Blood Cell Biophysical Trajectories Reveal Response to CAR-T Therapy in Diffuse Large B-Cell Lymphoma

Although chimeric antigen receptor T-cell (CAR-T) therapy has substantially improved outcomes in diffuse large B-cell lymphoma (DLBCL), disease relapse remains a major clinical challenge. Because treatment-associated immune remodeling evolves dynamically over time, complementary biomarkers capable of longitudinally monitoring immune-cell state changes are needed to evaluate therapeutic response and relapse following CAR-T therapy. In this study, we developed a label-free, high-throughput image cytometry platform for longitudinal, multiparametric profiling of peripheral blood mononuclear cells (PBMCs) collected from a cohort of 17 DLBCL patients undergoing CAR-T therapy. Using bright-field single-cell imaging, we quantified four representative biophysical parameters, including cell size, eccentricity, refractive index, and mean pixel intensity, from PBMC populations at base and post-treatment time points. These parameters were analyzed at both population and patient levels to characterize treatment-associated remodeling trajectories and biophysical heterogeneity. Static comparisons revealed population-level biophysical differences between the 10 relapsed (R) and 7 nonrelapsed (NR) patients. Longitudinal and patient-level analyses, performed by tracking serial PBMC samples collected from the same patients across treatment time points, demonstrated that treatment outcome was associated with coordinated remodeling trajectories in NR patients and greater patient-specific heterogeneity in R patients. Patient-level effect size and heterogeneity analyses further demonstrated that relapse was associated with altered remodeling dynamics that were not apparent from population-averaged measurements alone. These findings demonstrate that longitudinal biophysical profiling of PBMCs enables quantitative monitoring of treatment-associated immune remodeling and provides a complementary biomarker for assessing therapeutic response following CAR-T therapy.