Since there is accumulating evidence that STAT3 is constitutively activated in PDAC and important for the progression and maintenance of this disease, the goal of this thesis was to establish a PDAC model in the lab to further investigate and characterize if the synergistic effect of VSV with STAT3 inhibition would also be effective on this cancer type. Aiming to unravel as many characteristics as in vitro studies allow, this study utilized not just 2D and 3D systems, but also cocultural approaches on both molecular and morphological levels.
Pancreatic ductal adenocarcinoma (PDAC) is considered as a very severe diagnosis because of the poor survival rates and the aggressive nature. Further investigations show the high complexity of the mutational cascades in PDAC initiation and progression. The importance of the tumor microenvironment is clarified, but as different studies led to paradoxic results in targeting the stroma, it gives us evidence that it is just poorly understood yet. Given the limitations of current treatments and the failure of various novel approaches, a necessity of further investigating new therapeutic options is unquestionable.
Table of Contents
I. Introduction
1.1 Pancreatic cancer.
1.1.1 PDAC development and progression
1.1.2 Microenvironment
1.1.3 Current treatment options and their limitations
1.2 Oncolytic viruses
1.2.1 Vesicular stomatitis virus
1.3 Signal transducer and activator of transcription 3
1.4 Aim of this thesis
II. Material
2.1 Cell lines
2.2 Reagents
2.3 Antibodies
2.4 Consumables
2.5 Appliances
III. Methods
3.1 Cell culture
3.1.1 3D cell culture systems
3.1.2 Co-culture systems
3.2 Viability assay
3.3 Tissue culture infection dose
3.4 Western blotting
3.5 Flow cytometric analysis
3.6 Confocal microscopy
3.7 Luciferase assay
3.7.1 Plasmid DNA preparation
3.7.2 Transfection
3.7.3 Interferon induction and response
3.8 Scratch assay
IV. Results
4.1 PDAC cells are susceptible to rVSV-GFP-mediated oncolysis in vitro
4.2 S3I-201 causes reduction of PDAC cell viability in vitro
4.3 Combination therapy enhances PDAC killing and safety
4.4 Combinational therapy in the microenvironment
4.5 Effects of S3I-201 and VSV in co-culture
4.6 Combination therapy in 3D
4.7 Inhibition of migration
V. Discussion
Research Objectives and Topics
This master thesis investigates the therapeutic potential of combining oncolytic vesicular stomatitis virus (VSV) with STAT3 inhibition as a novel treatment strategy for pancreatic ductal adenocarcinoma (PDAC). The study focuses on the synergistic effects of these agents on tumor cell killing, the reduction of migratory and proliferative potential in cancer-associated fibroblasts, and the potential to enhance therapeutic safety in healthy primary pancreatic cells.
- Oncolytic virotherapy with rVSV-GFP
- STAT3 signaling inhibition using S3I-201
- PDAC microenvironment and tumor-stroma crosstalk
- 2D, 3D, and co-culture model development
- Mechanisms of tumor cell death and migration inhibition
Excerpt from the Book
1.1.2 Microenvironment
Besides genetic mutations also PDACs microenvironment plays a crucial role not just for its development but its progression and maintenance as well. A mature pancreatic cancer is histologically defined as tumor islands that are surrounded by its dense stroma. Hence, the desmoplastic regions can account for up to 90% of this disease (Xie and Xie 2015). Being a hallmark of cancer, these regions are highly heterogenous. They consist of cellular components like cancer associated fibroblasts (CAFs), pancreatic stellate cells (PSCs) and immune cells, as well as acellular extracellular matrix (ECM), growth factors and cytokines. All these components form a complex network and the interplay with each other as well as the cancer cells is crucial for cancer initiation and progression (Xu, Zhou et al. 2016).
