(D-128) Studying immunomechanics in a novel 3-D spheroid model of tumor-associated macrophages

Introduction:: Glioblastoma (GBM) is one of the most aggressive forms of cancer, typically killing patients within two years of diagnosis [1]. Despite recent treatment advances such as immunotherapies, which have been used to achieve cures for other cancer types, GBM remains incurable. This is largely due to the extremely immunosuppressive GBM tumor microenvironment (TME) [2]. This is mediated in large part by tumor-resident macrophages, which adopt an anti-inflammatory, pro-tumor phenotype [3]. Macrophages’ role as immune regulators, as well as their sheer quantity in brain tumors, make them attractive therapeutic targets. Most are bone-marrow derived and are recruited to the tumor via GBM-produced chemokines [3]. They secrete factors that contribute to tumor progression and treatment resistance as part of a positive feedback loop, further recruiting more macrophages. Importantly, these cells are key players in the suppression of cytotoxic T cell activity and the promotion of regulatory T cell expansion, preventing an effective anti-tumor immune response even with immunotherapies. The GBM TME also features mechanical abnormalities. As a tumor grows, it expands into the surrounding brain, generating compressive solid stress both within the tumor and in the surrounding tissue. Macrophages can contribute to up to half of the tumor mass and are known to respond to mechanical cues, allowing them to both contribute to and respond to solid stress. A systematic study of the interaction between macrophages and compressive stress in GBM would provide valuable information on the “immunomechanics” of GBM and how it can be targeted therapeutically.


Materials and Methods:: To isolate the contribution of macrophages on solid stress, we have optimized two protocols to create spheroids from RAW264.7 murine macrophages. The first is the hanging drop method, where droplets of cell suspension supplemented with methylcellulose are used to create uniform spheroids with tunable sizes, which can then be later embedded in agarose. The second method is to directly incorporate a single-cell suspension into a 1% agarose gel, where spheroids form over time as the cells proliferate in place. The resulting agarose-embedded spheroids were stained with calcein AM and propidium iodide for live and dead cells, respectively, to observe spheroid viability and morphology.

In upcoming experiments, we will embed fluorescent microbeads in the gel to trace the strain imposed on the agarose gel by the growing spheroids, as described by [4]. As the spheroid expands it compresses the surrounding gel, resulting in a local increase in microbead density (Fig. 1A). We will collect 3-D image stacks using a confocal microscope to quantify this effect and calculate the local strain imposed on the gel. We can then calculate the solid stress generated by the spheroid given the known mechanical characteristics of agarose gel. The incorporation of a murine glioblastoma cell line (such as GL261) in future experiments will allow us to compare the generation of solid stress when the two cell types interact.


Results, Conclusions, and Discussions:: We first verified the viability of the novel macrophage spheroids over time via calcein AM and propidium iodide staining. Spheroids formed by the hanging drop method, as well as those formed from single cells suspended in agarose, were viable for at least 7 or more days in culture. Preliminary experiments show that macrophage spheroids grow in a similar way to tumor spheroids. When embedded in 1% agarose, macrophage spheroids adopt either spheroidal or oblate shapes over time (Fig. 1B). This indicates that macrophage spheroids create a stress field in the agarose, sometimes resulting in microcracks that disrupt their spheroidal shape, as previously characterized with cancer cell lines [4]. In upcoming experiments, we will incorporate fluorescent microbeads to precisely quantify this stress field. We anticipate that the macrophage spheroids will generate local compressive stress in the same way that tumor spheroids have been observed to do. Future work will include macrophage and glioblastoma co-cultures to observe whether there is a synergistic effect on solid stress. This novel macrophage spheroid model will allow us to understand to what extent macrophages can directly and indirectly contribute to solid stress in the tumor.

These initial optimization studies have laid the groundwork for future experiments to assess the effect of compression on normal glial and immune cell populations in the brain. This work will elucidate key pathways involved in cancer “immunomechanics” and inform strategies to induce more effective anti-tumor immune responses and increase GBM treatment efficacy.


Acknowledgements (Optional): : National Institutes of Health, Berthiaume Institute for Precision Health, Warren Center for Drug Discovery, Indiana Clinical and Translational Sciences Institute


References (Optional): : [1] “Glioblastoma Multiforme – Symptoms, Diagnosis and Treatment Options.” https://www.aans.org/ (accessed Sep. 14, 2021).

[2] B. T. Himes, P. A. Geiger, K. Ayasoufi, A. G. Bhargav, D. A. Brown, and I. F. Parney, “Immunosuppression in Glioblastoma: Current Understanding and Therapeutic Implications,” Front Oncol, vol. 11, p. 770561, Oct. 2021, doi: 10.3389/fonc.2021.770561.

[3] A. Buonfiglioli and D. Hambardzumyan, “Macrophages and microglia: the cerberus of glioblastoma,” Acta Neuropathologica Communications, vol. 9, no. 1, p. 54, Mar. 2021, doi: 10.1186/s40478-021-01156-z.

[4] G. Cheng, J. Tse, R. K. Jain, and L. L. Munn, “Micro-environmental mechanical stress controls tumor spheroid size and morphology by suppressing proliferation and inducing apoptosis in cancer cells,” PLoS One, vol. 4, no. 2, p. e4632, 2009, doi: 10.1371/journal.pone.0004632.