Project Summary/Abstract Most tumors are not homogeneous, but rather comprised of highly distinct and heterogenous cancer cell populations. Adjacent cells within a tumor may harbor different genetic alterations and have different phenotypes. Thus, complex tumors are more 'than the sum of their parts', which contributes to aggressiveness, metastatic potential, and drug resistance. This complexity of intratumor heterogeneity is a major challenge in cancer therapy. In contrast to normal cells in which chromosome segregation is tightly controlled to maintain a diploid chromosome set, cancer cells are frequently aneuploid. Indeed, abnormal gains or losses of chromosomes are amongst the most common characteristics of cancer cells with nearly all solid cancers exhibiting some type of aneuploidy. A high degree of tumor aneuploidy also correlates with poor clinical outcomes. Although this strong correlation between aneuploidy and cancer is well known, causal relationships are incompletely understood. Especially in normal tissues and during early stages of carcinogenesis, chromosome segregation errors are rare and transient events that are notoriously difficult to study. To advance our understanding how chromosome segregation errors drive cancer initiation, development, evolution, and heterogeneity and enable novel types of model systems to, for example, explore aneuploidy as a therapeutic target in patient-derived cancer organoids, this pilot project proposes to develop a molecular and imaging toolkit to observe and manipulate chromosome segregation dynamics with high spatial and temporal accuracy in three-dimensional model and patient-derived cancer organoids. In Aim 1, we develop methods to identify, count and track specific chromosomes during multiple cell divisions in cancer organoids. Two proposed approaches involve endonuclease-deactivated dCas9 combined with gRNAs to create unique chromosome-specific spectral barcodes or using dCas9 imaging of repetitive DNA sequences to generate chromosome-specific fluorescence patterns, with the long-term goal of enabling "live karyotyping" in cancer organoids using deep learning networks. In Aim 2, we develop three alternative strategies to control chromosome dynamics and integrity using optogenetics. The proposed optogenetic actuators include a light-controlled kinetochore-microtubule interface, an optogenetic chromosome trap to immobilize specific chromosomes during cytokinesis, and photoactivated Cas9 to produce acentric chromosome arms or induce chromothripsis. The effectiveness of these strategies will be evaluated in mammary epithelial and breast cancer models, with the goal of understanding how acutely induced segregation errors or chromothripsis of specific chromosomes affects cancer cell dynamics and tumor evolution.

Project Narrative An abnormal number of chromosomes and associated copy number variations of the genetic material are hallmarks of cancer. However, how chromosome segregation errors and the resulting aneuploidy contribute to cancer initiation, malignancy, evolution, and heterogeneity is poorly understood. The goal of this project is to develop innovative imaging and optogenetics technology to observe and manipulate chromosome segregation dynamics with high spatial and temporal accuracy in patient-derived cancer organoids to ask fundamental questions about the complex relationship between aneuploidy and cancer and inform development of new and personalized therapies.