In a new study, Yale Cancer Center scientists have demonstrated a powerful method to analyze how tumor cells are altered as they metastasize to the brain. The research, published today in the journal Cell Reports, may eventually improve early diagnosis and treatment of metastatic brain cancer, whose incidence is climbing and whose treatments are typically limited.

“Once tumors metastasize in the brain, most drug therapies are no longer effective,” said senior author Don Nguyen, PhD, associate professor of pathology at Yale Cancer Center. “There’s a big question whether that's because those drugs are not getting into the brain, which is a difficult place for drugs to penetrate, or because additional mechanisms are at work.”

Emily Wingrove, a graduate student in his lab and co-lead author on the paper, took on this question with “xenograft” models of metastasis, in which human tumor cells were transferred into mice lacking an immune system. Wingrove and co-lead author Zongzhi Liu developed a system for extremely precise RNA sequencing of xenograft tissue samples to examine gene expression both in the human tumor cells and in the mouse cells. Gene expression drives the production of the proteins that shape a cell for its specific role in the body, such as a lung or muscle cell.

Experiments began in mice with a human lung cancer cell line that quickly metastasizes to the brain. The researchers analyzed how gene expression changed both for tumor cells and stromal cells as the tumors began to grow and compared that expression to healthy brain tissue.

Overall, the scientists discovered a striking set of alterations in gene expression. “We’re seeing changes in thousands of genes induced by the tumor microenvironment,” said Wingrove. “I would never have imagined that so many genes change just when you grow cells in a different context.

After validating some of these findings by examining human tissue samples, the researchers expanded the project by analyzing other xenograft models of brain metastatic melanoma and breast cancer.

More unexpectedly, the scientists found that two genes known as Tim-3 and Lag3 were significantly upregulated in the tumor microenvironment compared to healthy brains. Tim-3 and Lag3 proteins are best known for their roles on T cells as immune checkpoint receptors—basically switches that can dampen the T cell attack on a foreign substance.

The scientists went on to discover that these genes were highly expressed on two other types of cells that carry out immune tasks in the central nervous system. The researchers also validated this discovery in human samples and a mouse model of lung cancer with an intact immune system.

Additionally, the analyses pinpointed several biomarkers of brain inflammation, which might become useful in disease prognosis or treatment. Such biomarkers could be important, Nguyen noted, because neuroinflammation can significantly impair cognitive functions in patients or exclude them from clinical trials.

Another surprise came when the scientists wondered about how “plastic” tumor cells are in gene expression. When they took lung tumor cells back out of the mice and put them back into cell culture, expression of many genes reverted to baseline levels. “This result suggests that there may be a number of reversible biological pathways in brain metastasis that may represent promising targets for therapy,” said Nguyen.

The study also provides many early clues for identifying new biomarkers for early detection of brain metastasis. Today, brain cancer is typically advanced when it is detected clinically. Nguyen’s lab is also looking at potential opportunities to use existing drugs against some of the targets suggested by the research.