Modeling Early Urothelial Carcinoma Progression | Kristián Hordynskyj
Modelling early steps of urothelial carcinoma progression in a novel cell line progression series
Authors: Kristián Hordynskyj (1), Michaela Kripnerová (1), Barbora Vítovcová (1), Kateřina Houfková (1), Martin Pešta (1), Petra Vohradská (2), Jiří Hatina (1)
Supervisor: Michaela Kripnerová (1)
(1) Department of Biology, Faculty of Medicine in Pilsen, Charles University (2) Department of Medical Genetics, Faculty of Medicine in Pilsen, Charles University and University Hospital, Pilsen
State-of-the-Art: Bladder cancer is a common malignancy with high recurrence and progression rates. Its progression from non–muscle-invasive (NMIBC) to muscle-invasive bladder cancer (MIBC) marks a major clinical turning point, often linked to epithelial–mesenchymal transition (EMT) and increased invasiveness, metastasis, and therapy resistance. While models for metastatic progression of MIBC do exist, early progression stages — especially the pTa → pT1 transition — are missing. We address this gap using a unique spontaneous progression model based on the RT112 cell line and its more invasive derivative, RT112Heidelberg. This system provides valuable insight into the plasticity underlying early bladder cancer progression and offers a platform for identifying new therapeutic targets.
Objective: Our goal is to unravel the basic biology of early steps of bladder cancer progression and identify crucial biological hubs and potential therapeutic targets relevant for the NMIBC to MIBC transition.
Material and Methods: The model consisted of two human bladder cancer cell lines with the same genetic background: RT112, a widely used urothelial carcinoma cell line representing an early to intermediate disease stage, and its clonogenic, spontaneously progressive subline RT112Heidelberg. Five methodological approaches were used to assess genetic and phenotypic changes between the cell lines: oxygraphy for metabolic analysis, Incucyte® S3-videomicroscopy for evaluating stromal – cancer cell communication and doxorubicin resistance, conventional and fluorescence microscopy for assessment of morphology, motility and EMT-related traits, flow cytometry for assessment of dynamics of E-cadherin expression, and gene expression analysis of selected EMT- and NRF2-related genes by qPCR.
Results & Discussion: The progressive variant RT112Heidelberg features a highly aneuploid (hypertriploid) karyotype, with numerous structural and numerical aberrations. Its morphology oscilates between epithelial-like is sparse cultures to mesenchymal-like at confluency, with a highly dynamic E-cadherin expression. We didn’t see any dramatic change in overall E-cadherin expression level, but a marked increase in vimentin expression, reminiscent of a hybrid epithelial/mesenchymal phenotype. Accordingly, RT112Heidelberg display molitily of the collective type. In keeping with published results showing stabilization of the hybrid E/M phenotype by a constitutively activation of the Nrf2-regulatory circuit, we see both enhanced Nrf2 nuclear expression and increase expression of selected Nrf2 downstream genes.
Conclusion: The clonally related pair of cell lines RT112 – RT112Heidelberg can provide an important experimental platform to model early steps of urothelial carcinoma progression, including the NMIBC to MIBC transition, offering insights into biomarkers and targeted therapeutic strategies.
Funding: This study was supported by SVV-2024-260654, SVV-2025-260773, Academic Minigrant 4EU+ MA/25/F1/0/018 and by the League Against Cancer Prague.
Study program: Master´s degree – General Medicine | Year of study: 2
ID: 1139
Flubendazole Affects Microtubules of Glioblastoma | Eliška Šavlová
Repeated flubendazole treatment and its effect on glioblastoma multiforme cells
Author: Eliška Šavlová (1)
Supervisor: Barbora Vítovcová (1)
(1) Department of Biology, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: Glioblastoma multiforme (GBM) is a highly aggressive malignant tumour
disease of the central nervous system that is currently treated with maximal radical surgery followed
by a combination of radio- and chemotherapy. However, the current treatment protocol is insufficient and, therefore, efforts for its innovation are being made. One of the potentional repurposed drugs is flubendazole (FLU), an anthelmintic used mainly in veterinary medicine, which was proven by previous studies to have an inhibitory effect on various cancer cells. This effect is attributed to the FLU-caused inhibition on the microtubular cytoskeleton.
Objective: Our study aimed to test the effect of repeated FLU administration on GBM tumour
cells. We investigated the effect on the viability and overall morphology of GBM cells, but especially
on the microtubular cytoskeleton, after administration of FLU in differing quantities at repeating intervals.
Material and Methods: The study was performed on two GBM stabilized cell lines (U87MG and
U87MG-IDH1mut) that were separately influenced 1-3x every 24 hours by different concentrations
of FLU (200 nM, 500 nM, and 1000 nM). The effect on cell viability and proliferation was determined in real time using the IncuCyte device and a phase contrast microscopy. The effect on the microtubular cytoskeleton was then observed using fluorescent microscopy.
Results & Discussion: Our results showed that FLU effectively reduced the viability of GBM cells – this effect was even greater with multiple FLU administration. Furthermore, FLU inhibited microtubule polymerization, even with a lower dosage of the tested drug (200 nM). In addition, FLU significantly affected the overall morphology of GBM cells, including the structure and organization of microtubular network, while maintaining the effect even after multiple administrations of FLU.
