Influence of oncolytic vaccinia viruses on metastases of human and murine tumors: = Einfluss von onkolytischen Vaccinia Viren auf Metastasen von humanen und murinen Tumoren
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| Language: | English |
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| Subjects: | |
| Online Access: | Volltext Inhaltsverzeichnis |
| Physical Description: | 134 Blätter Illustrationen, Diagramme |
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| 245 | 1 | 0 | |a Influence of oncolytic vaccinia viruses on metastases of human and murine tumors |b = Einfluss von onkolytischen Vaccinia Viren auf Metastasen von humanen und murinen Tumoren |c vorgelegt von Juliane Meir aus Schleiz |
| 246 | 1 | 1 | |a Einfluss von onkolytischen Vaccinia Viren auf Metastasen von humanen und murinen Tumoren |
| 264 | 1 | |a Würzburg |c Juli 2015 | |
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| adam_text | JULIUS-MAXIMILIANS-UNIVERSITAET WUERZBURG
FAKULTAET FUER BIOLOGIE
INFLUENCE OF ONCOLYTIC VACCINIA VIRUSES ON
METASTASES OF HUMAN AND MURINE TUMORS
DISSERTATION
ZUR ERLANGUNG DES NATURWISSENSCHAFTLICHEN DOKTORGRADES DER
JULIUS-MAXIMILIANS-UNIVERSITAET WUERZBURG
VORGELEGT VON JULIANE MEIR
AUS SCHLEIZ
WUERZBURG, JULI 2015
SUMMARY.................................................................................................................................................
1
ZUSAMMENFASSUNG.................................................................................................................................
4
1
INTRODUCTION....................................................................................................................................7
1.1
CANCER.......................................................................................................................................7
1.2 FORMATION AND MEANING OF LYMPH NODE M
ETASTASES...........................................................8
1.2.1 THE LYMPHATIC SYSTEM
----------------------------------------------------------------------------
8
1.2.2 THE METASTATIC
PROCESS-------------------------------------------------------------------------10
1.2.3 CANCER STEM CELLS AND THE EPITHELIAL-MESENCHYMAL TRANSITION ARE
INVOLVED IN THE
METASTATIC
PROCESS-------------------------------------------------------------------------------12
1.2.4 METASTASES AS A THERAPEUTIC TARG ET
----------------------------------------------------------
15
1.3 VACCINIA
VIRUS.........................................................................................................................
16
1.3.1 VACCINIA VIRUS AS AN ONCOLYTIC A G EN T
---------------------------------------------------------
16
1.3.2
TAXONOMY-----------------------------------------------------------------------------------------
17
1.3.3
MORPHOLOGY---------------------------------------------------------------------------------------
18
1.3.4 REPLICATION
CYCLE----------------------------------------------------------------------------------19
1.3.5 THE VACV ISOLATE LIVP 1.1.1 AND RECOMBINANT VACCINIA VIRUS
CONSTRUCT GLV-LH68 21
1.4 TWO TUMOR MODELS AS EXAMPLES OF METASTATIC CANCER
......................................................
23
1.4.1 CERVICAL CARCINOMA
------------------------------------------------------------------------------
23
1.4.2
MELANOMA-----------------------------------------------------------------------------------------
27
1.5 AIMS OF THIS W
ORK................................................................. 30
2
MATERIAL........................................................................................................................................
33
2.1 CHEMICALS AND
ENZYMES........................................................................................................
33
2.2 BUFFERS AND
SOLUTIONS............................................................................................................
35
2.3 USED CELL LINES AND CELL CULTURE M
EDIA.................................................................................36
2.3.1 CELL
LINES-------------------------------------------------------------------------------------------
36
2.3.2 CELL CULTURE M EDIA
-------------------------------------------------------------------------------
36
2.4 SYNTHETIC
OLIGONUCLEOTIDES...................................................................................................37
2.5
KITS..........................................................................................................................................37
2.6
ANTIBODIES..............................................................................................................................
37
2.6.1 ANTIBODIES FOR
HISTOLOGY------------------------------------------------------------------------37
2.6.2 ANTIBODIES FOR FACS
ANALYSIS-------------------------------------------------------------------
38
2.7 RECOMBINANT VIRAL CONSTRUCT GLV-LH68 AND THE LIVP 1.1.1
CLONE.....................................38
2.8 LABORATORY
ANIMALS...............................................................................................................
38
2.9 CONSUMPTION
ITEMS...............................................................................................................
39
2.10
EQUIPMENT.............................................................................................................................
40
2.11
SOFTWARE.................................................................................................................................
41
3
METHODS.........................................................................................................................................
