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Abstract
Abstract A 3D bioprinted neurovascular unit (NVU) model was developed to study glioblastoma (GBM) tumor growth in a brain-like microenvironment. The NVU model included human primary astrocytes, pericytes and brain microvascular endothelial cells, and patient-derived glioblastoma cells (JHH-520) were used for this study. We used fluorescence reporters with confocal high content imaging to quantitate real-time microvascular network formation and tumor growth. Extensive validation of the NVU-GBM model included immunostaining for brain relevant cellular markers and extracellular matrix components; single cell RNA sequencing to establish physiologically relevant transcriptomics changes; and secretion of NVU and GBM-relevant cytokines. The scRNAseq revealed changes in gene expression and cytokines secretion associated with wound healing/angiogenesis, including the appearance of an endothelial mesenchymal transition (EndMT) cell population. The NVU-GBM model was used to test 18 chemotherapeutics and anti-cancer drugs to assess the pharmacological relevance of the model and robustness for high throughput screening. This article is protected by copyright. All rights reserved

Abstract
Bioprinting technologies are powerful new bioengineering tools that can spatially reproduce multiple microenvironmental cues in a highly controlled, tunable, and precise manner. In this study, microvalve bioprinting technology was successfully used to print in close proximity endothelial and tumor cells at higher concentrations than previously thought possible, while preserving their viability. We propose that the resulting multicellular models, bioprinted in a controlled extracellular matrix microenvironment, are well-suited to study endothelial and cancer cell crosstalk within a cancer niche. As proof of concept, microvalve bioprinting was applied to the bioengineering of a simplified glioblastoma model in which biological processes involved in tumor expansion, such as tumor cell invasion patterns, cell proliferation, and senescence could be easily visualized and quantified. In this model, U251 glioblastoma cells and primary human umbilical vein endothelial cells (HUVECs) exhibited good printability and high viability after printing. U251 cells formed physiologically relevant clusters and invasion margins, while HUVECs generated vascular-like networks when primary fibroblasts were added to the model. An oxidative stress mimicking the one encountered within a tumor microenvironment during radiotherapy or genotoxic chemotherapy was shown to both diminish endothelial cells proliferation and to increase their senescence. Results also suggested that stressed glioblastoma cells may alter normal endothelial cell proliferation but not impact their senescence. This data demonstrates the potential of microvalve bioprinting to fabricate in vitro models that can help decipher endothelial and tumor cell crosstalk, within controlled and modulable microenvironments, and can then be used to address critical questions in the context of cancer recurrence.