3D Bioprinted Mini-Brains

A Glioblastoma Model to Study Cellular Interactions and Therapeutics

  • Fig.1: Photographic image of the mini-brain bioprinting process. Fig.1: Photographic image of the mini-brain bioprinting process.
  • Fig.1: Photographic image of the mini-brain bioprinting process.
  • Fig.2: (Top) Schematic of the bioprinted mini-brain displaying tumor area surrounded by healthy brain. (Bottom) Photographic images of the complete mini-brains and cross-section of the frontal plane (scale bar = 5 mm).
  • Fig.2: (Top) Schematic of the bioprinted mini-brain displaying tumor area surrounded by healthy brain. (Bottom) Photographic images of the complete mini-brains and cross-section of the frontal plane (scale bar = 5 mm).
  • Fig.4: (A) Concentration-dependent metabolic activity and highlighted IC50 of GL261 cancer cells in 2D monolayer and GL261 cancer cells and RAW264.7 macrophage 3D monocultures 4 days post-bioprinting after treatment with BCNU in different concentrations for 48 h. (B) Metabolic activity of GL261 cancer cells after co-culture with RAW264.7 macrophages for 4 days and treatment with vehicle or BCNU for 48 h after separation of the two cell types on day 4. (C) Metabolic activity of GL261 cancer cells after co-culture with RAW264.7 macrophages for 4 days and treatment with vehicle, AS1517499 or BLZ945 on day 1 and 3 post-bioprinting and separation of the two cell types on day 4 (the same vehicle group was used in experimental setups (B) and (C)).

Glioblastoma-associated macrophages (GAMs) play a crucial role in the progression, invasion and chemoresistance of glioblastoma multiforme, a lethal form of brain tumor. However, so far, the cellular interactions between GAMs and tumor cells are not yet fully understood and current models are limited to pinpoint this exact interaction in a biologically relevant environment. Here we developed a novel 3D in vitro model using 3D bioprinting as an innovative way to study biological interactions between cells as well as response to cancer therapy similar to the clinical situation.

In glioblastoma multiforme (GBM), macrophages are one of the most important and abundant cell types in the tumor microenvironment (TME) [1]. These cells are actively recruited by tumor cells and polarized
to glioblastoma-associated macrophages (GAMs), which themselves support tumor progression, invasiveness and resistance to cancer therapy [2]. For the development of novel therapeutics, it is crucial to fully understand the interaction between GAMs and tumor cells as well as to test the performance of such therapeutics in a biologically relevant platform. In vivo models are arguably the closest to the realistic situation but are often too complex to pinpoint specific interactions between cells. Therefore 3D in vitro models offer a promising platform to study cellular interactions in a well-controlled and biologically relevant environment [3, 4]. In particular, 3D bioprinting has recently proven its capacity to fabricate biomimetic tissues, whose architecture, composition and functionality is close to natural tissues [5-7]. In this study we demonstrate the use of 3D bioprinting for the fabrication of a miniaturized brain (“mini-brain”, WxLxH: 4x6x5 mm), including a well-defined tumor area surrounded by macrophages, representing the healthy brain (fig. 2).

Biologically Relevant Interactions

To study their biologically relevant interactions, mouse glioblastoma cells (GL261) and macrophages (RAW264.7) were encapsulated into a bioink composed of 3 w/v % gelatin methacryloyl (GelMA) and 4 w/v % gelatin and bioprinted using a custom-modified commercially available 3D printing platform.

