Evaluation of the efficiency of double recombinant vaccinia virus VV-GMCSF-Lact against glioblastoma using 3D spheroid and cerebral organoid models

Abstract

Introduction: Human cerebral organoids (COs) are sophisticated three-dimensional (3D) models that successfully replicate the 3D cytoarchitecture of human brain development in its early stages. Co-cultivation of COs obtained from human-induced pluripotent stem cells with 3D U-87 MG glioblastoma cell spheroids (glioblastoma–CO assembloid [GCOA]) represents a robust strategy for modeling brain cancer behavior

Objectives: This study aims to evaluate the feasibility of using GCOAs and 3D U-87 MG glioblastoma cell spheroids as in vitro cell models to evaluate the cytotoxic effect of the oncolytic recombinant vaccinia virus-granulocyte–macrophage colony-stimulating factor (VV-GM-CSF)-Lact compared to the VV-GMCSF-del virus.

Methods: To assess the cytotoxicity of two virus strains, we used fluorescent and confocal microscopy, spectrophotometry, the MTT assay, and real-time polymerase chain reaction

Results: Glioblastoma cells died faster in the presence of VV-GMCSF-Lact virus, and 3D spheroids infected with this virus contained more caspase-3-positive cells. On the 10^th day in GCOA, glioblastoma cells began to invade the interior of the CO. Under conditions of 3D spheroid and GCOA infection with recombinant viruses, three differentially expressed genes – FSCN1, SCUBE2, and TNFRSF9 – associated with invasion were analyzed.

Conclusion: These 3D models of glioblastoma can be used in the development of antitumor drugs to study their cytotoxic effects.

Keywords

Glioblastoma / Cerebral organoids / Three-dimensional spheroids / Glioblastoma–cerebral organoid assembloid / VV-GMCSF-Lact / Oncolytic virus

Acknowledgements

We would like to thank the Microscopic Center of the Siberian Branch of the Russian Academy of Sciences for granting access to the microscopic equipment.

Funding

This study, including cell viability assays, polymerase chain reaction, immunohistochemistry, and quantitative polymerase chain reaction, was supported by the Russian Science Foundation (Grant No. 22-64-00041; available at https://rscf.ru/project/22-64-00041/, accessed on November 1, 2025). Cell culture maintenance was supported by the Russian state-funded project of the Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (Grant No.: 125012900932-4).

Conflict of interest

The authors declare they have no competing interests.

Author contributions

Conceptualization: Maya Dymova, Tatiana Shnaider, Vladimir Richter

Formal analysis: Elena Kuligina

Investigation: Maya Dymova, Gleb Petrov, Danil Drokov

Methodology: Maya Dymova, Gleb Petrov, Tatiana Shnaider, Sophia Yakovleva

Writing–original draft: Maya Dymova, Gleb Petrov

Writing–review & editing: Maya Dymova, Gleb Petrov

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data

Data are available from the corresponding author on reasonable request.

References

[1] Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024; 74(3): 229-263. doi: 10.3322/caac.21834

[2] Miller KD, Ostrom QT, Kruchko C, et al. Brain and other central nervous system tumor statistics, 2021. CA Cancer J Clin. 2021; 71(5): 381-406. doi: 10.3322/caac.21693

[3] Ma R, Taphoorn MJB, Plaha P. Advances in the management of glioblastoma. J Neurol Neurosurg Psychiatry. 2021; 92(10): 1103-1111. doi: 10.1136/jnnp-2020-325334

[4] Wu W, Klockow JL, Zhang M, et al. Glioblastoma multiforme (GBM): An overview of current therapies and mechanisms of resistance. Pharmacol Res. 2021; 171: 105780. doi: 10.1016/j.phrs.2021.105780

[5] Gimple RC, Bhargava S, Dixit D, Rich JN. Glioblastoma stem cells: Lessons from the tumor hierarchy in a lethal cancer. Genes Dev. 2019; 33(11-12): 591-609. doi: 10.1101/gad.324301.119

[6] Vargas López AJ. Glioblastoma in adults: A society for neuro-oncology (SNO) and European society of neuro-oncology (EANO) consensus review on current management and future directions. Neuro Oncol. 2021; 23(3): 502-503. doi: 10.1093/neuonc/noaa287

