Confined migration-derived breast cancer sublines reveal subtype‑specific responses to Arp2/3 inhibition

Introduction: Cancer cell migration through confined tissue spaces (CM) leads to the development of a pro-migratory phenotype. The Arp2/3 complex aids in cell movement, creating branched actin networks, and can be blocked by CK-666. We examined how CM‑derived sublines of low‑metastatic MCF7 and high‑metastatic MDA‑MB‑231 change their mechanics, proliferation, and collective migration in response to CK‑666.

Methods: Parental and CM sublines of MDA-MB-231 and MCF7 were generated by three rounds of CM through 8 μm pores. Young’s modulus was measured by scanning ion-conductance microscopy. Proliferation, fraction of DNA-replicating cells and collective migration weredetected by SRB, Ki67 immunofluorescence and wound healing assays, respectively, at 24 h after treatment.

Results: CM-derived MDA-MB-231 and MCF7 sublines were significantly softer compared to parental counterparts (1051 ± 624.2 vs. 786.4 ± 531 Pa and 2446 ± 1391 vs. 1614 ± 1198 Pa, respectively, all p < 0.05). CM-derived MCF7 cells showed 17.5% (p = 0.0097) increased viability relative to parental cells after CK-666 treatment, while MDA-MB-231 cells remained unaffected. CK-666 treatment reduced DNA-replicating fraction in MDA-MB-231 CM cells to 23.0 ± 2.4% (p = 0.0018) vs. 39.9 ± 1.54% in controls. No significant change was observed in parental cells or in MCF7 cells. CK-666 markedly decreased the migration of parental MDA-MB-231 cells (17.8 ± 5.25% vs. 34.3 ± 6.3% ) and stopped the migration of CM MDA-MB-231 cells (no closure vs. 25.4 ± 11.0% in controls). CK-666 treatment also diminished collective migration of parental MCF-7 cells (5.28 ± 3.8% vs. control 18.3 ± 1.1%), while CM MCF7 cells retained substantial wound closure (10.9 ± 1.37% vs. control 22.9 ± 3.2%).

Conclusions: CM‑related softening is accompanied by subtype‑specific functional responses to Arp2/3 inhibition. In high‑metastatic MDA‑MB‑231, CM and Arp2/3 blockade together shift cells into a viable but low‑proliferation, migration‑defective state. In contrast, in low‑metastatic MCF7, the softened CM subline preserves higher proliferation and substantial motility under CK‑666, suggesting more Arp2/3‑independent, mechanically adapted migration and a more drug‑tolerant phenotype.