Mosaic analysis with double markers reveals tumor cell of origin in glioma

Abstract

Cancer cell of origin is difficult to identify by analyzing cells within terminal stage tumors, whose identity could be concealed by the acquired plasticity. Thus, an ideal approach to identify the cell of origin is to analyze proliferative abnormalities in distinct lineages prior to malignancy. Here, we use mosaic analysis with double markers (MADM) in mice to model gliomagenesis by initiating concurrent p53/Nf1 mutations sporadically in neural stem cells (NSCs). Surprisingly, MADM-based lineage tracing revealed significant aberrant growth prior to malignancy only in oligodendrocyte precursor cells (OPCs), but not in any other NSC-derived lineages or NSCs themselves. Upon tumor formation, phenotypic and transcriptome analyses of tumor cells revealed salient OPC features. Finally, introducing the same p53/Nf1 mutations directly into OPCs consistently led to gliomagenesis. Our findings suggest OPCs as the cell of origin in this model, even when initial mutations occur in NSCs, and highlight the importance of analyzing premalignant stages to identify the cancer cell of origin.

Figure 1. MADM-based glioma model allows phenotypic analysis at single cell resolution

(A) Scheme of MADM-based glioma modeling. Inset illustrates how MADM concurrently mutates and labels cells (for full details, see Figure S1 and (Hippenmeyer et al., 2010; Zong et al., 2005)). (B) Representative confocal images show hGFAP-Cre-induced MADM labeling of four NSC-derived cell types in a 2-month old WT-MADM mouse. Arrows point to MADM-labeled cells expressing corresponding markers. Scale bars: 20μm; inset, 5μm. See also Figure S1.

Figure 2. MADM-mediated sporadic concurrent inactivation of p53 and NF1 in embryonic NSCs reveals the entire process of gliomagenesis

(A) Sagittal sections from brains of MADM mice at indicated ages. In WT-MADM mice (left column), both green and red cells are WT. In mutant-MADM mice (right column), green cells are p53 and NF1 double-null, red cells are WT. Tumor boundary is demarcated with dashed line. Scale bars: 2mm; Insets, 100μm. Ki67 staining shows that tumor cells are highly proliferative. (B) Systematic quantification of G/R ratios in mutant-MADM brains from P5 to P60. Total cell numbers counted are shown in parenthesis. Each number is the sum from three brains. (C) The percentage of BrdU+ cells in WT and mutant cell populations in the brain parenchyma of mutant-MADM mice at indicated ages. (D) The proportion of BrdU+ cells with genotypes “−/−“, “+/−“, and “+/+” in the brain parenchyma of mutant-MADM mice at indicated ages. “+/−“ includes both double-colored and colorless BrdU+ cells. In (C and D) BrdU was administered 1.5 hours prior to sacrifice. Error bars +/− SEM; N = 3 mice. See Experimental procedures for sampling scheme. * P<0.05, ** P<0.01, paired T-test. See also Figure S2.

Figure 3. Analysis at a pre-transforming stage of gliomagenesis suggests that OPCs rather than NSCs serve as the cell-of-origin

(A) Average G/R ratios of each cell type in brain parenchyma of P60 mutant-MADM mice. * P<0.05, ** P<0.01, paired T-test. “#” P<0.0001, one-way ANOVA. (B) Left chart: with a single BrdU injection (1.5 hours prior to sacrifice) at P60, BrdU-positive mutant (−/−) cells in the brain parenchyma consist entirely of OPCs (PDGFRα+). Right chart: upon BrdU administration by drinking water for one week, all BrdU-positive mutant (−/−) cells in the brain parenchyma belong to the oligodendrocytic lineage (Olig2+), and the majority of them are OPCs (PDGFRα+). (C–E) “4+1” channel staining shows that all BrdU+ mutant OPCs in a P60 mutant-MADM mouse brain belong to the oligodendrocyte lineage. (C) Without Olig2 staining, all MADM labeled BrdU+ cells are mutant (green). Notably, some BrdU+ mutant cells are PDGFRα negative (marked with cyan circles). (D) An adjacent section stained with Olig2 together with MADM, BrdU, and PDGFRα, shows that all mutant BrdU+ cells have red nuclei, indicating positive staining of Olig2. (E) Representative magnified confocal images show: mutant cells without Olig2 staining (left), mutant cells with Olig2 staining in red channel (middle), and heterozygous yellow cells (right). The orthogonal Z-axis is shown on the side of each panel. Scale bars: (C and D): 20μm, insets, 5 μm; (E): 5μm. (F) G/R ratios of BrdU+ cells in the SVZ and NeuN+ cells in the olfactory bulb (OB) from P60 mutant-MADM mice. BrdU was given in drinking water for 7 days. (G) Quantification of cells with indicated genotypes among all BrdU+ cells in the non-SVZ brain parenchyma (left chart) or the SVZ (right chart) after one week of BrdU administration. N=3 mice for all quantification in (A, B, F and G). See Figure S3 and Extended experimental procedures for detailed systematic sampling schemes. Error bars represent +/− SEM. Total cell numbers counted in (A, B, F and G) are shown in parenthesis. Each number is the sum from 3 brains. See also Figure S3.

