Primary Cilia Deficit in SCA1 Mice | Parvathi Satheesh
Primary Cilia Deficiency in a Mouse Model of Spinocerebellar Ataxia Type 1
Authors: Parvathi Satheesh (1), Jiří Moravec (2), Jana Tůmová (3)
Supervisor: Jan Tůma (1)
(1) Department of Pathological Physiology, Faculty of Medicine in Pilsen, Charles University
(2) Biomedical Center, Faculty of Medicine in Pilsen, Charles University (3) Department of Physiology,
Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: Primary cilia are tiny, antenna-like, non-motile sensory organelles found in most vertebrates. Their dysfunction leads to a class of disorders called ciliopathies and also occurs in many neurodegenerative diseases. Spinocerebellar ataxia type 1 (SCA1) is caused by an expanded CAG repeat in the ATXN1 gene; this translates into a polyglutamine (polyQ) ataxin-1 protein. Cellular aggregation of this mutant protein leads to progressive cerebellar degeneration, causing motor deficits such as ataxia and slurred speech. Ciliary dysfunction is observed in neurodegenerative diseases like SCA12 and another polyQ disorder, Huntington’s disease. However, the role of primary cilia in the pathogenesis of SCA1 has never been investigated.
Objective: The main objective of this study is to elucidate the role of ataxin-1 in the regulation of ciliogenesis and the role of primary cilia in the pathogenesis of SCA1.
Material and Methods: Primary skin fibroblast cultures of SCA1 and WT mice were established
according to the protocol by Bravo et al., 2021. Primary cilia are dynamic organelles, and they assemble maximally during interphase. Hence, to promote ciliogenesis, cells were cultured under serum-free conditions for 24 hours to impede mitosis. The cells were then fixed and immunostained for primary cilia markers, including acetylated α-tubulin and γ-tubulin, along with the mitotic marker PHH3, to accurately quantify cilia in non-dividing cells. In addition to cilia quantification, cell viability was measured using the MTS assay. Mitochondrial function was assessed by citrate synthase assay to analyze enzymatic function and basal respiration was studied using Seahorse extracellular flux analysis.
Results & Discussion: SCA1 fibroblasts showed a significant reduction in the number of primary
cilia under both serum-free and serum-containing growth conditions. Alongside this, we observed reduced cell viability in the SCA1 fibroblasts in the MTS assay. The results from DAPI staining during primary cilia quantification revealed no reduction in cell viability, contrary to MTS assay results, indicating possible mitochondrial dysfunction, as mitochondrial dehydrogenase activity is measured in the MTS assay. This is further supported by a decrease in citrate synthase activity and basal mitochondrial respiration. Taken together, our data suggest a defect in either the formation or maintenance of primary cilia in SCA1 fibroblasts, along with mitochondrial dysfunction.
Conclusion: The association between reduced ciliogenesis and mitochondrial dysfunction highlights
an unexplored aspect of SCA1 pathology worth investigating. Hence, the above experiments will be replicated in embryonic neural stem cells and neurons for a better understanding of primary cilia in SCA1 pathology.
Funding: Cooperatio Program (research areas NEUR) and GAUK #70124.
Study program: Doctoral study – Physiology and Pathological Physiology | Year of study: 2
ID: 1122
Biomarkers in Ovarian Cancer | Kamila Koucká
Notch signaling pathway and its role in ovarian cancer – possible biomarkers of therapeutic response from three perspectives
Authors: Kamila Koucká (1,2), Alzbeta Spalenkova (1,2), Karolina Seborova (1,2), Tereza Tesarova (1,2), Marie Ehrlichova (1,2), Ivona Krus (1), Lukas Rob (3), Martin Hruda (3), Jiri Bouda (4), Alena Bartakova (4), Vendula Smoligova (4), Iwao Ojima (5), Lei Chen (5), Hersch Bendale (5), Marcela Mrhalova (6), Katerina Kopeckova (7), Radka Vaclavikova (1,2)
Supervisor: Pavel Souček (1,2)
(1) Laboratory of Pharmacogenomics, Biomedical Center, Faculty of Medicine in Pilsen, Charles University (2) Toxicogenomics Unit, National Institute of Public Health, Prague, Czech Republic (3) Department of Gynecology and Obstetrics, Third Faculty of Medicine and University Hospital Kralovske Vinohrady, Prague, Czech Republic (4) Department of Gynecology and Obstetrics, Faculty of Medicine in Pilsen, Charles University and University Hospital, Pilsen (5) Institute of Chemical Biology & Drug Discovery, State University of New York at Stony Brook, Stony Brook, NY, United States (6) Department of Pathology and Molecular Medicine, Second Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic (7) Department of Oncology, Second Faculty of Medicine, Charles University and Motol
State-of-the-Art: Patients with epithelial ovarian cancer (EOC) face high mortality due to late diagnosis, recurrence, metastasis, and drug resistance. The main histological subtype is high grade serous carcinoma, which occurs in up to 80 % cases. One of the most used chemotherapy regimens is paclitaxel combined with platinum-based drugs. Resistance to therapy limits successful treatment and efforts are made to overcome it. NOTCH signaling pathway is significant for the onset and development of cancer. We have studied this pathway in samples of EOC patients and subsequently studied changes in this pathway after treatment with taxanes in in vitro and in vivo models.
