Three-dimensional attenuation coefficient analysis for the quantitative evaluation of CAR T cell cytotoxicity in tumor spheroids

Tumor spheroids, which closely replicate the tumor microenvironment in biological systems, can serve as a viable testing platform for assessing the effectiveness of immune cells in immunotherapy. However, evaluating the efficacy of the cell-based therapy in these models is challenging due to the lack of quantitative imaging tools that enable high-resolution, non-perturbative and real-time analysis for such three-dimensional structures. Herein, we present an optical coherence tomography (OCT)-based analysis for the quantitative evaluation of the kinetics and cytotoxicity of chimeric antigen receptor (CAR) T cells against tumor spheroids. In addition to the various physical features of the spheroids that can be measured by OCT systems (e.g., diameter, height, and volume), we performed Fourier-based extraction of the attenuation coefficient (AC) in three-dimensional space. These optical and physical parameters were then examined to correlate with and evaluate the kinetics and cytotoxic effects of human epidermal growth factor receptor 2 (HER2) CAR T cells on HER2-positive breast tumor cell spheroids.