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 5

Cell-free mtDNA in tumor-bearing state derives predominantly from nontumor cells.

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Cell-free mtDNA in tumor-bearing state derives predominantly from nontum...
(A) Relative recovery of mtDNA from HD and HPV+ HNC, HPV– HNC, and AML cancer patient plasma by DNA-IP. Dotted red line indicates the mean relative recovery of plasma nDNA. Dotted blue line indicates the mean relative recovery of mtDNA from cell line–conditioned media. (B and E) Concentration of protected and accessible cf-mtDNA in HD and patient plasma (B) or Cal33 xenograft plasma (E). Data are represented as box-and-whiskers plots displaying mean (+ symbol), median (horizontal line), minimum (lower whisker), 25th percentile (lower bound of box), 75th percentile (upper bound of box), and maximum (upper whisker). (C) Concentration of protected and accessible cf-mtDNA in cell line–conditioned media. (D) Plots of protected (left) and accessible (right) cf-mtDNA in HPV+ HNC patient plasma versus tumor burden (as measured by ctDNA). Data are fit with a nonlinear log-log regression and dashed lines represent 95% confidence interval. (F) An updated schematic of cfDNA origins and structure in tumor-bearing individuals reflecting our findings. Cell-free nDNA from both nontumor and tumor cells is not associated with EVs and exists as mono- and oligo-nucleosome particles. Conversely, a portion of cf-mtDNA from both tumor and nontumor cells is protected within membranous structures. However, cf-mtDNA from nontumor cells is more abundant, and a greater proportion is membrane protected than cf-mtDNA from tumor cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, nonparametric 2-sided t test with Mann-Whitney test (B and E); unpaired 2-sided t test (C); nonlinear log-log regression with Spearman correlation (D). Human cohorts: HD n = 50, HPV+ HNC n = 49, HPV– HNC n = 44, AML n = 6; n = 4 for mouse cohort.