Single-Molecule Systems for the Detection and Monitoring of Plasma-Circulating Nucleosomes and Oncoproteins in Diffuse Midline Glioma

Exploring the Applications of Single-Molecule Systems for Detection and Monitoring of Plasma-Circulating Nucleosomes and Oncoproteins in Diffuse Midline Glioma

Research Background and Problem Overview

Diffuse Midline Glioma (DMG) is a highly aggressive brain tumor primarily affecting children with extremely high mortality rates. This type of tumor typically arises in midline structures of the brain, such as the thalamus, pons, cerebellum, and spinal cord. Due to its specific location, invasive biopsy poses high risks. Consequently, its diagnosis and monitoring heavily rely on imaging techniques such as Magnetic Resonance Imaging (MRI). However, traditional imaging methods often fail to effectively guide treatment and accurately assess disease progression. For instance, MRI cannot reliably differentiate between true tumor progression and treatment-induced pseudoprogression. Additionally, although conventional biopsies can provide necessary molecular information, they come with significant invasive risks, particularly for pediatric patients. This highlights an urgent need for novel, non-invasive diagnostic tools that enable molecular-level tumor evaluation and dynamic monitoring.

Recently, liquid biopsy techniques, which analyze biomarkers from patients’ bodily fluids (such as blood or cerebrospinal fluid), have garnered significant attention. Specifically, in the context of brain tumors, biomarkers like cell-free DNA (cfDNA) and circulating free nucleosomes (cfnuc), released during tumor cell apoptosis, are emerging as potential non-invasive molecular markers. However, the exceedingly low concentration of cfDNA and related proteins in brain tumors that reach the plasma limits the sensitivity of detecting specific mutations or markers. Moreover, traditional cfDNA analysis, which relies on high-throughput sequencing, is costly and time-consuming, making it unsuitable for large-scale screening. In comparison, the ultrahigh sensitivity and resolution of single-molecule technologies may effectively address these challenges.

For this purpose, the study aims to apply innovative single-molecule technologies to conduct epigenetic mapping of circulating nucleosomes in the plasma of DMG patients, combined with the detection of tumor-specific oncoproteins, such as Lysine 27-to-Methionine substitution in Histone H3 (H3-K27M) and mutant p53. This novel system is not only designed for non-invasive diagnosis but also for evaluating treatment efficacy, providing a new approach to the molecular diagnosis and monitoring of this deadly pediatric tumor.

Paper Information and Research Team

This scientific study, titled “Single-Molecule Systems for the Detection and Monitoring of Plasma-Circulating Nucleosomes and Oncoproteins in Diffuse Midline Glioma,” was conducted by Nir Erez, Noa Furth, and their team, comprising members from several internationally recognized institutions such as the Weizmann Institute of Science and the University of Michigan. The article was published on January 21, 2025, in the open-access journal Cell Reports Medicine and was supported by multiple international research grants.


Research Workflow and Methods

1. Study Design and Technological Development

The core of the study lies in the application and optimization of single-molecule imaging technology. The researchers developed a platform named EpiNuC, which enables high-resolution epigenetic mapping of circulating nucleosomes isolated from plasma. The research workflow includes the following steps:

a) Sample Collection and Nucleosome Isolation

The authors collected plasma samples from multiple cohorts, including 19 DMG patients, healthy controls (33 individuals), and other cancer patient groups (colorectal cancer and pancreatic cancer patients). Nucleosome samples were enzymatically labeled and subsequently immobilized on surfaces coated with PEG (polyethylene glycol).

b) Epigenetic Characterization

Through Total Internal Reflection Fluorescence (TIRF) microscopy, single molecules were imaged by labeling nucleosome-specific histone post-translational modifications (e.g., H3K9ac, H3K27me3) using targeted antibodies. The experiment also integrated additional multi-modal epigenetic data, such as DNA methylation and protein biomarkers.

