Single-molecule imaging and tracking on clinical liquid biopsies reveals cancer biomarkers nanoscale organization and heterogeneity

Single-molecule imaging and tracking have revealed fundamental biological mechanisms at the molecular scale, yet their application to clinical research remains limited by technical complexity and sample preparation incompatible with patient-derived specimens. As a result, we lack information with molecular-scale resolution of clinically relevant biomarkers. Here, we develop a workflow enabling Points Accumulation in Nanoscale Topography (PAINT) combined with single-particle tracking (SPT) on clinical liquid biopsies, allowing analysis of biomarker nanoscale organization at the single-molecule level in cancer patients. Our approach features a sample preparation tailored to liquid biopsies and requires no fixation, covalent labelling, or genetic modification, making single-molecule imaging compatible with hospital clinical workflows. We demonstrate the methods diversity by imaging liquid biopsies from blood, bone marrow aspirates, and pleural effusions across different cancer types. PAINT-SPT captures both the expression and mobility of clinically relevant membrane receptor biomarkers, revealing pronounced inter- and intra-patient heterogeneity at the molecular and cellular levels. We discover that individual patients exhibit distinct molecular mobility fingerprints that reflect biomarker interaction states and correlate with clinical diagnostic readouts. Furthermore, these fingerprints distinguish healthy from cancer cells, enabling the development of a classifier that accurately identifies cancer cells based on their single-molecule behaviour. Together, our results establish a route to investigate patient-derived clinical samples at the single-molecule level and open new opportunities to understand cancer biology and biomarker function beyond ensemble-averaged measurements.