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Neurosphere and adherent culture conditions for malignant glioma stem cell lines

Permanent Link: http://ufdc.ufl.edu/UFE0041683/00001

Material Information

Title: Neurosphere and adherent culture conditions for malignant glioma stem cell lines
Physical Description: 1 online resource (49 p.)
Language: english
Creator: Rahman, Maryam
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: adherent, neurosphere, tumor
Clinical Investigation (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Understanding the cellular and molecular characteristics of malignant glioma that drive tumor growth is important for the development of more effective treatments to improve survival. Malignant glioma tumor cells are grown in culture for detailed study of the cellular mechanisms that drive growth. Although useful, cell culture also has the risk of selecting for certain cellular populations that may not be representative of the tumor from which they originated. These concerns have led to interest in developing glioma cell culture techniques that allow for propagation of cells that drive tumor growth with minimal changes to tumor cellular genetic and phenotypic profiles. Currently, the neurosphere assay (NSA), using serum-free media supplemented with growth factors, is the standard for growing and maintaining glioblastoma multiforme (GBM) tumor-initiating cells (TICs) in culture. Recently, serum free culture conditions supplemented with laminin have been described which grow neural stem cells (NSC) as a monolayer. Some of these data suggest that establishing cell lines from human gliomas is more efficient when the cells are grown as a monolayer and that the NSA is inferior to adherent culture in terms of apoptosis and differentiation of cells. We report the comparison of the NSA and laminin growth conditions for the growth of GBM cell lines in terms of growth, apoptosis, differentiation, clonal frequency and tumorigenicity in a xenotransplant model. The GBM cell lines did not demonstrate a difference in growth over time in either growth condition. Using multiple assays, the percentage of apoptotic cells was also equivalent in the NSA and laminin growth conditions. Flow cytometry for differentiation markers did not show statistically significant differences between NSA and laminin GBM cells. Clonal frequency, estimated using the limiting dilution assay, of the laminin cells was similar to published clonal frequency of GBM TICs grown using the NSA. Finally, cells grown in both conditions resulted in tumors when transplanted into the flanks of mice, and these mice had similar Kaplan-Meier survival curves. These data show that GBM cell lines grown in the NSA and laminin growth conditions are not different in terms of growth, apoptosis, differentiation, clonal frequency and tumorigencity. The development of alternative culture techniques for establishing cell lines is a useful addition to the existing tools for the study of human GBM (hGBM). hGBM cells grown in adherent conditions are not significantly different from cells grown with the traditional NSA.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Maryam Rahman.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Limacher, Marian C.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041683:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041683/00001

Material Information

Title: Neurosphere and adherent culture conditions for malignant glioma stem cell lines
Physical Description: 1 online resource (49 p.)
Language: english
Creator: Rahman, Maryam
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: adherent, neurosphere, tumor
Clinical Investigation (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Understanding the cellular and molecular characteristics of malignant glioma that drive tumor growth is important for the development of more effective treatments to improve survival. Malignant glioma tumor cells are grown in culture for detailed study of the cellular mechanisms that drive growth. Although useful, cell culture also has the risk of selecting for certain cellular populations that may not be representative of the tumor from which they originated. These concerns have led to interest in developing glioma cell culture techniques that allow for propagation of cells that drive tumor growth with minimal changes to tumor cellular genetic and phenotypic profiles. Currently, the neurosphere assay (NSA), using serum-free media supplemented with growth factors, is the standard for growing and maintaining glioblastoma multiforme (GBM) tumor-initiating cells (TICs) in culture. Recently, serum free culture conditions supplemented with laminin have been described which grow neural stem cells (NSC) as a monolayer. Some of these data suggest that establishing cell lines from human gliomas is more efficient when the cells are grown as a monolayer and that the NSA is inferior to adherent culture in terms of apoptosis and differentiation of cells. We report the comparison of the NSA and laminin growth conditions for the growth of GBM cell lines in terms of growth, apoptosis, differentiation, clonal frequency and tumorigenicity in a xenotransplant model. The GBM cell lines did not demonstrate a difference in growth over time in either growth condition. Using multiple assays, the percentage of apoptotic cells was also equivalent in the NSA and laminin growth conditions. Flow cytometry for differentiation markers did not show statistically significant differences between NSA and laminin GBM cells. Clonal frequency, estimated using the limiting dilution assay, of the laminin cells was similar to published clonal frequency of GBM TICs grown using the NSA. Finally, cells grown in both conditions resulted in tumors when transplanted into the flanks of mice, and these mice had similar Kaplan-Meier survival curves. These data show that GBM cell lines grown in the NSA and laminin growth conditions are not different in terms of growth, apoptosis, differentiation, clonal frequency and tumorigencity. The development of alternative culture techniques for establishing cell lines is a useful addition to the existing tools for the study of human GBM (hGBM). hGBM cells grown in adherent conditions are not significantly different from cells grown with the traditional NSA.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Maryam Rahman.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Limacher, Marian C.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041683:00001


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1 NEUROSPHERE AND ADHE RENT CULTURE CONDITI ONS FOR MALIGNANT GLIOMA STEM CELL LINES BY MARYAM RAHMAN A THESIS PRESENTED T O THE UNIVERSITY OF FLORIDA GRADUATE SCHOOL IN P ARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010

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2 2010 Maryam Rahman

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3 ACKNOWLEDGEMENTS I thank Dr. William Friedman and Dr. Brent Reynolds who have been tremendous mentors and have been committed to cultivating my research interests and guiding me during this process. I thank Dr. Marian Limacher, Dr. Nabih Asal, and Dr. Jane Douglas who have been indispensible for the APPCI program and who taught me during the early part of my academic career. I also thank the Florida Center for Brain Tumor R esearch and the Grey Ribbon Crusade for funding my research projects. I thank my research colleagues, specifically Dr. Loic Deleyrolle, Maria Caldeira, Dr. Hassan Azari, and Dr. Vinata Vedam Mai for their support, guidance and collaboration in the attempt to improve the lives of patients with brain tumors. Finally, I thank the brain tumor patients who donate their clinical information and tumor tissue for the search for a cure. On a personal note, I would like to thank my family especially my mother and my husband who have shaped me into who I am today and have supported me unconditionally.

