CTC Clusters More Likely to Cause Metastasis

Scientists know that circulating tumor cells (CTC) can clump together in groups of two to 50, but how these so-called CTC clusters form and their functional significance has remained unclear.

Now, new research shows that CTC clusters are tumor fragments, held together by the cell junction protein plakoglobin, that break off into the bloodstream. In breast cancers, these clusters are 23 to 50 times more likely to cause metastasis than single CTCs. These findings suggest that targeting pathways involving plakoglobin expression could be beneficial in reducing tumor dissemination (Cell 2014;158:1110–22).

“What kills patients is the metastasis,” says co–senior author Shyamala Maheswaran, PhD, an associate professor at the Center for Cancer Research at Massachusetts General Hospital Cancer Center in Boston. “We now have better insight into a mechanism that increases the efficiency of the metastatic process.”

In one experiment, researchers injected immunodeficient mice with a 1:1 mixture of human breast cancer cells engineered to express either a green marker or a red marker. Primary breast tumors developed and retained an equal distribution of green- and red-tagged cells. When scientists analyzed the CTC clusters, they found 91% were positive for both markers, suggesting the clusters do not result from the proliferation of a single tumor cell in the bloodstream.

To confirm that clusters are not the result of single CTCs coming together in the bloodstream, researchers injected green-tagged breast cancer cells into the right mammary fat pad and red-tagged breast cancer cells into the left, causing mice to develop two separate tumors. Investigators found 96% of CTC clusters were of a single color, indicating that these CTC clusters were not aggregating in the vasculature but originated from primary tumor fragments.

Although researchers found that CTC clusters make up only 2% to 5% of all CTCs, the clusters contributed to about half of lung metastases in breast cancer models.

Using CTC-Chip, a microfluidic device that captures CTCs from blood samples, the researchers found that patients with metastatic breast cancer and CTC clusters had reduced survival compared to those without clusters. Mean progression-free survival was 160.5 days for patients with only single CTCs compared with 32.6 days for patients with CTC clusters in more than three blood samples obtained at different times.

When researchers conducted RNA sequencing of single and clustered CTCs from breast cancer patients, they found that CTC clusters overexpressed plakoglobin, a component involved in cell-to-cell adhesion. Suppressing plakoglobin expression in breast cancer cells caused cell clusters to fall apart, disrupting cell-to-cell contact between breast cancer cells, but not normal breast cells, and reducing their metastatic potential.

Although researchers did not conduct RNA analysis of CTC clusters in other epithelial cancers, Maheswaran says plakoglobin might keep their CTC clusters together, too.

What remain unclear are the cues that drive clusters to be shed into the blood and the biological properties that enable them to be highly potent in initiating metastasis, researchers note.

“Prevention of metastasis is the holy grail in cancer,” says Maheswaran. “Our work provides a pathway that might potentially be targetable if we understood the mechanism in more detail.”

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