42
3.1 CELL BIOLOGICAL
METHODS.........................................................................................................
42
3.1.1 CULTURING OF ADHERENT GROWING CELLS
---------------------------------------------------------
42
3.1.2 CELL COUNTING VIA NEUBAUER HEMOCYTOMETER
----------------------------------------------
43
3.1.3 FREEZING AND THAWING OF
CELLS------------------------------------------------------------------
43
3.2 VIROLOGICAL M
ETHODS..............................................................................................................
43
3.2.1 INFECTION OF DIFFERENT CELLS LINES WITH VACCINIA
VIRUS--------------------------------------43
3.2.2 REPLICATION ASSAY
--------------------------------------------------------------------------------
44
3.2.3 STANDARD VIRAL PLAQUE
ASSAY--------------------------------------------------------------------44
3.2.4 CELL VIABILITY ASSAY
-------------------------------------------------------------------------------
45
3.3 MOUSE
EXPERIMENTS..............................................................................................................
46
3.3.1 SUBCUTANEOUS (S.C.) IMPLANTATION OF TUMOR
CELLS------------------------------------------46
3.3.2 FOOTPAD (F.P.) IMPLANTATION OF B16F10 MELANOMA CELLS
---------------------------------
46
3.3.3 ANESTHESIA OF MICE WITH
ISOFLURANE---------------------------------------------------------- 47
3.3.4 VACCINIA VIRUS INJECTION IN TUMOR-BEARING M
ICE--------------------------------------------47
3.3.4.1 INTRAVENOUS (I.V.) VIRUS INJECTION
.........................
47
3.3.4.2 INTRATUMORAL (I.T.) VIRUS
INJECTION..........................................................................47
3.3.5 INJECTION OF
IGG-----------------------------------------------------------------------------------
47
3.3.6 FLUORESCENCE IMAGING OF TUMORS AND M
ETASTASES-----------------------------------------48
3.4 ANALYSIS OF VIRAL TITERS IN TUMORS AND M
ETASTASES...............................................................
48
3.5 HISTOLOGICAL ANALYSIS OF TUMORS, LYMPH NODES AND ORGANS
................................................
49
3.5.1 CRYOSECTIONING OF TUMORS AND LYMPH NODES
-----------------------------------------------
49
3.5.2 IMMUNOHISTOCHEMICAL STAINING OF
CRYOSECTIONS-------------------------------------------49
3.5.3 AGAROSE SECTIONS OF LYMPH NODES AND TU M O
RS---------------------------------------------49
3.5.4 IMMUNOHISTOCHEMICAL STAINING OF AGAROSE
SECTIONS--------------------------------------50
3.5.5 ANALYSES OF DIGITAL FLUORESCENCE IMAGES
-----------------------------------------------------
50
3.5.5.1 QUANTIFICATION OF SECTION
LABELING........................................................................
50
3.5.5 2 MEASUREMENT OF FLUORESCENCE
INTENSITY..............................................................50
3.5 5.3 DETERMINATION OF LYMPH AND BLOOD VESSEL DENSITY
.............................................
51
3.6 RNA ISOLATION FROM TUMORS, METASTASES AND ORGANS AND ANALYSIS BY
RT-PCR..................51
3.6.1 RNA
ISOLATION-------------------------------------------------------------------------------------
51
3.6.2 DNA DIGESTION AND RNA CONCENTRATION M EASUREM
ENT-----------------------------------52
3.6.3 CDNA
SYNTHESIS-----------------------------------------------------------------------------------
52
3.6.4 POLYMERASE CHAIN REACTION
(PCR)------------------------------------------------------------- 53
3.6.5 DNA AGAROSE GEL
ELECTROPHORESIS-------------------------------------------------------------
54
3.7 PREPARATION OF SINGLE-CELL SUSPENSIONS AND FACS
ANALYSIS................................................55
3.7.1 PREPARATION OF SINGLE-CELL SUSPENSIONS
------------------------------------------------------
55
3.7.2 STAINING OF IMMUNE CELLS IN TUMOR AND LYMPH NODE SINGLE-CELL
SUSPENSIONS
--------
55
3.7.3 FLOW CYTOMETRIC
ANALYSES-----------------------------------------------------------------------56
3.7.4 DETERMINING THE GROWTH RATE OF CELLS OF A SINGLE-CELL SUSPENSION
---------------------
56
3.8 STATISTICAL
ANALYSIS..................................................................................................................56
RESULTS..........................................................................................................................................
57
4.1 CHARACTERIZATION OF METASTASES FORMATION AND VIROTHERAPY IN THE
HUMAN C33A
CERVICAL CANCER M
ODEL...........................................................................................................
