With CANINE, a collaboration between the School of Veterinary Medicine and the School of Arts and Sciences’ Biology Department, undergraduates are breaking new ground in immunology.
Mingyu Zhou of Friends Central and Penn rising sophomore Xiaotong Zhu practice their pipetting techniques as Rachel Orth, a rising senior, and John Wagner, a senior lecturer in biology, look on. Wagner and Penn Vet’s Jennifer Punt are guiding the students as they investigate blood samples from pet dogs to contribute to the field of canine immunology.
Shoulder to shoulder at a lab bench in the basement of Penn’s Levin Building, Sonia Luthra, Johanna Fowler, and Tracy Tran compare small microscope slides they’re preparing.
Fowler, a rising junior at Haverford College, and Tran, a rising sophomore at Penn, observed Luthra’s technique, drawing a sample of canine blood carefully across the slide to make a thin smear. The high school senior at Friends Central School had a leg up on the undergrads: whereas their 10-week project was only just beginning, Luthra had already logged a month in the lab.
Students get the hang of placing eosin dye on the plates, allowing different components of cells to stand out under the microscope.
Working under the supervision of Penn scientists Jennifer Punt and John Wagner, the students are doing more than just honing their scientific technique; they’re advancing the field of canine immunology.
Punt, an immunologist and associate dean at the School of Veterinary Medicine, and Wagner, a molecular biologist and senior lecturer in the School of Arts and Sciences, have termed the project CANINE, for cancer immunity in education. Both Punt and Wagner share a passion—and a track record—for getting students involved in “real” research, work that goes beyond rote experiments where the outcome is predetermined. CANINE is an expansion of those efforts, allowing students from high school, undergrad, and graduate levels to contribute to an emerging field of biology that may shed light on the role of the immune system in dogs with cancer.
“The really fun part of science, in my opinion, is the experimental design, the experimental interpretation,” says Wagner. “Yes, there’s the technical aspect that everybody learns, but the nice thing about this project is that it’s expanding the possibility of what students can do.”
The really fun part of science, in my opinion, is the experimental design, the experimental interpretation.
John Wagner
“I just adore working with undergraduates,” Punt adds. Permitting students to play a leading role in designing and carrying out the work builds their confidence, she’s observed. “If I can get the students to really relax into the research, I couldn’t be happier.”
Punt has been revamping educational offerings for vet students and fostering cross-school collaborations since she returned in 2017 to Penn Vet, where she had earned her VMD and Ph.D. in the school’s dual degree program more than 25 years earlier.
Punt’s earlier research had largely focused on the immune system’s T cells, and mice were her go-to animal model. But Punt has long sought alternative ways to pursue meaningful studies.
Overseeing the students’ work, Punt offers individualized advice and feedback. Since returning to Penn as an associate dean, she doesn’t have a lab of her own, but has continued to contribute to research and education through collaborations, such as this one with Wagner.
Back at Penn, she began spending time in the lab of Oliver Garden, chair of the Department of Clinical Studies at Penn Vet. A VMD/Ph.D. student in his lab, Sabina Hlavaty, focuses on myeloid-derived suppressor cells (MDSCs), which are believed to play a role in keeping the immune system restrained in certain contexts. Sometimes, the researchers believe, this restraint may come at the cost of compromising the immune system’s ability to fight off cancer. A recent emphasis in the lab has been characterizing MDSCs in dogs with cancer.
Putting together what she was learning from Garden’s lab with her desire to engage students in meaningful research, Punt generated an idea for a new line of inquiry. “I developed this mini-pilot around the question, ‘What can you learn from a small amount of blood?’”
Punt purchased a flow cytometer on eBay, allowing researchers to categorize and quantify cells by different characteristics. Data from the machine has been key to advancing the students’ research.
To be specific, she wanted to see if, using only a blood sample, students could make new insights about the role of the immune system in dogs with cancer. And she had a nearby source for samples: Penn Vet’s Ryan Hospital.
Last summer, Punt kicked off the pilot, with support from a Faculty Mentoring Undergraduate Research grant from Penn’s Center for Undergraduate Research and Fellowships (CURF), working with two undergraduates, Michelangella Arbocco from Penn and Trevor Esilu from Haverford, and then-high school student, Anjali Gupta, who has since matriculated to Penn. The research relied on a flow cytometer—a high-tech piece of equipment that enables researchers to sort and analyze the characteristics of a population of cells—that Punt purchased off eBay.
“Everyone thought I was totally nuts,” she recalls. “But it is in great shape, John found a spot for it, it runs as cheaply as you can possibly imagine, and the students have really thrived on it.”
