Development of a High-throughput Agar Colony Formation Assay to Identify Drug Candidates against Medulloblastoma

Abstract

Medulloblastoma (MB) is the most common malignant childhood brain cancer. High-risk MB tumours have a high incidence of metastasis and result in poor patient survival. Drug screens, commonly used to identify potential novel therapeutic agents against MB, focus on 2D cell proliferation and viability assays given that these assays are easily adaptable to high-throughput regimes. However, 2D models fail to address invasive characteristics that are crucial to MB metastasis and are thus not representative of tumour growth in vivo. In this study, we developed a 3D 384-well agar colony formation assay using MB cells of molecular subgroup 3 that is associated with the highest level of metastasis. Two fluorescence substrates, resazurin and glycyl-phenylalanyl-aminofluorocoumarin (GF-AFC) that measure cell viability via distinct mechanisms were used to assess the growth of MB cells in the agar matrix. The assay was optimised for seeding density, growth period, substrate incubation time and homogeneity of the fluorescent signals within individual wells. Our data demonstrate the feasibility to multiplex the two fluorescent substrates without detectable signal interference. This assay was validated by assessing the concentration-dependent effect of two commonly used chemotherapeutic agents clinically used for MB treatment, vincristine and lomustine. Subsequently, a panel of plasma membrane calcium channel modulators was screened for their effect on the 3D growth of D341 MB cells, which identified modulators of T-type voltage gated and ORAI calcium channels as selective growth modulators. Overall, this 3D assay provides a reproducible, time and cost-effective assay for high-throughput screening to identify potential drugs against MB.

Keywords: assay development; calcium signalling; drug discovery; high-throughput; medulloblastoma.

Figure 1

Optimisation of cell density and culture time for D341 cells. Representative images were acquired using an IN Cell Analyzer 2200 including (A) colony development over time at 10× magnification and (B) a single colony after 7 days incubation at 20× magnification. Quantitative relative fluorescence level of (C) resazurin, and (D) GF-AFC substrates from cells seeded at four different densities and cultured for 7, 10 and 14 days. Data expressed as mean ± standard deviation from three independent experiments with four replicates each. ns = not significant (p > 0.05), ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA with Dunnett multiple comparisons test compared with the no-cells group). Z’ = Z’-factor.

Figure 2

Optimisation of exposure times for the resazurin and GF-AFC substrates. Plates were incubated with (A) resazurin, or (B) GF-AFC substrates and analysed at different time points. Data are expressed as mean ± standard deviation from three independent assays with four replicates each. ns = not significant (p > 0.05), * p < 0.05, *** p < 0.001, **** p < 0.0001 (one-way ANOVA with Dunnett multiple comparisons test compared with the first timepoint group).

Figure 3

Multiplexing resazurin and protease assays in D341 cells. For the monoplex assay, agar plates were incubated with resazurin substrate (6 h) or GF-AFC substrate (3 h) alone. For the multiplex assay, plates were incubated with resazurin substrate for 3 h followed by GF-AFC substrate in the same well for another 3 h. Plates were analysed for (A) resorufin signal at 560/590 nm excitation/emission and (B) GF-AFC signal at excitation/emission wavelength of 380/505 nm. Data expressed as mean ± standard deviation from three independent experiments with four replicates each. ns = not significant (p > 0.05), t-test with two-tailed comparison.

Figure 4

Signal distribution across a well using resazurin substrate. Data expressed as mean ± standard deviation from three independent assays with four replicates each. (A) Measurements taken from different sections within one Z-position inside a single well (multiple read, 2 × 2 with 250 µm distance from border of the read regions) using Tecan Spark 20 M multimode microplate reader: ns = not significant (p > 0.05), *** p < 0.001, **** p < 0.0001, one-way ANOVA with Dunnett multiple comparisons test compared to reading from the centre. (B) Measurement at different Z-positions within a well ranging from 14600–35500 µm: ns = not significant (p > 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA with Dunnett multiple comparisons test compared to reading at Z-position of 14,600 μm. (C) Top versus bottom reading: **** p < 0.0001, t-test with two-tailed comparison test.

Figure 5

Cytostatic effect of vincristine and lomustine on medulloblastoma (MB) cells. D341 cells (10,000 cells/well in agar) were treated with different concentrations of vincristine (0–100 nM) or lomustine (0–100 µM). After 7 days, plates were analysed using the established multiplex assay and read using plate reader for (A) resazurin and (B) GF-AFC substrates. Data expressed as mean ± standard deviation from three independent assays with four replicates each. ns = not significant (p > 0.05), **** p < 0.0001 (one-way ANOVA with Dunnett multiple comparisons test compared with the non-treated 0 control group).

Figure 6

Effect of plasma membrane calcium channel modulators on substrate-independent growth of MB cells. D341 cells (10,000 cells/well in agar) were treated with DMSO (solvent control) or different concentrations of test compounds from 1 to 30 µM. After 7 days, plates were analysed by quantifying (A) resazurin and (B) GF-AFC substrate fluorescence. Data represent mean ± standard deviation from two independent assays with four replicates each. The control group for both assays is the same and 0.3% DMSO was used as solvent control for all the compounds except for NNC55-0396, where media was used. ns = not significant (p > 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001 (one-way ANOVA with Dunnett multiple comparisons test compared with the 0-control group).