Analysis of the white blood cell differential as part of a flow cytometry-based approach is a common routine diagnostic tool used in clinics and research. For human blood, the methodological approach, suitable markers, and gating strategies are well-established. However, there is a lack of information regarding the mouse blood count. In this article, we deliver a fast and easy protocol for reprocessing mouse blood for the purpose of flow cytometric analysis, as well as suitable markers and gating strategies. We also present two possible applications: for the analysis of the whole blood count, with blood from a cardiac puncture, and for the analysis of a certain leukocyte subset at multiple time points in the framework of a mouse experiment, using blood from the facial vein. Additionally, we provide orientation values by applying the method to 3-month-old and 24-month-old male and female C57BL/6J mice. Our analyses demonstrate differences in the leukocyte fractions depending on age and sex. We discuss the influencing factors and limitations that can affect the results and that, therefore, need to be considered when applying this method. The present study fills the gap in the knowledge related to the rare information on flow cytometric analysis of mouse blood and, thus, lays the foundation for further investigations in this area.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal forms of cancer, characterized by a highly desmoplastic tumor microenvironment. One main risk factor is chronic pancreatitis (CP). Progression of CP to PDAC is greatly influenced by persistent inflammation promoting genomic instability, acinar–ductal metaplasia, and pancreatic intraepithelial neoplasia (PanIN) formation. Components of the extracellular matrix, including immune cells, can modulate this progression phase. This includes cells of the innate immune system, such as natural killer (NK) cells, macrophages, dendritic cells, mast cells, neutrophils, and myeloid-derived suppressor cells (MDSCs), either promoting or inhibiting tumor growth. On one hand, innate immune cells can trigger inflammatory responses that support tumor progression by releasing cytokines and growth factors, fostering tumor cell proliferation, invasion, and metastasis. On the other hand, they can also activate immune surveillance mechanisms, which can limit tumor development. For example, NK cells are cytotoxic innate lymphoid cells that are able to kill tumor cells, and active dendritic cells are crucial for a functioning anti-tumor immune response. In contrast, mast cells and MDSCs rather support a pro-tumorigenic tumor microenvironment that is additionally sustained by platelets. Once thought to play a role in hemostasis only, platelets are now recognized as key players in inflammation and cancer progression. By releasing cytokines, growth factors, and pro-angiogenic mediators, platelets help shape an immunosuppressive microenvironment that promotes fibrotic remodeling, tumor initiation, progression, metastasis, and immune evasion. Neutrophils and macrophages exist in different functional subtypes that can both act pro- and anti-tumorigenic. Understanding the complex interactions between innate immune cells, platelets, and early precursor lesions, as well as PDAC cells, is crucial for developing new therapeutic approaches that can harness the immune and potentially also the coagulation system to target and eliminate tumors, offering hope for improved patient outcomes.

Pancreatic ductal adenocarcinoma (PDAC) ranks among the most aggressive malignancies and exhibits a higher prevalence and poorer prognosis in male patients. While both the importance of natural killer (NK) cells in anti-tumor immunity and sex differences in NK cell function are well established, the interplay between biological sex and NK cell-mediated responses in PDAC has not been systematically addressed. Here, we provide novel insights revealing that both the intrinsic cytotoxic capacity of NK cells and the tumor microenvironment differ between sexes and jointly modulate anti-tumor immunity in PDAC. Using in vitro assays with primary NK cells from male and female mice, we found that female-derived NK cells exhibit consistently superior cytotoxic activity and immune activation. Importantly, these findings were reflected and even amplified in vivo. In a syngeneic allograft PDAC model, female mice displayed enhanced NK cell function, and female tumors exhibited distinct structural characteristics supporting stronger NK cell activity. Notably, this occurred despite the use of the same cell line in both sexes, indicating that host sex significantly shapes the composition and architecture of the tumor microenvironment. Our results provide new evidence that sex differences in NK cell-mediated immunity, within the broader immune and stromal landscape, may differentially impact tumor control in male and female PDAC hosts. This study therefore underscores the importance of considering sex as a biological variable in the preclinical evaluation of pancreatic cancer therapies.