Cells shed a wealth of material during their lifecycle – RNA, DNA, lipids, and proteins – much of which is detectable in the blood. Disease processes can change materials that are shed providing clues as to the presence of a malady and how much a disease has advanced. To identify disease based on released factors, a major challenge lies in teasing apart which cell shed what, and why.

Research is beginning to show how shed material relates to patient outcomes. Small RNAs shed by bacteria appear to trigger rheumatoid arthritis, and proteins shed by cancer cells can trigger metastases. But for billions of other molecules, it’s not entirely clear what their role is.

One of the first steps in understanding the role of shed molecules is isolating them, says Robert Coffey Jr., M.D., Ingram Professor of Cancer Research at Vanderbilt University Medical Center. Coffey’s research team has been studying the molecular packages cancer cells shed into the blood—and finding promising disease biomarkers inside.

A New Paradigm

Most research into shed particles has centered around exosomes – membrane-bound vesicles that bud off from one cell and are absorbed by another. Researchers have found a host of signaling molecules inside exosomes, and they are thought to be a cornerstone of cell-to-cell communication.

“If you’re looking for DNA, exosomes are not your target.”

“It’s been a big deal that there is supposedly DNA in exosomes,” said Dennis Jeppesen, Ph.D., postdoctoral fellow in Coffey’s laboratory. “But if you’re looking for DNA, exosomes are not your target.”

In a pair of papers published in Cell and Cell Reports, Jeppesen and others from Coffey’s research team proposed a new model for how DNA is actively secreted by cells. The findings point to a novel molecular package for DNA – amphisomes – and completely reshape conventional knowledge about how cells share material.

Help from the Laboratory

The researchers designed a series of ultracentrifugation and high-resolution density gradient fractionation techniques to separate out materials cells shed into human plasma. They found extracellular RNA, RNA-binding proteins, lipids, and DNA are all secreted via different carriers; some constituents are associated with vesicles like exosomes and some are not.

When the researchers used immunocapture to analyze exosomes specifically, they did not find any DNA inside. Instead, DNA was targeted for secretion by hybrid organelles (amphisomes, a fusion between lysosomes and endosomes). Different proteins and lipids, on the other hand, were in exosomes and others in secreted packages called exomeres.

“We showed this for multiple cancer cell lines,” Jeppesen said. “We also see the same kind of thing for normal cells.”

The papers outline unique molecular profiles for each kind of secreted vesicle demonstrating vesicles and proteins could therefore represent new cancer biomarkers. The researchers also began to investigate functional consequences of the contents – the proteins they found inside the exomeres, for example, activated colon tumor growth in an organoid model.

Paving the Way for Liquid Biopsies

Coffey says the studies “set the field on a firmer foundation” to understand the role of shed material in disease progression.

Researchers can apply the new strategies to isolate different types of vesicles from blood, and use antigens on the surfaces to identify vesicle origins. Exomeres and amphisome contents secreted by cancer cells could reveal important information related to disease stage, or a cancer’s metastasis potential.

The research is a major advance for “liquid biopsies” that analyze secreted material in a person’s blood. Liquid biopsies have the potential to improve cancer diagnoses by identifying tumor-specific biomarkers found in certain vesicles.

Said Jeppesen, “Now it’s possible to say with greater precision what’s in what’s in these vesicles. Now you have an idea of what target you’re looking for.”

About the Expert

Robert Coffey Jr., M.D.

Robert Coffey Jr., M.D., is Ingram Professor of Cancer Research at Vanderbilt University Medical Center. He is also a provider in the Division of Gastroenterology, Hepatology and Nutrition. His research interests include regulation of protein trafficking in colon cancer, the role of cancer stem cells in initiating tumor growth and mediating drug resistance, and development of laboratory methods to identify extracellular signaling strategies used by cancer cells.