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Projekte

3D Human Tissue-Engineered Models for Drug Discovery

Targeting the tumor microenvironment of pancreatic cancer

Spatial distribution of infiltrated macrophages in our 3D spheroid using confocal laser scanning microscopy. The spheroid was stained for tumor cells (purple), cancer‑associated fibroblasts (blue) and M2 macrophages (green). White arrows indicate positive green signals. Infiltrated M2 macrophages are located in the fibrotic shell of the 3D spheroid.

Pancreatic Ductal Adenocarcinoma (PDAC), a highly aggressive form of pancreatic cancer, is known for displaying early metastasis and enhanced resistance against classical chemotherapeutic treatment regiments, leading to a very low overall survival rate. This troubling and detrimental pathophysiological feature, impeding and exacerbating patient’s treatment, is mainly caused by the characteristic fibrotic desmoplasia of the tumor microenvironment (TME), which mainly consists of so-called cancer-associated fibroblasts (CAF), extensive amounts of extracellular matrix and various other recruited types of tumor‑promoting cells.

In our research project, we established a 3D spheroid co-culture model using PDAC tumor cells and patient-derived cancer-associated fibroblasts to investigate the biological interactions and to analyse the efficacy and impact of novel drug candidates. A potential and promising therapeutical target is the significant cellular process of epithelial-mesenchymal transition (EMT), in which immobile tumor cells acquire migratory properties associated with increased malignancy, invasiveness and metastasis activity. The reversion of malignant tumor cells to their epithelial state is theorized to diminish chemoresistance and metastasis significantly, thereby improving the chemotherapeutic intervention and treatment. Further research includes an extension of our 3D spheroid co-culture model to include endothelial and immune cells, and a 3D tumor microenvironment-on-chip model for personalized drug testing.

Selected publications:

  1. Schiedlauske K, Deipenbrock A, Pflieger M, Hamacher A, Hänsel J, Kassack MU, Kurz T, Teusch NE. Novel Histone Deacetylase (HDAC) Inhibitor Induces Apoptosis and Suppresses Invasion via E-Cadherin Upregulation in Pancreatic Ductal Adenocarcinoma (PDAC). Pharmaceuticals. 2024; 17(6):752. https://doi.org/10.3390/ph17060752
  2. Xie B, Hänsel J, Mundorf V, Betz J, Reimche I, Erkan M, Büdeyri I, Gesell A, Kerr RG, Ariantari NP, Yu H, Teusch N, Mrsny RJ, Pseudopterosin and O-Methyltylophorinidine Suppress Cell Growth in a 3D Spheroid Co-Culture Model of Pancreatic Ductal Adenocarcinoma. Bioengineering, 2020, 7 (2), 57. DOI: 10.3390/bioengineering7020057
  3. Movahhed S, Westphal J, Kempa A, Schumacher C E, Sperlich J, Neudörfl J-M, Teusch N, Hochgürtel M, Schmalz H-G, Total Synthesis of (+)‐Erogorgiaene and the Pseudopterosin A−F Aglycone via Enantioselective Cobalt‐Catalyzed Hydrovinylation, Chemistry – A European

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal form of pancreatic cancer characterized by therapy resistance and early metastasis, resulting in a low survival rate. PDAC is accompanied by cancer-related alterations of intrapancreatic nerves, which play a pivotal role in tumor progression and metastasis. Substantial modifications of intrapancreatic nerves are concomitant with alterations in the ratio of autonomic and sensory fibres (neural remodelling) and an increased nerve size (hypertrophy) and density. Furthermore, cancer cells infiltrate intra-cancerous nerve fibres, a phenomenon referred to as perineural invasion (PNI) and serves as a conduit for cancer cell dissemination. PNI is a hallmark feature of PDAC, with the highest prevalence of all tumors, correlating with recurrence, contributing to massive pain, and significantly reducing survival rates. 

In the present study, we are developing a novel human-based model system emulating PNI in accordance with the 3R principles. To this objective, we integrate pancreatic cancer cells, iPSC-derived sensory neurons, and primary Schwann cells into a novel 3D multicellular model system. To assess the invasiveness of the pancreatic cancer cells, we employ a multifaceted approach that incorporates invasion determination, sequencing, and transcriptomic analysis.

© RG Teusch
(A) 3D spheroid stained for cancer cells (red) and Schwann cells (green), Scale bar 300 µm. (B) Migration of cancer cells (purple) to Schwann cells (green), Scale bar 500µm. (C) iPSC-derived sensory neurons (neurites purple, nucleus blue), Scale bar 100 µm.

