This feature was originally published in PittPharmacy.
When Shilpa Sant thinks about the way cells grow in the human body, she draws an analogy to people sitting in a classroom: Craving interaction, the cells—like restless students—start talking to each other, and those conversations shape the actions that follow.
“It’s all about the microenvironment,” ex- plains Sant, an assistant professor in the Departments of Pharmaceutical Sciences and Bioengineering.
Like the human beings they constitute, cells need communication—and they inter- act with, and are shaped by, the environment around them. at environment, a sort of scaffolding, informs the cells about what to do and when to do it. And, as with humans, the cells respond to the cues that come from stressors or changes in that environment.
That premise is the underpinning of a new three-dimensional model for breast cancer that was developed by Sant’s lab and featured in the journal Cancer Research. e idea was to more closely mimic the way cancer cells be- have in the human body, a process that Sant says is not completely duplicated in standard two-dimensional monolayer cultures.
Ultimately, the hope is that better models will help to re ne the development of drugs targeting the disease, Sant says.
Scientists already knew that for some drugs, the effect on actual tumors differed from the observed effect on cells in two-dimensional at models in which they were tested. Re- searchers wondered if perhaps only the outer cells of a tumor were affected, and whether the drug needed to get closer to inner tumor cells to work. Using the existing clinical data about how outcomes differed from expA path to the laboratoryectations, Sant designed her model in the lab.
A path to the laboratory
Sant’s methodology is influenced not only by her background in pharmaceutical sciences, but also by the complementary work she has done in bioengineering. After earning her bachelor’s and master’s degrees in her native India, she moved to Canada, where she earned a PhD in pharmaceutical technology from the University of Montreal.
She won a fellowship to train at the Wyss Institute for Biologically Inspired Engineering at Harvard and the Center for Bioengineering at Brigham and Women’s Hospital in Boston. She worked on tissue engineering and regenerative medicine, fascinated by how well the disciplines meshed together.
Initially, Sant’s passion lay with teaching, and she served as a full-time lecturer in pharmacy while she still lived in India.
“Interactions with the students—that’s what really keeps me going, I think,” she says. But as satisfying as she found teaching, she longed for something else: an opportunity to pursue new ideas in the field. It was this desire that prompted her to earn her PhD, and before she won her fellowship, she worked in the clinical research department at Labopharm, Inc. in Montreal.
Though she loved the job, she accepted the FQRNT fellowship from Canadian Government because she missed academia, particularly the students.
Personally, it was a challenging decision for Sant, who was married and had a young family. She celebrated her younger son’s first birthday and then immediately moved to Boston. Though her husband and children were extremely supportive, Sant knew that competing for grants and pursuing research had the potential to interfere with family life; so when her fellowship ended, she wanted to choose her next steps carefully.
When she interviewed with the Pitt School of Pharmacy, she already had an offer in hand from a Canadian institute. She was impressed, however, by the caliber of Pitt’s pharmacy and engineering schools, as well as the University’s highly interdisciplinary environment.
“It was the kind of research I was envisioning in my lab,” she says. “Pitt is a really collaborative environment.”
But what really set Pitt apart was the number of people who came to the University decades ago, and never left.
“That gave me a feeling that people really liked to be here,” says Sant. “I can still manage to have family time while having the great, collaborative environment.”
In fact, her family was so pleased with the University that her husband, Vinayak Sant, accepted a position as an assistant professor of pharmaceutical sciences. Today, their children are 8 and 13, and Shilpa Sant is able to pursue the research that is her passion.
A work in progress
Sant started her lab at Pitt in 2012, focusing on the breast cancer model. Cancer generally turns fatal via metastasis, spreading to other sites through the body. In breast cancer, oncologists considered many factors, one of them being tumor size, which has been shown to influence tumor progression to advanced stage and metastasis. With better modeling, Sant theorizes, the factors that influence tumor movement can be better understood.
“That was the rst question that we started asking,” Sant recalls. “What happens as the tumor grows? How do the cells behave?”
Bigger tumors mean more cells fighting for nutrients, creating a “survival of the fittest” situation, she says. Influenced by these stressors, the cells start looking for ways to survive, which is why they change biologically. The cells emit some signals that direct the blood supply to the tumors, and new blood vessels begin to grow. By figuring out how to survive and then adapting, the tumor becomes harder to kill.
