From Single Cells to the Circulating Tumor Microenvironment: Label‐Free Size‐Selection and a Morphological Taxonomy for Liquid Biopsy in Cytopathology

BACKGROUND: Liquid biopsy-here, the analysis of blood-has often focused on counting solitary circulating tumor cells (CTCs), a readout that omits the multicellular architecture governing metastasis. We summarize a hierarchical morphological taxonomy built on the circulating tumor microenvironment (cTME), then ask what an isolation method must deliver before that taxonomy can be applied at the microscope. METHODS: We appraised the literature on label-free size-selection (membrane filtration) of whole blood, comparing suction-driven, microfluidic, and gravity-driven platforms for recovery of intact multicellular units and for readability of the resulting preparations by routine cytologic stains and multiplex immunocytochemistry. The critique is paired with the authors' own single-center experience, reported in labeled subsections. RESULTS: Circulating tumor-associated cells (C-TACs) form the functional cellular tier of the cTME: a "mobile niche" comprising a malignant core-solitary CTCs and circulating tumor emboli (CTE)-together with non-malignant supporting partners. Antigen-dependent capture disrupts these units and misses non-epithelial partners, whereas size-selection retains them by size and deformability, independently of surface phenotype. We propose the driving force, and hence the shear imposed on captured cells, as the primary axis for classifying these platforms-a testable hypothesis rather than an established result. Chemo-response profiling of the intact collective by an independent group showed 97% concordance with radiological response in therapy-naïve patients; in our own series, perioperative CTE detection stratified adjuvant chemotherapy benefit in non-small cell lung cancer. CONCLUSION: Size-selection converts a liquid biopsy into a cytology slide, placing interpretation of the circulating tumor ecosystem within the existing competence of the diagnostic laboratory.