevance to tissue repair.

In contrast to cold blooded vertebrates, the ability to regenerate morphologically and functionally complex structures is limited in adult mammals. Recruitment of progenitor cells is a key step in the regenerative process. The possibility of repairing missing or diseased tissues in humans has been potentiated by the increasing understanding of somatic stem cells, their plasticity and the possibility of modulating it, that could be harnessed either to stimulate endogenous repair or to engineer the required tissue. Here, we focus on human mesenchymal stem cells (MSCs), important players in tissue homeostasis in healthy organisms, with a particular emphasis on those derived from the adipose tissue (ADSCs). While a mark of MSC identity is the ability to differentiate into osteoblasts, chondrocytes and adipocytes, there is evidence that their potential goes beyond these three mesenchymal lineages. We discuss some differentiation and modulatory properties of MSCs and provide an overview of our recent work on ADSCs from paediatric patients (pADSCs) that has shown their ability to give raise to non-mesenchymal cells, consistent with a significant plasticity. Finally, we present novel data indicating that both mesenchymal lineages (adipogenic, chondrogenic and osteogenic) and neural and epithelial lineages can originate from clonal lines that like the parental line express markers of pluripotency as well as the stromal cell marker, GREM1. Together these data support the existence of pADSC multipotent stem cells.