Marcel Heinrich1 Ruchi Bansal1 Yu Shrike Zhang2 3 4 Raymond Schiffelers5 Jai Prakash1

1, Department of Biomaterials Science & Technology, University of Twente, Enschede, , Netherlands
2, Division of Engineering in Medicine, Harvard Medical School, Brigham & Womens Hospital, Cambridge , Massachusetts, United States
3, Division of Health Sciences & Technology, Harvard-Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
4, Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, United States
5, Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht, , Netherlands

Glioblastoma-associated macrophages (GAMs) play a crucial role in the progression and invasiveness of glioblastoma multiforme (GBM), however, the exact crosstalk between GAMs and glioblastoma cells is not fully understood. Furthermore, there is lack of relevant in vitro models to mimic their specific interaction in a dynamic and relevant environment. 3D bioprinting offers a promising approach to culture cells with well-defined structure and composition. In this study, we aim to develop novel bioprinted mini-brains that display in vivo-like behavior of both glioblastoma cells and GAMs.
We bioprinted mini-brains (WxLxH; 4x6x3mm) comprised of either glioblastoma cells or macrophages or together to study their interaction in a realistic way. Both macrophages and glioblastoma cells remained viable in the mini-brains for at least 10 days. We observed an induction of glioblastoma-specific markers (e.g. Gfap up to 15-fold) compared to 2D cultures at mRNA level. Next, we studied whether tumor cells recruit macrophages to their site. To mimic that, we bioprinted mini-brains consisting of either macrophages or glioblastoma cells and cultured them in the same well. We found a 125-fold upregulation of Ccl2, a chemokine related to macrophages recruitment compared to 2D culture, indicating that tumor cells actively recruit macrophages in the 3D co-culture. Furthermore, we cultured mini-brains consisting of macrophages next to glioblastoma cells to confirm their migration. We found that macrophages significantly migrated towards the tumor site, indicating successful crosstalk between these cells. Next, we investigated the effect of direct cell-to-cell contact of tumor cells and macrophages in the 3D culture. We bioprinted mini-brains consisting of macrophages including a cavity containing glioblastoma cells mimicking the clinical situation. We investigated the gene expression of GAM-specific markers and observed a significant upregulation of markers for the GAM phenotype (Arg-1, Mmp2, Mmp9, Cd206), indicating that tumor cells polarized macrophages towards GAMs. In addition, by resecting the tumor area from the mini-brains and investigate the expression of glioblastoma-related markers, we found that markers for tumor progression (Gfap, Chil1) and tumor invasion (Mmp9, Vimentin) were significantly overexpressed in the co-culture, displaying how GAMs support glioblastoma progression and invasion. To examine the clinical relevance of this model, we performed transcriptomic analysis of 159 GBM patients using available database, which showed a significant upregulation of highly relevant markers such as Mmp2, Mmp9 or Chil1.
These data indicate that tumor cells induce recruitment of macrophages towards themselves and change their phenotype, as well as how GAMs support tumor progression and invasion. Altogether, our bioprinted mini-brains are a viable tool to study the interactions between different cell types and could potentially be used for drug screening purposes.