Formation of stable small cell number three-dimensional ovarian cancer spheroids using hanging drop arrays for preclinical drug sensitivity assays

Abstract

Background: Ovarian cancer grows and metastasizes from multicellular spheroidal aggregates within the ascites fluid. Multicellular tumor spheroids are therefore physiologically significant 3D in vitro models for ovarian cancer research. Conventional hanging drop cultures require high starting cell numbers, and are tedious for long-term maintenance. In this study, we generate stable, uniform multicellular spheroids using very small number of ovarian cancer cells in a novel 384 well hanging drop array platform.

Methods: We used novel tumor spheroid platform and two ovarian cancer cell lines (A2780 and OVCAR3) to demonstrate the stable incorporation of as few as 10 cells into a single spheroid.

Results: Spheroids had uniform geometry, with projected areas (42.60×10(3)μm-475.22×10(3)μm(2) for A2780 spheroids and 37.24×10(3)μm(2)-281.01×10(3)μm(2) for OVCAR3 spheroids) that varied as a function of the initial cell seeding density. Phalloidin and nuclear stains indicated cells formed tightly packed spheroids with demarcated boundaries and cell-cell interaction within spheroids. Cells within spheroids demonstrated over 85% viability. 3D tumor spheroids demonstrated greater resistance (70-80% viability) to cisplatin chemotherapy compared to 2D cultures (30-50% viability).

Conclusions: Ovarian cancer spheroids can be generated from limited cell numbers in high throughput 384 well plates with high viability. Spheroids demonstrate therapeutic resistance relative to cells in traditional 2D culture. Stable incorporation of low cell numbers is advantageous when translating this research to rare patient-derived cells. This system can be used to understand ovarian cancer spheroid biology, as well as carry out preclinical drug sensitivity assays.

Keywords: 3D culture; High throughput; Multicellular tumor spheroids; Ovarian cancer; Preclinical drug testing.

Fig. 1

Formation of small cell number A2780 spheroids on hanging drop array plates. (A) Representative phase contrast micrographs of A2780 spheroids on Day 1 and Day 7. Spheroids of A2780 cells were initiated with 10, 20, 50 and 100 cells per drop on hanging drop array plates. Spheroid formation was studied using live cell microscopy. Cells within hanging drops aggregated into a spheroid-like structure on Day 1. At Day 7, tight spheroids with clear boundaries were observed. Scale bar = 100 μm. (B) Projected area of A2780 spheroids. Calibrated images were used to obtain morphometric data at Day 1 and Day 7 to determine spheroid sizes. Areas of A2780 spheroids increased from Day 1 to Day 7 in hanging drop cultures, as a function of the initial cell seeding density. Projected 2D spheroid areas were significantly different (*p < 0.05, one-way ANOVA) on Day 7 between all cell densities. (C) Proliferation in A2780 spheroids. Proliferation within spheroids was assessed using a fluorescence-based alamarblue assay. A fold-increase in alamarblue fluorescence intensity was observed in all spheroids. Regardless of initial cell seeding densities, proliferation index varied non-significantly from 9 fold to 11 fold over 7 days in hanging drop culture.

Fig. 2

Formation of small cell number OVCAR3 spheroidson hanging drop array plates. (A) Representative phase contrast micrographs of OVCAR3 spheroidson Day 1 and Day 7. Spheroids of OVCAR3 cells were initiated with 10, 20, 50 and 100 cells on hanging drop arrays. Live cell microscopy was used to monitor spheroid formation, and calibrated images were used to obtain spheroid area measurements indicative of their size. Cells within hanging drops aggregated to form spheroid-like structures by Day 1, and continued to aggregate and form tightly packed spheroids with defined boundaries by Day 7. Scale bar = 100 μm. (B) Projected area of OVCAR3 spheroids. Area varied as a function of initial cell seeding density, and was significantly higher at Day 7 compared to Day 1 (*p < 0.05, one-way ANOVA). (C) Proliferation in OVCAR3 spheroids. Proliferation of cells within spheroids was assessed using a fluorescence-based alamarblue assay. Fold increase in alamarblue fluorescence varied non-significantly from 10 fold to 11 fold over 7 days in 3D culture.

