Written by 丨Xiao Ping
As the name suggests, extracellular free DNA (cell-free DNA, cfDNA) is composed of extracellular DNA fragments and can be obtained through plasma or serum. In healthy individuals, it mainly comes from the death of blood cells. cfDNA can change under different physiological and pathological states. For example, in cancer patients, a part of cfDNA comes from tumor cells. Sequence analysis of this part of DNA, known as circulating tumor DNA, ctDNA, , can reveal tumor-specific gene changes. liquid biopsy based on cfDNA analysis provides a new strategy for noninvasive diagnosis [1-3].
In plasma, cfDNA mainly exists in the form of nucleosome (cf nucleosome). Nucleosomes are the basic constituent unit of chromosomes, which are formed from ~150 bp DNA-encapsulated histone octamers. In addition to the DNA sequence information, the combination of different chemical modifications of histone imparts nucleosome tissue-specific epigenetic information and provides the state of intracellular gene expression and regulation. Evidence shows that some epigenetic information is retained on the cf nucleosome. Through ChIP-seq, the epigenetic information on it can be interpreted 【4】.
Although the current method can allow us to obtain epigenetic information of cf nucleosomes in plasma, they have great limitations. Mainly speaking, the samples for related detection are large, the detection range is limited, and the cost is high. At the same time, the information obtained is limited and the resolution is poor. For example, only a single observation information can be obtained (such as a single modified distribution, or nucleosome placeholding). Therefore, high-resolution detection methods that integrate information at different levels need to be established.
Recently, the research team of the Weizmann Institute of Science in Israel Efrat Shema research team published the Nature BiotechnologyMultiplexed, single-molecule, epigenetic analysis of plasma-isolated nuclearosomes for cancer Diagnostics research paper, developed a liquid biopsy method based on single-molecular imaging, EPINUC, a technology that can analyze multiple epigenetic information parameters from plasma samples with less than 1 mL. Further, the researchers used this assay method, combined with high sensitivity detection of protein biomarkers, to prove their important value in the diagnosis of colorectal cancer.
First, based on the previous establishment of imaging technology (Total internal reflection microscopy. TIRF) single-molecular imaging system that observes histone modification combination [5] . Researchers developed EPINUC (Epigenetics of plasma-isolated nuclearosomes) technology (See Figure 1, see the details of the process, see the notes of the figure) .
Figure 1 Technical roadmap. a. Sample preparation process of cf nucleosomes. This process consists of two enzymatic reactions to , which are repairing the DNA terminals through Klenow polymerase, and adding poly(A) tails through terminal transferase . A mixture of dATPs and fluorescently labeled dATPs (Cy3-dATPs) was used in this process to label nucleosomes. b. cf nucleosomes are immobilized on the PEGylated-poly(T) surface by dA:dT hybridization, and are then incubated with fluorescently labeled antibodies targeting different histone modifications for modified labeling. c. Record nucleosome locations using TIRF microscope to generate time-lapse imaging of antibody binding events.
researchers tested the specificity and binding linearity of the antibodies, and finally confirmed that six antibodies were suitable for the EPINUC system, namely H3K4me3, H3K36me3 and H3K9ac, which characterize transcriptional activation states, H3K9me3 and H3K27me3, which characterize transcriptional repression and heterochromatin states, and H3K4me1, which characterize enhancers. During the experiment, the nucleosomes were labeled with Cy3 (green) , and the combination of the two histone modifications could be labeled with AF488 (blue green) and AF647 (red) , respectively. Under the same observation system, the proportion of nucleosomes with specific histone modifications, the proportion of different histone modifications on the nucleosomes, and the common labeling of different histone modifications on the same nucleosomes can be recorded and analyzed. Through multiple tests of the same sample, the repetition/stability of this method has also been verified. EPINUC is the first technology to detect by using small volume samples (1 mL) for single-molecular precision nucleosome modification feature.
On this basis, in order to obtain more evaluation parameters, the researchers used a single-molecular system to increase the evaluation and quantification of protein biomarkers and DNA methylation (see Figure 2) .
Figure 2 Technology roadmap. a. Protein marker detection. First, the biotinylated antibodies targeting different proteins were anchored on the PEG-strepvidin surface, and then the corresponding protein in the plasma was captured by incubation, and finally the detection was performed using fluorescently labeled antibodies and TIRF imaging. This solution can complete the simultaneous detection of 3 proteins . b. DNA methylation detection. First, biotinylated MBD2 and Cy3-labeled cfDNAs were incubated. The former could specifically bind to the latter carrying methylated modification. Then, by incubation with PEG-streptavidin, the biotin-MBD2-methylated cfDNA was anchored on the PEG-streptavidin surface, and then TIRF imaging was performed. Each imaging point represents a single binding complex, the number of points corresponds to the level of DNA methylation in plasma.
has established the above detection platform. The researchers used EPINUC to obtain diagnostic information on three levels - histone modification, DNA methylation and protein biomarkers - from plasma samples from 33 healthy individuals and 40 advanced CRC patients (preoperative or after surgical resection and chemotherapy). Carcinoembryonic antigen (CEA) has an elevated plasma level in CRC patients and is a classic clinical biomarker 【6】. Through single-molecular imaging counting, the CEA level in CRC individuals was higher, and the CEA level in patients decreased after surgical resection. But it is worth noting that higher levels of CEA are also observed in a few healthy individuals. Simultaneous detection of MST1 and CEA/MST1 ratio measurement can better diagnose the samples, demonstrating the advantages of combined biomarker detection. In addition, the plasma CEA/MST1 ratio of patients after surgical resection was changed compared with those of unresected CRC patients and had higher similarity with plasma from healthy people, indicating the potential value of this test in monitoring treatment prognosis.
