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Fig. 3 | Journal for ImmunoTherapy of Cancer

Fig. 3

From: Radiotherapy enhances natural killer cell cytotoxicity and localization in pre-clinical canine sarcomas and first-in-dog clinical trial

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. bc. 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 ALDHbright 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

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