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4 TABLE OF CONTENTS page ACKNOWLEDGEMENTS ............................................................................................... 3 LIST OF FIGURES .......................................................................................................... 5 ABSTRACT ..................................................................................................................... 7 CHAPTER 1 INTRODUCTION ...................................................................................................... 9 Overview ................................................................................................................... 9 Malignant Glioma ...................................................................................................... 9 Tumor Stem Cells and Cell Culture ........................................................................ 11 Neurosphere Assay and Adherent Culture Conditions ........................................... 11 Summary ................................................................................................................ 13 2 MATERIALS AND METHODS ................................................................................ 15 Tum or Samples and Cell Culture ............................................................................ 15 Flow Cytometry ....................................................................................................... 16 Limiting Dilution Analysis ........................................................................................ 17 Xenotransplantation ................................................................................................ 18 3 RESULTS ............................................................................................................... 21 4 DISCUSSION ......................................................................................................... 37 Summary and Significance of Results .................................................................... 37 Study Limitations .................................................................................................... 40 Future Plans ........................................................................................................... 40 REFERENCES .............................................................................................................. 42 BIOGRAPHICAL SKETCH ............................................................................................ 49

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5 LIST OF FIGURES Figure page 2 1 hGBM cells in culture .......................................................................................... 20 2 2 hGBM cells stained with tryphan blue ................................................................. 20 3 1 N umber of cells obtained in vitro over multiple passages of GBM cell lines grown in NSA and adherent culture conditions ................................................... 23 3 2 Percent of cells positive for tryphan blue denoting cell death at the time of trypsinization of neurospheres or cells grown on laminin ................................... 23 3 3 Flow cytometry plots of laminin PI experiment .................................................... 24 3 4 Flow cytometry plots of NSA PI experiment ....................................................... 24 3 5 Flow cytometry plots of laminin DAPI experiment ............................................... 25 3 6 Flow cytometry plots of NSA DAPI experiment ................................................... 25 3 7 Summary of PI and DAPI experiments ............................................................... 26 3 8 Flow cytometry plots demonstrating annexin V and PI staining in cells grown in laminin conditions ........................................................................................... 26 3 9 Flow cytometry plots demonstrating annexin V and PI staining in cells grown in NSA conditions ............................................................................................... 27 3 10 Summary of annexin V experiment s ................................................................... 27 3 11 Flow cytometry plots demonstrating caspase staining in live cells grown in NSA and laminin culture conditions .................................................................... 28 3 12 Summary of activated caspase 3 experiments in live cells grown in laminin or NSA .................................................................................................................... 29 3 13 Flow cytometry histograms of cells grown in laminin or NSA conditions and subsequently fixed and staine d with an antibody to activated caspase 3 ........... 30 3 14 Summary of caspase 3 experiments using fixed cells ........................................ 31 3 15 Flow cytometry plot s showing DiIC staining in NSA and laminin grown cells ..... 32 3 16 Summary of DiIC experiments ............................................................................ 33 3 17 Summary of apoptosis markers by flow cytometry percent cells positive in NSA and laminin grown GBM cells ..................................................................... 33

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6 3 18 The geometric mean of laminin cells as a percent of NSA cell geometric mean from flow cytometry .................................................................................. 34 3 19 Limiting dilution assay of cells grown using laminin ............................................ 35 3 20 Mouse after implantation of GBM cells ............................................................... 36 3 21 Kaplan Meier graph demonstrating similar survival of NOD/SCID mice after implantation of hGBM cells grown in either NSA and laminin culture conditions ........................................................................................................... 36

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7 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science NEUROSPHERE AND ADHE RENT CULTURE CONDITI ONS FOR MALIGNANT GLIOMA STEM CELL LINES By Maryam Rahman May 2010 Chair: Marian Limacher Major: Medical Sciences Clinical and Translational Science Understanding the cellular and molecular characteristics of malignant glioma that drive tumor growth is important for the development of more effective treatments to i mprove survival. Malignant glioma tumor cells are grown in culture for detailed study of the cellular mechanisms that drive growth. Although useful, cell culture also has the risk of selecting for certain cellular populations that may not be representative of the tumor from which they originated. These concerns have led to interest in developing glioma cell culture techniques that allow for propagation of cells that drive tumor growth with minimal changes to tumor cellular genetic and phenotypic profiles. C urrently, the neurosphere assay (NSA), using serum free media supplemented with growth factors, is the standard for growing and maintaining glioblastoma multiforme (GBM) tumor initiating cells (TICs) in culture. Recently, serum free culture conditions supplemented with laminin have been described which grow neural stem cells (NSC) as a monolayer. Some of these data suggest that establishing cell lines from human gliomas is more efficient when the cells are grown as a monolayer and that the NSA is inferior t o adherent culture in terms of apoptosis and differentiation of cells.