Zhou, who completed a month in the lab as part of a final project for her high school, kept detailed records of her experiments in her lab notebook, and learned to use and interpret flow cytometry analyses.
The students began working with blood samples from pet dogs seen at Ryan. They narrowed in on the receptor for a growth factor, IGF-1, that is associated with size differences in dogs. Larger dogs, which have shorter lifespans and higher rates of cancer, tend to have more circulating IGF-1. “I decided to take that leap and say, ‘Could higher levels of circulating IGF-1 enhance the suppressive effects of white blood cells,’” says Punt, a suppression that may foster the growth of cancer?
To respond to IGF-1, a cell needs to have a receptor for it, so the students began to look for the receptor on different cell types. Pretty quickly, some interesting tidbits started to trickle in.
“One of my students, Trevor, had some very beautiful data that suggested all of the PMN [polymorphonuclear] MDSCs had one IGF-1 receptor,” Punt says.
They found the receptor on other subtypes of immune cells, but not all of them, opened up the possibility for examining how the receptor’s expression varies with a dog’s size, health status, cell type, and other traits. “It becomes a brand-new way of thinking,” says Punt.
Wagner and Orth prepare to spin down samples in a table top centrifuge. Orth has worked on the CANINE project since taking Wagner’s laboratory course last fall.
To begin to address some of those new queries, Punt teamed with Wagner, whose laboratory course for undergraduates and master’s students, Biology 425: Biochemistry and Molecular Genetics Superlab, takes up a new line of research each time it is offered. The two professors had worked together years ago, while each held a position at Haverford, and saw a mutual benefit in collaborating again.
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“From my side, it’s really nice to have someone like Jenni involved, who enjoys bringing the undergraduates into the research going on at the Vet School,” says Wagner.
Punt sees advantages for Penn Vet as well. “Penn’s campus and Penn Vet in particular is so interestingly and uniquely situated—literally across the street from an incredible undergraduate program—so it just seemed like an ideal place to get involved.”
During the fall 2018 semester, students in Wagner’s class, a group that included biology majors and non-majors, seniors as well as first-years, and even master’s students, successfully assayed a variety of cell types for the presence or absence of a receptor for IGF-1.
“We pretty much hit the marks on the stage for what you would have hoped to do in the first semester,” Wagner says.
Permitting students to play a leading role in designing and carrying out the work builds their confidence.
Jennifer Punt
One of the students in the class, Rachel Orth from Hummelstown, Pennsylvania, was captivated enough by the experience in class to continue working on CANINE as a work-study student through the spring, and will be in the lab this summer as well. “I’m planning to apply to vet school this summer and fall,” says the rising senior, noting that the experience in a lab so tightly connected to veterinary medicine should serve her well in that process.
If the line of inquiry continues to be productive, Punt and Wagner foresee students being coauthors on published research down the line. All this from the small but significant question, as Punt frames it, “What can you learn from a small amount of blood?”
Moving into the summer, bolstered by the momentum from Wagner’s class and Orth’s continued work, Punt and Wagner will be working with Fowler, Tran, and Orth as well as rising sophomore Xiaotong Zhu and second-year veterinary student Lisa Dourmashkin to sustain that progress. The group, supported by a CURF Team Grant for Interdisciplinary Activities, plans to begin putting together a database to look for associations between cancer types and their results looking at the IGF-1 receptor. With Wagner’s expertise in molecular biology, they also hope to begin looking at how stimulating IGF-1 affects gene expression and functions of immune cells.
Luthra and Mingyu Zhou, a fellow Friends Central student, presented on their studies for their senior project. Meanwhile Zhu, who is undecided on her major but is leaning toward physics, is looking forward to getting lab experience under her belt. “Hearing Dr. Punt speak about this work at my College House really drew me in,” say Zhu, who hails from China. “I’d worked at a lab in high school but this experience is going to be a lot different, a lot deeper.”
That is undoubtedly a prime goal for Punt and Wagner.
“I’ve been in labs with a very different philosophy, where the emphasis is on demonstrating techniques,” says Wagner. “This is a totally different thing. What we’re doing is potentially publishable. That’s a marker for showing what you’re doing is genuine, and the undergrads are doing it.”
Pet dogs, including Punt’s golden retriever Sammy, are the source of blood samples used in the lab. Punt is hopeful that the CANINE research will underscore the rich potential of doing impactful science without relying on mouse models. (Photo: Jennifer Punt)
Homepage photo: Weaving in lessons on immunology as she relays instructions for completing laboratory tasks, Jennifer Punt’s (center) love of teaching shines through as she works with high school students and undergraduates in a basement lab in the Levin Building.
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