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Karolinska Study Makes Case for Including Dying Cells in Proteomic Drug Response Experiments


NEW YORK (GenomeWeb) – Researchers at the Karolinska Institute have completed a study comparing the proteomes of dying and surviving cancer cells in drug response experiments.

Published last month in Molecular & Cellular Proteomics, the study indicates that including data on dying cells can significantly add to such analyses and highlights the complicating role cellular heterogeneity might play even in cell line-based experiments.

According to Roman Zubarev, a professor in the Department of Medical Biochemistry and Biophysics at Karolinska and senior author on the paper, the work stemmed from his group's research using mass spec-based proteomics to help identify the protein targets and mechanisms of action of various small molecule cancer drugs.

In this work, they collect protein expression data on cancer cell lines treated with drugs of interest, the idea being that protein abundance changes can provide insight as to the proteins and pathways a drug is targeting. Typically, the authors noted, these experiments look at cultures of matrix-attached cancer cells. During drug treatment, dying cells detach from this matrix, but, Zubarev said, mass spec analysis is usually done only on the still-attached cells.

This is because it was assumed that the detached cells had died and lost their structural integrity, meaning proteins and other molecules could leak out, radically distorting their proteomes, Zubarev said. "We hadn't looked at these cells before because we though there was nothing interesting there."

However, discussions with cell death experts including Karolinska researcher Boris Zhivotovsky, convinced Zubarev that these detached cells might actually contain useful information. In fact, he added, Zhivotovsky suggested that many of them would form a viable new population if removed from the drug and recultured — meaning they were not actually dead at all.

To test the idea that including the detached cells could improve their analysis, Zubarev and his colleagues profiled the proteomes of detached and attached HCT-116, A375, and RKO cells that had been treated for 48 hours with the chemotherapeutics 5-fluorouracil, methotrexate, and paclitaxel.

The researchers found that adding the data on the detached cells "significantly improved the target ranking for paclitaxel."

Perhaps most interesting, they identified a set of six proteins that were consistently up or down regulated between the attached and detached cells regardless of the cell type being analyzed or the drug used.

Zubarev said that at the outset he and his colleagues assumed protein expression changes differences due to cell type and drug treatment would prove more significant than those between detached and attached cells.

"But the reality was the opposite," he said. "The first [distinction] was dying versus living. The second was the treatment. And the third was the cell line."

The six proteins differentially regulated between the attached and detached cells in all cell lines and under all drug treatments were USP11, CTTN, ACAA2, EIF4H, UHRF1, and RNF40. Using siRNA knockdown to investigate the roles of these proteins in cell death and survival, the researchers found that silencing USP11, CTTN, ACAA2, and EIF4H inhibited proliferation, silencing UHRF1 made cells more drug-sensitive, and silencing RNF40 made cells less drug sensitive. The consist differences in these proteins between attached and detached cells suggests they could represent targets for future drug discovery efforts, the authors noted.

The researchers also identified a number of upregulated proteins in the detached cells previously linked to metastasis. One question raised by the study, Zubarev said, is whether the detached cells retain epigenetic changes that make them less sensitive to the drug treatment. This, along with the fact that they have detached from the larger attached population, suggest they might have metastatic potential.

"These are cells that are running away from the bulk of the other cells," he said. "So, they are very significantly affected by the drug, but they have also become detached. If they were in the body, they would be in the bloodstream."

Zubarev said that he and his colleagues found some discussion in the literature of whether metastases might develop from cells shed due to the effects of drug treatment or adverse conditions within the tumor environment, but he said he was not aware "of any significant, systemic work" exploring this question.

Zubarev said his lab is now looking more closely at the differences between detached and attached cells in hopes of better understanding why the two populations appear to respond differently to drug treatment even though they are all part of the same, presumably homogeneous, cell line culture.

"This is a very intriguing question," he said, suggesting that it could be due to the fact that such cultures likely contain a population of cancer stem cells as well as differentiated cancer cells and that these two groups may have different drug sensitivities.

Another possibility is that different cells within the culture may be at different points in their cell cycles, which could influence the effect of the drugs, Zubarev said.

"We're now actually trying to investigate this question, and we want to investigate it at a single-cell level," he said, adding that for this work his lab is using a single-cell mass spec method developed by Bogdan Budnik, director of proteomics at the Harvard University Mass Spectrometry and Proteomics Resource Laboratory and a former graduate student in Zubarev's lab.

"We want to know if these cells are different in terms of some of them will die and some will not," he said. "And when is this differentiation taking place? Two hours [after treatment]? Four hours? If you do a bulk analysis, you will see that the proteome hasn't changed very much, but some [individual] cells may start changing very quickly. But you can only learn that at the single-cell level."