HP-F3: Cancer Metabolism, Translational Proteomics and Epigenetics, Organoid Cancer Models
Type: Practical Course with Student Seminars
Part 1: Amino acid metabolism in cancer
Date: 25.-29. November 2024
Hosts/Supervisors: Tamara Prentzell, Christiane Opitz, and co-workers
Topics:
Historically, metabolism has been considered a type of rather passive housekeeping functions that are required for cell survival, but nowadays it is recognized of having an active and multifaceted role in modulating cell fate, function and disease. Amino acid metabolism refers to the plethora of biochemical pathways that produce, break down, and utilize amino acids. Disorders in amino acid metabolism have been linked with a number of pathological conditions, including metabolic disorders, cardiovascular diseases, and cancer. Furthermore, alterations in amino acid levels impair normal immune cell function and thereby also influence anti-tumor immunity. Our research focusses on tryptophan catabolism and metabolic signaling through the aryl hydrocarbon receptor (AHR). Tryptophan is the least abundant amino acid and can be degraded by the three enzymes indoleamine 2,3-dioxygenase 1 (IDO1), tryptophan 2,3-dioxygenase 2 (TDO2) and interleukin-4-induced 1 (IL4I1). Tryptophan catabolism generates immunosuppressive metabolites, creating a permissive environment for tumor growth and immune evasion. However, the specific mechanisms governing the interplay between tryptophan catabolism and the immune system remain unclear. The AHR is a ligand-activated transcription factor that enables cells to adapt to environmental, nutritional or metabolic changes. By modulating both tumor cell intrinsic malignant properties as well as anti-tumor immunity, the AHR is a critical regulator of tumor progression. We are employing a multidisciplinary approach, combining biochemical, molecular, and immunological techniques to comprehensively address the impact of tryptophan metabolites on AHR activation and the tumor microenvironment in general. We use high-throughput mass spectrometry techniques to profile the metabolites derived from tryptophan catabolism and combine this knowledge with comprehensive enzymatic assays as well as expression analyses of key enzymes involved in tryptophan metabolism.
Content:
This one-week practical course offers students the opportunity to learn and apply fundamental and advanced analytical methods in the field of cancer metabolism. The focus is on investigating metabolic processes in cancer cells and analyzing the expression and function of key metabolic enzymes in the frame of tryptophan catabolism.
Objectives of the internship:
- Understanding the basic concepts of cancer metabolism.
- Introduction to and practical application of modern analytical methods such as High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography coupled with Tandem Mass Spectrometry (LC-MS/MS).
- Conduct and evaluate specific enzymatic assays to investigate the activity of key enzymes in cancer metabolism.
Students will be supervised by experienced scientists and PhD students who will guide them through various experimental techniques of the department and who will also assist with data analysis and interpretation.
Part 2: Diving into proteomics to crack epigenetics and promote translational medicine
Date: 2.-6. December 2024
Hosts/Supervisors: Gianluca Sigismondo, Dennis Friedel, Fuat Kaan Aras, and co-workers
Topics:
As canonical example, the caterpillar and the butterfly share the exact same genome while they express different proteins sets during their life stage. Without impacting on the genetic code, epigenetics factors are indeed able to drive inter-organism differences by modifying the post translational modification (PTM) status of the fundamental building blocks of life: proteins, and nucleic acids, thereby regulating fundamental biological processes ranging from cell division and gene transcription to DNA damage repair and DNA replication. Especially for proteins, an increasing number of PTMs has been described in nature, and PTMs can regulate in time and space protein localization, function, as well as interaction partners, and stability. Protein PTMs therefore affect the chemico-physical properties of the target, but have a specific impact also on its mass. Quantitative mass spectrometry (MS)-based proteomics therefore represents the elective tool to study in an unbiased way the epigenetic repertoire and its regulation upon different cellular stimuli. Deregulation in the epigenetic landscape (or epigenome) has been strongly associated to different cellular disfunctions and it is a known factor driving cancer predisposition. For this reason, a growing number of drugs targeting epigenetic enzymes have been introduced into the clinic. Despite the initial efficacy, quite some patients still show refractory or resistant phenotype to such epigenetic drugs, thereby requiring on one side a thorough understanding of the epigenetic mechanisms and on the other side a detailed patient classification.
Content:
In this module, the students will be introduced to the most advanced strategies to study epigenetics via MS-based quantitative proteomics, and they will have hands-on experience covering from sample preparation and enrichment procedures ahead of mass spectrometry, to data acquisition, analysis of their own results and basic visualization. In this regard, samples from cell lines upon selective epigenetic perturbation will be used as playground for elucidating the biological effect of such specific insult in terms of pathways and targets deregulation.
Part 3: Creating cancer models using organoids and CRISPR/Cas9 base editors
Date: 9.-13. December 2024
Host/Supervisors: Jens Puschhof, Lena Schorr, Kyanna Ouyang, Kamil Moskal, Sahana Asokan, Aurelia Saftien
Topics:
Adult stem cells (ASCs) reside in most organs of the mammalian body, where they constantly renew tissue or replenish lost cells upon injury. This process is based on the stem cells' ability to self-renew and give rise to differentiated daughter cells. One of the most active ASCs is located at the bottom of the crypts in the intestine, from where it continuously replenishes all cell types of the epithelium every 5 days. In 2009, it was first shown that ASCs from the mouse small intestine can perform their functions in vitro as well. Upon embedding the intestinal stem cells in a gel of extracellular matrix proteins and providing them with medium containing the essential niche components, they started to self-renew, differentiate and organize into 3D structures which are referred to as organoids. These models can be propagated indefinitely, contain both ASCs and differentiated cell types and resemble the structure and function of the original organ. Organoids models are rapidly becoming a central workhorse of cancer research to study the cellular heterogeneity of tumors, test patient-specific drug efficacy, recapitulate the mutational processes active in a tumor and assess tumor-microenvironment interactions.
Content:
In this practical, organoids will be derived from mouse biopsies and engineered using CRISPR/Cas9 base editing techniques to recapitulate key mutations found in colorectal cancer. To achieve these goals, several key aspects of organoid experimentation will be introduced, including media preparation, passaging and plating of organoids, live imaging, electroporation, clonal expansion, DNA extraction and sanger sequencing to validate CRISPR success.