Spotlight

Staying Alive: New Technique Grows Normal and Cancer Cells Rapidly

Healthy prostate epithelial cells grown in culture. (Image courtesy of Dr. Richard Schlegel)

Healthy prostate epithelial cells grown in cell culture using a new technique developed by Georgetown and NIH researchers. (Image courtesy of Dr. Richard Schlegel)

A new technique for rapidly establishing cultures of normal cells and cancer cells from patient tissue samples, and keeping them alive for extended periods of time, could open important new avenues in research and patient care, according to the investigators who developed the culturing method.

In a study published online last month in the American Journal of Pathology, a team of researchers from the Georgetown Lombardi Comprehensive Cancer Center, the National Institute of Allergy and Infectious Diseases, and the Uniformed Services University of the Health Sciences reported a high success rate in establishing the cell cultures, in some cases doing so with as few as four viable cells.

The technique—which relies heavily on a drug called a Rho-associated kinase (ROCK) inhibitor and connective tissue cells called fibroblasts—not only keeps the cells alive and continually reproducing, but initial studies suggest that the cells appear to lack the abnormalities that commonly arise with other cell culturing methods.

Other researchers cautioned that, although the technique holds great promise, it remains to be seen whether other labs can achieve the same results with the method and how closely the cultured cells resemble cells as they exist and function in the body.

At first blush, though, the method "could represent a giant leap for cell culture technology," said Dr. Seema Agarwal of Yale University School of Medicine, who co-authored an editorial on the study.

The cultures have multiple potential uses, said the study's senior author, Dr. Richard Schlegel, chair of the Department of Pathology at Lombardi, such as determining whether patients' tumors are likely to respond to a prescribed treatment. "And as the disease evolves, we would be able to collect and analyze samples at different time points and modulate the treatment depending on the results," he continued.

Although they proposed some possibilities, the research team still needs to better understand the mechanisms by which the technique works, Dr. Schlegel acknowledged. And more sophisticated analyses of cells grown in this manner, including gene expression profiling and mutational analyses, are required to determine more definitively whether the technique fundamentally alters the cells in a way that could limit their use.

"To better understand what is happening will take some time," Dr. Schlegel stressed. "But we thought it was important to get the technique out there."

A Little Bit of This and a Little Bit of That

Human cancer cell lines have been used heavily in research for nearly six decades, dating back to the establishment of the HeLa cell line, as described in Rebecca Skloot's best-selling book The Immortal Life of Henrietta Lacks. But cell lines of some cancer types, such as prostate and lung cancers, have been difficult to establish and often can only be successfully derived from samples of aggressive tumors or metastases, and not from primary tumors, Dr. Schlegel explained. Many cell lines also undergo significant genetic changes during the culturing process.

Cultures of normal, healthy epithelial cells—the cells of origin for the large majority of cancers—are even more difficult to establish, since the cells often quickly become dormant or die. Currently, most success in "immortalizing" normal cells, explained Dr. Channing Der of the University of North Carolina (UNC) Lineberger Comprehensive Cancer Center, "involves irreversible, sometimes quite drastic, experimental manipulations," such as forcing cells to express the enzyme telomerase or the use of powerful viral oncogenes.

The fibroblasts used in the new culturing method, called feeder cells, are from mice; the ROCK inhibitor is similar to a drug approved in Japan to treat stroke patients. Both have been used previously to establish cell cultures, but this appears to be the first time they have been used together.

Once cells are isolated from tissue samples, they are added to a culture flask containing a layer of feeder cells, along with the ROCK inhibitor and other commonly used components of cell culturing media, and incubated. Every 3 days the feeder cells and the growing epithelial cells are separated and again combined with feeder cells and the ROCK inhibitor.

The researchers call the cells "conditionally reprogrammed cells," Dr. Schlegel explained, because they grow and thrive only under these conditions. If either the feeder cells or the ROCK inhibitor are removed, the cells quickly stop growing.

[This method] could represent a giant leap for cell culture technology.

—Dr. Seema Agarwal

In the reported study, the researchers grew normal cells and cancer cells taken from breast, prostate, pancreas, lung, and colon tumors with varying degrees of success, depending on the tissue type. The group has already improved its success rate, Dr. Schlegel said, by tweaking parts of the process, such as the incubation technique.

The potential to establish immortal normal cell and tumor cell cultures from the same patient "would be a powerful advance that we haven't had before," said Dr. Der.

For example, because screening finds a growing number of cancers at earlier stages, Dr. Agarwal noted, having matched normal cell and tumor cell cultures from the same patient could provide valuable insight into the initial stages of cancer development—research that has been extremely difficult to do.

"That is largely a black box for us right now," she said. "It could open up things about the biology of cancer that we don't understand yet."

Digging Deeper

There is still "a long way to go" to better understand the potential use of cell cultures established in this manner, Dr. Der cautioned. The ROCK inhibitor used in the study, for example, is not a "pure" ROCK inhibitor, he noted. It targets several other kinases, so the impact of the drug's more promiscuous activity has to be more clearly explained. Studies are also needed to analyze whether the mouse fibroblasts are causing unanticipated changes in the growing human cells at the genomic and protein levels, stressed Dr. Agarwal, who recently learned the technique at Lombardi.

The culturing process should also be tried with human fibroblasts as feeder cells as a validation step, she continued, "and if there is a difference [in the results], it raises a concern about whether the microenvironment niche created by using the mouse feeder layers is indeed different than the human microenvironment."

More studies are needed to determine the technique's impact on the epigenetic makeup of the cultured cells and whether the technique has the same effect on the expression of an important tumor suppressor gene, called p16, that is frequently altered in another common culturing technique, said Dr. Bernard Weissman, also of the UNC Lineberger Cancer Center.

Although there are still many unknowns, if the technique can be validated and refined by other labs, it "clearly has some big advantages" over current culturing methods, Dr. Weissman said. "We would have far more robust [in vitro] models than we currently do," he added, which could improve our understanding of the biology of cancer development and progression. "That's where I think this will have a big impact."

—Carmen Phillips