ENDOCRINE SOCIETY
OXFORD
Development and Characterization of 3-Dimensional Cell Culture Models of Adrenocortical Carcinoma
Sarah Feely,10 Nathan Mullen, 10D Padraig T. Donlon,1 Eileen Reidy,1 Ritihaas Surya Challapalli,1 Mariam Hassany,1 Anna Sorushanova,1 Eduardo Ribes Martinez,1,2 Peter Owens,3
Anne Marie Quinn,4 Abhay Pandit,2 Brendan Harhen,5 David P. Finn,1,2 Constanze Hantel,6 Martin O’Halloran,2,7 Punit Prakash,8 and Michael C. Dennedy 1,2(D
1Discipline of Pharmacology and Therapeutics, School of Medicine, University of Galway, Galway, H91 V4AY, Ireland
2Science Foundation Ireland (SFI) Research Centre for Research in Medical Devices (CURAM), Biomedical Science Building, University of Galway, Galway, H91 TK33, Ireland
3Centre for Microscopy and Imaging, Anatomy, School of Medicine, University of Galway, Galway, H91 TK33, Ireland
4Department of Anatomic Pathology, Galway University Hospital, Galway, H91 YR71, Ireland
5Biological Mass Spectrometry Core Facility, University of Galway, Galway, H91 TK33, Ireland
6Department of Endocrinology, Diabetology and Clinical Nutrition, University Hospital Zurich (USZ) and University of Zurich (UZH), 8091 Zurich, Switzerland
“Translational Medical Device Laboratory, University of Galway, Galway, H91 V4AY, Ireland
8Department of Electrical and Computer Engineering, Kansas State University, Manhattan, KS 66506, USA
Correspondence: Michael Conall Dennedy, MD, PHD, FRCPI, Discipline of Pharmacology and Therapeutics, 3.13 Lambe Institute for Translational Research, School of Medicine, University of Galway, Costello Road, Galway, H91 V4AY, Ireland. Email: dennedym@universityofgalway.ie.
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.
Key Words: 3D cell culture models, adrenocortical carcinoma, preclinical models, drug resistance
Abbreviations: 3D, 3-dimensional; ACC, adrenocortical carcinoma; Ang II, angiotensin II; ECM, extracellular matrix; FSK, forskolin; HPLC, high-performance liquid chromatography; STR, short tandem repeat.
Adrenocortical carcinoma (ACC) is a rare and aggressive ma- lignancy, with an incidence of 1 to 2 per million people annu- ally and a poor prognosis, with a median overall survival of approximately 24 months (1-3). Most cases are diagnosed at an advanced stage with local spread or metastases (4, 5). The only curative option is R0 surgical resection of limited dis- ease, ENS@T Stage 1 to 2 (1, 6). Recurrent disease is typically managed with adjuvant mitotane or systemic chemotherapy (cisplatin/etoposide/doxorubicin or streptozocin), although both regimens have low recurrence-free and overall survival rates (7, 8).
ACC presents several research and clinical challenges. Chemotherapy response rates are poor and obtaining clinical
samples or primary cells for research is difficult due to its rarity (7, 9-15). Moreover, primary cells do not adhere or proliferate easily in cell culture (9-15). While several steroidogenic ACC cell lines, such as MUC-1, H295R, and HAC15, have become available, only 2 are commercially accessible (H295R and HAC15) (16-22). Our laboratory works with these 3 cell lines, including MUC-1, which is notable for its resistance to mito- tane (18). Reliable preclinical ACC models remain limited, and currently rely mostly on monolayer cell culture or xeno- grafts (14, 17, 18, 23-32). There is therefore a pressing need for improved models, preferably animal-sparing, to advance drug screening and understand mechanisms of cell survival and death in ACC.