Conclusion: FLU shows a strong effect on GBM tumour cells by reducing their viability and proliferation
even at low doses of FLU, with the tested drug also affecting the microtubular cytoskeleton. Our results suggest that FLU is a promising drug with great potential and its effect needs to be further studied.
Study program: Master´s degree – General Medicine | Year of study: 2
ID: 1116
STAT3 in Glioblastoma after Fubendazole Treatment | Ladisla Čepička
Effect of Flubendazol on Glioblastoma multiforme cells with focus on ST AT3 molecule
Author: Ladislav Čepička (1)
Supervisor: Barbora Vítovcová (1)
(1) Department of Biology, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: Glioblastoma multiforme (GBM) belongs to the most common and most aggressive tumor diseases, and despite treatment, the average time of survival ranges between 12 and 15 months. The effects of the currently used treatment protocol are limited especially by the low accessibility of the commonly used drug temozolomide in the brain (mainly because of the blood-brain barrier) and the high overall resistance of GBM tumor cells, which is also connected with the increased expression and activation of STAT3 molecule. One of the drugs potentially inhibiting this signal STAT3 molecule is flubendazole (FLU), originally veterinary anthelmintic, which previously showed an inhibitory effect on a wide range of different tumor cells.
Objective: The aim of this work is to investigate the inhibitory effect of FLU on STAT3 molecule in GBM tumor cells, with special focus on the evaluation of FLU effect on tumor cells proliferation and the expression of STAT3 molecule.
Material and Methods: Two stabilized GBM cell lines were used for this project – U87MG and U87MG-STAT3-KO, in which specific STAT3 gene knock–out was created using the CRISPR/Cas9 method. This modification was performed at a collaborating workplace (Department of Medical Biology and Genetics, Faculty of Medicine in Hradec Králové). The proliferation and viability of cells, as well as their morphology, were evaluated in real-time by the Incucyte imaging system and subsequently by the phase-contrast microscope. Changes in STAT3 expression were observed via fluorescent microscopy.
Results & Discussion: FLU decreased the proliferation and viability of GBM cells, and this effect was observed already after administration of very low concentrations. Simultaneously, our results confirmed the inhibitory effect of FLU on the STAT3 molecule. Moreover, the specific knock–out of this molecule reduced the proliferation and also partially affected the morphology of the GBM cells. Additionally, our results showed a decrease in cell proliferation by the effect of FLU on cells with specific STAT3 knock-out, showing possible further affection of cell viability connected to this signal molecule.
Conclusion: FLU is a promising potential antitumor drug, which shows an inhibitory effect on GBM cells proliferation through decreasing the STAT3 expression. It effectively works already at significantly low concentrations, and it could be a promising direction for future research.
Study program: Master´s degree – General Medicine | Year of study: 2
ID: 1133
Glia in the Hippocampusof a Model of SCA 1 | Patricie Klosse
Densitometric analysis of glia in the hippocampus of a mouse model of spinocerebellar ataxia type 1
Authors: Patricie Klosse (1), Jan Látal (2), Zdeňka Purkartová (1), Štěpán Kápl (1), Dana Jelínková (1)
Supervisor: Jan Cendelín (1)
(1) Department of Pathological Physiology, Faculty of Medicine in Pilsen, Charles University
(2) Grammar School, Mikulášské náměstí, Plzeň
State-of-the-Art: Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant hereditary
neurodegenerative disease affecting the cerebellum and other parts of the central nervous system
including the hippocampus. Astrocytes and microglia are activated during various pathological
processes in the brain and are involved in pathogenesis of secondary changes in the diseased tissue. Glial cells are important components of the nervous tissue participating in local tissue niche that might have an impact on neural plasticity, residual tissue function as well as its neurogenic potential, and thereby influence response to cell-based and plasticity supporting therapies.
Objective: The aim of the study was to provide densitometric assessment of astrocytes and
microglia in the hippocampus of a mouse model of SCA1 in comparison with healthy mice.
Material and Methods: Paraformaldehyde fixed brains of 5 heterozygous SCA1 and 5 strainmatched
healthy 6-months-old mice were cryosectioned and processed for immunofluorescent detection of astrocytes and microglia. Astrocytes were labelled with anti-GFAP primary antibody. For identification of microglia, anti-IBA1 primary antibody was used. The sections were photodocumented using a fluorescent microscope. Intensity of fluorescent signal was evaluated in the hippocampi by means of densitometric approach in ImageJ software. Signal density was considered an indirect indicator of combined effect of glial multiplication and activation.
Results & Discussion: Density of fluorescent signals related to the astrocytic marker GFAP in the hippocampi of heterozygous SCA1 mice did not differ from that in healthy animals. Similarly, no significant differences between SCA1 and control mice were found in density of anti-IBA1
immunofluorescence, a microglial marker. Furthermore, the correlation between densities of GFAP and IBA1 was insignificant. SCA1 is a late onset spinocerebellar degeneration and neuropathology is less severe in the hippocampus than in the cerebellum in these mice. Thus, the moderate early primary hippocampal neuropathology does not probably induce glial activation in this mouse model.
Conclusion: The results suggest that hippocampal neuropathology is not accompanied by significant
proliferation and/or activation of astrocytes or microglia in SCA1 mice at the age of 6
months.
Funding: This work was supported by Cooperatio (NEUR and MED/DIAG research areas).
Study program: Master´s degree – General Medicine | Year of study: 4
ID: 1097