Scientists have used a technique known as mosaic analysis with double markers (MADM) in mice to identify what they claim is the cell type of origin in the brain cancer glioma. A team led by the University of Oregon’s Hui Zong, Ph.D., generated MADM mouse models that allowed them to track cell differentiation and proliferation in transformed neural stem cells (NSCs) and their differentiated progeny during the whole process of gliomagenesis, even before tumors developed.
The results showed that mutant NSC-derived OPCs exclusively displayed significant overexpansion and aberrant growth prior to evidence of malignancy. None of the other NSC-derived lineages or NSCs themselves displayed abnormal growth or proliferation.
Moreover, cancer cells in the tumors that subsequently developed in the MADM mice displayed evident OPC features. The findings strongly suggest that it is mutation-carrying OPCs derived from NSCs that appear to represent the true cell of origin in glioma and not the mutant NSCs themselves.
Reporting their work in Cell the researchers say the clincher was the finding that introducing the same mutations directly into OPCs derived from normal NSCs consistently led to gliomagenesis. Their paper is titled “Mosaic Analysis with Double Markers Reveals Tumor Cell of Origin in Glioma.”
MADM, a mouse genetic mosaic system, was developed by Dr. Zong and Liqun Luo at Stanford University back in 2005. MADM can be used to generate a small number of homozygous mutant cells in defined cell types in engineered mice, thus mimicking the sporadic loss of heterozygosity (LOH) of tumor suppressor genes (TSGs) in human cancers.
The process involves crossing separately engineered parent mice, each of which carries an incomplete mutation. In their offspring, the complete homozygous mutation in the targeted cell type is acquired only sporadically during recombination prior to cell division. Its acquisition by a cell is accompanied by the permanent activation of a green fluorescent protein (GFP) label. The wild-type (WT) sibling cell resulting from the same cell division acquires a red fluorescent protein (RFP) and acts as an effective internal control. The single-cell resolution afforded by the technique allows scientists to track mutant cells throughout the entire process of tumorigenesis, according to Dr. Zong and his team.
The researchers chose to generate an MADM mouse model in which both p53 and NF1 were sporadically inactivated in NSCs, as the two genes are among the most frequently mutated in human glioma patients. They then analyzed the overall expansion of cells derived from the resulting GFP-labeled mutant NSCs in mice at different ages.
What they found was that in animals with the mutant-MADM brains there was a progressive overrepresentation of green mutant cells from day five after birth to day 60. The formation of full-blown GFP+ tumors developed at about five months of age.
Because mutant (green) and wild-type (red) sibling cells originate from the same mother cell in equal numbers initially, the ratio of green to red cell numbers (G/R ratio) provides a quantitative measure of the extent to which the mutant cells have expanded in comparison with their nonmutant siblings.
In the MADM mouse model the average G/R ratio in the brain increased from about 4 to about 30 between day five and day 60, indicating a continuous overexpansion of the mutant cells. However, the increase of G/R ratio appeared to reach a plateau between days 30 and 60, suggesting that overexpansion of mutant cells had largely stopped prior to the formation of actual tumors. Further analyses showed that at this point the mutant cells were still dividing but slowly.
Even though mutant cell proliferation appeared to plateau by two months, by 4–5 months of age all the mutant-MADM mice developed brain tumors that were invariably positive for GFP, confirming that the tumors formed from the population of overexpanded MADM-induced mutant cells. Although the transcriptome profiles of these tumors were very similar, pathological analyses demonstrated significant heterogeneity in their appearance, with some demonstrating typical astrocytic features, and others malignant glioma features. Most were highly anaplastic.
The next stage was to determine the identity of the proliferating mutant cell type by evaluating the G/R ratios of all four mutant NSC-derived cell types (neurons, astrocytes, oligodendrocytes, and OPCs) at day 60, when mutant cell expansion had ceased but tumors had yet to develop. This suggested it was only OPCs that had overproliferated; by day 60 the G/R ratio of OPCs was over 130, significantly higher than the ratios of the other three cell types. Moreover, the mutation-carrying OPCs appeared to have a reduced capacity to give rise to mature oligodendrocytes, as the G/R ratio of oligodendrocytes was about 10 times less than that of OPCs.
Having identified OPCs as the major proliferating cell type at the pretransformation stage, the researchers went on to evaluate the expression patterns of cell-specific markers in tumors that developed in the MADM-glioma animals. They found enriched expression of OPC markers, and GFP+ proliferating cells expressed numerous OPC markers but not markers for neurons or astrocytes.
Global transcriptome comparison of tumor samples and all four neuroglial cell types in addition confirmed that tumor cells closely resembled OPCs, rather than any of the other neuroglial cell types. Significantly, and despite their morphological heterogeneity, the tumor cells also all shared almost identical molecular profiles, supporting the hypothesis that they were all derived from a common cell of origin. “Taken together, detailed comparison between mutant and WT cell behaviors in NSCs and all progeny lineages strongly suggests that NSCs function as the cell of mutation but fail to directly transform, whereas OPCs function as the cell of origin for glioma,” the authors conclude.
Up until this point the results indicated an overall gliomagenesis pathway by which NSCs carrying the p53/NF1 mutations gave rise to mutant OPCs, which then multiply and eventually transform into malignancies. What wasn’t yet clear, however, was whether the mutations must first occur in NSCs or whether OPCs could be directly transformed by the same set of mutations. To address this question the team generated an MADM mouse model in which the p53/NF1 mutations were directly introduced into OPCs.
In these animals the green mutant OPCs populated the entire brain and reached an average G/R ratio of over 300. In fact, the authors state, all lines of evidence including marker expression patterns, transcriptome profiles, tumorigenic capacity, and pathology indicated that tumors developing in both this model and the NSC-mutant model were intrinsically identical.
“Our study extends rather than contradicts previous reports,” the authors state. “Limited by the cellular resolution at pretransforming stages, previous studies demonstrated the transforming potential of NSCs but did not clearly distinguish cell of mutation, from cell of origin. MADM, on the other hand, offers a robust analytical paradigm for the identification of the cell of origin with both permanently GFP-labeled mutant cells and RFP-labeled WT internal control cells immediately after the initial mutations occurred.”
Dr. Zong and colleagues suggest MADM could in principle be used to help investigate other aspects of cancer biology, such as evaluating tumor architecture, tracking metastasized tumor cells, and studying tumor-niche interactions. “Broadly, the ability to perform sporadic single-cell genetic manipulations in unambiguously labeled cells should make MADM an invaluable analytical tool for fields such as developmental biology and neuroscience that rely heavily on in vivo analyses,” they conclude.