Exploring Innate Immunity Pathways in the Sex-Based Differences of Glioblastoma using Drosophila melanogaster Models Public

Abstract

Glioblastoma multiforme is the most common, primary malignant brain tumor in adults, accounting for 15% of all central nervous system cancer diagnoses. Previous studies have characterized a sex-based difference in human glioblastoma patients, in which biological males have a 60% higher chance of developing glioblastoma than do biological females. This sex-dependent disparity carries over into survival rates as well, where, regardless of age, race, or geographical location, biological males tend to have a worse prognosis. Even so, the median survival rate after a standard treatment of surgical resection, radiotherapy, and adjuvant chemotherapy with temozolomide, remains dismal at 15 months. A novel focus for developing glioblastoma treatment is the controlled manipulation of glial-based innate immune responses. Endogenous innate immunity is also stratified based on sex, with biological females having a more robust response to infection or injury than biological males. Considering these parallel sex-based differences, we aim to further investigate the impact of       innate immune response pathways on gliomagenesis.

Using a Drosophila model of glioblastoma, we conducted morphological screens of genes within the Toll signaling pathway, known to play a major role in Drosophila innate immune responses. Glial-specific RNAi-gene knockdown constructs and gene overexpression constructs identify Toll-9 and spatzle, which encode a Toll-pathway receptor and ligand, as candidate genes for further investigation for sex-based differences in neoplastic glial growth in our Drosophila glioblastoma model. These results provided us with a framework for future investigations into innate immunity genes that enhance or suppress the glioblastoma phenotype.

The data collected during this exploratory investigation provide preliminary support for the interaction between innate immunity signaling pathways and sex-based differences in glioblastoma. Further characterization of the sex-based phenotypic influences of downstream signaling effectors of the Toll signaling pathway may prove useful for identifying new therapeutic targets for glioblastoma treatments. 

Table of Contents

Introduction and Background 1

Clinical Characteristics of Glioblastoma Multiforme 2

Glioblastoma Biology 3

Sex-Based Differences in Glioblastoma 3

Innate Immunity in Cancer Treatment 4

Innate Immunity and Toll-Like Receptors in Glioblastoma 5

Sex-Based Differences in Glial-Based Innate Immunity 6

Drosophila melanogaster as a Model for Glial Neoplasia 7

Innate Immunity in Drosophila melanogaster and the Toll Signaling Pathway 10

Scope of the Thesis 13

Materials and Methods 14

Drosophila melanogaster Stock Maintenance 15

UAS-GAL4 Genetic System for Drosophila Crosses 15

Drosophila Larval Brain Dissection, Fixation, and Preservation 16

Immunohistochemical Staining of Drosophila Larval Brains 18

Confocal Microscopy and Image Analysis 18

Drosophila Larval Brain RNA Extraction and Preparation 18

RNA Sequencing 19

Results 20

A Morphological Screen of Innate Immunity Genes that Potentially Modify Glial Neoplasia in Drosophila 21

Innate Immunity Gene Knockdowns that Enhance Glial Neoplasia in a Drosophila GBM Model 25

Innate Immunity Gene Knockdowns that Suppress Glial Neoplasia in a Drosophila GBM Model 26

An Overexpression Screen Identified Innate Immunity Pathways as Cell-Autonomous Suppressors 27

Sex-Based Differences in Innate Immunity Gene Knockdowns in a Drosophila GBM Model 29

Controlling for Sex-Based Differences as a Result of Other Cell Signaling Pathways or Environmental Factors in Drosophila GBM Model 31

Discussion 35

References 40

Appendix I: Supplementary Figures 46

Appendix II: Protocols 49

About this Master's Thesis

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	M.S.
Submission	Master's Thesis
Language	English
Research Field	Health Sciences, Oncology
Mot-clé	Sex-Based Differences Toll Signaling Pathway Innate Immunity Glioblastoma Drosophila
Committee Chair / Thesis Advisor	Read, Renne, Emory University
Committee Members	Moberg, Kenneth H., Emory University Gilbert-Ross, Melissa, Emory University

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