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Molecular Imaging of Cancer-related Molecules in Cancer Cells and their Microenvironment

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dc.contributor.advisor김병수-
dc.contributor.author류주희-
dc.date.accessioned2017-07-13T08:37:12Z-
dc.date.available2017-07-13T08:37:12Z-
dc.date.issued2014-08-
dc.identifier.other000000021700-
dc.identifier.urihttps://hdl.handle.net/10371/119707-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 화학생물공학부, 2014. 8. 김병수.-
dc.description.abstractMolecular imaging is a key element of cancer management in the 21st century. Molecular imaging enables the visualization and characterization of cancer-specific events at the cellular and molecular levels in living systems. It allows cancerous lesions to be detected at an earlier stage, which is one of the most effective strategies for cancer treatment. In addition, it has a major impact on intra-operative cancer detection, the real-time validation of therapy, as well as the knowledge of the role of cancer-related components. Specifically, optical imaging is a highly sensitive and non-invasive molecular imaging technique, in contrast to anatomical imaging approaches.
Cancer research has been focused on studying and characterizing the genetically determined expression of biomolecules by cancer cells in the past several decades. For targeted cancer imaging, over-expressed receptors on the surface of cancer cells or over-produced enzymes by cancer cells have been utilized. However, genetic and epigenetic alterations in tumor cells have been found to be insufficient to offer tumor cells with malignant properties although they are prerequisites for malignant processes. In recent years, it has become obvious that other components of tumors, such as non-tumor cells (endothelial cells, fibroblasts), connective tissue, extracellular matrix (ECM), immune cells, and soluble factors play fundamental roles in tumor development and progression. These components are collectively known as the tumor microenvironment. Therefore, increased attention has been paid to the tumor microenvironment as well as cancer cells for cancer imaging.
In this study, imaging probes were first developed to target and visualize the components of cancer cell or the tumor microenvironment
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dc.description.abstractthe probes were then applied for cancer imaging. In Chapter 2, cathepsin B (CB), an over-produced enzyme by cancer cells was targeted and visualized in metastatic tumor models. The fact that the activity of CB is markedly linked to the metastatic process and that CB is found highly expressed in the pericellular regions in this process makes CB an attractive target for diagnosing metastases. A CB-sensitive nanoprobe (CB-CNP) was developed to include CB-sensitive fluorescence peptide probes conjugated onto the surface of tumor-targeting glycol chitosan nanoparticles (CNPs). The fluorescence intensity of the CB-CNP was strongly quenched in physiological condition. However, self-quenched CB-CNP boosted strong fluorescence signals in the presence of CB, not of cathepsin L or cathepsin D, due to the CB-specific cleavage of peptide probes. Importantly, the intravenously injected CB-CNP demonstrated the potential to discriminate metastases in vivo in three metastatic mouse models, including 4T1-luc2 liver metastases, RFP-B16F10 lung metastases and HT1080 peritoneal metastases. CB-CNPs may be useful for depicting metastases through the non-invasive molecular imaging of CB activity.
In Chapter 3, an over-expressed receptor on the surface of cancer cells, epidermal growth factor receptor (EGFR), was targeted for cancer imaging. Herein, an epidermal growth factor-based nanoprobe (EGF-NP) was developed for the in vivo optical imaging of EGFR. The self-quenched EGF-NP is fabricated by sequentially conjugating a near-infrared (NIR) fluorophore (Cy5.5) and a quencher (BHQ-3) to EGF, a low-molecular weight polypeptide (6.2 kDa), compared to EGFR antibody (150 kDa). The self-quenched EGF-NP presented great specificity to EGFR, and rapidly internalized into the cells. Importantly, the self-quenched EGF-NP boosted strong fluorescence signals upon EGFR-targeted uptake into EGFR-expressing cells, followed by lysosomal degradation, as confirmed by lysosomal marker cell imaging. Consistent with the cellular results, the intravenous injection of EGF-NP into tumor-bearing mice induced strong NIR fluorescence intensity in the target tumor tissue with high specificity against EGFR-expressing cancer cells. The signal accumulation of EGF-NP in tumors was much faster than that of EGFR monoclonal antibody (Cetuximab)-Cy5.5 conjugates due to the rapid clearance from the body and tissue permeability of low-molecular weight EGF. This self-quenched, EGF-based imaging probe can be applied to diagnose of various cancers.