57
4.1.1 ENLARGED LYMPH NODES AFTER SUBCUTANEOUS IMPLANTATION OF C33A
CELLS--------------57
4.1.2 COMPARISON OF GLV-LH68 INFECTION OF C33A AND C33A-RFP CELLS IN
VITRO-------------59
4.1.2.1 INSERTION OF RFP DOES NOT IMPAIR THE REPLICATION OF GLV-LH68 IN
C33A-RFP
CELLS.........................................................................................................................
59
4.1.2.2 INSERTION OF RFP DOES NOT IMPAIR THE GLV-LH68-MEDIATED LYSIS OF
C33A-RFP
CELLS
........................................................................
61
4.1.3 MONITORING OF TUMOR AND LYMPH NODE METASTASES FORMATION AFTER
SUBCUTANEOUS IMPLANTATION OF C33A-RFP CELLS
--------------------------------------------
62
4.1.3.1 TUMOR VOLUME IS INCREASING FROM WEEK TO WEEK AFTER IMPLANTATION
OF
C33A-RFP
CELLS.......................................................................................................
63
4.1.3.2 LUMBAR AND RENAL LYMPH NODE METASTASES APPEARED AND INCREASED
OVER
TIME ACCORDING TO TUMOR VOLUM
E.........................................................................64
4.1.3.3 LYMPH NODES AT DISTANT SITES CAN REVEAL RFP SIGNAL
............................................
65
4.1.3.4 TUMOR CELLS MIGRATE VIA THE LYMPHATIC AND HAEMATOGENOUS RO U TE
.................
67
4.1.3.5 RFP SIGNAL IS FOUND IN LUNGS AND KIDNEYS CONFIRMING THE
HAEMATOGENOUS
SPREAD OF TUMOR
CELLS............................................................................................69
4.1.3.6 HISTOLOGICAL ANALYSIS OF LUNGS AND KIDNEYS REVEALING THE ORIGIN
OF RFP SIGNAL. 70
4.1.4 COMBATING TUMORS AND METASTASES WITH THE ONCOLYTIC VACCINIA VIRUS
GLV-LH68* 72
4.1.4.1 INFLUENCE OF VACCINIA VIRUS GLV-LH68 ON C33A-RFP TU M O RS
...............................
72
4.1.4.2 INFLUENCE OF GLV-1H68 ON C33A-RFP LYMPH NODE M ETASTASES
.........................
74
4.1.4.3 REGRESSION OF RFP SIGNAL IN KIDNEYS AFTER VIRUS ADMINISTRATION
........................
76
4.1.5 TUMOR AND LYMPH NODE COLONIZATION BY GLV-LH68
--------------------------------------
77
4.1.6 HISTOLOGICAL AND FACS ANALYSIS OF C33A TUMORS AND LYMPH N O D ES
--------------------
78
4.1.6.1 HIGHER NECROTIC AREAS IN TUMORS COMPARED TO LYMPH
NODES..............................79
4.1.6.2 HIGHER PROLIFERATIVE STATUS IN LYMPH NODE METASTASES COMPARED TO
PRIMARY
TU M O
R.....................................................................................................................
79
4.1.6.3 IMMUNE CELL STATUS IN TUMORS AND LYMPH NODE M ETASTASES
.............................
81
4.1.6.4 LYMPH AND BLOOD VESSEL DENSITY IN TUMORS AND LYMPH NODE M
ETASTASES
.......
82
4.1.6.5 VASCULAR PERMEABILITY IN TUMORS AND LYMPH NODE M ETASTASES
........................
84
4.2 CHARACTERIZATION OF METASTASES FORMATION AND VIROTHERAPY IN THE
MURINE B16F10
MELANOMA
MODEL...................................................................................................................85
4.2.1 COMPARISON OF GLV-LH68 AND LIVP 1.1.1 INFECTION OF B16F10 CELLS
IN VITRO
----------
85
4.2.1.1 REPLICATION OF GLV-LH68 AND LIVP 1.1.1 IN B16F10 CELLS
.....................................
85
4.2.1.2 LIVP 1.1.1 LYSES B16F10 CELLS MORE EFFICIENTLY THAN
GLV-LH68...........................87
4.2.2
IN VIVO
EXPERIMENTS
-----------------------------------------------------------------------------
89
4.2.2.1 COMPARISON OF DIFFERENT IMPLANTATION TECHNIQUES: SUBCUTANEOUS VS
FOOTPAD...................................................................................................................
89
4.2.2.2 COMPARISON OF FOOTPAD IMPLANTATION IN FOXNL NUDE AND C57BL/6
MICE
..........
