Development and Characterization of 3-Dimensional Cell Culture Models of Adrenocortical Carcinoma

Abstract

Adrenocortical carcinoma (ACC) is a rare malignancy of the adrenal cortex that is associated with a poor prognosis. Developing effective treatment options for ACC is challenging owing to the current lack of representative preclinical models. This study addressed this limitation by developing and characterizing 3-dimensional (3D) cell cultures incorporating the ACC cell lines, MUC-1, HAC15, and H295R in a type I collagen matrix. ACC tissue samples were analyzed by immunohistochemistry to determine the presence of type I collagen in the tumor microenvironment. Cell viability and proliferation were assessed using flow cytometry and confocal microscopy. mRNA expression of steroidogenic enzymes and steroid secretion was analyzed by comparing the 3D and monolayer cell culture models. All cells were successfully cultured in a type I collagen matrix, which is highly expressed in the ACC tumor microenvironment and showed optimal viability until day 7. All 3 models showed increased metabolic and proliferative activity over time. Three-dimensional cell cultures were steroidogenic and demonstrated increased resistance to the gold standard chemotherapy, mitotane, compared with monolayer. The use of these models may lead to an improved understanding of disease pathology and provide a better representative platform for testing and screening of potential therapies.

Keywords: 3D cell culture models; adrenocortical carcinoma; drug resistance; preclinical models.

Figure 1.

ACC cells express type I collagen in their extracellular matrix. (A) Graphical representation of mean staining intensity in adrenocortical carcinoma (ACC) tissues (n = 4) vs skin (n = 3). (B) Graphical representation of the mean staining intensity in ACC tissues vs non-aldosterone–producing adenoma (NAPACA) (n = 2), phaeochromocytoma (PC/PGL) (n = 3), and aldosterone-producing adenoma (APA) (n = 3) tissues. (C) Immunohistochemistry images of skin, ACC, NAPACA, phaeochromocytoma, and APA. Data are represented as the mean ± SEM. Statistical comparisons were performed using (1) unpaired t test (skin vs ACC) and (2) 1-way ANOVA followed by post hoc analysis when comparing the expression between adrenal tissues.

Figure 2.

ACC cells in 3D cell culture maintain viability over 21 days in culture: (A-C) Cell viability in H295R, HAC15, and MUC-1 cells in 3D cell culture vs monolayer cell culture measured using flow cytometry analysis of Sytox Blue staining. Representative histograms and gating strategies are presented elsewhere (Fig. S1 (64)). (D) Representative confocal microscopy images using calcein (green) and propidium iodide (red/no stain) demonstrated high levels of cell viability in all 3 cell lines at day 7. HAC15 cells showed a visible necrotic core on day 14. Scale bar: 500 mm. Data are represented as the mean ±SEM. Statistical comparisons were performed using 2-way ANOVA followed by post hoc analysis compared to monolayer. Statistical significance is denoted as *P < .05, **P < .01, ****P < .0001.

Figure 3.

ACC cells are steroidogenic when cultured in 3D cell culture models: cortisol, aldosterone and androstenedione secretion following stimulation with FSK (10 mM) (cortisol and androstenedione secretion), or with ANGII (10 nM) (aldosterone secretion) measured using HPLC tandem mass spectrometry cortisol secretion for H295R (A) and HAC15 (B). Aldosterone secretion for H295R (C) and HAC15 (D). Androstenedione secretion for H295R (E), HAC15 (F), and MUC-1 (G). Data are represented as the mean ±SEM. Statistical comparisons were performed using 2-way ANOVA followed by post hoc analysis compared with monolayer. Statistical significance is denoted as *P < .05, **P < .01, ***P < .001, ****P < .0001.

Figure 4.

ACC cells proliferate and have high metabolic activity in 3D cell culture. (A-C) Metabolic activity in H295R, HAC15, and MUC-1 cells in 3D cell culture measured using alamarBlue staining. (D-F) Comparison between metabolic activity in monolayer vs 3D cell culture for each cell line measured using alamarBlue staining. Ki67 positive (+) and Ki67 (−) expression in (G and J) H295R, (H and K) HAC15, and (I and L) MUC-1 3D and monolayer cell culture models, respectively. Data are represented as mean±SEM. Statistical comparisons were performed using 2-way ANOVA followed by post hoc analysis. Statistical significance is denoted as *P < .05, **P < .01, ***P < .001, ****P < .0001. Data are representative of 3 independent experiments.

Figure 5.

ACC cells are resistant to mitotane when treated in 3D cell culture compared to monolayer cell culture: (A-C) % live cells of H295R, HAC15, and MUC-1 cells in 3D cell culture vs monolayer measured using flow cytometry analysis of Sytox blue staining. Data are represented as mean ± SEM. Statistical comparisons were performed using 2-way ANOVA followed by post hoc analysis compared with monolayer. Statistical significance is denoted as *P < .05, ****P < .0001. Data are representative of 3 independent experiments.