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Linköping University medical dissertations, No. 1149
Macrophage antigen expression in breast
and colorectal cancers
A consequence of macrophage - tumour cell fusion?
Ivan Shabo
Division of Surgery
Department of Clinical and Experimental Medicine
Faculity of Health Science, Linköping University
SE-58185 Linköping, Sweden.
Linköping 2009
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SUPERVISOR
Professor Joar Svanvik
Division of Surgery,
Department of Clinical and Experimental Medicine,
Faculty of Health Sciences, SE-581 85, Linköping,
Sweden
Copyright © Ivan Shabo 2009
Email: [email protected]
Front cover is designed by Ivan Shabo and illustrates the modell of macrphage-cancer cell fusion.
Printed by LiU-Tryck, Linköping, 2009
ISBN: 978-91-7393-545-6
ISSN 0345-0082
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”Människosjälen har två krafter,
en aktiv och en kontemplativ.
Med den förra går man framåt.
Med den senare kommer man fram.”
St. Augustinus (354-430)
To my beloved mother Sabiha, Josefin, To my beloved mother Sabiha, Josefin, To my beloved mother Sabiha, Josefin, To my beloved mother Sabiha, Josefin,
JulianJulianJulianJulian,,,, Sivan Sivan Sivan Sivan and the memory of mand the memory of mand the memory of mand the memory of my y y y
fatherfatherfatherfather....
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ABSTRACT
Carcinogenesis is a sophisticated biological process consisting of a series of progressive
changes in somatic cells from premalignant to malignant phenotype. Despite the vast
information available about cancer cells, the origin of cancer and cause of metastasis still
remain enigmatic. The hypothesis of cell fusion is one of several models explaining the
evolution of neoplasia into clinically significant cancer. This theory states that cancer cells
through heterotypic fusion with host cells generate hybrids expressing traits from both
parental cells, and acquire metastatic potentials and growth-promoting properties. The cell
fusion theory is still unproven and speculative, but cell fusion is a common biological process
in normal tissue. Accumulated evidence shows that macrophage-cancer cell fusion occurs in
vitro and in vivo and produces hybrids with metastatic potential, but the clinical significant of
cell fusion is unclear. The aim of this thesis is to test this hypothesis in clinical patient
materials and to explore the clinical significance of macrophage phenotype traits in solid
tumours.
Paraffin-embedded cancer and normal tissue specimens from patients with breast cancer
(n=133) and colorectal cancer (two different patient materials with totally 240 patients) were
immunostained for the macrophage-specific antigen, CD163. The expression of CD163 was
tested in relation to macrophage infiltration and tumour stage, survival time, irradiation, DNA
ploidy, cancer cell proliferation and apoptosis.
Phenotypic macrophage traits, such as the expression of CD163, were seen in both breast and
colorectal cancers, and were correlated to advanced tumour stages and poor survival. CD163
expression was more frequent in rectal cancer after irradiation and was associated with
decreased apoptosis. Cancer cell proliferation was correlated to both macrophage infiltration
and CD163 expression. Multivariate analysis showed that CD163 is a significant prognostic
factor in both breast and colorectal cancers
In an attempt to examine factors related to the function of macrophage fusion, the expression
of the signalling adaptor protein DAP12 was tested and related to CD163 expression in breast
cancers from 133 patients. DAP12 was shown to occur in breast cancer cells and was related
to high histologic tumour grade, skeletal and liver metastasis, and poor prognosis. The
findings in this thesis support the cell fusion theory and illustrate its clinical impact on tumour
progression and metastasis.
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LIST OF CONTENTS
ABSTRACT ...........................................................................................................................................5
LIST OF CONTENTS...........................................................................................................................7
ABBREVIATIONS................................................................................................................................9
LIST OF PAPERS...............................................................................................................................11
INTRODUCTION ...............................................................................................................................13
THE BIOLOGY OF CANCER ..................................................................................................................13
The somatic mutation theory ..........................................................................................................13
Epigenetics and cancer...................................................................................................................15
The tissue organization field theory ...............................................................................................16
The cell fusion theory .....................................................................................................................17
The roles of macrophages in tumour biology.................................................................................18
The macrophage antigen CD163....................................................................................................19
DAP12 ............................................................................................................................................20
BREAST CANCER.................................................................................................................................21
Pathology........................................................................................................................................21
Aetiology and risk factors...............................................................................................................22
Therapy and prognostic factors......................................................................................................23
COLORECTAL CANCER .......................................................................................................................24
Pathology........................................................................................................................................24
Aetiology and risk factors...............................................................................................................25
Therapy and prognostic factors......................................................................................................25
AIMS OF THE THESIS .....................................................................................................................27
MATERIALS AND METHODS........................................................................................................28
PATIENT AND TISSUE SAMPLES ..........................................................................................................28
TISSUE MICROARRAY .........................................................................................................................29
IMMUNOHISTOCHEMISTRY .................................................................................................................29
ANALYSIS OF APOPTOSIS....................................................................................................................30
STATISTICAL ANALYSIS......................................................................................................................30
RESULTS.............................................................................................................................................30
PAPERS I AND IV ................................................................................................................................32
PAPER II..............................................................................................................................................39
PAPER III ............................................................................................................................................41
DISCUSSION.......................................................................................................................................46
Macrophage traits in rectal cancer cells and irradiation ..............................................................47
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Macrophage traits in cancer cells and macrophage infiltration....................................................48
Macrophage fusion antigen is expressed by breast cancer cells....................................................49
CONCLUSIONS..................................................................................................................................51
SAMMANFATTNING PÅ SVENSKA..............................................................................................52
ACKNOWLEDGEMENTS ................................................................................................................54
REFERENCES ....................................................................................................................................56
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ABBREVIATIONS
APC adenomatosis polyposis coli
BMDC Bone marrow derived cell
c-myc cellular myelocytomatosis gene
c-src Cellular sarcoma oncogene
CD Cluster of differentiation
CIS Carcinoma in situ
CRC Colorectal cancer
CXCL-14 Chemokine (C-X-C motif) ligand 14
DAP12 DNAX-activating protein of molecular mass 12 kilodaltons
DRFS Distant recurrence free survival
EGF Epidermal growth factor
ER-α Oestrogen receptor α
ER-β Oestrogen receptor β
HER-2 Human Epidermal growth factor Receptor 2
HNPCC Hereditary nonpolyposis colorectal cancer
IHC Immunohistochemistry
IL Interleukine
ITAM Immunoreceptor tyrosine-based activation motifs
K-ras Kirsten rat sarcoma viral oncogene homolog
M-CSF Macrophage colony-stimulating factor
MGC Multinucleated Giant cell
MMP-9 Matrix metallopeptidase 9
PI3K phosphatidylinositol 3-kinase
PLOSL Polycystic Lipomembranous Osteodysplasia with Sclerosing
Leukoencephalopathy
RANKL Receptor activator of nuclear factor kappa B ligand
SMT Somatic mutation theory
SRCR Scavenger receptor cysteine-rich
Syk spleen tyrosine kinase
TAM Tumour associated macrophages
TGF-β Transforming growth factor beta
TMA Tissue microarray
TNM Tumour - Node - Metastasis
TOFT Tissue organization theory
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TREM-2 Triggering receptor expressed on myeloid cells 2
UICC International Union Against Cancer
ZAP-70 Zeta-chain-associated protein kinase 70
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LIST OF PAPERS
1- Breast cancer expression of CD163, a macrophage scavenger receptor, is related to
early distant recurrence and reduced patient survival. Shabo I, Stål O, Olsson H, Doré
S, Svanvik J. Int J Cancer. 2008 Aug 15;123(4):780-6.
2- Expression of the macrophage antigen CD163 in rectal cancer cells is associated
with early local recurrence and reduced survival time. Shabo I, Olsson H, Sun XF,
Svanvik J. Int J Cancer. 2009 Apr 14.
3- Tumour cell expression of CD163 is an independent prognostic factor in colorectal
cancer patients. Shabo I, Olsson H, Elkarim R, Arbman A, Sun XF, Svanvik J.
Submitted.
4- DAP12, a macrophage fusion receptor, is expressed in breast cancer cells and
associated with skeletal and liver metastases and poor survival. Shabo I, Olsson H,
Stål O, Svanvik J. Manuscript.
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INTRODUCTION
The biology of cancer
Cancer is a common cause of morbidity and mortality, and represents a major public health
problem in many parts of the world. The origin of cancer remains enigmatic. In spite of the
progress in cancer diagnosis, treatment, and improved survival, cancer still causes higher
mortality than heart diseases in patients under the age of 85 years 1. The malignant
transformation consists of series of progressive changes from premalignant precursor lesions
to the malignant phenotype. The somatic cells progressively acquire properties of malignant
traits such as autonomous generation of growth signals, evasion of apoptosis,
immortalization, induction of angiogenesis and the capacity of invasion and metastasis.
Carcinogenesis is a sophisticated biological process that is thought to be evolved from genetic
and epigenetic events, which together with selective tissue microenvironment produce clonal
cell populations with specific characteristics. This process occurs over several years, which is
consistent with epidemiological data indicating that cancer incidence increases with age.
Morphologically, cancer develops in focal proliferative lesions, such as papillomas, polyps
and adenomas. It is still unclear why and how these proliferative lesions arise and how they
develop towards cancer. Current models of carcinogenesis based on the gene mutation
hypothesis have limitations in explaining many aspects of cancer. In recent decades, several
new theories have been proposed to explain the malignant transformation of somatic cells.
The somatic mutation theory
During the past 50 years, the somatic mutation theory (SMT), first proposed by Theodor
Boveri in 1914, has been recognized as a predominant explanation for malignant
transformation. The theory states that cancer is derived from a single cell that, in a process of
sequential accumulation, has acquired somatic mutations in genes that control cell growth,
differentiation, apoptosis and maintenance of genomic integrity. SMT is based on the idea
that somatic cells are, in the absence of regulatory stimuli, in a proliferative quiescence 2-4.