An important role in these interactions play cancer associated fibroblasts, which are known to promote the progression and metastasis of PDAC. This is achieved by the secretion of growth factors and inflammatory cytokines, the mediation of ECM formation and suppressing the immune system. Vice versa, CAFs are activated by the cancer cells. As a consequence, α-SMA, expressed by activated stromal cells, belongs to negative prognostic markers for PDAC in the clinics (von Ahrens, Bhagat et al. 2017).
As shown in Figure 1, SMAD4 deficiency as well as Kras hyper activation play important roles in PDAC. This is also attributed to the interaction with its stroma. It was shown for this combinational mutation that it leads to an constitutive activation of the TGF-β, resulting in accelerated CAF and PSC proliferation, enhanced ECM production and eventually induced angiogenesis via the VEGF pathway (Ahmed, Bradshaw et al. 2017). Furthermore, the constitutive IL-6 production of the stromal cells over activates the STAT3 pathway in tumor cells in a paracrine manner. This pathway is commonly known for its role in oncogenesis as well as its capability of initiating metastasis via the induction of epithelial to mesenchymal transition (EMT) (Nagathihalli, Castellanos et al. 2016).
Summary of Chapters
I. Introduction: Provides an overview of pancreatic ductal adenocarcinoma, the role of oncolytic viruses, the function of STAT3 signaling, and the specific goals of this thesis.
II. Material: Lists the cell lines, reagents, antibodies, consumables, and laboratory equipment used throughout the experiments.
III. Methods: Details the experimental procedures, including cell culture techniques (2D, 3D, and co-culture), viability assays, viral titer determination, western blotting, and microscopy methods.
IV. Results: Presents the findings regarding the synergistic effects of rVSV-GFP and STAT3 inhibitor S3I-201 in various PDAC models, including infection efficacy, cell viability, and migration potential.
V. Discussion: Evaluates the experimental results, interprets the synergy between the two therapeutic agents, discusses implications for future clinical applications, and addresses limitations and future research directions.
Keywords
Pancreatic ductal adenocarcinoma, PDAC, oncolytic virus, vesicular stomatitis virus, VSV, STAT3, S3I-201, cancer-associated fibroblasts, CAFs, tumor microenvironment, synergistic therapy, apoptosis, cell migration, tumor spheroids, immunotherapy.
Frequently Asked Questions
What is the core focus of this research?
The research explores the potential of a combinational therapy using oncolytic viruses and STAT3 inhibitors to improve the treatment of pancreatic ductal adenocarcinoma.
What are the primary themes covered in this thesis?
The thesis covers tumor biology, viral oncology, signaling pathways like STAT3, the tumor microenvironment, and advanced in vitro modeling.
What is the main research question or goal?
The goal is to determine if the combination of rVSV-GFP and the STAT3 inhibitor S3I-201 exhibits synergistic anti-tumor effects in PDAC cells while maintaining or improving safety for healthy cells.
Which scientific methods are employed?
The study utilizes 2D cell cultures, 3D tumor spheroids, transwell co-culture systems, MTT viability assays, flow cytometry, western blotting, and confocal microscopy.
What is addressed in the main body of the work?
The main body details the materials and methods used for cell culture and assays, followed by extensive results on cell viability, viral replication, signaling pathway inhibition, and migration studies.
Which keywords define this work?
Key terms include PDAC, oncolytic virus, STAT3 inhibition, tumor microenvironment, and combinatorial therapy.
How does STAT3 inhibition impact the virus-PDAC interaction?
The study indicates that STAT3 inhibition by S3I-201 enhances the cytotoxic effect against PDAC cells without interfering with viral replication in tumor cells.
What was observed regarding healthy primary pancreatic cells?
The combination therapy was found to be significantly safer for healthy primary cells, as the inhibitor reduced viral titers in healthy tissue, suggesting a potential improvement in therapeutic safety.
- Arbeit zitieren
- Simon Dedic (Autor:in), 2017, In vitro characterization of oncolytic virus in combination with STAT3 inhibition for improved therapy against pancreatic ductal adenocarcinoma, München, GRIN Verlag, https://www.grin.com/document/420553
