AccScience Publishing / IJB / Online First / DOI: 10.36922/IJB026330357
Cite this article
23
Download
633
Views
Journal Browser
Volume | Year
Issue
Search
News and Announcements
View All
RESEARCH ARTICLE

A scalable 3D-printed brain tumour model for high-throughput screening glioblastoma biology and drug response

Panthipa Suwannakot1,2 ,  Eva Tomaskovic-Crook1,2,3* ,  Kimberley L. Alexander2,4,5 ,  Jeremy Micah Crook1,2,3*
Show Less
1 Arto Hardy Family Biomedical Innovation Hub, Chris O’Brien Lifehouse, Sydney, NSW 2050 , Australia
2 School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, NSW 2006 , Australia
3 Intelligent Polymer Research Institute, Australian Institute of Innovative Materials, Innovation Campus, University of Wollongong, NSW 2500 , Australia
4 Brain Cancer Research, Chris O’Brien Lifehouse, Sydney, NSW 2050 , Australia
5 Department of Neuropathology, Royal Prince Alfred Hospital, Sydney, NSW 2050 , Australia
Received: 19 December 2025 | Revised: 13 August 2026 | Accepted: 26 August 2026 | Published online: 26 August 2026
© 2026 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
Abstract

Glioblastoma (GBM) is a highly aggressive brain tumour characterized by its rapid infiltrative growth patterns, marked intra- and inter-tumoural heterogeneity, and resistance to conventional treatments. GBM has a median overall survival of 15 months and a 5-year survival rate of approximately 5%. Current treatment modalities, including surgery and chemoradiotherapy, have shown limited success. To address the need for more effective therapeutic strategies and a deeper understanding of GBM biology, we utilised droplet printing technology to fabricate a high-throughput 3D brain tumour model. We first optimized the printability of the GBM cell-laden gelatin methacrylate (GelMA) bioink, tailoring its mechanical properties to mimic the stiffness of human brain tissue. Next, we employed metabolic and immunofluorescence assays to investigate the response of the printed GBM droplets to the standard-of-care chemotherapeutic agent temozolomide (TMZ). Furthermore, using digital droplet PCR (ddPCR), we assessed the expression of microRNAs typical for paediatric GBM (miR-490, miR-876-3p, miR-876-5p, miR-448, miR-137, and miR-501-3p), with miR-191-5p used as the endogenous control. Our approach provides proof-of-concept for high-throughput screening of GBM drug response. Moreover, our 3D-printed brain tumour model could be used for patient-personalised tumour screening, encompassing theranostics for GBM diagnostics, treatment, and monitoring.

Keywords
Glioblastoma
Drug resistance
Drug screening
3D bioprinting
Gelatin methacrylate
Hydrogel
MicroRNA
Funding
This work was funded by Arto Hardy Family, SurFebruary Cancer Research Fund, Tour de Cure, Tregaskis Foundation, Yip family, Ian Potter Foundation and NSW Office for Health and Medical Research.
Conflict of interest
Jeremy Crook is an Editorial Board Member of this journal, but was not in any way involved in the editorial and peer-review process conducted for this paper, directly or indirectly. The authors declare they have no competing interests.
