These are 3D live-cell imaging datasets of gastric tumor organoids in co-culture with primary human Natural Killer (NK) cells. The datasets were analyzed by a new, deep learning-based 3D image analysis software tool, SiQ-3D, which we developed and presented in the paper titled "Deep Learning-based 3D single-cell imaging analysis pipeline for quantifying cell-cell interaction dynamics in the tumor microenvironment". Interested users can download the SiQ-3D software code from GitHub (https://github.com/simonlbd1/SiQ-3D) or Code Ocean (https://codeocean.com/capsule/6676007/tree/v2), analyze the 3D image datasets locally, and cross-check the results with the SiQ-3D quantified results that we provided here.