Cell-free DNA topology depends on its subcellular and cellular origins in cancer
Ethan Z. Malkin, Steven De Michino, Meghan Lambie, Rita Gill, Zhen Zhao, Ariana Rostami, Andrea Arruda, Mark D. Minden, Scott V. Bratman
Ethan Z. Malkin, Steven De Michino, Meghan Lambie, Rita Gill, Zhen Zhao, Ariana Rostami, Andrea Arruda, Mark D. Minden, Scott V. Bratman
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Resource and Technical Advance Cell biology Oncology

Cell-free DNA topology depends on its subcellular and cellular origins in cancer

Abstract

Cancer cells release large quantities of cell-free DNA (cfDNA) into the surrounding tissue and circulation. As cfDNA is a common source of biomarkers for liquid biopsy and has been implicated as a functional mediator for intercellular communication, fundamental characterization of cfDNA topology has widespread biological and clinical ramifications. Whether the topology of cfDNA is such that it exists predominantly in membrane-bound extracellular vesicles (EVs) or in nonvesicular DNA-protein complexes remains poorly understood. Here, we employed a DNA-targeted approach to comprehensively assess total cfDNA topology in cancer. Using preclinical models and patient samples, we demonstrate that nuclear cfDNA is predominantly associated with nucleosomal particles and not EVs, while a substantial subset of mitochondrial cfDNA is membrane protected and disproportionately derived from nontumor cells. In addition, discrimination between membrane-protected and accessible mitochondrial cfDNA added diagnostic and prognostic value in a cohort of head and neck cancer patients. Our results support a revised model for cfDNA topology in cancer. Due to its abundance, nuclear cfDNA within nucleosomal particles is the most compelling liquid biopsy substrate, while EV-bound and accessible mitochondrial cfDNA represent distinct reservoirs of potential cancer biomarkers whose structural conformations may also influence their extracellular stability and propensity for uptake by recipient cells.

Authors

Ethan Z. Malkin, Steven De Michino, Meghan Lambie, Rita Gill, Zhen Zhao, Ariana Rostami, Andrea Arruda, Mark D. Minden, Scott V. Bratman

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Figure 2

Cell-free nDNA from conditioned media is nucleosomal and not vesicle associated.

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Cell-free nDNA from conditioned media is nucleosomal and not vesicle ass...
(A) Left: Schematic of permeabilization/degradation assay on membrane-protected DNA. Samples were treated with either PBS or Triton X-100 (i.e., the condition) followed by subsequent treatment with either PBS mock or DNase I (i.e., the treatment), before being subjected to DNA-IP. Right: HCT116 cells were used as a positive control for membrane-protected DNA subjected to permeabilization/degradation assay. Values were normalized to the PBS/mock treatment. (B) Permeabilization/degradation assays on conditioned media from MCF10A, B16F10, and HCT116 cells. Values were normalized to their respective PBS/mock treatment. (C) DNA-IP was performed on conditioned media from MCF10A, B16F10, and HCT116 cells, and lipid content in each fraction was quantified using a modified phospho-sulfo-vanillin assay. Values were normalized to their respective input fraction. (D) Immunoblotting of common EV markers HSP90, CD63, flotillin1, and syntenin1, as well as histone H3, after DNA-IP of conditioned media. Representative image of blot from HCT116-conditioned media. See complete unedited blots in the supplemental material. (E) Cell-free DNA from untreated conditioned media (top) or MNase-treated media (bottom) was purified, and fragment sizes were quantified by BioAnalyzer. Shown are both the BioAnalyzer electropherogram and gel image for each treatment. The green lower marker (LM) is 35 bp, and the purple upper marker (UM) is 10,380 bp. Representative data from HCT116-conditioned media. ns, not significant; **P < 0.01, ***P < 0.001, ****P < 0.0001, ordinary 2-way ANOVA with Tukey’s multiple-comparison test (A and B); unpaired 2-sided t test with Welch’s correction (C).