ACC tumors are large, typically over 6 cm, often necrotic, highly proliferative (Ki67-high), and steroidogenic (5, 33-35). Hormonal excess, particularly cortisol, leads to Cushing syndrome, worsening prognosis due to complications like hyper- tension and immunosuppression (36). Androgen excess is also common, while mineralocorticoid excess is rare (37). Radiological diagnosis of ACC is aided by biochemical testing of steroid excess including urine steroid metabolomics, which de- tect adrenocortical steroid precursors (38-41). Histologically, ACC is evaluated using the Weiss Score and Ki67 score, both im- portant prognostic markers, where higher scores indicate higher recurrence and worse outcomes overall (1, 33, 42).
Preclinical models of ACC should reflect the disease’s hetero- geneity, including steroidogenesis, varied proliferation rates, central necrosis, and chemotherapy resistance. Current research still relies heavily on monolayer cell cultures (H295R and HAC15), which fail to fully mimic the tumor microenvironment and are limited in studying key interactions between tumor cells, stroma, and the extracellular matrix (ECM) (43, 44). Monolayer cell cultures also lack areas of confluent necrosis and do not ef- fectively model invasion or migration (45, 46). In contrast, 3-di- mensional (3D) cell culture models better replicate the tumor microenvironment, exhibiting more representative growth pat- terns, necrosis, and hypoxia, and allow for modeling of increas- ing complexity which can mimic interactions between cancer cells and immune, stromal, and stem cells (46-50). Complex microfluidic systems can also be used to further model vascular invasion and the endothelial barrier and to improve the transla- tional relevance of these models for drug screening (51).
While recent advances have led to the development of 3D spheroid and coculture models for ACC, a significant knowledge gap remains. Few models incorporate ECM components or com- plex multicellular interactions (28, 52-63). Previous studies have used only Matrigel and hyaluronic acid/gelatin to model the ECM and there has been no previously published work which has modeled collagen as a more biologically relevant ECM (55, 57, 59). In the current study we address this knowledge gap by using type I collagen, abundant in ACC, to offer a more transla- tionally relevant system for evaluating tumor response to therapy with the aim of providing an animal-sparing approach to drug testing. We have specifically developed a 3D cell culture model for ACC which uses 3 human ACC cell lines embedded in a type I collagen matrix which is cross-linked to represent tumor stiffness. We describe a model which offers the potential for the addition of greater complexity to incorporate stromal and im- mune elements to better represent the tumor microenvironment as an animal sparing preclinical drug screening model of ACC.
Materials and Methods
Clinical Sample Collection
Paraffin-embedded tumor tissue and clinical details were col- lected from patients undergoing adrenalectomy at University Hospital Galway between 2014 and 2024, including samples from ACCs, adrenal adenomas, and pheochromocytomas. All samples were obtained with informed consent, and ethics ap- proval was provided by the University Hospital Galway Research Ethics Committee (ref. C.A. 3194).
Immunohistochemistry
Collagen expression in normal and tumor tissues was evaluated via immunohistochemistry. Nonspecific binding was blocked
using horse serum (Vector Laboratories, catalog # S-2012-50) for 1 hour and sections were stained with a type I collagen antibody (Thermofisher, catalog # PA1-26204, RRID:AB_ 2260734). Slides were incubated with a horseradish peroxidase- conjugated secondary antibody (Vector Laboratories, catalog # MP-7500, RRID:AB_2336534) and visualized with DAB (Vector Laboratories, catalog # SK-4105, RRID:AB_2336520) before hematoxylin counterstaining. A histopathologist, blinded to diagnosis, reviewed all slides.
Cell Culture
Monolayer
H295R (primary ACC tumor) (NCI-H295R, RRID: CVCL_0458), HAC15 (H295R subclone) (CRL-3301, RRID: CVCL_S898), and MUC-1 (metastatic ACC) cell lines were maintained in different media specific to each line (16, 18-22). Cells were cultured in a humidified atmosphere with 5% CO2 at 37 ℃ and used between specified passages. All cell lines were validated using short tandem repeat (STR) profiling.