The assumption that the default state of cells is quiescence likely arose from the fact that
historically it was difficult to propagate metazoan cells in vitro in defined media; this may
have led researchers to look for positive control factors (growth factors) to stimulate the cells 5, 6.
It is estimated that at least 4-7 mutated genes are required for transformation of normal
somatic cells to malignant cells. Each shift in cellular phenotype during the histopathological
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transitions in tumour development reflects a new mutation sustained in the genome of the
evolving premalignant cell population (Fig. 1). This conceptual idea of cancer was a form of
Darwinian evolution by which the sequential mutation in growth-controlling genes will
increase the proliferative capacity and hence selective advantage in cells with mutant genes 7,
8. Besides that, the discovery of a large number of carcinogenic and mutagenic chemicals and
transforming genes (oncogenes) further favoured SMT as the main theory of carcinogenesis.
Figure 1. Multistep clonal development of malignancy. The illustration shows a series of cumulative mutations
evolved during the malignant transformation.
Oncogenes are mutated forms of normal genes, called proto-oncogenes, which normally
control cell division and differentiation. When a proto-oncogene mutates into an oncogene, it
becomes permanently activated, acquires dominant function and causes increased cell
proliferation. The more than 100 oncogenes now recognized are divided into five different
classes: growth factors, growth factor receptors, signal transducers, transcription factors and
apoptosis regulators 9.
Tumour suppressor genes encode proteins that normally inhibit cell growth. When such genes
are mutated or deleted, they lose their function. The result is increased cell growth and cancer.
The genes were first identified by induction of cell hybrids between tumour cells and normal
cells. The major difference between oncogenes and tumour suppressor genes is that
oncogenes result from the activation of proto-oncogenes, whereas tumour suppressor genes
are inactivated in carcinogenesis. Another difference is that the majority of oncogenes
develop from mutations in normal genes (proto-oncogenes) during the life of the individual
(acquired mutations), whereas abnormalities of tumour suppressor genes can be both inherited
and acquired. About 30 tumour suppressor genes have been identified, including p53,
BRCA1, BRCA2, APC, and RB1. There are several types of tumour suppressor genes: Genes
that control cell division, such as RB1 (retinoblastoma) gene, DNA repair genes, and
apoptosis genes, such as p53 genes.
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Information gained during the past decade indicates that genetic alternations cannot alone
explain malignant transformation of somatic cells. Briefly, the main objections to SMT are:
(a) experimental studies have shown that overexpression of a given oncogene may enhance
growth in one cell type but inhibit growth or induce apoptosis in another. This observation
contradicts the original concept of oncogenes as dominant gain-of-function mutants of normal
cell growth genes 10; (b) The notion that quiescence is the default state of cells does not fit
with evolutionary theory. Proliferation is assumed to be the default state of prokaryotes; it has
been suggested that unicellular eukaryotes and growth factors act as survival factors or
substances that neutralize the effects of inhibitor factors 11-15; (c) Neoplastic phenotypes are
adaptive and can be normalized in spite of DNA mutation. Experimental studies showed that
teratocarcinoma cells injected into early embryos generated normal tissues, and hepatocellular
carcinoma cells injected into normal livers became normal hepatic tissue 14, 16.
Epigenetics and cancer
Gene expression and thereby the development of diseases, including cancer, may not only be
caused by mutations in genes but even by chemical modifications that alter how the genes
function. Genetic changes alone cannot explain the diversity of phenotypes within a
population despite their identical DNA sequences, e.g., monozygotic twins exhibit different
phenotypes and different susceptibilities to a disease 17.
Epigenetics refers to heritable alternations in gene expression that occur without alteration in
DNA sequence. These changes may remain through cell divisions for the rest of cell life and
can last for multiple generations. For example, a tumor suppressor gene, which normally
regulates cell growth and death, can be silenced by an epigenetic modification, rather than by
a mutation of the gene itself. Unlike genetic alterations, epigenetic changes are potentially
reversible. There are three main epigenetic processes in relation to cancer - DNA methylation,
covalent modification of histone and genomic imprinting. The first two mechanisms are
related to how DNA is packed in the nucleus. DNA methylation is a process of covalent
addition of methyl group to 5th position of cystosine within CpG islands, which are frequent
in the promoter region of genes. In cancer cells, hypermethylation is frequently detected in the
promoter regions of genes that control several cellular processes, such as proliferation,
apoptosis, DNA repair, and immortalization. The silencing of genes that regulates these
processes can therefore promote tumour formation and growth. For example,
hypermethylation of CpG islands at tumour suppressor genes switches off these genes,
whereas hypomethylation leads to genome instability and inappropriate activation of
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oncogenes. Histone covalent modifications are post translational changes of the amino acids
that make up histone proteins and include histone acetylation, methylation and
phosphorylation. Once the amino acids are changed, the shape of the histone sphere may be
modified and thereby alter chromatin structure and with that influencing gene expression.
Genomic imprinting is the silencing, or relative silencing of one parental allele compared with
the other parental allele. It is maintained by differentially methylated regions within or near
imprinted genes 18, 19.
Genetic mutations are common during early stages of the malignant transformation and these
are likely to be associated with tumour initiation. In contrast, few specific genetic mutations
have been linked to tumour progression. It is proposed that DNA mutations leads to cellular
transformation (cancer initiation), but induced epigenetic changes in the transformed cell
enhance their ability of metastasizing (tumour progression) 20.
Epigenetic changes can be induced by environmental and life style factors, like diet 17, 21.
Several factors influence the DNA methylation levels of a cell without requiring a change in
DNA sequence. With aging, the genome in certain tissues tend to become hypomethylated
whereas certain CpG islands become hypermethylated These age related epigenetic alteration
are similar to that found in cancer cells 22.
The tissue organization field theory
The tissue organization field theory (TOFT) states that proliferation is the default state of
cells, as in unicellular organisms or in the developing embryo, and carcinogenesis results
from alternation of normal tissue structure and the microenvironment rather than from genetic
or cellular damages. Sonnenschein and Soto postulate that tissue stroma is the primary target
of carcinogens, and that mutations are consequences, not causes, of the malignant phenotype23
. It is proposed that, in normal tissue, the proliferative activity of somatic cells is repressed by
biochemical systems of normal tissue organization. The malignant transformation evolves
when pathogens or carcinogens disrupt the normal biological restrictions, and somatic cells
proliferate (regain their default state) and result in hyperplasia. As the exposure to
carcinogens persists, the architecture of tissue stroma may be disrupted and can even adopt a
different tissue pattern (metaplasia). The suggestion that this process is reversible is supported
by the observation of spontaneously regressing tumours.23-27.
Criticism of TOFT is based on the assumption that cancers have clonal progression from a
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single cell and that neoplasm arises via epigenetic alternation in the tissue microenvironment.
Epigenetic differential gene expression may select clones of cells with increased proliferative
capacity and competitive preponderance 5, 12.
The cell fusion theory
The theory of cell fusion in cancer, first proposed by Aichel in 1911, states that cancer cells
may produce hybrids with metastatic phenotype when they spontaneously fuse with migratory
leukocytes (Fig. 2). These hybrids acquire genetic and phenotypic characteristics from both
mother cells. They gain the functional ability to migrate from leukocytes and uncontrolled
growth from the original cancer cells, and thereby adopt higher growth-promoting abilities
than their maternal cancer cell 28-31.
Cell-cell fusion may result in two forms of hybrids: heterokaryons or synkaryons. When bi- or
multinucleated hybrids are generated, the parental genomes are located in segregated different
nuclei (heterokaryons). Hybrids with parental genomes mixed in a single nucleus are called
synkaryons. Cell fusion is a common biological process that has long been known to produce
viable cells and to play major roles in mammalian development and differentiation. Cell-cell
fusions occur during embryogenesis and morphogenesis32. Skeletal muscle consists of a
syncytium that arises from the fusion of mesodermal cells. In the placenta, trophoblasts fuse
to form syncytiotrophoblasts. Osteoclasts and MGCs (multinucleated giant cells) are
generated from the fusion of macrophages 33-35. Cell fusion also contributes to tissue repair.
Bone-marrow-derived (BMD) cells contribute to liver and intestinal regeneration by cell
fusion 36-38.
Initially, cell fusion will generate multinuclear hybrids (heterotypic fusion). These hybrids are
capable of cell divisions that result in daughter cells expressing both parental sets of
chromosomes in a single nucleus 39. Fusion of two cells in G1 results in a binucleated cell
with two centrosomes, and duplication during post-fusion incubation will lead to four
centrosomes. Centrosome duplication occurs with normal kinetics in fused binucleated cells,
and the division may lead to diploid cells. If the cells are in different phases of the cell cycle,
chromatin will condense and disappear 40-42. Heterotypic cell fusion between bone marrow
derived-cells (BMDCs) and somatic cells, such as hepatocytes, cardiomyocytes and skeletal
muscle cells, has been demonstrated in several studies 30, 43, 44. Inflammation and irradiation
have been shown to induce frequent heterotypic cell fusion between myeloid/lymphoid cells
and non-hematopoietic cells such as cardiomyocytes, skeletal muscle and hepatocytes45.
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Figure 2. Macrophage – cancer cell fusion model. The fusion between macrophage and cancer cell generates a
hybrid with genetic and phenotypic traits from both parental cells.
Recent reports present evidence that cell fusion (both in vivo and in vitro) may occur in
cancer46-53. Cell fusion has even been documented in two human cases with tumours
occurring in transplanted organs 50. BMDCs have been suggested as a source of multiple
tumour types 54. Fusion occurs between BMDCs and intestinal epithelial cells in the stem cell
niche of the small intestine and might be involved in tumour genesis of intestinal mucosa 36.
Fusion between tumour associated macrophages (TAM) and cancer cells may cause hybrids
with increased metastatic potential in animal models 42, 46-49, 55-59. Macrophage traits in cancer
cells might therefore be explained by cell fusion between myeloid and cancer cells 60, 61.
Although these data do not formally prove that tumour–host cell (macrophage) hybrids do
generate during the malignant transformation, the combined results from some of the above-
mentioned studies in vitro and in vivo present compelling evidence that cell fusion may occur
in tumours 29.
