Researchers at the Dana-Farber Cancer Institute have developed a technology for screening thousands of drugs in freshly isolated human cancer cells, to help identify which drugs are most likely to be effective against those cancers.
The new approach, known as high-throughput dynamic BH3 profiling (HT-DBP), is a scaled-up version of a method created by Dana-Farber researchers that gauges how close tumor cells are to apoptosis after treatment with cancer drugs. The scientists hope that by using tumor cells collected directly from human patients, their technique may prove to be more accurate than traditional drug-screening approaches, which can use laboratory cell models that may be weeks or even years removed from their origin in patients. The new method could feasibly be used to improve physicians’ ability to personalize treatment to individual patients, and help scientists to uncover vulnerabilities in cancer cells that can be targeted by new drugs.
“Cancer cells that are cultured for extended periods of time can undergo a variety of changes and may not be representative of the tumor cells that are actually in a mouse or human,” said Patrick Bhola, PhD, of Dana-Farber, who is first author of the team’s published paper in Science Signaling. “The challenge has been to create a drug-screening technique that shrinks the gap between tumor cells in the body and the cells we do the screening on. The technique we’ve developed helps to accomplish that.” Bhola, and colleagues from Dana-Farber, Harvard Medical School, and the Broad Institute, reported on initial tests with the HT-DBT technology, in a paper titled, “High-throughput dynamic BH3 profiling may quickly and accurately predict effective therapies in solid tumors.”
Despite decades of research, it is still not always possible to predict, based solely on molecular markers, whether a patient’s tumor will be sensitive to an individual drug, the authors noted. The best way to test this response would be to put drug and cancer cells together. “To determine how a patient’s tumor will respond to a drug, it is hard to imagine a more practical way to do this than to put the living cancer cell in contact with that drug.” However, as the researchers pointed out, prior attempts at determining chemosensitivity ex vivo have demonstrated inadequate accuracy, and this has “… limited enthusiasm” for the general approach. Older strategies often tested only small numbers of drugs, commonly in long-term cultures that might not maintain true cancer phenotypes, and used readouts that weren’t capable of single cell resolution. “The tools were simply not good enough to drive clinical decision-making, and enthusiasm waned,” Bhola and colleagues commented.
Many chemotherapies applied to cancer cells effectively change the balance of pro-death and anti-death molecules at mitochondria. Once the activity of pro-death molecules outweighs the activity of anti-death molecules, mitochondria release toxic substances that destroy the cancer cell. The Dana-Farber-led team has developed a technology that can determine how close the cell is to the brink of apoptosis, a property scientists have dubbed “apoptotic priming,” by adding segments of pro-death proteins to mitochondria and directly measuring the release of toxic proteins. The segments are known as BH3 domains, hence the name “dynamic BH3 profiling” or DBP.
When a drug is put on a patient’s cancer cells, DBP indicates whether, and how fully, the drug switches on the pro-death program. Tumor cells that show a significant increase in apoptotic priming after being treated with a particular drug are likely to respond to that drug in the lab as well as in patients. “The method identifies chemical inducers of mitochondrial apoptotic signaling, a mechanism of cell death,” the team commented. “HT-DBP requires only 24 hours of ex vivo culture, which enables a more immediate study of fresh primary tumor cells and minimizes adaptive changes that occur with prolonged ex vivo culture.”
One of the useful features of the first iteration of the DBP technology was that it generated results quickly—less than a day in many cases. But it was also limited by its ability to screen only 10–20 drugs at a time—a significant constraint given the myriad drugs now available to treat many kinds of cancer. “With a limitation of 10 to 20 compounds per sample, we could only perform hypothesis-driven drug testing and could not perform unbiased screening of chemical libraries to discover new therapies or new indications for existing therapies,” the investigators said. To improve on this the Dana-Farber researchers joined with colleagues at the Broad Institute of MIT and Harvard, and the Laboratory of Systems Pharmacology at Harvard Medical School to miniaturize and automate the DBP technology so it could screen hundreds or thousands of drugs, creating a high-throughput (HT) model of the technique. The increased capacity meant investigators could conduct unbiased screenings of drugs in patient or mouse tumor cells.
