Combination radioimmunotherapy with adoptive NK transfer targets cancer stem cells in canine models of bone and soft tissue sarcoma.

Methods Dog NK cells were isolated from fresh PBMCs using Ficoll separation and CD5 depletion. Isolated NK cells (CD3+, CD5dim, TCR-) were expanded via co-culture with irradiated (100Gy) K562-C9-mIL-21 for 2-3 weeks in 100IU/ mL recombinant human IL-2. Canine osteosarcoma (OSCA) tumor lines and fresh canine primary sarcomas were evaluated for susceptibility to NK killing before/after RT in vitro and in xenograft experiments with NSG mice. NK cytotoxicity was assessed in 4-16 hour killing assays by Flow cytometry using a BD Fortessa cell sorter (BD Biosciences) with 7-Aminoactinomycin as cell viability marker.