The roles of macrophages in tumour biology
Macrophages are a heterogeneous population of cells derived from monocytes. Macrophages
originate from mesoderm. During embryogenesis, they appear first in the yolk sac, then in the
liver, and finally in bone marrow. Large populations of tissue macrophages exist in the small
intestine, liver (Kupffer cells), and lungs. Blood monocytes arise in the marrow from
precursor cells (monoblasts) and enter inflamed or infected tissues, where they can mature
into macrophages and increase resident macrophage populations. Monocytes can also mature
into dendritic cells that present antigens to T cells.
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Fusion is an important function of macrophages and results in the formation of osteoclasts
and multinucleated giant cells (MGC). Osteoclast formation is stimulated by RANKL
(Receptor Activator of Nuclear factor Kappa B Ligand) and the macrophage colony-
stimulating factor (M-CSF). Macrophage fusion is even induced by interleukin-4 (IL-4), IL-
13 and interferon γ 34.
Macrophages show two different polarization states, M1 and M2 macrophages, in response to
different microenvironmental signals. M1 macrophages are proinflammatory and
characterized by the release of mainly inflammatory cytokines and microbicidal/tumoricidal
activity. M2 macrophages have an immunosuppressive phenotype and are polarized by anti-
inflammatory molecules such as IL-4, IL-13, and IL-10, apoptotic cells, and immune
complexes. M2 macrophages release anti-inflammatory cytokines and have scavenging
potentials as well as supporting angiogenesis and tissue repair. Monocyte/macrophage cells
are important for tumour cell migration, invasion and metastasis. Tumour associated
macrophages (TAM) represent the M2-type and promote tumour progression 62-66.
Monocytes are actively recruited to the tumour stroma, and high infiltration of TAMs in many
tumour types correlates with lymph node involvement and distant metastasis 67. Experimental
studies show that inhibition of macrophage infiltration in tumours may inhibit metastasis and
progression of secondary tumours 68, 69. The clinical significance of macrophage infiltration in
tumour stroma, however, is still controversial. High infiltration of TAMs is correlated to poor
prognosis in breast, prostatic, ovarian and cervical carcinoma 70. In colorectal cancer, there
are conflicting data about the clinical significance of macrophage infiltration, but several
studies show that low macrophage density in tumour stroma is associated with an unfavorable
prognosis 70-72.
TAMs contribute to angiogenesis, lymphangiogenesis and tumour progression by expressing
pro-angiogenic growth factors such as MMP-12, IL-1, VEGF and IL-8 70, 73, 74. Clinical
studies have shown that increased infiltration of TAMs in solid tumours is associated with
high micro-vessel density and poor prognosis. These data are particularly strong for hormone-
dependent cancers, such as breast cancer.
The macrophage antigen CD163
CD163, formerly known as M130 or RM3/1 antigen, is a transmembrane scavenger receptor
for the haptoglobin-haemoglobin (Hp-Hb) complex and is encoded by a gene on chromosome
12, location p13.375. It is specifically expressed by non-neoplastic monocytes/macrophages
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and by neoplasms with monocytic/histiocytic differentiation but not by other normal tissues
and usually not by cancer cells. 75-78. CD163 is 130-kDa glycoprotein with an amino-terminal
signal element, 9 scavenger receptor cysteine-rich (SRCR) domains, one transmembrane
element, and a short cytoplasmic tail76. Stable Hp–Hb complexes are delivered to the
reticuloendothelial system by CD163 receptor-mediated endocytosis followed by lysosomal
proteolysis of globin and conversion of haem to iron and bilirubin-ligand complex. The
expression of CD163 is upregulated by the acute phase mediator IL-6, glucocorticoids and IL-
10, whereas the proinflammatory lipopolysaccharide (LPS), IL-4, TGF-β and interferon-γ
downregulate the CD163 expression79-81.
It has also been suggested that CD163 is a monocyte/ macrophage differentiation antigen.
Tissue macrophages have higher CD163 expression than monocytes, which has been
proposed as part of monocyte maturation to a phagocytic macrophage 82. CD163 is expressed
in M2 macrophages83. Almost all TAMs express CD163, indicating the phenotypic shift
towards M2 macrophage84, 85.
DAP12
The signalling adaptor protein DAP12 (DNAX activating protein of 12 kD), also known as
KARAP (killer cell activating receptor-associated protein), plays a crucial role in macrophage
fusion during osteoclast formation 86, 87. It has been suggested that DAP12 may also be
involved in macrophage fusion, leading to the formation of multinucleated giant cells and
constituting an essential target to control the resolution of inflammatory disorders based on
monocytes/macrophages and neutrophils 88. Whether DAP12 is also involved in macrophage
fusion with other type of cells is unclear.
By activation of the DAP12 receptor, the tyrosine residues in the DAP12-ITAM are
phosphorylated, leading to the recruitment and activation of the protein tyrosine kinases Syk
and ZAP70, which in turn lead to the activation of phosphatidylinositol 3-kinase (PI3K) 89, 90.
Several DAP12-associated receptors are presented on macrophages and other myeloid cells.
In human, mutations of DAP12 or TREM-2 lead to polycystic lipomembranous
osteodysplasia with sclerosing leucoencephalopathy (PLOSL), which is associated with bone
lesions and osteoporotic features. This phenotype is based on impaired osteoclast
differentiation and function 87, 91.
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Breast cancer
Pathology
Breast cancer is the leading cancer type in females and the most common cause of death from
cancer in women aged 40 to 44 years. The incidence of breast cancer is 4-5 times higher in
the industrial world than in developing countries and in Native Americans in USA. In
Sweden, breast cancer incidence in women increased by 1.2 % annually during the past 20
years. In the recent 10-year period, however, the annual increase has been 0.8 per cent 1, 92, 93.
Breast cancers arise from the epithelial cells in the terminal duct lobular unit and depending
on whether cancer cells exceeded through the basement membrane classifies into Carcinoma
in situ (CIS) and invasive carcinoma. CIS is a preinvasive form in which malignant cells have
not invaded through basement membrane of glandular epithelium. Both in situ and invasive
cancers are classified mainly into ductal or lobular types. The histopathological classification
of breast cancer is described in Box 1.
Currently, the staging of breast cancer is determined according to guidelines from the
International Union Against Cancer (UICC). This system is a clinical and pathological
staging, based on the TNM (Tumour-Node-Metastasis) system, and contains additional
information about tumour size, node status and metastasis (Box 2).
Box1 – Classification of primary breast cancer
Non invasive epithelial cancers
- Lobular carcinoma in situ (LCIS)
- Ductal carcinoma in situ (DCIS)
o DCIS COMEDO type.
o DCIS Non-COMED typre.
- Papillary carcinoma in situ
Invasive breast cancer
- Ductal carcinoma
o Paget disease of the nipple (uncommon)
- Lobular carcinoma
- Uncommon breast cancer
o Mucinous carcinoma
o Tubular carcinoma
o Medullary carcinoma
o Metaplastic carcinoma
o Invasive papillary carcinoma
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Box 2 – Correlation of UICC and TNM classification of breast cancer.
UICC Stage TNM Classification
0 Tis N0 M0
I T1 N0 M0
IIA T1 N1 M0
T2 N0 M0
IIB T2 N1 M0
T3 N0 M0
III
any T, N2-3, M0;
T3, any N, M0;
T4, any N, M0
IV any T, any N, M1
Scoring Tumour Size (T):
- T1 = 0-2 cm - T2 = 2-5 cm - T3 =>3 cm - T4 = ulcerated or attached
Scoring Node Status (N):
- N0 = negative nodes - N1 = positive nodes
Scoring Metastasis:
- M0 = no spread of tumour - M1 = tumour has spread
Aetiology and risk factors
The aetiology of breast carcinoma is unknown but is assumed to be multifactorial. It has been
suggested that genetic factors exist because of the strong familial tendency. On the other
hand, no significant inheritance pattern has been found, which indicates that sporadic breast
cancers are due either to the action of multiple genes or to similar environmental factors
acting on members of the same family or patient group. A family history (limited to first-
degree relatives) of breast carcinoma increases the risk twofold to threefold. The occurrence
of carcinoma in one breast increases the risk of carcinoma in the other breast about sixfold.
Women without any of the above risk factors still have a high incidence of breast cancer 94, 95.
Most hereditary breast cancer is said to arise from mutations of BRCA1 and BRCA2.
Mutation of either of these genes results in a 37%-85% lifetime risk of breast cancer. The
frequency of the BRCA-1 gene, located on chromosome 17q, is between 1/500 and 1/800.
BRCA-1 is thought to encode a tumour suppressor protein. The frequency of BRCA-2,
located on chromosome 13q, is lower in the general population.
Age is also an important risk factor in all forms of breast cancer. Females with inherited
predisposition with BRCA1 and BRCA2 alternations commonly incur breast cancer after the
age of 20, whereas breast cancer occurs in women without hereditary predisposition mainly in
postmenopausal years. Individual breast cancers have an average of 12 driving gene
mutations. Exposure to ionizing radiation increases the risk of breast cancer and is greatest if
exposure occurs before the age of 30 94, 96.
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Hormones are widely believed to be important in breast cancer aetiology as several studies
linked breast cancer to age at menarche, menopause, first pregnancy after age of 30 and
postmenopausal obesity. In general, hormonal risk factors are associated with 1.5 to 2.0
relative risk of developing breast cancer. Oestrogen is the most frequently studied hormone in
relation to breast cancer because epidemiological data indicate that prolonged oestrogen
exposure (early menarche, late menopause, null parity, and delayed pregnancy) increases the
risk of breast cancer. The genomic actions of oestrogens are mediated via oestrogen receptors,
which bind oestrogens with high affinity and specificity. These receptors are members of a
family of nuclear hormone receptors that bind steroids, thyroid hormone, and retinoids. These
receptors function as ligand-modulated nuclear transcription factors 97, 98. Two oestrogen
receptor molecules have been identified: the original oestrogen receptor alpha (ER-α), and the
oestrogen receptor beta (ER-β). Evidence linking oral contraceptives to breast carcinoma is
controversial. A few studies suggest a very slightly increased incidence in women who use
oral contraceptives. Other reports indicate a decreased risk of breast cancer after
discontinuation of combined hormone therapy 99, 100.
