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Cell-of-origin susceptibility to glioblastoma formation declines with neural lineage restriction

Nature Neuroscience (2019) | Download Citation

Abstract

The contribution of lineage identity and differentiation state to malignant transformation is controversial. We have previously shown that adult neural stem and early progenitor cells give origin to glioblastoma. Here we systematically assessed the tumor-initiating potential of adult neural populations at various stages of lineage progression. Cell type–specific tamoxifen-inducible Cre recombinase transgenes were used to target glioblastoma-relevant tumor suppressors Nf1, Trp53 and Pten in late-stage neuronal progenitors, neuroblasts and differentiated neurons. Mutant mice showed cellular and molecular defects demonstrating the impact of tumor suppressor loss, with mutant neurons being the most resistant to early changes associated with tumor development. However, we observed no evidence of glioma formation. These studies show that increasing lineage restriction is accompanied by decreasing susceptibility to malignant transformation, indicating a glioblastoma cell-of-origin hierarchy in which stem cells sit at the apex and differentiated cell types are least susceptible to tumorigenesis.

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Data Availability

Sequence data supporting the findings in this paper are publicly accessible in Gene Expression Omnibus as GSE117258. The data supporting the findings of this study are available from the corresponding authors upon request.

Additional information

Journal peer review information Nature Neuroscience thanks Martine Roussel, Mario Suva and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Acknowledgements

The authors thank D. Laks for processing of the RNA sequencing data and members of the Parada laboratory for helpful suggestions and discussion. We thank N. Socci, E. Feng and V. Boyko for their help in various analyses, and the MSKCC Genomics and Bioinformatics Cores and the Weill Cornell Genomics and Epigenomics Cores for their assistance. This work was supported in part by the Children’s Tumor Foundation Young Investigator Award and National Institutes of Health (NIH) T32 Postdoctoral Training Grant (2T32CA124334-06; PI: Jerry Shay) to S.A.L. L.F.P. is a recipient of NIH R01 grant CA131313-01A1 and National Cancer Institute R35 grant CA210100.

Author information

Affiliations

  1. Brain Tumor Center, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    • Sheila Alcantara Llaguno
    • , Daochun Sun
    • , Alicia M. Pedraza
    • , Elsa Vera
    • , Zilai Wang
    •  & Luis F. Parada
  2. Cancer Biology & Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    • Sheila Alcantara Llaguno
    • , Daochun Sun
    • , Alicia M. Pedraza
    • , Elsa Vera
    • , Zilai Wang
    •  & Luis F. Parada
  3. Department of Pathology, Section of Neuropathology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    • Dennis K. Burns
  4. Department of Neurosurgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    • Luis F. Parada
  5. Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    • Luis F. Parada

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Contributions

S.A.L. and L.F.P. conceptualized the study. S.A.L., D.S., A.M.P. and E.V. performed experiments. Z.W. contributed reagents and material. S.A.L., D.S., A.M.P., E.V., D.K.B. and L.F.P. analyzed the data. S.A.L and L.F.P. wrote the manuscript.

Competing interests

The authors declare the following financial competing interests: L.F.P. has advisory or consulting relationships with Bio-Thera Pharmaceuticals (2013–2018), Howard Hughes Medical Institute Scientific Advisory Board (2006–2023) and the National Cancer Institute Board of Scientific Advisors (2013–2018).

Corresponding authors

Correspondence to Sheila Alcantara Llaguno or Luis F. Parada.

Integrated supplementary information

  1. Supplementary Figure 1 Histologic and molecular analysis of iCK-cre mutants.

    a. Immunofluorescence staining for NeuN in different brain regions. b. Quantification of indicated neuronal lineage markers in iCK-cre mutant (M) vs. control (C) brains. (Left-most panel) Parvalbumin, n=12M,14C, p=0.2930; (Middle-left panel) Calretinin, n=9M,9C, p=0.3138; (Middle-right panel) GABAARα, n=12M,9C, p=0.0173; (Right-most panel) vGlut2, n=7M,7C, p=0.6140. *p<0.05. Two-tailed unpaired Student’s t-test. Data is presented as mean +/- SEM. c. Gene ontology analysis of gene events (n=239 unique genes; listed in Supplementary Table 1) in iCK-cre mutants (n=3) compared to controls (n=3) by PANTHER, showing functional annotations by biologic process (left panel) and molecular function (right panel) using Fisher’s Exact Test with false discovery rate adjustment. d. Quantification of mean telomere length (left panel) and % of short telomeres (right panel) of cortical and dentate gyrus (dg) neurons in iCK-cre mutants (cortex: n=2770; dg: n=45996) vs. controls (cortex: n=3093; dg: n=37722). ****p<0.0001 using two tailed unpaired Students t-test and Chi-square test, respectively. Telomere length is presented as mean+/- SEM, while % short telomeres is presented as a ratio of number of short telomeres (below 25th percentile) over total number of telomeres. e. Telomere FISH images of iCK-cre mutants vs. controls in the cortex and dentate gyrus. All scale bars=100 μm. In a and e, experiments were independently repeated with similar results at least n=3 times using at least n=3 different mouse tissue samples for each group.