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8 We report the comparison of the NSA and laminin growth conditions for the growth of GBM cell lines in terms of growth, apoptosis, differentiation, clonal frequency and tumorigenicity i n a xenotransplant model. The GBM cell lines did not demonstrate a difference in growth over time in either growth condition. Using multiple assays, the percentage of apoptotic cells was also equivalent in the NSA and laminin growth conditions. Flow cytometry for differentiation markers did not show statistically significant differences between NSA and laminin GBM cells. Clonal frequency, estimated using the limiting dilution assay, of the laminin cells was similar to published clonal frequency of GBM TICs grown using the NSA. Finally, cells grown in both conditions resulted in tumors when transplanted into the flanks of mice, and these mice had similar KaplanMeier survival curves. These data show that GBM cell lines grown in the NSA and laminin growth con ditions are not different in terms of growth, apoptosis, differentiation, clonal frequency and tumorigencity. The development of alternative culture techniques for establishing cell lines is a useful addition to the existing tools for the study of human GB M (hGBM). hGBM cells grown in adherent conditions are not significantly different from cells grown with the traditional NSA.

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9 CHAPTER 1 INTRODUCTION Overview Gliomas represent the most common primary brain tumor in the U.S. with approximately 10,000 new c ases of malignant glioma diagnosed each year.1 Gliomas originate from transformed glial cells (i.e. astrocytes, ol igodendrocytes and ependymal cells), and usually result in diffuse infiltration of brain tissue. Understanding the cellular and molecular characteristics of malignant glioma that drive tumor growth is important for the development of more effective treatme nts to improve survival. Tumor cells are grown in culture to 1) study growth patterns, and 2) provide an endless supply of cells for animal transplantation and other experiments. Although very useful, cell culture also has the risk of selecting for a certain cellular population or changing the genetic or phenotypic expression profile over time.2,3 Therefore, tumor cells grown in culture may not be truly representative of the tumor from which they originated. These concerns have led to interest in developing glioma cell culture techniques that allow for propagation of cells that drive tumor growth with minimal changes to tumor cellular genetic and phenotypic profiles. Malignant Glioma Gliomas are classified by the Worl d Health Organization (WHO) as grade I IV depending on histologic features.4 Malignant gliomas are defined as WHO grade III and IV gliomas. The median survival with treatment for WHO grade III gli omas (anaplastic glial tumors) is only 24 months and for WHO grade IV gliomas (glioblastoma multiforme) is a dismal 14 months.1,5 Treatment for malignant gliomas includes surgical resection, chemotherapy and radiation. However, despite aggressive treatment, all survivors have

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10 tumor recurrence. The median survival after development of recurrence for malignant glioma is 7 months.6,7 For patients with recurrent glioblastoma mul tiforme (GBM) the median survival is approximately 24 months.7 9 Management of recurrence can involve supportive care alone palliative resection, radiation, and chemotherapy or participation in clinical trials. The poor prognosis of malignant glioma, especially GBM, has been attributed to the limitations of our current treatment regimens. Current treatment includes radiation (XRT) and temozolomide (TMZ). Limited treatment efficacy for malignant glioma has been attri buted to tumor cellular and genetic heterogeneity and limited delivery of therapy to the tumor site.10 Understanding the cellular make up of a particular tumor may allow for tailored therapy. An example of this phenomenon is the discovery of the role of O6methylguanineDNA methyltransferase (MGMT), a DNA repair gene, in GBM tumor resistance. GBM patients with epigenetic silencing of MGMT, d ue to methylation of the promoter, have improved survival regardless of treatment.11 Additionally, GBM patients with silenced MGMT have significantly more improvement in survival after treatment with XRT with concom itant temozolomide (TMZ), now the standard of care for initial treatment of GBM, compared to patients with active MGMT.12, 13 The discovery of MGMT status influencing treatment response to TMZ highlights one of t he major hurdles in the treatment of malignant glioma tumor cellular and genetic heterogeneity.14,15 Cell surface protein expression and molecular defects vary dramatically among and within tumors.1622 This heterogeneity represents a significant therapeutic challenge and most likely complements the de novo and acquired resistance of these tumors, especially with monotherapy. Blocking proliferation of the

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11 entire tumor popula tion has been the challenge in the development of novel therapies for malignant glioma. This challenge has led to interest in combinatorial therapy approaches as well as tumor cellular assessment to determine which treatments will be most effective.10 Understanding cellular and molecular malignant glioma defects requires studying these cells in culture and using cultured cells for further ex periments. Tumor Stem Cells and Cell Culture The origin, maintenance, and resistance of solid tissue malignancies, including malignant gliomas, is attributed to transformed precursors that have the cardinal properties of stem cells.23,24 These transformed cells with stem cell like properties are hypothesized to be resistant to conventional therapy based on the notion that conventional therapy targets the heterogeneous body of cancer cells in a relatively nonspecifi c fashion and spares the tumor stem cells due to their unique properties.2527 A body of evidence now exists suggesting that brain tumors contain this relatively rare subpopulation of tumor initiating cells that exhibit stem cell characteristics,24,2830 that this population may be responsible for treatment resistance31 and targeting this population may be an important therapeutic strat egy in treating patients with brain tumors.24,32 Therefore, studying malignant glioma in culture requires the culture conditions to maintain the tumor initiating cells (TICs) that are hypothesized to drive tumor gro wth, as well as preserve the genetic and phenotypic properties of these cells. Only with these criteria can cell culture results be relevant for patients with malignant glioma. Neurosphere Assay and Adherent Culture Conditions The demonstration of adult neural stem cells (NSC) grown as neurospheres in the 1990s,33,34 confirmed findings of previous investigators3543 and led to widespread