Therapy and prognostic factors
Surgery is the primary treatment of breast cancer and includes breast conservation (excision
of tumours <4 cm with a 1 cm margin of normal tissue combined with postoperative
irradiation) or a mastectomy. There are no significant differences in overall survival or
disease-free survival between the patients who underwent total mastectomy and breast
conservative surgery 101, 102. Certain clinical and pathological factors influence selection for
breast conservation or mastectomy because of their impact on local recurrence after breast
conserving therapy. These include young age, the presence of an extensive in situ component,
the presence of multiple tumours, the presence of lymphatic or vascular invasion, and
histological grade. Young patients (< 35) appear to have a higher risk of developing local
recurrence than older patients do.
Nonsurgical breast cancer therapies consist of radiation therapy, hormonal therapy and
chemotherapy. Radiation therapy is used for all stages of breast cancer. Patients receive
radiotherapy to the breast after wide local excision or quadrantectomy. Doses of 40-50 Gy are
delivered in daily fractions over three to five weeks. After mastectomy, radiotherapy should
be considered for patients at high risk of local recurrence.
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Endocrine therapy is a complex medical field. Hormone receptors are detectable in more than
90% of well-differentiated ductal and lobular invasive cancers. The most widely studied
hormone receptors are the oestrogen receptor and progesterone receptor. About 75% of
invasive breast cancer express oestrogen receptor. Clinical responses to anti-oestrogen
therapy (Tamoxifen) are evident in more than 60% of women with hormone receptor–positive
breast cancers, but in less than 10% of women with hormone receptor–negative breast
cancers. A rare long-term risk of tamoxifen use is endometrial cancer. The major advantage of
tamoxifen over chemotherapy is the absence of severe toxicity. HER2/neu expression is used
for prognostic purposes. Anti-HER-2/neu therapy (trastuzumab, Herceptin) is used for
patients with HER-2/neu overexpression. Patients with cancers that overexpress HER2/neu
may benefit if trastuzumab is added to paclitaxel chemotherapy 100, 103, 104. Chemotherapy
consists of adjuvant and neoadjuvant chemotherapy as well as chemotherapy against distant
metastases.
Clinically, prognostic factors are used to determine which patients have such a favourable
outcome after local/surgical therapy that adjuvant systematic therapy is not needed. Tumour
stage according to the TNM system is the most reliable indicator of prognosis. Factors that
are in routine clinical use include axillary lymph node status, tumour size, histological type,
nuclear and histological grade (Nottingham Histologic Grade and Nottingham Prognostic
Index), HER-2 and ER and PR status. Markers such as S-phase fraction, Ki-67, apoptosis,
angiogenesis and CA 15-3 are other prognostic factors used in several institutions but are still
controversial 94, 105, 106.
Colorectal cancer
Pathology
Colorectal cancer (CRC) is one of the most common cancers in Western countries. The
incidence of this disease in Sweden is stable, although colon cancer in women has increased
by 1.4 per cent per year on average during the past decade. CRC was about 12% of all new
cancer cases diagnosed in Sweden during 2007 92. Carcinoma of the colon, particularly the
right colon, is more common in women, and carcinoma of the rectum is more common in
men. Multiple synchronous colonic cancers, i.e. two or more carcinomas occurring
simultaneously, are found in 5% of the patients. Metachronous cancer is a new primary lesion
in a patient who has had a previous resection for cancer. The mean cumulative risk of
metachronous CRC was 6-10%. Ninety-five per cent of malignant tumours of the colon and
rectum are adenocarcinomas.
25
Aetiology and risk factors
Aging is the main risk factor for colorectal cancer. Its incidence increases steadily after the
age of 50, with about 90% of cases diagnosed at an age older than 50 years. Individuals of any
age, however, may develop colorectal cancer. Approximately 85% of colorectal cancers occur
sporadically, whereas 15% of cases are hereditary. The most common form of hereditary
colorectal cancer is hereditary nonpolyposis colorectal cancer (HNPCC). First-degree
relatives of patients with sporadic colorectal cancer have 2-3 times higher risk of large bowel
cancer, and it is estimated that 15–20% of cancers of the large bowel are due primarily to an
inherited genetic defect 93, 107.
Epidemiological observations show that environmental factors are significantly associated
with colorectal cancer. Populations that consume diets high in animal fat and low in fibres
have high incidence of colorectal cancer, which in turn leads to the hypothesis that dietary
factors contribute to carcinogenesis. A diet high in oleic acid (olive oil, coconut oil, fish oil)
does not increase risk. Obesity and a sedentary lifestyle dramatically increase cancer-related
mortality in a number of malignancies, including colorectal carcinoma. Chronic inflammation
as in ulcerative colitis, Crohn’s colitis and the presence of an ureterocolostomy are other
conditions that predispose to CRC92, 93, 107-110.
Carcinogenesis in the large bowel is a long multistep process involving multiple genetic
alterations, including oncogene activation (K-ras point mutation, c-myc amplification and
overexpression, and c-src kinase activation), and inactivation of tumour-suppressor genes
(point mutations in the APC gene and P53).
Cancer of the colon and rectum spreads through direct extension, haematogenic metastasis or
and lymphatic metastasis. Transperitoneal metastasis occurs when the tumour has extended
through the serosa and tumour cells enter the peritoneal cavity. Intraluminal metastasis occurs
when cancer cells spread from the surface of the tumour along in the faecal current and may
implant more distally on intact or damaged mucosa.
Therapy and prognostic factors
Surgical resection is the primary treatment of CRC. Tumour resection should involve
segmental removal of the involved colon or rectum including regional lymph nodes along the
vascular pedicles. If complete resection of all gross disease is achieved, the risk of relapse can
be estimated based on pathologic staging. Metastatic CRC is a result of residual microscopic
disease that is not evident at the time of surgery. Patients with high risk of relapse based on
26
clinical or pathologic staging, may be considered for additional local therapy in form of
preoperative or postoperative radiotherapy, systemic adjuvant chemotherapy, or both.
Resection of the primary tumour may be indicated even if distant metastases have occurred,
as preventing obstruction or bleeding may offer palliation for long periods. For rectal cancer,
the choice of operation depends on factors such as the distance of the lesion above the anal
verge, the gross extent of the tumour, the degree of differentiation, and the patient’s habits
and general condition. Preservation of the anal sphincter and avoidance of colostomy are
desirable if possible.
The TNM classification system (Table 1) is used as pathologic staging in modern
management of CRC and provides prognostic information. Adequate lymphadenectomy and
subsequent pathologic assessment of lymph nodes are both important components of
multidisciplinary care.
Table 1. TNM staging of colorectal cancer
TNM Stage Primary Tumour Lymph Metastasis Distant Metastasis
0 TIS N0 M0
I T1 N0 M0
T2 N0 M0
T3 N0 M0 II
T4 N0 M0
IIIA T1,2 N1 M0
IIIB T3,4 N1 M0
IIIC Any T N2 M0
IV Any T Any N M1
27
THE AIMS OF THIS THESIS
The background of this thesis is the hypothesis of cell-cell fusion. We propose that:
Heterotypic fusion between macrophages and tumour cells occurs in cancer
progression and metastasis.
Fusion between these two cell types results in hybrids with increased metastatic
potentials.
According to the cell fusion model, the hybrids gain phenotypic characteristics from
both mother cells. Macrophage-specific antigen CD163 is a macrophage phenotype
expressed by hybrids after fusion between macrophage and cancer cells.
The specific aims of the studies included in this thesis are:
To demonstrate the presence of macrophage traits in cancer cells from breast and
colorectal cancers.
To investigate the clinical significance of CD163 expression in cancer cells.
To correlate CD163 expression to other biological processes in malignancy, including
apoptosis, cancer cell proliferation and angiogenesis.
To explore whether macrophage phenotype traits in cancer cells are affected by
macrophage infiltration in the colorectal tumour stroma.
To investigate the influence of radiotherapy on CD163 expression in tumour cells.
To demonstrate the presence and clinical significance of DAP12 in solid tumour and
how it may correlate to CD163 expression.
28
MATERIALS AND METHODS
Patient and tissue samples
The studies in this thesis are based on paraffin-embedded specimens from patients with breast
and colorectal cancers. In Papers I and IV, breast cancer specimens from 133 women were
used. These specimens were previously collected in a tissue microarray, and the TNM stage,
Nottingham histological grade (NHG), oestrogen receptor status, DNA ploidy of the tumour,
time for distant metastases and survival were known. The patients were diagnosed and treated
by conventional methods at the surgical departments in southeastern Sweden. All patients
were in Stage II according to the UICC and all received adjuvant tamoxifen therapy. In Paper
I, normal breast tissue was selected from 20 patients operated upon with breast reduction.
Normal adjacent breast tissue samples from 20 out of the 133 patients with breast cancer were
also included; 10 specimens with normal (adjacent) breast tissue from patients with positive
CD163 breast cancers and 10 specimens with normal (adjacent) breast tissue from patients
with negative CD163 breast cancers.
In Paper II, 163 patients with primary rectal cancer from southeastern Sweden were included.
Specimens were collected from distal normal mucosa (N=119), adjacent normal mucosa
(N=81) and primary tumours (N=139). Some 101 biopsies, collected prior to treatment, from
primary tumours were also included in the present study. Specimens from both preoperative
biopsy and primary tumours from the same patient were available in 88 cases. The patients
were previously included in the Swedish rectal cancer trial 111 and followed up during 1987-
2004 with a median period of 71 months. Briefly, all patients were randomized to either
preoperative radiotherapy (5 x 5 Gy delivered in 1 week), followed by surgery within the next
week (radiotherapy group N=76), or to surgery with no additional radiotherapy (Non-
radiotherapy group N=87). None of the patients received adjuvant chemotherapy. Curative
anterior resection was performed in all patients. Follow-up was performed by matching all
patients against the Swedish Cancer Register and the Cause of Death Register until 2004.