References
  1. Ostrom QT, Cioffi G, Gittleman H, et al. CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the United States in 2012–2016. Neuro-oncology. 2019;21(Supplement_5):v1-v100. doi: 10.1093/neuonc/noz150
  2. Llaguno SA, Chen J, Kwon C-H, et al. Malignant astrocytomas originate from neural stem/progenitor cells in a somatic tumor suppressor mouse model. Cancer cell. 2009;15(3):240. doi: 1016/j.ccr.2009.02.008
  3. Lee JH, Lee JE, Kahng JY, et al. Human glioblastoma arises from subventricular zone cells with low-level driver mutations. Nature. 2018;560(7717):243-247. doi: 10.1038/s41586-018-0389-3
  4. Gilbert MR, Dignam JJ, Armstrong TS, et al. A randomized trial of bevacizumab for newly diagnosed glioblastoma. N Engl J Med. 2014;370(8):699-708. doi: 10.1056/NEJMoa1308573
  5. Stupp R, Taillibert S, Kanner A, et al. Effect of tumor-treating fields plus maintenance temozolomide vs maintenance temozolomide alone on survival in patients with glioblastoma: a randomized clinical trial. J Am Med Assoc. 2017;318(23):2306-2316. doi: 10.1001/jama.2017.18718
  6. Zanders ED, Svensson F, Bailey DS. Therapy for glioblastoma: is it working? Drug Discov Today. 2019;24(5):1193-1201. doi: 10.1016/j.drudis.2019.03.008
  7. Foglizzo V, Cocco E, Marchiò S. Advanced cellular models for preclinical drug testing: from 2D cultures to organ-on-a-chip technology. Cancers. 2022;14(15):3692. doi: 10.3390/cancers14153692
  8. Jensen C, Teng Y. Is it time to start transitioning from 2D to 3D cell culture? Front Mol Biosci. 2020;7:33. doi: 10.3389/fmolb.2020.00033
  9. Nii M, Lai JH, Keeney M, et al. The effects of interactive mechanical and biochemical niche signaling on osteogenic differentiation of adipose-derived stem cells using combinatorial hydrogels. Acta Biomater. 2013;9(3):5475-5483. doi: 10.1016/j.actbio.2012.11.002
  10. DeForest CA, Sims EA, Anseth KS. Peptide-functionalized click hydrogels with independently tunable mechanics and chemical functionality for 3D cell culture. Chem Mater. 2010;22(16):4783-4790. doi: 10.1021/cm101391y
  11. Tomaskovic-Crook E, Crook JM. Clinically amendable, defined, and rapid induction of human brain organoids from induced pluripotent stem cells. In: Methods in Molecular Biology. New York, NY: Springer New York; 2017:13-22. doi: 10.1007/7651_2017_95
  12. Tomaskovic-Crook E, Higginbottom SL, Zhang B, Bourke J, Wallace GG, Crook JM. Defined, simplified, scalable, and clinically compatible hydrogel-based production of human brain organoids. Organoids. 2023;2(1):20-36. doi: 10.3390/organoids2010002
  13. Tang M, Xie Q, Gimple RC, et al. Three-dimensional bioprinted glioblastoma microenvironments model cellular dependencies and immune interactions. Cell research. 2020;30(10):833-853. doi: 10.1038/s41422-020-0338-1
  14. Giunti L, Da Ros M, De Gregorio V, et al. A microRNA profile of pediatric glioblastoma: The role of NUCKS1 upregulation. Mol Clin Oncol. 2019;10(3):331-338. doi: 10.3892/mco.2019.1795
  15. Gaur A, Jewell DA, Liang Y, et al. Characterization of microRNA expression levels and their biological correlates in human cancer cell lines. Cancer Res. 2007;67(6):2456-2468. doi: 10.1158/0008-5472.CAN-06-2698
  16. Godlewski J, Nowicki MO, Bronisz A, et al. Targeting of the Bmi-1 oncogene/stem cell renewal factor by microRNA-128 inhibits glioma proliferation and self-renewal. Cancer Res. 2008;68(22):9125-9130. doi: 10.1158/0008-5472.CAN-08-2629
  17. Kim H, Huang W, Jiang X, Pennicooke B, Park PJ, Johnson MD. Integrative genome analysis reveals an oncomir/oncogene cluster regulating glioblastoma survivorship. Proc Natl Acad Sci USA. 2010;107(5):2183-2188. doi: 10.1073/pnas.0909896107
