A new single-cell technology is giving scientists their clearest view yet of immune cell behavior—capturing not just genetic intent but real-time activity. By measuring RNA and proteins simultaneously, it reveals cytokine activity with greater accuracy, strengthening the foundation for understanding cancer, inflammation and treatment resistance. Published in the April 8 issue of Scientific Reports, the findings could ultimately sharpen immunotherapy design and improve predictions of patient response.

Researchers at Sylvester Comprehensive Cancer Center, part of the University of Miami Miller School of Medicine, working with collaborators at the University of California, San Francisco, and the Helen Diller Family Comprehensive Cancer Center, developed CIPHER-seq, which enables simultaneous measurement of RNA and proteins within the same immune cell for a more complete view of immune responses.

Five-layer multimodal architecture of CIPHER-seq. Schematic representation of the five information layers captured by CIPHER-seq: (1) whole-transcriptome scRNA-seq, (2) cell-surface protein profiling, (3) intracellular cytokine detection, (4) intracellular protein measurement, and (5) sample multiplexing via oligo-tagged barcodes and unique RNA probes. Distinct color-coded antibodies illustrate simultaneous detection of extracellular markers, cytokines, and other intracellular proteins within the same cell.

“RNA gives us clues about where a cell is headed,” said Justin Taylor, M.D., a Sylvester physician-scientist and co-senior author of the study. “Proteins show us where it actually arrives, and this clearer picture could help scientists design better immunotherapies and help clinicians predict which patients are most likely to benefit from them.”

Single‑cell RNA sequencing has transformed biomedical research. It allows scientists to study thousands of immune cells at once and see which genes are turned on or off. But RNA is only a set of instructions. Proteins are the molecules that carry out those instructions.

That gap becomes especially important when scientists study cytokines—small but powerful proteins that immune cells use to communicate. Cytokines help control inflammation, direct immune attacks and shape how tumors grow or shrink. Yet RNA levels for cytokines often do not correlate with the amount of protein a cell actually produces.

“In immune cells, RNA and protein don’t always rise and fall together,” said co-senior author Emiliano Cocco, Ph.D., Miller School assistant professor of biochemistry and molecular biology.

That mismatch isn’t an error. It’s biology. RNA can appear quickly and disappear just as fast, while proteins take time to build up and may linger longer. Studying RNA alone can miss this timing, Cocco added.

CIPHER‑seq—short for Cytokine Intracellular Protein High‑throughput Expression with RNA sequencing—was built to close that gap. The method allows researchers to gently preserve cells, enabling them to measure multiple layers of information at once.

From a single immune cell, CIPHER‑seq captures:

• RNA from across the genome
• Proteins on the cell surface
• Proteins inside the cell
• Cytokines trapped inside before they are released

This creates a fuller, more reliable snapshot of immune activity.

One challenge with studying cells is that the process itself can stress them. Some existing methods damage cells during preparation, triggering artificial stress responses that distort results.

When the research team compared CIPHER‑seq with standard approaches, they found that CIPHER‑seq caused far less cellular stress. Cells processed with other methods showed signs of damage, especially in their mitochondria—the cell’s energy centers. Those stress signals can interfere with accurate measurements, making it harder to distinguish true immune behavior from technical artifacts.

“We wanted a method that lets cells stay as close as possible to their natural state,” said Taylor, a member of Sylvester’s Translational and Clinical Oncology Program and The Pap Corps Endowed Professor in Leukemia.

To test the platform, 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 known to play important roles in immune defense and cancer biology. Just as importantly, the technology showed when these signals appeared.

Using a computational approach that orders cells along a timeline of activation, researchers found that RNA signals rose first, followed shortly by protein levels. The delay was small, but consistent.

“It’s like seeing the plan before the action,” said first author, Avni Bhalgat, Ph.D. “Cytokines help determine whether immune cells attack cancer, ignore it or even help tumors grow. Understanding how and when immune cells produce these signals is critical.”

When both RNA and protein are measured simultaneously, CIPHER-seq allows researchers to track immune responses step by step, rather than relying on a single layer of data, Taylor explained, and this provides a stronger foundation for studying cancer, inflammation and treatment resistance.

“The platform helps us move beyond inference and toward understanding how immune responses truly unfold—one cell at a time,” he said.

Source – University of Miami Miller School of Medicine