Intravenous vs. Subcutaneous prophylactic vaccination of protein transferred 4TO7TMVs against 4TO7-HER2 murine breast cancer Open Access

Caoyonan, Brianne Emily (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/xd07gt33n?locale=en
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Abstract

There are many different avenues of dealing with breast cancer today, including: early detection, surgery, radiation, chemotherapy, and hormone therapy for ER, PR, and HER2 positive breast cancer. However, many of these hormone therapies lead to resistance and sometimes relapse or progression. Immunotherapy provides another level of defense by activating the immune system against tumor cells and, thereby, minimizing effects on healthy tissue. In this study we aimed to develop tumor membrane vesicles (TMVs) modified by protein transfer to express HER2, with or without additional glycosyl phosphatydlionsitol anchored immunostimulatory molecules (GPI-ISMs), in order to activate a HER2 specific response against 4TO7RGhHER2 murine breast cancer cells. These different 4TO7 TMVs, were made from 4TO7RG tumors, modified by protein transfer with GPI-anchored HER2, IL-12, and B7-1, and injected intravenously or subcutaneously for prophylactic vaccination against 4TO7RGhHER2 experimental metastasis. Mice vaccinated with 4TO7ts TMVs protein transferred with HER2 induced greater IgG responses against 4TO7RGhHER2 cells and greater protection against lung metastasis compared to mice vaccinated with unmodified 4TO7ts TMVs or soluble GPI-hHER2. Subcutaneous vaccination was also found to be more highly immunogenic overall compared to intravenous vaccination based on increase IgG responses against 4TO7HER2 cells and decreased lung metastasis. These results are promising for therapeutic studies aimed to activate HER2 specific immune responses in order to achieve tumor regression.

Table of Contents

Introduction

Breast Cancer…………………………………………………………………………...…1

Cancer and the Immune System…………………………………………………..………1

TMVs and Protein Transfer of GPI-ancored Immunostimulatory Molecules………….…3

Experimental Design………………………………………………………………………4

Materials and Methods

Cell culture………………………………………………………………………………...7

Harvesting roller bottle cells……………………………………………………………....8

Fluorescence-activated cell sorting (FACS)………………………………………………8

GPI-protein purification using affinity chromatography………………………………….9

Western Blot (WB) analysis……………………………………………………………..11

Part I……………………………………………………………………………...11

Part II…………………………………………………………………………….12

Silver Stain (SS) analysis………………………………………………………………...14

Part I……………………………………………………………………………...14

Part II………………………………………………………………………….....14

Polyvinylpyrrolidone mediated protein concentration…………………………………...15

Dialysis mediated protein purification…………………………………………………...16

Protein transfer of GPI-protein onto sheep erythrocytes………………………………...16

Bicinchoninc assay (BCA) for estimation of protein concentration……………………..17

Protein transfer of GPI-ISMs onto 4TO7ts Tumor Membrane Vesicles (TMVs)……….17

Quantification of GPI-protein incorporated onto TMVs………………………………...18

Western Blot……………………………………………………….…………….18

ELISA (enzyme-linked immunosorbent assay)………………………………….18

In vivo studies using BALB/cJ mice…………………………………………………….19

Model…………………………………………………………………………….19

Prophylactic vaccination using 4TO7ts TMVs…………………………………..20

Intravenous live 4TO7RGhHER2 cell challenge……………………………...…20

Serum antibody assay………………………………………………………...….20

Clonogenic assay…………………………………………………………….…..21

Live cell count……………………………………………………………………23

Kinetics of 4TO7RGhHER2 metastasis………………………………………….23

Results

Surface protein expression profile of 4TO7RGhHER2 culture cells…………………….24

Kinetics of 4TO7RGhHER2 metastasis………………………………………………….25

GPI-hHER2 protein analysis……………………………………………………………..28

GPI-Protein incorporation onto 4TO7ts TMVs………………………………………….29

FACS analysis of GPI-ISM incorporation onto 4TO7ts TMVs…………………………31

Weight changes post 4TO7RGhHER2 challenge………………………………………..32

Serum antibody response is specific to hHER2………………………………………….33

Quantification of 4TO7RGhHER2 metastasis…………………………………………...36

HER2 expression on lung metastatic cells……………………………………………….39

Discussion………………………………………………………………………………………..40

Conclusion……………………………………………………………………………………….43

References………………………………………………………………………………………..44

Figures

Figure 1: Overall experimental design…………………………………………………………….6

Figure 2: Surface protein expression profile of 4TO7hHER2 culture cells………………...…....24

Figure 3: Kinetics of 4TO7RGhHER2 metastasis clonogenic assay…………………………….26

Figure 4: Kinetics of 4TO7RGhHER2 metastasis protein expression changes………………….27

Figure 5: GPI-hHER2 protein analysis…………………………………………………………..28

Figure 6: GPI-Protein incorporation onto 4TO7ts TMVs………………………………………..30

Figure 7: FACS analysis of GPI-ISM incorporation onto 4TO7ts TMVs……………………….31

Figure 8: HER2 expression on 4TO7RGhHER2 cells used for intravenous challenge after prophylactic vaccination…………………………………………………………………………32

Figure 9: Weight changes post 4TO7RGhHER2 challenge……………………………………...33

Figure 10: Serum antibody responses to 4TO7RG vs 4TO7RGhHER2…………………………34

Figure 11: Quantification of 4TO7hHER2 metastasis…………………………………………...38

Figure12: HER2 expression on lung metastatic cells………………………………………...….39

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