A Heterotypic Tridimensional Model to Study the Interaction of Macrophages and Glioblastoma In Vitro

Abstract

Background: Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor, and macrophages account for 30–40% of its composition. Most of these macrophages derive from bone marrow monocytes playing a crucial role in tumor progression. Unraveling the mechanisms of macrophages-GBM crosstalk in an appropriate model will contribute to the development of specific and more successful therapies. We investigated the interaction of U87MG human GBM cells with primary human CD14+ monocytes or the THP-1 cell line with the aim of establishing a physiologically relevant heterotypic culture model. Methods: primary monocytes and THP-1 cells were cultured in the presence of U87MG conditioned media or co-cultured together with previously formed GBM spheroids. Monocyte differentiation was determined by flow cytometry. Results: primary monocytes differentiate to M2 macrophages when incubated with U87MG conditioned media in 2-dimensional culture, as determined by the increased percentage of CD14+CD206+ and CD64+CD206+ populations in CD11b+ cells. Moreover, the mitochondrial protein p32/gC1qR is expressed in monocytes exposed to U87MG conditioned media. When primary CD14+ monocytes or THP-1 cells are added to previously formed GBM spheroids, both invade and establish within them. However, only primary monocytes differentiate and acquire a clear M2 phenotype characterized by the upregulation of CD206, CD163, and MERTK surface markers on the CD11b+CD14+ population and induce alterations in the sphericity of the cell cultures. Conclusion: our results present a new physiologically relevant model to study GBM/macrophage interactions in a human setting and suggest that both soluble GBM factors, as well as cell-contact dependent signals, are strong inducers of anti-inflammatory macrophages within the tumor niche.

3D Coculture between Cancer Cells and Macrophages: From Conception to Experimentation

“…Some cell lines spontaneously aggregate when adherence to the substrate is restricted by using specific nonadherent culture plates. This easy, inexpensive technique is particularly suitable for the generation of spheroids and has already been used with different cancer cell lines, including breast (4T1), , intestinal (HCT116), bladder (MGH-U3/T24), ovarian (OVCAR3), pancreatic (HPAC), and brain (U87MG) cell lines. Different types of culture plates can be used, such as NUNCLON SPHERA 96-well U bottom plates, ultralow attachment 96-well plates, , ultralow attachment 6-well plates, or NanoCulture 96-well plates with a microsquare pattern .…”

“…All of these plates have a specific coating to prevent cell adhesion and allow the formation of homogeneous spheroids. Hanging drop plates can also be used, , in which case 20 μL drops are deposited on the lid. Another technique is to use conventional plates (96 U-well and 6-well), subsequently treated with an agent, such as poly-2-hydroxyethyl methacrylate (poly-Hema), to prevent cell adhesion and allow good spheroid formation .…”

“…Two types of lines are mainly used: (i) human monocytes isolated from peripheral blood mononuclear cells (PBMCs) from healthy donor buffy coats ,,− ,,,,, or (ii) murine monocytes extracted from mouse femurs and tibias and subsequently transformed into bone marrow-derived macrophages (BMDMs). ,,,,,,− (i) Primary human monocytes isolated from PBMCs are generally used to study the genetic and phenotypic response of monocytes to different stimuli and are a source of potential biomarkers for numerous pathologies, including inflammatory diseases and cancers. The dynamics of their subpopulations underlie the appearance and progression of many pathologies, such as cancer .…”

Cell-Based Therapy for the Treatment of Glioblastoma: An Update from Preclinical to Clinical Studies

“…Utilizing a similar method, another study was able to deliver monocytes that were loaded with conjugated polymer nanoparticles into GB cells and that allowed for the expansion and improvement of photodynamic therapy for GB [ 43 ]. Additionally, another experiment by Gattas and colleagues, [ 44 ] used primary monocytes that were cultured in the presence of U87MG-conditioned media, and co-cultured with GB spheroids. They found that monocytes differentiated and acquired clear M2 phenotypes, while also inducing alterations in the cell cultures.…”

“…They found that monocytes differentiated and acquired clear M2 phenotypes, while also inducing alterations in the cell cultures. The fact that these monocytes upregulated CD206, CD163 and MERTK surface markers on the CD11b and CD14 populations made them strong inducers of anti-inflammatory macrophages [ 44 ]. These three studies can be referenced together to show the range of ways that immune cells can be used to provide care to those with life-devastating tumors.…”