The role of BMP9 signaling during tumor progression: = Die Rolle des BMP9 Signalwegs in der Tumorprogression
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| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
Erlangen ; Nürnberg
2017
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | 114 Seiten Illustrationen, Diagramme 30 cm |
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MARC
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| 100 | 1 | |a Brand, Verena Maria Hedi |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a The role of BMP9 signaling during tumor progression |b = Die Rolle des BMP9 Signalwegs in der Tumorprogression |c Verena Maria Hedi Brand |
| 246 | 1 | 3 | |a Die Rolle des BMP9 Signalwegs in der Tumorprogression |
| 246 | 1 | 1 | |a Die Rolle des BMP9 Signalwegs in der Tumorprogression |
| 264 | 1 | |a Erlangen ; Nürnberg |c 2017 | |
| 300 | |a 114 Seiten |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
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| 502 | |b Dissertation |c Friedrich-Alexander-Universität Erlangen-Nürnberg |d 2017 | ||
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Record in the Search Index
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| adam_text | TABLE OF CONTENT
1. SUMMARY
...............
2.
ZUSAMMENFASSUNG..............................................................................................................................................
9
3.
INTRODUCTION.......................................................................................................................................................
11
3.1. TUMOR
PROGRESSION..................................................................................................................................
11
3.2. BMP9, A MEMBER OF THE TGFSS
SUPERFAMILY..........................................................................................12
3.3. BMP9
SIGNALING.........................................................................................................................................14
3.4. EPITHELIAL TO MESENCHYMAL TRANSDUCTION (EMT) AND THE POTENTIAL
IMPACT OF BM P9
....................
16
3.5. BMP9 IN
ANGIOGENESIS.............................................................................................................................18
3.5.1. TUMOR
ANGIOGENESIS.......................................................................................................................
18
3.5.2. THE ROLE OF BMP9 DURING ANGIOGENESIS
........................................................................................
22
3.6. THE ROLE OF BMP9 IN
CANCER....................................................................................................................
23
3.7. THE TGF SUPERFAMILY AND BMP9 AS THERAPEUTIC TARGET FOR CANCER
THERAPY.....................................25
3.8. AIMS OF THE
STUDY.....................................................................................................................................
26
4. M
ATERIAL..............................................................................................................................................................27
4.1.
CHEMICALS..................................................................................................................................................
27
4.2. CELL
CULTURE...............................................................................................................................................
27
4.2.1. CELL
LINES............................................................................................................................................27
4.2.2. MEDIA, BUFFER AND
SOLUTIONS..........................................................................................................29
4.2.3.
CONSUMABLES...................................................................................................................................
30
4.2.4.
KITS.....................................................................................................................................................30
4.2.5. RECOMBINANT
PROTEINS....................................................................................................................31
4.3.
ANTIBODIES.................................................................................................................................................
32
4.3.1. PRIMARY
ANTIBODIES.........................................................................................................................
32
4.3.2. SECONDARY
ANTIBODIES.....................................................................................................................34
4.4. IMMUNOFLUORESCENCE AND
IMMUNOHISTOCHEMISTRY............................................................................
34
4.5. IN VITRO CELL
IMAGING.................................................................................................................................35
4.6. SDS-PAGE AND WESTERN
BLOTTING...........................................................................................................35
4.7. ACEA
ASSAY................................................................................................................................................
36
4.8. PCR REAGENTS AND
PRIMERS......................................................................................................................36
4.9. SURFACE PLASMON RESONANCE
(SPR)........................................................................................................37
4.10. PLASMID PREPARATION
(MEGAPREP)..........................................................................................................37
4.11.
TRANSFECTION................................................................................................................................................38
4.12.
CRISPR/CAS9...............................................................................................................................................38
4.13.
ANIMALS........................................................................................................................................................38
4.14. COMPOUNDS FOR IN VIVO
USE......................................................................................................................
38
4.15.
SOFTWARE.....................................................................................................................................................
39
5. M
ETHODS.............................................................................................................................................................40
5.1. CELL
CULTURE..................................................................................................................................................40
5.1.1. CELL CULTURE
CONDITIONS.....................................................................................................................40
5.1.2. SUB CULTURING, FREEZING AND THAWING OF
CELLS................................................................................
40
5.1.3. CELL
COUNTING.....................................................................................................................................
40
5.1.4. IN VITRO CELL
IMAGING.........................................................................................................................
