Fig. 2

Expanded natural killer (NK) cells show high cytotoxicity against breast cancer cell lines and primary tumours in vitro. a, b Expanded NK cells from breast cancer patients (red) or healthy donors (blue) were plated with tumour cell lines at different NK cell to tumour cell ratios. After 4–5 h, cells were stained with viability dye and then percent cell death was assessed using flow cytometry. a NK cell cytotoxicity against the triple negative MDA-MB-231/luc cell line. b NK cell cytotoxicity against the HER2-positive MDA-MB-453 cell line. Percent-specific lysis was calculated and graphed (n = 4, mean ± SEM). Unpaired Student’s t test was used for statistical comparison between the groups. c, d MDA-MB-231 and MDA-MB-453 cells were stained for the expression of major histocompatibility complex (MHC) class I (anti-HLA-A,B,C–PE antibody) and assessed by flow cytometry. Results are shown in c histogram and d geometric mean. e–g Expanded NK cells from autologous breast cancer patient (black) or allogenic breast cancer patient (red) were carboxyfluorescein succinimidyl ester (CFSE)-labelled and plated with patient-derived breast cancer tumour cells at different NK cell to tumour cell ratios, and incubated and assessed as in a and b. Cells were gated on singlets, then tumour cells were selected using a CFSE negative gate (e) or in a hCD56 negative gate (f) and finally analysed for cell death. e Experiment was carried out using patient-derived tumour from breast cancer patient B (BCP-B), presenting with oestrogen receptor- and progesterone receptor-positive (ER⁺/PR⁺/HER2^–) breast cancer. NK cells were CFSE-labelled prior to the assay. Data show the average from three independent experiments using the same patient-derived tumour sample. Unpaired Student’s t test was used for statistical comparison between the two groups. f Percent-specific lysis from one experiment carried out using patient-derived sample from breast cancer patient A (BCP-A) presenting with TNBC. NK cells were stained with hCD56. g An example of the gating used for e