Chemotactic factors underlying tumor infiltration by immunocompetent cells in human colorectal ...

144 Pages·2017·11.75 MB·English

Chemotactic factors underlying tumor infiltration by
immunocompetent cells in human colorectal cancer

Inauguraldissertation

Zur

Erlangung der Würde eines Doktors der Philosophie
vorgelegt der
Philosophisch-Naturwissenschaftlichen Fakultät
Der Universität Basel

von

Cremonesi Eleonora

aus Italien, Lodi

Basel, 2017

Original document stored on the publication server of the University of Basel
edoc.unibas.ch

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0
International License.
Genehmigt von der Philosophisch-Naturwissenschaftlichen
Fakultät

auf Antrag von

Prof. Dr. Christoph Hess
Prof. Dr. med. Giandomenica Iezzi
Prof. Dr. med. Alfred Zippelius

Basel, 22.03.2016

Prof. Dr. J Schibler
Dekan
During my PhD training I have extensively investigated the role of chemotactic factors
involved in the recruitment of beneficial immune cells in human colorectal cancer (CRC).
This thesis consists of an introduction highlighting the clinical relevance of immune cell
infiltration in CRC, and providing an overview of CRC microenvironment determinants and
their possible influence on immune cell migration. A complete description of methods used
and results obtained is then included. Finally, major findings and their implications are
reviewed in the discussion.
The results of this study have been included in a manuscript currently under preparation.
Beside my main research project, I have also been involved in additional projects of our group
addressing the prognostic significance of a number of immune cells markers in human CRC,
including interleukin-17A (IL-17), granulocyte macrophage colony-stimulating factor (GM-
CFS), tumor necrosis factor receptor superfamily, member 4 (TNFRSF4, also known as
OX40) and programmed death-ligand 1(PD-L1), and the development of innovative
tridimensional systems for culturing human CRC cells in vitro. Publications resulting from
these studies are included in the appendix.
To my family…
Summary
Colorectal cancer (CRC) is a common digestive tract malignancy and a major cause of cancer
mortality. Several studies have convincingly shown that CRC infiltration by
immunocompetent cells and, in particular, cytotoxic CD8+ T cells (CTLs), IFN-γ-producing
T-helper 1 cells (Th1), Foxp3+ regulatory T cells (Tregs), and CD16+ MPO+ neutrophils, is
significantly associated with prolonged patient survival. However, the chemotactic factors
driving these cell populations into the tumor site, their cellular sources and their
microenvironmental triggers remain to be elucidated.
During my PhD training I have investigated the chemokine/chemokine receptor network
promoting CRC infiltration by immune cells associated to favorable prognosis.
In particular, I addressed:
1. The expression of immune cell markers and their correlation with chemokine
expression in primary CRC tissues;
2. The identification of chemokine receptors relevant for CRC infiltration by beneficial
immune cells;
3. The chemokine sources in CRC;
4. The microenvironmental stimuli triggering chemokine production in CRC tissues;
5. The effects of chemokine production on immune cell recruitment into CRC.
The expression of a panel of genes encoding 39 chemokines and 7 markers specific for
defined immune cell populations was assessed by quantitative PCR array in 62 samples of
freshly excised primary CRC and autologous healthy colonic tissue. Correlations between
expression of chemokine genes and immune cell markers were then evaluated.
Furthermore, chemokine receptor profiles were analysed by flow cytometry on cell
suspensions obtained upon digestion of clinical specimens or on corresponding cell
populations from autologous peripheral blood. Based on chemokine receptor expression on
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tumor infiltrating cells and correlations between expression of chemokines and immune cell
markers, I could identify for each immune cell subset a putative “chemokine signature”:
1) CCL3, CCL5, CCL8 CXCL9, CXCL10 and CXCL12, associated with recruitment of
cytotoxic CTLs;
2) CCL5, CCL22, CXCL9, and CXCL12 correlating with infiltration by Th1;
3) CCL22 and CXCL12 potentially attracting Tregs;
4) CXCL2 and CXCL5 promoting chemotaxis of CD16+ MPO+ neutrophils.
I have further investigated potential chemokine sources and stimuli leading to chemokine
release within CRC tissues. I found that CRC cells purified from primary tumor specimens
express many of the genes encoding identified immune cell recruiting chemokines, including
CCL3, CCL5, CXCL2, CXCL5, CXCL9 and CXCL10. In vitro experiments showed that
chemokine production by CRC cells is triggered upon their exposure to microbial stimuli,
such as Toll-like receptor agonists, or CRC-associated bacteria, including Fusobacterium
nucleatum, Bacteroides Fragilis, Bacteroides vulgatus, and Escherichia Coli, thus suggesting
that components of the gut flora may critically influence chemokine production in CRC
tissues. This was indeed confirmed by “in vivo” experiments showing that chemokine gene
expression in xenografts, generated upon injection of human CRC cells in immunodeficient
NSG mice, appeared to be related to the presence of commensal bacteria. In particular,
chemokine gene expression levels in intracecal xenografts, were found to be ≥10 fold higher
as compared to those of subcutaneous xenografts, and they were significantly reduced upon
antibiotic treatment of tumor bearing mice.
Most importantly, a correlation between extent of immune cell infiltration and bacterial load
was also observed in human CRC samples. Indeed, CRC samples characterized by high
expression of chemokine and immune cell markers, displayed significantly higher bacterial
loads, as assessed by analysis of bacterial 16S ribosomal RNA, as compared to samples
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showing low chemokine expression and immune cell infiltration. In addition, a significant
correlation between bacterial load and expression of the Th1 marker IRF1, CCL3 and CCL5,
was also detected.
Our in vitro and in vivo results cumulatively suggest that bacteria-induced chemokine
production by tumor cells may lead to tumor infiltration by beneficial immune cells.
Consistent with this hypothesis, in preliminary “in vitro” experiments, I found that
supernatants of bacteria-stimulated CRC cells promote chemotaxis of CTLs and Th1 cells to a
higher extent than untreated tumor cells.
Additional “in vivo” studies are clearly warranted. In particular, I plan to evaluate
intratumoral recruitment of CRC-derived CTLs and Th1 cells upon adoptively transfer into
intracecal xenografts-bearing mice.
Bacterial species or strains mostly contributing to high chemokine expression and immune
cell infiltration in human CRC samples also remain to be identified. Microbiome analysis of
CRC samples characterized by high or low immune cell infiltration might be envisaged in
future studies.
The results of the present work together with the proposed additional studies will contribute to
the understanding of the interplay occurring between gut flora and immune system in CRC,
and may pave the way towards innovative treatments aimed at modifying the gut flora in
order to promote CRC infiltration by beneficial immune cell subsets.

