Investigators at the Baylor College of Medicine have developed a high-throughput microscopy technique that has enabled them to quantify androgen receptor subcellular and subnuclear distribution and transcriptional reporter-gene activity on a cell-by-cell basis.
Simultaneous analysis of DNA content allowed them to determine cell cycle position and enabled them to analyze cell cycle-dependent changes in androgen receptor function in unsynchronized cell populations.
According to its inventors, the technology could provide a rapid systems-level analysis of compounds or RNAi that may differentially affect wild-type androgen receptors or clinically relevant androgen receptor mutations.
Several cell-based assays have to date been developed to screen potential androgen receptor agonists or antagonists, most of which are based on using a luciferase reporter gene. Researchers place the reporter gene under the control of an androgen receptor promoter or a UAS promoter.
However, this approach has several limitations. The data are intrinsically based on cumulative data derived from thousands or millions of cells that vary in terms of cell cycle and androgen receptor level. In addition, luciferase assays provide single read-outs, and yield information only on the transcriptional reporter gene activity of the androgen receptor. They do not provide information on how various compounds affect androgen receptor distribution, subnuclear organization and mobility, promoter occupancy, and chromatin modeling.
But according to the Baylor researchers, a single cell-based multiplex assay could overcome some of these disadvantages and more directly provide information on the mechanism of action of novel androgen receptor agonists and antagonists.
“Going fast has some advantages and some disadvantages: The faster you go, the more biology you give up, in terms of quantity and quality of the data,” Michael Mancini, an associate professor of molecular and cellular biology at Baylor and director of the college’s integrated microscopy core, said this week.
“We are in the middle [and] we are trying to identify a sweet spot that gives us a lot of biology. We give up some speed, but it is certainly faster than doing five different experiments to get the same amount of information,” he added.
This high-throughput imaging-based multiplex screening will provide a rapid systems-level analysis of compounds or RNAi that may differentially affect wild-type androgen receptors or clinically relevant androgen receptor mutations, Mancini told CBA News.
He added that many people talk about how “the cost of going fast has been tremendous, but it has not really netted a corresponding increase in new drug discovery.”
Mancini and his colleagues are trying to develop complex systems biology-oriented, imaging-based screens that “may not be as fast, but the biological readout is incredible, and getting much, much better.”
The Baylor team’s work appears online this week in Public Library of Science One.
“There is a really nice shift that I am seeing to more complex screens, where they look at cell cycle, they look at translocation, they look at toxicity, protein levels, all at the same time.”
Mancini’s lab made stable cell lines that express tagged or untagged versions of the antigen receptor, both wild-type androgen receptor and some clinical mutations, and “we have roboticized the whole thing,” said Mancini. “We look for several parameters, one being the protein expression level, which is akin to a Western blot. We also look for translocation, which again, generally, the biochemical equivalent would be done by fractionating cells and running a Western [blot] – very inaccurate, but useful.”
As part of their screening assay, the researchers look for patterns of the receptor inside the nucleus. In particular, “there is a speckled pattern that we are interested in that is consistent with transcriptional activity — not always guaranteeing it, but consistent with it,” said Mancini.
They built in a reporter gene system so that they can visualize reporter gene products, instead of doing luciferase assays, “where you never know what cell is being measured, its all a biochemical average. We actually measure all of these functions on a cell-by-cell basis,” said Mancini.
The scientists also built into the system a cell cycle analyzer that allows them to determine what part of the cell cycle the cell is in. That component is in one well. “So instead of running multiple experiments to assess functional readouts, we are increasingly doing more and more of that one well at a time.”
Within the PLoS paper, there is a sampling of some known compounds that affect androgen receptors, some antagonists, and some agonists, and “there are some environmental compounds, which we are increasingly getting interested in,” Mancini said.