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12 recognition of adult mammalian neurogenesis. Mitotically active stem and progenitor cells were found in discrete regions of the mature brain that could be grown in serum free culture conditions. The differentiated cells would rapidly die in the neurosphere culture conditions, whereas the NSC w ould proliferate and generate spheres. Useful for the maintenance and study of somatic stem cells, the neurosphere assay (NSA) was subsequently used to isolate tumor cells.4446 Specifically, the NSA was used to is olate and expand cells from human brain tumors.28 The NSA is one of the most frequently adopted methods for the enrichment and expansion of both somatic neural stem cells (NSC) and brain TICs. The NSA is the current standard for identifying and maintaining brain TICs in culture.47 Brain TICs have been found to be more genetically representative of the original tumor genetically when grown in the NSA compared to serum conditions.48,49 Furthermore, GBM cells in the NSA bear similarity to st em cells demonstrating potential for indefinite self renewal, ability for terminal differentiation into glial and neuronal lineages, genetic expression profiles similar to neural stem cells, and genetic stability over serial passage.48 Since the cells are maintained in serum free media, their environmental cues are limited to the surrounding cells.50 The extrinsic cues can be manipulated easil y to study the genetic and molecular control of these cells. However, this assay has notable limitations such as variable composition of cells and overestimation of the proportion of neural stem cells. First, the NSA can produce variable composition of ce ll types depending on the media,51 frequency of passaging and whether dissociation is performed before cell differentiation.52 Moreover, the assay can overestimate the number of stem cells.53 A 1:1 relationship between stem cell and

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13 neurosphere does not exist and the NSA overestimates the proportion of neural stem cells (usually <5% of the overall NSA population) by an order of magnitude53. Additionally, neurospheres are not always clonal since they are mobile and can merge with one another.54 To overcome these limitations, modifications to the NSA have been proposed. The colony forming assay is a semi solid culture with collagen that has been described to prevent the migration and fusion of mouse NSC spheres.55 Only cells from the large colonies (>2mm) demonstrated stem cell characteristics and the capability for long term self renewal (>7 passages).55 Similarly with human brain TICs, the NSA has been supplemented with methylcellulose in an attempt to decrease sphere motility.28 Recently, serum free culture conditions supplemented with lamini n have been described to grow NSC as a monolayer.5659 These techniques were extended to brain TICs with an extracellular matrix60 and subsequently laminin.61 Some of these data suggest that establishing cell lines from human gliomas is more efficient when the cells are grown as a monolayer and that the NSA is inferior to adherent culture methods in terms of higher percentages of apopto sis and differentiation of cells.61 Summary Malignant glioma, specifically GBM, has a dismal prognosis despite aggressive treatment with surgery, XRT and chemotherapy. The development of novel therapies requires a comprehensive understanding of GBM TICs, their genetic/phenotypic profiles and what drives their growth. Therefore, developing cell culture techniques that maintain tumor cells that are representative of the original tumor and are relevant for tumor growth is of paramount importance. The NSA had replaced culture media with serum to grow GBM cells since it maintains TICs and preserves characteristics of the original

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14 tumor. Recently, however, the use of serum free media supplemented with laminin to create adh erent cultures has been identified as a possible improvement over the NSA in terms of cellular differentiation and apoptosis. Based on limitations of these data we hypothesized that the NSA and adherent culture condition are not significantly different. We tested this hypothesis by comparing the NSA and adherent culture conditions for the growth of GBM stem cell lines.

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15 CHAPTER 2 MATERIALS AND METHOD S Tumor Samples and Cell Culture Fresh brain tumor samples were obtained at the time of surgical excision fro m patients after obtaining informed consent. GBM tumor samples were dissociated into single cells using trypsin and cultured in the NSA at a cell density of 100,000 to 200,000 cells/mL. These samples were established as cell lines by undergoing long term p ropagation (>10 passages) and were subsequently used for NSA vs. adherent culture comparisons ( Figure 2 1). The NSA growth media included: Neural stem cell medium and human proliferation supplement (Stem Cell) with 20ng/mL epidermal growth factor (EGF) (R &D), 10ng/mL basic fibroblast growth factor (bFGF) (R&D) and 2 g/mL heparin (Sigma). Cultures were grown in T25 flasks. The adherent culture required coating of flasks with laminin (Sigma) (diluted 1:10 in neural stem cell medium) for at least 3 hours at 3 7 C with 5% CO2. The same medium was used for the adherent and NSA culture conditions. The NSA cells were grown until formation of 100 micron spheres and the adherent cells were grown until cells were confluent. For passage of cells, the cells were dissoci ated with 0.05% trypsinEDTA (Gibco) for 2 minutes at 37C, counted and re plated at a density of 50,000 cells/mL. Cells were stained with 0.04% tryphan blue at the time of counting to identify dead cells (tryphan blue +) ( Figure 2 2). These cells were cou nted and the percentage of dead cells was calculated for each growth condition at the time of each passage. These values were compared using the Students t test and a p value of 0.05 was considered significant.

