October 1, 2010 (Vol. 30, No. 17)

Vicki Glaser Writer GEN

Technique Gaining Ground and Leading to Novel Insights on a Genomic Scale

Over the past several years, RNAi screening went through a period of technology development aimed at gaining a better understanding of its advantages and limitations, and there is now an “established set of guidelines and good practices,” says Steven Haney, Ph.D., an associate fellow in Pfizer’s applied quantitative genotherapeutics unit.

Development in the field is now more focused on how RNAi technology can be used to probe fundamental biology, he observes. One example is Pfizer’s use of siRNA image-based screening to identify kinase genes that allow cancer cells to adapt to and survive in a low-oxygen environment. Dr. Haney will be one of the presenters at CHI’s upcoming “RNAi for Functional Screens” conference.

Unlike proliferative cancer cells that grow unchecked in the oxygen-rich periphery of tumors, cells in the interior of a tumor must adapt to an acidic and hypoxic environment. It is in this interior compartment that researchers believe metastatic cells acquire their ability to leave the primary tumor and where some sort of tumor stem cell or tumor-initiating cell is believed to reside, at least in some tumor types.

Under limiting oxygen conditions, cells adapt to the hypoxic environment by undergoing various types of changes, including aberrations in nuclear morphology.

To identify which genes in the kinase family are associated with these changes in nuclear morphology, Dr. Haney’s group designed a high-content screen that incorporates multiple siRNAs per kinase gene and uses 4´,6-diamidino-2-phenylindole (DAPI)—a fluorescent stain that binds to DNA—to visualize changes in the size and shape of nuclei. They chose siRNA screening because it yields information on the direct link between gene expression and cellular adaptability to hypoxic stress.

“RNAi screening is a very valuable method for collecting information about what controls a process,” he says. To generalize their findings, his group is screening multiple different cell lines from tumor types known to leverage their ability to adapt to hypoxic stress to drive their aggressive growth. These include colon cancer, renal cell carcinoma, and melanoma.

A critical bottleneck in cancer research and therapeutics development has been translating the behavior of tumor cells grown in culture to what occurs in vivo, in Dr. Haney’s view. Advances in high-throughput screening strategies aim to “push the biology,” he says, and to develop methods for doing in vitro screening in a setting that most closely models the in vivo environment.

High-content assay technology is creating new opportunities for studying cells using biological assays to measure more subtle changes in cell activity and viability than traditional apoptosis endpoints.

Nuclei of colon cancer cells grown under normoxia and anoxia (>1% oxygen): The difference in nuclei shape and texture between these two conditions formed the basis of a screening endpoint to identify genes that interfere with how cancer cells adapt to the hypoxic environment within a tumor. These differences were quantified through digital imaging and automated analysis. siRNAs that caused statistically significant differences were noted, and genes with several siRNAs causing such changes were identified as hits. [Pfizer]