Background: VPA is an antiepileptic drug used to treat tumor-related seizures. TTFields is an antimitotic therapy that is FDA-approved for GBM. Since GBM patients may use both therapies, we examined the combination of TTFields and VPA in vitro using GBM cells isolated from a newly-diagnosed GBM patient. Cells proliferation and clonogenic potential were assessed after cells were exposed to VPA during TTFields application.

Methods: A single-cell suspension was generated from a patient tumor (male, age 64, IDH-WT, no 1p19q deletion, EGFR not amplified, unmethylated MGMT promoter). Cells were cultured in DMEM/F12 media with 10% FBS and gentamicin. Cells were plated on plastic coverslips (1×104cells/coverslip) and incubated overnight. Coverslips were divided into 8 groups (Control, 250 µM, 500 µM, 1 mM VPA, ± TTFields; n=5/group). TTFields were applied at 200 kHz with a field intensity of ~1.6 V/cm for 14 days with VPA in the media as indicated. Cells were harvested, stained and counted with a Cellometer K2 (Nexcelom Bioscience, Lawrence, MA) that provides viable cell counts. For each group, 2000 cells each from 3 coverslips were plated for the clonogenic assay (CA). After 2 weeks, cells were crystal violet stained, imaged and counted. Groups were compared by ANOVA (GraphPad Prism v9, GraphPad Software, San Diego, CA) with significance at P<0.05.

Results: Cell count and CA data are in the Table. TTFields alone reduced cell counts by >50%, and reduced colony counts and area in the CA. VPA alone was only effective at the 1 mM concentration and reduced the cell count by 40%. Combination of TTFields and 1 mM VPA reduced the cell counts by 95%. However, VPA alone and combined with TTFields increased colony number and area in the CA.

Conclusions: Although the combination of VPA and TTFields (200 kHz) produced a greater decrease in GBM cell proliferation, VPA increased the clonogenic potential and may hinder the response to TTFields.

Cell counts and clonogenic assay data

Cell countsControl250 μM VPA500 μM VPA1 mM VPATTFieldsTTFields + 250 μM VPATTFields + 500 μM VPATTFields + 1 mM VPAANOVA P-value
Mean 206100 212100 231400 123400 77220 42160 45290 9193 <0.0001 
Std. Deviation 73065 70637 71366 21001 38736 7754 35910 8910  
Clonogenic assay colony counts          
Mean 31.29 43.89 40.56 63.56 18.33 11.67 30.56 36.33 <0.0001 
Std. Deviation 29.06 17.69 19.55 14.94 8.66 10.82 20.25 16.56  
Clonogenic assay colony area (pixels)          
Mean 1177 2494 1977 3070 394.8 202.9 787.6 854.7 <0.0001 
Std. Deviation 1485 1250 1371 987.5 225.9 206.9 548.3 438.8  
Cell countsControl250 μM VPA500 μM VPA1 mM VPATTFieldsTTFields + 250 μM VPATTFields + 500 μM VPATTFields + 1 mM VPAANOVA P-value
Mean 206100 212100 231400 123400 77220 42160 45290 9193 <0.0001 
Std. Deviation 73065 70637 71366 21001 38736 7754 35910 8910  
Clonogenic assay colony counts          
Mean 31.29 43.89 40.56 63.56 18.33 11.67 30.56 36.33 <0.0001 
Std. Deviation 29.06 17.69 19.55 14.94 8.66 10.82 20.25 16.56  
Clonogenic assay colony area (pixels)          
Mean 1177 2494 1977 3070 394.8 202.9 787.6 854.7 <0.0001 
Std. Deviation 1485 1250 1371 987.5 225.9 206.9 548.3 438.8  

Citation Format: Sharon K. Michelhaugh, Susan Coombe, Katherine E. Degen, Sandeep Mittal. Valproic acid (VPA) combined with Tumor Treating Fields (TTFields) in vitro decreases cellular proliferation and increases clonogenic potential of glioblastoma (GBM) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1037.