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16 Additionally, the fold expansion at each passage was compared between NSA and the laminin growth conditions. The cells were passaged every 410 days depending on the cell line. The NSA and laminin cells were passaged simultaneously for each line for fair comparison (n=4 in each group). The difference was compared to zero using the Students t test and p values were calculated. A p value of 0.05 was considered significant. Flow Cytometry For apoptosis assays, live cells were evaluated for caspase 3 (1:300) (Calbiochem), annexin V (1:100) (Invitrogen), 1,1,3,3,3,3 hexamethylindodicarbocyanine iodide (DiIC) (1:1000) (Invitrogen), propidium iodide (PI) (1:1000) (Sigma), and 4,6 diamidino 2 phenylindole (DAPI) (1:2000) (Invitrogen). Cell death was evaluated using PI and DAPI. Subsequently, late, mid and early markers of apoptosis were tested. Annexin V, a marker of late apoptosis, binds phosphatidylserine (PS) which is normally confined to the inner plasma membrane, but becomes exposed with apoptosis. The annexin V cell experiments were followed by t esting for activated caspase 3 (activated death protease that catalyzes the cleavage of key cellular proteins6264) expression in live and fixed cells. For fixed cells, analysis with caspase 3 (1:1000) (BD) was perf ormed with cells fixed with 90% methanol at 20C for 15 minutes. An early measure of cell death is the integrity of the mitochondrial membrane.65 Cyanine dyes, such as DiIC, penetrate eukaryotic cell membranes and accumulate in the mitochondria with ac tive membrane potentials. When this potential is disrupted, there is less DiIC staining. The hGBM cell lines were maintained in NSA and adherent culture conditions. To evaluate the purity of the stem cell population in each growth condition, expression of

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17 differentiation markers was tested. Cells were tested for GFAP (astrocytic differentiaton), beta tubulin III (neuronal differentiation), and nestin (stem cell marker) .28 Additionally, cells were tested f or proliferative potential with Ki67 and MCM2. The cells were fixed and stained with antibodies against the markers. Flow cytometry was performed and percent cell staining and geometric mean were measured. Nestin (1:1000) (Chemicon), glial fibrillary acidi c protein (GFAP) (1:2000) (BD), beta tubulin III (1:2000) (Promega), MCM2 (1:500) (Santa Cruz), and Ki67 (1:750) (Invitrogen) were used for analysis of differentiation. Flow cytometry was performed using the LSR II (Becton Dickinson ). Flow cytometry anal ysis was performed with Flow Jo (Treestar, Inc.). The percent cells positive and geometric mean values are reported as mean standard error. The values were compared using the Students t test and computing p values. Given the use of multiple assays to te st apoptosis, to reduce the risk of an alpha error, significance was determined to be a p value of 0.05 divided by the number of assays (6) resulting in a significant p of 0.008. Limiting Dilution Analysis Since reliable molecular or cellular markers of ne ural or tumor stem cells do not exist, these cells are defined with functional criteria. Included in these criteria is clonogenic capacity, or the ability of one founder cell to create a colony consisting of stem cells and more differentiated cells.66 The clonal frequency of hGBM cells was tested in vitro with the limiting dilution assay67. Cells were grown in 96 well plates coated with laminin with dilutions of 110 cells/well. The wells that became confluent or close to confluent were considered to have clonogenic cells. For the NSA, cells were

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18 plated and wells were evaluated for number of neurospheres (sphere forming frequency). A limiting dilution analysis was performed using laminin coated 96 well plates. The wells were coated with laminin for at least 3 hours at 37C with 5% CO2. Medium with GBM cells was serially diluted to plate 110 cells/well using the standard medium described above. Cultures were analyzed after 3 weeks. Wells with confluent growth or at least full by visual inspection were considered full. The percent of wells with less than 75% growth were considered empty and the negative log ( log) of the proportion of negative wells was calculated for each cell density. This result was plotted to determine the clonal frequency of the adherent culture conditions using the extreme limiting dilution analysis (ELDA) software67 and 95% confidence intervals are reported. For the NSA, sphere forming frequency was used as an estimate of clonal frequency. Similarly, cells were serially diluted and plated in 96 well plates. After 3 weeks of growth, the wells were evaluated for the number of spheres at least 100 m in size. The number of spheres was divided by the number of cells plated and multiplied by 100 to obtain a sphere forming percentage of cells. These data are reported as a mean with standard error. Xenotransplantation All animal work was approved by the Institutional Animal Care and Use Committee (IACUC). Non obese diabetic/severe combined immunodeficient (NOD/SCID) female mice 8 10 weeks of age were anesthetized a nd received a flank subcutaneous injection of 1x106 cultured GBM cells in 200 microliters of medium and 100 microliters of Matrigel (BD). The cells had been passaged in either NSA or adherent conditions for at least 5

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19 passages prior to xenotransplantation. The animals were divided into two equal groups based on receipt of cells from NSA (n=5) or those from adherent culture (n=5). The animals were monitored for tumor growth and were euthanized once tumors reached 1.5cm in size. A KaplanMeier survival analys is was performed, ratio generated for the NSA and laminin group, and the 95% CI reported to determine the relationship between type of tumor cell culture and length of survival.

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20 A B Figure 2 1. hGBM cells in culture. A) Cells grown as a monolayer in laminin. B) Cells grown as spheres in the NSA. Figure 2 2. hGBM cells stained with tryphan blue. Only the dead cells were positive for the dye (asterisk).

