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Dissection of tumor heterogeneity in vivo

Periodic Reporting for period 4 - CancerHetero (Dissection of tumor heterogeneity in vivo)

Reporting period: 2019-12-01 to 2020-05-31

It is now widely accepted that tumors are composed of heterogeneous population of cells, which contribute to many aspects of treatment resistance observed in clinic. Despite the acknowledgment of the tumor cell heterogeneity, little evidence was shown about complexity and dynamics of this heterogeneity in vivo, mainly because of lacking flexible genetic tools which allow sophisticated analysis in primary tumors. We recently developed a very efficient mouse somatic brain tumor model which have a full penetrance of high grade glioma development. Combination of this model with several transgenic mouse lines allow us to isolate and track different population of cells in primary tumors, most importantly, we also confirmed that this can be done on single cell level. Here I propose to use this set of valuable genetic tools to dissect the cellular heterogeneity in mouse gliomas. First we will perform several single cell lineage tracing experiment to demonstrate the contribution of brain tumor stem cell, tumor progenitors as well as the relatively differentiated cells, which will provide a complete data sets of clonal dynamics of different tumor cell types. Second we will further perform this tracing experiment with the presence of conventional chemotherapy. Third we will perform single cell RNA sequencing experiment to capture the molecular signature, which determines the cellular heterogeneity, discovered by single cell tracing. This result will be further validated by analysis of this molecular signatures in human primary tumors. We will also use our established in vivo target validation approach to manipulate the candidate molecular regulators to establish the functional correlation between molecular signature and phenotypic heterogeneity. This project will greatly improve our understanding of tumor heterogeneity, and possibly provide novel approaches and strategies of targeting human glioblastomas.
It is the first report since the start of the project. The goal of the project is to analyse cellular heterogeneity of primary glioblastoma. Since the beginning of the project, we have made the following progresses which are within the planed time line.
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
The quantitative analysis of clonal growth of brain tumors in vivo was never performed before, these results collected by the current progress may form the basis of quantitative analysis of tumor growth, which will provide new insights into treatment of the glioblastoma.
Brain tumor stem cells marked by Tlx-GFP reporter