40
5.2. SURFACE PLASMON RESONANCE
(SPR).......................................................................
41
5.3.
ELISAS...........................................................................................................................................................41
5.3.1. BMP9
ELISA........................................................................................................................................41
5.3.2. ENDOTHELIN-1
ELISA..........................................................................................................................
43
5.3.3. INTERLEUKIN-6 ELISA (HUMAN AND
MOUSE)......................................................................................43
5.4. FUNCTIONAL ELECTRONIC CELL SENSOR ASSAY
(XCELLIGENCE).......................................................................
43
5.5. WESTERN BLOT
ANALYSES..............................................................................................................................43
5.5.1. CELL LYSATE
PREPARATION....................................................................................................................43
5.5.2. TUMOR LYSATE PREPARATION
.....................................
43
5.5.3. DETERMINATION OF PROTEIN CONCENTRATION
.....................................................................................
44
5.5.4. SDS-PAGE AND WESTERN
BLOTTING...................................................................................................44
5.6.
RT-PCR........................................................................................................................................................
44
5.6.1. PRIMER
DESIGN....................................................................................................................................
44
5.6.2. RNA
ISOLATION....................................................................................................................................
45
5.6.3. REVERSE
TRANSCRIPTION......................................................................................................................45
5.6.4. REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION (RT-PCR)
.....................................................
45
5.6.5. AGAROSE GEL
ELECTROPHORESIS..........................................................................................................
45
5.7. RNA
SEQUENCING........................................................................................................................................
45
5.7.1. CELL LYSATE
PREPARATION...................................................................................................................
45
5.7.2. RNA
SEQUENCING................................................................................................................................46
5.8. TRANSFECTION &
CRISPR/CAS9...................................................................................................................
46
4
5.8.1. PLASMID
PREPARATION.......................................................................................................................
46
5.8.2. TRANSFECTION OF MURINE 10T1/2
CELLS.............................................................................................46
5.8.3. FACS -
SORTING..................................................................................................................................46
5.8.4.
RE-CULTIVATION..................................................................................................................................46
5.8.5. CELL LYSATE PREPARATION FOR WESTERN BLOT ANALYSES
.....................................................................
47
5.9. WOUND CLOSURE
ASSAY.....................................................................................................................47
5.10. PERICYTE - ENDOTHELIAL CELL ATTACHMENT
ASSAY............................................................................
47
5.11. CELLTITER-GLO* LUMINESCENT CELL VIABILITY
ASSAY..................................................................................47
5.11.1. ENDOTHELIAL CELLS AND
PERICYTES.....................................................................................................
47
5.11.2. RENAL CELL CARCINOMA CELLS
A-498.................................................................................................47
5.12. CASPASE-GLO 3/7
ASSAY.........................................................................................................................
48
5.13. ANIMAL
EXPERIMENTS...............................................................................................................................
48
5.14.
ULTRAMICROSCOPY......................................................................................................................................
48
5.15.
IMMUNOHISTOCHEMISTRY..........................................................................................................................
48
5.15.1. CHROMOGENIC DETECTION OF CD31 OR ALK1 ON PARAFFIN EMBEDDED TUM
ORS.............................48
5.15.2. FLUORESCENT DETECTION OF CD31/NG2/LECTIN ON FRESH FROZEN TUMOR
SAMPLES
........................
49
5.15.3. FLUORESCENT DETECTION OF DESMIN/A-SMA/LECTIN, PHOSPHO-HISTONE
H3, CLEAVED
....................
CASPASE-3, VIMENTIN OR E-CADHERIN ON PARAFFIN EMBEDDED TUMORS
.....................................
49
5.15.4. FLUORESCENT DETECTION OF SNAIL ON PARAFFIN EMBEDDED
TUMORS................................................50
5.16. STATISTICAL
ANALYSES..................................................................................................................................50
6.
RESULTS...............................................................................................................................................................
51
6.1. DEVELOPMENT OF A MONOCLONAL ANTIBODY SELECTIVELY TARGETING
BMP9............................................51
6.2. BMP9 AFFECTS ANGIOGENESIS BY INDUCTION OF SIGNALING IN ENDOTHELIAL
CELLS AND PERICYTES
............
53
6.2.1. ENDOTHELIAL CELLS AND PERICYTES EXPRESS RECEPTORS FOR
BMP9...................................................53
6.2.2. SMAD 1/5/8 PHOSPHORYLATION IS INDUCED BY BMP9 IN ENDOTHELIAL
CELLS
.....................................