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21 CHAPTER 3 RESULTS Cells were counted at every passage and expansion curves for the NSA and adherent culture conditions were not different by comparing difference in fold expansion with zero using the Students t test (p=0.24) ( Figure 3 1). Similarly, cell death was measured at the time of passage by counting the number of cells positive for tryphan blue during the cell counts. Cell death between the two groups was the same by Students t test (p=0.7) (n=9) ( Figure 3 2). Cells were also analyzed for markers of death, late phases of dying and early markers of dying. Initially dead cells were identified using either PI or DAPI to stain cells with compromised cell membranes.6870 Similar to the tryphan blue data, no statistical difference was found between NSA or laminin cells in PI (p=0.27) (Figures 33, 3 4) or DAPI s taining (p=0.93) (Figures 35, 3 6). These experiments are summarized in Figure 3 7. Using a marker of late apopotosis, annexin V, was evaluated in live cells71. Percentage of cells expressing annexin V in laminin c ells was 7.6% 1.8 ( Figure 3 8) compared to 10.1% 1.5 in NSA cells ( Figure 3 9) (p=0.32). These experiments are summarized in Figure 3 10. Using a caspase 3 inhibitor conjugated to a flurochrome (PE), activated caspase 3 was quantified in live cells. T he staining was equivalent between NSA (5.9% 1.0) and laminin cells (7.2% 1.2) (p=0.41) ( Figure 3 11, 3 12). Conversely, measuring the amount of antibody staining activated caspase 3 in fixed cells, the laminin cultured cells demonstrated a trend for i ncreased immunoreactivity (4.4% 0.6) when compared to NSA culture (2.2% 0.6) (p=0.026) ( Figure 3 13, 314).

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22 Live cells were tested for DiIC, identifying apoptotic cells as those without DiIC staining. No statistically significant difference between the NSA and laminin cells was found (p=0.55) ( Figure 3 15, 316). Based on multiple markers of apoptosis, no difference exists in terms of apoptosis between GBM cells grown in NSA or laminin culture conditions ( Figure 3 17). The NSA and adherent cells had no significant differences in GFAP, Nestin, beta tubulin III, Ki67 and MCM2 staining using the Students t test although there was a trend for more GFAP and beta tubulin III staining and less Ki67 and MCM2 staining in the cells grown with laminin ( Figure 3 18). The data shows the laminin cell staining as a percent of the NSA cell staining. Based on our data, assuming an difference in staining of 50% between cells grown in the NSA and laminin conditions, with an alpha error of 0.05 and power of 80%, we would require at least 50 experiments with both culture conditions to show statistical significance in the trends we found. Given the strong evidence of no differences in the culture conditions, we did not pursue this. Using a standard calculation for clonogenic frequency67 (by taking the negative log of the proportion of empty wells) the clonal frequency of the cell lines grown in laminin was determined to be 5.5% or 1 cell out of 18.1 (95% CI 15.9 20.6) ( Figure 3 19). This value was similar to the mean clonal frequency of hGBM cells grown in the NSA of 7.1% 0.4 (data not shown). These results are similar to the reported clonal frequency of hGBM cell lines grown in the NSA.29, 72 The growth of the tumors and s urvival analysis was the same for hGBM cells grown in the NSA (median survival 78 days) and those grown with laminin (median survival 71 days) (ratio 1.099, 95% CI 0.8 to 1.4) ( Figure 3 20, 321).

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23 Figure 3 1. N umber of cells obtained in vitro over multi ple passages of GBM cell lines grown in NSA and adherent culture conditions. Standard error is shown with the error bars. The fold expansion over time in the two different conditions was not statistically different (p=0.24). Figure 3 2. Percent of cells positive for tryphan blue denoting cell death at the time of trypsinization of neurospheres or cells grown on laminin. Standard error is shown with the error bars. No statistically significant difference was found (p=0.7)

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24 A B Figure 3 3 Flow cytometry plots of laminin PI experiment. A) Laminin cells with no PI. Using the PI autofluorescence of the cells, a gate was identified (pink line) to separate PI positive and PI negative cells. B) Applying this gate to the cells stained with PI, 3.21% of the cells were found to be PI positive/dead. A B Figure 3 4. Flow cytometry plots of NSA PI experiment. A) NSA cells with no PI. Using the PI autofluorescence of the cells, a gate was identified (pink line) to separate PI positive and PI negative cells. B) Applying this gate to the cells stained with PI, 1.59% of the cells were found to be PI positive/dead.

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25 A B Figure 3 5. A) Flow cytometry plots of laminin DAPI experiment. Laminin cells with no DAPI are shown on the left. Using the DAPI autofluorescence of the cells, a gate was identified (pink line) to separate DAPI positive and DAPI negative cells. B) Applying this gate to the cells stained with DAPI, 1.02% of the cells were found to be DAPI positive/dead. A B Figure 3 6. Flow cytometry plots of NSA DAPI experiment. A) NSA cells with no DAPI. Using the DAPI autofluorescence of the cells, a gate was identified (pink line) to separate DAPI positive and DAPI negative cells. B) Applying this gate to the cells stained with DAPI, 1.14% of the cells were found to be DAPI positive/dead.

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26 Figure 3 7 Summary of PI and DAPI experiments A) Percent cells positive for PI. B) Percent cells positive for DAPI. The mean and standard error are shown in the graphs. No statistical differences were found between cel ls grown in NSA and laminin culture conditions. A B Figure 3 8 Flow cytometry plots demonstrating annexin V and PI staining in cells grown in laminin conditions. A) Cells stained with PI only to identify dead cells. Using the autoflurorescence of paci fic blue (the fluorochrome conjugated to the annexin V antibody) of these cells, a gate was identified (pink lines) to separate live/dead cells (PI /+) and annexin V /+ cells (pacific blue /+). B) This gate was applied to cells stained with PI and an ant ibody to annexin V conjugated to pacific blue. 6.52% of the cells were positive for annexin V and apoptotic. 0.87% of the cells were positive for annexin V but also positive for PI and therefore already dead.