Three-dimensional cell culture
Three-dimensional cell culture models were generated by em- bedding cells in a bovine type I collagen matrix(Collagen Solutions, catalog # FS22005), mixed with 4arm PEG succini- midyl glutarate (pentaerythritol) (Jenkem USA, catalog # A7031) and cells, forming a gel. Each cell line was seeded in the collagen matrix and incubated at 37 ℃ with 5% CO2. The composition of the 3D culture matrix was based on pre- viously optimized concentrations (unpublished work).
Cellular viability
Cell viability in 3D cell cultures was assessed using Calcein-AM (Merck, catalog # 17783) (8 mM) and propidium iodide (Miltenyi Biotec, catalog # 130-093-233) (5 µg/mL) and visual- ized with confocal microscopy. Flow cytometry using Sytox blue (Invitrogen catalog S 34857) was employed for quantita- tive analysis of cell death, with monolayer and 3D cell cultures dissociated and stained at days 3, 7, 14, and 21. Flow cytometry data was analyzed using De Novo software FCS express V 7.14.0020 or FlowJo v10.8 software (BD Life Sciences). The gating strategy is illustrated elsewhere (Fig. S1 (64)).
Proliferation and metabolic activity
Ki67 antibody (Biolegend, catalog # 151210, RRID: AB_2716008) staining was used to evaluate proliferation in both monolayer and 3D cell cultures, followed by flow cy- tometry. Metabolic activity was assessed using alamarBlue (ThermoFisher, catalog # DAL1025) fluorescence assays, comparing the activity of monolayer and 3D cell cultures spec- trophotometrically excited at a wavelength of 590/40 nm us- ing the Hidex Sense Microplate Reader.
Steroid quantification
Steroid secretion (aldosterone, cortisol, and androstenedione) was stimulated with 10 nM angiotensin II (Ang II) (Merck, cata- log # A9525-1MG) and 10 µM forskolin (FSK) (Merck, catalog # F6886, 10MG) for 24 hours in monolayer and 3D cell cultures, with media supernatants collected and stored for ana- lysis as previously described (16, 65). Steroid levels were quanti- fied via high-performance liquid chromatography (HPLC)
coupled to tandem mass spectrometry, with internal standards and standard curves used for concentration calculations.
Gene expression
Semiquantitative reverse transcription polymerase chain reac- tion was used to measure steroidogenic enzyme expression (CYP11B1, CYP11B2, and StAR) as previously described (65). RNA was isolated from monolayer and 3D cell cultures, reverse-transcribed, and analyzed using real-time polymerase chain reaction with SYBR green detection. Analysis was per- formed using the 44CT method on the QBase + analytical software, Version 3.3 (Qiagen), against a geometric mean of 2 reference genes (RPLPO and PMM1) in monolayer. Data are represented as the fold change in mRNA relative to the monolayer cell culture control at day 7.
Drug treatments
Cells in monolayer and 3D cell cultures were treated with vari- ous concentrations of mitotane (Merck, catalog SML1885) for 24 or 48 hours, respectively. A DMSO (Merck, catalog # D2650) vehicle control was included, and treatment was ad- ministered after 5 days of 3D cell culture.
Statistical Analysis
Data are presented as mean ± SEM. Paired sample analyses were conducted using 1-way analysis of variance (ANOVA), with multiple-group comparisons analyzed using 2-way ANOVA and post hoc tests. Statistical significance was set at P <. 05 unless stated otherwise, indicated on graphs as *P <. 05, ** P <. 01, and *** P <. 001.
Detailed methodology is available elsewhere (Supplementary Materials section 1 (64)).
Results
ACC Cells Express Type I Collagen in Their Extracellular Matrix
Type I collagen is 1 of the most highly expressed proteins in the tumor microenvironment in various cancer types and rep- resents the most suitable matrix for generating 3D cancer models (66). To verify its suitability for 3D cell culture of ACC, we measured the expression of type I collagen in human ACC and benign adrenal adenomas using immunohistochem- istry. Type I collagen was strongly expressed in the tumor microenvironment of ACC (Fig. 1A). There was no difference in staining intensity between the ACC tissues and control skin tissue samples (Fig. 1A). Type I collagen was also equally ex- pressed in the tumor stroma of non-aldosterone-producing adenomas, phaeochromocytomas, and aldosterone producing adenomas, with no significant difference between each tissue type (Fig. 1B). However, expression of type I collagen was not observed in normal adrenal tissue.
