NEW YORK – An international team led by investigators at Weill Cornell Medicine, Memorial Sloan Kettering Cancer Center, and Yonsei University College of Medicine in South Korea has teased out a cancer protective role for small DNA-containing extracellular vesicles (EVs).
In a study published in Nature Cancer on Tuesday, EVs appeared to reduce the risk of cancer metastasis by packaging damaged tumor DNA and using it to trigger antitumor immune mechanisms at distant sites.
"We previously showed that EVs contain DNA (EV-DNA) representing the entire genome," the authors wrote in their paper. "However, the mechanism of genomic EV-DNA packaging and its role in cancer remain elusive."
With that in mind, the investigators started by using double-stranded DNA digestion and protection experiments, sequencing, mass spectrometry, and other approaches to characterize the source, location, and distinct chromatin structure of double-stranded DNA found in and on the surface of EVs in human colorectal cancer (CRC), breast cancer, lung cancer, and leukemia cell lines.
From there, the team used CRISPR-based editing to systematically lop out more than 18,900 genes to screen for the genes and pathways that influenced EV-DNA packaging in four human cancer cell lines — a search that led to 612 EV-DNA packaging-related genes.
"Collectively, our findings identified key regulators of EV-DNA packaging and provide insight into the molecular mechanisms by which high levels of tumor-derived EV-DNA modify the [pre-metastatic niche] to activate antitumor immunity and prevent metastatic progression," the authors explained.
The team's pathway analyses pointed to key roles for innate immune system components and developmental pathway players, for example, while genes involved in the cell cycle, cell division, and DNA repair appeared to dampen processes involved in EV-DNA packaging.
With their follow-up experiments — including cell line-based studies to validate genes from the initial screen using a combination of secondary screening and deep sequencing — the researchers confirmed the importance of immune system- and development-related genes, including the apoptotic peptidase activating factor 1 gene APAF1 and the neutrophil cytosolic factor 1 gene NCF1.
Consistent with the increase in CRC metastasis reported in the absence of the APAF1 gene in the past, the team's flow cytometry, immune cell profiling, RNA sequencing, and other analyses of mouse models and human cell lines pointed to an uptick in metastasis when the APAF1 gene was missing.
That, in turn, suggested EV-DNA packaging could help in reducing such metastases, prompting a series of experiments to explore EV-DNA effects on more distant immune cells.
In particular, the investigators detected ramped-up DNA damage response (DDR) activity in "Kupffer" immune cells that took up EV-DNA in the liver, apparently leading to lower liver metastasis risk via altered cytokine production and the advent of anti-tumor immunity-related tertiary lymphoid structures.
In contrast, DDR was dialed down in Kupffer cells in the liver in the absence of EV-DNA, while metastasis-associated cytokine secretion was bumped up.
Similarly, the investigators explained, higher-than-usual levels of EV-DNA secretion in biopsy samples from CRC patients coincided with a reduced risk of metastasis to the liver, lung, lymph nodes, and peritoneum.
Based on the dip in EV-DNA packaging in tumor cells with pronounced responses to DNA damage, meanwhile, the authors suggested that EVs function as a way to package and remove damaged DNA from cancer cells before carting it off to educate immune cells at possible metastasis sites.
"[A]ccording to our research findings, EV-DNA has a dual role in clearing damaged DNA in cancer cells and activating innate immune function in distant organs," they wrote, adding that the "unique structure of EV-DNA could be exploited to develop an advanced DNA vaccine to stimulate immune responses and boost antitumoral immunity and immunotherapy efficacy in persons with cancer, thereby reducing metastasis."