  18. Cui Q, Yu Z, Purisima EO, Wang E. Principles of microRNA regulation of a human cellular signaling network. Mol Syst Biol. 2006;2(1):46. doi: 10.1038/msb4100089
  19. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116(2):281-297. doi: 10.1016/S0092-8674(04)00045-5
  20. Piwecka M, Rolle K, Belter A, et al. Comprehensive analysis of microRNA expression profile in malignant glioma tissues. Mol Oncol. 2015;9(7):1324-1340. doi: 10.1016/j.molonc.2015.03.007
  21. Wang M, Xie H, Stellpflug W, et al. BTECH: a platform to integrate genomic, transcriptomic and epigenomic alterations in brain tumors. Neuroinformatics. 2011;9:59-67. doi: 10.1007/s12021-010-9091-9
  22. Belter A, Rolle K, Piwecka M, Fedoruk-Wyszomirska A, Naskręt-Barciszewska MZ, Barciszewski J. Inhibition of miR-21 in glioma cells using catalytic nucleic acids. Sci Rep. 2016;6(1):24516. doi: 10.1038/srep24516
  23. Zhang S, Han L, Wei J, et al. Combination treatment with doxorubicin and microRNA-21 inhibitor synergistically augments anticancer activity through upregulation of tumor suppressing genes. Int J Oncol. 2015;46(4):1589-1600. doi: 10.3892/ijo.2015.2841
  24. Palizkaran Yazdi M, Barjasteh A, Moghbeli M. MicroRNAs as the pivotal regulators of Temozolomide resistance in glioblastoma. Mol Brain. 2024;17(1):42. doi: 10.1186/s13041-024-01113-6
  25. Tolue Ghasaban F, Maharati A, Zangouei AS, Zangooie A, Moghbeli M. MicroRNAs as the pivotal regulators of cisplatin resistance in head and neck cancers. Cancer Cell Int. 2023;23(1):170. doi: 10.1186/s12935-023-03010-9
  26. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat methods. 2012;9(7):676-682. doi: 10.1038/nmeth.2019
  27. Tomaskovic‐Crook E, Zhang P, Ahtiainen A, et al. Human neural tissues from neural stem cells using conductive biogel and printed polymer microelectrode arrays for 3D electrical stimulation. Adv Healthc Mater. 2019;8(15):1900425. doi: 10.1002/adhm.201970062
  28. James EC, Tomaskovic‐Crook E, Crook JM. Engineering 3D Scaffold‐Free Nanoparticle‐Laden Stem Cell Constructs for Piezoelectric Enhancement of Human Neural Tissue Formation and Function. Adv Sci. 2024;11(40):2310010. doi: 10.1002/advs.202470239
  29. Sun M, Sun X, Wang Z, Guo S, Yu G, Yang H. Synthesis and properties of gelatin methacryloyl (GelMA) hydrogels and their recent applications in load-bearing tissue. Polymers. 2018;10(11):1290. doi: 10.3390/polym10111290
  30. Liu W, Heinrich MA, Zhou Y, et al. Extrusion bioprinting of shear‐thinning gelatin methacryloyl bioinks. Adv Healthc Mater. 2017;6(12):1601451. doi:10.1002/adhm.201770062
  31. Padilla C, Quero F, Pępczyńska M, et al. Understanding the molecular conformation and viscoelasticity of low sol-gel transition temperature gelatin methacryloyl suspensions. Int J Mol Sci. 2023;24(8):7489. doi: 10.3390/ijms24087489
  32. Yu K, Zhang X, Sun Y, et al. Printability during projection-based 3D bioprinting. Bioact Mater. 2022;11:254-267. doi: 10.1016/j.bioactmat.2021.09.021
  33. Park HE, Gasek N, Hwang J, Weiss DJ, Lee PC. Effect of temperature on gelation and cross-linking of gelatin methacryloyl for biomedical applications. Phys Fluids. 2020;32(3). doi: 10.1063/1.5144896
  34. Budday S, Nay R, de Rooij R, et al. Mechanical properties of gray and white matter brain tissue by indentation. J Mech Behav Biomed Mater. 2015;46:318-330. doi: 10.1016/j.jmbbm.2015.02.024
  35. Kaster T, Sack I, Samani A. Measurement of the hyperelastic properties of ex vivo brain tissue slices. J Biomech. 2011;44(6):1158-1163. doi: 10.1016/j.jbiomech.2011.01.019
