Cell‐Free DNA as a Biomarker of Cell Death and Treatment Response for On‐Chip Three‐Dimensional Tumor Models

On-chip 3D cancer models are important tools to investigate treatment response and tumor biology. With their increasing adoption, there is a need for non-destructive methods that enable the sequential monitoring of cell death and tumor response over time. This study presents quantifying cell-free DNA (cfDNA) as a proxy for cell death for on-chip 3D sarcoma models. qPCR analysis of media cfDNA from spheroids and micro-dissected tumor explants (MDTs) generated from SK-LMS-1 and MCA-205 cell lines cultured on microfluidic chips showed a correlation between baseline cfDNA concentration and on-chip tumor burden. Furthermore, cfDNA levels increased in a dose-dependent manner following cytotoxic treatments, including staurosporine and hydrogen peroxide. Single-dose radiation therapy also raised cfDNA levels in media from MCA-205 but not SK-LMS-1 MDTs, in accordance with their 2D culture response. Finally, cfDNA release within the first days of culture was shown to reflect patient-derived MDT survival, addressing a critical unmet need for ex vivo culture. This study suggests that media cfDNA quantification could be used as a reliable, non-disruptive, and rapidly quantifiable measure of cell death for on-chip 3D models, which facilitates more robust use of 3D model systems for pre-clinical assessment of treatment-induced cell death and biological response.