Fig. 3

Viability of cells within multicellular ovarian cancer spheroids. (A–D) Live/Dead staining on A2780 spheroids with varying cell densities, with minimal red/dead cell staining. Following 7 days in hanging drop array culture, A2780 or OVCAR3 spheroids were incubated with calcein-AM and ethidium homodimer. Live cells within spheroids were indicated by green fluorescence for calcein-AM, while dead cells were indicated by red fluorescence for ethidium homodimer. Confocal microscopy was used to image calcein and ethidium homodimer fluorescence through the height of the spheroids. (E) Quantification of Live/Dead staining in A2780 spheroids. A bar graph representation of the percentage of live and dead cells within the different spheroids is depicted. Excellent viability was observed, with <15% of cells staining red. (F–I) Live/dead staining on OVCAR3 spheroids with varying cell densities. (J) Quantification of Live/Dead staining in OVCAR3 spheroids. Bar graph representation of the percentage of live and dead cells within the OVCAR3 spheroids is depicted. On an average, <12% of the cells stained red for ethidium homodimer. Scale bar = 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Three-dimensional structure of multicellular ovarian cancer spheroids. (A–D) Three-dimensional structure of A2780 spheroids plated with 10, 20, 50 or 100 cells/drop. Actin cytoskeletons of cells within spheroids were stained at Day 7 by incubation with fluorescently conjugated phalloidin. Nuclei were counter stained with DAPI. Stained spheroids were observed on the confocal microscope, and merged images are presented. (E–H) Three-dimensional structure of OVCAR3 spheroids plated with 10, 20, 50 or 100 cells/drop. Three-dimensional morphology observed with cortical actin cytoskeleton within cells and spanning cell–cell interactions. DAPI immunostaining confirms the self-assembly into tight spheroid three-dimensional structure. Scale bar = 100 μm.

Fig. 5

Chemoresistance in A2780 spheroids in response to cisplatin treatment. (A) Decrease in projected area in cisplatin treated A2780 spheroids. A2780 spheroids on hanging drop arrays were treated with varying doses of cisplatin. Morphological changes in spheroids were observed using live cell microscopy, and morphometry was performed to note a change in spheroid area with cisplatin treatment. Spheroid areas were reduced in cisplatin treated spheroids compared to untreated control A2780 spheroids. At the 50 μM cisplatin dose, 10 cell spheroids and 20 cell spheroids dropped significantly in area by 30% and 22.27% respectively (***p < 0.001, one-way ANOVA) of the control untreated spheroids. (B) Viability and chemoresistance in A2780 spheroids. Alamarblue fluorescence was utilized to monitor viability in A2780 spheroids treated with cisplatin compared to untreated A2780 spheroids. Compared to the 2D conventional 96-well cultures, three-dimensional spheroids demonstrated higher viability (***p < 0.001, two-way ANOVA) at the 50 μM cisplatin dose. (C) Representative phase contrast micrographs of untreated control and 50 μM cisplatin treated spheroids within hanging drop arrays.

Fig. 6

Chemoresistance in OVCAR3 spheroids in response to cisplatin treatment. (A) Decrease in projected area in cisplatin treated OVCAR3 spheroids. OVCAR3 spheroids on hanging drop arrays were treated with varying doses of cisplatin. Morphological changes in spheroids were observed using live cell microscopy, and morphometry was performed to note a change in spheroid area with cisplatin treatment. Spheroid areas were reduced in cisplatin treated spheroids compared to untreated control OVCAR3 spheroids. At the 50 μM cisplatin dose, 10-, 20-, and 50-cell spheroids dropped significantly in area by 15–35% (**p < 0.01, one-way ANOVA) of the control untreated spheroids. (B) Viability and chemoresistance in OVCAR3 spheroids. Alamarblue fluorescence was utilized to monitor viability in OVCAR3 spheroids treated with cisplatin compared to untreated OVCAR3 spheroids. At the 50 μM cisplatin dose, compared to the 2D conventional 96-well cultures, three-dimensional spheroids demonstrated higher viability (*p < 0.05, **p < 0.01, two-way ANOVA). (C) Representative phase contrast micrographs of untreated control and 50 μM cisplatin treated spheroids within hanging drop arrays.