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27 A B Figure 3 9. Flow cytometry plots de monstrating annexin V and PI staining in cells grown in NSA conditions. A) Cells stained with PI only to identify dead cells. Using the autoflurorescence of pacific blue (the fluorochrome conjugated to the annexin V antibody) of these cells, a gate was identified (pink lines) to separate live/dead cells (PI /+) and annexin V /+ cells (pacific blue /+). B) This gate was applied to cells stained with PI and an antibody to annexin V conjugated to pacific blue. 12.6% of the cells were positive for annexin V and apoptotic. 0.81% of the cells were positive for annexin V but also positive for PI and therefore already dead. Figure 3 10. Summary of annexin V experiments. The percentage of cells positive for annexin V are shown for both growth conditions. The mean and stardard error are shown. No statistically significant differences were found.

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28 Figure 3 11. Flow cytometry plots demonstrating caspase staining in live cells grown in NSA and laminin culture conditions. A) Cells without caspase 3 inhibitor. Using t he PE (fluorochrome conjugated to the caspase 3 inhibitor) autofluorescence of cells, a gate was identified (pink line) to separate PE /+ cells. B) Applying this gate to cells with caspase 3 inhibitor, 2.16% of laminin cells were found to express activated caspase 3. C) Similarly, a gate was chosen using NSA control cells. D) Applying this gate to cells with caspase 3 inhibitor, 1.67% of cells were found to express activated caspase 3.

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29 Figure 3 12. Summary of activated caspase 3 experiments in live cell s grown in laminin or NSA. Mean and standard error of percent cells positive are shown. No statistically signficant differences were found.

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30 Figure 3 13. Flow cytometry histograms of cells grown in laminin or NSA conditions and subsequently fixed and stained with an antibody to activated caspase 3. A,C) Using the autofluorescence of pacific blue (fluorochrome conjugated to the caspase 3 antibody) of the cells with no antibody, a gate was identified to separate pacific blue /+ cells. B,D) Applying these g ates to cells with the antibody to activated caspase 3, 10.8% of laminin and 4.63% of NSA cells were found to express activated caspase 3.

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31 Figure 3 14. Summary of caspase 3 experiments using fixed cells. The percent cell staining is shown with mean and s tandard error. Although there was a trend for increased activated caspse 3 expression in cells grown in laminin, this did not reach statistical significance.

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32 Figure 3 15. Flow cytometry plots showing DiIC staining in NSA and laminin grown cells. A, C) Cells stained with DAPI only were used to chose a gate (pink lines) to separate live/dead cells (DAPI /+) and DiIC /+ (AF633 fluorochrome /+). B, D) These gates were applied to cells stained with DAPI and DiIC conjugated to AF633. 86% of laminin grown ce lls and 97% of NSA grown cells were positive for DiIC (nonapoptotic).

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33 Figure 3 16. Summary of DiIC experiments. The percent cells positive for each experiment, mean and standard error are shown. No significant differences were found between laminin and NSA grown hGBM cells. Figure 3 17. Summary of apoptosis markers by flow cytometry percent cells positive in NSA and laminin grown GBM cells. p= NS for all comparisons. The mean and standard error are shown in the graph.

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34 F igure 3 18. The geometric mean of laminin cells as a percent of NSA cell geometric mean from flow cytometry. A) Proliferative markers Ki67 (p=0.22) and MCM2 (p=0.51). B) Differentiation markers GFAP (p=0.44), Nestin (p=0.52), and beta tubulin III (p=0.18). The mean and standard error are shown in the graphs. A B

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35 Figure 3 1 9. Limiting dilution assay of cells grown using laminin. Determination of clonogenic capacity was based on ability of cell grown on laminin to fill a well. A) Full well after growth of cel ls on laminin. B) Example of a well considered not full after growth of cells on laminin. C) Graph of the log of the number of nonfull wells and the dose of the cells plated.

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36 Figure 3 20. Mouse after implantation of GBM cells. Example of a subcutaneous (SQ) tumor growing in the flank of a mouse. Figure 3 21. Kaplan Meier graph demonstrating similar survival of NOD/SCID mice after implantation of hGBM cells grown in either NSA and laminin culture conditions. Mice were euthanized once tumors reached 1 .5cm in any dimension.

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37 CHAPTER 4 DISCUSSION Summary and Significance of Results The NSA is the current standard for identifying and maintaining brain TICs in culture.47 However, this assay has notable limitations including variable composition of cells51,52 and overestimation of proporti on of neural stem cells.53 To overcome these limitations, modifications to the NSA have been proposed, including the addition of laminin to grow cells flat. Some advantages of adherent culture conditions suggested by these reports inc lude a more pure stem cell population, decreased differentiation, increased tumorigenicity, and decreased cellular apoptosis.61 Our experiments are the first to directly compare the NSA and laminin culture conditio ns for hGBM cell lines using multiple in vitro and in vivo assays. To test potential differences in the proportion of CSCs, we used cell culture techniques, as stem cells are defined by functional criteria. Growing our hGBM cell lines with the two different culture conditions demonstrated equivalent growth curves with long term expansion. These growth data were supported by the finding that NSA and laminin cells had no differences in expression of proliferation markers (Ki67 and MCM2). Furthermore, cells gr own in both conditions had similar clonal frequency (ability of a single cell to generate a large number of progeny). These results in sum show that cells grown in the NSA and laminin culture conditions have no statistically significant difference in the proportion of stem cells and progenitors.67,73 Recent reports claim that cells from NSA spheroids have increased differentiation although this finding was not statistically analyzed.61 As CSCs differentiate, they lose their stem cell characteristics such as self renewal, generation of a large number of