AS WELL AS
PERICYTES.........................................................................................................................54
6.2.3. SMAD 2/3 PHOSPHORYLATION PATTERNS ARE DIFFERENT IN ENDOTHELIAL
CELLS A N D .............................
PERICYTES UPON BMP9
STIMULATION................................................................................................55
6.2.4. PRE-INCUBATION OF BMP9 WITH MAB BMP9-0093 INHIBITS BMP9-INDUCED
..................................
SMAD
PHOSPHORYLATION..................................................................................................................
56
6.2.5. RNA SEQUENCING CONFIRMS DIFFERENT GENE REGULATION IN HUVEC AND
PERICYTES
.....................
56
6.2.6. EFFECTS OF BMP9 ON PROLIFERATION AND MIGRATION BEHAVIOR OF
ENDOTHELIAL CELLS
.......................
AND
PERICYTES...................................................................................................................................57
6.2.7. BMP9 REDUCES ATTACHMENT OF PERICYTES TO
HUVEC...................................................................60
6.2.8. GENE KNOCKOUT BY CRISPR/CAS9 REVEALS AN IMPORTANT ROLE OF BMPRII
D U RIN G .........................
BMP9-INDUCED SMAD 1 /5 /8 PHOSPHORYLATION IN 1011/2 CELLS
.................................................
62
6.3. BMP9 DIRECTLY AFFECTS TUMOR CELLS AND INDUCES EPITHELIAL TO
MESENCHYMAL TRANSITION IN ...............
RENAL CELL CARCINOMA
CELLS.........................................................................................................................66
6.3.1. BMP9 RECEPTORS ARE EXPRESSED ON RENAL CELL CARCINOMA CELL LINES
AND MEDIATE
......................
BMP9-INDUCED SMAD 1 /5/8
PHOSPHORYLATION..............................................................................
66
6.3.2. BMP9 INDUCES EPITHELIAL TO MESENCHYMAL TRANSITION IN RENAL CELL
CARCINOMA CELLS
..................
IN
VITRO.................................................................................................................................................67
6.3.3. INCREASED EXPRESSION OF SNAIL MEDIATES CELL PROTECTIVE EFFECTS IN
VITRO B Y
...............................
REDUCING CASPASE ACTIVITY IN A-498
CELLS.......................................................................................
68
6.3.4. BMP9 DOES NOT DIRECTLY AFFECT PROLIFERATION OF A-498 IN CONTRAST
TO ET-1 AND IL-6...............69
6.4. INHIBITION OF BMP9 IN VIVO BY MAB BMP9-0093 DEMONSTRATES THAT BMP9
EXERTS DIFFERENT
...........
EFFECTS ON TUMOR
PROGRESSION.................................................................................................................71
6.4.1. INHIBITION OF BMP9 IN VIVO BY TREATMENT WITH MAB BMP9-0093 DELAYS
TU M O R........................
GROWTH OF A-498 RENAL CELL CARCINOMA XENOGRAFTS
......................................................................
71
6.4.2. THE VASCULAR PATTERN REMAINS UNAFFECTED BY MAB BMP9-0093, IN
CONTRAST TO .........................
TREATMENT WITH
B20.4.1...................................................................................................................71
6.4.3. VASCULAR COVERAGE IS INCREASED AFTER ANTI-BMP9 AND ANTI-VEGF
TREATM ENT
...........................
74
6.4.4. HIGHER VESSEL COVERAGE BY MAB BMP9-0093 TREATMENT IS ABLE TO
REDUCE VESSEL
...................
LEAKINESS............................................................................................................................................
76
6.4.5. EX VIVO ANALYSES DEMONSTRATE DECREASED PROLIFERATION AND ENHANCED
CASPASE
.....................
ACTIVITY IN MAB BMP9-0093 TREATED
TUMORS................................................................................
77
6.4.6. REDUCED ET-1 LEVELS IN ANTI-BMP9 TREATED TUMORS MAY CAUSE
DECREASED PROLIFERATION
....
79
6.4.7. TREATMENT WITH MAB BMP9-0093 DOES NOT INHIBIT THE INDUCTION OF
EMT IN VIVO
.................
80
6.5. COMPENSATION OF BMP9 NEUTRALIZATION BY
BMP10.............................................................................81
7.
