Vertically Integrated System for Tracking and Assessing cell-cycle aware phenotypes under confinement

Quantitative cell biology often studies migration and the cell-cycle (CC) in separate assays, limiting mechanistic insights, particularly under geometric confinement. Here, we introduce a vertically integrated platform for simultaneously tracking single-cell migration and assessing CC under confinement. Our system integrates cell engineering via multiplexed sensors for cell-cycle, actin, and tubulin, as well as photopatterned engineered extracellular matrix (ECM) islands of defined sizes. It also features an automated, high-throughput pattern-aware imaging pipeline (Fab2Mic) that enables on-pattern, joint migration-CC assessment in the same live cells. Since the local microenvironment plays a critical role in metastasis by constraining cell behaviors within spatial boundaries, we used an HT1080 fibrosarcoma model as an illustrative case. Where static phenotyping yielded 40% G1 and 60% S/G2/M, with larger cell areas and tubulin spread in the S/G2/M phase, dynamic phenotyping via live-cell imaging confirmed CC-linked motility, with faster instantaneous velocities in G1, exemplifying the CC-migration correlations. These phenotypes were modulated by the spatial confinement imposed by the engineered ECM islands. Stronger confinement reduced cell area and tubulin spread and increased the frequency of abnormal CC events, particularly Long G1 states on smaller engineered ECM islands. It also induced a confinement-specific S/G2/M-G1 mitotic slippage, observed only under our confined conditions. Together, this vertically integrated system suggests that confinement may continuously tune migration–CC coupling and provides a deployable pipeline for CC-aware mechanobiology and screening. Moreover, we stress how dynamic imaging provides access to variables that are difficult or impossible to infer from static snapshots, including velocity and CC timing.