HT-DBP can be used as both a scientific tool and as a means of rapidly matching patients with the drugs best able to corral their cancer. In their reported study in Science Signaling, the investigators used HT-DBP to screen 1,650 drugs in fresh samples of breast cancer tissue from mice. They selected six of the drugs, including three that showed activity in DBP and three that did not, which they then tested in the mice.
The findings confirmed that the three drugs flagged as active in the HT-DBT screen did shrink the animals’ tumors or delay tumor growth. In contrast, the three drugs that had shown no signs of activity on DBP had no discernible effect on the tumors in the mice. The researchers also performed similar screens on mouse avatars of colorectal cancer and identified a drug combination that delayed tumor growth in one of the mouse models.
The overall results point to the advantages of performing direct functional drug testing on freshly isolated tumor tissue, the study authors said. “HT-DBP provides valuable chemical vulnerability information on the actual tumor without intervening model creation.”
Dana-Farber’s Anthony Letai, MD, PhD, senior author of the Science Signaling paper, added, “Laboratory specimens of tumor tissue are widely used to extract information on the molecular makeup of tumors—the DNA, RNA, proteins, and other components of cells. While these studies have had a major impact on cancer treatment, they provide a static picture of the tumor cell, rather than the kind of functional information we need to understand how tumor cells actually interact with drugs. Our approach involves putting living cancer cells in contact with drugs to assess their potential.”
The investigators also explored whether tumor cells grown in culture conditions for an extended period of time differed from fresh cells in their vulnerability to specific cancer drugs. To evaluate the effect of extended culture on tumor cells, the investigators performed HT-DBP on freshly collected tumor cells from breast cancer tissue from mice, and on tumor cells taken from the animals that had then been grown in a lab for a month. They found that while some drug vulnerabilities were preserved during the extended culture, other vulnerabilities were artificially lost or gained. Importantly, a drug vulnerability that was lost during extended culture was able to delay tumor growth in mice, whereas a vulnerability that was gained during extended cultured had no effect on the tumors. These results suggested that performing drug screens on extended cultures of cancer cells may miss potentially useful therapies.
“Here, by developing a high-throughput method to evaluate drug sensitivity of tumors within 24 hours of excision, we provide data that the very process of prolonged ex vivo propagation rapidly alters chemical vulnerabilities compared to the primary tumor,” the scientists noted.
The researchers suggest that the technique, when applied to patient tissue, could be used to personalize therapy and improve the translation of therapies from the bench to the bedside. “With HT-DBP, the drug could be screened on a tumor sample only recently removed from a patient,” Letai said. “By using tissue samples with greater fidelity to tissue within the body, this technique provides a more accurate representation of what actually happens when a drug meets a tumor.”
To evaluate its potential in customizing treatment, investigators performed HT-DBP on colon cancers directly removed from patients, rather than on cells that had first been cultured in a lab or modeled in a mouse. The test identified several agents that increased apoptotic signaling in human colon cancer cells, making them potential candidates as treatments for the cancer.
The HT-DBP approach could in addition be used in clinical trials to identify patients most likely to benefit from investigational therapies. It might also be used in the lab to gain new insights into how cancer cells are active at the molecular level. If HT-DBP reveals that a drug targeting a particular signaling pathway that pushes a set of tumor cells toward apoptosis, it’s a sign that the cells are depending on that pathway for their growth and survival.
The technology might also enable the identification of combination therapies for hard-to-treat cancers. “A potential use of HT-DBP will be the assembly of combination regimens for typically chemorefractory tumors,” the authors commented. “Evaluating combination therapies requires a factorial increase in the number of screening wells, which is not amenable to freshly isolated tumors, or primary tumors. One of the theoretical advantages of DBP over conventional measures of the cell death is the ability to identify compounds that sensitize cells for apoptosis but may not induce frank cell death, rendering them invisible by most other techniques. These compounds that sensitize cells for apoptosis, but do not kill cells, could be effective in combination.”