They also displayed a disturbed expression of E-cadherin, one of the caretakers of the epithelial phenotype, compared with spheroids. Although selleck the role of EMT in carcinoma dissemination is controversial (Christiansen and Rajasekaran, 2006), downregulation of E-cadherin has been linked to invasion and metastasis in CRC (Bates and Mercurio, 2005). These observations suggest that colosphere-forming cells maintain EMT potential. The putative involvement of these cells in cancer spread was underlined by the colospheres’ ability to migrate into Matrigel. Indeed, one of the first events that metastatic cells have to complete is the local invasion of stroma, a process that requires a degradation of extracellular matrix components by proteolytic enzymes including MMPs (Fidler, 2002; Pantel and Brakenhoff, 2004).
One particular group of MMPs, the gelatinases A and B, also known as MMP-2 and MMP-9, are of particular interest with respect to the development and progression of CRC (Garbett et al, 1999; Mook et al, 2004). Gelatinase analysis showed that ex vivo colospheres displayed MMP-2 and MMP-9 activity, as did parental xenograft tissue, whereas paired cell line spheroids were devoid of such proteases. More importantly, as shown by a subrenal capsule assay, colospheres displayed undoubtedly a high tumorigenicity and metastatic potential, compared with the spheroid model, suggesting that these 3D structures contained more cancer-initiating cells. It could be hypothesised that the different tumorigenicity is related to a higher passage number into mice for xenografts giving rise to colospheres than for xenografts leading to the CT320X6 cell line.
Nevertheless, the presumed selection of more aggressive cells through in vivo passage is not so clear. We have determined that the CT320 cell line, established directly from a patient GSK-3 sample without any passage into mice, and the CT320X6 cell line, derived from the xenograft XenoCT320, display a similar growth curve in nude mice (unpublished data). It is also noteworthy that tumours obtained after injection of colospheres were well-differentiated adenocarcinomas, whereas tumours derived from a cell-suspension injection were poorly differentiated carcinomas, illustrating, as largely observed (personal communication, Heiner Fiebig), that cancer cell lines established from differentiated human tumours lead to poorly differentiated tumours, whereas xenografts directly from tissue tumour specimens maintain their differentiation level. The differences observed between colospheres and spheroids have to be put in line with the different growth conditions (ex vivo vs in vitro), which in turn involve different microenvironments.