Background Dogs are an outbred species that develop spontaneous cancers with striking similarities to humans and can serve as a crucial link to optimize immunotherapy treatments. We have completed canine immunotherapy trials using overlapping but distinct methods to stimulate natural killer (NK) cells in dogs with solid tumors. Our objective was to investigate changes in NK cell populations in response to immunotherapy and compare gene expression profiles between dogs receiving three separate NK-targeting treatments.

Methods Samples were available for a total of seven dogs treated for cancer at UC Davis (UCD Trial #1, n=2, UCD Trial #2 n=1) and University of Wisconsin (UW, n=4) in IACUC-approved trials. Dogs in UCD Trial #1 received two injections of autologous NK cells in combination with inhaled rhIL-15. Dogs in UCD Trial #2 underwent palliative radiotherapy (RT) in addition to infusion of PBMC-derived allogeneic NK cells. Dogs in the UW cohort received low-dose molecular targeted radionuclide therapy (MTRT) with external beam RT and intratumoral injection of IL-2 fusion cytokine. Matched pre- and post-treatment samples were submitted for single-cell RNA sequencing and integrated, data visualization was performed using R packages, Seurat and ggplot2. We hypothesized that trials would have distinct NK-activation signatures in response to unique treatment.

Results NK cells were able to be identified in each dataset by expression of NK-related genes, including NCR3, and lack of CD3 expression. Cells within the NK cluster following treatment with RT and allogeneic transfer had significantly increased expression of GZMA with an over two-fold increase in the percentage of NK cells post-treatment, reaching 14% of PBMCs. During treatment with RT and allogeneic transfer, NK cells retained steady expression of NK activating and functional genes although we also observed a distinct peak in KLRA1 expression 7 days after NK transfer. Dogs receiving MTRT therapy had increased expression of several activation markers, including CD16, NCR1, KLRA1 and CD69, in post-treatment NK cells compared to pretreatment. Notably, the MTRT therapy-specific NK gene activation signature in response to treatment was similar across patients in the cohort and distinct from dogs receiving adoptive NK transfer.

Conclusions This scRNASeq analysis provides insight into the diverse mechanisms of NK activation during unique NK-targeting immunotherapy regimens in dogs with cancer. Our transcriptomic analysis of first-in-dog clinical trials serves as a basis for investigating biomarkers of NK responses in innovative canine immunotherapy combinations.

Ethics Approval The animal studies were reviewed and approved by IACUC and UC Davis Clinical Trials Review Board. Written informed consent was obtained from the owners for the participation of their animals in this study.