Lineage tracing of metastasis in a mouse model for Non-small cell lung cancer (NSCLC):
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
|
| Schlagworte: | |
| Online-Zugang: | Volltext Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. und engl. Sprache. - Online erschienen: 2014 |
| Beschreibung: | 166 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Lineage tracing of metastasis in a mouse model for Non-small cell lung cancer (NSCLC) |c von Chitra Thakur |
| 246 | 1 | 3 | |a Untersuchung metastatischer Prozesse durchgenetische Zellmarkierung in einem Mausmodelldes nichtkleinzelligen Lungenkarzinoms (NSCLC) |
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Datensatz im Suchindex
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| adam_text | LINEAGE TRACING OF METASTASIS IN A MOUSE MODEL FOR
NON-SMALL CELL LUNG CANCER (NSCLC)
UNTERSUCHUNG METASTATISCHER PROZESSE DURCH
GENETISCHE ZELLMARKIERUNG IN EINEM MAUSMODELL
DES NICHTKLEINZELLIGEN LUNGENKARZINOMS (NSCLC)
DOCTORAL THESIS FOR A DOCTORAL DEGREE
AT THE GRADUATE SCHOOL OF LIFE SCIENCES,
JULIUS-MAXIMILIANS-UNIVERSITAT WURZBURG,
SECTION INFECTION AND IMMUNITY
SUBMITTED BY
CHITRA THAKUR
FROM
BHOPAL, INDIA
WURZBURG 2012
HTTP://D-NB.INFO/1054939217
TABLE OF CONTENTS
TABLE OF CONTENTS 1
ZUSAMMENFASSUNG 6
SUMMARY 8
11NTRODUCTION 10
1.1
NON SMALL
CELL LUNG
CANCER (NSCLC) 10
1.1.1 THE MITOGENIC CASCADE
AND RAS/RAF SIGNALING 11
1.1.1.1 RAS/RAF SIGNALING 12
1.2
MOLECULAR ABNORMALITIES
FOUND IN HUMAN LUNG
CANCER 14
1.2.1 RAF PROTEIN 15
1.2.2 THE RAS GENES 16
1.2.3 THE MYC PROTO-ONCOGENES 16
1.2.4 TUMOR SUPPRESSOR GENES 17
1.3 METASTASIS 17
1.3.1 COLLECTIVE MIGRATION 18
1.4 CELLULAR
ARCHITECTURE OF THE
LUNG 19
1.4.1 CELL POPULATIONS INVOLVED
IN THE HOMEOSTASIS AND INJURY REPAIR OF THE DISTAL LUNG 21
1.5 GENETIC LINEAGE
TRACING 24
1.5.1 WHAT IS LINEAGE TRACING? 24
1.5.2 NEED OF LINEAGE TRACING STRATEGY
IN PULMONARY/ONCOLOGY RESEARCH 25
1.5.3 REPORTER MOLECULES EMPLOYED FOR LINEAGE LABELING EXPERIMENTS 26
1.5.3.1 FLUORESCENT REPORTER PROTEINS 26
1.5.3.2 ADVANTAGE OF FLUORESCENT REPORTERS OVER CONVENTIONAL MARKING
SYSTEMS 26
1.5.3.3 RED FLUORESCENT PROTEIN (DSRED) 27
1.6 IN VIVO IMAGING 28
1.7 MOUSE
MODELS FOR STUDYING
TUMOR PROGRESSION
AND METASTASIS 29
1.7.1 TETRACYCLINE-CONTROLLED TRANSCRIPTIONAL ACTIVATION 30
1.7.1.1 TET OFF 30
1.7.1.2 TETON 31
1
1.7.2 THE CRE-LOXP TECHNOLOGY 32
2 EXPERIMENTAL
DESIGN AND GOALS OF THE PROJECT 35
2.1 GOALS OF THE PROJECT 36
2.2 TRANSGENIC
MICE USED FOR THE STUDY 36
3 MATERIALS 40
3.1
INSTRUMENTS 40
3.2 CHEMICAL
REAGENTS 42
3.3 ANTIBODIES 45
3.3.1 PRIMARY ANTIBODIES 45
3.3.2 SECONDARY ANTIBODIES 46
3.3.3 ANTIBODIES FOR IMMUNOBLOTTING 47
3.4 ENZYMES 47
3.5 KITS 48
3.6 OLIGONUCLEOTIDES ....49
3.7 OLIGOS
FOR EXPRESSION
ANALYSIS... 50
3.8
SOLUTIONS
AND BUFFERS 52
4 METHODS 54
4.1
MOLECULAR BIOLOGY
METHODS 54
4.1.1 MEASUREMENT OF DNA CONCENTRATION 54
4.1.2 POLYMERASE CHAIN REACTION (PCR) 54
4.1.3 AGAROSE-GEL ELECTROPHORESIS OF DNA MOLECULES 54
4.1.4 ISOLATION OF RNA 55
4.1.5 CDNA SYNTHESIS 55
4.1.6 REAL-TIME PCR 56
4.2 BIOCHEMISTRY
METHODS 56
4.2.1 PREPARATION OF WHOLE PROTEIN LYSATES FROM CELLS AND TISSUES 56
4.2.2 MEASUREMENT OF PROTEIN CONCENTRATION 56
4.2.3 SDS PAGE 57
4.2.4 IMMUNOBLOTTING 58
4.2.5 MEMBRANE STRIPPING 58
2
4.2:6 EUKARYOTIC CELL CULTURE TECHNIQUE 59
