Single-cell buoyant mass reveals CD8⁺ T-cell fitness and predicts melanoma checkpoint response

Introduction/Rationale: Reliable, label-free evaluations of systemic T-cell fitness are lacking. Most biomarkers are tumor-intrinsic (e.g., TMB, PD-L1) or rely on molecular readouts (gene expression, cytokines) that provide only partial insight into a T cell’s integrated functional potential. We hypothesized that an intrinsic physical property of resting CD8⁺ T cells could integrate composition and state to reveal functional heterogeneity. We therefore asked whether single-cell buoyant mass can resolve hidden CD8⁺ states and predict response to immune checkpoint blockade (ICB) in a clinical study.

Methods: We engineered a live-cell, label-free SMR microfluidic sorter and paired it with transcriptomic/proteomic profiling. Ex vivo functional assays quantified proliferation, memory re-expression, and exhaustion propensity. In a neoadjuvant melanoma cohort, we profiled pre-treatment circulating CD8⁺ T cells and computed two features: mean heavy-cell mass and fraction of heavy cells.

Results: Resting CD8⁺ T cells showed a bimodal buoyant-mass distribution of light and heavy populations. Heavy cells were biosynthetically primed, expanded faster, re-expressed CD62L more readily, and resisted exhaustion; light cells accumulated mitochondrial mass, proliferated more slowly, and exhausted earlier after delayed activation. In a neoadjuvant melanoma cohort, pre-treatment heavy-cell metrics predicted pathologic response to checkpoint blockade and outperformed tumor mutational burden. Predictive information resided in subtle heavy-population shifts captured by SMR.

Conclusion: Buoyant mass resolves hidden T-cell heterogeneity and provides an integrated, stimulation-independent biomarker of systemic immune fitness. This fast, label-free readout supports patient stratification and broader immunotherapy applications. As the first/lead author, I will present these findings and the translational path to a clinical diagnostic.