위암의 암성복수 마우스모델을 이용한 방사면역치료에서 64Cu-DOTA-Cetuximab의 복강내주사 또는 정맥내주사 투여 후의 생물학적 분포 비교
Biodistribution Studies of 64Cu-DOTA-Cetuximab after Intraperitoneal and Intravenous Injection in a Malignant Ascites Mouse Model of Human Gastric Carcinoma
- 주제(키워드) Stomach neoplasm , epidermal growth factor receptor , ascites , intraperitoneal , radioimmunotherapy , radioimmunoconjugates , cetuximab , 64Cu-DOTA-cetuximab , positron emission tomography , biodistribution
- 발행기관 아주대학교
- 지도교수 한상욱
- 발행년도 2014
- 학위수여년월 2014. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000016000
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Intraperitoneal (i.p.) radioimmunotherapy (RIT) may be a promising treatment strategy for intraperitoneally-confined malignant diseases including malignant ascites (MA) in gastric cancer. We conducted a comparative biodistribution study of 64Cu-DOTA-cetuximab in subcutaneous (SQ) tumor and MA mouse models. A mouse xenograft model of bilateral SQ tumors that express epidermal growth factor receptor (EGFR) was established by SQ injections of NUGC-4 and MKN-45 gastric cancer cells into either shoulder of female BALB/c nude mice. The MA model was produced by i.p. injection of NUGC-4 cells. Cetuximab was conjugated to a bifunctional chelator (p-SCN-Bn-DOTA) and labeled with Copper-64 (T1/2 = 12.7 hours). Radiolabeled cetuximab was then administered intravenous (i.v.) or i.p. injection. Positron emission tomography (PET) imaging and biodistribution studies were conducted at the indicated time points. In the bilateral SQ tumor model, the peak uptake of radiolabeled cetuximab in the NUGC-4 and MKN-45 tumors was detected at 24 h post-injection of 64Cu-DOTA-cetuximab by PET imaging and biodistribution analysis. NUGC-4 tumor uptake of radiolabeled cetuximab was significantly higher than MKN-45 tumor uptake at all time points. The EGFR expression levels of the two cell lines were similar according to in vitro experiments. However, based on flow cytometry analyses at different incubation times with primary antibody (cetuximab) ranging from 3 min to 3 h, the EGFR expression levels were increased over time, and the increasing slope was higher in NUGC-4 cells than in MKN-45 cells. In the i.v.-induced MA model mice, the biodistribution of radiolabeled cetuximab in the blood pool and liver was high at 1 h and 3 h after i.v. injection and decreased over time. In the i.p.-induced MA model mice, the percent injected dose per gram (% ID/g) of ascites was very high and more than 8% at 1, 3, 24, and 48 h post injection; however, the % ID/g in all normal organs was equal to or less than 6% at all time points. In addition, all peak uptakes of normal organs and i.p.-induced NUGC-4 tumors appeared at 24 h after injection. In addition, the uptake ratios of tumor, peritoneum, and ascites to normal organs after i.p. injection were greater than after i.v. injection, particularly at the early time points. Finally, autoradiography studies using excised peritoneum and mesentery indicated several hot spots corresponding to peritoneal seeding. The results suggest that using the i.p. route for RIT in malignant ascites models shows advantages over the i.v. route with regard to tumor targeting and half-life prolongation of radioimmunoconjugates in the peritoneal cavity.
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ABSTRACT ⅰ
TABLE OF CONTENTS ⅲ
LIST OF FIGURES ⅴ
LIST OF TABLES ⅶ
Ⅰ. INTRODUCTION 1
A. Gastric Cancer and Malignant Ascites 1
B. Intraperitoneal Radioimmunotherapy 3
C. Purpose of the Study 6
Ⅱ. MATERIALS AND METHODS 7
A. Materials 7
1. Cell Lines 7
2. Subcutaneous Tumor and Malignant Ascites Mouse Models 7
3. Preparation of Radioimmunoconjugates 8
B. Methods 10
1. Western Immunoblot Analysis 10
2. Flow Cytometry Analysis 11
3. In vitro Cytotoxicity of Cetuximab 12
4. Immunofluorescence Microscopy 12
5. In vitro Stability Study of 64Cu-DOTA-Bn-NCS-Cetuximab 13
6. Immunoreactivity Assay 13
7. MicroPET Imaging 14
8. Biodistribution Studies 15
9. Peritoneal Membrane Autoradiography 15
10. Statistical Analysis 16
Ⅲ. RESULTS 17
A. EGFR Expression Levels in Gastric Carcinoma Cells 17
B. Subcellular Localization of Cetuximab-bound EGFR 19
C. Cetuximab Cytotoxicity in Gastric Cancer Cells 20
D. Conjugation and 64Cu Labeling 21
E. In Vitro Properties of 64Cu-DOTA-Cetuximab 23
F. Bilateral Subcutaneous Tumor and Malignant Ascites Mouse Models 26
G. PET Imaging of Bilateral Subcutaneous Tumor Model Mice 28
H. Biodistribution of 64Cu-DOTA-Cetuximab in Bilateral Subcutaneous Tumor Model Mice 29
I. EGFR expression level affected by incubation periods 33
J. PET Imaging of Malignant Ascites Model Mice after I.V. or I.P. Administration of 64Cu-DOTA-Cetuximab 35
K. Biodistribution of 64Cu-DOTA-Cetuximab in Malignant Ascites Model after I.V. or I.P. Administration 37
L. Localization of 64Cu-DOTA-Cetuximab in Peritoneal Seeding 43
Ⅳ. DISCUSSION 44
Ⅴ. CONCLUSION 49
REFERENCES 50
국문요약 61
