Finding the Cell of Origin in Medulloblastoma

Amanda BaheThe University of Arizona

Hui Zong LabMentor: Brit Ventura

What is Medulloblastoma?

• Tumor of the Cerebellum• Affects Children– Ataxia: Balance Problems

• Treatment– Chemo/Radiation Therapy– Surgery

Genetic Components of Medulloblastoma

Gorlin Syndrome

• Ptc Mutation

--> OVERPROLIFERATION

• Children with this disease have a higher incidence of MB

Mariano S. TRENDS in Molecular Medicine Vol.11 No.1 January 2005

Proliferation

Cerebellar Development• 2 Germinal Zones– VZ: Neural Stem Cells (NSCs) reside– Rhombic Lip/EGL: Granule Neuron Precursors

(GNPs) reside

Region Specific Markers:- BLBP: Bergmann Glia from VZ cells- Olig2: Transcription factor in GCPs of RL- Math1 is in RL and EGL

Markers

BLBP/Math1-GFP

BLBP

RLVZ

EGL

RLVZ

EGL

RLVZ

EGL

RLVZ

EGL

RLVZ

EGL

From Kimee Kunibe

Olig2/Math1-GFP

Olig2

RL

RL

Our Question:• Do tumor cells express stem cell markers?

Origin of Medulloblastoma:Neural Stem Cells (NSCs)

vs.Granule Cell Precursors (GCPs)

TOOL:

Mosaic Analysis with Double Markers in Mice (MADM)

MADM System

Tumor

Math1-Cre generated tumor

Many greens cells co-label with glial marker.

MADM: p53-/- BLBP

Experimental Design

• Dissociation of MB Tissue– Dissection of mice and cerebellum

• Cell Culture– Tumors of different cell lines: 8619 and 9130

• Antibody Staining– Use of markers: BLBP and Olig2

• Confocal Imaging/Analysis

Tissue Dissociation

AnalysisCell Culture Antibody

StainingConfocal Imaging

8619: Math1-driven Medulloblastoma

- Properties of VZ cells? VZ -> BG

_x000c_Mutant BLBP+ _x000e_ Mutant BLBP -0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

BLBP

BLBP PrimaryBLBP Secondary

Rise in MUTANT BLBP+ cells from 1° to 2°

8619: Math1-driven Medulloblastoma

- Properties of RL/GCP cells?

_x000d_Mutant Olig2+ _x000f_ Mutant Olig2 -0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

Olig2

Olig2 PrimaryOlig2 Secondary

Rise in MUTANT Olig2+ cells from 1° to 2°

9130: Math1-driven Medulloblastoma

_x000c_Mutant BLBP+ _x000d_ Mutant BLBP-0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

BLBP

BLBP PrimaryBLBP Secondary

- Properties of VZ cells? VZ -> BG

Rise in MUTANT BLBP+ cells from 1° to 2°

9130: Math1-driven Medulloblastoma

_x000d_Mutant Olig2+ _x000e_ Mutant Olig2-0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

Olig2

Olig2 PrimaryOlig2 Secondary

- Properties of RL/GCP cells?

Rise in MUTANT Olig2+ cells from 1° to 2°

Summary

• Increase in MARKER+ cells from primary to secondary is PROPORTIONAL to decrease in MARKER- cells from primary to secondary.

• This proportion is maintained between the tumors.

• Though the proportion is similar, the INITIAL EXPRESSION LEVELS in the cells vary.

Conclusion•Level of Olig2 expression may correlate with

malignancy.–Expression (9130) = MORE robust–Expression (8619) = LIMITED life span

•Heterogeneity among tumors–Oncogenic mutations in GNPs led to different cell

survival rates/status in culture experiments.

•Observations in cultured cells mimic that of cells in vivo.

Acknowledgements

•Zong LabDr. Hui ZongBrit Ventura

•SPUR ProgramPeter O’DayChelsie Fish

•REU Program•Everyone who made SPUR possible