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.