Scientists from The Scripps Research Institute say they have developed a new method to screen for potential cancer drugs. The technique makes use of tiny, three-dimensional, ball-like aggregates of cells called spheroids, which can be used to interrogate hundreds or even thousands of compounds rapidly using high-throughput screening (HTS).

Using this approach, the team has already identified one potential drug for a cancer gene and reported its results (“A Novel Three-Dimensional High-Throughput Screening Approach Identifies Inducers of a Mutant KRAS Selective Lethal Phenotype”) in Oncogene.

“The RAS proteins are the most frequently mutated oncogenes in cancer, with the highest frequency found in pancreatic, lung, and colon tumors. Moreover, the activity of RAS is required for the proliferation and/or survival of these tumor cells and thus represents a high-value target for therapeutic development. Direct targeting of RAS has proven challenging for multiple reasons stemming from the biology of the protein, the complexity of downstream effector pathways and upstream regulatory networks. Thus, significant efforts have been directed at identifying downstream targets on which RAS is dependent. These efforts have proven challenging, in part due to confounding factors such as reliance on two-dimensional adherent monolayer cell cultures that inadequately recapitulate the physiologic context to which cells are exposed in vivo,” write the investigators.

“To overcome these issues, we implemented an HTS approach using a spheroid-based 3-dimensional culture format, thought to more closely reflect conditions experienced by cells in vivo. Using isogenic cell pairs, differing in the status of KRAS, we identified Proscillaridin A as a selective inhibitor of cells harboring the oncogenic KRasG12V allele. Significantly, the identification of Proscillaridin A was facilitated by the 3D screening platform and would not have been discovered employing standard 2D culturing methods.”

The researchers used a technique called confocal microscopy to confirm that the cell lines were forming spheres. Here is the BxPC-3-KRASG12V cell line. [Kota et. al./The Scripps Research Institute]
“What’s important about this research is that we’re able to do studies using a form of cancer cells that are more physiologically relevant and better recapitulates how these cells appear in the body,” says Timothy Spicer, Ph.D., director of lead identification discovery biology and HTS on Scripps Research’s Florida campus and one of the study’s corresponding authors.

“Until now, most of the research to screen for cancer drugs has used cells that are growing flat on a plate,” adds Louis Scampavia, Ph.D., director of HTS chemistry and technologies at Scripps Research and one of the study’s co-authors. “With these 3D spheroids, we emulate much more closely what’s found in living tissues.”

The spheroids are 100 to 600 μm in diameter, equivalent to the thickness of a few sheets of paper. In contrast to single layers of cells normally used to screen for drugs, which tend to all grow at the same rate because they get the same exposure to oxygen and nutrients, the spheroids mimic what might happen in a tumor: Some cells are on the outside and some are on the inside.

“In the past, KRAS has been a very tricky protein to target. People have spent several decades trying, but so far there has been little success,” says Joseph Kissil, Ph.D., a professor at Scripps Research Institute and the other co-corresponding author. “The KRAS protein is relatively small, and that’s made it hard to attack it directly. But the method of screening that we used in this study allowed us to come at the question in a different way.”

The investigators performed a phenotypic screen, looking for drugs that had an effect on cell growth. “We came at this in an unbiased way,” Dr. Kissil explains. “We were not trying to design something to attack a specific part of the KRAS protein. We were just looking for something that acted on some part of the pathway that’s driving cell growth.”

The compound the scientists identified, Proscillaridin A, is similar to a class of drugs used to treat some heart conditions. Although the team says this particular drug is unlikely to be developed as a cancer treatment, it validates the approach of conducting drug screenings using spheroids. “It’s unlikely we would have discovered this connection using standard 2D methods,” Dr. Scampavia says.

“From our perspective, this is a proof-of-principle study,” adds Dr. Kissil. “It shows you can look at libraries of drugs that have already been approved for other diseases, and find drugs that may also work for cancer. In theory, you could use this screening method for any line of cancer cells, and any mutation you want.”

“We would love to use this research to create a pipeline for new oncology drugs,” says Dr. Spicer. “Many of the most promising compounds may be overlooked with 2D screening. This study provides direct evidence that screening for drugs using 3D structures of cancer cells may be more appropriate.”

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