Pharmacological and toxicological compound assessment in a novel 3D immune competent skin model

Distribiution of dermal dendritic cells (red signal) in our human immune competent skin model after sensitizer treatment. Scale Bar = 20 µm.

As the largest organ of the body, the skin is permanently exposed to environmental hazards, radiation, chemical substances and biological agents, thereby forming the first line of immunological defense in the body. However, these factors can lead to serious skin reactions such as skin irritation, inflammation, allergies or cancer. Hence, there is a substantial need to screen and assess the toxicity of agents and the identification of efficacious novel drug candidates for the skin.

We are currently developing a novel human 3D immune competent full-thickness skin model according to the 3R principles (“replace”, “reduce” and “refine”), for pharmacological and toxicological compound assessment. For this, langerhans cell surrogates are integrated as central initiators of the immune reaction in the skin, implementing skin sensitization and inflammation in the 3D model. To assess the tested compounds, inflammatory pathways are investigated via sequencing and transcriptomic analysis and threshold values will be determined for putative compound classifications and perspectively for high throughput screenings.

Further research includes the integration of additional cell types e.g. melanocytes, melanoma cells or vascularization, allowing distinct disease modelling for drug discovery.

Selected publications:

  1. Hölken JM, Wurz A-L, Friedrich K, et al. Incorporating immune cell surrogates into a full-thickness tissue equivalent of human skin to characterize dendritic cell activation. Scientific Reports. 2024/12/04 2024;14(1):30158. doi:10.1038/s41598-024-81014-9
  2. Holken JM, Friedrich K, Merkel M, et al. A human 3D immune competent full-thickness skin model mimicking dermal dendritic cell activation. Front Immunol. 2023;14:1276151. doi:10.3389/fimmu.2023.1276151
  3. Holken JM, Teusch N. The Monocytic Cell Line THP-1 as a Validated and Robust Surrogate Model for Human Dendritic Cells. Int J Mol Sci. Jan 11 2023;24(2)doi:10.3390/ijms24021452

Natural Product Research

Workflow starting from fungi isolation till fermentation
Workflow after obtaining a total extract of the fungus till conducting different bioassays on structurally characterized natural products

Since antiquity, nature has served as a source of remedies for many diseases of mankind. Notwithstanding the development of many modern synthetic drugs, there is a huge need for exploring bioactive natural products which could serve as lead compounds for drug discovery and development. Although the early days of natural products’ discovery efforts focused on the isolation of interesting secondary metabolites from plants, sponges, soft or hard corals, …. etc, by time attention turned into smaller creatures, such as endophytic and marine fungi, which have proven themselves as rich sources of remarkable biologically active secondary metabolites. One of the undergoing research projects conducted in our group comprises the study of fungi from different habitats (terrestrial and marine habitats) as potential producers of new bioactive natural products. The workflow starts with collecting samples followed by isolation and purification of several fungi. The isolated fungi are then identified and cultivated on suitable culture media. The obtained crude extracts are then tested against several bioassays in a bioactivity-guided approach. Afterwards, the biologically active extracts are fractionated via different chromatographic techniques including column chromatography utilizing different stationary phases, liquid chromatography and HPLC (High Performance Liquid Chromatography) till single pure compounds are obtained. The structures of isolated compounds are deduced based on a combination of 1D and 2D NMR spectroscopy and HRESIMS data as well as by comparison with the literature. For full characterization of compounds, absolute configurations should be assigned based on optical rotations (OR) measurements and comparison with the literature values, modified Mosher’s method (MMM), electronic circular dichroism (ECD) calculations or single-crystal X-ray diffraction analysis (XRD). The last step includes the evaluation of different bioactivities of the isolated structurally characterized compounds including cytotoxicity against different human cell lines, anti-inflammatory activity and many other activities.

Selected publications:

