Abstract
Brain tumors are dynamic complex ecosystems with multiple cell types. To model the brain tumor microenvironment in a reproducible and scalable system, we developed a rapid three-dimensional (3D) bioprinting method to construct clinically relevant biomimetic tissue models. In recurrent glioblastoma, macrophages/microglia prominently contribute to the tumor mass. To parse the function of macrophages in 3D, we compared the growth of glioblastoma stem cells (GSCs) alone or with astrocytes and neural precursor cells in a hyaluronic acid-rich hydrogel, with or without macrophage. Bioprinted constructs integrating macrophage recapitulate patient-derived transcriptional profiles predictive of patient survival, maintenance of stemness, invasion, and drug resistance. Whole-genome CRISPR screening with bioprinted complex systems identified unique molecular dependencies in GSCs, relative to sphere culture. Multicellular bioprinted models serve as a scalable and physiologic platform to interrogate drug sensitivity, cellular crosstalk, invasion, context-specific functional dependencies, as well as immunologic interactions in a species-matched neural environment.
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Data availability
All raw sequencing data and selected processed data is available on GEO at the accession number GSE147147 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE147147). There are no restrictions on data availability, and all data will be made available upon request directed to the corresponding authors. All biological materials used in this manuscript will be made available upon request to the corresponding authors. Distribution of human patient-derived GSCs may be distributed following completion of a material transfer agreement (MTA) with the appropriate institutions if allowed.
Code availability
All computational algorithms utilized in the manuscript have been referenced in the corresponding figure legend and described in the methods section. Additional details can be made available upon request.
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Acknowledgements
This work was supported by grants provided by the National Institutes of Health: CA217065 (R.C.G); CA217066 (B.C.P.); DK099810 and DK114785 (B.F.C); CA197718, CA154130, CA169117, CA171652, CA238662, NS087913, NS089272, NS103434 (J.N.R); CA243296 (D.L.); R01EB021857, R21AR074763, R33HD090662 (S.C.), and the National Science Foundation: 1644967, 1937653 (S.C.). H.J. is a Biogen fellow of the Life Sciences Research Foundation. A.R.M. is supported by the California Institute for Regenerative Medicine (DISC2-09649) and by the National Institutes of Health (MH107367, N5105969). We thank the UCSD School of Medicine Microscopy Core, which is supported by a NINDS P30 grant (NS047101), for use of their confocal microscopes. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1650112 (J.S.). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. Portions of individual panels were prepared in part using images from Servier Medical Art by Servier (https://smart.servier.com/), which is licenced under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).
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M.T., Q.X., and R.C.G designed the research, performed in vitro and in vivo experiments, wrote the manuscript, and generated figures. M.T., Z. Zhong, T.T., J. Tian, J.S., P.W., X.W., and B.S. performed technical development of 3D bioprinting and constructed 3D models. J.S., P.W., and B.S. synthesized and characterized the bioprinting materials. R.C.G., R.L.K, Q.W., D.D. and L.Z. performed in vivo mouse experiments including establishment of orthotopic xenografts, monitoring, and drug treatments. R.C.G., B.C.P., Z.Q., and J. Tang performed data analysis for drug response prediction, RNA-seq analysis, CRISPR screening, and patient database analyses. Z. Zhu, P.M., H.J., B.F.C., and A.R.M generated primary human macrophages and human induced pluripotent stem cell derived macrophages. M.T., Q.X., R.C.G., Z. Zhong, T.T., J. Tian, A.Y., D.L., and M.H.L. performed cell culture experiments including genome editing, drug viability screening, and viral production. M.T., R.C.G., S.B., and X.W. performed immunofluorescence assays. T.E.M., B.F.C., Q.X., A.R.M., S.C. and J.N.R designed, supervised the research, and edited the manuscript.
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A.R.M. is a co-founder and has equity interest in TISMOO, a company dedicated to genetic analysis focusing on therapeutic applications customized for the autism spectrum disorder and other neurological disorders origin genetics. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. The remaining authors declare no potential conflicts of interest.
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Tang, M., Xie, Q., Gimple, R.C. et al. Three-dimensional bioprinted glioblastoma microenvironments model cellular dependencies and immune interactions. Cell Res 30, 833–853 (2020). https://doi.org/10.1038/s41422-020-0338-1
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DOI: https://doi.org/10.1038/s41422-020-0338-1
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