92
4.2.2.3 CHARACTERIZATION OF TUMOR AND LYMPH NODE METASTASES FORMATION
AFTER
FOOTPAD IMPLANTATION OF B16F10 CELLS
.................................................................
93
4.2.2.4 COMPARISON OF DIFFERENT VIRUS ADMINISTRATION ROUTES: INTRAVENOUS
VS.
INTRATUMORAL..........................................................................................................
96
DISCUSSION.....................................................................................................................................98
5.1 CHARACTERIZATION OF METASTASES FORMATION AND VIROTHERAPY IN THE
HUMAN C33A
CERVICAL CANCER M ODEL
..................
.............
.
.........................................................................
98
5.1.1 THE HUMAN CERVICAL CANCER CELL LINE C33A AS A NEW METASTATIC
MODEL----------------98
5.1.2 INSERTION OF RFP IN C33A CELLS HAS NO EFFECTS ON INFECTION,
REPLICATION AND CELL
LYSIS BY GLV-LH68
--------------------------------------------------------------------------------
99
5.1.3 C33A-RFP CELLS FORM TUMORS AND MIGRATE TO LYMPH NODES AFTER
SUBCUTANEOUS
IMPLANTATION-------------------------------------------------------------------------------------100
5.1.4 C33A CELLS USE THE LYMPHATIC AS WELL AS THE HAEMATOGENOUS ROUTE OF
MIGRATION 101
5.1.5 SUCCESSFUL THERAPY OF PRIMARY C33A-RFP TUMORS AND METASTASES WITH
THE
ONCOLYTIC VACCINIA VIRUS
GLV-LH68-----------------------------------------------------------103
5.1.6 PREFERENTIAL COLONIZATION OF PRIMARY TUMORS COMPARED TO LYMPH NODE
METASTASES BY GLV-LH68
---------------------------------------------------------------------
104
5.1.7 HISTOLOGICAL ANALYSIS REVEAL THE DIFFERENCES BETWEEN PRIMARY TUMOR
AND LYMPH
NODE M
ETASTASES-------------------------------------------------------------------------------
105
5.1.8
CONCLUSION----------------------------------------------------------------------------------------107
5.2 B16F10 AS A PROMISING TOOL TO ANALYZE ONCOLYTIC VIRUS THERAPY IN AN
IMMUNE
COMPETENT MOUSE M
ODEL...................................................................................................
108
5.2.1 LIVP 1.1.1 IS MORE EFFICIENTLY INFECTING, REPLICATING IN AND
LYSING B16F10 CELLS
COMPARED TO GLV-LH68 IN CELL
CULTURE------------------------------------------------------108
5.2.2 IMPLANTATION OF B16F10 CELLS INTO THE FOOTPAD OF IMMUNOCOMPETENT
C57BL/6
MICE AS A MODEL TO STUDY M
ETASTASES------------------------------------------------------109
5.2.3 FORMATION OF TUMORS AND SENTINEL LYMPH NODE METASTASES AFTER
IMPLANTATION OF
B16F10 CELLS IN THE FOOTPAD
------------------------------------------------------------------
110
5.2.4 INTRATUMORAL VIRUS INJECTION LEADS TO HIGHER TITERS IN TUMORS AND
LYMPH NODE
METASTASES COMPARED TO INTRAVENOUS INJECTION
-----------------------------------------
111
5.2.5 O
UTLOOK-------------------------------------------------------------------------------------------112
6
REFERENCES..................................................................................................................................113
7
APPENDIX.....................................................................................................................................
127
7.1
ABBREVIATIONS.......................................................................................................................
127
7.2
DANKSAGUNG.........................................................................................................................
130
7.3 EIDESSTATTLICHE
ERKLAERUNG....................................................................................................
132
7.4
LEBENSLAUF............................................................................................................................