Objective: The aim was to analyse and compare the deregulations in the gene expression profile of the NOTCH signaling pathway in samples from patients with EOC and paclitaxel resistant and sensitive SKOV-3 ovarian cancer cell lines in vitro, as well as in in vivo models.
Material and Methods: RNA, DNA, and protein were isolated from tumors of patients with EOC and non-malignant ovarian tissue samples. cDNA was synthesized from RNA and RT-PCR was subsequently performed. Statistical evaluation of gene expression levels stratified by clinical data was done by the SPSS program. For the in vitro study, paclitaxel-sensitive and resistant ovarian cancer cell sublines SKOV-3/sen and SKOV-3/res were used. Expression of NOTCH signaling pathway was analysed after treatment with different concentrations of taxanes – paclitaxel or Stony Brook taxanes (SB-Ts). For in vivo experiments, SKOV-3/sen and SKOV-3/res cell-derived xenografts were established in immunodeficient nude mice. After tumors developed, mice were treated with paclitaxel, SB-Ts, or their combinations i.p. twice a week.
Results & Discussion: In tumors from EOC patients, a notable upregulation of NOTCH1/3/4, and JAG2, along with a downregulation of the NOTCH2 gene, was observed in comparison with control tissues. We found a significant correlation between low NOTCH4 expression and the presence of peritoneal metastasis as well as a shortened platinum-free interval. In the in vitro resistant cell subline model, treatment with experimental SB-Ts led to a significant upregulation of NOTCH3, demonstrating high efficacy against paclitaxel-resistant ovarian tumor cells. Additionally, the administration of SB-Ts resulted in NOTCH3 upregulation in an effective combination regimen with paclitaxel, compared to paclitaxel alone and untreated controls, in the in vivo cell-derived xenograft mouse model of resistant ovarian cancer.
Conclusion: Based on our results, we suggest the NOTCH3 gene as a potential target for preclinical
studies in resistant ovarian cancer. The current study also highlights the NOTCH4 gene as a potential predictive biomarker of therapeutic response in EOC.
Funding: Supported by the Czech Health Research Council grant NU22-08-00186, the Czech Science
Foundation grant 21-14082S, the Czech Ministry of Education, Youth and Sports program INTER-EXCELLENCE, sub-program INTER-ACTION, project no. LUAUS23164, and the NIH, U.S.A.
grant R01 CA103314.
Study program: Doctoral study – Experimetnal Surgery | Year of study: 4
ID: 1120
Liver Fibrosis and Liver Tissue Engineering | Ekaterina Panova
Connections between mild liver fibrosis model and liver tissue engineering
Authors: Ekaterina Panova (1,2), Sandra Clara-Trujillo (2), Maria Stefania Massaro (1), Richard Pálek (1), Manuel Salmeron Sanchez (2), M. Teresa Donato (3), Laia Tolosa (3), Gloria Gallego-Ferrer (2)
Supervisor: Vladimíra Moulisová (1)
(1) Biomedical Center, Faculty of Medicine in Pilsen, Charles University (2) Center for Biomaterials and Tissue Engineering, Universitat Politècnica de València, Valencia, Spain (3) Experimental
Hepatology Unit, Health Research Institute La Fe, Valencia, Spain
State-of-the-Art: Decellularized tissues represent a very promising material for tissue engineering. After all cells are gently removed from the original tissue, the remaining extracellular matrix preserved in its native tissue-specific architecture provides a perfect environment for repopulation with new cells, ideally harvested from individual patients. Hepatic is an important target for such an application, due to the liver being one of the organs with the highest demand for both transplantation and in vitro models, as it is the one in charge of blood detoxication and its disease and failure is the 11th major death cause worldwide.
Objective: The aim of my work is to follow up on my study focused on the design of mild liver fibrosis model, and implement the knowledge, skills and optimized protocols during the development of liver tissue in vitro. Decellularized liver will be used as the biomaterial for tissue engineering applications.
