Simulating cell-free chromatin using preclinical cancer models for liquid biopsy applications [cfChIPseq]

Cell-free DNA circulates in blood bound to nucleosomes, forming cell-free chromatin (cfChromatin) that retains epigenetic features, including nucleosome positioning and histone modifications. cfChromatin provides a rich source of cancer biomarkers; however, low abundance of tumor-derived cfChromatin and limited availability of clinical samples pose challenges for liquid biopsy research. To address this, we developed a framework to simulate cfChromatin nucleosomal distributions using nuclease-treated conditioned media from tissue cultures. Whole-genome sequencing confirmed that inferred nucleosome positioning reflected cell-type-specific gene expression and chromatin accessibility patterns, and comparisons with plasma cfChromatin from xenografted mice revealed concordant nucleosome profiles. Notably, simulated cfChromatin displayed stronger tumor-specific nucleosome profiles than patient plasma, where hematopoietic-derived cfChromatin dilutes signal. We further leveraged simulated cfChromatin to advance cell-free chromatin immunoprecipitation and sequencing methods, identifying repressive and bivalent chromatin domains predictive of transcriptional activity. Altogether, our results demonstrate utility of simulated cfChromatin as a scalable preclinical tool for liquid biopsy research. Chromatin Immunoprecipitation DNA sequencing (ChIP-Seq) of media cell-free chromatin (cf-chromatin) from SU-DHL-6 cells, at varying input concentrations (H3K4me3, 30ng and 300ng; H3K27me3, 10ng, 30ng, and 300ng) after 30 minutes of micrococcal nuclease (MNase) digestion. Biological replicates were generated per condition.