(M1230-06-33) Evaluation of Anti-Proliferative Effect of Vorinostat (a pan-HDAC Inhibitor) on Glioblastoma Multiforme Using 3-D Cell Culture Model of T98G

Purpose: Glioblastoma Multiforme (GBM) is a Malignant brain tumor, accounting for a total of 47.7% of CNS tumors with a critically high incidence rate of 3.2-4 per 100,000 persons/ year. The tumor originates within astrocytes due to mutations in Isocitrate dehydrogenase (IDH) leading to altered native substrate affinity, its conversion to harmful metabolite and subsequent effects like blockading dioxygenases to HIF-⍺ activation (Hanif et al., 201701). Current treatments involve craniotomy (bulk tumor removal), radiation therapy & chemotherapy (with Temozolomide, Lomustine & Bevacizumab). Despite the presence of standardized regimens, GBM still has high mortality, with a patient median survival rate of 1-1.5 years post prognosis (Davis, 2016). Most pre-clinical models evaluate chemotherapeutic efficacy of molecular leads against GBM via use of monolayer cultures of cell lines like T-98G, U251 & U87MG. While these studies do give an idea of cytotoxicity towards specific Glioblastoma genotypes, they do not examine how leads counter tumor proliferation or epigenetically alter disease progression (Davis, 2016). 3-D cell culture presents an attractive alternative to characterize prospective anti-neoplastic leads in terms of tumor growth, ECM invasion ability, necrotic core occurrence and others. 3D cultures also exhibit better biomarker expression as well as heterogenicity and histopathology that is more comparable to in-vivo conditions, making them better fits for mechanistic studies (Phon et al., 2022). Vorinostat is a class I & II HDAC (Histone deacetylase) inhibitor approved for cutaneous T-Cell lymphoma and is currently being investigated for efficacy in other malignancies (Richon, 2006). Vorinostat functions via multiple mechanisms but its role in countering GBM remains to be fully explored. Our project aims to investigate the mechanism by which Vorinostat exhibits anti-proliferative action towards T-98G spheroids developed using Rotatory Cell Culture Systems (RCCS).

Methods: T-98G glioblastoma cell line was cultured on DMEM media at 37^oC & 5% CO₂. Vorinostat was dissolved in DMSO to prepare 10 mg/ml solution. Various working solutions in media were prepared via serial dilutions. MTT cytotoxicity assay was performed by seeding wells of a 96-well plate at a seeding density of 5000 cells per well. After overnight incubation, the cells were treated with either media or working solutions of Vorinostat in DMEM (n = 3). After predetermined timepoints (48 & 72 hours), media is changed in all wells, along with addition of 10 µl MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reagent. After 4 hours of incubation at 37^oC, 100 µl detergent reagent is added to each well (to breakdown formazan), & place in dark for 4 hours. Finally, absorbance is measured at 570 nm. For RCCS culture, T98G cells were trypsinized and viable cells are counted using Trypan Blue method. A 10-12 ml cell suspension with seeding density of 10⁶ cells/ ml is made and slowly drawn into a 10 ml High Aspect Rotating Vessel (HARV) using a syringe. The HARV is then placed inside a rotator within a CO₂ incubator and rotated at 15 rpm for a minimal of 72 hours or till spheroids are visible (media changed at 75% change rate). The spheroids were transferred via encapsulation of cell suspension with Vitrogel hydrogel solution in a 1:2 ratio, followed by letting the gel set at 37^oC for 15 mins. Media is added from top and changed at 50-85% change rate. (Figure1) Fluorescence Microscope: The effect of various concentrations of Vorinostat on 3-D embedded spheroids was assessed using both Acridine Orange (AO) – Propidium Iodide (PI) staining & cytotoxicity assay. The fluorescence staining performed by treating spheroids with designated concentrations of Vorinostat, followed by AO–PI dye treatment. Images were taken from Nikon Widefield Fluorescence microscope & analyzed using ImageJ. Fluorescence intensity was calculated for regions of interest (ROI) having the spheroids, and then measuring mean grey values for split channels of green and red (for AO & PI respectively). The time rate change of % ratio of green to red intensity was used to assess viability over time. Cytotoxicity assay for spheroid culture was done similar to above mentioned MTT assay.

Results: The cytotoxicity experiments on 2-D culture generated an IC50 of 11.34 µM via linear regression. The 3-D culture showed higher IC50 of 33.92 µM (Figure 2). The RCCS showed successful spheroid formation at 72 hours. The spheroids were transferred with Vitrogel and cultured overnight before vorinostat treatment. The 8.5 µM, 11.34 µM vorinostat treatments showed up to 40% viability reduction between 48 & 72 hours. The 14.18 µM vorinostat treatment showed linear decrease in in percentage viability till 48 hours, followed by a sharp increase.

Conclusion: Vorinostat did show viability reduction of T98G 3-D cell culture at the dose 14.18 µM (Treatment 3) specifically at 48-hour time point. In future, doses above 33.92 µM need to be tested on T-98G spheroids for live-dead imaging. Its anti-neoplastic effect can be further characterized using other parameters of Fluorescence Morphometry like Lacunarity, Circularity & Nucleus-to-Cytoplasm (N/C) ratio.

References: 1. Hanif, F., Muzaffar, K., Perveen, K., Malhi, S., & Simjee, S. (201701). Glioblastoma multiforme: A review of its epidemiology and pathogenesis through clinical presentation and treatment. APJCP, 18(1). https://doi.org/10.22034/APJCP.2017.18.1.3
2. Davis, M. (2016). Glioblastoma: Overview of disease and treatment. Clinical Journal of Oncology Nursing, 20(5), S2–S8. https://doi.org/10.1188/16.cjon.s1.2-8
3. Zalles M, Towner RA. Pre-Clinical Models and Potential Novel Therapies for Glioblastomas. In: Debinski W, editor. Gliomas [Internet]. Brisbane (AU): Exon Publications; 2021 Apr 30. Chapter 1. Available from: https://www.ncbi.nlm.nih.gov/books/NBK570707/ doi: 10.36255/exonpublications.gliomas.2021.chapter1
4. Phon, B. W. S., Kamarudin, M. N. A., Bhuvanendran, S., & Radhakrishnan, A. K. (2022). Transitioning pre-clinical glioblastoma models to clinical settings with biomarkers identified in 3D cell-based models: A systematic scoping review. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 145, 112396. https://doi.org/10.1016/j.biopha.2021.112396
5. Richon, V. M. (2006). Cancer biology: Mechanism of antitumour action of vorinostat (suberoylanilide hydroxamic acid), a novel histone deacetylase inhibitor. British Journal of Cancer, 95(S1), S2–S6. https://doi.org/10.1038/sj.bjc.6603463

Acknowledgements: Financial support from School of Pharmacy and Health Professions, Creighton University.
Conflict of Interest Statements: We confirm that there are no conflicts of interest associated with this work.

Figure 1: (a) Representative workflow of RCCS experiment. (b) Images of formed spheroids within HARV.
(c) Inverted microscope image of spheroids encapsulated in Vitrogel, transferred to cell culture ware. They appear with artefacts from spheroids in adjacent Vitrogel layers.


Figure 2: (a) MTT cytotoxicity assay of Vorinostat on 2-D culture of T-98G cells. (IC50 = 11.34 µM). (b) MTT cytotoxicity assay of Vorinostat on 3-D culture of T-98G spheroids (IC50 = 33.92 µM). (c) Fluorescent images of spheroids from different groups after 72 hours exposure to Vorinostat. (d) Live-dead assay characterized using %G/R fluorescence over time. Fluorescence intensity calculated using ImageJ software.