Effectiveness of NKG2D CAR-NK92 cells in targeting solid tumors: novel dynamic cytotoxicity analysis via real-time...

Lung, colorectal, and liver cancers are among the most prevalent and lethal solid tumors globally. However, traditional therapeutic modalities and even current immunotherapeutic strategies such as chimeric antigen receptor (CAR)-T cell therapy have failed to deliver durable clinical benefits. This limitation is primarily attributed to the challenges posed by tumor heterogeneity, off-target toxicity, and, critically, to the inadequate assessment of the dynamic anti-tumor efficacy of these treatments. A major bottleneck in the preclinical evaluation of CAR-based immunotherapies for solid tumors is the overreliance on static endpoint cytotoxicity assays, including lactate dehydrogenase (LDH) release and chromium-51 (⁵¹Cr) release assays. These conventional methods merely quantify the final extent of tumor cell lysis and are unable to capture critical kinetic parameters-such as response latency, cytotoxicity rate, and the capacity for complete tumor elimination in vitro-all of which are pivotal for predicting clinical applicability. To address this gap, we employed real-time cell analysis (RTCA), a label-free and continuous monitoring technology, to systematically evaluate the anti-tumor potency of NKG2D CAR-engineered NK92 cells against lung (H1299), colorectal (HCT116), and liver (Hep3B) cancer cell lines. RTCA's unique capability to track tumor cell viability at 15-min intervals revealed that NKG2D CAR-engineered NK92 cells initiated cytotoxicity immediately without a lag phase, exhibited steeper cytotoxicity slopes, and achieved complete target cell eradication at an effector-to-target (E:T) ratio of 5:1-dynamic insights that remained undetectable with static assays. Notably, using this advanced cell killing detection method, we found that NKG2D CAR-engineered natural killer (NK) cells displayed cytotoxicity kinetics comparable to those of five times the number of unmodified NK cells against H1299, HCT116 and Hep3B cell lines. At E:T ratios of 5:1, the engineered cells also secreted significantly higher levels of pro-inflammatory cytokines (IFN-γ and TNF-α) compared to unmodified NK92 cells. This confirmed robust immune activation that coincided with real-time tumor killing. These findings not only demonstrate that NKG2D CAR-engineered NK92 cells represent a promising immunotherapeutic candidate for solid tumors, but also highlight RTCA as a useful and informative platform for monitoring cytotoxicity kinetics in real time and advancing the translational development of solid tumor immunotherapies.