Information about local and distant recurrences was obtained from patient medical records.
In Paper III, specimens from a new patient material, consisting of 77 colorectal patients, were
used. Whole tissue sections were obtained from formalin-fixed paraffin-embedded tissue from
primary tumours (n=77) and distal normal mucosa (n=25). Fifteen of the specimens from
primary tumours and distal normal mucosa were matched samples from the same patients. All
patients were diagnosed by conventional clinical methods and underwent surgical resection at
the University Hospital in Linköping and Vrinnevi Hospital in Norrköping (Sweden). The
diagnosis of colorectal cancer was confirmed by routine histopathology. Clinical-pathologic
29
data were obtained from surgical and pathologic patient records.
Tissue microarray
Tissue microarray (TMA) is an efficient and validated method for collecting several minute
specimens on one slide instead of one section/specimen per slide. These slides are prepared
by transferring paraffin tissue cores from many “donor” blocks to one “recipient” archival
block 112-114.
In Papers I, II and IV, paraffin-embedded tumour specimens collected in TMA were used.
Briefly, three morphologically representative regions were chosen in each block, and three
cylindrical core tissue specimens (0.6 mm in diameter) were taken from these areas and
mounted in a recipient block. The tissue microarrays were constructed using a manual arrayer
(Beecher Inc, WI). Liver samples were included on each tissue block as a control.
Immunohistochemistry
Immunohistochemistry (IHC) is used to identify a tissue structure in situ by means of a
specific antigen–antibody interaction in which the antibody has been tagged with a visible
label. Different markers, such as fluorescent dye, enzyme, radioactive element or colloidal
gold, visualize the antigen-antibody interactions.
The immunostaining in all studies included in this thesis was performed according to the
EnVision protocol, Dako, Denmark. Serial sections of 5 µm were obtained, deparaffinized in
xylene and hydrated in a series of graded alcohols. Heat-induced antigen retrieval was carried
out by water bath pre-treatment in Tris EDTA before staining with primary antibodies.
Detection was carried out using the DAKO Envision system. Positive internal control staining
was characterized as positive staining of tissue macrophages (monocytes and histiocytes). As
negative controls, the primary antibody was replaced by an irrelevant isotype – antimouse
IgG1.
The macrophage markers used in Paper I were CD163 (clone 10D6, Novocastra, England),
CD68 (PG-N1, DakoCytomation), and MAC387 (MO747, DakoCytomation). Polyclonal
antibodies against IL-10 (sc-7888) and TGF-β (sc-146) were obtained from SDS Bioscience
(Santa Cruz Biotechnology, CA). CD163 was also used in Papers II and III. In Paper IV,
DAP12 expression was characterized by anti-human rabbit polyclonal antibody DAP12 (FL-
113) from Santa Cruz, USA. Immunohistochemical staining in Papers II and III with Ki-67 as
well as angiogenic marker CD31, CD34 and D2-40 was performed in previous studies115-117.
These antibodies are well characterized in previous reports 118-127. In each study, the histology
30
and immunostaining were independently evaluated by at least two of the authors. Inter-
observer agreement was measured according to Cohen κ. 128.
Analysis of apoptosis
Apoptosis was detected by the TUNEL assay. The Apop-Tag in situ apoptosis detection kit
(Oncor, Gaithersburg, MD) was used to detect apoptosis. The percentage of apoptotic cells
was determined by counting approximately 1,000 tumour cells. Cases are considered negative
if apoptotic cells constitute <5% of the tumour cells 115, 116.
Statistical analysis
All statistical analyses were performed using STATISTICA version 7 (StatSoft, Inc).
Expression of IHC markers was studied in relation to patient clinical data and tumour
characteristics. Person’s Chi-square test and Spearman rank correlation test were used for the
comparisons. Survival curves were estimated according to life table methods, and hazard
ratios were calculated by Cox regression analysis, both in univariate and multivariate analysis.
A P value less than 5% was considered statistically significant
RESULTS
Prior to the studies presented in the thesis, a pilot study was conducted to find out if cancer
cells expressed macrophage markers in solid tumours. Initially, five cases of each colon,
gallbladder, breast, and prostate cancers were immunostained with commonly used,
macrophage markers CD14, CD68, CD163, vimentin (ViM) and MAC387. Solitary cases of
each type of these tumours showed expression for CD163 and MAC387 in cancer cells but
none of the other markers were expressed in cancer cells. On the other hand, macrophage in
tumour stroma expressed all these markers. In order to find out if the expression of these
macrophage antigens was different in metastases compared to primary tumours, specimens
from breast cancer (n=5), colorectal cancers (n=5), breast cancer lymph node metastases
(n=5) and colorectal liver metastases (n=5) were stained with macrophage markers. In about
20-50 % of cases, cancer cells in both primary tumours and their corresponding metastases
expressed CD163 and to some extend even MAC387. Specimens where CD163 and MAC387
expression occurred in more than 25% of cancer cells were considered as positive. The other
macrophage markers were again not expressed by tumour cells (Table 2). In the light of these
data, CD163 and MAC387 were henceforth used as macrophage antigen to detect
macrophage phenotype in cancer cells.
31
According to the cell-fusion model, we hypothesized that macrophage-cancer cell hybrids
expressing CD163 and/or MAC387 would be more frequent in tetraploid than diploid
tumours. In an attempt to explore this, diploid (n=10) and tetraploid (n=10) primary colorectal
cancers were stained with CD163 and MAC387. Both cancers showed similar CD163 and
MAC387 expression pattern (Table 3)
Table 2 – Expression of macrophage antigen CD14, CD68, VD163, MAC387 and Vimentin (ViM) in breast
cancer (BRC), breast cancer lymph node meastases, colorectal cancer (CRC) and colorectal liver metastases. A
pilot study.
BRC BRC Lymph node
metastases CRC
CRC liver
metastases
CD14
Negative 5 5 5 5
Positive 0 0 0 0
CD68
Negative 5 5 5 5
Positive 0 0 0 0
CD163
Negative 2 5 3 2
Positive 3 0 2 3
MAC387
Negative 4 5 5 5
Positive 1 0 0 0
ViM
Negative 5 5 5 5
Postive 0 0 0 0
Table 3 – Expression of macrophage antigen CD163 and MAC387 in diploid and tetraploid colorectal cancer. A
pilot study.
Diploid CRC Tetraploid CRC
CD163
Negative 7 8
Positive 3 2
MAC387
Negative 10 9
Positive 0 1
32
Papers I and IV
Tissue microarray (TMA) with breast cancer tissue samples from 133 patients was used for
immunohistochemical staining with CD163, MAC387, CD68, IL-10 and TGF-β (Paper I) as
well as DAP12 (Paper IV). Six samples in TMA used in Paper I and four samples in TMA
used in Paper IV could not be assessed technically and were excluded from further analysis.
The immunostaining of CD163, MAC387 and DAP12 was characterized by granular
cytoplasmic, or cytoplasmic and membrane staining patterns. The tumour cells which were
pleomorphic and atypical with large nuclei and nucleoli were easy to distinguish from
macrophages. Other cells such as fibrocytes and lipocytes were not stained (Fig. 3). One of
the 20 patients with normal breast tissue showed weak expression of CD163, whereas the
other cases exhibited neither CD163 nor MAC387 expression.
Expression of CD163, MAC387 and CD68 in non-neoplastic tissue samples was limited to
the constituent macrophages of each respective site. CD163 staining was localized
predominantly to the membrane and was scored on a 5-tiered score as follows: 0%, 1-25%,
26-50%, 51-75% and 76-100% of the lesional cells. During further analysis, CD163
expression was graded in two categories; if more or less than 25 per cent of the cancer cells
were stained. Cancer cells in breast cancer sections showed generally weak staining for
DAP12, whereas several clones of cancer cells exhibited strong DAP12 staining. These cells
were considered significantly positive, and their incidence in each section was scored in three
grades: grade 0, no cancer cells with strong DAP12 staining; grade 1, 1-10% of cancer cells
with strong DAP12 staining; and grade 2, >10% of cancer cells with strong DAP12 staining.
Samples with cancer cells expressing DAP12 in >10% were considered positive in further
analysis. No tumour cells expressed CD68. The macrophages in the tissue stained for all 4
antibodies. The inter-examiner agreement (κ) in evaluating immunostaining of DAP12 and
CD163 according to Cohen was 0.634 and 0.88, respectively.
Out of 127 cancers, 15 (12%) expressed MAC387, and 61 (48%) expressed CD163 in more
than 25% of cancer cells. Moreover, in cancers where the fraction of cancer cells expressing
CD163 was the highest, expression of MAC387 (P=0.0012) was more common. DAP12
expression was positive in 85 (66%) cases out of 129.
TGF-β was expressed in all breast cancer cases; IL-10 in 128/131. The staining intensity of
IL-10 was inversely related to CD163 expression (P=0.022), but was not related to MAC387
expression (P=0.157). TGF-β was preferentially expressed in CD163 (P=0.009) and MAC387
(P < 0.001) positive tumours. Neither IL-10 nor TGF-β expression was linked to clinical
variables such as ER expression, lymph node status, NHG grade, DNA ploidy or S-phase.
33
The breast cancer cell lines (BT-474, T47D, MCF-7, SKBR-3 and ZR-751) did not express
CD163 under basal conditions or after stimulation with IL-10 or TGF-β.
Figure 3. Immunostaining of cancer cells in breast cancer with CD163, MAC387 and DAP12
CD163 (p=0.010), DAP12 (p=0.015) and MAC387 (p=0.00026) expression are more
common in histologically advanced breast cancers as their expression increases proportionally
to NHG grade. Expressions of CD163 and MAC387 are more common in oestrogen receptor
negative cancers (p=0.0001). DAP12 expression is not correlated to oestrogen receptor status,
lymph node status or DNA ploidy (Table 4).