ACC Cells in 3D Cell Culture Maintain Viability Over 21 Days in Culture
Next, we evaluated the viability of each ACC cell line (MUC-1, H295R, and HAC15) in 3D cell culture on days 7 and 14 using confocal imaging and on days 3, 7, 14, and 21 using flow cytometry. Flow cytometry analysis demon- strated that all 3 cell lines were viable in 3D cell culture, with the highest viability observed during the first 7 days. The viability of H295R and MUC-1 cells decreased
significantly at days 14 and 21 (Fig. 2A and 2C), while HAC15 cells demonstrated lower baseline viability than the other cell lines but remained stable over 21 days (Fig. 2B). Confocal microscopy confirmed cell viability in all 3 cell lines (Fig. 2D), though an increase in the number of dead cells was observed over time. H295R cells showed areas of con- fluent necrosis on days 7 and 14. Interestingly, HAC15 cells demonstrated a necrotic core in 3D cell culture. However, this was not observed when the cells were analyzed by flow cytom- etry, indicating that the necrotic core is lost upon extraction from the collagen matrix (Fig. 2D).
When comparing the viability of cells in 3D cell cultures vs monolayer cell cultures, H295R and HAC15 cells showed lower viability in 3D cell culture at all time points (Fig. 2E and 2F). MUC-1 cells also showed lower viability in 3D cell culture, but only at days 14 and 21 (Fig. 2E).
H295R and HAC15 ACC Cells are Steroidogenic When Cultured in 3D Cell Culture Models
Steroidogenesis occurs in most ACC patients in vivo (35, 38). In monolayer cell culture, HAC15 and H295R ACC cell lines are well described as steroidogenic at baseline and under stimulated conditions (65). Steroidogenesis in MUC-1 cells is less well characterized with conflicting results in the litera- ture (16, 18). We investigated whether each cell line demon- strated a steroidogenic milieu in monolayer and in 3D cell culture by measuring the secretion of cortisol, androstene- dione, and aldosterone, as well as mRNA expression of steroi- dogenic enzymes/mediators.
Under FSK stimulated conditions, on 1 hand, H295R and HAC15 cells secreted significantly more cortisol in monolayer than 3D cell culture for most timepoints measured (Fig. 3A and 3B). MUC-1, on the other hand, did not demonstrate ster- oidogenesis for cortisol or aldosterone in either monolayer or 3D cell culture under stimulated or unstimulated conditions. Androstenedione was present in all 3 cell lines under stimu- lated conditions and was either similar (H295R at all time- points; HAC15 D14, MUC-1 D14) or higher (HAC15 D7, MUC-1 D7) in 3D cell culture than monolayer cell culture. (Figure 3F-3H). Under Ang II stimulation, aldosterone secre- tion was significantly higher in 3D cell culture for H295R (D14) and HAC15 (all timepoints) cells than in monolayer (Fig. 3D and 3E).
Expression of key steroidogenic enzymes, including CYP11B1 and CYP11B2, was measured under stimulated conditions for all cell lines in monolayer and 3D cell culture following 7 and 14 days in cell culture. No significant differ- ence was demonstrated for the expression of these enzymes or StAR protein between monolayer and 3D cell culture or across the measured timepoints (Fig. S2A-H (64)). In MUC-1 cells, there was no expression of either enzyme in line with the absence of steroidogenesis for cortisol and aldos- terone. StAR expression was seen however, with a higher ex- pression in monolayer cell culture compared to 3D cell culture at day 14 only (Fig. S2I (64)).