Material and Methods: For liver fibrosis model, the scaffold was generated by crosslinking tyramine-grafted gelatin with HRP with additional crosslinking with glyoxal. Initially, the material properties were tested for pore size (SEM), stiffness (rheology measurements), and swelling capacity. Then, cytocompatibility was tested by culturing both scaffold types with HepG2 cells, and eventually more detailed in vitro study with HUVECs and porcine hepatocytes was conducted to assess the effect of different scaffold stiffness on primary cell behaviour. Here, the methods included immunofluorescence staining of specific protein markers as well as gene expression assays, both being used for quantification of individual protein expression. Cell cultures were tested individually as well as in the co-culture.
Results & Discussion: Gelatin-tyramin (Gel) scaffold as control healthy liver tissue and Gel rehydrated in glyoxal (Gel-GlyO) scaffold mimicking F1 fibrosis were compared. GlyO addition increased the scaffolds’ stiffness from 2 kPa to 5 kPa. Pore size reduced in Gel-GlyO scaffolds and swelling ratio was higher for Gel. Cell viability was 86% at day 5 for HepG2 in Gel-GlyO, 90% for HUVECs in both materials, and 60% on day 7 for porcine hepatocytes (pHeps) in Gel-GlyO, while 30% at all timepoints in Gel. Cell functionality showed increased angiogenesis markers (vWF and CD31) for HUVECs in Gel-GlyO at days 1 and 3. Co culture had enhanced pro-fibrotic proteins activity (VEGFR-2 and CYP2E1) in Gel-GlyO at days 1 to 7. After 7 days, pHeps did not show changes in SLC2A2 gene expression in Gel-GlyO compared to Gel.
Conclusion: A mild fibrosis model was successfully designed and tested having a potential to be used as in vitro diagnostic and/or drug testing platform. Moreover, it provided new knowledge and experimental tools that are utilized in my current TE project, cell repopulation of decellularized liver scaffold.
Funding: The work was funded by the Spanish Ministry of Science and Innovation through PID2022-136433OB-C21 and -C22 grants, the National Institute for Cancer Research (Programme EXCELES, No. LX22NPO5102, Next Generation EU), and the COOPERATIO-207043 project (Surgical disciplines), Charles University.
Study program: Doctoral study – Experimetnal Surgery | Year of study: 1 ID : 1137
Salivary Gland Oncocytic Lesions | Bacem Khalele Othman
Diagnostic Algorithm for salivary gland oncocytic lesions
Author: Bacem Khalele Othman (1)
Supervisor: Alena Skálová (1,2)
(1) Department of Pathology, Faculty of Medicine in Pilsen, Charles University and University
Hospital, Pilsen (2) Bioptická laboratoř, s.r.o., Pilsen
State-of-the-Art: Rendering a supervised machine-learning-based diagnostic algorithm that
combines morphological, immunohistochemical, and molecular data into a diagnostic tool is
practically useful. Using dimensionality reduction to distil dominant oncocytic architectural
phenotypes with statistically validated clustering fidelity and its application to a large, registry-
based, real-world dataset improve its robustness.
Objective: Diagnosing salivary gland lesions with oncocytic differentiation is challenging as it
encompassing 21 reactive and neoplastic lesions. We propose a diagnostic algorithm to suggest
differentials based on analyzing oncocytic detection in the salivary gland lesion in AS’ registry
(501 out of 6383 SGL).
Material and Methods: We developed a weighted probabilistic scoring algorithm incorporating
dimensional reduction and hierarchical clustering analysis of morphological features, immunohistochemical profiles, and molecular labelling that represents the most significant weighted
values. If molecular information is missing, the algorithm computes the values corresponding to
morphology and immunohistochemical vectors.
Results & Discussion: Salivary gland lesions with oncocytic differentiation comprise malignant neoplasms (e.g., acinic cell carcinoma, salivary duct carcinoma, myoepithelial carcinoma), benign neoplasms (e.g., pleomorphic adenoma, Warthin tumor, oncocytoma), cystic lesions and reactive process (e.g. multifocal oncocytic metaplasia), with different extent of oncocytic differentiation, either extensive, focal, or wall-lining, in which oncocytes are positive for MIA. Principal component analysis revealed three distinct oncocytic architectural patterns with eigenvalues 1.0: extensive (λ₁=2.84), focal (λ₂=1.92), and wall-lining (λ₃=1.37). Hierarchical clustering demonstrated discrete entity groupings with a cophenetic correlation coefficient of 0.89.
Conclusion: The algorithm demonstrates acceptable performance metrics in differential diagnosis
generation. Although oncocytic differentiation can theoretically occur in any lesion, the
salivary oncocytic lesions were confined to real data with adequate molecular investigation.
Funding: The work was supported by the grant SVV (No. 260 773).
Study program: Doctoral study – Pathology | Year of study: 3
ID: 1094