Single-cell ribosome profiling to monitor the translational landscape of tumor-initiating cells

Objective

Temporal control of gene expression is critical for cancer cells. However, while transcriptional control has been extensively studied in cancer, it has become evident that translational control is a major determinant of protein abundance and cellular function. Therefore, protein synthesis pathways have immense, hitherto untapped potential to understand tumorigenesis and may provide novel entry points for cancer therapies. Tumors are heterogeneous tissues, where a small subset of tumor-initiating cells plays a key role in cancer progression and therapy resistance. Tumor-initiating cells exhibit low protein synthesis rates, a hallmark of stemness, yet how translational control impacts their function remains enigmatic. Here, we will develop an in vivo strategy to comprehensively monitor the translational landscape of tumor-initiating cells at single-cell resolution. Leveraging our recently established in vivo single-cell ribosome profiling strategy, ultrasound-guided in utero lentiviral microinjections and an elegant mouse model of cancer, we plan to track and perturb tumor-initiating cells in their native environment. First, we will develop and validate a method to annotate the in vivo single-cell translational efficiency landscape of tumor-initiating cells. Second, focusing on translationally altered genes in tumor-initiating cells, we will identify translational programs driving tumor progression and therapy resistance in vivo. Third, we will molecularly characterize key translational efficiency genes and integrate spatial transcriptomics to map tumor-initiating cell behavior. Collectively, the unique combination of advanced tools will offer unprecedented and systematic insights into the translational landscape of tumor-initiating cells, identify novel translationally regulated genes driving cancer progression and therapy resistance and lay the foundation for developing novel therapies that specifically target tumor-initiating cells in cancer patients.