ACC Cells Proliferate and Have High Metabolic Activity in 3D Cell Culture
Proliferation and metabolic activity of ACC cells were eval- uated in monolayer and 3D cell cultures. Metabolic activity in- creased in all 3 cell lines over time, with the highest fluorescence readings observed at day 14 (Fig. 4A-4C). When comparing
A Type I Collagen Expression in Adrenocortical carcinoma Vs Skin
B Type I Collagen Expression in Adrenal Tumours
C
Mean Intensity (10^3) (AU)
Mean Intensity (10^3) (AU)
ns
25
ns
25
ns
20
20
ns
15
15
10
10
5
5
0
ACC
0
Skin
ACC
NAPACA
PC/PGL
APA
Skin
Adrenocortical Carcinoma
-
.
Non-Aldosterone Producing Adenoma
Phaeochromocytoma
Aldosterone Producing Adenoma
monolayer and 3D cell cultures, all 3 cell lines demonstrated lower metabolic rates in 3D cell culture, except for MUC-1 at day 21 (Fig. 4D-4F).
Cellular proliferation was measured using Ki67 expression, with all 3 cell lines showing a broadly similar proliferation pattern between monolayer and 3D cell cultures. H295R cells in 3D cell culture showed a higher proportion of actively pro- liferating cells across all time points (Fig. 4G). In HAC15 cells, a higher proportion of proliferating cells was observed at day 14 and 21 in 3D cell culture (Fig. 4H), while MUC-1 cells showed greater proliferation in 3D cell culture at days 7 and 14 (Fig. 4I). In contrast, monolayer cell culture showed a
decrease in proliferation over time in MUC-1 and H295R cell lines (Fig. 4J and 4L). This decrease was only seen at day 7 in HAC15 cells (Fig. 4K).
ACC Cells are Resistant to Mitotane When Treated in 3D Cell Culture Compared With Monolayer Cell Culture
Previous studies have demonstrated that cancer cells grown in 3D cell cultures display greater chemotherapy resistance than monolayer cell cultures (67, 68). In this study, the response of each ACC cell line to mitotane was evaluated in 3D cell culture
A
H295R
B
HAC15
C
MUC-1
ns
ns
**
ns
ns
ns
ns
ns
100
*
%Viability
100
100
80
%Viability
Monolayer
80
%Viability
80
60
60
60
3D
40
40
40
20
20
20
0
0
0
3
7
14
21
3
7
14
21
3
7
14
21
Time (Days)
Time (Days)
Time (Days)
D
Day 7
Day 14
H295R
500 pm
500 m
HAC15
MUC-1
500 pm
500 um
and compared with monolayer cell culture. In monolayer cell culture, H295R cells were more sensitive to mitotane than HAC15 or MUC-1 cells (Fig. 5). However, in 3D cell culture, lethal doses of mitotane displayed significantly lower cytotox- icity across all cell lines, requiring much higher doses to achieve cell death (Fig. 5A-5C). Cell death in 3D cell cultures was equal- ly distributed throughout the collagen matrix (Fig. S3 (64)).
Discussion
In this study, we developed and characterized a novel 3D cell culture model of ACC using 3 steroidogenic ACC cell lines
(H295R, HAC15, and MUC-1) embedded in a type I collagen ECM. We validated the use of type I collagen by confirming its expression in the ECM of human adrenocortical tumors, in- cluding ACC. The 3D cell culture model was analyzed for via- bility, steroidogenesis, proliferative activity, and chemotherapy response. While baseline cellular viability in 3D cell culture was lower than in monolayer cell culture, proliferation was comparable, indicating high cell turnover. H295R and HAC15 cell lines remained steroidogenic in 3D cell culture, as demonstrated by the expression of steroidogenic enzymes and steroid secretion. In our hands the metastatic ACC model, MUC-1 cells were steroidogenic for androstenedione only and
Cortisol
A
H295R
B
HAC15
(nmol/L/ 100,000 Cells)
(nmol/L/ 100,000 Cells)
ns
ns
30
60
40
Monolayer
20
20
3D
15
10
10
5
5
0
0
7
14
7
14
Time (Days)
Time (Days)
Aldosterone
C
H295R
D
HAC15
(p/mol/L/100,000 Cells)
400
(p/mol/L/100,000 Cells)
Monolayer
ns
20000
300
3D
10000
200-
100
1000
0
0
7
14
7
14
Time (Days)
Time (Days)
Androstenedione
E
H295R
F
HAC15
G
MUC-1
(nmol/L/ 100,000 Cells)
(nmol/L/ 100,000 Cells)
1.0-
15
(nmol/L/ 100,000 Cells)
ns
ns
*
ns
3.
