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Mixed media messaging: Different cell culture conditions influence drug activity in cancer cells

The choice of the ideal cell culture medium decides whether a cell line lives or dies. This growth environment mimics the conditions faced by cells in a living organism and provides the nutrients the cells need to thrive.

Jason Cantor
Jason Cantor

Morgridge researcher Jason Cantor and his colleagues are studying how changes in the cell culture environment could lead to a better understanding of the biological mechanisms that lead to disease—and the treatments that can stop or prevent them.

In a new study published today Scientific advancesThe researchers used high-throughput chemical screening to show how different cell culture medium conditions affect the effectiveness of drugs.

“We're trying to study the modeling capacity of the nutrient conditions that cells in the body might see,” says Cantor, who is also an assistant professor of biochemistry at UW-Madison. “If you can understand a mechanism and the underlying causes of things, that's probably the most powerful way to inform how you approach what's going on downstream.”

The Cantor Lab previously developed Human Plasma-Like Medium (HPLM), a cell culture medium systematically designed to more accurately represent the nutrient environment in human blood. In 2021, Thermo Fisher Scientific made it commercially available to scientists worldwide.

In this study, they performed high-throughput chemical screens on leukemia cell lines grown under three different medium conditions: unmodified RPMI (a cell culture medium commonly used to culture human blood cells), dialyzed serum-modified RPMI, and dialyzed serum-modified HPLM .

They screened a comprehensive library of thousands of compounds from different drug classes, including antivirals, antibiotics, antihistamines, and those specifically targeting diseases such as cancer, Alzheimer's, and diabetes.

Kyle Flickinger

“It uses drugs that are already approved, drugs in clinical trials, drugs that are in the trial phase and aren't even in the pipeline yet – so it really covers the entire range of possible drugs,” says Kyle Flickinger, a PhD student in the UW-Madison Integrated Program in Biochemistry and first author of the article.

Their analysis revealed 117 compounds with different drug sensitivities based on media conditions. Three strong responses have been identified for drugs linked to purine metabolism, the various signaling pathways that affect guanine – one of the four nucleotide building blocks that make up DNA and RNA. These drugs also interacted with hypoxanthine, a purine derivative present in serum-modified media.

“The cells basically save the poison and can die,” says Flickinger. “But under conditions where hypoxanthine is present, it competes with this drug for the ability of cells to take it up, thereby protecting them.”

This suggests that available nutrients in the environment can either protect cells from the effects of drugs or make them more vulnerable.

The researchers also identified a unique mechanism with an antiviral drug, brivudine. When this compound was added to leukemia cell lines cultured in HPLM, the cancer cells stopped growing.

“Basically, we see that it has an anti-cancer activity that is literally in our hands that has been completely obscured in the conventional media, and we don't know why,” Cantor says. “At this point, based on what we know, we can say that we have identified a drug-nutrient interaction. Maybe it points to where we need to look to understand biology.”

Going back to basics, they examined various avenues and designed experiments—including gene knockouts, CRISPR screening, and mass spectrometry—to determine the mechanistic effect of this interaction.

They found that brivudine impairs cell growth by targeting two enzymes involved in folate metabolism. In addition, they found that it was due to the concentration of folic acid in the different media that interacted with the drug.

Folic acid is abundant in both human blood and mouse blood. Building on their data from in vitro experiments, they collaborated with Rebecca Richards and her laboratory at the Wisconsin Blood Cancer Research Institute to test brivudine treatment in vivo in mice with leukemia transplants. The treatment took effect after a week, showing a 50 percent reduction in signaling markers that track tumor growth.

“There are always ways to improve our models and make them more complex. Considerations about other aspects of the environment are always ripe for consideration.”

Jason Cantor

Cantor says this collaborative research was essential in demonstrating that their workflow can provide a proof of concept for downstream applications.

“You get these two experimental examples: you have confirmation that something is on track with what you already know, and then you have the discovery that something is off target or non-canon,” he adds .

This comparative approach – which examines the conditionality between nutrients, genes, drugs and other environmental factors – opens the door to drug discovery and finds ways to expand the therapeutic window of existing drugs, thereby significantly improving outcomes.

“To get a drug approved, thousands of compounds go through an initial screening phase, and that just costs time and money,” says Flickinger. “If we can improve this in vitro condition by even 1-2%, that’s still time, money and lives saved.”

Different models in research will always have a balance of advantages and disadvantages, as it is not yet possible to fully capture the complexity of what happens inside a human body. The Cantor lab continues to pursue the unknown, with future research expanding to modify cell culture conditions as well as the use of different cell lines.

“There are always ways to improve our models and make them more complex,” says Cantor. “Considerations of other aspects of the environment are always ripe for selection.”

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