Oncolytic therapy with vaccinia virus GLV-1h68 [Elektronische Ressource] : comparative microarray analysis of infected xenografts and human tumor cell lines / vorgelegt von Andrea Worschech

Viktoria Szirmai

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 38
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

Oncolytic Therapy with Vaccinia Virus
GLV-1h68
- Comparative Microarray Analysis of Infected
Xenografts and Human Tumor Cell Lines-





Dissertation

Zur Erlangung des naturwissenschaftlichen Doktorgrades der
Bayerischen Julius-Maximilians-Universität Würzburg


vorgelegt von

Andrea Worschech
aus Bad Brückenau


Würzburg, Januar 2010























meiner Familie












Eingereicht am: ............................................................................


Mitglieder der Promotionskommission:
Vorsitzender: Prof. Dr. T. Dandekar
Gutachter: Prof. Dr. A. A. Szalay
Gutachter: Prof. Dr. G. Krohne


Tag des Promotionskolloquiums: .................................................

Doktorurkunde ausgehändigt am: ..............................................


Erklärung

Hiermit erkläre ich, dass die vorliegende Arbeit selbstständig und nur
unter der Verwendung der angegebenen Quellen und Hilfsmittel
angefertigt wurde.

Weiterhin versichere ich, dass die Dissertation bisher nicht in gleicher
oder ähnlicher Form in einem anderen Prüfungsverfahren vorgelegen hat,
und ich bisher keine akademischen Grade erworben oder zu erwerben
versucht habe.


Würzburg, Januar 2010



Andrea Worschech





















Die Neugier steht immer am Anfang eines Problems, das
gelöst werden will.

Galileo Galilei











Table of Contents


1 SUMMARY .................................................................................................................................................. 3
2 ZUSAMMENFASSUNG ............................. 9
3 INTRODUCTION ..................................................................................................................................... 15
3.1 THE COMPLEXITY OF CANCER BIOLOGY ......................... 15
3.2 TISSUE REJECTION REQUIRES A SWITCH FROM A CHRONIC TO AN ACUTE INFLAMMATION – REVIEW
1 .................................................................................................................................................... 17
3.3 THE IMMUNOLOGIC ASPECTS OF POXVIRUS ONCOLYTIC THERAPY – REVIEW 2 .............................. 25
3.4 CONSTITUTIVE ACTIVATION OF INTERFERON-SIGNALING PATHWAYS IN HUMAN CANCERS REVEALS
TWO DISTINCT TAXONOMIES .......................................................................................................... 34
3.4.1 Heterogeneous inflammatory phenotype in various cancer types ........................................ 34
3.4.2 Antiviral state in pancreatic cancers leads to various permissivity to Adenovirus replication
in vitro........................................... 35
3.5 REFERENCES .................................................................. 36
4 AIM OF THE STUDY............................................................... 37
5 RESULTS ................................................................................................................... 39
5.1 SIGNATURES ASSOCIATED WITH REJECTION OR RECURRENCE IN HER-2/NEU-POSITIVE MAMMARY
TUMORS - RESEARCH ARTICLE 1 .................................................................................................... 39
5.2 SYSTEMIC TREATMENT OF XENOGRAFTS WITH VACCINIA VIRUS GLV-1H68 REVEALS THE
IMMUNOLOGIC FACET OF ONCOLYTIC THERAPY – RESEARCH ARTICLE 2 ....... 52
5.3 IN VITRO PERMISSIVITY OF 75 HUMAN CANCER CELL LINES TO ADENOVIRUS 5 AND ONCOLYTIC
VACCINIA VIRUS – MANUSCRIPT FOR RESEARCH ARTICLE 3............................................................ 76
5.3.1 Background .......................................................................................... 77
5.3.2 Materials and Methods ......................................... 80
5.3.3 Results .. 83
5.3.4 Discussion .......................................................... 104
5.3.5 References ................................................................ 107
6 DISCUSSION ........................................................................... 110
6.1 REFERENCES ................................ 116
7 LIST OF ABBREVIATIONS ................................................................................. 118
8 PUBLICATIONS ..................................................................... 119
9 ACKNOWLEDGEMENTS .................................................... 120


