For years, scientists have been studying how molecular subtypes of breast cancer, specifically triple-negative breast cancer (TNBC) and luminal A and B cancers, may improve planning treatment and developing new therapies. Although the discovery of immunotherapy has become one of the most promising advances in cancer therapy, only a fraction of patients with these subtypes responds to immunotherapy, and efficacious treatment has so far been largely limited to select tumor types.
Research presented at the 2022 CRI-ENCI-AACR International Cancer Immunotherapy Conference in New York City focused on understanding the role of myeloid subsets within hormone-driven malignancies to identify and develop therapeutic targets and approaches. conducted at the Moffitt Cancer Center Tampa in The Ruffell Lab, showed the results led by Brian Ruffell, PhD.
"TNBC and luminal B breast cancers are the focus types for our research, knowing that therapeutic options are limited," noted Brian Ruffell, PhD, Associate Member in the Department of Immunology at the H. Lee Moffitt Cancer Center and Research Institute. "We are searching for ways to bring combination immunotherapy to these patients."
Importance of Molecule TIM-3
The element of surprise during the research process, according to Ruffell, was discovering the importance of the molecule TIM-3 on dendritic cells. "These cells provide instructions for how the immune systems should respond to cancer and infection," he noted. "The focus in my lab is identifying new targets in dendritic cells. While knowing the target was already in trials for hitting T cells, because it was highly expressed, we wanted to see whether it was important in dendritic cells as well. And it was especially important in our models, primarily breast cancer where the TIM-3 molecule is poorly expressed by T cells."
This was surprising given the focus in the field on the role of this molecule in T cells, according to Ruffell. "We found that TIM-3 prevented dendritic cells from reacting to exogenous tumor DNA. In addition to our own study, other studies have highlighted how this one molecule, TIM-3, could regulate dendritic cell function through multiple pathways."
For example, the laboratory of Vijay Kuchroo, DVM, PhD, at Harvard Medical School and Brigham and Women's Hospital, where the TIM-3 molecule was first identified, recently identified another mechanism of action.
"One of our key steps was to figure out how to target this gene, which is based on their research," Ruffell said. "If the drug gets approved for certain cancers, we were hoping to demonstrate we don't need to find cancer where the molecule is expressed at high levels of T cells, since dendritic cells throughout the body appear to express TIM-3 at high levels. Another potential takeaway from the research is that we need to understand how to use the appropriate combination therapy. If we use the drug as a single agent, nothing happens, so we really had to bring in chemotherapy to allow this drug to function in our preclinical models."
Measuring Uptake of DNA
The research led by Ruffell also concluded that DNA uptake can occur spontaneously without the use of transfection reagents. "The key step in reaching the desired outcome was to figure out a way to measure uptake of DNA by the dendritic cells," he explained.
To execute their research, the team designed a protocol to track exogenous DNA uptake by dendritic cells by first measuring exogenous DNA uptake using supernatant containing cancer cell debris, which allows for DNA uptake in the absence of transfection reagents. Noting that, while inhibitors or antibodies that alter the process can be added, either flow cytometry or fluorescent microscopy can be used to measure DNA uptake.
"The protocol was originally intended to mimic the exposure of dendritic cells to dying cells in the tumor microenvironment or other pathological conditions of high cellular death," he said.
Ruffell and his team inadvertently realized that, if they "treated dendritic cells with killed tumor debris, they could replicate their in vivo findings related to TIM-3 and work backwards from there."
"Previous studies that looked at how dendritic cells are activated by tumor DNA had been unable to replicate the DNA entering cells in vitro," he said. "They either had to do a transfection step or they injected DNA into tumors. We didn't know we were looking at DNA in the first place, but were accidently able to model DNA uptake by leaving the DNA in an unpurified state, similar to how it might be within tumors."
The next step in research for The Ruffell Lab is to explore three new areas. "First, we want to understand how DNA gets inside the cells without the need for liposomes, as we think this has the potential to lead to new therapeutic approaches," Ruffell explained. "Second, we want to identify other types of therapy that can be combined with TIM-3 antibodies, as this could expand the number of patients who would benefit from this relatively safe and effective treatment approach. And, lastly, we are searching for additional therapeutic targets on dendritic cells with support from the Cancer Research Institute, a nonprofit organization that funds immunotherapy research."
Amy Gallagher is a contributing writer.