A Patient-Derived Glioblastoma Organoid Model that Preserves Intra- and Intertumoral Heterogeneity

Öz

Glioblastoma (GB) is an aggressive brain tumor characterized by extensive heterogeneity. We aimed to generate patient-derived GB organoids preserving histological features and to establish a biobank for future studies. Materials and Methods: Tumor tissues from GB patients were dissected into 0.5–1 mm fragments and cultured for 14 days in organoid medium under orbital shaking. Immunohistochemical analysis was performed to assess GFAP, OLIG2, IDH-1, p53, ATRX, and Ki67 expression before and after organoid formation. Results: All tumor samples formed spherical organoids within 14 days. Histological analysis showed preservation of GB-specific morphological features, including nuclear atypia and pleomorphism. GFAP, OLIG2, and ATRX expression patterns were concordant between parental tumors and organoids in cases. IDH-1 expression was consistent in three patients. Ki67 and p53 expression levels showed similar patterns between organoids and corresponding tumors. Conclusion: Patient-derived GB organoids retain key histopathological features and may serve as a platform for GB modeling.

Anahtar Kelimeler

• Glioblastoma
• organoid culture
• tumor heterogeneityse

Destekleyen Kurum

We greatly acknowledge to the Scientific and Technological Research Council of Turkey (TÜBİTAK) for supporting author Cagla Tekin within the scope of the 2211-A doctoral program scholarship.

Proje Numarası

TAY-2022-584

Etik Beyan

Ethical approval for this study was granted by the Bursa Uludag University Ethics Committee (Dated 20.06.2021, decision no: 2021-9/14). All patients were fully informed about the study objectives, confidentiality measures, and their right to withdraw at any time, and written informed consent was obtained prior to participation. The study was conducted in full accordance with the Declaration of Helsinki.

Teşekkür

We thanks to Prof. Dr. Şükrü Yıldırım from Istanbul Maltepe University, Prof. Dr. Şahsine Tolunay and Assoc. Prof. Dr. Mine Ozsen from Bursa Uludag University for their support in IHC analysis. We would also like to thanks to Prof. Dr. Bahattin Hakyemez for special assistance with MRI imaging.

İntra- ve İntertümöral Heterojeniteyi Koruyan Hasta Kaynaklı Glioblastoma Organoid Modeli

Öz

Amaç: Glioblastoma (GB), yaygın heterojenite ile karakterize edilen agresif bir beyin tümörüdür. Amacımız, histolojik özellikleri koruyan hasta kaynaklı GB organoidleri üretmek ve gelecekteki çalışmalar için bir biyobanka oluşturmaktır. Materyal ve Metot: GB hastalarından alınan tümör dokuları 0,5-1 mm'lik parçalara ayrıldı ve 14 gün boyunca organoid ortamında, orbital çalkalama altında kültüre edildi. Organoid oluşumundan önce ve sonra GFAP, OLIG2, IDH-1, p53, ATRX ve Ki67 ekspresyonunu değerlendirmek için immünohistokimyasal analiz yapıldı. Bulgular: Tümör örneklerinin tamamı 14 gün içinde küresel organoidler oluşturdu. Histolojik analiz, nükleer atipi ve pleomorfizm de dahil olmak üzere GB'ye özgü morfolojik özelliklerin korunduğunu gösterdi. GFAP, OLIG2 ve ATRX ekspresyon paternleri, ana tümörler ve organoidler arasında uyumluydu. IDH-1 ekspresyonu üç hastada tutarlıydı. Ki67 ve p53 ekspresyon seviyeleri, organoidler ve karşılık gelen tümörler arasında benzer dağılım gösterdi. Sonuç: Hasta kaynaklı GB organoidleri, temel histopatolojik özellikleri korur ve GB modellemesi için bir platform olarak hizmet edebilir.

Anahtar Kelimeler

• Glioblastoma
• organoid kültürü
• tümör heterojenitesi

Destekleyen Kurum

Bilimsel ve Teknolojik Araştırma Konseyi'ne (TÜBİTAK) yazar Çağla Tekin'e 2211-A doktora programı bursu kapsamında destek verdikleri için büyük teşekkür ediyoruz.

Proje Numarası

TAY-2022-584

Etik Beyan

Bursa Uludağ Üniversitesi Etik Kurulu bu çalışmayı onaylamıştır (2021-9/14).

Teşekkür

İstanbul Maltepe Üniversitesi'nden Prof. Dr. Şükrü Yıldırım, Bursa Uludağ Üniversitesi'nden Prof. Dr. Şahsine Tolunay ve Doç. Dr. Mine Özsen'e IHC analizindeki destekleri için teşekkür ederiz. MRI görüntüleme konusunda özel yardımı için Prof. Dr. Bahattin Hakyemez'e de teşekkür etmek isteriz.