EPINUC also provides quantitative measurements of the ratios including the total number of cf nucleosomes, 6 histone modifications and their pairwise combinations in each plasma sample. Consistent with relevant reports, the plasma cf nucleosomes in CRC patients were higher than those in healthy control groups. It can be seen that although most epigenetic modifications of and its parameters have not changed, there are several parameters that have significantly different differences: the levels of nucleosomes modified by H3K27me3, H3K9me3, H3K9ac and H3K4me1 in CRC patients are higher; the ratio of H3K9ac/H3K4me1 is higher. It is worth noting that in CRC, the proportion of nucleosomes jointly modified by H3K9me3 and H3K36me3 decreased, and the proportion of "bivalent'" nucleosomes marked by H3K4me3 and H3K27me3 increased, which coincides with classical cognition. In addition, the reduced DNA methylation level in CRC samples is consistent with previous studies.
The changes in epigenetics and biomarkers of late CRC prompted researchers to focus on whether EPINUC is suitable for the diagnosis of early CRC (phase I and II). Using 17 plasma samples, they found that, like advanced CRC, there were significant differences in DNA methylation levels, CEA, and CEA/MST1 ratios between early patients and healthy individuals. It is worth noting that the levels of H3K27me3 and H3K9me3 modified nucleosomes in the plasma of patients with stage I and II CRC are the same as those observed in advanced CRC. Additionally, we did not observe an increase in cf nucleosome levels in early CRC, which may be associated with lower tumor burden. Although the levels of H3K4me1 and H3K9ac modified nucleosomes did not differ significantly from the healthy group, the proportion of co-modified nucleosomes in early CRC patients was lower than that in healthy individuals. The above results show that various epigenetic information changes have occurred in the cf nucleosomes in early CRC patients. Furthermore, the detection and analysis of plasma samples of patients with pancreatic ductal adenocarcinoma prove that EPINUC can help diagnose a variety of cancers with its advantages of multi-level and multi-dimensional information acquisition.
To visually present the above results, the researchers conducted principal component analysis (PCA). PCA showed spatial distances between groups, where samples of early CRC patients were located between healthy individuals and samples of advanced CRC patients, possibly reflecting a transitional relationship. Immediately afterwards, the researchers used machine learning to use machine learning, the best prediction model showed diagnostic performance that was better than purely relying on protein biomarkers, biomarker combination DNA methylation or biomarker combination histone modification, with a sensitivity of 92% (95% CI 89.3-94.7), specificity of 85% (95% CI 80.2-89.8), and accuracy of 92% (95% CI 89.7-94.3).
Figure 3 Technology roadmap. Obtaining histone information is as described above. In short, cf nucleosomes are anchored through dA:dT hybridization, and then incubated and imaged by carrying fluorescently labeled histone-specific modified antibodies. Immediately afterwards, the histones were removed, the intact DNA was exposed in situ, and a single-molecule sequencing process that relies on TIRF imaging was carried out to obtain DNA sequence information.
Finally, the researchers combined single-molecular DNA sequencing technology to develop EPINUC-seq technology (see Figure 3). uses the differences in epigenetic modifications of different tissues to explore the possibility of judging the origin of the cf nucleosome by detecting and confirming the tissue of the origin of the cf nucleosome.
This work has developed EPINUC single-molecular detection technology, applied it to liquid biopsy, and used single-molecular accuracy to detect multi-layered diagnostic information, including histone and DNA modifications and protein biomarkers, which can distinguish CRC patients and healthy populations with highly specific and sensitive. Obviously, this technology greatly broadens the field of liquid biopsy and has the potential to be applied to early diagnosis and prognostic monitoring of tumors.
original link:
https://www.nature.com/articles/s41587-022-01447-3
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references
1. Heitzer, E., Haque, I. S., Roberts, C. E. S. Speicher, M. R. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat. Rev. Genet. 20, 71–88 (2019).
2. Lo, Y. M. D., Han, D. S. C., Jiang, P. Chiu, R. W. K. Epigenetics, fragmentomics, and topology of cell-free DNA in liquid biopsies. Science 372, eaaw3616 (2021).
3. Shen, S. Y. et al. Sensitive tumour detection and classification using plasma cell-free DNA methylomes. Nature563, 579–583 (2018).
4. Sadeh, R. et al. ChIP–seq of plasma cell-free nuclearosomes identifies gene expression programs of the cells of origin. Nat. Biotechnol. 39, 586–598 (2021).
5. Shema, E. et al. Single-molecule decoding of combinedatorially modified nuclearosomes. Science 352, 717–721 (2016).
6. Tiernan, J. P. et al. Carcinoembryonic antigen is the preferred biomarker for in vivo colorectal cancer targeting. Br. J. Cancer108, 662–667 (2013).
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