When physician Noha Mousa left Egypt for Toronto to pursue a Ph.D., she didn't foresee wrestling with common extraction protocols to find a practical and patient-friendly method to measure estrogen levels in breast tissue. "It was very time-consuming and very messy," Mousa says. "We really needed a miniature method to do it, and nothing was available."
Nor did she expect that she would have to seek collaborators to develop new technology. But that's what it took: Mousa and her mentor Robert Casper, an endocrinologist and professor in the Department of Obstetrics and Gynecology at the University of Toronto in Canada, teamed up with the lab of Aaron Wheeler, an assistant professor of chemistry, also at Toronto, to develop a "lab on a chip" technique that prepares and purifies tiny samples of breast tissue and blood so scientists can easily measure estrogen levels. The technique, which in the future may help identify women at increased risk for breast cancer, was published in October in the inaugural issue of Science Translational Medicine.
This collaboration has been the most fun part of my own research work because I am learning so much. You can really see how different people can work together.
The story of how this collaboration addressed a critical clinical problem highlights the serendipity, hard work, good will, and communication needed to translate exciting technologies to the clinic. "This collaboration has been the most fun part of my own research work because I am learning so much," Mousa says. "You can really see how different people can work together."
An untestable hypothesis
When Mousa was in medical school in Egypt, she knew she wanted to be an obstetrician/gynecologist. She became interested in the health of menopausal women while doing her residency at Assiut University in Egypt and pursuing a master's degree there in natural hormone therapy. She knew she also wanted to do research while practicing medicine. So, when her husband, Mohamed Abdelgawad, was accepted to the Ph.D. program in mechanical engineering at the University of Toronto, Mousa applied for the Ph.D. program there as well and chose to work in Casper's lab.
Casper, a physician-investigator, studies reproductive science broadly, with a goal of translating basic discoveries into clinical interventions. Mousa joined the lab to work on the role of estrogen and anti-estrogen therapies in reducing the risk of breast cancer among postmenopausal women.
Casper and Mousa posit that local concentrations of estrogen in the breast play a far more crucial role in cancer risk than circulating levels. "If the circulating levels of estrogen were a critical component of breast cancer risk, we would see the risk drop as women get older," Casper says. "But we know that doesn't happen--breast cancer risk increases in a straight-line fashion as women get older."
One explanation--the one the researchers embrace--is found in the fact that breast tissue can manufacture its own estrogen via the enzyme aromatase. Drugs that inhibit aromatase are routinely given to breast cancer patients to stymie the growth of estrogen-fueled breast cancers. "If 90% of the risk of cancer comes from local exposure [in the breast], when we add aromatase inhibitors to hormone [replacement] therapy, we should be able to reduce the incidence of breast cancer while preventing the side effects of aromatase inhibitors such as hot flashes and the loss of bone density," Casper says. "It would really be the best of both worlds."
As they embarked on testing this clinical hypothesis, they discovered, much to their surprise, that existing technology for measuring estrogen in breast tissue was onerous and impractical, requiring patients to submit to biopsies under sedation and requiring the collection of a rather large amount of tissue. After that, several purification steps are required before estrogen can be detected.
"Patients would never agree to such an invasive process, and we wouldn't be able to monitor estrogen levels in the breast well enough," Mousa says. They had to find a better way to measure local estrogen levels--work that was well outside their areas of expertise. Casper urged Mousa to seek collaborators who could help.
"One of the things that I learned when I was a fellow was that science is big and you can't be good at everything," Casper says. "You have to focus on what you do well and try to find people who are good at what they do and see if you can find some sort of interaction."
Answers across the dinner table
Each night, as Mousa ate dinner with her husband, who was pursuing a Ph.D. in Wheeler's analytical chemistry lab, the couple talked about their research. Abdelgawad was working with digital microfluidic (DMF) devices, which use electrical charges to move minute volumes of liquid on the surface of a microchip smaller than a postage stamp.
Mousa thought the technology might be able to solve her problem because the device required only a miniscule droplet of fluid and offered complete control of the fluid's behavior and movement. "Theoretically, that meant we could miniaturize any traditional lab experiment," Mousa says. Furthermore, miniaturization would make it possible to use a very small breast tissue sample, such as a fine-needle aspirate. "Fine-needle aspirates and biopsies are frequently used when doctors suspect breast cancer," Mousa says. "Patients tolerate them very well."
