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A new single-cell method called CIPHER-seq is giving scientists an unprecedented view of immune cell behavior, connecting genetic signals to real-time protein activity. Developed by researchers at the Sylvester Comprehensive Cancer Center at the University of Miami Miller School of Medicine, in collaboration with teams at the University of California, San Francisco, and the Helen Diller Family Comprehensive Cancer Center, the approach simultaneously measures RNA and proteins within the same cell. The study, published in Scientific Reports, highlights how this dual readout strengthens the understanding of cancer, inflammation, and treatment resistance.
Single-cell RNA sequencing has become a vital tool for decoding immune functions, revealing which genes are turned on or off in thousands of cells at once. But RNA alone represents potential—it outlines a cell’s intention, not necessarily its current state. Proteins, conversely, perform cellular tasks and provide a direct measure of activity. The gap between RNA and protein levels becomes especially important when analyzing cytokines, small proteins that orchestrate immune responses and govern inflammation, tumor growth, or regression. As Emiliano Cocco, co-senior author of the study, noted, “In immune cells, RNA and protein don’t always rise and fall together.” RNA fluctuations happen quickly, while protein changes unfold more slowly and persist longer.
To bridge this gap, CIPHER-seq—short for Cytokine Intracellular Protein High-throughput Expression with RNA sequencing—was engineered to capture multiple data layers from the same immune cell: RNA transcripts, surface proteins, intracellular proteins, and cytokines before release. This integrated approach yields a more complete and accurate picture of how immune cells function.
Importantly, the method minimizes stress on cells during sample preparation. When compared with conventional techniques, CIPHER-seq preserved cell integrity far better, avoiding mitochondrial damage and artificial stress signals that can distort results. “We wanted a method that lets cells stay as close as possible to their natural state,” said Justin Taylor, co-senior author. Testing confirmed that the platform effectively tracked cytokine responses such as interferon-gamma and tumor necrosis factor, including the timing of their appearance and progression.
By combining transcriptomic and proteomic data, CIPHER-seq reveals the sequential steps of immune activation—showing RNA signals first and protein changes shortly after. “It’s like seeing the plan before the action,” said first author Avni Bhalgat. “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.”
Publication details: Bhalgat, A., Micin, K., Affer, M. et al. CIPHER-seq enables intracellular multimodal profiling of cytokine responses in single immune cells. Sci Rep 16, 9693 (2026). https://doi.org/10.1038/s41598-026-44946-y
Single-cell RNA sequencing has become a vital tool for decoding immune functions, revealing which genes are turned on or off in thousands of cells at once. But RNA alone represents potential—it outlines a cell’s intention, not necessarily its current state. Proteins, conversely, perform cellular tasks and provide a direct measure of activity. The gap between RNA and protein levels becomes especially important when analyzing cytokines, small proteins that orchestrate immune responses and govern inflammation, tumor growth, or regression. As Emiliano Cocco, co-senior author of the study, noted, “In immune cells, RNA and protein don’t always rise and fall together.” RNA fluctuations happen quickly, while protein changes unfold more slowly and persist longer.
To bridge this gap, CIPHER-seq—short for Cytokine Intracellular Protein High-throughput Expression with RNA sequencing—was engineered to capture multiple data layers from the same immune cell: RNA transcripts, surface proteins, intracellular proteins, and cytokines before release. This integrated approach yields a more complete and accurate picture of how immune cells function.
Importantly, the method minimizes stress on cells during sample preparation. When compared with conventional techniques, CIPHER-seq preserved cell integrity far better, avoiding mitochondrial damage and artificial stress signals that can distort results. “We wanted a method that lets cells stay as close as possible to their natural state,” said Justin Taylor, co-senior author. Testing confirmed that the platform effectively tracked cytokine responses such as interferon-gamma and tumor necrosis factor, including the timing of their appearance and progression.
By combining transcriptomic and proteomic data, CIPHER-seq reveals the sequential steps of immune activation—showing RNA signals first and protein changes shortly after. “It’s like seeing the plan before the action,” said first author Avni Bhalgat. “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.”
Publication details: Bhalgat, A., Micin, K., Affer, M. et al. CIPHER-seq enables intracellular multimodal profiling of cytokine responses in single immune cells. Sci Rep 16, 9693 (2026). https://doi.org/10.1038/s41598-026-44946-y