Flow cytometry is a widely applied approach for exploratory immune profiling and enables the identification of individual cells in a cell population, such as a tumor cell suspension, by staining cell markers with antibodies conjugated with a fluorophore. The markers allow for the differentiation of a large variety of immune cell populations, their change in frequency, and activation status upon treatment. Conventional flow cytometers, which use bandpass filters and need conventional compensation matrices, are limited by the number of parameters that can be simultaneously analyzed, restricting their utility. Spectral analysis technology promises a greater flexibility for panel design, a higher number of analyzed parameters, and more accurate visualization of results. Our standard all-in-one flow cytometry panel uses all possible fluorescent detectors of the flow analyzer BD Fortessa, which permits the differentiation of the main immune cell populations in the tumor, such as T cells (CD4+, CD8+, Treg), B and NK cells, macrophages (M1/M2), MDSCs (granulocytic and monocytic), and dendritic cells. However, in addition to immune cell frequency, it would also be interesting to investigate cell surface activation and memory markers like PD-1, CD40, CD44, or CD62L or intracellular activation markers like IFN-gamma, perforin, granzyme B, or Ki67. The 32-channel spectral flow analyzer SP6800 ZE from Sony allows the combination of both. Flow cytometry data of different tumor samples will be presented, as well as the comparison of the results and resolution of both flow analyzer systems. Auto-fluorescence of cells, which causes fluorescent signal contamination of other fluorescent markers in the conventional flow cytometer, can be subtracted using spectral technology to see the true fluorescent population, thereby allowing dim signal detection in rare populations. A comprehensive overview will be given regarding the pros and cons of the spectral analyzer system and difficulties switching from the one system to the other. Citation Format: Philipp Metzger, Muriel Malaisé, Cynthia Obodozie, Holger Weber. Benefit of using a spectral flow analyzer for the analysis of immune cell populations in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 629.

Checkpoint inhibitor treatment has become a common therapy of various cancer types. Still, there is a need for well-characterized preclinical mouse models, as clinical data indicates that patients only partially respond to immune modulatory regiments. When compared to the classical subcutaneous or subQperior࣪ (implantation into the mammary fat pad) syngeneic mouse models, orthotopic models are considered more predictive, since implantation of tumor cells into the organ of origin allows organotypical interaction between tumor cells and the surrounding stroma, including immune cells. Up until now, our standard procedure for an orthotopic Hepa1-6 liver model is the injection of Hepa1-6 cells into the spleen with the subsequent migration of the injected Hepa1 6 cells into the liver for five minutes via Vena lienalis. A major drawback of this method for a syngeneic model in immune-competent mice is the following resection of the spleen, a prominent secondary lymphoid organ. Therefore, we tested two other implantation methods: the direct injection of Hepa1-6 cells into a liver lobe using Matrigel࣪ and the injection of Hepa1-6 cells into the portal vein. In contrast to the standard procedure, both new test methods have in common an excessive bleeding potential which must be prevented. As the Hepa1-6 cells are transduced with luciferase, their growth can be monitored in vivo by bioluminescence imaging and the growth characteristics will be compared. In addition to the growth, the composition of the immune populations will be analyzed staining with the all-in-one flow cytometry panel which permits the differentiation of the main immune cell populations in the tumor, such as T cells (CD4+, CD8+, Treg), B and NK cells, macrophages (M1/M2), MDSCs (granulocytic and monocytic), and dendritic cells. The data will help to select the most suitable method for testing new drug candidates in an orthotopic liver tumor environment. Citation Format: Cynthia Obodozie, Sandra Moor, Gojko Bijelic, Muriel Malaisé, Philipp Metzger, Holger Weber. Comparison and consequences of different implantation techniques on the orthotopic growth of syngeneic Hepa1-6 liver cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1621.