Study Shows Oxygen-starved Cancer Cells have a Survival Advantage Promoting Cancer Spread

Cells in green are hypoxic cells that are permanently marked so they can be followed throughout the metastatic process. The red cells have not experienced hypoxia. Photo Courtesy: Yu Jung Shin
Cells in green are hypoxic cells that are permanently marked so they can be followed throughout the metastatic process. The red cells have not experienced hypoxia. Photo Courtesy: Yu Jung Shin

Low-oxygen conditions, or hypoxia, are known to be detrimental in cancer and contributes to its development.

Using cells from human breast cancers and mouse breast cancer models, researchers at the Johns Hopkins Kimmel Cancer Center have found significant new evidence that tumor cells exposed to low-oxygen conditions have an advantage when it comes to invading and surviving in the bloodstream.

The experiments mapping the “fate” of the cells in two- and three-dimensional lab-created tissue systems and in live animals specifically showed that cells from a primary cancer exposed to low-oxygen levels have a four times greater probability of becoming viable circulating tumor cells—and likely spreading to distant tissues—than those under normal oxygen conditions.

The results were published in the October 24, 2019 edition of Nature Communications.

Daniele Marie Gilkes Ph.D., assistant professor of oncology and researcher in the breast and ovarian cancer program of the Johns Hopkins Kimmel Cancer Center, focuses her research on the role of the extracellular matrix in cancer metastasis, hypoxia and the tumor microenvironment and transcriptional regulation by hypoxia-inducible factors. Photo Courtesy: 2019
Daniele Marie Gilkes Ph.D., assistant professor of oncology and researcher in the breast and ovarian cancer program of the Johns Hopkins Kimmel Cancer Center, focuses her research on the role of the extracellular matrix in cancer metastasis, hypoxia and the tumor microenvironment and transcriptional regulation by hypoxia-inducible factors. Photo Courtesy: 2019

“Our findings also show that these post-hypoxic cells have six times the probability of forming lung metastases, suggesting that oxygen starvation enhances their metastatic capabilities,” noted study leader Daniele M. Gilkes, Ph.D., assistant professor of oncology and researcher in the breast and ovarian cancer program of the Johns Hopkins Kimmel Cancer Center.

Post-hypoxic cells
Gilkes and her team also identified a pattern of genetic expression in post-hypoxic cells that appears to help the cells survive oxidative stress when they enter the bloodstream. Some tumor cells retain parts of this genetic signature as a “hypoxic memory” even after they have been reoxygenated, the researchers found.

“Cancer cells tend to become more aggressive as they adapt to low oxygen levels,” Gilkes said, “but we were surprised to find that cells that were exposed to hypoxia in the primary tumor maintained their aggressive features even when they were reoxygenated in the blood.”

Biomarker
In the future, the unique features of the hypoxic cells might be used as biomarkers to identify patients at risk for metastasis, or might be targeted directly by therapies to prevent or limit metastasis, the research team suggested.

Hypoxia occurs in 90% of solid tumors and is known to have an adverse impact on a patient’s prognosis. However, little is known about how tumor cells change in response to low oxygen. Gilkes said that most research teams — including her own — grow and experiment with tumor cells using the same oxygen concentrations as normal air.

“This is actually a much higher level of oxygen than what is found in our bodies,” Gilkes explained.

“For example, the average concentration of oxygen in breast tissue is on the order of 6% to 8%, whereas solid breast tumors have a gradient of oxygen concentrations that reach much less than 1% oxygen in some regions,” she added.

Switch
For their new experiments, designed to capture the changes that occur as normal breast cells become malignant, Gilkes and colleagues developed an experimental system that uses oxygen as a switch to make tumor cells “light up” with a fluorescent marker after they are exposed to low oxygen conditions of 0.5% or less, comparable to the levels measured in human tumors.

The study’s first author and member of Gilkes’ lab, Inês Godet, used this marker to follow the fate of these cells as they multiplied and moved around within 2D and 3D tissue “spheres “ and “mini-organs” created in the laboratory, as well as in live mouse models of breast cancer.

Using fluorescence activated cell sorting to capture red or green (oxygen deprived) breast cancer cells, followed by RNA sequencing, the team found that the expression of many gene products, including integrin alpha 10 (ITGA10) and ceruloplasmin (CP) are induced in cells that experienced hypoxia within tumors, but not in cells exposed to hypoxia in the lab.

Predictor of Survival
The tumor-based hypoxia pattern was also better at predicting the survival of patients free of distant metastases, they concluded after studying similar genetic expression data from primary tumors from more than 1,000 patients with breast cancer.

In fllow-up studies, the researchers want to answer questions whether post-hypoxic tumor cells at metastatic sites are more resistant to chemotherapy than other cells. The also want to understand whether targeting these post-hypoxic cells will be beneficial for treating patients with metastatic cancers.

The study was supported by the National Institutes of Health (NIH), The V Scholar Foundation, Susan G. Komen Foundation, the Jayne Koskinas Ted Giovanis Foundation for Health and Policy, the Cindy Rosencrans Fund for Metastatic Triple-Negative Breast Cancer, the Emerson Collective and the SKCCC Core Grant.

Reference
Godet I, Shin YJ, Ju JA, Ye IC, Wang G, Gilkes DM. Fate-mapping post-hypoxic tumor cells reveals a ROS-resistant phenotype that promotes metastasis. Nat Commun. 2019 Oct 24;10(1):4862. doi: 10.1038/s41467-019-12412-1.