Phenotypic impact of keratin 8/18 downregulation in colonic in vitro 3D cultures
Fagersund, Jimmy (2022)
Fagersund, Jimmy
2022
Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi-fe2022062048078
https://urn.fi/URN:NBN:fi-fe2022062048078
Tiivistelmä
The second deadliest and third most common type of cancer is colorectal cancer (CRC). The disease mechanism is to a large extent unknown and the number of cases is on the rise in westernized countries. Interestingly, keratins [subfamily of intermediate filaments] have shown to be involved in development of both colitis and colorectal cancer. Keratins (K) occur as two different subtypes (type I and II) which form obligate heteropolymeric filaments that influence essential cellular functions such as proliferation, cell death and differentiation. Further insight is needed for the functionality of keratins; especially when it comes to colitis and CRC development. Traditional 2D culture and animal models have been utilized extensively in keratin research, but neither is ideal. In vitro 3D modelling is an alternative approach. By suspending cells in extracellular matrix, the cells are given an in vivo-like microenvironment. The aim for this project was to achieve standardized 3D models, substituting 2D culture and mouse models, and compare phenotypical differences between 2D and 3D culture caused by K8/K18 loss. Both 2D culture and 3D culture of Caco-2 cells and organoids were used to study viability, morphology, proliferation, apoptotic signaling and differentiation; and if any changes occur as a response of K8/K18 downregulation. K18 shRNA Caco-2 cells showed a slower proliferation but no viability difference in 2D culture, simultaneously as they possessed K8 and K80-positive aggregates. In 3D culture, the same cells showed an eccentric lumen formation and hampered growth. Moreover, the K18 shRNA cells showed increased protein levels of villin (in both 2D/3D). Surprisingly, K8 knockout Caco-2 cells merely showed modest differences in morphology, viability and proliferation. Finding K8-positive aggregates in K18 knockdown cells suggests that they could have caused the observed phenotypes. Further assessment is needed to determine if the aggregates are cancer-specific or if they could be an undescribed in vivo phenotype. The results show behavioral differences between 2D culture and 3D culture, such as proliferation and morphology, which suggests that in vitro 3D cultures could be a better approach to conventional 2D culture.