Staminalità e dormienza delle cellule del cancro al seno nel microambiente osseo

This work investigated the role of N - cadherin in the interaction between dormant tumor cells and a particular subpopulation of osteoblasts, called SNOs (Spindle-shape N-cadherin positive Osteoblast) located at the level of the endosteal niche. Since the N-cadherin transduction signal requires a homophilic interaction, mouse BrCa cell line 4T1 were used to establish an interaction with primary mouse osteoblasts in vitro and in vivo. On these cells, the Cdh2 gene encoding for N-cadherin was removed via CRISPR / Cas9, obtaining 4T1Cdh2KO-GFP cells. Subsequently, 4T1Cdh2KO-GFP were transfected with a murine Cdh2-turboGFP vector to restore the expression of N-cadherin in the KO cells obtaining 4T1Cdh2Res-GFP cells. We evaluated the expression of some markers typical of Hematopoietic Stem cells, including CXCR4, SCA1 and TIE2. Only CXCR4 was expressed, with a reduced expression in 4T1Cdh2Res-GFP compared to 4T1Cdh2KO-GFP cells. To evaluate the stem capacity and the self-renewal ability of these cells we investigated the formation of primary and secondary mammospheres, respectively, observing a lower volume and number in the 4T1Cdh2Res-GFP compared to 4T1Cdh2KO-GFP cells. The formation of primary and secondary mammospheres was investigated also in 4T1 cells sorted by MACS into 4T1 N-CadherinHigh and 4T1 N-CadherinLow obtaining in the primary mammospheres a lower volume and number in 4T1 N-CadherinHigh compared to 4T1 N-CadherinLow cells. The number of secondary mammospheres was similar while a lower volume was observed in 4T1 N-CadherinHigh compared to the 4T1 N-CadherinLow cells. Finally, we observed that the transcriptional expression of N-cadherin did not affect the mRNA expression of Notch2, a receptor relevant to induce dormancy in BrCa cells lodging the endosteal niche. The effect of N-cadherin expression in 4T1 cells was evaluated in vivo using four-week-old Balb/c wild-type mice intratibially injected with 4T1 N-cadherinHigh and 4T1 N-cadherinLow cells, noting the absence of significant differences in the number of cells colonizing the endosteal niche.