Physics-informed decoding of photoacoustic flow cytometry waveforms reveals in vivo melanoma CTC states

Circulating tumor cells (CTCs) are informative biomarkers of hematogenous dissemination and metastatic risk, but conventional assays still capture only a limited and discontinuous fraction of the circulation. In vivo photoacoustic flow cytometry (PAFC) offers a label-free route for monitoring melanoma CTCs, yet most analyses still treat PAFC primarily as a thresholding and counting tool. Here we used an end-to-end simulation framework to determine how vascular position, cell size, cell-vessel coupling, cluster morphology, and sampling conditions reshape PAFC waveforms across both the single-pulse and event domains. Position mainly controlled pulse visibility and defined a robust central operating window of about -4.2 to 4.2 μm, cluster morphology broadened events from about 2.8 ms in the compact state to 5.6 ms in the single-file state, cell size most clearly altered pulse width and N-wave structure, and contrast-optimal coupled states did not coincide with peak-optimal states. Flow speed together with beam width then determined whether the event sequence was sampled densely enough for reliable decoding. These results convert PAFC waveforms from simple count signals into interpretable signatures of melanoma CTC state and provide a mechanistic basis for longitudinal monitoring and diagnostic support.