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.

Background and Objectives: Epidemiological investigations have shown positive correlations between increased diesel exhaust particles (DEP) in ambient air and adverse health outcomes. DEP are the major constituent of particulate atmospheric pollution and have been shown to induce proinflammatory responses both in the lung and systemically. Here, we report the effects of DEP exposure on the properties of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs), including stemness, regeneration, and immunomodulation. Methods and Results: Non-apoptotic concentrations of DEP (10 μg/ml) inhibited the migration and osteogenic differentiation capacity of WJ-MSCs. Gene expression profiling showed that DEP increased intracellular reactive oxygen species (ROS) and expression of pro-inflammatory and metabolic-process-related genes including cFos. Furthermore, WJ-MSCs cultured with DEP showed impaired suppression of T cell proliferation that was reversed by inhibition of ROS or knockdown of cFos. ERK inhibition assay revealed that DEP-induced ROS regulated cFos through activation of ERK but not NF-κB signaling. Overall, low concentrations of DEP (10 μg/ml) significantly suppressed the stemness and immunomodulatory properties of WJ-MSCs through ROS/ERK/cFos signaling pathways. Furthermore, WJ-MSCs cultured with DEP impaired the therapeutic effect of WJ-MSCs in experimental colitis mice, but was partly reversed by inhibition of ROS. Conclusions: Taken together, these results indicate that exposure to DEP enhances the expression of pro-inflammatory cytokines and immune responses through a mechanism involving the ROS/ERK/cFos pathway in WJ-MSCs, and that DEP-induced ROS damage impairs the therapeutic effect of WJ-MSCs in colitis. Our results suggest that modulation of ROS/ERK/cFos signaling pathways in WJ-MSCs might be a novel therapeutic strategy for DEP-induced diseases.

The glioblastoma is a complex ecosystem with multiple cell types and an extracellular matrix (ECM) unique to the brain. Dynamic interactions between tumor cells and other non-neoplastic cell types drive the progression of cancer and continuously remodel the local microenvironment. Some major non-neoplastic players in the glioblastoma microenvironment include blood vessels that support tumor growth, several resident central nervous system (CNS) cells such as astrocytes, neurons, and microglia, as well as tumor-associated macrophages, the most substantial non-neoplastic component of glioblastoma. While animal models retain the genomic signature and transcriptome of the original patient tumor tissue, the use of immunocompromised animals inherently limits investigation of the role of immune components within the glioblastoma tissue. Here, we developed a rapid 3D-bioprinting method to construct a clinically relevant multicellular in vitro model to recapitulate the complexity of the glioblastoma microenvironment. The 3D models made of brain-specific materials were constructed with a central core of glioblastoma stem cells, with or without macrophages, surrounded by the resident CNS cells, which served to mimic the brain parenchyma surrounding the tumor tissue. Gene expression and transcriptome analysis demonstrated that both the glioblastoma stem cells and the macrophage precursors responded to the 3D-bioprinted glioblastoma microenvironment and better resembled their counterparts in patient tumor tissue compared to sphere or suspension culture. Furthermore, the four-cell model with macrophages closely resembled patient transcriptional profiles predictive of patient prognosis and drug sensitivity, and better recapitulated the glioblastoma invasiveness and stemness compared to three-cell models without macrophages or sphere cultures. Finally, the 3D-bioprinted models also enabled whole genome CRISPR screening to identify unique functional dependencies not identified in sphere culture controls. The 3D-bioprinting method is highly scalable and reproducible. The multicellular glioblastoma model combines fine spatial control of brain-specific materials and multiple cell types to create a sophisticated human species-matched model that contains both neoplastic and non-neoplastic regions. Citation Format: Min Tang, Qi Xie, Ryan C. Gimple, Briana C. Prager, Zhixin Qiu, Jacob Schimelman, Pengrui Wang, Derrick Lee, Aaron Yu, Tyler E. Miller, Reilly L. Kidwell, Xueyi Wan, Jing Tang, Trevor Tam, Jing Tian, Bingjie Sun, Shaochen Chen, Jeremy Rich. 3D-bioprinting of biomimetic multicellular glioblastoma tissues enable modeling of tumor-immune interactions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 320.

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