[7] Seker-Polat F, Pinarbasi Degirmenci N, Solaroglu I, Bagci-Onder T. Tumor cell infiltration into the brain in glioblastoma: From mechanisms to clinical perspectives. Cancers (Basel). 2022; 14(2): 443. doi: 10.3390/cancers14020443

[8] Bose A, Datta S, Mandal R, Ray U, Dhar R. Increased heterogeneity in expression of genes associated with cancer progression and drug resistance. Transl Oncol. 2024; 41: 101879. doi: 10.1016/j.tranon.2024.101879

[9] Qiu J, Sun M, Wang Y, Chen B. Identification of hub genes and pathways in gastric adenocarcinoma based on bioinformatics analysis. Med Sci Monit. 2020; 26: e920261. doi: 10.12659/msm.920261

[10] Kapałczyńska M, Kolenda T, Przybyła W, et al. 2D and 3D cell cultures - a comparison of different types of cancer cell cultures. Arch Med Sci. 2016; 14(4): 910-919. doi: 10.5114/aoms.2016.63743

[11] Paolillo M, Comincini S, Schinelli S. In vitro glioblastoma models: A journey into the third dimension. Cancers (Basel). 2021; 13(10): 2449. doi: 10.3390/cancers13102449

[12] Heinrich MA, Huynh NT, Heinrich L, Prakash J. Understanding glioblastoma stromal barriers against NK cell attack using tri-culture 3D spheroid model. Heliyon. 2024; 10(3): e24808. doi: 10.1016/j.heliyon.2024.e24808

[13] Rybin MJ, Ivan ME, Ayad NG, Zeier Z. Organoid models of glioblastoma and their role in drug discovery. Front Cell Neurosci. 2021; 15: 605255. doi: 10.3389/fncel.2021.605255

[14] Kelava I, Lancaster MA. Dishing out mini-brains: Current progress and future prospects in brain organoid research. Dev Biol. 2016; 420(2): 199-209. doi: 10.1016/j.ydbio.2016.06.037

[15] Pine AR, Cirigliano SM, Nicholson JG, et al. Tumor microenvironment is critical for the maintenance of cellular states found in primary glioblastomas. Cancer Discov. 2020; 10(7): 964-979. doi: 10.1158/2159-8290.cd-20-0057

[16] Bian S, Repic M, Guo Z, et al. Genetically engineered cerebral organoids model brain tumour formation. Nat Methods. 2018; 15(8): 631-639. doi: 10.1038/s41592-018-0070-7

[17] Ogawa J, Pao GM, Shokhirev MN, Verma IM. Glioblastoma model using human cerebral organoids. Cell Rep. 2018; 23(4): 1220-1229. doi: 10.1016/j.celrep.2018.03.105

[18] Weth FR, Peng L, Paterson E, Tan ST, Gray C. Utility of the cerebral organoid glioma ‘GLICO’ model for screening applications. Cells. 2022; 12(1): 153. doi: 10.3390/cells12010153

[19] Linkous A, Balamatsias D, Snuderl M, et al. Modeling patient-derived glioblastoma with cerebral organoids. Cell Rep. 2019; 26(12): 3203-3211.e5. doi: 10.1016/j.celrep.2019.02.063

[20] Fedorova V, Pospisilova V, Vanova T, et al. Glioblastoma and cerebral organoids: Development and analysis of an in vitro model for glioblastoma migration. Mol Oncol. 2023; 17(4): 647-663. doi: 10.1002/1878-0261.13389

[21] Vasileva N, Ageenko A, Byvakina A, et al. The recombinant oncolytic virus VV-GMCSF-lact and chemotherapy drugs against human glioma. Int J Mol Sci. 2024; 25(8): 4244. doi: 10.3390/ijms25084244

[22] Kochneva G, Sivolobova G, Tkacheva A, et al. Engineering of double recombinant vaccinia virus with enhanced oncolytic potential for solid tumor virotherapy. Oncotarget. 2016; 7(45): 74171-74188. doi: 10.18632/oncotarget.12367

