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Broad Institute Validates New Cell Array Platform in Drug-Resistance Study

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A team of scientists at the Broad Institute has developed a new cell microarray and validated it as a potential functional genomics tool by using it to screen cells for drug resistance in melanoma.

According to the researchers, the tool has several advantages over other cell array technologies, including a reduced cell and reagent requirement and the elimination of cell migration.

The researchers, who are hoping their technology can be widely adopted for functional genomics screening, discussed the new chip and the validation study in a paper published recently in Science Signaling.

Lead author Kris Wood said that the group set out to develop the tool in part to take advantage of the Broad's library of RNAi reagents that target every gene in the human and mouse genomes. While scientists had the reagents at their disposal, traditional screening methods were too costly to support large-scale projects.

"Doing screens is an expensive process using the traditional methods," said Wood, who is a postdoc in the lab of David Sabatini, also an author on the paper. "For a multi-well plate, where each well is treated with an individual reagent, the cost is about $1 per well, which can become prohibitively expensive if you want to screen thousands of cells," he told BioArray News this week.

For the past four years, the Broad team has worked on developing a new cell microarray platform to overcome challenges associated with current methods and with other previously described cell array technologies. The result is the MicroSCALE platform, which stands for microarrays of spatially confined adhesive lentiviral features.

To make the arrays, the team used an Aushon Biosystems 2470 arrayer to print adhesive spots onto polyacrylamide hydrogel-coated glass slides supplied by Surmodics. To these spots they added lentiviruses engineered to deliver RNAi or open reading frames into cells.

According to the paper, the resulting arrays can be stored in a freezer until needed. After the arrays are incubated with cells for a few days, allowing the viruses to turn on or off cellular genes, researchers can expose the cells to a drug or environmental condition and scan the slides to quantify features that have been fluorescently tagged.

Wood said that the Broad's array overcomes several limitations of other cell array platforms. One is the tendency of cells to migrate after they have been deposited.

"In most prior reports, people have printed onto planar glass slides," Wood said. "When cells land on those surfaces, they can migrate and all may not be treated with the reagent you think they were treated with," he said. "With our approach, cells stick to the surfaces and cannot migrate."

By relying on lentiviruses to deliver RNAi and ORFs into the cells, the team also avoided transient transfection. "Lentiviruses can integrate genetic material into a host genome, and allow stable instead of transient transfection," said Wood. "We can also use an antibiotic solution to remove all untreated cells from the array so that it is clean and there is no background of untreated cells," he added.

Mechanisms of Action

Though cell arrays have been previously described, Wood claimed that not many have been shown to do "bona fide functional screening." To validate its platform, the Broad team decided to look at drug resistance in melanoma, which Wood said was an "interesting biological question" to attempt to answer.

"There are a large number of targeted therapies working their way through pharma pipelines," Wood said. "While they can have a positive effect, after some time the patient becomes drug resistant. The tumors come raging back, and there is very little in a clinic that can be done for that patient," he said. "It's a bit of a black box and we don't understand what those mechanisms could be."

Wood and fellow researchers collaborated with Broad senior associate member Levi Garraway to screen cells against vemurafenib, marketed by Daiichi Sankyo and Roche as Zelboraf and designed to target BRAF mutations in melanoma cells.

In doing so, Wood and colleagues rediscovered genes and pathways known to play a role in resistance to Zelboraf. The team also used the array to attempt to identify new resistance genes. They seeded melanoma cells onto microarrays featuring ORF-carrying viruses that overexpress genes from protein kinases, known for their involvement in cancer. They treated each array with six separate drugs related to Zelboraf, and observed the same pattern of surviving cells on each microarray.

According to Wood, the team found that drugs targeting similar biological processes resulted in profiles of resistance-conferring genes. Clinicians often try to overcome drug resistance by giving first-line and second-line treatments targeting the same pathway, but the team argued in the paper that using a combination of drugs targeting independent pathways could be a better way to avoid drug resistance.

"Our evidence suggests that drugs targeting common biochemical nodes or pathways will exhibit highly similar modifier profiles, reflecting their shared mechanisms of action, whereas those targeting distinct pathways will exhibit unique profiles, reflecting distinct mechanisms of action," the authors wrote. Wood noted that the results suggest that combination therapies could "inadvertently be causing drug resistance rather than preventing it."

'A Good Problem'

While the Broad's cell array is still just a prototype, the institute is looking to make the platform more widely available. In the paper, the team noted MicroSCALE's advantages, claiming that cell and reagent requirements are between "10- and 25-fold lower" than for analogous 384-well plate-based screens. The team also claimed that MicroSCALE screens can be performed in "any standard biological laboratory without requiring specialized facilities."

Potential settings for the new array include screens involving phenotypes with low frequency that require large numbers of cells; screens involving non-adherent cells; and screens that require physical isolation of the medium associated with each perturbation, such as those involving secreted factors, the authors wrote in the paper.

Drug modifier screens are another potential application of MicroSCALE because they "have the potential to systematically reveal the genes and pathways that modulate drug sensitivity," the researchers wrote in their paper. They also noted that they performed 45,000 discrete ORF overexpression and drug treatment measurements during the melanoma drug resistance study — a "scale that would be difficult and costly to achieve with existing technologies" — to generate kinome-wide drug modifier profiles across multiple classes of inhibitors.

"We would like to make this tool useful to the broadest number of scientists as possible and we are currently working through the financial and IP issues to make that happen," Wood said, without elaborating. He said that the team is now looking to further develop the platform and will take a "broader look at anticancer drugs to predict optimal drug targets from real human tumor."

A number of other labs, both within industry and academia, have already expressed a desire to work with the Broad team. "I think there is a healthy interest in collaborating with us," he said.
Wood added that his team's main issue now is deciding what projects to pursue. "It's a good problem to have," he said.

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