**
ns
Monolayer
10
0.5
2
3D
5
1
0.0
7
14
0
7
14
0
7
14
Time (Days)
Time (Days)
Time (Days)
did not express CYP11B1 or CYP11B2. Importantly, the cells in 3D cell culture showed reduced mitotane-induced cell death compared to monolayer cell cultures, reflecting a more repre- sentative pharmacological response of ACC in the tumor microenvironment.
ACC is a rare cancer with limited models available for re- search (44). While several steroidogenic ACC cell lines exist, only H295R and HAC15 are available commercially (16-22). More recently MUC-1 cells have provided a better in vitro model for chemotherapy-resistant metastatic disease, and in this study, we demonstrate that HAC15 cells also show mito- tane resistance (18). Primary cell culture remains challenging, and clinical studies are difficult to recruit due to the low inci- dence of ACC (7, 9-15). Finally, few animal models of ACC ex- ist, and most are reliant upon the use of human cell line xenografts (44, 69). Overall, current models of drug screening, especially with monolayer cultures, have not translated well to improved patient outcomes, demonstrating the need to explore novel and improved methodologies for drug screening in this disease (11, 70-72). Three-dimensional cell culture addresses some limitations of monolayer cell culture by offering a more representative tumor microenvironment that includes cancer
cells, stromal cells, and the ECM (43). Collagen, particularly type I collagen, is the most abundant ECM protein, associated with tumor growth and invasion in other cancer models (73). However, this ECM protein had not previously been used to model 3D cell cultures in ACC, which favored the use of Matrigel and hyaluronic acid/gelatin (55, 57, 59). After verify- ing the expression of type I collagen in ACC, we determined that the bovine collagen ECM would be an ideal matrix for 3D ACC cell culture model, which was then used for 3 available cell lines which differed in their chemotherapy response and steroidogenic milieu. In our study, all 3 cell lines continued to proliferate and maintained steroidogenesis in 3D cell culture over 21 days, although demonstrating lower viability than monolayer cultures. The presence of confluent necrosis, par- ticularly in HAC15 cells, mirrored the in vivo tumor environ- ment. The reduced baseline viability observed in 3D cell cultures likely results from cellular overcrowding, leading to competition for nutrients and oxygen, particularly in the core of the cultures (67). This is consistent with the hypoxic and nu- trient gradients that lead to necrosis in tumors (46, 74). While cortisol production was lower in 3D cell culture, there was higher aldosterone and androgens production than monolayer
AlamarBlue 3D Cell Culture
A
H295R
B
HAC15
C
MUC-1
ns
ns
Fluoresence
800000
**
600000
Fluoresence
800000
600000
600000
Fluoresence
400000
400000
400000
200000
200000
200000
0
0
3
21
0
7
14
21
3
7
14
3
7
14
21
Time (Days)
Time (Days)
Time (Days)
Monolayer V 3D Cell Culture
D
H295R
E
HAC15
F
MUC-1
Fluoresence
1500000
**
Fluoresence
1500000
Fluoresence
1500000
ns
**
Monolayer
1000000
1000000
1000000
3D Cell Culture
500000
500000
500000
0
3
7
14
21
0
3
7
14
21
0
3
7
14
21
Time (Days)
Time (Days)
Time (Days)
Ki67 3D Cell Culture HAC15
G
H295R
H
I
MUC-1
100
ns
ns
100
100
ns
%Gated Cells
80
%Gated Cells
80
%Gated Cells
80
Ki67 (+)
60
60
60
Ki67 (-)
40
40
40
20
20
20
0
0
3
7
14
21
3
7
14
21
0
3
7
14
21
Time (Days)
Time (Days)
Time (Days)
Ki67 Monolayer Cell Culture
J
H295R
K
HAC15
L
MUC-1
**
ns
%Gated Cells
100
**
%Gated Cells
100
%Gated Cells
100
Ki67 (+)
80
80
80
Ki67 (-)
60
60
60
40
40
40
20
20
20
0
3
7
14
21
0
3
0
7
14
21
3
7
14
21
Time (Days)
Time (Days)
Time (Days)
cell culture. This is consistent with a prior study of ACC cell cul- tures in a spheroid model (61). However, the mechanism for this has not yet been studied. Therefore, our overall findings show that 3D ACC cell culture models demonstrate numerous disease characteristics, making them useful for drug screening and studies of cytotoxic chemotherapy.