  36. Jagielska A, Norman AL, Whyte G, Vliet KJV, Guck J, Franklin RJ. Mechanical environment modulates biological properties of oligodendrocyte progenitor cells. Stem Cells Dev. 2012;21(16):2905-2914. doi: 10.1089/scd.2012.0189
  37. Polio SR, Kundu AN, Dougan CE, et al. Cross-platform mechanical characterization of lung tissue. PloS one. 2018;13(10):e0204765. doi: 10.1371/journal.pone.0204765
  38. Levental KR, Yu H, Kass L, et al. Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell. 2009;139(5):891-906. doi: 10.1016/j.cell.2009.10.027
  39. Pötschke R, Gielen G, Pietsch T, et al. Musashi1 enhances chemotherapy resistance of pediatric glioblastoma cells in vitro. Pediatr Res. 2020;87(4):669-676. doi: 10.1038/s41390-019-0628-9
  40. Gaspar N, Marshall L, Perryman L, et al. MGMT-independent temozolomide resistance in pediatric glioblastoma cells associated with a PI3-kinase–mediated HOX/stem cell gene signature. Cancer Res. 2010;70(22):9243-9252. doi: 10.1158/0008-5472.CAN-10-1250
  41. Weller M, Stupp R, Reifenberger G, et al. MGMT promoter methylation in malignant gliomas: ready for personalized medicine? Nat Rev Neurol. 2010;6(1):39-51. doi: 10.1038/nrneurol.2009.197
  42. Kaina B, Christmann M, Naumann S, Roos WP. MGMT: key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents. DNA repair. 2007;6(8):1079-1099. doi: 10.1016/j.dnarep.2007.03.008
  43. Agnihotri S, Burrell K, Buczkowicz P, et al. ATM regulates 3-methylpurine-DNA glycosylase and promotes therapeutic resistance to alkylating agents. Cancer Discov. 2014;4(10):1198-1213. doi: 10.1158/2159-8290.CD-14-0157
  44. Mathews LA, Cabarcas SM, Farrar WL. DNA repair: the culprit for tumor-initiating cell survival? Cancer Metastasis Rev. 2011;30:185-197. doi: 10.1007/s10555-011-9277-0
  45. Trivedi RN, Almeida KH, Fornsaglio JL, Schamus S, Sobol RW. The role of base excision repair in the sensitivity and resistance to temozolomide-mediated cell death. Cancer Res. 2005;65(14):6394-6400. doi: 10.1158/0008-5472.CAN-05-0715
  46. Pu J, Yuan K, Tao J, et al. Glioblastoma multiforme: an updated overview of temozolomide resistance mechanisms and strategies to overcome resistance. Discover Oncol. 2025;16(1):731. doi: 10.1007/s12672-025-02567-3
  47. Kochanowski P, Catapano J, Pudełek M, et al. Temozolomide induces the acquisition of invasive phenotype by o6-methylguanine-dna methyltransferase (Mgmt)+ glioblastoma cells in a snail-1/cx43-dependent manner. Int J Mol Sci. 2021;22(8):4150. doi: 10.3390/ijms22084150
  48. Chen M, Cheng Y, Yuan Z, Wang F, Yang L, Zhao H. NCK1-AS1 increases drug resistance of glioma cells to temozolomide by modulating miR-137/TRIM24. Cancer Biother Radiopharm. 2020;35(2):101-108. doi: 10.1089/cbr.2019.3054
  49. Lu Y, Guo G, Hong R, et al. LncRNA HAS2-AS1 promotes glioblastoma proliferation by sponging miR-137. Front Oncol. 2021;11:634893. doi: 10.3389/fonc.2021.634893
  50. Li D-M, Chen Q-D, Wei G-N, et al. Hypoxia-induced miR-137 inhibition increased glioblastoma multiforme growth and chemoresistance through LRP6. Front Oncol. 2021;10:611699. doi: 10.3389/fonc.2020.611699
  51. Tunca B, Tezcan G, Cecener G, et al. Olea europaea leaf extract alters microRNA expression in human glioblastoma cells. J Cancer Res Clin Oncol. 2012;138(11):1831-1844. doi: 10.1007/s00432-012-1261-8
  52. Xiao J, Peng F, Yu C, et al. microRNA-137 modulates pancreatic cancer cells tumor growth, invasion and sensitivity to chemotherapy. Int J Clin Exp Pathol. 2014;7(11):7442-7450.