DISCUSSION............................................................................................................................................................83
7.1. BMP9 REDUCES MIGRATION AND ATTACHMENT OF PERICYTES TO ENDOTHELIAL
CELLS
.................................
83
7.2. EFFECTS OF SELECTIVE INHIBITION OF BMP9 ON ANGIOGENESIS IN VIVO ARE
LIMITED TO VASCULAR.................
COVERAGE.....................................................................................................................................................
85
7.3. BMP9 SIGNALING IN RENAL CELL CARCINOMA CELL LINES INDUCES
EPITHELIAL TO MESENCHYMAL
...................
TRANSITION IN VITRO AND MEDIATES PROLIFERATIVE AND CELL PROTECTIVE
EFFECTS
.......................................
87
7.4. COMPENSATION OF BMP9 BY BMP10 CAN BE CONSIDERED AS A POTENTIAL
CAUSE FOR THE LA C K ...............
OF EFFECTS ON TUMOR
VASCULATURE.............................................................................................................
88
8.
CONCLUSION..........................................................................................................................................................
89
9.
REFERENCES.........................................................................................................................................................
91
10.
ABBREVIATIONS..................................................................................................................................................107
11.
DANKSAGUNG.....................................................................................................................................................
I L L
12. CURRICULUM
VITAE.............................................................................................................................................
113
|
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| language | English |
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| spelling | Brand, Verena Maria Hedi Verfasser aut The role of BMP9 signaling during tumor progression = Die Rolle des BMP9 Signalwegs in der Tumorprogression Verena Maria Hedi Brand Die Rolle des BMP9 Signalwegs in der Tumorprogression Erlangen ; Nürnberg 2017 114 Seiten Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Friedrich-Alexander-Universität Erlangen-Nürnberg 2017 Knochen-Morphogenese-Proteine (DE-588)4305932-6 gnd rswk-swf Tumorzelle (DE-588)4186433-5 gnd rswk-swf Tumorpromoter (DE-588)4332428-9 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Knochen-Morphogenese-Proteine (DE-588)4305932-6 s Tumorzelle (DE-588)4186433-5 s Tumorpromoter (DE-588)4332428-9 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030094211&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Brand, Verena Maria Hedi The role of BMP9 signaling during tumor progression = Die Rolle des BMP9 Signalwegs in der Tumorprogression Knochen-Morphogenese-Proteine (DE-588)4305932-6 gnd Tumorzelle (DE-588)4186433-5 gnd Tumorpromoter (DE-588)4332428-9 gnd |
| subject_GND | (DE-588)4305932-6 (DE-588)4186433-5 (DE-588)4332428-9 (DE-588)4113937-9 |
| title | The role of BMP9 signaling during tumor progression = Die Rolle des BMP9 Signalwegs in der Tumorprogression |
| title_alt | Die Rolle des BMP9 Signalwegs in der Tumorprogression |
| title_auth | The role of BMP9 signaling during tumor progression = Die Rolle des BMP9 Signalwegs in der Tumorprogression |
| title_exact_search | The role of BMP9 signaling during tumor progression = Die Rolle des BMP9 Signalwegs in der Tumorprogression |
| title_full | The role of BMP9 signaling during tumor progression = Die Rolle des BMP9 Signalwegs in der Tumorprogression Verena Maria Hedi Brand |
| title_fullStr | The role of BMP9 signaling during tumor progression = Die Rolle des BMP9 Signalwegs in der Tumorprogression Verena Maria Hedi Brand |
| title_full_unstemmed | The role of BMP9 signaling during tumor progression = Die Rolle des BMP9 Signalwegs in der Tumorprogression Verena Maria Hedi Brand |
| title_short | The role of BMP9 signaling during tumor progression |
| title_sort | the role of bmp9 signaling during tumor progression die rolle des bmp9 signalwegs in der tumorprogression |
| title_sub | = Die Rolle des BMP9 Signalwegs in der Tumorprogression |
| topic | Knochen-Morphogenese-Proteine (DE-588)4305932-6 gnd Tumorzelle (DE-588)4186433-5 gnd Tumorpromoter (DE-588)4332428-9 gnd |
| topic_facet | Knochen-Morphogenese-Proteine Tumorzelle Tumorpromoter Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030094211&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT brandverenamariahedi theroleofbmp9signalingduringtumorprogressiondierolledesbmp9signalwegsindertumorprogression AT brandverenamariahedi dierolledesbmp9signalwegsindertumorprogression |