Sant uses tumor cell lines that are derived from primary, non-invasive breast tumors. Every time the lab conducts its experiments, it uses the same cell lines when creating the range of sizes and shapes for her 3D tumor models. Borrowing a concept from the microelectronic industry, she created cylindrical holes of different sizes and surrounded them with a gel that the cells dislike, so they try to avoid sticking to it. Cells in the larger wells began moving together as a whole tumor on top of the gel, crawling away. It reminded Sant of a scene from Spider-Man 3- when Peter Parker gets his black suit.
“The surprising thing was we never expected them to move,” she said. And yet they did: In fact, the entire tumor moved, and did the next time she ran the experiment, and when a student ran it, too. Small tumors popped completely out of the well after five or six days, when they ran out of space, while larger tumors started crawling out of the well and wound up half inside the well and half outside.
“Cells need communication – and they interact with, and are shaped by, the environment around them.”
That got Sant to thinking: For tumors to spread in metastasis, they need to be able to break away from the matrix around them and find a second home where they can grow. So they began looking at the biological factors as the migrating tumors changed their phenotype.
An important factor in Sant’s model is that both early stage and migratory tumors were made from the same non-invasive, non-migratory cell line, allowing them to study the changes in cell phenotype in real time. Many other models in the literature use different non-invasive and invasive cell lines to model different stages of tumor progression such as early stage vs. migratory aggressive stage.
“I think that was the most exciting part of this research,” she says, because it more closely simulates the disease in people.
When they tried the same process in cell lines from the triple negative breast cancer, the larger tumors of these cell lines didn’t move, which validated the clinical observation of poor correlation between tumor size and aggressive phenotype in patients with hormone receptor negative breast tumors.
Importantly, Sant’s model also revealed time-dependent changes in expression of some receptors due to changes in the surrounding microenvironment in large tumors. For example, only cells grown as large tumors lost expression of estrogen receptors while those grown as small tumors did not. All these cells, when grown as flat monolayers express these receptors. When testing potential drug therapies, these biological changes are significant. When the Sant lab tested a clinically used drug, 4-hydroxytamoxifen, targeting estrogen receptors in small vs. large microtumors grown in the lab, they found that although the drug was effective in inhibiting the growth of small non-migratory tumors, it could not effectively inhibit growth of large microtumors which lost the target expression. Similarly, Sant adds, “A molecule targeting a late-stage tumor won’t work in early-stage patients if the target isn’t even present in those cells. But researchers wouldn’t know that if they tested it in a two-dimensional model that doesn’t differentiate the cell behavior that is apparent in the 3D model. And cancer cells in mouse models might behave differently than human cells do.
“So we get physiologically more relevant data, and that’s the goal,” Sant says.
What lies ahead
Some challenges remain for the model to achieve its full potential. Sant hopes the pharmaceutical industry will adopt the 3D prototype, but her method does not allow for the screening of multiple compounds simultaneously in a high throughput manner, as the industry prefers. So Sant hopes to create that capability.
In addition, because cancer is such a complex disease, many factors influence the patient’s outcome—not just the tumor size, which is what Sant’s model represents. For example, patients with very dense breast tissue are more prone to cancer, but science still doesn’t know why. Sant would like to know what factors exist in the dense microenvironment that make it more hospitable to the disease.
“The surprising thing was we never expected them to move,” she said. And yet they did: In fact, the entire tumor moved.”
Also, many patients carry microcalcifications, prompting them to get second mammograms to look for possible malignancies. Very little is known about where the microcalcifications come from or whether they play a role in advancing cancer.
“We definitely would like to now move forward to look at these other prognostic factors,” she says, using bioengineering tools to build models in vitro. Her lab is building synthetic matrices to reproduce dense tissue and other factors.
“We are trying to build complexity in the microenvironment adding matrices and different cell types that are seen in the patient tumors” she says, adding that she is pursuing additional grant funding.
Sant’s hope is that her models will fill the gaps left by 2D and animal models. In addition, she hopes to obtain access to cells derived from specific patients to create a personalized therapy approach, growing a person’s cells in real time to see whether they would respond to a particular therapy.
If she is able to do that—the type of personalized medicine that is frequently predicted for the future of health care—Sant envisions a significant potential benefit for the patient.
And that, she says, “would be really cool.”