Impact on immunity and metastasis

New research reveals how EV-DNA packaging influences antitumor immunity and highlights its potential as a predictive biomarker for colorectal cancer metastasis.

Study: Unique structural configuration of EV-DNA primes Kupffer cell-mediated antitumor immunity to prevent metastatic progression. Image Credit: shisu_ka/Shutterstock.com

In a recent study published in Nature Cancer, a team of researchers investigated the unique structure of deoxyribonucleic acid (DNA) in extracellular vesicles (EVs) and its role in cancer progression.

The study examined how EV-DNA, through its association with histones, influences immune cell responses and impacts the pre-metastatic niche. It also explored how it could serve as a predictive biomarker for metastasis, especially in colorectal cancer.

Background

Extracellular vesicles are nano-sized structures secreted by cells to mediate communication, especially in disease contexts such as cancer.

These vesicles carry diverse molecular particles, including DNA, proteins, and ribonucleic acid (RNA). Previous research has shown that EVs are involved in preparing distant organs for tumor colonization by forming pre-metastatic niches.

However, while many of the proteins and RNA in EVs have been linked to tumor progression, the role of EV-DNA remains less clear.

Some studies have suggested that EV-DNA activates immune responses through recognition pathways, but the packaging mechanisms and functional implications of EV-DNA in cancer are not fully understood. Moreover, the variability in EV-DNA detection methods has led to inconsistent findings, further complicating our understanding of its significance.

About the study

As genomic DNA constitutes a significant fraction of EV-DNA, it presents a potential marker for tumor-specific mutations. The gap in knowledge about the functions and mechanisms of EV-DNA in cancer also necessitates further research to understand its influence on cancer metastasis and immune regulation, as well as its potential clinical applications.

The present study employed a combination of cellular, molecular, and in vivo techniques to investigate the role of EV-DNA in cancer progression. Using ultracentrifugation, extracellular vesicles were isolated from various cancer cell lines, including colorectal and breast cancer models.

Subsequently, the DNA distribution on EVs was analyzed with super-resolution microscopy and biochemical assays, confirming its predominant localization on vesicle surfaces.

Western blotting and mass spectrometry were also conducted to examine the unique post-translational modifications and cleavage patterns in EV-associated histones compared to those in cellular histones.

Additionally, a genome-wide knockout screening using clustered regularly interspaced short palindromic repeats (CRISPR) was conducted across multiple cell lines to identify genes involved in EV-DNA biogenesis.

In vivo experiments in mouse models further examined the functional consequences of EV-DNA loss, focusing on metastasis and immune responses.

Kupffer cells or liver macrophages were also identified as primary recipients of EV-DNA, and their immune activation was assessed through transcriptomic profiling after EV exposure.

Additionally, EV education experiments were performed by administering EVs with varying DNA levels to mice. This approach evaluated the influence of EV-DNA on immune cell behavior and metastatic outcomes.

Quantitative methods such as RNA sequencing and cytokine analysis were used to determine the molecular pathways activated by EV-DNA. Histological techniques were also employed to visualize immune structures, such as tertiary lymphoid structures, induced by EV-DNA in the liver.

Major findings

The findings suggested that EV-DNA has a critical role in inhibiting metastasis through immune activation. The researchers identified the primarily genomic and chromatinized EV-DNA as a key trigger for immune responses when taken up by liver macrophages or Kupffer cells.

After taking up EV-DNA, these cells exhibited activation of DNA damage response pathways, leading to the production of cytokines that promote antitumor immunity.

Kupffer cell activation also facilitated the formation of tertiary lymphoid structures, enhancing local immune surveillance in the liver. Additionally, CRISPR screening identified neutrophil cytosolic factor 1 (NCF1) and apoptotic protease activating factor-1 (APAF-1) as essential genes for EV-DNA packaging.

Furthermore, the loss of these genes resulted in reduced EV-DNA levels, weakened immune activation, and a significant increase in metastasis in colorectal cancer models. Importantly, EVs with low DNA content failed to induce the immune responses necessary for metastasis suppression.

The in vivo experiments also reported that mice receiving EVs with high DNA levels showed a marked reduction in liver metastases compared to those treated with EVs lacking DNA.

Additionally, the RNA sequencing revealed that EV-DNA induced specific cytokines, such as C-X-C motif chemokine ligand (CXCL)10 and tumor necrosis factor (TNF), which are known for their roles in antitumor immunity.

Conversely, low-DNA EVs promoted immune-invasive environments by inducing cytokines such as interleukin-4 and C-C motif chemokine ligand 1 (CCL1). The clinical analyses supported these findings and showed that higher EV-DNA levels in colorectal cancer patients correlated with a reduced risk of distant metastases.

Conclusions

Overall, the study established EV-DNA as an important factor in activating immune defenses against metastasis and highlighted its unique chromatin structure. The findings demonstrated that EV-DNA levels are inversely correlated with metastatic potential, offering a promising biomarker for assessing cancer prognosis.

By elucidating the mechanisms of EV-DNA’s impact on immune activation and metastasis, the study paved the way for innovative therapeutic strategies targeting EVs in cancer treatment.