Dual-parameter tomographic imaging of attenuation and backscattering coefficients for quantitative evaluation of immune cell-mediated cytotoxicity in tumor spheroids

<b>Rationale:</b> Quantitative, non-perturbative assessment of immune cell-mediated cytotoxicity in tumor spheroids remains challenging due to the lack of real-time label-free analytical tools. Conventional methods such as fluorescence imaging or biochemical assays often require labeling and provide limited longitudinal analysis, which prohibits dynamic monitoring of therapeutic responses. This study presents a dual-parameter tomographic analysis method that simultaneously quantifies attenuation coefficient (AC) and backscattering coefficient (BSC) from optical coherence tomography (OCT) datasets, enabling dynamic evaluation of therapeutic responses in three-dimensional (3D) tumor spheroids. <b>Methods:</b> We developed a 3D Gabor transform-based algorithm to extract depth-resolved AC and BSC metrics from OCT volumetric datasets. Unlike conventional strategies, our method enables simultaneous voxel-wise measurements of AC and BSC values, with superior noise robustness. Experiments with intralipid solutions across a range of concentrations revealed that the Gabor-based approach yielded AC and BSC estimations with more than three times greater precision than prior methods. This approach enables high-resolution measurements of structural and optical property changes associated with apoptosis, allowing spatial and temporal mapping of treatment-induced cytotoxicity in HER2-positive breast tumor spheroids treated with AZD4547 and HER2-targeted chimeric antigen receptor (CAR) T cells. <b>Results:</b> In AZD4547-treated spheroids, AC increased dose-dependently from 0.39 to 0.64, reflecting a 64% rise, while BSC increased from 0.09 to 0.12, an approximate 33% increase. CAR T cell treatment induced a rapid, spatially progressive increase in both AC and BSC, originating at the spheroid periphery and advancing inward. Over 12 hours, AC rose from 0.40 to 0.82 (2-fold increase) and BSC from 0.09 to 0.20 (2.2-fold increase). While AC and BSC individually correlated with spheroid viability, their combined analysis consistently achieved a higher coefficient of determination (R² = 0.98) across both treatment modalities. <b>Conclusions:</b> This dual-parameter OCT-based assay framework provides a sensitive, label-free method for distinguishing between immune- and drug-induced apoptosis in tumor spheroids. Its strong correlation with viability and capacity to resolve spatially resolved dynamics underscore its potential for robust, <i>in situ</i> assessment of immunotherapeutic efficacy.