Glioma is still difficult to treat because of its high malignancy, high recurrence rate, and high resistance to anticancer drugs. An alternative method for research of gliomagenesis and drug resistance is to use in vitro tumor model that closely mimics the in vivo tumor microenvironment. In this study, we established a 3D bioprinted glioma stem cell model, using modified porous gelatin/alginate/fibrinogen hydrogel that mimics the extracellular matrix. Glioma stem cells achieved a survival rate of 86.92%, and proliferated with high cellular activity immediately following bioprinting. During the in vitro culture period, the printed glioma stem cells not only maintained their inherent characteristics of cancer stem cells (Nestin), but also showed differentiation potential (glial fibrillary acidic protein and β-tubulin III). In order to verify the vascularization potential of glioma stem cells, tumor angiogenesis biomarker, vascular endothelial growth factor was detected by immunohistochemistry, and its expression increased from week one to three during the culture period. Drug-sensitivity results showed that 3D printed tumor model was more resistant to temozolomide than 2D monolayer model at TMZ concentrations of 400-1600 μg ml. In summary, 3D bioprinted glioma model provides a novel alternative tool for studying gliomagenesis, glioma stem cell biology, drug resistance, and anticancer drug susceptibility in vitro.

Three-dimensional (3D) bioprinting of living structures with cell-laden biomaterials has been achieved in vitro, however, some cell-cell interactions are limited by the existing hydrogel. To better mimic tumor microenvironment, self-assembled multicellular heterogeneous brain tumor fibers have been fabricated by a custom-made coaxial extrusion 3D bioprinting system, with high viability, proliferative activity and efficient tumor-stromal interactions. Therein, in order to further verify the sufficient interactions between tumor cells and stroma MSCs, CRE-LOXP switch gene system which contained GSCs transfected with “LOXP-STOP-LOXP-RFP” genes and MSCs transfected with “CRE recombinase” gene was used. Results showed that tumor-stroma cells interacted with each other and fused, the transcription of RFP was higher than that of 2D culture model and control group with cells mixed directly into alginate, respectively. RFP expression was observed only in the cell fibers but not in the control group under confocal microscope. In conclusion, coaxial 3D bioprinted multicellular self-assembled heterogeneous tumor tissue-like fibers provided preferable 3D models for studying tumor microenvironment in vitro, especially for tumor-stromal interactions.

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Glioma stem cells (GSCs) are thought to be the root cause of tumor recurrence and drug resistance in glioma patients. In‐depth study of GSCs is of great significance for developing the treatment strategies of glioma. Unfortunately, it is difficult and takes complicated process to obtain GSCs. Therefore, establishing an ideal in vitro model for enriching GSCs will greatly promote the study of GSCs. In this study, the stemness properties of glioma cells were enhanced in three‐dimensional (3D) bioprinted tumor model. Furthermore, the possible molecular mechanism of GSCs enrichment: epithelial–mesenchymal transition (EMT) was explored. Compared with two‐dimensional cultured cells, the proportion of GSCs and EMT‐related genes in 3D cultured cells were significantly increased. Moreover, the 3D cultured glioma cells with improved stemness properties resulted in higher drug resistance in vitro and tumorigenicity in vivo. Taken together, 3D bioprinted glioma cell‐laden scaffold provides a proper platform for the enrichment of GSCs and it is expected to further promote the research on glioma drug resistance. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 383–391, 2019.

Currently, there is still a lack of appropriate in vitro model for studying lung cancers, especially for recapitulating their invasion and metastasis properties. To develop an appropriate in vitro model for lung cancer research, low-temperature molding principle of biological manufacturing and 3D bioprinting was used in this study to fabricate a cell-laden hydrogel grid scaffold structure, using gelatin-sodium alginate-lung cancer cell A549/95-D suspension as the bio-ink. Cells distributed evenly in this model with high viability, and can be cultured sustainably. This model can be cultured for up to 28 days and maintained its structural integrity. Histology, gene analysis, and scratch test showed that 3D printed cells had enhanced invasion and migration capability compared to those cultured in 2D environment, indicating that the in vitro model developed in this study was more biomimetic compared to 2D models, and it is highly valuable in biomedical research.

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Nucleolar and spindle-associated protein (NUSAP1) is a microtubule and chromatin-binding protein that stabilizes microtubules to prevent depolymerization, maintains spindle integrity. NUSAP1 could cross-link spindles into aster-like structures, networks and fibers. It has also been found to play roles in progression of several cancers. However, the potential correlation between NUSAP1 and clinical outcome in patients with glioblastoma multiforme (GBM) remains largely unknown. In the current study, we demonstrated that NUSAP1 was significantly up-regulated in GBM tissues compared with adult non-tumor brain tissues both in a validated cohort and a TCGA cohort. In addition, Kaplan-Meier analysis indicated that patients with high NUSAP1 expression had significantly lower OS (P = 0.0027). Additionally, in the TCGA cohort, NUSAP1 expression was relatively lower in GBM patients within the neural and mesenchymal subtypes compared to other subtypes, and associated with the status of several genetic aberrations such as PTEN deletion and wild type IDH1. The present study provides new insights and evidence that NUSAP1 over-expression was significantly correlated with progression and prognosis of GBM. Furthermore, knockdown of NUSAP1 revealed its regulation on G2/M progression and cell proliferation (both in vitro and in vivo). These data demonstrate that NUSAP1 could serve as a novel prognostic biomarker and a potential therapeutic target for GBM.