Using Spectral Flow Cytometry to Characterize Anti-Tumor Immunity in Orthotopic and Subcutaneous Mouse Models of Cancer.

Mouse models remain at the forefront of immuno-oncology research, providing invaluable insights into the complex interactions between the immune system and developing tumors. While several flow cytometry panels have been developed to study cancer immunity in mice, most are limited in their capacity to address the complexity of anti-cancer immune responses. For example, many of the panels developed to date focus on a restricted number of leukocyte populations (T cells or antigen-presenting cells), failing to include the multitude of other subsets that participate in anti-cancer immunity. In addition, these panels were developed using blood or splenic leukocytes. While the immune composition of the blood or spleen can provide information on systemic immune responses to cancer, it is in the tumor microenvironment (TME) that local immunity takes place. Therefore, we optimized this spectral flow cytometry panel to identify the chief cell types that take part in cancer immunity using immune cells from cancer tissue. We used pancreatic tumors implanted both orthotopically and subcutaneously to demonstrate the panel's flexibility and suitability in diverse mouse models. The panel was also validated in peripheral immune districts (the blood, spleen, and liver of tumor-bearing mice) to allow comparisons between local and systemic anti-tumor immunity. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tumor induction-Orthotopic Alternate Protocol: Tumor induction-Subcutaneous Basic Protocol 2: Preparation of single-cell suspensions from the tumor, spleen, liver, and blood of tumor-bearing mice Basic Protocol 3: Staining single-cell suspensions from the tumor, spleen, liver, and blood of tumor-bearing mice.

Accumulation of tissue-resident natural killer cells, innate lymphoid cells, and CD8+ T cells towards the center of human lung tumors.

Lung cancer is a leading cause of cancer-related death worldwide. Despite recent advances in tissue immunology, little is known about the spatial distribution of tissue-resident lymphocyte subsets in lung tumors. Using high-parameter flow cytometry, we identified an accumulation of tissue-resident lymphocytes including tissue-resident NK (trNK) cells and CD8+ tissue-resident memory T (TRM) cells toward the center of human non-small cell lung carcinomas (NSCLC). Chemokine receptor expression patterns indicated different modes of tumor-infiltration and/or residency between trNK cells and CD8+ TRM cells. In contrast to CD8+ TRM cells, trNK cells and ILCs generally expressed low levels of immune checkpoint receptors independent of location in the tumor. Additionally, granzyme expression in trNK cells and CD8+ TRM cells was highest in the tumor center, and intratumoral CD49a+CD16- NK cells were functional and responded stronger to target cell stimulation than their CD49a- counterparts, indicating functional relevance of trNK cells in lung tumors. In summary, the present spatial mapping of lymphocyte subsets in human NSCLC provides novel insights into the composition and functionality of tissue-resident immune cells, suggesting a role for trNK cells and CD8+ TRM cells in lung tumors and their potential relevance for future therapeutic approaches.