Periodic Reporting for period 4 - CancerHetero (Dissection of tumor heterogeneity in vivo)
Reporting period: 2019-12-01 to 2020-05-31
1) Single cell tracing of brain tumor stem cells: To visualize the in vivo contribution of the quiescent Tlx-GFP positive cells in mouse high grade gliomas, we will make use of our established Nestin-TVA;Tlx-GFP;Tlx-CreERT2;Confetti animal model. Tumors will be induced by injection RCAS vectors expressing PDGFB+AKT+Luciferase to the mice brain carry the following genetic modification: Nestin-TVA;Tlx-GFP;Tlx-CreERT2;Confetti. With tracing of single Tlx positive cells and their progenies, we found these cells are long-term clonal forming cells in vivo. We also collected strong evidence of exsitance of two distinct Tlx positive cells in vivo.
2) Sing cell lineage tracing of fast-dividing tumor cells: We demonstrate that only around 5% the proliferating tumor cells express Tlx-GFP, to determine the contribution of the proliferating population in vivo, we will perform single cell tracing experiment in the proliferating population. It is known that retrovirus infects only the proliferating population, which makes it a very valuable tool to deliver fluorescent proteins to the proliferating tumor cells in tumor bearing Nestin-TVA;Tlx-GFP mice. By injecting retroviral-GFP construct to tumor bearing mice, we confirmed that the infected cells are actively dividing cells and they are Tlx-GFP-negative. Initial observations show that these cell loose proliferation capacity during tumor progression. A quantitative description of these cells was completed and showed these cells are short lived in vivo
3) Single cell RNA seq experiment demonstrates that mouse glioblstoma cells are extremely hetergeneous but form a stem cell hierarchy in vivo