DAP12 expression was associated with skeletal and liver but not lung metastasis. It was
highly expressed in 45% of patients with skeletal metastases (p=0.067) and 60% of patients
with liver metastases (p=0.046). No differences in DAP12 expression were found in patients
with or without lung metastases (p=0.997). DAP12 expression was not associated with
expression of CD163, MAC387, IL-10 or TGF-β.
Patients with breast cancers expressing CD163 (n=61) had significantly reduced distant
recurrence-free survival (DRFS) when they were compared with patients with CD163
negative expression (n=66) (HR = 1.9, 95% C.I. 1.1–3.4, p = 0.024) (Fig. 4a).
CD163 MAC387 DAP12
Negative expression
Positive expression
34
In an attempt to study more homogenous tumour subgroups, 73 patients with NHG 1–2 (Fig.
4b) and 43 patients with DNA diploid breast cancers (Fig. 4c) were examined. In both groups
the DRFS was significantly shorter in patients with tumours that expressed CD163 [patients
with NHG 1–2: HR = 3.0 (1.3–6.9), p=0.0094, and patients with DNA diploid breast cancers:
HR=5.3 (1.5–19.0), p=0.0048]. In the group with diploid breast cancer, 8 patients had
metastases in the skeleton; in 7 of these the primary tumours were CD163 positive.
Patients with tumours expressing DAP12 acquired skeletal and liver metastases earlier than
patients with negative/low DAP12 expression (p=0.023) (Figure 5a). The same pattern was
observed in patients with liver metastases (p= 0.028) (Figure 5b). Interestingly, patients with
lung metastases showed no differences in DRFS rates according to DAP12 expression
(p=0.64) (Figure 5c).
To investigate how an interaction between DAP12 and CD163 expression may affect DRFS
rates, breast cancer patients were allocated in four groups: group 1 with high DAP12 and
Table 4. Univariate analysis comparing the expression of CD163, MAC387 and DAP12 in 127 patients with
breast cancer in relation to DNA ploidy and clinicopathological variables.
CD163 MAC387 DAP12
Negative Positive p Negative Positive p Negative Strong
N (%) N (%) N (%) N (%) N (%) N (%) p
NHG Score
NHG 1 14 (67) 7 (33) 21 (100) 0 (0) 19 (86) 3 (14)
NHG 2 31 (60) 21 (40) 50 (96) 2 (4) 37 (71) 15 (29)
NHG 3 21 (39) 33 (61) 0.000 41 (76) 13 (24) <0.001 32 (58) 23 (42) 0.015
ER Status
Positive 56 (58) 41 (42) 94 (97) 3 (3) 19 (63) 11 (37)
Negative 10 (33) 20 (67) 0.019 18 (60) 12 (40) <0.001 69 (70) 30 (30) 0.516
Tumour size
≤ 2 cm 21 (50) 21 (50) 39 (93) 3 (7) 32 (74) 11 (26)
>2 cm 45 (53) 40 (47) 0.75 73 (86) 12 (14) 0.25 56 (65) 30 (35) 0.288
DNA ploidy
Diploid 23 (53) 20 (47) 40 (93) 3 (7) 31 (72) 12 (28)
Aneuploid 43 (51) 41 (49) 0.81 72 (86) 12 (14) 0.23 57 (66) 29 (34) 0.508
35
positive CD163 expression; group 2 with high DAP12 and negative CD163 expression; group
3 with low DAP12 and positive CD163 expression; and group 4 with low and negative
CD163 expression. Patients with high DAP12 and/or CD163 expression had significantly
lower survival than patients with breast cancer expressing neither CD163 nor low/negative
DAP12 expression (p=0.00077) (Figure 6b).
NHG score, tumour size, oestrogen receptor status and lymph node status are important
clinicopathological prognostic factors in breast cancer. To investigate the prognostic value of
CD163 expression, multivariate analysis adjusting for these prognostic factors was
performed. MAC387 was also included in the multivariate analysis because its expression
was associated with tumour stage in univariate analysis (Paper I). Multivariate analysis shows
that CD163 is a significant prognostic factor in relation to distant recurrence (p=0.053) and
breast cancer mortality (p=0.043) rates (Table 5)
In Paper IV, multivariate analysis adjusted for clinicopathological variables revealed that
DAP12 expression had a significant prognostic impact as an independent factor associated
with skeletal metastases (HR=2; 95% CI=1-4,46; P=0.049) and DRFS (HR=1.,8; 95% CI=1-
3.35; P=0.037).
36
Figure 4. (a) Distant recurrence-free survival (DRFS) in 127 patients with breast cancer. (b) DRFS in the 73
patients with breast cancers with Nottingham Histologic Grade (NHG) 1-2. (c) DRFS in the 43 patients with
diploid breast cancer. Patients with tumours that express CD163 in more than 25% of the cells have lower DRFS
in all these groups.
DNA diploid cancer
0 2 4 6 8 10 12 14 16 18 20
Years
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
Dis
tant
recurr
ence-f
ree s
urv
ival
P=0.0048
CD163 negative (n=23)
CD163 posi tive (n=20)
c
NHG 1-2
0 2 4 6 8 10 12 14 16 18 20
Years
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
Dis
tant
recurr
ence-f
ree s
urv
ival
P=0.0094
CD163 positive (n=28)
CD163 negative (n=45)
b
All patients
0 2 4 6 8 10 12 14 16 18 20
Years
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
Dis
tant
recurr
ence-f
ree s
urv
ival
P=0.024
CD163 negative (n=66)
CD163 positive (n=61)
a
37
Figure 5. Kaplan-Meier analysis of survival in patients with breast cancer in relation to DAP12 and the presence
of skeletal, liver and lung metastases. (a) Patients with skeletal metastasis and high DAP12 expression have
lower survival rates (P=0.023). (b) The presence of high DAP12 expression in patients with liver metastases is
also associated with lower survival (P=0.0028). (c) Patients with lung metastases show no differences in survival
rates in relation to the expression of DAP12 (P=0.,64).
Pulmonary metastasis
0 2 4 6 8 10 12 14 16 18
Years
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
Dis
tant
recurr
ence-f
ree s
urv
ival
P=0.64
Negative/weak DAP12 expression (n=88)
Strong DAP12 expression (n=41)
c
Liver metastases
0 2 4 6 8 10 12 14 16 18
Years
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
Dis
tant
recurr
ence-f
ree s
urv
ival
P=0.028
Negative/weak DAP12 expression (n=88)
Strong DAP12 expression (n=41)
b
Skeletal metastases
0 2 4 6 8 10 12 14 16 18
Years
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
Dis
tant
recurr
ence-f
ree s
urv
ival
P=0.023
Negative/weak DAP12 expression (n=88)
Strong DAP12 expression (n=41)
a
38
Figure 6. Kaplan-Meier analysis of survival in patients with breast cancer in relation to DAP12 and CD163
expression. (a) DRFS in all patients according to the presence of DAP12 expression. (b) Survival in patients with
breast cancer expressing both or either CD163 or DAP12.
Table 5 Multivariate analysis of distant recurrence and breast cancer related death in relation to CD163, MAC387
and clinical used prognostic factors by proportional hazard
Distant recurrence Breast cancer death
Hazard ratio
(95% C.I.)
Test for
significance
Hazard ratio
(95% C.I.)
Test for
significance
Lymph node status
N - 1.0 1.0
N+ 2.1 (1.0-4.6) P=0.048 1.9 (0.89-4.2) P=0.094
Tumour size (mm)
≤ 20 1.0 1.0
> 20 1.3 (0.67-2.4) P=0.46 1.7 (0.83-3.5) P=0.15
ER Status
ER - 1.0 1.0
ER + 0.70 (0.34-1.5) P=0.34 0.68 (0.32-1.5) P=0.33
NHG score
NHG 1 1.0 1.0
NHG 2 1.6 (0.59-4.5) P=0.048 2.2 (0.61-7.6) P=0.022
NHG 3 2.6 (0.93-7.2) 3.8 (1.1-13.3)
MAC387 Expression
MAC387 - 1.0 1.0
MAC387 + 0.48 (0.18-1.3) P=0.14 0.42 (0.15-1.2) P=0.11
CD163 Expression
CD163 <25% 1.0
CD163 >25% 1.8 (1.0-3.3) P=0.053 2.0 (1.0-3.8) P=0.044
CD163 and DAP12 expression
0 2 4 6 8 10 12 14 16 18
Years
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
Cum
ula
tive P
rop
ort
ion
Su
rviv
ing
Positive CD163 and/or DAP12 (n=78)
Negative CD163 and DAP12 (n=48)
P=0.00077
bAll patients
0 2 4 6 8 10 12 14 16 18
Years
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
Dis
tant
recurr
ence-f
ree
su
rviv
al
Strong DAP12 expression (n=41)
Negative/weak DAP12 expression (n=88)
P=0.0028
a
39
Paper II
Out of rectal cancer specimens from 169 patients, cancer cells expressed CD163 in 23%
(n=32) of the primary tumours (n=139) and in 10 % (n=10) of the pre-treatment biopsies.
Epithelial cells in 81 normal adjacent and 119 distal mucosal specimens did not show any
CD163 expression (Fig.7). Inter-observer variation calculated according to Cohen was κ
=0.834.
Regardless of preoperative radiotherapy, the patients with initially CD163 positive cancers
had an earlier local recurrence (P=0.044) (Fig. 8a) and a lower survival time (p=0.045) (Fig.
8b). The survival time was related to CD163 expression independently of distal metastasis
(P=0.0036). Postoperative rectal cancer specimens from patients (n=61) treated with
preoperative radiotherapy had a significantly (p=0.044) higher proportion of cancer cells
expressing CD163 compared with corresponding specimens from patients not treated with
preoperative radiotherapy. Tumours with CD163 positive cancer cells were correlated to
metastases and poor survival. This correlation, however, was not further influenced by the
fraction and staining intensity of the positive cancer cells.