Figure 4. MADM-generated glioma cells exhibit many OPC features

(A) Representative image of a mutant-MADM brain carrying a GFP+ glioma. (B–D) Adjacent H&E staining of tumor regions shows typical glioma features, including necrotic areas (“N “ in B), multinucleated giant cells (C), and peri-vascular satellitosis (D). Scale bars: (A), 2mm; (C and D), 200μm. Magnification in (B), 400×. (E) Representative low magnification images show elevated expression of a panel of well-established glioma markers in tumor regions. All staining was done with adjacent sections from the same tumor. Tumor boundary is demarcated by dashed lines. T, tumor mass. Scale bars: 100μm except for CD9 staining, where it is 50μm. (F) Quantitative RT-PCRs confirm the over-expression of OPC markers in MADM-generated glioma. (G–L) Confocal images at high magnification show that proliferating (Ki67+) green tumor cells express markers for OPCs (G–I, pointed by arrows) but not for other cell types (J–L). The signals of cell-type marker staining in the right column of (J–L) were converted to red for better examination of their co-localization with GFP. Some Ki67-negative green cells were CC1+ (circled in L). Scale bars: 20μm. (M) Transcriptome comparison between tumor samples and four neuroglial cell types (top four rows), and the four subtypes of human GBMs defined by TCGA (bottom four rows) with the single sample Gene Set Enrichment Analysis (ssGSEA) method. N and O represent tumor samples from mutant-MADM mice induced by NSC-Cre (Nestin-Cre or hGFAP-Cre) and by NG2-Cre, respectively; PDGFRα+ indicates primary tumor cells enriched by anti-PDGFRα immunopanning method. Color key: red to blue indicates significantly similar to dissimilar. See also Figure S4.

Figure 5. Spatial analyses of early lesions based on perineuronal cytoarchitecture as a landmark implicate that glioma initiate at brain regions away from the SVZ

(A–C) Immunofluorescent staining of mutant-MADM brains at distinct tumorigenic stages. Neuronal nuclei were stained with NeuN and are marked as “*”. Arrows point to perineuronal pre-transforming OPCs or tumor cells. The proliferating status of perineuronal mutant cells is shown by Ki67 staining (yellow arrows in the bottom row). (D) H&E staining of the adjacent section of (C) shows perineuronal satellitosis. (E) Proportion of perineuronal structures with distinct mutant OPC-to-neuron ratios (O:N) either in pre-transforming MADM mutant brains (P30 and P60, N=3 brains each) or in tumors (T, N=4). (F) Schematic summary of lesion sites (green spots), which are defined by O:N≥3 perineuronal structure together with MADM labeling, Ki67 staining, and also pathology in most cases. Brains devoid of any detectable lesions are shown in light gray. The analysis is based on a cohort of mutant-MADM mice induced by hGFAP-Cre. (G–K) Representative brain images from (F) with gliomas from medium to small sizes. Insets show Ki67 staining of the tumor regions. The glioma identity in these brains was confirmed by pathological criteria except for (K), in which the perineuronal structure of ≥ 3 mutant OPCs suggests that it should be a lesion at its early stage. Scale bars: (A–D), 20μm; (G–K): left column, 2mm; middle and right columns, 50μm. See also Figure S5.

Figure 6. OPCs can be directly transformed into malignant glioma

(A–D) In the MADM system NG2-Cre transgene labels OPCs and oligodendrocytes but not astrocytes or neurons. Arrows point to MADM-labeled cells expressing indicated markers. (E–H) Mutant OPCs over-expand at pre-transforming stages. Brain sections from P60 WT-MADM (E) or mutant-MADM (F) mice induced by NG2-Cre. (G) G/R ratios within each cell lineage in P60 Mutant-MADM brains induced by NG2-Cre. NA: not applicable. Error bars represent +/− SEM. Total cell number being counted are shown in parenthesis. (H) Percentage of OPCs (PDGFRα+) versus oligodendrocytes (CC1+) within mutant and WT cell populations. N=3 mice in (G) and (H). * P<0.05, T-test. (I and J) Representative gross images of malignant glioma in NG2-Cre induced mutant-MADM mice, either locating around hypothalamus with invasion into the subarachnoid space (I) or residing within the brain parenchyma (J). Arrows in (I) point to tumor cells spreading along the meninges. Ki67 Staining in insets shows that tumor cells are highly proliferative. Scale bars: 2mm; Inset, 100μm. See also Figure S6.

Figure 7. Comparative pathological analyses of NSC- and NG2-Cre induced tumors suggest that tumor cell morphology is highly dependent on the location rather than initially mutated cell types

(A and B) Regardless of the Cre lines used, tumors at the same location exhibit indistinguishable pathological features. (C) Transplantation of tumor cells from NG2-Cre induced glioma with subarachnoid invasion (top row) into the brain parenchyma of NOD/SCID mice to generate secondary tumors (bottom row). Pathological features of the primary and the secondary tumors mimic tumor features in (B) and (A) respectively. Tumor boundaries are demarcated by dashed lines. The magnification of images in the middle columns is 400×. Images from the right column are 2.5× digital zoom-in of the corresponding middle-column ones.