Multiscale mechanobiology of circulating tumor cells: microfluidics and atomic force microscopy

Cancer progression is shaped by coupled molecular, biochemical, and mechanical changes across multiple scales, from altered signaling and cytoskeletal organization to cellular deformation, adhesion, and metastatic dissemination. Circulating tumor cells (CTCs) are rare and heterogeneous in blood, occurring as single cells and multicellular clusters that provide access to evolving tumor phenotypes through liquid biopsy. Their apparent elasticity, deformability, adhesion, and viscoelastic behavior may reflect invasive potential, treatment response, and phenotypic state, while cytoskeletal proteins, membrane lipids, cell surface glycans, and receptors provide molecular context for these mechanical signatures. This review examines the complementary roles of microfluidics and atomic force microscopy (AFM) in multiscale CTC mechanobiology. Microfluidic platforms support label-free and affinity-based enrichment, capture, positioning, and recovery of viable cells. AFM provides localized measurements of apparent elasticity, deformation, adhesion, viscoelastic response, and nanoscale surface architecture in liquid, while functionalized probes enable molecular recognition and molecule-specific force measurements. We evaluate physical, affinity-based, cluster-preserving, open-space, and artificial intelligence-assisted microfluidic strategies together with reversible device assembly, automated AFM targeting, fluidic probes, advanced mapping modes, and treatment-response applications. Existing studies establish feasibility, including direct AFM measurements of individual patient-derived CTCs and automated analysis following microfluidic enrichment in model rare-cell mixtures. Clinical evidence remains limited, many integrated workflows rely on cultured cells or spiked samples, and within the literature identified in this review, direct AFM characterization of intact patient-derived CTC clusters was not found. Clinical translation will require confirmation of tumor identity, standardized acquisition, analysis, and reporting, increased throughput, statistical analysis that preserves the hierarchy of patients, cells, and force curves, and validation in independent patient cohorts. Integrated AFM-microfluidic platforms could extend CTC liquid biopsy beyond enumeration toward functional mechanobiological analysis relevant to metastasis, therapeutic response, and biomarker development.