The Role of the CXCL12/CXCR4 Signaling Pathway in Regulating Cellular Migration

Abstract We investigated the CXCL12/CXCR4 signaling pathway as a regulator of adipose-derived stem cell (ADSC) self-assembling toroidal constructs using collagen hydrogels. ADSCs formed toroid rings when cultured on hydrogel surfaces but failed to do so when mixed within the matrix. Gene expression profiling revealed significant upregulation of the CXCL12/CXCR4 pathway in toroid-forming conditions, supported by immunofluorescence studies that confirmed CXCL12 presence in toroids but not in mixed-in cultures. Early toroid formation was marked by the emergence of CXCL12 expression, correlating with cell migration. Targeted inhibition experiments identified the PI3K pathway as a critical regulator, delaying cell migration by ∼16 h, while N-Cadherin, Ras/Raf, and ERK1/2 inhibition either reduced or halted migration over extended periods. Through Western blot analysis, altered expression of α-Smooth muscle actin and focal adhesion kinase under PI3K inhibition was highlighted thus emphasizing their roles in toroid formation. Lastly, initial coculture studies with 4T1 breast cancer cells unexpectedly showed CXCL12 localization primarily in 4T1 cells within mixed toroids, suggesting modified chemotactic signaling. Our findings establish CXCL12/CXCR4 as crucial for ADSC toroid formation and reveal the pathway's complex involvement in cellular organization and migration, presenting a robust model for exploring cell–cell and cell–matrix interactions relevant to tissue engineering and cancer research.