  53. Wang Y, Chen R, Zhou X, et al. miR-137: a novel therapeutic target for human glioma. Mol Ther-Nucl Acids. 2020;21:614-622. doi: 10.1016/j.omtn.2020.06.028
  54. Luo M, Wu L, Zhang K, et al. miR-137 regulates ferroptosis by targeting glutamine transporter SLC1A5 in melanoma. Cell Death Differ. 2018;25(8):1457-72.
  55. Sun G, Cao Y, Shi L, et al. Overexpressed miRNA-137 inhibits human glioma cells growth by targeting Rac1. Cancer Biother Radiopharm. 2013;28(4):327-334. doi: 10.1089/cbr.2012.1380
  56. Velasco MX, Kosti A, Guardia GD, et al. Antagonism between the RNA-binding protein Musashi1 and miR-137 and its potential impact on neurogenesis and glioblastoma development. Rna. 2019;25(7):768-782. doi: 10.1261/rna.069211.118
  57. Zhu X, Li Y, Shen H, et al. miR-137 restoration sensitizes multidrug-resistant MCF-7/ADM cells to anticancer agents by targeting YB-1. Acta Biochim Biophys Sin. 2013;45(2):80-86. doi: 10.1093/abbs/gms099
  58. Takwi AA, Wang Y-M, Wu J, Michaelis M, Cinatl J, Chen T. miR-137 regulates the constitutive androstane receptor and modulates doxorubicin sensitivity in parental and doxorubicin-resistant neuroblastoma cells. Oncogene. 2014;33(28):3717-3729. doi: 10.1038/onc.2013.330
  59. Chen L, Wang X, Wang H, et al. miR-137 is frequently down-regulated in glioblastoma and is a negative regulator of Cox-2. Eur J Cancer. 2012;48(16):3104-3111. doi: 10.1016/j.ejca.2012.02.007
  60. Li D, Shan W, Fang Y, Wang P, Li J. miR-137 acts as a tumor suppressor via inhibiting CXCL12 in human glioblastoma. Oncotarget. 2017;8(60):101262. doi: 10.18632/oncotarget.20589
  61. Zhang Z, Song X, Tian H, et al. MicroRNA-137 inhibits growth of glioblastoma through EGFR suppression. Am J Transl Res. 2017;9(3):1492-1499.
  62. Woo PY, Li Y, Chan AH, et al. A multifaceted review of temozolomide resistance mechanisms in glioblastoma beyond O-6-methylguanine-DNA methyltransferase. Glioma. 2019;2(2):68-82. doi: 10.4103/glioma.glioma_3_19
  63. He Y, Kaina B. Are there thresholds in glioblastoma cell death responses triggered by temozolomide? Int J Mol Sci. 2019;20(7):1562. doi: 10.3390/ijms20071562
  64. Yang F, Zhao WJ, Jia CL, Li XK, Wang Q, Chen ZL. MicroRNA-876-3p functions as a tumor suppressor gene and correlates with cell metastasis in pancreatic adenocarcinoma via targeting JAG2. Am J Cancer Res. 2018;8(4):636-649.
  65. Ma B, Xu J, Chen G, et al. miR-876-5p exerts tumor suppressor function by targeting TWIST1 and regulating the epithelial-mesenchymal transition in glioblastoma. Int J Clin Exp Med. 2020;13:1454-1463.
  66. Li X, Wang N, Leng H, Yuan H, Xu L. Hsa_circ_0043949 reinforces temozolomide resistance via upregulating oncogene ITGA1 axis in glioblastoma. Metab Brain Dis. 2022;37(8):2979-2993. doi: 10.1007/s11011-022-01069-3
Share
Back to top
International Journal of Bioprinting, Electronic ISSN: 2424-8002 Print ISSN: 2424-7723, Published by AccScience Publishing