Getting to the core of cancer research

In 2024, more than two million estimated new cases of cancer will be diagnosed in the United States and at minimum 600,000 people will die from the disease. With cancer treatments in high demand, a group of researchers at the University of Pittsburgh developed a new technology to evaluate the effectiveness of cell-based immunotherapy.

A popular treatment for advanced blood cancers such as leukemia and lymphoma is chimeric antigen receptor (CAR) T cell immunotherapy. CAR T cells, which are produced by collecting T cells from the patient and re-engineering them in the laboratory to produce proteins on their surface, bind to specific antigens and kill cancer cells. Although CAR T immunotherapy has been highly effective in treating blood cancers, it is less effective against solid tumors, according to Ioannis Zervantonakis, assistant professor of bioengineering at Pitt’s Swanson School of Engineering. 

“In solid masses such as breast cancer, tumor-fighting immune cells need to navigate through the solid tissue barriers.” Zervantonakis said. “One of the thoughts in the field is that T cell-based immunotherapies are not as effective for solid cancers because T cells cannot effectively migrate into the tumor mass and kill cancer cells.” 

To study solid cancers and mimic the environment of a tumor, Zervantonakis collaborated with Jason Lohmueller, assistant professor of surgery at Pitt’s School of Medicine, and modeled CAR T therapy in three-dimensional spheroids instead of a typical two-dimensional model. Spheroids are believed to mimic tumor behavior more effectively than 2D cell cultures because, like tumors, they contain both surface-exposed and deeply buried cells, proliferating and non-proliferating cells, and a hypoxic center with a well-oxygenated outer layer of cells. 

“When we're modeling cancer, you can grow cancer cells on a flat surface, which is typically how most of this work is done,” Zervantonakis said. “But you can also force the cancer cells to organize into a three dimensional spheroid and study how the T cells move in this 3D environment during the process of cancer cell killing.”

Not only does this method more accurately reflect the complex tumor architecture, but using a spin-coated microwell is also crucial for better imaging results, according to Youngbin Cho, postdoctoral fellow in the Zervantonakis lab. 

“It is pretty difficult to capture the cell dynamics inside the core of the spheroid.” Cho said. “So we used a transparent microwell instead of a typical cell culture plate to immobilize the spheroid, which allowed us to use our microscope to capture what’s going on in the core.”

Their results, “CAR T cell infiltration and cytotoxic killing within the core of 3D breast cancer spheroids under the control of antigen sensing in microwell arrays,” were recently published in APL Bioengineering (doi.org/10.1063/5.0207941). This study was led by Cho, PhD students Matthew Laird, Ruxuan Li, Dorota Jazwinska, undergraduate student Teddi Bishop, and Research Assistant Professor Elisa Ruffo.The findings revealed a quantitative threshold for effective antitumor CAR T function in vitro and that increasing CAR T cell seeding density resulted in higher CAR T cell infiltration and cancer cell elimination in the spheroid core.

“Testing the efficacy of cell-based immunotherapies using spheroids has important implications for the design of protocols that promote immune cell trafficking in solid tumors and attack cancer cells in the spheroid core,” Zervantonakis said. “We found that there is a ratio of spheroid size to CAR T cell numbers that predicts complete cancer cell elimination and that sensing of HER2 (breast cancer) antigen is necessary for the CAR T cells to traffic to the spheroid core. ”

Zervantonakis used universal SNAP-CAR T cells previously developed by Lohmueller’s lab and licensed by pharmaceutical company Coeptis Therapeutics in his models. SNAP-CAR is a CAR technology that can be easily adapted to target multiple types of cancer and lower the chance of unwanted side effects by pairing the CAR directly with an antibody. 

“SNAP-CAR allows us to make a highly programmable and tunable CAR T cell therapy.” Lohmueller said. “CAR T cells are very precious and expensive to make, but high doses can cause toxicity. So with SNAP-CAR what we can do is give a high dose of T cells, but tune therapy with antibodies to make sure the treatment is effective without toxicity.” 

Paired together, SNAP-CAR treatment and Zervantonakis’ spheroid modeling have broad implications for fine-tuning CAR T therapy, understanding how CAR T cells infiltrate a tumor, and studying and treating a broader range of solid cancers. 

“Having systems where we can evaluate cancer treatments outside of animal models enables rapid testing and engineering to improve CAR T therapy into tumors.” Lohmueller said. “There's also an opportunity here to test more complex systems that involve other types of immune cells common in tumors with organoid models, which will help get us even closer to human disease and help us to figure out how we can overcome all of the things that tumors are doing to thwart CAR T cell treatments.”