CD44 has been described in many malignancies as a marker of cancer stem cells (CSCs). Several techniques can be used to detect these cells. Here we detail CD44 detection by flow cytometry, a precise technique allowing to determine the percentage of positive cells and the mean fluorescent intensity reflecting the CD44 expression by cells in the samples. The protocol explained here can be used to detect CD44 from cell suspensions prepared from tissues or in vitro cell cultures.
© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Galectins are a group of carbohydrate-binding proteins with a presumed immunomodulatory role and an elusive function on antigen-presenting cells. Here we analyzed the expression of galectin-1 and found upregulation of galectin-1 in the extracellular matrix across multiple tumors. Performing an in-depth and dynamic proteomic and phosphoproteomic analysis of human macrophages stimulated with galectin-1, we show that galectin-1 induces a tumor-associated macrophage phenotype with increased expression of key immune checkpoint protein programmed cell death 1 ligand 1 (PD-L1/CD274) and immunomodulator indoleamine 2,3-dioxygenase-1 (IDO1). Galectin-1 induced IDO1 and its active metabolite kynurenine in a dose-dependent manner through JAK/STAT signaling. In a 3D organotypic tissue model system equipped with genetically engineered tumorigenic epithelial cells, we analyzed the cellular source of galectin-1 in the extracellular matrix and found that galectin-1 is derived from epithelial and stromal cells. Our results highlight the potential of targeting galectin-1 in immunotherapeutic treatment of human cancers.
© 2023 The Authors.

Aim: To assess the effect of physical training on the selected parameters of the immune system regarding CD3, CD4, CD8, CD11, CD161, CD45A cell counts in rats treated with N-methyl-N-nitrosourea (MNU). Material and Methods: Thirty-eight female Sprague-Dawley rats were injected intraperitoneally with MNU and were divided into three groups, i.e., sedentary control (SC), the group of moderate-intensity training (MIT) and the group of high-intensity training (HIT). Physical training was supervised immediately after MNU administration and was conducted 5 days per week for 12 weeks on a three-position treadmill. Results: A significant difference was found between SC and training groups in terms of the number of induced tumors per rat (1.57 vs. 0.4, p = 0.05) and in the following lymphocyte subpopulations: CD4+/CD8+ (p = 0.01), CD3−/CD11b+ (p = 0.02), CD3−/CD161+ (p = 0.002), CD3−/CD161− (p = 0.002), CD3+/CD45RA+ (p = 0.003) and CD3−/CD45RA+ (p = 0.005). In terms of the intensity of physical training, the highest efficacy was found for MIT and the following lymphocyte subpopulations: CD3−/CD11b+ (SC vs. MIT, p < 0.001), CD3−/CD161+ (SC vs. MIT, p = 0.002), CD3−/CD161− (SC vs. MIT, p = 0.002), CD3+/CD45RA+ (SC vs. MIT, p = 0.02) and CD3−/CD45RA+ (SC vs. MIT, p < 0.001, MIT vs. HIT, p = 0.02). Furthermore, negative correlations were found between the number of apoptotic cells and CD3−/CD11b (r = −0.76, p = 0.01) in SC and between the number of induced tumors and CD3+/CD8+ (r = −0.61, p = 0.02) and between their volume and CD+/CD8+ (r = −0.56, p = 0.03) in the group of rats undergoing training. Conclusions: Physical training, particularly MIT, affected immune cell function and an altered immune response can be considered a mechanism underlying the effect of exercise on breast cancer development.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with poor patient outcomes, highlighting the unmet clinical need for targeted therapies and better model systems. Here, we developed and comprehensively characterized a diverse biobank of normal and breast cancer patient-derived organoids (PDO) with a focus on TNBCs. PDOs recapitulated patient tumor intrinsic properties and a subset of PDOs can be propagated for long-term culture (LT-TNBC). Single cell profiling of PDOs identified cell types and gene candidates affiliated with different aspects of cancer progression. The LT-TNBC organoids exhibit signatures of aggressive MYC-driven, basal-like breast cancers and are largely comprised of luminal progenitor (LP)-like cells. The TNBC LP-like cells are distinct from normal LPs and exhibit hyperactivation of NOTCH and MYC signaling. Overall, this study validates TNBC PDOs as robust models for understanding breast cancer biology and progression, paving the way for personalized medicine and tailored treatment options.
A comprehensive analysis of patient-derived organoids of TNBC provides insights into cellular heterogeneity and mechanisms of tumorigenesis at the single-cell level.
©2022 American Association for Cancer Research.

P-selectin is a key surface adhesion molecule for the interaction of platelets with leukocytes. We have shown previously that the N-terminal domain of Staphylococcus aureus extracellular fibrinogen-binding protein (Efb) binds to P-selectin and interferes with platelet-leukocyte aggregate formation. Here, we aimed to identify the minimal Efb motif required for binding platelets and to characterize its ability to interfering with the formation of platelet-leukocyte aggregates.
Using a library of synthetic peptides, we mapped the platelet-binding site to a continuous 20 amino acid stretch. The peptide Efb68-87 was able to bind to resting and, to a greater extent, thrombin-stimulated platelets in the absence of fibrinogen. Dot blots, pull-down assays and P-selectin glycoprotein ligand-1 (PSGL-1) competitive binding experiments identified P-selectin as the cellular docking site mediating Efb68-87 platelet binding. Accordingly, Efb68-87 did not bind to other blood cells and captured platelets from human whole blood under low shear stress conditions. Efb68-87 did not affect platelet activation as tested by aggregometry, flow cytometry and immunoblotting, but inhibited the formation of platelet-leukocyte aggregates (PLAs). Efb68-87 also interfered with the platelet-dependent stimulation of neutrophil extracellular traps (NETs) formation in vitro.
We have identified Efb68-87 as a novel selective platelet-binding peptide. Efb68-87 binds directly to P-selectin and inhibits interactions of platelets with leukocytes that lead to PLA and NET formation. As PLAs and NETs play a key role in thromboinflammation, Efb68-87 is an exciting candidate for the development of novel selective inhibitors of the proinflammatory activity of platelets.
© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.