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

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 ( 51 Cr) 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.