NK cells limit early stages of metastasis in ex vivo models of breast cancer. (A) Dot plot of GFP⁺ K14⁺ and K14⁻ tumor cells stained for MHC class I expression. (B) Schema of hNK cell-tumor organoid coculture. Tumor organoids were isolated from dissected MMTV-PyMT mammary tumors, and hNK cells were isolated from the spleens of FVB/n mice. Tumor organoids were cultured alone or in coculture with hNK cells in collagen I gels. (C) Representative DIC images of MMTV-PyMT tumor organoids alone (top) or in coculture with hNK cells (bottom) at 0 and 24 h. Scale bar, 50 µm. (C′ and C″) Boxplots of inverse circularity (C′) and area fold change (C″) of MMTV-PyMT tumor organoids alone or in coculture with hNK cells. Error bars represent 5th to 95th percentile. ****, P < 0.0001 by Mann–Whitney U test. (D) Schema of hNK cell-tumor cluster coculture. Tumor clusters were isolated from dissected MMTV-PyMT mammary tumors, and hNK cells were isolated from the spleens of WT mice. Tumor clusters were cultured alone or in coculture with hNK cells in Matrigel. (D′) Representative DIC images of MMTV-PyMT tumor colonies alone (top) or in coculture with hNK cells (bottom) at 24 h. Scale bar, 50 µm. (D″) Quantification of normalized colony formation count from MMTV-PyMT tumor clusters cultured alone or in coculture with hNK cells. Colony count was normalized to control. Mean is represented; ***, P < 0.001 by Mann–Whitney U test.