In Chapter 4, the detection of lysyl oxidase (LOX) activity in ECM was attempted using gold nanoprobes. Stiffness of ECM is significantly associated with cancer progression and malignancy. LOX is considered as an important factor to affect stiffness of ECM among various factors. In addition, LOX has been validated for a prognostic marker for metastasis and survival in multiple cancer types including head and neck squamous cell carcinoma. Therefore, a sensitive and simple assay for detecting the activity of LOX is needed. The plasmon coupling-based color change using AuNPs has been widely used to detect the presence or amounts of analytes that cause the selective aggregation of AuNPs. The AuNPs were functionalized with LOX-sensitive hexapeptides (LOX-AuNPs) to detect LOX. LOX-AuNPs turn from their original pinkish red to purple and the absorption spectra of the LOX-AuNPs red-shifted in correlation with the LOX concentration. In addition, LOX-AuNPs showed high specificity for LOX as presented in the experiments with LOX inhibitor or Control-AuNPs. LOX-AuNPs demonstrated the potential to detect LOX in cancer cells and tumor tissues including different LOX contents. LOX detection by LOX-AuNPs in cancer cells was more sensitive compared to those by a commercially available LOX assay or Western blot analysis. LOX levels measured using LOX-AuNPs in various tumor tissues were correlated directly with their collagen contents and their ECM stiffness. Therefore, LOX-AuNPs may be applied to detecting LOX activity and further ECM stiffness of tumor tissues.
Successful surgical procedures for cancer treatment depend on the accurate and rapid localization of tumor tissues, following their correct resection. In Chapter 5, tumor tissue-specific imaging by topical application was attempted for rapid tumor visualization. It was hypothesized that tissue-permeable probes can differently accumulated in tumor or normal tissue due to the structural difference between them. To make tissue-permeable probes, the factor of high volume of distribution (Vd) was utilized. The Vd can be calculated as the ratio of the total amount of a drug in the body and its concentration in the blood at a steady state. A higher Vd indicates that the drug is more diluted than it should be in the blood, suggesting that a larger amount of the drug has been distributed in the tissue, not in blood. Accordingly, the drug with high Vd can be tissue-permeable. Four drug-dye conjugates were synthesized by directly coupling the NIR dye, FCR675, with 4 drugs with different Vd values, including raloxifene (2348 l/kg), scopolamine (3.1 l/kg), ampicillin (0.38 l/kg), and ibuprofen (0.15 l/kg). Drug-dye conjugates were topically applied via spraying on the liver surface of the HCT116 liver tumor-bearing mice and transanally applied via the anus into the descending colon of colon tumor-bearing mice. Topically applied Ralo-FCR675 also rapidly penetrated to the tumors maximum depth and, more importantly, Ralo-FCR675 preferentially accumulated in the tumor lesions within 3 min. Vd could be a factor to differentiate between tumor tissue and non-tumor tissue. Fluorescently labeled drugs with high Vd, such as raloxifene may be useful for the rapid visualization of tumors, contributing to successful surgical procedures for cancer treatment.
Finally, in Chapter 6, tumor-associated macrophage (TAM) was targeted and visualized. Specifically, TAM infiltration into tumors has been strongly correlated with poor prognosis in multiple cancer types. Hence, the imaging of TAM infiltration in tumors could be helpful in clinical applications for the prognosis and treatment of tumors. Scavenger receptors class A (SR-A) is known to be highly expressed in the TAMs of multiple cancer types and to be recognized with various negatively charged macromolecules including dextran sulfate (DS). Herein, FPR675-labeled DS nanoprobes (DSNPs) were developed to detect TAMs. DSNPs were significantly uptaken into RAW264.7 macrophage cells and isolated TAMs, not in MDA-MB-468 cancer cells. In an MDA-MB-468 subcutaneous tumor model, DSNPs provide high fluorescence signal both in tumor and liver sites. Further immunohistochemical analyses demonstrated that DSNPs mainly target CD204-positive TAMs. Therefore, DSNPs could be potentially utilized to visualize TAM infiltration in tumors.
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dc.description.tableofcontentsTable of contents

