Expanded CD56^superbrightCD16⁺ NK Cells from Ovarian Cancer Patients Are Cytotoxic against Autologous Tumor in a Patient-Derived Xenograft Murine Model

Antitumor functions of expanded NK cells increase with increasing CD56 brightness. Expanded NK cell population was stratified by flow cytometry analysis based on CD56 expression intensity. A, Percent (n = 6 donors), (B) MFI (n = 6 donors), and (C) representative flow plots of IFNγ expression. D, Expanded NK cell percent CD107a expression following 5-hour incubation with OVCAR8 target cells (n = 5 donors). E, Representative flow plots of CD107a expression. Results are from two independent experiments and were analyzed via matched one-way ANOVA; *, P < 0.05; **, P < 0.01.

Expanded OCP PB- and ascites-NK cells reduce tumor burden and improve survival in a cell-line xenograft model of human OC. Expanded OCP PB-NK, OCP ascites (Asc)-NK, or HD PB-NK cells were adoptively transferred intraperitoneally to xenograft mice beginning 2 days following injection of OVCAR8-Luciferase (Luc) ovarian cancer cells. A, Schematic timeline of NK cell treatments. B, Mice were imaged at indicated days and tumor burden was quantified via bioluminescence (radiance). Results were analyzed via two-way ANOVA; ****, P < 0.0001 (n = 5 mice per group from one experiment). C, Images of mice at day 16 with color scale standardized across images. D, Survival of mice compared across groups and analyzed using the log-rank (Mantel–Cox) test followed by Bonferroni correction for multiple comparisons; **, P < 0.0083 (n = 4–5 mice per group from one experiment).

Expanded OCP-NK cells reduce burden of established ovarian cancer tumor. Expanded OCP PB-NK cells were adoptively transferred intraperitoneally to NRG mice beginning 8 days following injection of OVCAR8-Luc ovarian cancer cells. A, Schematic timeline of NK cell treatments. B, Mice were imaged at indicated days and tumor burden was quantified via bioluminescence (radiance). Results were analyzed via two-way ANOVA; **, P < 0.01 (n = 5 mice per group from one experiment). C, Images of mice at days 8 and 11 with color scale standardized across images.

Establishment of patient-derived xenograft (PDX) model of human ovarian cancer. A, Schematic of model establishment: primary ovarian cancer cells in indicated volume of either PBS or ascites fluid were injected intraperitoneally into NRG mice. Tumor burden was assessed at endpoint. B, Survival of mice following injection of indicated doses of ovarian cancer cells in PBS or ascites fluid. C, Short-term survival of mice based on volume of ascites fluid injected to assess volume-based toxicity. D, CA-125 levels quantified in ascites of PDX mice compared with peritoneal fluid of control NRG mice. Results were analyzed via an unpaired t test; *, P < 0.05 (control NRG mice n = 3; PDX mice n = 5 from 2 experiments). E–G, Representative histologic tumor cross-sections stained with H&E. E, Image of tumor core taken at 20× objective magnification; scale bar, 200 μm. F, Image encompassing tumor edge and core taken at 4× objective magnification; scale bar, 500 μm. G, Image of tumor edge taken at 20× objective magnification; scale bar, 200 μm.

Expanded NK cells maintain a cytotoxic phenotype in an autologous ovarian cancer microenvironment. Expanded OCP PB-NK, OCP ascites (Asc)-NK, or HD PB-NK cells were adoptively transferred intraperitoneally to PDX mice with visible signs of ascites or control NRG mice with no tumor. Forty-eight hours following adoptive NK cell transfer, ascites or peritoneal fluid was collected from mice and cells were stained with NK cell markers for flow-cytometric analysis of NK cell phenotype. A, Representative flow plot of gating strategy. NK cells were identified as human (h)CD45⁺hCD56⁺hCD3⁻. Proportion of (B) CD56^superbright NK cells, (C) CD16 expression, (D) NKp46 expression, and (E) NKp30 expression on NK cells. Results were analyzed via two-way ANOVA; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (tumor group n = 5; no tumor control group n = 3 from one experiment).