New single-cell technology gives scientists the clearest view yet of immune cell behavior, capturing not only their genetic intent but also their real-time activity. Simultaneous RNA and protein measurements reveal cytokine activity more precisely and strengthen the basis for understanding cancer, inflammation, and treatment resistance. Published in the April 8th issue scientific reportthis discovery could ultimately sharpen the design of immunotherapy and improve prediction of patient response.
Researchers at the Sylvester Comprehensive Cancer Center, part of the University of Miami Miller School of Medicine, in collaboration with collaborators at the University of California, San Francisco and the Helen Diller Family Comprehensive Cancer Center, developed CIPHER-seq, which can simultaneously measure RNA and proteins in the same immune cells to provide a more complete picture of immune responses.
RNA gives us clues about where our cells are headed. A protein shows where it actually ends up, and this clearer picture can help scientists design better immunotherapies and help clinicians predict which patients are most likely to benefit from that immunotherapy. ”
Justin Taylor, MD, a physician-scientist at Sylvester and co-senior author of the study
Single-cell RNA sequencing has transformed biomedical research. This allows scientists to study thousands of immune cells at once and see which genes are turned on or turned off. But RNA is just a set of instructions. Proteins are molecules that carry out those instructions.
This gap is especially important when scientists study cytokines, small but powerful proteins used by immune cells to communicate. Cytokines help control inflammation, direct immune attacks, and determine how tumors grow or shrink. However, cytokine RNA levels often do not correlate with the amount of protein that cells actually produce.
“In immune cells, RNA and proteins do not always rise and fall together,” said co-senior author Emiliano Cocco, Ph.D., assistant professor of biochemistry and molecular biology at the Miller School.
That mismatch is not an error. It’s biology. RNA appears quickly and disappears just as quickly, but proteins take longer to build and may persist longer. Cocco added that if you only study RNA, you may miss this opportunity.
CIPHER-seq (short for Cytokine Intracellular Protein High-Throughput Expression and RNA-Seq) was built to fill that gap. This method allows researchers to gently preserve cells and measure multiple layers of information at once.
CIPHER‑seq captures from a single immune cell:
- RNA from the entire genome
- cell surface proteins
- proteins in cells
- Cytokines are trapped inside before being released
This creates a more complete and reliable snapshot of immune activity.
One of the challenges of studying cells is that the process itself can stress the cells. Some existing methods can damage cells during preparation, causing artificial stress responses and skewing results.
When the research team compared CIPHER-seq to standard approaches, they found that CIPHER-seq caused much less cellular stress. Cells treated with other methods showed signs of damage, particularly to mitochondria, the energy centers of cells. These stress signals can interfere with accurate measurements, making it difficult to distinguish between true immune behavior and technical artifacts.
“We wanted a way to get the cells as close to their natural state as possible,” said Taylor, a member of Sylvester’s Translational Clinical Oncology Program and the Pap Corps Endowed Professor in Leukemia.
To test the platform, the researchers activated immune cells and tracked their responses. CIPHER-seq clearly captured increases in key cytokines, including interferon gamma and tumor necrosis factor signaling pathways, which are known to play important roles in immune defense and cancer biology. Equally important, technology has shown us when these signals appear.
Using a computational approach that orders cells along a timeline of activation, the researchers found that RNA signals rise first, followed quickly by protein levels. The lag was slight, but consistent.
“It’s like looking at the plan before you take action,” said lead author Dr. Avni Bhargat. “Cytokines help immune cells decide whether to attack cancer, ignore it, or even help it grow. It’s important to understand when and how immune cells produce these signals.”
By measuring both RNA and protein simultaneously, CIPHER-seq allows researchers to track immune responses in stages rather than relying on a single layer of data, Taylor explained, providing a strong foundation for studying cancer, inflammation, and treatment resistance.
“This platform will help us move beyond speculation and understand how the immune response actually unfolds, one cell at a time,” he said.
sauce:
University of Miami Miller School of Medicine
Reference magazines:
Balgat, A. Others. (2026). CIPHER-seq enables intracellular multimodal profiling of cytokine responses in single immune cells. scientific report. DOI: 10.1038/s41598-026-44946-y. https://www.nature.com/articles/s41598-026-44946-y