Kaynakça

1. 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 Oncol. 2019;21:v1-v100. doi:10.1093/neuonc/noz150
2. 2. Xu C, Yuan X, Hou P, et al. Development of glioblastoma organoids and their applications in personalized therapy. Cancer biology & medicine. 2021;20(5), 353–368. doi:10.20892/j.issn.2095-3941.2023.0061
3. 3. Neftel C, Laffy J, Filbin MG, et al. An integrative model of cellular states, plasticity, and genetics for glioblastoma. Cell. 2019;178:835-849. doi:10.1016/j.cell.2019.06.024
4. 4. Haddad AF, Young JS, Amara D, et al. Mouse models of glioblastoma for the evaluation of novel therapeutic strategies. Neuro-Oncol Adv. 2021;3:vdab100. doi:10.1093/noajnl/vdab100
5. 5. Zhang C, Jin M, Zhao J, Chen J, Jin W. Organoid models of glioblastoma: advances, applications and challenges. American journal of cancer research. 2020;10(8):2242-2257.
6. 6. Zhang DY, Song H, Ming G. Modeling neurological disorders using brain organoids. Semin Cell Dev Biol. 2021;111:4-14. doi:10.1016/j.semcdb.2020.05.026
7. 7. Veninga V, Voest EE. Tumor organoids: opportunities and challenges to guide precision medicine. Cancer Cell. 2021;39:1190-201. doi:10.1016/j.ccell.2021.07.020
8. 8. Xu H, Jiao D, Liu A, Wu K. Tumor organoids: applications in cancer modeling and potentials in precision medicine. J Hematol Oncol. 2022;15:1-20. doi:10.1186/s13045-022-01278-4

9. 9. Jin MZ, Han RR, Qiu GZ, Ju XC, Lou G, Jin WL. Organoids: an intermediate modeling platform in precision oncology. Cancer Lett. 2018;414:174-180. doi:10.1016/j.canlet.2017.11.021
10. 10. Behjati S, Huch M, van Boxtel R, et al. Genome sequencing of normal cells reveals developmental lineages and mutational processes. Nature. 2014;513:422-425. doi:10.1038/nature13448
11. 11. Duarte AA, Gogola E, Sachs N, et al. BRCA-deficient mouse mammary tumor organoids to study cancer-drug resistance. Nat Methods. 2018;15:134–140. doi:10.1038/nmeth.4535
12. 12. Jacob F, Salinas RD, Zhang DY, et al. A patient-derived glioblastoma organoid model and biobank recapitulates inter- and intra-tumoral heterogeneity. Cell. 2020;180:188-204.e22. doi:10.1016/j.cell.2019.11.036
13. 13. Qin LX, Tang ZY, Ma ZC, et al. P53 immunohistochemical scoring: an independent prognostic marker for patients after hepatocellular carcinoma resection. World J Gastroenterol. 2002;8(3):459-463. doi:10.3748/wjg.v8.i3.459
14. 14. Wang C, Sun M, Shao C, et al. A multidimensional atlas of human glioblastoma-like organoids reveals highly coordinated molecular networks and effective drugs. npj Precis. Onc. 2024;8:19. doi:10.1038/s41698-024-00500-5
15. 15. Moscow JA, Fojo T, Schilsky RL. The evidence framework for precision cancer medicine. Nat. Rev. Clin. Oncol. 2018;15, 183–192. doi:10.1038/nrclinonc.2017.186
16. 16. Marquart J, Chen EY, Prasad V. Estimation of the percentage of US patients with cancer who benefit from genome-driven oncology. JAMA Oncol. 2018;4:1093-1098. doi:10.1001/jamaoncol.2018.1660
17. 17. Brennan CW, Verhaak RG, McKenna A, et al. TCGA Research Network: The somatic genomic landscape of glioblastoma. Cell. 2013;24;157(3):753. doi:10.1016/j.cell.2013.09.034
18. 18. Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 2008;455(7216):1061–1068. doi:10.1038/nature07385
19. 19. Louis DN, Perry A, Wesseling P,et al. The 2021 WHO Classification of Tumors of the Central Nervous System: a summary. Neuro Oncol. 2021;23(8):1231-1251. doi:10.1093/neuonc/noab106
20. 20. Wang LB, Karpova A, Gritsenko MA, et al. Clinical Proteomic Tumor Analysis Consortium: Proteogenomic and metabolomic characterization of human glioblastoma. Cancer Cell. 2021;39(4):509-528.e20. doi:10.1016/j.ccell.2021.01.006
21. 21. Venteicher AS, Tirosh I, Hebert C, et al. Decoupling genetics, lineages, and microenvironment in IDH-mutant gliomas by single-cell RNA-seq. Science. 2017;355(6332):eaai8478. doi:10.1126/science.aai8478
22. 22. Wang X, Sun Y, Zhang DY, Ming GL, Song H. Glioblastoma modeling with 3D organoids: progress and challenges. Oxf Open Neurosci. 2023;2:kvad008. doi:10.1093/oons/kvad008.
23. 23. Shamir ER, Ewald AJ. Three-dimensional organotypic culture: experimental models of mammalian biology and disease. Nat Rev Mol Cell Biol. 2014;15(10):647-664. doi:10.1038/nrm3873
24. 24. Hubert CG, Rivera M, Spangler LC, et al. A three-dimensional organoid culture system derived from human glioblastomas recapitulates the hypoxic gradients and cancer stem cell heterogeneity of tumors found in vivo. Cancer Res. 2016;76:2465-77. doi:10.1158/0008-5472.CAN-15-2402
25. 25. Taurin S, Alzahrani R, Aloraibi S, Ashi L, Alharmi R, Hassani N. Patient-derived tumor organoids: A preclinical platform for personalized cancer therapy. Transl Oncol. 2025;51:102226. doi:10.1016/j.tranon.2024.102226
26. 26. Bleijs M, van de Wetering M, Clevers H, Drost J. Xenograft and organoid model systems in cancer research. EMBO J. 2019;38(15):e101654. doi: 10.15252/embj.2019101654