Three-dimensional hydrogel culture systems support growth and determination of chemosensitivity of feline sarcoma and carcinoma cell lines

Abstract

Objective: To evaluate feline injection site-associated sarcoma (FISAS) and oral squamous cell carcinoma (FOSCC) cells in 3-D hydrogel-based cell cultures to determine chemosensitivity to carboplatin at concentrations comparable to those eluted from carboplatin-impregnated calcium sulfate hemihydrate (C-ICSH) beads.

Sample: 2 immortalized cell lines, each from a histologically confirmed primary FISAS and FOSCC.

Procedures: Hydrogels (10% wt/vol) were formed via UV exposure from methacrylamide-functionalized gelatin dissolved in PBSS. For each cell line, approximately 100,000 cells were encapsulated per hydrogel. Three cell-seeded 3-D hydrogels were evaluated for each carboplatin concentration (0, 150, 300, 450, and 600 µM) across 3 experiments. Drug efficacy was assessed by luminescence assay 72 hours after treatment. Growth of tumor cells treated with 300 µM or 600 µM carboplatin was evaluated using live-cell morphology imaging and confocal microscopy at 3, 7, and 14 days after treatment.

Results: Mean half-maximal inhibitory concentration (IC50) values for FISAS and FOSCC cells ranged from 123 to 171 µM and 155 to 190 µM, respectively, based on luminescence assay. Viability at 3, 7, and 14 days for both cell lines at 300 µM carboplatin was 50%, 25%, and 5% and at 600 µM carboplatin was 25%, 10%, and < 5%.

Clinical relevance: 3-D hydrogel cell culture systems supported growth of feline tumor cells for determination of in vitro chemosensitivity. IC50s of each cell line were within the range of carboplatin concentrations eluted from C-ICSH beads. Cells from FISAS and FOSCC cell lines treated with carboplatin showed dose-dependent and time-dependent decreases in viability.

Figure 1—

Mean ± SE luminescence by carboplatin dose for feline injection site-associated fibrosarcoma (FISAS) and feline oral squamous cell carcinoma (FOSCC) cells at 72 hours after carboplatin treatment. Higher concentrations of carboplatin affected the viability of FISAS and FOSCC cells at 72 hours after treatment. Luminscence decreased in all treatment groups with increased carboplatin concentration.

Figure 2—

Dose-response curves and half-maximal inhibitory concentration (IC₅₀) values for FISAS and FOSCC cells at 72 hours. Carboplatin induced dose-dependent decreases in the viability of FISAS and FOSCC cells. The IC₅₀ values for both FISAS (A) and FOSCC (B) cell lines at 72 hours are indicated.

Figure 3—

Laser confocal images of FISAS cells stained with calcein AM (green, live cells) and ethidium homodimer-1 (EthD-1; red, dead cells). Untreated cells (A), cells treated with 300 μM (B), and cells treated with 600 μM (C) carboplatin at 3 days. Untreated cells (D), cells treated with 300 μM (E), and cells treated with 600 μM (F) carboplatin at 14 days. Note the decrease in live cells staining green and increase in dead cells staining red with increasing carboplatin dose and time. Scale = 200 μm.

Figure 4—

Laser confocal images of FOSCC cells stained with calcein AM (green, live cells) and EthD-1 (red, dead cells). Untreated cells (A), cells treated with 300 μM (B), and cells treated with 600 μM (C) of carboplatin at 3 days. Untreated cells (D), cells treated with 300 μM (E), and cells treated with 600 μM (F) of carboplatin at 14 days. Note the decrease in live cells staining green and increase in dead cells staining red with increasing carboplatin dose and time. Scale = 200 μm.