A Vertically Integrated System for Tracking and Assessing Cell-Cycle-Aware Phenotypes under Confinement

Quantitative cell biology often examines migration and cell-cycle progression separately, limiting insights into their interplay under spatial constraints. Here, we present a vertically integrated platform combining multiplexed fluorescent reporters for cell-cycle phases, actin, and tubulin with photopatterned extracellular matrix islands of defined sizes, alongside an automated imaging pipeline (Fab2Mic) for high-throughput, live-cell tracking of migration and cell-cycle dynamics under confinement. Using HT1080 fibrosarcoma cells, we observed that confinement progressively reduced cell area and cytoskeletal spread, altered cell-cycle phase distributions, and increased abnormal cell-cycle events, including prolonged G1 and mitotic slippage, which is unique to confined conditions. Dynamic imaging revealed cell-cycle-dependent motility variations, with faster migration in G1. This system enables systematic, cell-cycle-aware mechanobiology studies under controlled confinement,providing access to dynamic phenotypes inaccessible to static assays and offering a scalable approachfor mechanistic investigations and screening applications.