4.2.7 FREEZING CELL
LINES 59
4.2.8 ANALYSIS OF CULTURED CELLS VIA IMMUNOCYTOCHEMISTRY 60
4.3 HISTOLOGICAL
METHODS 60
4.3.1 PARAFFIN-EMBEDDING AND SECTIONING OF TISSUE 60
4.3.2 OPTIMAL CUTTING TEMPERATURE (O.C.T.) - EMBEDDING AND
CRYOSECTIONING OF TISSUE 61
4.3.3 HAEMATOXYLIN AND EOSIN STAINING 61
4.3.4 IMMUNOHISTOCHEMICAL STAININGS (ENZYMATIC BASED DETECTION) 62
4.3.5 ALCIAN BLUE STAINING 65
4.3.6 IMMUNOFLUORESCENCE STAININGS 66
4.3.7 X GAL STAINING ON FROZEN SECTIONS 66
4.3.8 X GAL STAINING OF FACS SORTED DSRED
CELLS 67
4.3.9 X-GAL: WHOLE MOUNT TISSUE PREPARATION 67
4.3.10 FACS SORTING OF DSRED CELLS FROM TRIPLE
TRANSGENIC REPORTER MICE 68
4.4 ANIMAL
EXPERIMENTS 69
4.4.1 GENERATION OF REPORTER TRANSGENIC MOUSE LINES FOR LINEAGE TRACING
EXPERIMENTS 69
4.4.2 BREEDING OF MICE 69
4.4.3 GENOTYPING OF TRANSGENIC MICE 70
4.4.4 SALIFICATION OF MICE 71
4.4.5 TRANSPLANTATION - EXPERIMENTS 71
4.5 STATISTICAL
ANALYSIS 72
5 RESULTS 73
5.1
TRANSGENIC REPORTER
MICE FOR
LINEAGE LABELING
STUDIES IN A MURINE NSCLC
MODEL 73
5.1.1 THE RED FLUORESCENT PROTEIN (RFP) MICE 73
5.1.2 GENERATION OF COMPOUND TRANSGENIC REPORTER MICE FOR LINEAGE
TRACING EXPERIMENTS 73
5.1.3 TESTING OF THE COMPOUND REPORTER TRANSGENIC MICE GENERATED FOR THE
EXPRESSION OF THE REPORTER
GENE 77
5.1.3.1 LUNG TARGETED EXPRESSION OF THE REPORTER GENE IN THE DSRED
MICE 77
5.1.3.2 FACS SORTED
DSRED CELLS CULTURED ON MATRIGEL 82
5.1.4 LABELING TUMOR CELLS INDUCED BY ONCOGENIC C RAF 84
5.1.4.1 LUNG TUMOR FORMATION IN C RAF
BXB-DSRED INDUCED MICE
EXHIBITING CUBOIDAL
TUMOR CELL
PHENOTYPE 85
5.1.4.2 EVALUATION OF THE TUMOR FOR THE EXPRESSION OF THE LINEAGE MARKER
AND THE TRANSGENE 86
3
5.1.4.3 LABELING SMALL
CRYPTIC LESIONS AT EARLIER TIME POINTS 87
5.1.4.4 C RAF BXB-DSRED TRANSGENIC MICE
EXHIBIT PROLIFERATING BRONCHIOLAR HYPERPLASIA 88
5.1.5 LABELING
TUMOR CELLS GENERATED IN A REPORTER METASTATIC NSCLC MODEL 90
5.1.6 INTRODUCTION OF MYC INDUCES A NEW PHENOTYPE: PREMALIGNANT LESIONS
93
5.1.7 EVALUATION AND CHARACTERIZATION OF THE PRIMARY TUMOR 95
5.1.8 TUMOR PROGRESSION CONCOMITANT WITH ANGIOGENESIS IN THE ABSENCE OF
EMT 96
5.1.9 LABELING EFFICIENCY IN THE TRANSGENIC COMPOUND REPORTER MICE 98
5.1.10 PROLIFERATION AND APOPTOSIS IN THE PRIMARY TUMOR 99
5.1.11 STATUS OF P53
IN THE COMPOUND TRANSGENIC REPORTER ANIMALS 101
5.1.12 SCREENING FOR DSRED POSITIVE CELLS VIA FLOW
CYTOMETRY 102
5.1.13 LOSS OF SPC AND
TTF-1
IN THE PRIMARY TUMORS: HALLMARKS FOR METASTATIC PROGRESSION 103
5.1.14 EVIDENCE OF CIRCULATING BRONCHIOALVEOLAR STEM CELLS
(BASCS) IN THE TUMORS 105
5.1.15 DETECTION OF THE TUMOR SPECIFIC TRANSCRIPTS
IN COMPOUND TRANSGENIC REPORTER MICE VIA GENE
EXPRESSION ANALYSIS 107
5.1.16 GENE EXPRESSION PROFILING OF THE PRIMARY LUNG TUMOR 109
5.1.17 SURVIVAL CURVE AND TUMOR BURDEN OF THE REPORTER TRANSGENIC
ANIMALS ILL
5.2
ANALYSIS OF LIVER METASTASES 114
5.2.1 ANATOMICAL AND
MORPHOLOGICAL CHARACTERIZATION OF THE METASTATIC LESIONS 114
5.2.2 SPC EXPRESSION IS DETECTED
IN CYSTIC METASTASES 117
5.2.3 DSRED IS DETECTED IN THE SINGLE CLUSTER OF CELL INCLUDING
THE CYSTIC AND
THE PAPILLARY LESIONS
OF
METASTATIC COLONIZATION 118
5.2.4 EVALUATION OF METASTASES FOR THE EXPRESSION OF PROGENITOR
MARKERS/LINEAGE SELECTORS 120
5.2.5 CLARA CELL EMERGES AT THE MIXED CYSTIC PAPILLARY
STAGE OF LIVER METASTASES 123
