Cancer is a disease of genetic mosaicism because cancerous cells harbor genetic mutations that are absent in normal cells within the same individual. In familial cancer patients, even though initial mutations exist in every cell, in most cases only specific cell types can progress into malignancy. Those cell types are called cancer cell-of-origin. Such a cell type-specific susceptibility implies the existence of a permissive or even synergizing signaling context in the cell-of-origin for particular genetic mutations to cause cancer formation. Therefore, identification of the cancer cell-of-origin would provide critical insights for understanding tumorigenic mechanisms and for designing rational therapeutic strategies.
Despite such importance, identification of the cell-of-origin for most cancers has been a daunting task (Visvader, 2011
). The reliability of revealing cell identity solely based on molecular and cellular analyses of late stage tumors are often confounded not only by infiltrated bystander cells but also by the acquired plasticity often found in terminal cancerous cells. To circumvent these issues, genetically engineered mouse models have been widely used to determine the tumorigenic potential of a specific cell type by initiating mutations with a cell-type-specific Cre transgene. However, it is critical to note that cells initially acquiring mutations (cell-of-mutation) may not be the cell-of-origin. When mutations are introduced in stem/progenitor cells, it is extremely difficult to distinguish whether initial mutant cells directly transform, or if they merely pass on mutations to more restricted progeny that then transform. In the latter scenario, the mutated stem/progenitor cell is simply the cell-of-mutation while the transforming progeny is the actual cell-of-origin (Visvader, 2011
The cell-of-origin for malignant glioma, a type of deadly brain cancer, remains controversial. Successful isolation of tumor cells with stem cell features (known as cancer stem cells) from human gliomas (Singh et al., 2004
) implies neural stem cells (NSCs) as the cell-of-origin. However, such NSC-like features of malignant glioma cells could be acquired during transformation rather than reflect the nature of the original cell type (Visvader, 2011
). Further evidence supporting the NSC-origin of glioma was obtained from mouse genetic studies. For example, the inactivation of tumor suppressor genes (TSGs) p53
and neurofibromatosis 1
) or the expression of a mutant form of p53
in NSCs consistently led to glioma formation in mouse models; and the physical locations of tumors appeared to associate with the subventricular zone (SVZ), where adult NSCs reside (Alcantara Llaguno et al., 2009
; Wang et al., 2009
; Zhu et al., 2005
). However, other studies suggest that NSC-derived progeny such as astrocytes or oligodendrocyte precursor cells (OPCs) might directly transform (Bachoo et al., 2002
; Lindberg et al., 2009
; Persson et al., 2010
). This unresolved controversy partially stems from the distinct oncogenic mutations used in these models that make the direct comparison difficult, and more importantly from the lack of high-resolution analyses of cellular aberrations during the transforming process.
MADM (Mosaic Analysis with Double Markers), a mouse genetic mosaic system (Zong et al., 2005
), could in principle be used to analyze aberrations in individual cell lineages prior to the final transformation, and should thus be suitable for identifying cancer cell-of-origin. Via Cre/loxP-mediated mitotic inter-chromosomal recombination, MADM generates a small number of homozygous mutant cells thus mimicking the sporadic loss-of-heterozygosity (LOH) of TSGs in human cancers (Knudson, 1971
). MADM also permanently labels these mutant cells with green fluorescent protein (GFP) and their sibling wild type (WT) cells with red fluorescent protein (RFP) within an otherwise unlabeled heterozygous mouse ( and S1A
). The single cell resolution benefited from the sparse labeling (0.1–1%, or much lower) (Zong et al., 2005
) enables one to track mutant cells throughout the entire process of tumorigenesis. The sibling red WT cells serve as internal controls for green mutant cells, thereby greatly facilitating detailed analyses of cellular aberrations of all lineages in their native environment. MADM can provide features that are indispensable for a robust analytical paradigm to identify a cell-of-origin.
MADM-based glioma model allows phenotypic analysis at single cell resolution
Here we report the application of MADM to glioma modeling. After initiating p53/NF1 mutations sporadically in neural stem cells (NSCs), we analyzed mutant NSCs and all their progeny at pre-malignant stages. We found significant overexpansion and aberrant growth specifically in OPCs but not NSCs or other lineages. Upon tumor formation, marker staining and transcriptome analysis confirmed the OPC nature of tumor cells. Finally, introducing the same mutations into OPCs consistently led to gliomagenesis. Our findings reveal OPCs as the cell-of-origin for glioma even when initial p53/NF1 mutations occur in NSCs, thus resolving the current controversy by distinguishing cancer cell-of-mutation from cell-of-origin. Importantly, although our studies focused on glioma, the analytical paradigm with MADM developed here could be applied to identifying cellular origins for many other cancers.