Fig 1.
The Collected Drug Library HTS in RB Y79 and retinal epithelial RPE-1 cells. a Schematic of the primary screening workflow.
Dose-response curves of (b, c) gemcitabine and (d, e) irinotecan in (b, d) Y79 and (c, e) RPE-1 cells at 37 °C (black) and 42°C (yellow). N = 3, mean and SD values are shown.
Table 1.
Cytotoxicity (expressed as IC50 values) of the 32 primary hit drugs.
Table 2.
ZIP average synergism scores (δavg) of the selected drug combinations in Y79 and RPE-1 cells under normal and hyperthermic conditions in comparison with the clinically used combinations.
Fig 2.
Identification of the most potent drug combination for the treatment of RB.
Heat maps of cell viability inhibition (left) and ZIP synergy scores (right) for the combinations of carboplatin with (a) gemcitabine and (b) etoposide in Y79 cells. c Cell viability inhibition (bars) and ZIP synergy scores (dots) for the combination of 3.3 µM carboplatin and 2.5 µM of either gemcitabine, irinotecan, aminacrine, trifluridine, etoposide or vincristine in Y79 and RPE-1 cells. d Cell viability of 1 µM carboplatin with 0–100 nM gemcitabine in Y79 and RPE-1 cells; unpaired t-test. e Cell viability of 0 - 100 µM carboplatin with 0–100 nM gemcitabine in Y79 cells; two-way ANOVA with Dunnett test. f Heat maps of cell viability inhibition (left) and ZIP synergy scores (right) for the combination of 0–1000 nM carboplatin and 0–100 nM gemcitabine in Y79 cells. N = 3, p < 0.05(*), mean and SD values are shown.
Fig 3.
Effects of gemcitabine and carboplatin on DNA synthesis. A Inhibition of DNA synthesis determined using the EdU incorporation assay for gemcitabine and carboplatin alone and their combination, cell nuclei (blue) and EdU (green) are shown, scale bars 100 µm, representative images of three independent experiments are shown. b Quantification of the EdU incorporation, more than 100 cells per condition were analyzed using Fiji software.
Mean and SEM are shown, N = 3, unpaired t-test, p < 0.05 (*).
Fig 4.
Efficacy of gemcitabine and carboplatin-gemcitabine. a Eye survival curves of Y79 xenografts after intravitreal melphalan (blue), intravitreal gemcitabine (red), intravitreal gemcitabine plus systemic carboplatin (orange), and vehicle (black). b Proportion of tissues infiltrated with retinoblastoma cells according to treatment groups.
A total of 5 animals (5 brains, 10 optic nerves) were analyzed per treatment group.