The tumor preventive effect of polyIC and the importance of Toll-like receptor-mediated signal transduction in hepatocarcinogenesis
Hematology, Oncology
Final Report Abstract
This collaborative research between the Calvisi laboratory at the University of Regensburg and the Feng laboratory at the University of California San Diego focused on the role of CD133 in intercellular signal transduction. The core hypothesis suggested that a proliferative signal deficit (SHP2 deletion in the mouse liver), followed by a pro-proliferative stimulus (partial hepatectomy), led to the emergence of regional clusters of actively proliferating CD133-positive cells. Interestingly, these clusters were not clonal but originated from diverse neighbouring hepatocytes. Single-cell RNA sequencing (scRNAseq) revealed an exchange of Immediate Early Gene (IEG) transcripts within CD133-positive colonies, resulting in higher intracellular diversity (entropy). This led to the idea of a unique CD133-dependent intercellular transport mode for IEGs. Based on a distinct staining pattern with a monoclonal antibody (86781; CST) displaying particles in a filament-like pattern spanning adjacent cells within the PLC human cancer cell line, a hypothesis was formulated: a novel type of signaling organelle, different from exosomes and microvesicles, governs the intercellular transport of IEG transcripts, inducing stemness and potentially explaining CD133's function as a stem cell marker. The research aimed to study this purported signaling organelle's nature. An experiment involving coplating CD133-GFP overexpressing cells with cytoplasmic mCherry-positive cells demonstrated particle transport between clonally unrelated neighboring cells. Correlative light and electron microscopy with improved resolution verified the presence of freely moving CD133-GFP-positive particles in adjacent cell cytoplasm. The particles' exact nature, whether protein aggregates or lipid-enclosed vesicles, remained undetermined. To delve deeper, techniques suitable for live-cell imaging were developed, relying on the Dog- Catcher Dog-Tag system and the ALFA-Tag ALFA-nanobody system. Creating knock-ins of small molecular tags into the CD133 genomic sequence using CRISPR-Cas9 was essential, given an aberrant distribution pattern observed with CD133 overexpression compared to wild type. Successful knockins were obtained, and mass spectrometry on the ALFA-Tag knockins, confirmed by immunoprecipitation, hinted at novel interaction partners of CD133, including major vault protein (MVP) and Transmembrane protein 205 (TMEM205).
Publications
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Identification of CD133+ intercellsomes in intercellular communication to offset intracellular signal deficit. eLife, 12.
Kaneko, Kota; Liang, Yan; Liu, Qing; Zhang, Shuo; Scheiter, Alexander; Song, Dan & Feng, Gen-Sheng
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Complex Roles of PTPN11/SHP2 in Carcinogenesis and Prospect of Targeting SHP2 in Cancer Therapy. Annual Review of Cancer Biology, 8(1), 15-33.
Scheiter, Alexander; Lu, Li-Chun; Gao, Lilian H. & Feng, Gen-Sheng
