Fig. 3

Expanded Dog NK Cells are Cytotoxic against Canine Patient-Derived Specimens including Cancer Stem Cells. a. The schema depicts generation of canine patient-derived xenografts from a freshly excised grade III soft tissue sarcoma. Three mm³ fragments were allowed to grow to approximately 15–20 mm in maximal dimension for ex vivo killing assays or for passaging into additional mice. Representative photomicrographs from passages 0 and 2 of PDX #465049 demonstrate characteristic spindle cell tumor morphology, hyperchromatic nuclei, and increased nuclear-cytoplasmic ratio consistent with canine soft tissue sarcoma which is preserved over serial passaging. Scale bar = 50 μM. b – c. When dog PDX tumors reached approximately 20 mm in size from indicated passages, they were excised and digested to create single cell suspensions. Tumor cells were then incubated with ⁵¹Cr and labelled cells were co-cultured with activated allogeneic dog NK cells for 4 h. Tumor cell lysis was assessed with a scintillation counter, and percent cytotoxicity was calculated using standard formulas. Representative data from 2 to 3 experiments are shown. d. Graphic depicts schema for in vivo experiment assessing therapeutic efficacy of intra-tumoral allogeneic dog NK cells with intra-tumoral rhIL-2 similar to our first-in-dog clinical trial protocol. P2 PDX mice received two injections one week apart of intra-tumoral NK plus 100,000 IU rhIL-2 versus control injections. Tumor volume was measured twice weekly by external caliper. e. A passage 2 flank PDX tumor (#450554) was excised and digested. Representative flow cytometry shows staining for diethylaminobenzaldehyde (DEAB) to control for background fluorescence and staining for ALDH^bright cells, representing the putative CSC sub-population. f. Day 14–21 activated allogeneic dog NK cells were co-cultured for 12–16 h with ex vivo digested dog PDX sarcoma tumor cells using the ALDEFLUOR system to measure the putative CSC population. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 via one-way ANOVA with Tukey’s post-test