Simulating cell-free chromatin using preclinical models for cancer-specific biomarker discovery

Cell-free chromatin (cf-chromatin) is a rich source of biomarkers across various conditions, including cancer. Tumor-derived circulating cf-chromatin can be profiled for epigenetic features, including nucleosome positioning and histone modifications that govern cell type-specific chromatin conformations. However, the low fractional abundance of tumor-derived cf-chromatin in blood and constrained access to plasma samples pose challenges for epigenetic biomarker discovery. Conditioned media from preclinical tissue culture models could provide an unencumbered source of pure tumor-derived cf-chromatin, but large cf-chromatin complexes from such models do not resemble the nucleosomal structures found predominantly in plasma, thereby limiting the applicability of many analysis techniques. Here, we developed a robust and generalizable framework for simulating cf-chromatin with physiologic nucleosomal distributions using an optimized nuclease treatment. We profiled the resulting nucleosomes by whole genome sequencing and confirmed that inferred nucleosome positioning reflected gene expression and chromatin accessibility patterns specific to the cell type. Compared with plasma, simulated cf-chromatin displayed stronger nucleosome positioning patterns at genomic locations of accessible chromatin from patient tissue. We then utilized simulated cf-chromatin to develop methods for genome-wide profiling of histone post-translational modifications associated with heterochromatin states. Cell-free chromatin immunoprecipitation and sequencing (cf-ChIP-Seq) of H3K27me3 identified heterochromatin domains associated with repressed gene expression, and when combined with H3K4me3 cfChIP-Seq revealed bivalent domains consistent with an intermediate state of transcriptional activity. Combining cfChIP-Seq of both modifications provided more accurate predictions of transcriptional activity from the cell of origin. Altogether, our results demonstrate the broad applicability of preclinical simulated cf-chromatin for epigenetic liquid biopsy biomarker discovery. ### Competing Interest Statement S.V.B. is inventor on patents related to cell-free DNA mutation and methylation analysis technologies that are unrelated to this work and have been licensed to Roche Molecular Diagnostics and Adela, respectively. S.V.B. is a co-founder of, has ownership in, and serves in a leadership role at Adela. M.M.H. is an inventor on a patent application related to cell-free methylation analysis licensed to Adela. D.W.C reports consulting/advisory roles with AstraZeneca, Exact Sciences, Eisai, Gilead, GlaxoSmithKline, Inflex, Inivata, Merck, Novartis, Pfizer, Roche and Saga; research funding to institution from AstraZeneca, Guardant Health, Gilead, GlaxoSmithKline, Inivata, Knight, Merck, Pfizer, and Roche and Patent (US62/675,228) for methods of treating cancers characterized by a high expression level of spindle and kinetochore associated complex subunit 3 (ska3) gene. R.K. reports research funding from Roche and Abbvie. All other authors declare no conflicts.