Extracellular vesicles are the primary source of blood-borne tumour-derived mutant KRAS DNA early in pancreatic cancer

Abstract

Up to now, the field of liquid biopsies has focused on circulating tumour DNA and cells, though extracellular vesicles (EVs) have been of increasing interest in recent years. Thus, reported sources of tumour-derived nucleic acids include leukocytes, platelets and apoptotic bodies (AB), as well as large (LEV) and small (SEV) EVs. Despite these competing claims, there has yet to be a standardized comparison of the tumour-derived DNA associated with different components of blood. To address this issue, we collected twenty-three blood samples from seventeen patients with pancreatic cancers of known mutant KRAS G12 genotype, and divided them into two groups based on the time of patient survival following sampling. After collecting red and white blood cells, we subjected 1 ml aliquots of platelet rich plasma to differential centrifugation in order to separate the platelets, ABs, LEVs, SEVs and soluble proteins (SP) present. We then confirmed the enrichment of specific blood components in each differential centrifugation fraction using electron microscopy, Western blotting, nanoparticle tracking analysis and bead-based multiplex flow cytometry assays. By targeting wild type and tumour-specific mutant KRAS alleles using digital PCR, we found that the levels of mutant KRAS DNA were highest in association with LEVs and SEVs early, and with SEVs and SP late in disease progression. Importantly, we established that SEVs were the most enriched in tumour-derived DNA throughout disease progression, and verified this association using size exclusion chromatography. This work provides important direction for the rapidly expanding field of liquid biopsies by supporting an increased focus on EVs as a source of tumour-derived DNA.

Keywords: apoptotic bodies; cancer diagnostics; ctDNA; digital PCR; exosomes; extracellular vesicles; liquid biopsy; microvesicles; pancreatic cancer; platelets.

FIGURE 1

Differential centrifugation of whole blood efficiently separates different cell and vesicle populations. (a) Schematic of the differential centrifugation protocol used to separate red (RBC) and white blood cells (WBC), platelets (PL), apoptotic bodies (AB), large (LEV) and small extracellular vesicles (SEV), as well as soluble protein (SP) and flow through (FT). (b) Representative transmission electron microscopy images of vesicles enriched in AB, LEV and SEV fractions. Corresponding scale bars are inset. (c) Representative Western blots of blood components for histone H3 (H3), CD42A, cleaved Caspase 9 (CASP9), BAX, CD9 and CD81. A CD9 blotted membrane with vesicular and SP components was additionally overexposed. Molecular weight is indicated beside each blot. (d) Nanoparticle tracking analysis data showing the concentration of particles/ml at sizes ranging from 0–1 μm in LEV, SEV and SP fractions. Inset is a quantification of the concentration of particles from 400–600 nm found in each fraction. (d) Median CD9/63/81 fluorescence for 35 capture antibody coated beads from the MACSplex vesicle surface protein flow cytometry assay in the different blood components. The platelet fraction was diluted 1:10 before analysis

FIGURE 2

Blood components are associated with distinct amounts of wild type and mutant KRAS DNA in PDAC patients. (a) Kaplan‐Meier curves showing the days of survival following blood sampling for 14 early and 13 late group samples. (b) Copies/μl dPCR reaction mix of wild type (left) and mutant (right) KRAS DNA found in all blood components of early and late group patients. (c) Patient normalized copies/μl of mutant KRAS DNA found in each blood component in early (top) and late (bottom) group patients. (d) Ratio of mutant : wild type KRAS DNA copies/μl for each blood component and platelet poor plasma (PPP) in early patients. (e) Particle count and protein concentration for the five SEC fractions derived from two pooled SEV fractions. (f) Copies/μl of mutant KRAS DNA found in each SEC fraction of patient SEVs. (g) Copies/μl of mutant KRAS DNA found in each SEC fraction of patient SP. Statistics for a&b are derived from two‐tailed unequal variance t‐tests between indicated groups. Statistics for c–g are derived from two‐tailed unpaired Mann‐Whitney‐Wilcoxon tests between indicated groups