Mousa asked her husband whether he thought DMF devices would work for her. "He kept telling me that they needed to perfect the device before they could think about finding an application," Mousa says. "But, I thought, maybe if you have the application you want to use it for, you could use that to perfect the device." Abdelgawad relented and told Mousa to contact Wheeler about her ideas.
When Mousa met with Wheeler, she drew a diagram showing all the steps that were required in the traditional method of analyzing estrogen levels in breast tissue. Wheeler was skeptical but knew an exciting problem when he saw one. "I've learned that I need to enthusiastically support students when they have new, creative ideas," he says. "They don't always work, but when they do, it's really worth it."
Wheeler invited Mousa into the lab and paired her with first-year graduate student Mais Jebrail. "I have to admit to being really intimidated working with a physician when I was a first-year student," Jebrail says. "And one who was married to my colleague."
Learning new tricks
Mousa wanted to develop the device to accept minute amounts of raw tissue or fluid--in this case, breast aspirate or blood--and then perform any necessary purification steps needed to detect estrogen levels. The collaborators had to work together on every step.
"I think many times I would tell them that we needed the device to do something, and they would tell me, 'You know, that sounds easy, but it is really very complicated,' " Mousa says, laughing. "I would see the clinical side of things, and it would take them a very long time to explain to me how things work on their side!"
For example, one of the first hurdles to overcome was the need to perform a liquid–liquid extraction to clean up the samples. That process is similar to making vinaigrette: Vinegar and oil are shaken up together, and as the vinaigrette settles, the vinegar (a water-based liquid) settles to the bottom and the oil floats on top. With a liquid-liquid extraction, the sample is the water-based liquid and an organic solvent serves as the uncharged liquid. When the liquids separate, contaminants in the sample move to the organic solvent, whereas the steroid hormones such as estrogen stay in the water-based liquid.
"We were really pushing the device to a different level by making it accommodate processes such as liquid–liquid extraction," Jebrail says. Although the DMF device could easily move water around the chip, organic solvents have no charge and cannot be manipulated in this way. They solved that problem by developing a "wall" on the chip with holes in it to contain the organic solvent but allow the water-based sample to move into the solvent and back out again.
Once they fine-tuned the DMF device to extract and purify estradiol, the detectable form of estrogen, the team put the samples in a mass spectrometer to quantify the estrogen levels. The tests revealed that they had created a reliable method for measuring estrogen in tiny samples of breast tissue or fluid.
"This collaboration worked so well because Mais is one of the most creative problem solvers I've ever seen, and Noha is just fearless," Wheeler says. "As a clinician, she walks into my lab and takes over working in areas she has never worked before. We did get lucky. It was just a perfect match of problems, people, and technology."
As Mousa works to finish up her Ph.D., she and her collaborators are refining the technique further. They believe the technique could be developed as a test that can be performed in a doctor's office to help assess breast cancer risk and determine whether breast cancer therapies are working. The technique could be used to measure other hormones and may in time prove useful in monitoring fertility treatments or risks of other cancers.
"We've filed a patent on the device, and we are working to quickly move this into the hands of people who know how to commercialize it," Wheeler says. "A couple of my students are graduating, and they are looking at spinning out a company that would develop this for market."
In addition, a clinical trial is under way, testing whether aromatase inhibitors can reduce the risk of breast cancer in menopausal women on hormone replacement therapy.
Mousa, who is also working as a clinical fellow in obstetrics and gynecology at Mount Sinai Hospital, and her husband plan to return to Egypt where they will pursue academic careers at Assiut University when they complete their studies. "I hope we will be able to continue this work and collaborate with our wonderful team in Canada," she says.
While the project will hopefully one day have a profound impact on women's health, it has left an indelible mark on the careers of the collaborators.
"I really loved that this was a fun project to do," Jebrail says. "I got to meet different groups, but it also allowed me to appreciate what I am doing in the lab. It is so easy to get caught up with research and not see how your work fits into the big picture, and I got to do that at a very early point in my career."