[23] Vasileva N, Ageenko A, Dmitrieva M, et al. Double recombinant vaccinia virus: A candidate drug against human glioblastoma. Life (Basel). 2021; 11(10): 1084. doi: 10.3390/life11101084

[24] Gridina MM, Matveeva NM, Fishman VS, et al. Allele-specific biased expression of the CNTN6 gene in iPS cell-derived neurons from a patient with intellectual disability and 3p26.3 microduplication involving the CNTN6 gene. Mol Neurobiol. 2018; 55: 6533-6546. doi: 10.1007/s12035-017-0851-5

[25] Yakovleva S, Knyazeva A, Yunusova A, et al. Functional divergence of NOTCH1 and NOTCH2 in human cerebral organoids reveals receptor-specific roles in early corticogenesis. Int J Mol Sci. 2025; 26(15): 7309. doi: 10.3390/ijms26157309

[26] Liu H, Zhang Y, Li L, et al. Fascin actin-bundling protein 1 in human cancer: Promising biomarker or therapeutic target?. Mol Ther Oncolytics. 2021; 20: 240-264. doi: 10.1016/j.omto.2020.12.014

[27] Glorieux C, Huang P. CD137 expression in cancer cells: Regulation and significance. Cancer Commun (Lond). 2019; 39(1): 70. doi: 10.1186/s40880-019-0419-z

[28] Goodspeed A, Heiser LM, Gray JW, Costello JC. Tumor-derived cell lines as molecular models of cancer pharmacogenomics. Mol Cancer Res. 2016; 14(1): 3-13. doi: 10.1158/1541-7786.mcr-15-0189

[29] Wang X, Sun Y, Zhang DY, Ming GL, Song H. Glioblastoma modeling with 3D organoids: Progress and challenges. Oxford Open Neurosci. 2023; 6(2): kvad008. doi: 10.1093/oons/kvad008

[30] Keller F, Bruch R, Schneider R, Meier-Hubberten J, Hafner M, Rudolf R. A scaffold-free 3-D Co-culture mimics the major features of the reverse warburg effect in vitro. Cells. 2020; 9(8): 1900. doi: 10.3390/cells9081900

[31] Kannan S, Murugan AK, Balasubramanian S, Munirajan AK, Alzahrani AS. Gliomas: Genetic alterations, mechanisms of metastasis, recurrence, drug resistance, and recent trends in molecular therapeutic options. Biochem Pharmacol. 2022; 201: 115090. doi: 10.1016/j.bcp.2022.115090

[32] Semenov DV, Vasileva NS, Dymova MA, et al. Transcriptome changes in glioma cells upon infection with the oncolytic virus VV-GMCSF-lact. Cells. 2023; 12(22): 2616. doi: 10.3390/cells12222616

[33] Semenov DV, Vasileva NS, Menyailo ME, et al. Single-cell transcriptomic changes in patient-derived glioma and U87 glioblastoma cell cultures infected with the oncolytic virus VV-GMCSF-lact. Int J Mol Sci. 2025; 26(14): 6983. doi: 10.3390/ijms26146983

[34] Lamb MC, Tootle TL. Fascin in cell migration: More than an actin bundling protein. Biology (Basel). 2020; 9: 403. doi: 10.3390/biology9110403

[35] Guo E, Liu H, Liu X. Overexpression of SCUBE2 inhibits proliferation, migration, and invasion in glioma cells. Oncol Res. 2017; 25(3): 437-444. doi: 10.3727/096504016x14747335734344

[36] Lin YC, Sahoo BK, Gau SS, Yang RB. The biology of SCUBE. J Biomed Sci. 2023; 30: 33. doi: 10.1186/s12929-023-00925-3

[37] Ali H. SCUBE2, vascular endothelium, and vascular complications: A systematic review. Biomed Pharmacother. 2020; 127: 110129. doi: 10.1016/j.biopha.2020.110129

[38] Blank AE, Baumgarten P, Zeiner P, et al. Tumour necrosis factor receptor superfamily member 9 (TNFRSF9) is up-regulated in reactive astrocytes in human gliomas. Neuropathol Appl Neurobiol. 2014; 41(2): e56-e67. doi: 10.1111/nan.12135