Introduction: : Macrophages can encompass up to 50% of tumor mass, and their presence correlates with poor patient prognosis [1]. Macrophages within the tumor microenvironment tend to be anti-inflammatory, which aids in creating an immunosuppressive microenvironment and promoting tumor progression. During a perfunctory course of infection, macrophages are often one of the first lines of defense against a pathogen and exhibit cell surface markers that facilitate antigen presentation and production of pro-inflammatory cytokines such as IFN- γ, TNF-⍺, IL-1, and IL-6 [2]. Once the immune response attenuates and the pathogen is cleared, macrophages shift their phenotype toward an M2 phenotype that exhibits cell surface markers that assist in the phagocytic function of macrophages and secretion of anti-inflammatory cytokines such as IL-10 and TGF-β [2]. The current thought is that the majority of macrophages that reside in a tumor have an M2 macrophage phenotype and help create an immunosuppressive environment [1]. Therefore, understanding the dynamic effects of fluid shear stress (FSS) on macrophage polarization states can provide valuable insight into how tumor-associated macrophages function as they enter the bloodstream during metastasis and opening up new therapeutic targets to prevent metastatic nodes from forming. In this work, we exposed macrophages to shear stress throughout the differentiation and polarization process and observed a range of responses in macrophage polarization dependent on their initial state.
Materials and
Methods: : Monocytes were isolated, using a pan monocyte isolation kit, from peripheral whole blood collected from the Gulf Coast Regional Blood Center. Isolated monocytes were plated in 10cm petri dishes at 500,000 cells/mL and exposed to macrophage-colony stimulating factor (M-CSF) at a concentration of 100nM. The cells were incubated with M-CSF for 6 days, where fresh media and M-CSF was added 72 hours after the initial addition of M-CSF. Once the monocytes have been exposed to M-CSF for 6 days, the cells should be adhered to the bottom of the petri dish and the cells should appear to be spindly and the M-CSF-media should be removed and replaced with M-CSF-free media. After 24hr incubation with M-CSF-free media, the M2 macrophages were exposed to 30 minutes of cyclical fluid shear stress in the Integra Viaflow at 3 different accelerations at the tip of the needle: 11.63, 25.04, and 62.30 dyne/cm^2. Our lab has created specific pipette tips that fuse the body of a pipette with the blunt needle of a syringe in order to generate greater shear forces at the tip of the syringe, even if cells are only exposed to those forces for a few seconds [3]. The sheared M2 macrophages were then allowed to incubate for 24, 48, and 72 hours. Following the incubation period, cells were prepped for phagocytic assays, flow cytometry, and the media frozen for ELISA assays to measure phagocytic activity, key cell surface marker expression, and cytokine secretion.
Results, Conclusions, and Discussions:: Results M2 macrophages showed increased phagocytic activity 24 hours following fluid shear stress (FSS) exposure compared to static conditions (Fig1A,B). In addition to increased phagocytic activity, M2 macrophages did not secrete TNF-⍺, but secreted high levels of TNF-⍺ after the addition of lipopolysaccharide (LPS), which is a known promoter of M1-characteristics(Fig1C). There was a general increase of secreted TNF-⍺ in the sheared groups with LPS, where sheared group 2 had the highest level of secreted TNF-⍺(Fig1C). M2 macrophages without the addition of LPS, was found to secrete IL-10, a known anti-inflammatory cytokine, where shear group 2 had secreted the most in the groups without LPS(Fig1D). The addition of LPS not only increased the secretion of TNF-⍺, but also dramatically increased the secretion of IL-10, where shear groups 1 and 2 had more IL-10 secreted (Fig1D). Sheared M2 macrophages had lower CD206 expression, where shear groups 1 and 2 had the lowest expression(Fig1E). The sheared M2 macrophages had increased expression of CD86 and CD80, M1-like markers, that corresponded to a decrease in CD206 expression(Fig1F,G). Discussion and Conclusion Shear stress can affect subpopulations of macrophages differently, which indicates that FSS could be affecting macrophages within circulating tumor clusters (CTCs) in different ways that may make one population more successful in forming metastatic tumors. Since the vast majority of macrophages within the tumor microenvironment are likely to be M2-like in nature, we wanted to first explore the effects of FSS on polarized M2 macrophages since these cells would be more likely to form clusters with tumor cells to form CTCs. Following FSS exposure, in both LPS groups and groups without LPS), there was a retention of M2-like characteristics, such as increased phagocytic activity, IL-10 secretion, and CD206 expression remained fairly high throughout. The sheared M2 macrophages also gained M1-like characteristics, such as CD80 and CD86 expression. Ongoing research is addressing the effects of macrophage polarization as it relates to cancer metastasis by co-culturing macrophages and cancer cells to understand the effects macrophages have on cancer cell survival and model the circulating tumor clusters that are found in the bloodstream of cancer patients.
Acknowledgements and/or References (Optional):: References 1. Poh, A.R. and Ernst, M. (2018) “Targeting macrophages in cancer: From bench to bedside,” Frontiers in Oncology, 8. https://doi.org/10.3389/fonc.2018.00049. 2. Yao, Y., Xu, X.-H. and Jin, L. (2019) “Macrophage polarization in physiological and pathological pregnancy,” Frontiers in Immunology, 10. https://doi.org/10.3389/fimmu.2019.00792. 3. Fabiano, A.R. et al. (2024) ‘Multiplex, high-throughput method to study cancer and immune cell mechanotransduction’, Communications Biology, 7(1). doi:10.1038/s42003-024-06327-x.