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i
List of figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv
List of tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxvii

Chapter 1. Introduction

1.1. Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2. Molecular imaging in cancer management . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3. Cancer imaging modalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.4. Targets of cancer cells for cancer imaging . . . . . . . . . . . . . . . . . . . . . . . . 11
1.5. Targets of the tumor microenvironment for cancer imaging . . . . . . . . . . . 13
1.5.1. Cellular components of the tumor microenvironment . . . . . . . . . . . 15
1.5.2. Non-cellular components of the tumor microenvironment . . . . . . . . 17
1.6. Scope of this thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1.7. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Chapter 2. Imaging of cathepsin B activity with activatable nanoprobes

2.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.2. Experimental section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.2.1. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.2.2. Preparation of the cathepsin B-sensitive nanoprobe . . . . . . . . . . . . . . 31
2.2.3. Characterization of cathepsin B-sensitive nanoprobe . . . . . . . . . . . . 32
2.2.4. In vitro enzyme specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.2.5. Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.2.6. Cellular uptake and immunocytochemistry . . . . . . . . . . . . . . . . . . . . . 35
2.2.7. Tumor models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.2.8. In vivo and ex vivo fluorescence imaging . . . . . . . . . . . . . . . . . . . . 37
2.2.9. Histological and Western blot analyses . . . . . . . . . . . . . . . . . . . . . . 38
2.2.10. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.3.1. Development and characterization of cathepsin B-sensitive nanoprobe . 40
2.3.2. Advantages of nanoprobes for imaging of cathepsin B activity . . . 46
2.3.3. Cellular uptake and cathepsin B distribution . . . . . . . . . . . . . . 47
2.3.4. Probe specificity for cathepsin B in vivo . . . . . . . . . . . . . . . . . . . . . 50
2.3.5. In vivo imaging of cathepsin B activity in three different metastatic mouse models . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
2.4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
2.5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
2.6. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Chapter 3. Epidermal growth factor receptor-targeted imaging of tumors

3.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
3.2. Experimental section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.2.1. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.2.2. Synthesis and characterization of epidermal growth factor-based nanoprobe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
3.2.3. Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
3.2.4. Binding studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
3.2.5. Cellular imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
3.2.6. In vivo and ex vivo fluorescence imaging . . . . . . . . . . . . . . . . . 77
3.2.7. Histological and Western blot analyses . . . . . . . . . . . . . . . . . . . . . . 78
3.2.8. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.3.1. Preparation of epidermal growth factor-based nanoprobe . . . . . . . . 79
3.3.2. Characterization and cellular imaging of epidermal growth factor-based nanoprobe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
3.3.3. Probe specificity for epidermal growth factor receptor . . . . . . . . . 85
3.3.4. Intracellular location of activated epidermal growth factor-based nanoprobe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
3.3.5. In vivo fluorescence imaging in tumor-bearing mice . . . . . . . . . . . . . 87
3.4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
3.5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
3.6. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Chapter 4. Detection of lysyl oxidase activity in extracellular matrix using gold nanoprobes

