Multicolour flow cytometry protocol for dogs v1

Although immunotherapy is becoming a standard approach of human cancer treatment, only a minor fraction of patients responds to the therapy. It is therefore required to determine the sub-populations of patients who will respond to immunotherapies along with developing novel strategies to improve efficacy of anti-tumor immune reactions. Development of novel immunotherapies is currently heavily relying on mouse models of cancer. These models are important for better understanding of mechanisms behind tumor immune escape and investigation of novel strategies to overcome it. Nevertheless, the murine models do not necessarily represent the complexity of spontaneously occurring cancers in humans. Dogs spontaneously develop a wide range of cancer types with an intact immune system under similar environment and exposure to humans, which can serve as translational models in cancer immunotherapy research. To date though, there is still relatively limited amount of information regarding immune cell profiles in canine cancers. One possible reason could be the lack of established method to isolate and simultaneously detect different immune cell types in neoplastic tissues. Here we describe a protocol for multi-color flow cytometry to distinguish immune cell types in blood, lymph nodes, and neoplastic tissues from dogs with cancer. Our results demonstrate that a 9-color flow cytometry panel enables to characterize different cell subpopulations including myeloid cells. We also show that the panel allows to detect minor/aberrant subsets within a mixed population of cells in various neoplastic samples including blood, lymph node and solid tumors. To our knowledge, this is the first simultaneous immune cell detection panel applicable for solid tumors in dogs. This multi-color flow cytometry panel has the potential to inform future basic research focusing on immune cell functions in translational canine cancer models.