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Clinical Trial
, 289 (7), 3869-75

Identification of Double-Stranded Genomic DNA Spanning All Chromosomes With Mutated KRAS and p53 DNA in the Serum Exosomes of Patients With Pancreatic Cancer

Affiliations
Clinical Trial

Identification of Double-Stranded Genomic DNA Spanning All Chromosomes With Mutated KRAS and p53 DNA in the Serum Exosomes of Patients With Pancreatic Cancer

Christoph Kahlert et al. J Biol Chem.

Abstract

Exosomes are small vesicles (50-150 nm) of endocytic origin that are released by many different cell types. Exosomes in the tumor microenvironment may play a key role in facilitating cell-cell communication. Exosomes are reported to predominantly contain RNA and proteins. In this study, we investigated whether exosomes from pancreatic cancer cells and serum from patients with pancreatic ductal adenocarcinoma contain genomic DNA. Our results provide evidence that exosomes contain >10-kb fragments of double-stranded genomic DNA. Mutations in KRAS and p53 can be detected using genomic DNA from exosomes derived from pancreatic cancer cell lines and serum from patients with pancreatic cancer. In addition, using whole genome sequencing, we demonstrate that serum exosomes from patients with pancreatic cancer contain genomic DNA spanning all chromosomes. These results indicate that serum-derived exosomes can be used to determine genomic DNA mutations for cancer prediction, treatment, and therapy resistance.

Keywords: Chromosomes; DNA; Double-stranded genomic DNA; Exosomes; KRAS; Mutations; Pancreatic Cancer; Serum; p53.

Figures

FIGURE 1.
FIGURE 1.
Exosomes contain >10-kb fragments of double-stranded genomic DNA. A and B, the presence and concentration of exosomes from human pancreatic cancer cell lines and human serum samples from patients with pancreatic cancer were determined by using a NanoSight® LM10 (A) and electron microscopy (B). C and D, exosomes were characterized by the exosome-specific expression of CD9, TSG101, and CD63 by FACS analysis (C) and the exosome-specific expression of TSG101 and CD63 by Western blotting (D). E, to exclude RNA contamination after exosome lysis and DNA extraction, the DNA eluate from two cell lines (Panc-1 and T3M4) and the DNA eluate from corresponding exosomes were treated with DNase I and RNase A. Subsequently, the eluate was run on a 2% agarose gel. F, the presence of double-stranded DNA from cellular Panc-1 exosomes and human serum exosomes was confirmed by a double-stranded detection kit (representative figure for exosomal DNA from Panc-1, one healthy donor, and one patient with pancreatic cancer). PDAC, pancreatic ductal adenocarcinoma. SSC-A, side scatter detector A.
FIGURE 2.
FIGURE 2.
Exosomes contain mutated KRAS and p53 DNA. A, by PCR, we amplified a 466-bp fragment of KRAS spanning exon 2 and intron 2 and a 1564-bp fragment of p53 spanning 4 exons and 3 introns. B, Sanger sequencing of genomic DNA from Panc-1 cells and corresponding exosomes revealed the same heterozygous mutation of KRAS on codon 12 (GGT to GAT) and the similar homozygous mutation of p53 on codon 273 (CGT to CAT). T3M4 cells and corresponding exosomes displayed the same homozygous mutation of p53 on codon 220 (TAT to TGT). C, PCR amplification provided evidence for long fragments of DNA in circulating exosomes from two healthy donors and two patients with pancreatic cancer. We could retrieve a 466-bp fragment of KRAS DNA and 609-bp fragment of p53 DNA spanning exons 7 and 8 and intron 7. When serum samples depleted of exosomes were subjected to PCR, no KRAS or p53 amplicon could be detected. PDAC, pancreatic ductal adenocarcinoma. D, Sanger sequencing of serum exosome-derived DNA was able to detect DNA with a KRAS mutation on codon 22. In a second patient, Sanger sequencing revealed a KRAS mutation on codon 12 and a p53 mutation on codon 273.
FIGURE 3.
FIGURE 3.
Serum-derived exosomes contain genomic DNA spanning all chromosomes. A and B, whole genome sequencing was conducted on serum-derived, exosomal DNA and corresponding primary tumor from two patients. BIC-seq control-free log2 copy-number profile across all human chromosomes, bin size 1000 bp; RAW profile, black; segmented, red line. Profiles demonstrate somatic chromosomal gains (upper) and losses (lower), as well as normal polymorphism. In the second case (B), a lack of structural chromosomal rearrangement expected for pancreatic ductal adenocarcinoma is explained due to the possible low number of cancer cells in the sample. Sequencing revealed that circulating exosomes contain genomic DNA spanning all chromosomes.

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