Age, gender, TNM stage, angiogenesis, proliferation and differentiation grade were not
correlated to CD163 expression. In the non-radiotherapy group, infiltrative growth was more
common in the CD163 positive than in the CD163 negative group (p=0.022). In general,
CD163 expression was inversely correlated to apoptosis (p=0.018). In tumours from patients
treated with preoperative radiotherapy (n=54) there was significantly higher apoptosis in
Normal rectal epithelium Rectal cancer with CD163 expression
Figure 7 – CD163 expression in rectal cancer. Note that the normal epithelial cells do not exhibit CD163
(left panel) while macrophages in stroma stain for CD163. In right panel, a clone of rectal (right panel)
and stromal macrophages is stained with CD163.
40
CD163 negative than positive tumours (p=0.028). No correlation between CD163 expression
and apoptosis was found in the 73 patients not given preoperative radiotherapy (p=0.165)
(Table 6).
The proliferative activity in cancer cells (expressed as Ki-67), Lymphangiogenesis (D2-40
expression) and neoangiogenesis (CD34 expression) did not differ between CD163 positive
and negative tumours (Table 6).
Figure 8 - Kaplan-Meier survival curves for 101 patients with rectal cancer (biopsies). Patients who express
CD163 acquire (a) earlier local recurrence (p=0,044) and have (b) poor survival compared (p=0,045).
41
CD163 expression was more frequent in specimens from tumours removed at the operation
from patients in preoperative radiotherapy group (31%) than from patients in the non-
preoperative radiotherapy group (17%) (p=0.044). In 88 of the patients with preoperative
biopsies, tissue samples from the cancer taken at the operation were also available. These
surgical specimens were also analyzed for CD163. In those with negative preoperative
biopsies, 12 had turned positive.
Paper III
The aims of this study were to investigate whether the density of macrophages in tumour
stroma (macrophage infiltration) could influence the expression of macrophage antigen
CD163 in colorectal cancer cells. CD163 expression by tumour cells and macrophage
infiltration were examined in whole surgical specimens (not TMA) to evaluate a larger area of
tumour sections. Sections from paraffin-embedded blocks from primary tumours (n=77) and
distal normal mucosa (n=25) from 91 patients with colorectal cancer were included.
Specimens from both primary tumours and distal normal colorectal mucosa were collected
from 11 patients. The cancer cells expressed CD163 in 18% (n=8) of the cases with colon
cancer and 16% (n=5) with rectal cancer. CD163 was not expressed in either adjacent normal
mucosa (n=77) or distal mucosal specimens (n=25).
Patients with tumours expressing CD163 and high macrophage infiltration had shorter
survival than those with no CD163 expression and low macrophage infiltration (Table 7 and
Fig. 10). Cox regression analysis revealed that regardless of tumour stage, the CD163
expression (p=0.015) and macrophage infiltration (P=0.058) had a prognostic impact in
relation to survival time.
CD163 expression is related to advanced stages of colorectal cancer (p=0.007). Colorectal
tumours in 16 patients (21%) showed a high infiltration level of TAM. Macrophage
infiltration was not correlated to CD163 expression, tumour stage, age, and gender or tumour
localization. The expression levels of angiogenesis (CD31) and lymphangiogenesis (D2-40)
markers were not correlated either to CD163 expression in colorectal cancer cells or to
macrophage infiltration in tumour stroma (Table 8).
42
Table 6. Characteristics of patients with rectal cancer in relation to radiotherapy and CD163 expression
Non-Radiotherapy Radiotherapy
CD163-
N (%)
CD163+
N (%) p
CD163-
N (%)
CD163+
N (%) p
Age
<=69 years 36 (88) 5 (12) 24 (71) 10 (29)
>69 years 29 (78) 8 (21) 0.264 18 (67) 9 (33) 0.742
Gender
Female 31 (91) 3 (9) 14 (61) 9 (39)
Male 34 (77) 10 (23) 0.102 28 (74) 10 (26) 0.294
TNM stage
I 20 (91) 2 (9) 13 (76) 4 (24)
IIA + IIIA 20 (77) 6 (23) 14 (64) 8 (36)
IIIB + IIIC 21 (81) 5 (19) 12 (71) 5 (29)
IV 4 (100) 0 (0) 0.457 3 (60) 2 (40) 0.814
Growth pattern
Expansive 49 (87.5) 7 (12,5) 19 (66) 10 (34)
Infiltrative 10 (62.5) 6 (37,5) 0.022 14 (70) 6 (30) 0.742
Differentiation
Low 51 (85) 9 (15) 32 (71) 13 (29)
High 14 (78) 4 (22) 0.470 10 (62.5) 6 (37.5) 0.522
Apoptosis
Negative 16 (73) 6 (27) 6 (46) 7 (54)
Positive 44 (86) 7 (14) 0.165 32 (78) 9 (22) 0.028
Proliferation
Ki-67<30% 25 (93) 2 (7) 17 (65) 9 (35)
Ki-67>60 27 (75) 9 (25) 0.069 14 (70) 6 (30) 0.741
Angiogenesis
CD34 negative 29 (85) 5 (15) 16 (64) 9 (36)
CD34 positive 27 (82) 6 (18) 0.701 17 (65) 9 (35) 0.918
Lymphangiogenesis
D2-40 negative 28 (82) 6 (18) 14 (67) 7 (33)
D2-40 positive 29 (88) 4 (12) 0.525 18 (64) 10 (17) 0.862
43
The proliferation activity in cancer cells, expressed as Ki-67 expression and S-phase fraction,
was significantly associated with CD163 expression. In tumours with high macrophage
density, 10 (83%) exhibit S-phase fraction exceeding 8% (P= 0.007). Although Ki-67
expression was increased in the majority (>90%) of tumours, this was not statistically
significant (p=0.437) in relation to macrophage density in the tumour stroma (Table 8).
To examine the interaction between CD163 expression and macrophage density in relation to
survival time, the patients were classified in four groups. Group 1: patients having tumours
with negative CD163 and low macrophage infiltration; group II: CD163 negative with high
macrophage infiltration; group III: CD163 positive with low macrophage infiltration; and
group IV: CD163 positive tumours with high macrophage infiltration. Patients with positive
CD163 tumours had lower survival time independently of macrophage infiltration level
(P=0.007) (Fig. 10c).
44
Table 7. Survival rates in patients with colorectal cancer estimated in relation to CD163 expression and
macrophage infiltration in tumour stroma.
Survival rates
Mean (SD) 1- year (%) 5 - year (%) p
CD163 expression
Negative 70 (±39) 90 64
Positive 34 (±45) 70 21 0.034
TAM infiltration
Low 69 (±46) 88 61
High 43 (±40) 81 38 0.058
Figure 10. Kaplan-Meier survival curves for 77
patients with colorectal cancer. Patients with positive
CD163 expression (a) have poor survival
independently to tumour stage (p=0,034).
Macrophage density in tumour stroma is related to
lower survival time but statistically not significant
(P=0,058). CD163 expression in colorectal cancer as
prognostic factor is not correlated to macrophage
infiltration (p= 0,007)
45
Table 8. CD163 expression and macrophage infiltration in relation to clinical and biological data.
CD163 expression in colorectal cancer
cells
Macrophage infiltration with CD163 as a
macrophage marker
Negative Positive Low High
No. of
Samples (%)
No. of
Samples (%) p
No. of Samples
(%)
No. of
Samples (%) p
Gender
Male 34 (54) 8 (57) 32 (52.5) 10 (62.5)
Female 29 (46) 6 (43) 0.829 29 (47.5) 6 (37.5) 0.473
Age (year)
<=59 9 (82) 2 (18) 9 (15) 2 (13)
60-69 16 (89) 2 (11) 14 (23) 4 (25)
70-79 24 (80) 6 (20) 24 (39) 6 (37)
>=80 14 (78) 4 (22) 0.832 14 (23) 4 (25) 0.992
Localization
Right Colon 26 (81) 6 (19) 26 (44) 6 (37.5)
Left Colon 11 (85) 2 (15) 9 (15) 4 (25)
Rectum 25 (83) 5 (17) 0.956 24 (41) 6 (37,5) 0.654
Stage
I 9 (14) 0 (0) 8 (13) 1 (6)
II 27 (43) 1 (7) 9 (32) 6 (38)
III 19 (30) 8 (64) 8 (30) 5 (31)
IV 8 (13) 5 (29) 0.007 4 (31) 4 (25) 0.612
CD31
<median 20 (45) 7 (70) 20 (51) 7 (47)
> median 24 (55) 3 (30) 0.161 19 (49) 8 (53) 0.761
D2-40
<median 20 (45) 7 (80) 20 (51) 8 (53)
>median 24 (55) 3 (20) 0.161 19 (49) 7 (47) 0.761
Ki-67
Low expression 1 (2) 2 (20) 2 (4) 1 (9)
High expression 54 (98) 8 (80) 0.011 52 (96) 10 (91) 0.437
S-phase
<8% 29 (58) 3 (25) 30 (60) 2 (17)
>=8% 21 (42) 9 (75) 0.039 20 (40) 10 (83) 0.007
46
DISCUSSION
According to the hypothesis of cell fusion, somatic cell hybridization between non-metastatic
cancer cells and macrophages results in hybrid cells manifesting phenotypic properties of both
mother cells. These hybrids may acquire new growth and metastatic properties and generate
highly malignant tumour variants. TAMs play an important role in the tumour
microenvironment, supporting tumour growth and progression by inducing neoangiogenesis
and stroma production as well as producing tumour-stimulating growth factors 129, 130. In most
cancers, a high density of TAMs predicts poor outcome. In vitro co-culture of macrophages
with tumour cells results in accelerated tumour growth 131. The biological cascade of
metastasis involves loss of cellular adhesion, increased motility and invasiveness, entry and
survival in the circulation, exit into new tissue, and eventual colonization of a distant site 132,
133. These functional and phenotypic characteristics are similar to the normal function of
macrophages, including cell-spreading capabilities, migration and matrix invasion.
