8468 Development And Characterisation Of Three-Dimensional Cell Culture Models Of Adrenocortical Carcinoma
Authors: Feely Sarah , Mullen Nathan , Donlon Padraig , Hantel Constanze , Dennedy Michael C 
Published: October 2024

Abstract
Disclosure: S. Feely: None. N. Mullen: None. P. Donlon: None. C. Hantel: None. M.C. dennedy: None.
Adrenocortical carcinoma (ACC) is a rare malignancy carrying a poor prognosis and limited treatment options. Surgical resection is the only option for cure; but unsuitable for advanced disease. Mitotane is the approved medical therapy, but associated with treatment resistance and adverse effects. Newer therapies, such as immune checkpoint inhibitors, have limited success. Development of improved therapies is limited by current pre-clinical disease models. No available model reflects the tumour micro-environment. Three-dimensional (3D) cell culture models are increasingly used in cancer research (6), with a paucity of developed models in ACC. In the current study, we developed novel 3D models of ACC using a type-1 collagen matrix. We then characterised these as representative ACC disease models . 3D cell culture models of ACC were optimised by embedding ACC cell lines, MUC-1, HAC15 and H295R within a type-1 Collagen matrix at differing cell numbers. Expression of type-1 collagen in ACC primary tumours was determined using immunohistochemistry (n=4). Cell viability for each model was assessed by evaluating sytox blue staining by Flow Cytometry. Metabolic activity of ACC cells within the matrix was determined using AlamarBlue staining. Prolferative activity was assessed using Ki67. Steroidogenesis was evaluated by measuring cortisol, aldosterone and androstenedione using LC-MS/MS.. Expression of CYP11B1, CYP11B2 CYP17, and StAR was measured using RT-qPCR. Type 1 collagen is strongly expressed in ACC tumour microenvironment. MUC-1, H295R and HAC15 cells were successfully cultured in a type 1 collagen matrix. MUC-1 cells cultured in the collagen matrix remain viable throughout the period of experimentation with optimum viability between 14 and 21 days. H295R cells also remain viable with optimum viability at an earlier stage of 7-14 days. HAC15 cells demonstrate high resilience in 3D cell culture with maximum viability throughout the entire period of experimentation up to 21 days. All three models increase their metabolic and proliferative activity over time. All three steroids are expressed by all three models with an increase in aldosterone secretion observed in 3D HAC15 and H295R models compared to monolayer (p&gt;0.001) at days 7 and 14. All models express key steroidogenic enzymes. 3D ACC cell culture models were developed successfully using collagen type 1 abundant in ACC. Typical characteristics of the ACC tumour environment included (i) the presence of live and necrotic cells (ii) high metabolic activity and proliferation, reflective of cell turnover.. Capacity of cells to spread and proliferate within the 3D cell culture models was limited by available collagen substrate. Steroidogenic capacity was demonstrated in 3D cell culture. This model therefore retained key characteristics of ACC and shows promise as an animal-sparing, pre-clinical model.
Presentation: 6/1/2024