c) Development of Tumor-Specific Oncoprotein Detection Methods

To specifically detect the highly tumor-specific H3-K27M mutation associated with DMG, the team developed a capture methodology using PEG-streptavidin-coated surfaces. By utilizing biotinylated, H3-K27M-specific antibodies, the researchers enriched these low-abundance circulating nucleosomes and detected them via fluorescent tagging. Similarly, mutant p53 protein detection employed a similar single-molecule immunoassay, quantifying its abundance and mutation ratio using antibodies targeting total and mutant-specific p53.

d) Data Analysis

Machine learning algorithms were employed to classify the collected data, evaluating the precision of single-molecule imaging techniques in molecular diagnostics. Moreover, traditional methods such as MRI imaging and droplet digital PCR (ddPCR) measurements were used as benchmarks to validate the efficacy of the proposed methodology.


2. Experimental Data and Findings

a) DMG-Specific Features Identified via Epigenetic Mapping

EpiNuC revealed significant epigenetic alterations in DMG patients’ plasma, such as elevated levels of H3K9ac and H3K4me3, compared to healthy controls. These epigenetic patterns are closely aligned with DMG-specific tumor abnormalities, laying the groundwork for molecular diagnosis.

Principal component analysis (PCA) further delineated the spatial distribution of DMG samples compared to healthy controls and other cancer groups. Although some modification patterns overlapped with other malignancies, specific alterations, such as elevated H3K27me3, were more pronounced in DMG samples.

b) Detection of H3-K27M Mutation

Using antibodies targeting H3.3-K27M and H3.1-K27M, the researchers demonstrated significantly higher single-molecule imaging signals in mutant nucleosome samples compared to wild-type (WT) controls. Furthermore, plasma from DMG patients carrying H3-K27M mutations exhibited substantial signal enhancement compared to healthy individuals and patients with other cancers.

c) Mutant p53 Detection and Treatment Monitoring

In serial samples from specific DMG patients, the study found that the proportion of mutant p53 in the plasma dynamically correlated with treatment responses. p53 levels initially decreased after chemotherapy or radiation therapy but rose during tumor relapse phases.

d) Dynamic Monitoring of Multi-Modal Biomarkers

The continuous monitoring of multiple DMG patients demonstrated strong correlations between levels of H3-K27M mutant nucleosomes, cfDNA, and tumor area measurements on MRI. Notably, in some patients, H3-K27M nucleosome levels outperformed MRI in reflecting tumor dynamics at earlier stages.


Conclusion and Significance

This study provides the first evidence that single-molecule imaging systems can precisely and conveniently detect brain tumor-associated biomarkers, offering a novel non-invasive diagnostic tool for Diffuse Midline Glioma. This innovative technology surpasses traditional molecular diagnostic methods in sensitivity and specificity, while also facilitating treatment monitoring and providing clinicians with more comprehensive decision-support data. Additionally, the system’s high throughput and low cost present promising practical applications.

The H3-K27M capture and detection methodology developed in this study also offers a technical foundation that could be extended to detect other critical oncogenic proteins (e.g., KRAS and BRAF mutations). By integrating cfDNA analysis, nucleosome epigenetics, and protein detection, multi-modal liquid biopsies hold great potential for precision medicine approaches in brain tumors.


Research Highlights

  1. Non-Invasiveness and High Sensitivity: The study enables highly accurate detection of ultra-low concentration tumor biomarkers using blood samples.
  2. Integration of Multi-Dimensional Biomarkers: The system achieves combined detection of epigenetic markers, mutant H3, and mutant p53.
  3. Dynamic Monitoring Capability: The single-molecule technology performs better than traditional MRI in precision and response speed for treatment monitoring.
  4. Wide Application Potential: The method is applicable not only for DMG but also for other tumor types in liquid biopsy.

Through this research, the authors have opened a new technological avenue for the molecular diagnosis and treatment monitoring of Diffuse Midline Glioma. This groundbreaking platform holds the promise of far-reaching impacts on brain tumor diagnostics and therapy globally.