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Table of content

Summary ........................................................................................................................................ 1
I. INTRODUCTION ......................................................................................................................... 6
1.Human colorectal cancer .................................................................................................................. 7
1.1 Epidemiology and etiopathogenesis .......................................................................................... 7
1.2 Staging and prognosis................................................................................................................ 9
1.3 Current treatment guidelines ................................................................................................... 10
2.CRC immune contexture ................................................................................................................ 11
2.1 Definition of immune contexture ............................................................................................ 11
2.2 Impact of immune contexture in CRC ..................................................................................... 12
3. Immune cell trafficking in CRC .................................................................................................... 13
3.1 Chemokines and chemokine receptors .................................................................................... 13
3.2 Role of chemokines in leukocyte trafficking ........................................................................... 17
3.3 Gut homing receptors .............................................................................................................. 17
3.4 Prognostic significance of chemokine expression in colorectal cancer ................................... 18
4. Pathophysiology of CRC microenvironment ................................................................................ 18
4.1 Physiology of normal colonic mucosa .................................................................................... 18
4.2 The gut microbiota .................................................................................................................. 20
4.3 Interactions between gut flora and normal colonic epithelium ............................................... 21
4.4 Physiopathology of gut epithelium in CRC ............................................................................. 25
II. AIM OF THE STUDY ................................................................................................................ 28
III. MATERIALS AND METHODS ................................................................................................ 30
1. Clinical specimen collection and processing ............................................................................. 31
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2. Cell lines .................................................................................................................................... 31
3. Bacteria ...................................................................................................................................... 31
4. Flow cytometry .......................................................................................................................... 32
5. Real-time reverse transcription PCR assays .............................................................................. 33
6. CRC cell stimulation with TLR agonists and bacteria .............................................................. 34
7. Migration assay ......................................................................................................................... 35
8. Generation of CRC xenografts .................................................................................................. 35
9. Statistical analysis ..................................................................................................................... 36
IV. RESULTS ..................................................................................................................................... 37
1. Expression of immune cell markers in primary CRC ................................................................ 38
2. Chemokine gene expression in primary CRC ........................................................................... 41
3. Correlations between expression of immune cell markers and chemokine genes in CRC ........ 41
4. Chemokine receptor expression on CRC infiltrating beneficial immune cells ......................... 42
5. Chemokine signatures underling immune cell recruitment in CRC .......................................... 47
6. Chemokine sources in primary CRC ......................................................................................... 47
7. Effects of microbial stimulation on chemokine production by CRC cells in vitro.................... 49
8. Effects of microbial stimulation on chemokine production in vivo .......................................... 52
9. Effects of chemokine production on immune cell recruitment into CRC tissues ...................... 54
V. DISCUSSION AND OUTLOOK ............................................................................................... 56
1. Discussion ..................................................................................................................................... 57
2. Outlook .......................................................................................................................................... 62
VI. BIBLIOGRAPHY ....................................................................................................................... 64
VII. APPENDIX .................................................................................................................................. 74

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I. INTRODUCTION

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