Parallel microfluidic platform enables efficient label-free capture of p16+ circulating tumor cells in oropharyngeal cancer

Circulating tumor cells (CTCs) are clinically significant biomarkers for cancer detection and monitoring due to their role in metastasis. However, their extreme rarity in peripheral blood and phenotypic heterogeneity, particularly following epithelial-to-mesenchymal transition (EMT), pose challenges for reliable isolation. Conventional epitope-dependent capture methods often fail to detect mesenchymal-like CTCs. Label-free microfluidic platforms offer a promising alternative by exploiting biophysical properties such as size and deformability. Building on our prior work, we developed a high-throughput multi-flow microfluidic (MFM) device to isolate diverse CTCs from clinically relevant blood volumes, with a focus on oropharyngeal cancer (OPC), where CTCs exhibit favorable size characteristics for inertial separation. A parallel microfluidic device comprising six inertial separation channels was fabricated using dry film photolithography and PDMS molding. Two versions were constructed: Chip A (20 mm in length) and Chip B (25 mm in length), both with a 150 µm x 50 µm cross-section. Device performance was evaluated using fluorescent microparticles (10.3, 15.5, and 18.7 µm) and head and neck cancer cell lines CAL27, SCC9, and HPV16-positive SCC2. Samples were processed at 600 µL/min with a buffer flow of 1.2 mL/min. Cell recovery and purity were assessed by high-content imaging and immunofluorescence. SCC2 DNA enrichment was quantified by qPCR. Clinical validation involved 20 p16+ oropharyngeal cancer patients. The MFM device achieved a 5-fold increase in throughput compared to the earlier single-channel design, while maintaining high separation efficiency. Chip A (~14 µm cutoff) recovered 90-94% of CAL27 and SCC9 cells with <17% WBC contamination. Chip B (~12 µm cutoff) improved recovery to 92%-97%, but with slightly higher WBC carryover. PCR analysis of HPV16-positive SCC2 cells confirmed >35-fold enrichment of tumor DNA in the CTC fraction. In clinical samples, CTCs were detected in 60% of OPC patients, including epithelial, mesenchymal-like, and dual CK/CSV-positive phenotypes. Notably, dual CK/CSV-positive CTCs, indicative of partial EMT, were observed in 25% of patients. p16 expression was observed in CTCs from 4 of 14 patients, suggesting potential concordance with tumor HPV status. The device preserved cell integrity, enabling downstream molecular analysis. The parallel MFM platform enables robust, label-free isolation of phenotypically diverse CTCs from clinically relevant blood volumes. Its ability to detect EMT-associated phenotypes and HPV16/p16 expression supports its utility in head and neck oncology. Compatibility with molecular workflows such as qPCR and successful clinical validation in OPC patients highlight its translational potential for liquid biopsy applications, including disease monitoring and precision oncology.