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Duke researchers figure out how to screen for aptamers that target tumors
DURHAM, N.C.—In what is being billed as "the first live targeting of tumors with RNA-based technology," Duke University Health System researchers have devised a way to screen for aptamers that could target living tumor tissue.
The technology could offers way to deliver the right therapies directly to tumors, which is important, given that finding and treating a tumor often means disturbing normal tissue—sometimes even the most helpful therapies can be invasive and harsh, the researchers point out. But by screening a large pool of aptamers in a rodent model with liver cancer using the new technology, the Duke team was able to find the best candidate molecule that bound a tumor protein.
"We are already exploring the ability to attach chemicals to the aptamers, so the aptamer molecules could deliver tumor-killing agents where they are needed, which is the next phase of our research," says Dr. Bryan Clary, chief of the Division of Hepatopancreatobiliary and Oncologic Surgery, senior author for the study that the team had published in Nature Chemical Biology online Nov. 29, under the title "In vivo selection of tumor-targeting RNA motifs."
"Most importantly, it's not necessary to have detailed knowledge of protein changes in the disease before the selection process," says lead author Dr. Jing Mi, assistant professor in the Duke Department of Surgery. "This greatly simplifies the process of molecular probe development. The selected aptamers can be used to discover proteins not previously linked with the disease in question, which could speed up the search for effective therapies."
Generally, aptamer offer ease of use because they can be easily regenerated and modified and therefore have increased stability over some other agents, such as protein-based antibodies, and they have a very low chance of immune-system interference.
Clary says that in their work with the rodent model, they hypothesized that the RNA molecules that bind to normal cellular elements would be filtered out, and that was indeed what happened.
"In this way, we found the RNA molecules that went specifically to the tumor," Clary says. The researchers removed the tumor, extracted the specific RNA in the tumor, amplified these specific molecules to create a greater amount, and reinjected the molecules to learn which bound most tightly to the tumor. They repeated this process 14 times to find a good candidate.
The team found a tumor-targeting RNA aptamer that specifically bound to RNA helicase p68, a nuclear protein produced in colorectal tumors.
"This aptamer not only binds to p68 protein in cell culture, but also preferentially binds to cancer deposits in a living animal," Mi notes. She and the rest of the team say the process could be repeated with different types of tumors.
For example, a scientist might take a breast cancer line and grow it in the lung as a metastasis model and then perform in vivo selection to identify RNAs specifically binding to the lung tumor.
The team reports that the discovery that p68 was the target was initially unexpected, "given that RNA helicases are largely reported to be proteins resident in the nucleus."
But they also wrote that cytoplasmic staining of the p68 RNA helicase has been reported in colon and ovarian cancer cell lines previously. Nucleolin, for example, another RNA helicase involved in ribosome biogenesis, reportedly functions as a cell surface receptor and is thought to act as a "shuttling protein" to help coordinate extracellular and nuclear events. An aptamer has been developed against nucleolin that, like RNA 14-16 in the Duke research, is readily taken up into cancer cells.
"In addition to the potential inhibitory properties of these nucleic acids, their ability to gain access to the cytoplasmic and nuclear compartments may serve as a mechanism to escort radiologic or therapeutic moieties to these sites," the Duke researchers wrote.
In contrast to work that identifies tumor vasculature, the Duke teams reports that its process identified an intracellular target protein within the tumor compartment.
"In contrast to in vitro selection (SELEX) of RNA binding motifs against defined tumor proteins or whole cell preparations, the in vivo process recognizes the in situ context of potential targets and leads to RNA molecules that are less likely to bind nontarget proteins in vivo," they wrote. "This strategy has potentially broad applications in creating reagents that allow for the discovery of targets that distinguish tissues of interest and in the creation of reagents that may be useful for target inhibition and in vivo escort to these tissues."
Mi says the new technology streamlines the screening process and increases confidence.
"The novelty of our work isn't the tumor specificity of the aptamer but the in vivo targeting," she points out. "When you use in vitro technologies to identify an aptamer, you may find out later when you do in vivo work that the aptamer doesn't work because of differences in tumor structure in vivo compared to in vitro."
Other authors besides Clary and Mi included Yingmiao Liu, Johannes Urban and Bruce A. Sullenger of the Duke Department of Surgery; Zahid N. Rabbani of the Duke Department of Radiation Oncology; and Zhongguang Yang of the Moses Cone Memorial Hospital Department of Internal Medicine.