  1. Reimche I, Yu H, Ariantari NP, Liu Z, Merkens K, Rotfuß S, Peter K, Jungwirth U, Bauer N, Kiefer F,  Neudörfl JM, Schmalz HG, Proksch P, Teusch N, Phenanthroindolizidine Alkaloids Isolated from Tylophora ovata as Potent Inhibitors of Inflammation, Spheroid Growth, and Invasion of Triple-Negative Breast Cancer. International Journal of Molecular Sciences, 2022, 23(18), 10319. DOI: DOI: 10.3390/ijms231810319
  2. El-Kashef DH, Youssef FS, Reimche I, Teusch N, Müller WEG, Lin W, Frank M, Liu Z, Proksch P, Polyketides from the marine-derived fungus Aspergillus falconensis: In silico and in vitro cytotoxicity studies. Bioorganic & Medicinal Chemistry, 2021, 29, 115883. DOI: 10.1016/j.bmc.2020.115883
  3. El-Kashef DH, Youssef FS, Hartmann R, Knedel TO, Janiak C, Lin W, Reimche I, Teusch N, Liu Z, Proksch P, Azaphilones from the Red Sea Fungus Aspergillus falconensis. Marine Drugs, 2020, 18 (4), 204. DOI: 10.3390/md18040204
  4. Xie B, Hänsel J, Mundorf V, Betz J, Reimche I, Erkan M, Büdeyri I, Gesell A, Kerr RG, Ariantari NP, Yu H, Teusch N, Mrsny RJ, Pseudopterosin and O-Methyltylophorinidine Suppress Cell Growth in a 3D Spheroid Co-Culture Model of Pancreatic Ductal Adenocarcinoma. Bioengineering, 2020, 7 (2), 57. DOI: 10.3390/bioengineering7020057
  5. Yu H, Sperlich J, Höfert SP, Janiak C, Teusch N, Stuhldreier F, Wesselborg S, Wang C, Kassack M, Dai H, Liu Z, Proksch P, Azaphilone pigments and macrodiolides from the coprophilous fungus Coniella fragariae. Fitoterapia, 2019, 137, 104249. DOI: 10.1016/j.fitote.2019.104249
Structures of (M)-viriditoxin and (P)-viriditoxin, the “M” and “P” refer to the atropisomer confirmation. B: UV-vis spectra of viriditoxin. C: 1H-NMR spectrum (600 MHz) of viriditoxin in chloroform-d. D: 13C-NMR spectrum (150 MHz) of viriditoxin in chloroform-d.

Viriditoxin (VDT) is a classic fungal secondary metabolite of a C-6/C-6’ linked dimeric naphthopyranone. It exhibits intriguing biological activities, initially identified as a potent antibacterial agent through its inhibition of the bacterial protein FtsZ. Subsequently, VDT was explored as a moderate tubulin inhibitor in various solid tumor cell lines.

Notably, recent research has uncovered VDT’s therapeutic potential in leukemia and lymphoma cells in contrast to solid tumor cells, but only low cytotoxic effects in human hematopoietic stem cells and peripheral blood lymphocytes. The mechanism of VDT to directly activate the mitochondrial apoptosis pathway represents a promising therapeutic approach for the treatment of therapy-resistant leukemia and lymphoma.

Our ongoing research not only focuses on elucidating the molecular mechanism of VDT but also involves the isolation and characterization of novel VDT derivatives from endophytic fungi. The absolute configurations of all isolated derivatives are determined and correlated with their bioactivity to gain deeper insights into their structure–activity relationship (SAR). Additionally, to generate novel VDT analogs, OSMAC (One Strain, Many Compounds) and precursor feeding approaches are employed to activate silent biosynthetic gene clusters and enhance the chemical diversity of secondary metabolites.

Neuropathic Pain

Peripheral neuropathy is a serious complication caused by various conditions, including chemotherapy, diabetes mellitus, traumatic injury, infections and many more. It is caused by damage to the somatosensory nervous system manifesting in sensory sensations such as tingling, burning, numbness, and the most severe symptom – pain. Approximately 800 million people worldwide suffer from neuropathic pain and it is becoming a significant burden for both patients and for their health care providers. Unfortunately, there are no disease-modifying therapies available and symptomatic treatment strategies show only limited efficacy and clinical success, leading to neuropathic pain negatively impacting the quality of the patient’s life and considerably increasing healthcare costs.

The aim of this interdisciplinary project is to identify, isolate and characterize natural product-based new drug candidates to find novel therapeutic options for the treatment of peripheral neuropathy. In order to achieve this, we plan to establish cell models of peripheral neuropathy of different pathological genesis. Therefore, we will use differentiated neuronal cell lines as well as primary human Schwann cells and iPSC derived peripheral sensory neurons. Finally, we aim to integrate fully functional and differentiated peripheral sensory neurons in our 3D full thickness skin model to reproduce the cutaneous-immuno-neuronal crosstalk and gain further insight into the pathology of neuropathy in the context of the local human environment. These models will be used to screen extracts of various natural origin for their bioactivity in a bioactivity-guided approach.

Selected publications:

  1. Wiegand V, Gao Y, Teusch N. Pharmacological Effects of Paeonia lactiflora Focusing on Painful Diabetic Neuropathy. Planta Med. Dec 2024;90(15):1115-1129. doi:10.1055/a-2441-6488