To show how their assay can be used as a screening tool, the researchers tested over a range of concentrations known androgen receptor agonists, including R1881 (a synthetic androgen), mibolerone, and dihydrotestoerone. They found that all three induced GFP-AR nuclear translocation, nuclear hyperspeckling, and dsRED2skl transcriptional reporter gene activity in a dose-dependent manner.
The scientists found that the half maximal effective concentration, or EC50, for hyperspeckling and transcriptional reporter gene activity were both approximately 30-fold higher than that of nuclear translocation, indicating that androgen receptor translocation is a distinct response from hyperspeckling and transcriptional reporter gene activity, and that the assay can yield highly quantifiable data.
The Need for Speed
“We have got RNAi experiments where we have about 25 384-well plates, which gets us through the entire kinase collection,” said Mancini.
If his lab had a few more microscopes, or much faster microscopes, then it could “certainly tackle 10,000 to 50,000 compounds per day, but we are not set up for that at this point,” he said.
Mancini went on to say that his lab is starting collaborations with undisclosed big pharma companies to screen such a large number of compounds, though he pointed out that many pharma labs are already set up to go faster.
“There is a really nice shift that I am seeing to more complex screens, where they look at cell cycle, they look at translocation, they look at toxicity, protein levels, all at the same time.” The prevailing method is to repeatedly set up five or 10 types of assays that each tell you different things about a cell, and then try to piece together the disparate data into something that makes sense, Mancini said.
Mancini declined to elaborate on his lab’s collaborations with pharmaceutical companies, but said that “I think there is a lot of commercial interest in something like this. In a couple of different ways, both in the cell lines that we are developing, which are amenable to more sophisticated readouts, and in the analytical tools to read them.”
Mancini said that he is hoping to set up a company around this technology, but that is still in the discussion stages.
Mancini said that his lab has done a lot of custom imaging development because “we have not been really happy with the commercial products out there.” He did mention that “we have had great success with the Beckman-Coulter [Cytoshop] platform.”
The lab also uses the Pipeline Pilot system from Accelrys. “What I saw at the beginning when I first got into this five or six years ago, was that very few, if any, software companies were allowing you to customize anything. We have found that most of the newer software platforms offer more customization. We have complete customization with the software that we are using now,” said Mancini, referring to Pipeline Pilot.
Mancini and his colleagues have been developing single-cell assays for nuclear receptors for about 10 years. “In the last five or six or so years, we have started moving towards automated ways of doing it, and the last few papers have included automated microscopy or high-throughput microscopy.”
The lab runs its assays in 384-well plate format, using four colors, and does image analysis and automated image acquisition. Mancini said this work has led to two PLoS One papers, one that was an estrogen receptor study published in May 2008, and the current androgen receptor study.
“The main goal is to define as many functional readouts as we can, per image, per cell, and avoid the general trend or the necessary trend in biochemical experiments, where there is no cellular appreciation of what is going on, it is all averaged data from thousands or millions of cells, with the heterogeneity that is intrinsic to really all cultures.”
There are quite detectable differences from cell to cell, whether it be expression level, cell cycle, et cetera, said Mancini. “We are very specific about the biology that we are interested in looking at, and we throw away a lot of cells that would always get averaged in biochemical studies. We are not interested in doing that any more.”
On occasion, he said, “We still have to do it that way, but whenever we can do a single cell image analysis that allows us to use high-speed automated quantitation, that is what we do.”
Mancini said his lab has a number of ongoing RNAi experiments using this assay, and that it is developing “much more elaborate cell lines that would not only look at protein levels, cell cycle, and translocations and transcriptions, but would give you a readout on a DNA binding, chromatin modeling, and protein-protein interactions.”
Such cell lines have already been created for the work on estrogen receptors that was published earlier this year, and now the lab is building something similar for androgen receptors.
“Further down the road is just simply moving further and further into the bioinformatics, so that we can start interrogating data pools in ways that help us to find total cellular responsiveness based upon a lot measurements of one cell at a time, rather than trying to look at responsiveness from a DNA-binding assay, or responsiveness in a transcription or cell cycle assay,” Mancini said.