Finding the Cell of Origin in Medulloblastoma Amanda Bahe The University of Arizona Hui Zong Lab Mentor: Brit Ventura The goal of the lab that I worked in this summer is to find the cell origin of Medulloblastoma.

What is Medulloblastoma? Tumor of the Cerebellum Affects Children Ataxia: Balance Problems Treatment Chemo/Radiation Therapy Surgery So what is Medulloblastoma? It’s a developmental tumor in the brain – meaning it develops as the cerebellum is developing. Because of this, it is seen most often in children. The cerebellum is responsible for motor control and function so children who present symptoms like ataxia/balance problems or other motor defects are often given an MRI to confirm that MB is the cause of these symptoms. As seen in this example, the physicians are then able to see the tumor and diagnose the patient with MB. One of the treatments for MB is chemo or radiation therapy which targets rapidly dividing, or overproliferating, cells. The problem with this is, if there is a stem cell component to MB, these cells would not be targeted because stem cells divide slowly. Another form of treatment is surgery. The obvious caveat here is that when the cancer is taken out, so is part of the developing cerebellum which poses a risk to the child’s long-term development. Transition: In addition to these patients, there are also patients who have a pre-disposed risk to MB – they have what is called Gorlin Syndrome.

Genetic Components of Medulloblastoma Gorlin Syndrome Ptc Mutation --> OVERPROLIFERATION Children with this disease have a higher incidence of MB It has been found that patients with Gorlin Syndrome have a higher incidence of MB. Genetic testing of tissue from patients with this disease have shown us a very important pathway needed to model MB in animals – this is called the Sonic Hedgehog Pathway. The ultimate goal of this process is to promote proliferation within the cell; patients, like those with Gorlin Syndrome, lack the Ptc receptor that binds to ShH which leads to overproliferation and ultimately, a tumor. Understanding this will be important later in my presentation. Transition: Because MB is a developmental tumor of the cerebellum it is important to briefly introduce you to basic cerebellar development. Proliferation Mariano S. TRENDS in Molecular Medicine Vol.11 No.1 January 2005

Cerebellar Development 2 Germinal Zones VZ: Neural Stem Cells (NSCs) reside Rhombic Lip/EGL: Granule Neuron Precursors (GNPs) reside There are two germinal zones in the cerebellum during development. The first region is called the ventricular zone and this is where neural stem cells reside. The other region is what is called the rhombic lip – where granule neuron precursors are housed, which then migrate outward to form the EGL. GNPs then differentiate into granule neurons and migrate to the IGL These two cell populations can be traced with the use of markers specific to the region that they reside in: BLBP marks Bergmann Glia which come from VZ cells; Olig2 marks cells that are in the RL and Math1 marks the cells that have migrated from the RL into the EGL. Transition: In order to use BLBP and Olig2 as distinguishing markers it is important to show their distinct expression patterns during development. Region Specific Markers: - BLBP: Bergmann Glia from VZ cells - Olig2: Transcription factor in GCPs of RL - Math1 is in RL and EGL

Markers EGL VZ RL BLBP/Math1-GFP BLBP Olig2/Math1-GFP Olig2 From Kimee Kunibe Olig2/Math1-GFP Olig2 On this slide are confocal images – on the left are cells marked with BLBP (red) shown to reside in the VZ. On the right are cells expressing Olig2 (red) that reside in the RL. Math1 cells, also expressing GFP, can be seen in both the VZ and the RL as well as along the EGL. Math1 is expressed in both populations because it is a marker for cells that migrate from the RL upward to form the EGL. Transition: Now that we have established that these markers can be used to distinguish between NSC and GNP identities, this brings us to our working hypothesis.

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) Because there are two cell populations that have been shown in the development of the cerebellum, it is hard to say which of these - either NSCs or GNPs - give rise to MB. So, we have developed a tool called MADM to track the mutant cells within the tumor in order to study tissue cells for either NSC or GCP characteristics.

MADM System This is how the MADM system works. Using the information from the patient genetic analysis, we use a Ptc +/- background to sensitize the mice to MB formation. On homologous chromosomes there are lox-P sites that are recognized by the Cre-recombinase which is driven under cell-type specific promoters, like Math1 in GNPs, and initiates interchromosomal recombination during the cell cycle. Upon cell division, there are three possible cell types resulting from this recombination event: 1) a mutant cell which has lost both copies of the tumor suppressor gene p53 and is labeled with GFP and 2) a sister cell that is wildtype for p53 and expresses RFP or 3) a possible sister cell that is heterozygous for p53 and is yellow (not shown here). This sporadic recombination event in select cells allows us to create random mutant cells that may have the potential to be cancerous and also trackable; it also allows us to use the red wildtype cells as an internal control. This system allows us to stain the MB tissue and be able to track those cells that are mutant (green) and positive for other markers of interests, like BLBP and Olig2. Transition: Using MADM to help us investigate our hypothesis it first necessary to establish that we can indeed form a medulloblastoma using this genetic system.

Tumor Math1-Cre generated tumor These are images showing the MB in the cerebellum that are green as a result of the MADM system (1st). The top image shows the green tumor cells that were derived from Math1-expressing GNPs. What is surprising to note is that these green cells also express BLBP, a marker of Bergmann glia not granule neurons! Transition: With proof that the MADM system is capable of inducing medulloblastoma, we can now begin to address the previously mentioned hypothesis. Many greens cells co-label with glial marker. MADM: p53-/- BLBP

Experimental Design Dissociation of MB Tissue Cell Culture Antibody Staining Confocal Imaging Tissue Dissociation Analysis 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 In order to put the MADM system to use, we dissociated tumor tissue from the mice who were most likely to have MB given the symptoms they showed. We dissected the brains of these mice and removed the cerebellum. Our tumor was derived from two separate cell lines – both of which are Math1-Cre driven. After dissociation of the tumor-containing tissues, the cells were purified through the process of cell culture. Depending on when these cells were cultured (primary or secondary passage), we stained them for different markers. Upon staining of the cells, confocal images were taken and analysis of the mutant cells and their markers were counted.

8619: Math1-driven Medulloblastoma - Properties of VZ cells? VZ -> BG Rise in MUTANT BLBP+ cells from 1° to 2°

8619: Math1-driven Medulloblastoma - Properties of RL/GCP cells? Rise in MUTANT Olig2+ cells from 1° to 2°

9130: Math1-driven Medulloblastoma - Properties of VZ cells? VZ -> BG Rise in MUTANT BLBP+ cells from 1° to 2°

9130: Math1-driven Medulloblastoma - 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. Our data suggests some key findings.

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. So what does this mean? Future: - Analyze more tumors. - More complete marker staining.

Everyone who made SPUR possible Acknowledgements Zong Lab Dr. Hui Zong Brit Ventura SPUR Program Peter O’Day Chelsie Fish REU Program Everyone who made SPUR possible