133
7.5
PUBLIKATIONSLISTE...................................................................................................................134
|
| any_adam_object | 1 |
| author | Meir, Juliane 1986- |
| author_GND | (DE-588)1116286866 |
| author_facet | Meir, Juliane 1986- |
| author_role | aut |
| author_sort | Meir, Juliane 1986- |
| author_variant | j m jm |
| building | Verbundindex |
| bvnumber | BV043827907 |
| collection | ebook |
| ctrlnum | (OCoLC)962085095 (DE-599)BVBBV043827907 |
| dewey-full | 610 |
| dewey-hundreds | 600 - Technology (Applied sciences) |
| dewey-ones | 610 - Medicine and health |
| dewey-raw | 610 |
| dewey-search | 610 |
| dewey-sort | 3610 |
| dewey-tens | 610 - Medicine and health |
| discipline | Medizin |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV043827907 |
| illustrated | Illustrated |
| indexdate | 2024-07-10T07:36:09Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-029238759 |
| oclc_num | 962085095 |
| open_access_boolean | 1 |
| owner | DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-12 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 DE-860 DE-2174 |
| owner_facet | DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-12 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 DE-860 DE-2174 |
| physical | 134 Blätter Illustrationen, Diagramme |
| psigel | ebook |
| publishDate | 2015 |
| publishDateSearch | 2015 |
| publishDateSort | 2015 |
| record_format | marc |
| spelling | Meir, Juliane 1986- Verfasser (DE-588)1116286866 aut Influence of oncolytic vaccinia viruses on metastases of human and murine tumors = Einfluss von onkolytischen Vaccinia Viren auf Metastasen von humanen und murinen Tumoren vorgelegt von Juliane Meir aus Schleiz Einfluss von onkolytischen Vaccinia Viren auf Metastasen von humanen und murinen Tumoren Würzburg Juli 2015 134 Blätter Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier Dissertation Julius-Maximilians-Universität Würzburg 2015 Zusammenfassung in deutscher und englischer Sprache Vaccinia-Virus (DE-588)4187306-3 gnd rswk-swf Krebs Medizin (DE-588)4073781-0 gnd rswk-swf Metastase (DE-588)4125915-4 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Krebs Medizin (DE-588)4073781-0 s Metastase (DE-588)4125915-4 s Vaccinia-Virus (DE-588)4187306-3 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-118530 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-118530 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=029238759&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Meir, Juliane 1986- Influence of oncolytic vaccinia viruses on metastases of human and murine tumors = Einfluss von onkolytischen Vaccinia Viren auf Metastasen von humanen und murinen Tumoren Vaccinia-Virus (DE-588)4187306-3 gnd Krebs Medizin (DE-588)4073781-0 gnd Metastase (DE-588)4125915-4 gnd |
| subject_GND | (DE-588)4187306-3 (DE-588)4073781-0 (DE-588)4125915-4 (DE-588)4113937-9 |
| title | Influence of oncolytic vaccinia viruses on metastases of human and murine tumors = Einfluss von onkolytischen Vaccinia Viren auf Metastasen von humanen und murinen Tumoren |
| title_alt | Einfluss von onkolytischen Vaccinia Viren auf Metastasen von humanen und murinen Tumoren |
| title_auth | Influence of oncolytic vaccinia viruses on metastases of human and murine tumors = Einfluss von onkolytischen Vaccinia Viren auf Metastasen von humanen und murinen Tumoren |
| title_exact_search | Influence of oncolytic vaccinia viruses on metastases of human and murine tumors = Einfluss von onkolytischen Vaccinia Viren auf Metastasen von humanen und murinen Tumoren |
| title_full | Influence of oncolytic vaccinia viruses on metastases of human and murine tumors = Einfluss von onkolytischen Vaccinia Viren auf Metastasen von humanen und murinen Tumoren vorgelegt von Juliane Meir aus Schleiz |
| title_fullStr | Influence of oncolytic vaccinia viruses on metastases of human and murine tumors = Einfluss von onkolytischen Vaccinia Viren auf Metastasen von humanen und murinen Tumoren vorgelegt von Juliane Meir aus Schleiz |
| title_full_unstemmed | Influence of oncolytic vaccinia viruses on metastases of human and murine tumors = Einfluss von onkolytischen Vaccinia Viren auf Metastasen von humanen und murinen Tumoren vorgelegt von Juliane Meir aus Schleiz |
| title_short | Influence of oncolytic vaccinia viruses on metastases of human and murine tumors |
| title_sort | influence of oncolytic vaccinia viruses on metastases of human and murine tumors einfluss von onkolytischen vaccinia viren auf metastasen von humanen und murinen tumoren |
| title_sub | = Einfluss von onkolytischen Vaccinia Viren auf Metastasen von humanen und murinen Tumoren |
| topic | Vaccinia-Virus (DE-588)4187306-3 gnd Krebs Medizin (DE-588)4073781-0 gnd Metastase (DE-588)4125915-4 gnd |
| topic_facet | Vaccinia-Virus Krebs Medizin Metastase Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-118530 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029238759&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT meirjuliane influenceofoncolyticvacciniavirusesonmetastasesofhumanandmurinetumorseinflussvononkolytischenvacciniavirenaufmetastasenvonhumanenundmurinentumoren AT meirjuliane einflussvononkolytischenvacciniavirenaufmetastasenvonhumanenundmurinentumoren |