Onderzoek naar uitzaaiingen bij colorectale kanker: proteomische inzichten belicht door fluorescentiemicroscopie

Cancer is the second leading cause of death worldwide, responsible for one in six deaths globally, significantly impacting both individuals and society. Patients' morbidity and mortality is mainly due to metastasis, a complex multi-step process where cancer cells spread from the primary tumor to other organs, forming secondary tumors. Despite efforts to promote preventive screening, cancer is often diagnosed at advanced stages, complicating treatment and negatively impacting patients' outcomes and well-being. Consequently, identifying and characterizing novel molecular targets for early diagnosis or therapy is indispensable for developing more effective strategies to combat cancer. This PhD thesis leverages the analytic capacity of quantitative proteomics, a powerful tool that can examine thousands of proteins simultaneously, providing insight into their expression, regulation, function and interactions on a large scale, therefore enhancing our understanding of their roles in complex and heterogeneous systems like cancer. Furthermore, validating and comprehensively analyzing dysregulated proteins and their functions, is essential to determine their potential in targeting cancer progression. Here we focus on colorectal cancer (CRC), the second deadliest cancer, often diagnosed at later stages and therefore associated with a worse prognosis. To elucidate mechanisms involved in CRC metastasis, we investigated CRC cell models consisting of established isogenic cells lines with low and high metastatic potential (KM12 and SW models). The problem statement and objectives of this thesis are detailed in Chapter 1. Chapter 2 provides a literature review of key concepts, including general cancer biology (section 2.1), clathrin as a potential target (section 2.2), and the main analysis tools used in this work: proteomics, hydrogels, and fluorescence microscopy (sections 2.3-2.5). Chapter 3 is dedicated to an in-depth fluorescence-based analysis of clathrin, the primary component of clathrin-mediated endocytosis, which is responsible for the uptake of receptors and ligands from the extracellular environment. Clathrin was selected because vesicle trafficking is known to be altered in cancer. Additionally, proteomic analyses of the CRC cell models found a massive upregulation of proteins involved in vesicle trafficking in highly versus poorly metastatic cells. Using fluorescence microscopy, we discovered that organization of clathrin at the membrane changes with metastatic potential. More specifically, alternative structures where clathrin forms large and planar assemblies, known as flat clathrin lattices, were more predominant in highly versus poorly metastatic cells. We demonstrated that these structures exhibit a high stability and persist at the plasma membrane for over eight hours. While fluorescence imaging and subsequent analysis provided insights into the link between clathrin and metastasis, the experiments were conducted on two‑dimensional glass or plastic substrates, overlooking the complex biochemical and biophysical cues in the heterogeneous tumor microenvironment. Among the overlooked factors present in the tumor microenvironment is the extracellular matrix stiffness, a key aspect of solid tumors' progression that has only recently received significant attention. To better mimic the in vivo situation and investigate the impact of stiffness on cell behavior, we developed a robust platform for synthesizing and characterizing polyacrylamide hydrogels with varying stiffnesses that can be adjusted for bulk cell culture applications in Chapter 4. The developed library included hydrogels that mimic the stiffness of both healthy tissue and advanced stages of tumor progression, such as adenoma and adenocarcinoma, which were selected to explore how substrate stiffness affects intracellular and secreted proteins in CRC. In Chapter 5, we performed TMT‑based quantitative proteomics to identify dysregulated proteins, and microarrays to survey cytokines. We determined that substrate stiffness significantly impacts secreted proteins, but surprisingly does not affect whole-cell intracellular protein levels, despite observable changes in cell morphology. Additionally, functional assays on cell migration and matrix remodeling indicated an interesting, yet unexplored, role of secreted factors in cancer progression. The overall findings of this thesis are discussed in Chapter 6, along with suggestions to further this research, emphasizing the need for increasingly complex and relevant cancer models.