The cytotoxicity of mitotane was tested in all 3 cell lines, showing the expected resistance in MUC-1 cells (75) and, for the first time, mitotane resistance in HAC15 cells in mono- layer. In 3D cell cultures, all 3 cell lines exhibited lower sensi- tivity to mitotane than monolayer, aligning with results seen in other 3D cancer models including breast and bowel cancer (67, 68, 76). The enhanced resistance in 3D cell cultures
highlights the advantage of using 3D cell culture models for drug screening, as they better replicate in vivo responses to chemotherapy, which are typically less robust than the re- sponse seen in monolayer cell culture (77).
The use of 3D cell culture models to study cancer is increas- ingly recognized as a standard (43, 68). However, technical challenges remain, such as accurately measuring cell viability and proliferation within 3D cell cultures (78). In our study, we utilized both real-time imaging and flow cytometry to validate our results, ensuring consistency between intact gels and ex- tracted cells. Further refinement of assays to minimize cell ex- traction will enhance future research using 3D cell culture models.
A
H295R
B
HAC15
100
100
%Live Cells
80
50µM
%Live Cells
80
50μΜ
*
ns
60
200μΜ
60
200μΜ
40
400μΜ
40
600μΜ
20
20
0
0
Monolayer
3D
Monolayer
3D
Monolayer
3D
Monolayer
3D
Monolayer
3D
Monolayer
3D
C
MUC-1
ns
ns
ns
100
%Live Cells
80
100μΜ
60
200μΜ
40
600µM
20
0
Monolayer
3D
Monolayer
3D
Monolayer
3D
In conclusion, we characterized a 3D cell culture model of ACC using 3 steroidogenic cell lines in type I collagen, which re- tained key characteristics of ACC. This model demonstrated greater resistance to mitotane cytotoxicity than monolayer cul- tures, offering a more complex and representative model for in- vestigating ACC. We propose that this model could serve as an animal-sparing platform for drug screening and for studying the mechanisms of chemotherapy resistance in ACC. Additionally, it provides a foundation for the development of more complex models incorporating stromal and immune cells.
Funding
This work was funded by National Institutes of Health (NIH) (R01EB028848) and Science Foundation Ireland (SFI) (20/US/ 3676) and (13/RC/2073 P2). C.H. receives funding from Swiss 3R Competence Centre.
Author Contributions
C.D. and S.F. created the experimental design. C.D. and S.F. wrote the paper. S.F. performed the experiments under the guidance and assistance of N.M., P.T.D., E.R., O.C., R.S.C., M.H., and M.C. E.R.M. aided in the RT-qPCR experiments. P.O. aided in the imaging using confocal microscopy. B.H. and D.F. aided with the running of Mass Spectrometry. A.M.Q. evaluated the histology slides. C.H. provided the MUC-1 cell line for these experiments.
Disclosures
No potential conflict of interest was reported by the authors.
Data Availability
Some or all datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.
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