1.19 ALDH marks a population of canine cancer stem cells which are preferentially targeted by dog NK cells

R. J. Canter5,6, E. Ames6, S. Mac6, S. Grossenbacher6, M. Kent3, W. Culp3, M. Chen4, W. J. Murphy6  3UC Davis School Of Veterinary Medicine,Surgical And Radiological Sciences,Davis, CA, USA 4University Of California – Davis,Pathology And Laboratory Medicine,Sacramento, CA, USA 5University Of California – Davis,Surgery/Surgical Oncology,Sacramento, CA, USA 6University Of California – Davis,Laboratory Of Cancer Immunology,Sacramento, CA, USA

Introduction: Aldehyde dehydrogenase (ALDH) is a common cancer stem cell (CSC) marker in diverse solid human tumors. We hypothesized that ALDHbright cells would demonstrate the CSC phenotype in dog soft tissue sarcomas (STS) and that these dog CSCs could be preferentially targeted by dog NK cells.  

Methods: ALDHbright cell populations from canine tumor lines and fresh canine primary STS were evaluated for long term colony outgrowth and their ability to form tumors in NOD-SCID IL2 receptor gamma chain null (NSG). STS tissue was obtained from primary dog STS samples and canine patient-derived xenografts (PDX) and evaluated by immunohistochemy (IHC) and flow cytometry for CSC markers including CD24, CD44, and ALDH.  Stained slides were reviewed by a blinded pathologist and scored for percentage and intensity of ALDH-positive cells. Flow cytometry was performed using a BD Fortessa cell sorter (BD Biosciences), and cell viability was analyzed using 7-Aminoactinomycin (7-AAD). Dog NK cells were isolated from leukocyte filters obtained from healthy donors at the School of Veterinary Medicine. NK cytotoxicity was assessed by chromium release and flow cytometry. Parametric and non-parametric statistical tests were performed as appropriate.

Results: ALDHbright canine tumor cells displayed properties of CSCs, including selective tumor formation in NSG mice after cell sorting into ALDHbright and ALDHdim populations and long term colony outgrowth in methylcellulose. Using positive selection with magnetic beads, we observed that canine NK cells are responsive to human cytokines, including IL-2, IL-12, and IL-18 with a 3 – 10-fold expansion in NK cells over 14 days. Ex vivo activated dog NK cells demonstrated 35 – 45% cytotoxicity and 57 – 62% cytotoxicity against dissociated dog STS tumors at effector-to-target ratios of 10:1 and 20:1, respectively. IHC staining of dog PDX specimens showed a marked reduction in ALDH score (P<0.05) after intra-tumoral injection of allogeneic dog NK cells compared to controls.

Conclusion: ALDHbright cells exhibit CSC properties in dog STS, and dog NK cells appear to possess an intrinsic ability to recognize and target them. Dog STS appear to be a valuable model to facilitate clinical translation of NK immunotherapy and targeting of CSCs.