Browsing by Subject "Organoids"
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(2021)Ovarian cancers (OCs) are gynecological malignancies that cause the most gynecological cancer related deaths due to asymptomatic early-stage development and late diagnosis. The treatment of OC has not improved significantly during the last decades, and challenges are often caused by chemoresistance and the heterogeneity of cancer cell populations. Therefore, there is an urgent need to improve OC treatment outcome and implement new targeted therapies that could address the subtype specific characteristics. The most common type of OC is epithelial ovarian cancer (EOC), that can be further divided into five subtypes with distinct molecular and histological characteristics. High-grade serous subtype represents majority of cases with up to 75% of EOC patients, while other subtypes such as low-grade serous, mucinous, clear cell and endometrioid OC being less common. Considerable progress has been made in cancer treatment via precision oncology, in which individual cancer biology and tumor molecular features are investigated and used to improve treatment decisions. For this purpose, the development of patient-derived cancer cells (PDCs) offers a good opportunity to study cancer biology in vitro and to build models for preclinical molecular profiling and functional testing. PDCs can be used to establish 2D and 3D models, and most recently, wide interest has been focused on patient-derived organoids (PDOs), that offer a better model of tumor and its microenvironment, while allowing long-term culture, cryopreservation, modification and high-throughput opportunities. In this study, the aim was to establish PDO cultures using tumor cells from low-grade serous OC patients for molecular profiling and functional drug testing. PDOs were generated from both fresh and frozen tumor tissue or ascitic samples resulting to successful development of long-term PDOs from three of the five models. In order to identify optimal culturing conditions for low-grade serous OC PDOs, two previously unpublished growth mediums were tested in parallel. The more complex of the mediums showed slightly better PDO growth in general. The immunohistochemistry staining with pan-cytokeratin and PAX8 was used to confirm the epithelial and ovarian origin of PDOs. In addition, cancer panel sequencing was performed to identify mutation profiles. Importantly, the small-scale drug testing, which was performed using conventional chemotherapeutics cisplatin and paclitaxel and targeted drugs gedatolisib and trametinib, showed sample-specific responses. In conclusion, the results from this project show that PDOs are good models for ex vivo precision medicine functional studies. Importantly, we managed to establish PDOs from frozen tumor cells, suggesting that PDOs could be initiated from living biobank samples. However, the challenges related to culturing of PDOs for functional assays included slower growth rate compared to 2D cancer cell cultures and technical challenges related to Matrigel, limiting the possibilities of high-throughput drug testing. By improving these factors, PDOs will offer an efficient 3D model for preclinical use.
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(2023)The developing human hindbrain and its role in neuronal pathogenesis have been relatively difficult to study for ethical reasons. By using the dual-SMAD inhibition and WNT signalling induction, a new method to culture brain organoids to resemble the human hindbrain has recently been established. In this study the new method has been used to detect the developing hindbrain’s response to flaviviral infection. Model virus used in this study is the Zika virus (ZIKV) which is known to alter the development of central nervous system and cause microcephalia. Pathogenic activity of the virus is measured by detecting the morphology of the organoids during infection as well as screening the organoids activation against oxidative stress, in a form of KEAP1/Nrf2-ARE pathway activation. Three different clones of ZIKV, which differ from each other by one amino acid in their non-structural protein 1 (NS1) gene, were used in the infections as well as two different time points of development. Controversially to previous findings on ZIKV infections to brain organoids, our findings show that developing hindbrain-like organoids do not change in shape or size during ZIKV infection. There are no differences in the lack of morphological changes between one-month olds or two months old organoids or between the different ZIKV clone infected organoids. The activation of the KEAP1/Nrf2-ARE pathway was measured by screening the two final products of the pathway, Nqo1 and HO-1. By screening the mRNA levels of these two genes, it showed that different ZIKV clones affect the activation of the KEAP1/Nrf2-ARE pathway in different levels at different times of development. Also, the expression of the same gene can be altered by the age of the organoids. Additionally, the expression of the two genes were different from each other at given time points and in response to the different clones. These findings suggest that the different isoforms of NS1 of ZIKV may alter the developing hindbrain’s response to oxidative stress. Findings also show that the time of the infection can additionally play a critical role to the ZIKV infection. The altered response to oxidative stress may contribute to microcephaly: the oxidation homeostasis of the developing hindbrain is modified, and apoptotic cell death can take place.
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