  2. Supplementary Figure 2 Expression analysis of iND-cre Tomato reporter and mutant vs. control brains.

    a. Tomato staining of iND-cre; R26-stop-tdTomato reporter and control at 4 weeks post-induction. b. Immunostaining of iND-cre reporter with lineage markers at 5 months post-induction c. Immunofluorescence staining of iND-cre mutant vs. control at 10 weeks post-induction. d. Quantification of % BrdU-positive cells in aged iND-cre mutants (n=3) vs. controls (n=3) in each of the indicated brain regions. Data is presented as mean +/- SEM. e. Immunofluorescence staining of aged iND-cre mutant vs. control at >6 months post-induction. All scale bars=100 μm. In a, b, c and e, experiments were independently repeated with similar results at least n=3 times using at least n=3 different mouse tissue samples for each group.

  3. Supplementary Figure 3 Expression analysis of iDlx-cre Tomato reporter.

    a. Tomato staining of tamoxifen- and vehicle-treated iDlx-cre;R26-stop-tdTomato reporter at 1 month post-induction. b. Immunostaining of iDlx-cre;R26-stop-tdTomato reporter with lineage markers at 5 months post-induction. All scale bars=100 μm. In a and b, experiments were independently repeated with similar results at least n=3 times using at least n=3 different mouse tissue samples for each group.

  4. Supplementary Figure 4 Molecular and histologic analysis of iDlx-cre mutants.

    a. Quantification of % BrdU-positive cells in aged iDlx-cre mutants (n=3) vs. controls (n=3) in each of the indicated brain regions. Data is presented as mean +/- SEM. b. Immunostaining of aged iDlx-cre mutants and controls with lineage markers. c. Western blot analysis of MAPK and PI3K pathway components in iDlx-cre mutant (M) and control (C) brains. Mouse GBM (mGBM) and HeLa cell lysates were used as positive controls. d. Quantification of mean telomere length (left panel) and % of short telomeres (right panel) of dentate gyrus neurons in iDlx-cre mutants (n=50144) vs. controls (n=65974). e. Quantification of mean telomere length (left panel) and % of short telomeres (right panel) of dentate gyrus neurons in iDlx-cre (n=33693) vs. iCK-cre (n=45988) mutants. In d and e, telomere length is presented as mean +/- SEM, while % short telomeres is presented as a ratio of the number of short telomeres (below 25th percentile) over total number of telomeres. ****p<0.0001 using two tailed unpaired Student’s t-test and Chi-square test, respectively. h. Telomere FISH images of iDlx-cre and iCK-cre mutants and controls in the dentate gyrus. All scale bars=100 μm. In b, c, and h, experiments were independently repeated with similar results at least n=3 times using at least n=3 different mouse tissue samples for each group.

  5. Supplementary Figure 5 Molecular profiling of Syn-cre tumors.

    RNA sequencing analysis of Syn1-cre, Nestin-creERT2, and NG2-creERTM tumors. a. Dimension reduction analysis of Syn1-cre (n=3), Nestin-creERT2 (n=3), and NG2-creERTM (n=3) mutant tumors. b. Heat map showing expression of NG2-creERTM tumor signature genes (also listed in Supplementary Table 2) in Syn1-cre (n=3), Nestin-creERT2 (n=3), and NG2-creERTM (n=3) tumors.

Supplementary information

  1. Supplementary Figures 1–5

    Supplementary Figures 1–5

  2. Reporting Summary

  3. Supplementary Table 1

    Gene events in iCK-cre mutants.

  4. Supplementary Table 2

    NG2-creERTM tumor signature genes.

  5. Supplementary Note 1

    Antibodies used in the study and related information.

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DOI

https://doi.org/10.1038/s41593-018-0333-8