PO_II_L: Pathology and Oncology II. Lectures
Krokker Lilla1,2, Szabó Borbála1,2, Németh Kinga1, Tóháti Rebeka2, Mészáros Katalin1, Paku Sándor3, Ildikó Krencz3, Patócs Attila1,2, Butz Henriett1,2
1Hereditary Tumours Research Group, Hungarian Academy of Sciences and Semmelweis University
2Department of Laboratory Medicine, Semmelweis University
31st Department of Pathology and Experimental Cancer Research, Semmelweis University
Introduction. There is a considerable difference among tumour cell behaviour of the monolayer in vitro models and solid tumours as 2D cultures do not represent the complexity of human tumours reliably. Adrenal carcinoma (ACC) is a rare tumor with poor prognosis. Mitotane is the main base of the medical treatment of ACC, however toxic side effects and deprived tumor response limit its clinical value.
Aim. To investigate the role of in vitro 3D ACC model in characterization of differences in mitotane therapy response among different tumor models.
Materials and methods. In vitro monolayer (2D) and 3D H295R cultures were generated by ultra-low attachment plate and extracellular matrix matrigel. In vitro functional assays assessed viability, proliferation, and dead cell ratio. In vivo ACC subcutaneous xenograft model was developed in SCID mice. Proliferation was evaluated by tumor measurements by caliper and Ki-67 immunostaining after sacrifice. In vitro and in vivo cortisol production was detected by HPLC-MS/MS. Mitotane treated and control in vitro 2D, 3D and in vivo xenograft samples were investigated by global transciptome analysis using whole RNA sequencing on Illumina platform.
Results. Cell proliferation was significantly lower in in vitro 3D models compared to monolayer cultures. Dead cell ratio was increased in 2D cultures compared 3D models upon mitotane treatment. Mitotane had more potential to decrease cortisol production in 2D compared to 3D models. In xenograft model mitotane treatment decreased serum cortisol level of mice. Transcriptome sequencing revealed differences in steroid hormone biosynthesis and metabolism, TGFβ and extracellular matrix related signalling pathways following mitotane treatment between different tumor models.
Conclusion. There is significant difference between in vitro ACC 2D and 3D tumor model characteristics and mitotane therapy response. By comparing to in vivo xenograft models, our innovative 3D model combining spheroid formation with extracellular matrix may represent a better in vitro model for ACC, and it can also be useful for investigation of ACC pathogenesis, therapy response and to reveal potential new therapeutic targets in the future.
Funding: STIA-KF-19 and NKFIH FK 135065 to H.B.; NKFIH K125231 to A.P.
Program: Hormonal Regulations
Supervisor: Dr. Henriett Butz
Semmelweis University, Károly Rácz Doctoral School of Clinical Medicine