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38 progeny and multipotency.74 Consequently, CSC culture should maintain relatively undifferentiated CSCs that are multipotent (retain the ability to differentiate when exposed to a differentiation agent). In our study, the NSA and laminin cells were not statistically different with regard to staini ng for nestin (relatively undifferentiated cells), GFAP (astrocyte differentiation) or beta tubulin III (neuronal differentiation). The differences between our results and other reports may be due to overgrowth of the neurospheres in other studies, resulti ng in increased cellular differentiation.75 In addition to the purity of the CSC population, increased tumorigenicity was described as an advantage of laminincultured cells. Laminin has also been implicated in hGBM invasion and migration based on in vitro migration anal yses.76,77 However, in our xenograft model, hGBM cells grown in laminin did not show statistically significant increased tumorigenicity or invasion. The animals implanted with NSA and laminincultured cells had simi lar tumor growth and survival times. The growth of the cells in vivo may have been affected by the use of matrigel.78 However, since both groups of cells grew in vivo equally with the same growth conditions, the likelihood of a difference between the cells is low. Moreover, our hGBM cells grown in NSA and adherent culture conditions did not show statistically significant differences in cell death or dying as quantified by apoptotic markers. The previous culture studies demonstrated increased apoptosis in cells grown with NSA compared to cells grown in adherent conditions.60,61 This literature demonstrated increased apoptosis in NSA cells using annexin V and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (detecting fragmented DNA) assays56,79 The decreased apoptosis with laminin was hypothesized to be due to

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39 laminin preventing cellular apoptosis that is secondary to detachment from the extracellular membrane.56 However, this difference was not quantified statistically nor confirmed with other techniques. Cell death is a heterogeneous process involving multiple an d sometimes overlapping pathways.80 Therefore, we used multiple apoptosis assays to quantify the difference in cell dying and death between cells in the NSA and laminin growth conditions. Immunofluorescence with a f low cytometer was used for several reasons.80 First, fluorescence has higher sensitivity due to improved signal to noise ratio compared to chromogenic techniques. Since this detection method does not involve an enzy matic reaction, variables that can change results such as buffer and pH are not relevant. Secondly, flow cytometry has the distinct advantage of providing both single cell and entire population analyses. Lastly, using fluorescent antibodies to the targets of interest allows for the use of several flurochromes with different absorption/emission spectra and the testing of three to four apoptotic assays simultaneously. Given the heterogeneity of apoptotic pathways and the numerous assays available to measure apoptosis, using multiple tests to quantify apoptosis is prudent. We used a robust analysis of early and late apoptosis as well as cell death using multiple assays and demonstrated no significant difference between NSA and laminin cultured cells. The previ ously reported differences in apoptosis are limited by testing of only one or two apoptosis measures. Based on functional criteria such as our long term proliferation data, cells grown in the NSA and laminin culture conditions have no differences in the proportion of stem cells and progenitors. Laminin grown cells also do not show increased clonal frequency

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40 in vitro or increased tumorigenicity in a xenograft model compared to the NSA. Furthermore, cells grown in the NSA and laminin have no differences in c ell death or dying. These data show no advantage of the laminin culture method to the NSA. Study Limitations The goal of this study was to study the NSA and laminin growth conditions for differences in cellular growth, apoptosis, clonal frequency and tumorigenicity. Limitations of the study include lack of genomic comparison between GBM TICs grown in the two different growth conditions. This study demonstrated that the clonal frequency of GBM TICs grown in laminin is similar to those grown in NSA. These d ata should be confirmed with a limiting dilution analysis in vivo with a xenotransplantation model. The in vivo SQ flank model would allow for evaluation of tumor initiating capacity, not just clonal frequency. Additionally, the in vivo limiting dilution a ssay could be repeated with an intracranial model. This model would evaluate tumor initiating capacity in a model more representative of GBM tumor growth and avoid the possible confounding factor of matrigel. Future Plans These data need to be supplemented with genomic comparisons. Genomic data would reveal if the two different growth conditions result in selection for cells with differing genotypes. These types of experiments are part of our future plans. Also, further analysis of the adherent, serum fr ee growth conditions should be completed using other types of tumor cells in vitro before its benefits can be determined. Developing novel and effective therapy for hGBM is challenging due to tumor heterogeneity, robustness and imperfect tumor models. The development of alternative culture techniques for establishing cell lines is a useful addition to the existing tools for

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41 the study of hGBM. Although potentially more convenient for certain applications, such as migration analyses, hGBM cells grown in adherent conditions are not significantly different from cells grown with the traditional NSA.

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49 BIOGRAPHICAL SKETCH Maryam Rahman, MD, was born and raised in Florida. She obtained her BA in chemistry from Johns Hopkins University, where she was a member of the varsity women's fencing team. She earned her MD from the University of Florida in 2005 with membership in AOA and the Chapman Humanism Society. She completed her general surgery internship at the University of Florida in 2006 and is scheduled to finish her neurosurgical residency in 2013. She is currently a member of the American Association of Neurological Surgery (AANS), Congress of Neurological Surgeons (CNS) and the Shands Cancer Committee. She was awarded the Chuck Shank award in 2009 for Excellence in Neurosurgery. Dr. Rahman is also the codirector of the neuroanatomy course for the first year medical students. She is pursuing her research interest in neurooncology with a 2year fellowship in the laborat ory of Dr. Brent Reynolds studying tumor stem cells and their role in developing novel therapy for glioblastoma multiforme. Additionally, earned her Master of Science in Medical Sciences with a concentration in clinical and translational research through t he Advanced Postgraduate Program in Clinical Investigation (APPCI), an NIH sponsored program. She has been awarded three research grants to support her work and was awarded the Department of Surgerys Resident Research Award in 2009.