First, we proved that the bioprinted cells were alive and active in the mini-brains for a total duration of 10 days based on Live/ Dead staining and a metabolic activity assay. Second, it was shown that cells were able to attach to the given matrix facilitating migration of these cell throughout the construct. Third, we investigated the migration behavior using a custom-designed migration assay based on cells evading the mini-brains towards a monolayer of the opposite cell type. On the one hand, we found that macrophages migrated in higher numbers towards cancer cells, indicating active recruitment of these cells towards the tumor sites. On the other hand, co-culture of cancer cells with macrophages increased their migratory behavior demonstrating the increased invasiveness of cancer cells based on the close proximity to macrophages. Forth, to investigate the exact interactions based on direct cell-to-cell contact (juxtacrine signaling) we bioprinted mini-brains, as previously described and cultured them under conventional culture conditions. With the help of the well-defined location of the tumor in this bioprinted model, we resected the tumor part and investigated the gene expression profiles of cancer cells and macrophages. Interestingly, we found that gene markers upregulated in our mini-brain model were in line of the overexpressed genes in glioblastoma patients [1, 2] (fig.3). In particular, macrophage markers related to angiogenesis, ECM-remodeling as well as GAM-specific markers displayed an up to 1000-fold higher upregulation in the mini-brains compared to conventional 2D cultures. Furthermore, cancer cells displayed increased expression of ECM-remodeling, macrophage recruitment and tumor invasion markers in the mini-brains. In addition, it could be shown that these highly expressed markers further have a significant effect on patient survival based on a gene-dependent survival analysis using publicly available data of 577 GBM patients, supporting the clinical relevance of our bioprinted model.

Resistance to Chemo- and Immunotherapy

To further demonstrate the capability of the novel mini-brains to be used as a platform for drug screening, we investigated the performance of the commonly used chemotherapy, temozolomide (BCNU), and known immunomodulatory drugs, AS1517499 and BLZ945, which have been previously reported to in particular inhibit macrophage polarization and tumor-supporting function [8, 9]. We found that the half maximal inhibitory concentration (IC50) of BCNU in cancer cells in a 3D monoculture was significantly higher (581 µM) compared to the 2D monolayer culture (139 µM) (Fig.4A), indicating that cancer cells are resistant in the 3D culture compared to conventionally used culture methods. In addition, by resecting the tumor area from the co-cultured mini-brain as aforementioned, we were able to investigate the response to the drug for each cell type individually. We found that macrophages significantly increased the growth rate of cancer cells in the mini-brains, which could drastically be inhibited by BCNU (Fig.4B). The use of the immunomodulatory drug BLZ945 in the mini-brains displayed significantly reduced growth of cancer cells (Fig.4C) based on macrophages inhibition combined with a reduced expression of GAM expression markers, similar to results reported in literature by Joyce et al. [8], who investigated this drug in vivo. Furthermore, AS1517499 treatment also displayed a reduced expression of GAM phenotypic markers in the macrophages of the mini-brain, similar to the results reported in the same study, confirming the potential of the mini-brains to be used as screening platform for chemo- as well as immunomodulatory therapeutics.

Conclusion

In summary, this study reveals a novel 3D bioprinted mini-brain model which is biologically relevant as well as has capability to be used as a platform to investigate the effects of drugs on different cell types. This proves the suitability of bioprinted tumor models to replicate the clinical situation and complex interactions within the TME.

Acknowledgments

The authors acknowledge funding from the Dutch Technology Foundation and the Dutch Cancer Society (STW/KWF; project no. 15204).

Authors:

Marcel A. Heinrich1 and Jai Prakash1

Affiliation
1Department of Biomaterials Science and Technology, Targeted Therapeutics Section
Technical Medical Centre, University of Twente, Enschede, The Netherlands.

 

Contact
Prof. Jai Prakash
Department of Biomaterials Science and Technology
University of Twente
Enschede, The Netherlands
j.prakash@utwente.nl

 

More on Tissue engineering!

 

References

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7. W. Jia, P.S. Gungor-Ozkerim, Y.S. Zhang, K. Yue, K. Zhu, W. Liu, Q. Pi, B. Byambaa, M.R. Dokmeci, S.R. Shin, A. Khademhosseini, Direct 3D bioprinting of perfusable vascular constructs using a blend bioink. Biomaterials, 2016. doi:10.1016/j.biomaterials.2016.07.038.

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