Cell-free DNA (cfDNA) and Exosome Profiling from a Year-Long Human Spaceflight Reveals Circulating Biomarkers

Abstract

Liquid biopsies based on cell-free DNA (cfDNA) or exosomes provide a noninvasive approach to monitor human health and disease but have not been utilized for astronauts. Here, we profile cfDNA characteristics, including fragment size, cellular deconvolution, and nucleosome positioning, in an astronaut during a year-long mission on the International Space Station, compared to his identical twin on Earth and healthy donors. We observed a significant increase in the proportion of cell-free mitochondrial DNA (cf-mtDNA) inflight, and analysis of post-flight exosomes in plasma revealed a 30-fold increase in circulating exosomes and patient-specific protein cargo (including brain-derived peptides) after the year-long mission. This longitudinal analysis of astronaut cfDNA during spaceflight and the exosome profiles highlights their utility for astronaut health monitoring, as well as cf-mtDNA levels as a potential biomarker for physiological stress or immune system responses related to microgravity, radiation exposure, and the other unique environmental conditions of spaceflight.

Keywords: Omics; Space Medicine.

Graphical abstract

Figure 1

Size Distribution of cfDNAs in Ambient Return, Ambient Return Simulation, and Fresh Samples (A) Ambient return simulation samples (control and ground samples with yellow border) show a highly similar pattern as observed for inflight samples (blue box with yellow border). Long cfDNA fragments likely originate from blood cells damaged during transport. (B) Ambient return samples show an increased fraction of cfDNA with fragment length >300bp compared to fresh samples. Our experimental procedure does only allow interrogation of DNA fragments up to a length of 500bp; thus, the content of long mtDNA fragments contained in intact circulating mitochondria is not reflected in this analysis.

Figure 2

Size Distribution of cf-mtDNAs We observed a wider range of cf-mtDNA lengths compared to total cfDNA (from 100 to 600bp). (A) cf-mtDNA size distributions are similar in ground, flight, and control samples and are not affected by ambient return (AR) or AR simulation. (B) Average length of cf-mtDNA is significantly longer than the average length reported for chromosomal cfDNA (~250bp vs. ~160bp). The average length of cf-mtDNA is not affected by sample type (control, flight, ground) or sample handling (fresh, AR, AR simulation)

Figure 3

Analysis of Normalized cfDNA Read Counts by Chromosome, including the Mitochondrial Genome (A) TW exhibits a significant increase in cell-free mtDNA during space flight compared to TW and HR ground samples. Counts are reads per kilobase per million reads or RPKM. (B) Chromosomes do not show any change in RPKM during space flight, as exemplified using chr21. (C) Q-PCR-based validation of increased cf-mtDNA fraction in plasma during space flight. (D) Normalized cf-mtDNA fraction and fraction of reads mapping to chr21 for 12 time point during the mission (T4-T6 = space flight). The highest increase in cf-mtDNA fraction is observed during the first months on the ISS. (E) Ambient return simulation using two control samples showed no increase in cf-mtDNA compared to fresh samples but a slight reduction. (F) Ambient return (AR) simulation using one HR ground sample did not show a significant increase in cf-mtDNA fraction. Two outliers within the fresh samples (FR) indicate that other conditions (e.g. stress, disease, immune reaction) could have influenced cf-mtDNA levels of HR on the ground.

Figure 4

cfDNA Nucleosome Footprinting (A) Nucleosome depletion in cfDNA around transcription start sites (TSSs) is highly correlated with the expression of the respective genes and can therefore be used to estimate promoter activity and gene expression. (B) t-SNE based on genome-wide promoter nucleosome footprint of cfDNA samples reveals no clustering of flight subject and ground subject samples.

Figure 5

Tissue of Origin Deconvolution (A) Correlation coefficients (multiplied by −1) for each tissue in each sample, clustered by sample and by tissue. The highest signals are, expectedly, from cells of hematopoietic origin. Spaceflight-dependent dynamics of tissue signal are confounded by the effect of ambient return, as suggested by ambient return samples tending to cluster together regardless of other features. (B) Clustering of samples using TSS protection in cfDNA as a measure of gene expression (lower protection correlates to higher expression). Ambient return samples cluster tightly together and uncover two major clusters of genes whose expression differs significantly from other samples, suggesting transport-related degradation processes or nucleosome detachment. Distribution of mean TSS protection per gene in ambient return and freshsamples is significantly different (t-test p<1e-3).

Figure 6

Characterization of Plasma-Derived Exosomes Isolated from HR and TW (A–E) Plasma samples were collected 3 years (TW) and 9 years (HR) post-flight. Nanosight profiles showing size distribution for exosomes isolated from the plasma of (A) control, (B) HR, and (C) TW. Median size of exosomes (D) and exosome concentration (E) in TW (n = 1), HR (n = 1), and controls (n = 6). (F) Venn diagram of exosomal proteins identified by mass spectrometry in plasma isolated from HR, TW, and age-matched healthy controls. (G–J) (G) Heatmap of plasma-derived exosomal proteins for HR, TW, and age-matched healthy controls. Pathway analysis of exclusive plasma-derived exosomal proteins from (H) TW, (I) HR, and (J) age-matched healthy controls.