Investigation of a Xenogenic Vaccine in a Murine Breast Cancer Model 公开

He, Sara (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/2b88qc88q?locale=zh
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Abstract


Table of Contents
Abstract
Introduction

1-15
Introduction
1-2
The Immune System and Escaping Immune Surveillance
2-4
GPI-anchored Cytokines and Vaccine Strategies
4-7
Developing Effective Cancer Immunotherapies
7-9
Xenogeneic Vaccines
9-10
Cross Priming
10-12
CHO cell Vaccine Model
12-13
HER2/neu
13-14
Rational and Hypothesis
14
Experimental Design
14-15
Materials and Methods
15-23
Cell Culture
15
DNA constructs
16
Establishment of CHO Cell Lines Expressing ISMs
16
Magnetic Bead Isolation
16-17
Cell Panning
18
Flow Cytometry Analysis
19
PIPLC Treatment
20
Membrane Preparation
20-21
Protein Estiation
21
Animals
21
Mouse Vaccinations and Challenge
22-23
Mouse Serum Analysis
23
Results
24-54
CHOhHER2 Transfectants
24-27
PIPLC Treatment
27-31
Results from Cell Membrane Preparation
32
Mouse Vaccine Studies Timeline
33
Live Vaccine Results
34-40
Irradiated Cell Vaccine Results
40-44
Membrane Vaccine Studies Results
45-56
Analysis of Serum
46-56
Discussion
56-61
Conclusion
61-62
References
62-77


Abstract
Tumor cells escape immune system surveillance by inducing immune tolerance. Therefore,
finding ways to break the tumor-induced immune tolerance is a fundamental aspect in
developing effective cancer therapies. Tumor modification with immunostimulatory molecules
(ISMs) such as co-stimulatory molecules and cytokines has successfully been shown to induce
an antitumor response. Xenogeneic immunization using non-self heterogeneic proteins with the
goal of inducing a cross-reactive response against a shared antigen through shared epitopes has
been proven to be an effective strategy for breaking immune tolerance against tumor associated
antigens. In our studies, CHO cells expressing human HER2 along with cytokines, IL-2, IL-12,
GM-CSF, and transmembrane protein B7-1 are used as a xenogeneic vaccine against a murine
mammary tumor cell line, TUBO. Since xenogeneic cells cannot stimulate T cells directly, the
ultimate goal is to determine whether vaccination with a xenogeneic CHO cell line expressing
xenogeneic human HER2 along with immunostimulatory molecules can induce a potent
antitumor immune response through indirect-priming. The study investigates the effects of B7-1,
and glycosyl-phosphatidylinositol (GPI)-anchored IL-2, IL-12, and GM-CSF on indirect priming
using a breast cancer model. Because the delivery method of a vaccine has proven to be
important and can affect the immune response, vaccines were delivered in a live cell, irradiated
cell, or cell membrane form. Mice were vaccinated and challenged 30 days later with BALB/c
mice derived mammary carcinoma, TUBO. Tumors grew in vaccinated mice at a reduced rate
compared to unvaccinated mice. Tumors grew at the slowest rate in mice vaccinated with the
live cell vaccine. However, no difference was seen in mice vaccinated with the irradiated cell
vaccine as compared to unvaccinated mice. Tumor incidence was also reduced in several groups
vaccinated with the live and irradiated cell vaccine. However, statistical tests showed there was
no significant difference in average tumor size between vaccinated and unvaccinated groups of
mice. The present study established a xenogeneic vaccine model system which can be used to
study the effectiveness of various vaccination strategies in inducing indirect and cross priming.

Table of Contents

Introduction 1-15
Introduction 1-2
The Immune System and Escaping Immune Surveillance 2-4
GPI-anchored Cytokines and Vaccine Strategies 4-7
Developing Effective Cancer Immunotherapies 7-9
Xenogeneic Vaccines 9-10
Cross Priming 10-12
CHO cell Vaccine Model 12-13
HER2/neu 13-14
Rational and Hypothesis 14
Experimental Design 14-15
Materials and Methods 15-23
Cell Culture 15
DNA constructs 16
Establishment of CHO Cell Lines Expressing ISMs 16
Magnetic Bead Isolation 16-17
Cell Panning 18
Flow Cytometry Analysis 19
PIPLC Treatment 20
Membrane Preparation 20-21
Protein Estiation 21
Animals 21
Mouse Vaccinations and Challenge 22-23
Mouse Serum Analysis 23
Results 24-54
CHOhHER2 Transfectants 24-27
PIPLC Treatment 27-31
Results from Cell Membrane Preparation 32
Mouse Vaccine Studies Timeline 33
Live Vaccine Results 34-40
Irradiated Cell Vaccine Results 40-44
Membrane Vaccine Studies Results 45-56
Analysis of Serum 46-56
Discussion 56-61
Conclusion 61-62
References 62-77

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