4.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
4.2. Experimental section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
4.2.1. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
4.2.2. Synthesis of gold nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
4.2.3. Preparation of peptide-functionalized gold nanoparticles . . . . . . . . . 106
4.2.4. Characterization of peptide-functionalized gold nanoparticles . . . . . 107
4.2.5. Sensitivity and specificity of peptide-functionalized gold nanoparticles for lysyl oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
4.2.6. Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
4.2.7. Commercially available lysyl oxidase assay . . . . . . . . . . . . . . . . . . . 109
4.2.8. Tumor models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
4.2.9. Histological and Western blot analyses . . . . . . . . . . . . . . . . . . . . . . . 110
4.2.10. Compressive modulus measurements . . . . . . . . . . . . . . . . . . . . . . . 111
4.2.11. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
4.3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
4.3.1. Reactivity of lysyl oxidase-peptide for lysyl oxidase . . . . . . . . . . . . 112
4.3.2. Characterization of peptide-functionalized gold nanoparticles . . . . . 112
4.3.3. Detection of lysyl oxidase in vitro . . . . . . . . . . . . . . . . . 114
4.3.4. Detection of lysyl oxidase in cancer cells . . . . . . . . . . . . . . . . . . . . . 116
4.3.5. Detection of lysyl oxidase in tumor tissues . . . . . . . . . . . . . . . . . . . 119
4.3.6. Analysis of extracellular matrix remodeling in tumors . . . . . . . . . . . . 121
4.4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
4.5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
4.6. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

Chapter 5. Tissue-permeable drug-dye conjugates for tumor visualization
5.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
5.2. Experimental section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
5.2.1. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
5.2.2. Synthesis of drug-dye conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
5.2.3. Characterization of drug-dye conjugates . . . . . . . . . . . . . . . . . . . . . . . 136
5.2.4. Cytotoxicity and cellular uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
5.2.5. Liver tumor model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
5.2.6. Topical application via spraying of drug-dye conjugates . . . . . . . . . . . 138
5.2.7. In vivo and ex vivo fluorescence imaging . . . . . . . . . . . . . . . . . . . . . . 139
5.2.8. Azoxymethane-induced colon cancer model . . . . . . . . . . . . . . . . . . . 140
5.2.9. Histological analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
5.3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
5.3.1. Characterization of drug-dye conjugates . . . . . . . . . . . . . . . . . . . . . 141
5.3.2. Preferential accumulation of drug-dye conjugates in tumors . . . . . 145
5.3.3. Analysis of the distribution of drug-dye conjugates . . . . . . . . . . . . . . 148
5.3.4. Transanal application to colon cancer model . . . . . . . . . . . . . . . . . . 151
5.4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
5.5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
5.6. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159


Chapter 6. Imaging of tumor-associated macrophages using dextran sulfate-based nanoprobes

6.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
6.2. Experimental section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
6.2.1. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
6.2.2. Preparation of dextran sulfate-based nanoprobes . . . . . . . . . . . 167
6.2.3. Characterization of dextran sulfate-based nanoprobes . . . . . . . . . . 168
6.2.4. Isolation of tumor-associated macrophages . . . . . . . . . . . . . . . . . . . . 168
6.2.5. Cytotoxicity and cellular uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
6.2.6. In vivo and ex vivo fluorescence imaging . . . . . . . . . . . . . . . . . . . . . 171
6.2.7. Histological analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
6.3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
6.3.1. Characterization of dextran sulfate-based nanoprobes . . . . . . . . . . . . 173
6.3.2. Cellular uptake of dextran sulfate-based nanoprobes . . . . . . . . . . . . 173
6.3.3. In vivo distribution of dextran sulfate-based nanoprobes . . . . . . . . . 174
6.3.4. Immunohistochemistry of tumor tissues . . . . . . . . . . . . . . . . . . . . . . 176
6.4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
6.5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
6.6. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
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dc.formatapplication/pdf-
dc.format.extent5220821 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectmolecular imaging-
dc.subjectcancer imaging-
dc.subjectoptical imaging-
dc.subjecttumor microenvironment-
dc.subjectdrug delivery system-
dc.subject.ddc660-
dc.titleMolecular Imaging of Cancer-related Molecules in Cancer Cells and their Microenvironment-
dc.typeThesis-
dc.description.degreeDoctor-
dc.citation.pagesxxvii, 189-
dc.contributor.affiliation공과대학 화학생물공학부-
dc.date.awarded2014-08-
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College of Engineering/Engineering Practice School (공과대학/대학원)Dept. of Chemical and Biological Engineering (화학생물공학부)Theses (Ph.D. / Sc.D._화학생물공학부)
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