PDGFB Is A Potent Inflammatory Driver In Pediatric High-Grade Glioma Pubblico
Ross, James (Fall 2019)
Abstract
Pediatric high-grade gliomas (pHGG) account for the most cancer-related deaths in children. Despite aggressive therapeutic strategies, tumor recurrence is inevitable and the median survival is just 12-15 months. One promising avenue of research is the development of novel therapies targeting the properties of non-neoplastic cell-types within the tumor such as tumor associated macrophages (TAMs). Much is known about TAMs in adult high-grade gliomas (aHGG), however very little is known about them in the pediatric setting. TAMs can make up 30-40% of the total tumor cell mass in aHGG, promoting tumor growth and immune evasion. This raises the question of whether pHGGs possess a distinct constituency of TAMs due to their unique genetic and epigenetic landscapes. To uncover the composition and behavior of TAMs in pHGG we utilize RCAS/tva, a somatic cell-type specific gene transfer system which allows us to recapitulate all major subtypes of pHGG in newborn immunocompetent mice, including histone wild-type and histone-mutant tumors. These tumors are induced in Nestin-positive cells by overexpression of PDGFA or PDGFB along with other driver mutations, each in their respective locations found in the human population. Tumors driven by PDGFB have a significantly lower median survival compared to PDGFA-driven tumors and have increased infiltration of lymphocytes and TAMs, specifically inflammatory monocytes. NanoString analysis indicates PDGFB-driven tumors have a highly inflammatory microenvironment. In vitro bone marrow derived monocyte and microglial cultures demonstrate the BMDM population is most responsible for the production of inflammatory chemokines and angiogenic factors in the tumor microenvironment. Using human pHGG tissue samples, we demonstrate PDGFB and PDGFRβ strongly correlate with TAM infiltration. We also show pHGGs are relatively barren for CD3+ and CD8+ T-cells. Further, using NanoString we show brainstem pHGGs are more inflammatory compared to hemispheric pHGGs. Lastly, using knockout mice deficient for individual chemokines, we demonstrate the feasibility of reducing TAM infiltration and prolonging survival in both PDGFA and PDGFB-driven tumors. Together, these studies provide strong evidence for the potent inflammatory effects PDGFB has in pHGGs and lays the foundation needed for the development of novel therapeutics targeting these tumors.
Table of Contents
Chapter 1
Epidemiology1
Genetic Alterations in pHGG 1
Therapeutic Strategies 5
PDGF Signaling in Pediatric Glioma 6
Chapter 2 12
Tumor Microenvironments and Cell Types Involved in pHGG 12
Tumor Associated Macrophages in High-Grade Glioma 14
Chapter 2 Methods 27
Chapter 3 35
Inflammatory Microenvironment of Human pHGGs 35
NanoString Analysis of Human pHGGs 36
Neuropathological Characterization of Human pHGGs 39
Chapter 3 Methods 42
Chapter 4 44
Mouse Modeling of PDGF-Driven HGGs 44
Inflammatory Microenvironment of PDGF-Driven Mouse HGGs 49
Flow Cytometry of PDGF-Driven Murine HGGs 54
qPCR Molecular Characterization of PDGF-Driven HGGs 58
In vitro Stimulation of BMDMs and Microglia with PDGF Ligands 62
Chemokine Knockout Mice Extend Survival in PDGF-Driven HGGs 66
Chapter 4 Methods 71
Chapter 5 77
Histone Mutations Confer Unique pHGG Subgroups 77
NanoString Analysis of Histone Mutant Murine HGGs 79
Survival Analysis of Histone Mutant Murine HGGs 86
Chapter 5 Methods 88
Table of Contents
Chapter 6 89
Future Proposed Experimental Studies 89
Tumor Location Versus Tumor Molecular Identity 89
Cell Sorting of Murine pHGGs 90
Pharmacologic Inhibition of PDGF Pathway 93
T-Cell Profiling of Murine pHGGs 94
In vivo Time Course Experiments For TAM Infiltration 94
Concluding Remarks 95
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