Background <p>In the developing human brain, forebrain pericytes of neural crest origin are proposed to exert their function at the leading front of growing vessels. The combined sprouts are made up by an endothelial tip cell and a more advanced pericyte extended tunnelling nanotube (TNT), which forms ‘intervascular bridges’ during vessel branching. To better understand of the process of pericyte TNTs (P-TNTs)-guided vessel growth, we have immunolocalized the c-KIT (CD117) receptor and its ligand SCF, as a possible ‘non canonical’ signaling pathway involved in endothelium-pericyte interactions during the pericyte-driven mode of sprouting.</p> Methods <p>Immunofluorescent high-resolution confocal microscopy and 3D modelling were applied on human developing brain and glioblastoma (GB) sections. TNTs were revealed by sequential scanning of 20-µm tissue sections, leveraging on NG2 proteoglycan as a marker of immature/re-activated pericytes and on collagen type IV (COL IV) as a TNT-associated ECM molecule. Confocal-aided morphometry was applied to evaluate the density of NG2⁺ TNTs on brain and tumor vascular networks. The cellular and subcellular immunolocalization of c-KIT and SCF was achieved by double staining with NG2 and the endothelial cell-marker CD31.</p> Results <p>In both developing brain and GB samples, P-TNTs are seen associated with pericytes budding from parental vessels or advancing at the distal edge of combined vessel sprouts. The number of TNTs largely prevails in fetal brain, compared with peripheral GB areas, while it significantly reduces in tumor core areas. c-KIT⁺/SCF⁺ endothelial cells lie close to c-KIT⁻/SCF⁺ pericytes in both fetal and tumor samples. In endothelial cells of fetal brain microvessels, c-KIT shows a dual cell membrane and nuclear localization, the latter being barely detectable on endothelial cells of tumor vessels.</p> Conclusions <p>P-TNTs, which are tightly associated in situ with endothelial–pericyte combined sprouts, appear to play a dual role during vessel collateralization by bridging the gap between distant vessels and guiding vascular outgrowth. The complementary cellular distribution of c-KIT and SCF observed in endothelial cells and pericytes suggests that both endothelial autocrine/paracrine SCF/c-KIT signaling and pericyte-derived paracrine/juxtacrine SCF cues may contribute to the pericyte-driven mode of vessel branching. Similar observations in GB samples further suggest a potential involvement of pericytes and their P-TNTs in tumor vascularization, although sprouting endothelial cells displayed distinct subcellular patterns of c-KIT expression in fetal versus GB tissues.</p>