2
Summary
1 Summary

It is commonly accepted that chronic infections and inflammations may lead to
mutagenesis of normal tissue and eventually facilitate development of cancerous
lesions. A well-described example is cervical cancer which often is associated with
an infection with human papilloma virus 16 or 18. On the contrary, newly gained
insight into tumor biology has shown that the host’s immune system can be a helpful
tool for tumor therapy when triggered and stimulated accordingly. In this case, the
chronic fostering inflammation in the tumor microenvironment is changed into an
acute one and tissue rejection can occur. One such stimulus are oncolytic viruses
that can be utilized to target manifested cancers and promote tumor regression due
to their selective ability to infect and kill human tumor cells.

Aim of this thesis was to study the contribution of the host`s immune system
during tumor regression. A wild-type rejection model was studied in which tumor
regression is mediated through an adaptive, T cell host response (Research article
1). Additionally, the relationship between VACV infection and cancer rejection was
assessed by applying organism-specific microarray platforms to infected and non-
infected xenografts. It could be shown that tumor rejection in this nude mouse model
was orchestrated solely by the host`s innate immune system without help of the
adaptive immunity. In a third study the inflammatory baseline status of 75 human
cancer cell lines was tested in vitro which was correlated with the susceptibility to
VACV and Adenovirus 5 (Ad5) replication of the respective cell line (Manuscript for
Research article 3).

Research article 1
HER2/neu (also known as ErbB-2) stands for "Human Epidermal growth factor
Receptor 2" and is a proto-oncogene whose overexpression is associated with worse
prognosis and increased relapse rate in breast cancer patients. Wild-type FVB mice
are capable of rejecting neu-overexpressing mammary carcinomas (MMC) within
three weeks because of specific recognition of rat neu protein by their T cells as
opposed to their transgenic counterparts, FVBN202, which tolerate rat neu protein
and fail to reject MMC. After subcutaneous MMC inoculation all mice rejected MMC

3
Summary
within three weeks after the challenge. However, a fraction of these animals (eight
out of 15 mice) developed recurrent tumors at the site of inoculation and these
relapsed tumors had lost neu expression under immune pressure. Total RNA from
both FVB and FVBN202 carrier mice as well as total RNA from spontaneous tumor in
FVBN202 mice was hybridized to 36k whole genome mouse arrays and statistical
analysis was performed. The top categories of genes that were up-regulated in
primary rejected MMC tumors were cytokine-cytokine interaction, mitogen-activated
protein kinase signaling, cell adhesion–related transcripts and axon guidance, T-cell
receptor, STAT and TLR signaling pathways. NK cell–mediated cytotoxicity and
calcium signaling pathways were also enriched in up-regulated genes. In contrast,
very little evidence of immune activation could be observed in either category of
nonregressing tumors, suggesting that lack of immune rejection is due to absent or
severely hampered immune responses in the tumor microenvironment independent
of the mechanisms leading to this resistance. From this analysis it became clear that
T-cell infiltration into tumors was associated with activation of various pathways
leading to the expression of IFN-α, IFN-γ, and several ISGs, including IRF4, IRF6,
and STAT2. In addition, several cytotoxic molecules were overexpressed, including
calgranulin A, calgranulin B, and granzyme B, all of them representing classic
markers of effector T-cell activation. Although the transcriptional patterns
differentiating regressing from nonregressing tumors were striking and in many ways
representative of previous observations in humans differences among MMC tumors
nonregressing in FVBN202 mice and those relapsing after regression in FVB mice
were subtle. The paradoxical relationship between adaptive immune responses
against cancer antigens and rejection or persistence of antigen bearin