USING ORGANOIDS TO STUDY CELL FATE IN HOMEOSTASIS AND CANCER

Stem cells are found in many organs, where they play essential roles in tissue repair and normal renewal. They have two defining properties: the ability to self-renew and the ability to differentiate into specialized cell types. This process is tightly regulated. Disruptions in stem-cell proliferation or differentiation can contribute to disease, aging, and cancer.

Such disruptions may result from mutations. Some mutations provide cells with advantages, such as faster growth or the ability to evade the immune system. Over time, cells carrying these cancer-associated mutations may multiply excessively, eventually developing into tumors. Importantly, individual cells within the same tumor often carry different mutations, a phenomenon known as intratumor heterogeneity. A mature tumor may contain hundreds of distinct mutations, while patients with clinically similar tumors may have different genetic abnormalities. Understanding the effects of these mutations could improve diagnosis and enable more targeted treatments.

In this thesis, we investigate the differences between normal tissue differentiation and its deregulation in cancer, using two technologies: organoids and CRISPR. Organoids are miniature, lab-grown versions of organs that enable the study of cell composition and differentiation in a controlled environment. CRISPR allows precise genetic editing, making it possible to investigate the effects of specific genes and cancer-associated mutations.

Chapter 1 reviews the use of CRISPR in human organoids for cancer research, focusing on the functional analysis of tumor-associated genetic changes. Chapter 2 describes protocols for differentiating human and mouse small-intestinal organoids into most mature cell types, including neuroendocrine cells. These rare cells regulate essential processes, including digestion and insulin release, through hormone production.

Because human intestinal neuroendocrine cells are scarce and difficult to study, Chapter 3 presents a method to increase their numbers in organoids by overexpressing Neurogenin3, a transcription factor that promotes neuroendocrine differentiation while halting proliferation. CRISPR was also used to generate genetic reporters were also developed to isolate specific neuroendocrine subtypes and to investigate the signals controlling their formation. Chapter 4 provides detailed protocols for Neurogenin3 overexpression and CRISPR-mediated genetic modification of intestinal organoids.

Chapter 5 examines neuroendocrine neoplasms, rare tumors that share features with neuroendocrine cells. Using CRISPR, we introduced mutations in MEN1, RB1, and TP53 into organoids and found that these mutations synergistically affect differentiation and proliferation. We also investigated the molecular function of menin, the MEN1 protein, identifying differences from previous studies that warrant further research.

Chapter 6 explores how different tumor types arise in the same tissue, focusing on the lung. Using CRISPR and lung organoids, we modeled tumor development from human airway stem cells and studied how distinct mutation combinations affect tumor initiation, differentiation, genome stability, and treatment response.

Overall, this dissertation establishes organoid systems and protocols for studying cell specification in health and disease. By introducing specific mutations into normal cells, we reveal their effects on tumor behavior and provide insights that may support the development of more precise and targeted cancer therapies.