5.2.6 PRESENCE OF BRONCHIOALVEOLAR STEM CELLS (BASCS) IN THE
METASTASES 125
6 DISCUSSION 127
6.1
NOVEL TRANSGENIC
MURINE MODEL
FOR LINEAGE
TRACING
OF ALVEOLAR TYPE
II CELLS IN NSCLC
METASTASIS 127
6.2 ALVEOLAR
TYPE
II CELLS IN NSCLC TUMOR
INITIATION; PERSPECTIVE AND FACTS 129
6.3
CONTRIBUTION OF ALVEOLAR TYPE
II CELLS
IN THE METASTASES; AN INSIGHT 134
6.4
PUTATIVE METASTATIC
INITIATING
CELLS; CONTRIBUTION
OF THE
ALVEOLAR LINEAGE 137
7 CRITICAL PARAMETERS, TROUBLESHOOTING AND TIME CONSIDERATIONS 140
8 APPENDIX 144
4
9 REFERENCES. 147
10 CURRICULUM VITAE 159
ACKNOWLEDGEMENTS 163
AFFIDAVIT 166
5
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| spelling | Thakur, Chitra 1982- Verfasser (DE-588)1053808208 aut Lineage tracing of metastasis in a mouse model for Non-small cell lung cancer (NSCLC) von Chitra Thakur Untersuchung metastatischer Prozesse durchgenetische Zellmarkierung in einem Mausmodelldes nichtkleinzelligen Lungenkarzinoms (NSCLC) 2012 166 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. und engl. Sprache. - Online erschienen: 2014 Würzburg, Univ., Diss., 2012 Metastase (DE-588)4125915-4 gnd rswk-swf Lungenkrebs (DE-588)4036668-6 gnd rswk-swf Maus (DE-588)4169148-9 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Lungenkrebs (DE-588)4036668-6 s Metastase (DE-588)4125915-4 s Maus (DE-588)4169148-9 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-85420 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-85420 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027422613&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Thakur, Chitra 1982- Lineage tracing of metastasis in a mouse model for Non-small cell lung cancer (NSCLC) Metastase (DE-588)4125915-4 gnd Lungenkrebs (DE-588)4036668-6 gnd Maus (DE-588)4169148-9 gnd |
| subject_GND | (DE-588)4125915-4 (DE-588)4036668-6 (DE-588)4169148-9 (DE-588)4113937-9 |
| title | Lineage tracing of metastasis in a mouse model for Non-small cell lung cancer (NSCLC) |
| title_alt | Untersuchung metastatischer Prozesse durchgenetische Zellmarkierung in einem Mausmodelldes nichtkleinzelligen Lungenkarzinoms (NSCLC) |
| title_auth | Lineage tracing of metastasis in a mouse model for Non-small cell lung cancer (NSCLC) |
| title_exact_search | Lineage tracing of metastasis in a mouse model for Non-small cell lung cancer (NSCLC) |
| title_full | Lineage tracing of metastasis in a mouse model for Non-small cell lung cancer (NSCLC) von Chitra Thakur |
| title_fullStr | Lineage tracing of metastasis in a mouse model for Non-small cell lung cancer (NSCLC) von Chitra Thakur |
| title_full_unstemmed | Lineage tracing of metastasis in a mouse model for Non-small cell lung cancer (NSCLC) von Chitra Thakur |
| title_short | Lineage tracing of metastasis in a mouse model for Non-small cell lung cancer (NSCLC) |
| title_sort | lineage tracing of metastasis in a mouse model for non small cell lung cancer nsclc |
| topic | Metastase (DE-588)4125915-4 gnd Lungenkrebs (DE-588)4036668-6 gnd Maus (DE-588)4169148-9 gnd |
| topic_facet | Metastase Lungenkrebs Maus Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-85420 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027422613&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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