Cell fusion is common in biology 32. Cancer cells may fuse spontaneously with several types
of somatic cells 44, 54, 134, 135. During the past decade, several experimental studies have
reported evidence suggesting that macrophage traits in cancer cells could result from
macrophage-tumour cell fusion 29, 43, 48, 136-141. Fusion is an important function of macrophages
and results in the generation of osteoclasts in bone tissue and giant cells in chronic
inflammation. In vitro fusion between macrophages and malignant cells generates hybrids
with enhanced metastatic ability 42, 55, 56, 59. Heterotypic cell fusion between BMDCs and
somatic cells has been demonstrated in several studies 39, 43-45, 142 and might be a source of
multiple tumour types 54.
A key, but challenging, issue in cell fusion research is to identify hybrids generated after
fusion between cells from two or several tissue types. To show that a specific cell type might
originate from two different cell or tissue origins, specific markers from both donor cells must
be demonstrated. CD163 is exclusively expressed in the monocyte-macrophage system 78, 143,
144 and was therefore used in the present thesis to investigate macrophage traits in breast and
colorectal cancers.
In Paper I, the cancer cell expression of CD163 is reported in about 48% of 127 patients with
breast cancer. Patients with CD163 positive breast cancer had distant metastases earlier and
had shorter survival. CD163 showed a significant impact as a prognostic factor in relation to
survival rates. These results are, to our knowledge, the first reported clinical data
demonstrating macrophage traits in solid tumours and thus substantiate previous preclinical
47
evidence demonstrating fusion between cancer cells and macrophages.
Breast cancer cells expressed even MAC387 in 12% of the patients. MAC387 is another
antigen expressed by granulocytes, monocytes and tissue macrophages145 but is reported even
in other tissues, including melanocytes, keratinocytes and epithelial cells 146-148. Thus,
MAC387 is not considered an exclusively macrophage-specific antigen and therefore was not
used in the other studies in the present thesis.
CD163 expression in breast and colorectal cancer cells can result from severe histological
aberrations due to genomic instability with expression of many heterotypic proteins. Another
feasible explanation is that cancer cells tend to be less differentiated and maintain their stem
cell properties with high cellular plasticity. Because of factors in the tumour
microenvironment, such as the presence of IL-10 80, 81, 149, 150, cancer cells may adopt
macrophage phenotypes. Recent studies indicate that the extracellular factors or cell – cell
interaction might be sufficient for reprogramming adult cells into a more pluripotent status 151-
154. Considering these findings, CD163 expression was tested in 5 breast cancer cell lines,
with and without stimulation by IL-10 and TGF-β. No expression of CD163 was found during
basal conditions or after stimulation with Il-10 or TGF-β. It should, however, be noted that
adjacent fibrocytes, lipocytes and normal ductal epithelium do not express this antigen.
CD163 was not expressed by normal breast epithelium from patients with or without breast
cancer.
Aneuploidy, the alteration in the number of chromosomes, is a fundamental property of
cancer. Both mutations and cell fusion can cause aneuploidy in cancer cells 31, 155. Cell fusion
generates bi- or multinucleated hybrids that, in later stages, undergo division into daughter
cells containing genetic material from both parental cells 39, 156. In Paper I, comparing CD163
expression with DNA ploidy showed that 7 of 8 patients with skeletal metastases and diploid
breast cancers had CD163 positive tumours. Tentatively, this observation may indicate cell
fusion as an explanation for aneuploidy in cancer.
Macrophage traits in rectal cancer cells and irradiation
The aim of the second paper was to study the presence of CD163 in rectal cancer and its
correlation to irradiation. CD163 expression in cancer cells was found in 17% of the surgical
specimens not treated with preoperative irradiation, compared with 31% of those given
preoperative radiotherapy. Neither adjacent nor distant normal rectal epithelium showed any
expression of CD163.
48
Recently, Johansson and Nygren independently reported increased heterotypic cell fusion in
response to chronic inflammation45, 142. Chronic inflammation is associated with several types
of cancers. This evidence may explain why the CD163 expression reported in Paper II is more
frequent in irradiated rectal cancers. Another explanation might be that CD163 positive cells
are less apoptotic and more resistant to irradiation than CD163 negative cancer cells. Note
that apoptosis after radiotherapy is more common in CD163 negative than in CD 163 positive
tumours, whereas in non-irradiated tumours, the presence of apoptosis was associated with
CD163.
Expression of CD163 in the cancer cells is associated with earlier local recurrence and death.
Radiotherapy is associated with increased CD163 expression but with later local recurrence
and death111. The explanation to these contradictory findings must be that radiotherapy
induces several cytotoxic effects besides causing expression of CD163.
Macrophage traits in cancer cells and macrophage infiltration
In Paper III, CD163 expression was examined in relation to macrophage infiltration in tumour
stroma to examine whether the number of macrophages could influence their phenotypes in
cancer cells. In this paper, the presence of CD163 in cancer cells is reported in a third patient
material consisting of 77 new patients with colorectal cancer. CD163 expression is associated
with advanced tumour stage and poor survival. These observations coincide with findings in
Papers I and II, which confirm that the presence of CD163 and its association with
clinicopathological variables, as well as its prognostic significance, are reproducible in three
different patient groups with three different types of solid tumours.
TAMs have complex dual functions in their interaction with neoplastic cells and are part of an
inflammatory process that promotes tumour progression 157. High infiltration of TAMs in
many tumour types is correlated with lymph node involvement and distant metastasis 67.
Inhibition of macrophage infiltration in tumours may inhibit metastasis and the progression of
secondary tumours 68, 69. The clinical significance of macrophage infiltration in tumour
stroma, however, is not clear. A high infiltration of TAMs has an unfavourable prognostic
impact in breast, prostatic, ovarian and cervical carcinoma 31, 70, 158, but is still controversial in
colorectal cancer 70-72. In Paper III, no correlation was found between macrophage infiltration
and tumour stage. The use of different macrophage markers and scoring systems to evaluate
macrophage infiltration may explain the contradictory findings of macrophage infiltration in
clinical studies.
49
CD163 expression and macrophage infiltration in cancer cells are also significantly associated
with tumour cell proliferation. Interaction between neoplastic cells and the tumour
microenvironment is crucial to tumour genesis. In vitro co-culture of macrophages with
tumour cells results in accelerated tumour growth 131, probably because TAMs express several
factors such as EGF, MMP-9, TGF-β and CXCL-14 that stimulate tumour cell proliferation
and survival 129, 130. The prognostic significance of CD163 expression in the cancer cells is not
correlated to the macrophage density in the tumour stroma. These results suggest that
macrophages may promote tumour growth and progression by heterotypic fusion and/or an
autocrine interaction with the cancer cells. The prevalence of macrophage-cancer cell fusion
is probably independent of macrophage intensity in tumour stroma.
Macrophage fusion antigen is expressed by breast cancer cells
DAP12 is essential for macrophage fusion, and the production and function of osteoclasts 159-
162. To determine whether the DAP12 macrophage phenotype was clinically correlated to
factors involved in macrophage function and fusion, immunostaining with DAP12 was
performed in 133 breast cancer patients. Tumour cells in the bone microenvironment
stimulate osteoclast and osteoblast recruitment and activation. Cancer cells also activate the
release of growth factors from stromal cells and from the bone matrix, which further promote
tumour growth in bone tissue 163, 164. Activated osteoclasts promote the growth of tumour cells
in the bone microenvironment 165, whereas osteoclast dysfunction results in decreased bone
metastasis and tumour-associated bone destruction 166, 167. To our knowledge, the role of
DAP12 in the fusion of macrophages with cancer cells has not previously been investigated.
DAP12 is significantly associated with skeletal and liver metastasis but not with lung
metastasis.
Consistent with experimental studies, the results in Paper IV indicate that DAP12 expression
in breast cancer cells (in vivo) promotes the interaction and fusion of cancer cells with
macrophages. High expression of DAP12 in breast cancer cells may also constitute a
phenotypic advantage for these cells to fuse with macrophages and form hybrids with
extended metastatic capacity. The significant association between DAP12 expression and
skeletal metastasis in breast cancer might thus be explained by a “seed and soil hypothesis”.
Briefly, the concept of this theory is that certain tumour cells (seeds) colonize selectively
distant organs (soil) with a favourable environment that facilitates the survival of tumour
cells. Breast cancer cells with high expression of DAP12 and phenotypic similarity to
macrophages may favour metastatic spread preferentially to bone and liver tissue, in which
macrophages are an important cellular component.
50
51
CONCLUSIONS
Breast and colorectal cancer cells express macrophage traits, illustrated by the
macrophage-specific antigen CD163.
CD163 expression is correlated to metastasis and poor survival in these tumours.
CD163 is a significant prognostic factor in breast and colorectal cancers.
CD163 expression is more frequent and inversely correlated to apoptosis after
irradiation, indicating that CD163 positive cells may increase or/and are more resistant
to irradiation than CD163 negative cancer cells.
The expression of CD163 in colorectal cancer is not correlated to the degree of
macrophage infiltration.
Cancer cell proliferation is correlated to both macrophage infiltration and CD163
expression in cancer cells.
DAP12 is expressed in breast cancer and significantly associated with skeletal and
liver metastasis as well as poor prognosis, suggesting that DAP12 expression may
promote fusion between breast cancer cells and macrophages and/or constitute a factor
that favours cancer cell homing preferentially to bone and liver tissue.
Proposing that CD163 expression is caused by macrophage-cancer cell fusion, the
findings in this thesis support the hypothesis of cell fusion and illustrate spontaneous
tumour-macrophage hybridization in vivo and its clinical impact on tumour
progression and metastasis
52
SAMMANFATTNING PÅ SVENSKA
Utvecklingen av cancer (carcinogenes) är en avancerad biologisk process som består av en
rad progressiva cellulära förändringar från premalign till malign fenotyp. Det är oklart varför
cancer metastaserar. En förklaring är att cancercellen utvecklar metastaseringsförmågan
genom en progressiv ansamling av somatiska genetiska förändringar (t.ex. genmutationer)
som styr bl.a. cellens tillväxt, differentiering och apoptos (programmerad celldöd).
En alternativ förklaring är att cancercellen omprogrammeras genom sammanslagning (fusion)
med celler, t.ex. makrofager, som redan har egenskaper att sprida sig längs blod- och lymfkärl
och att invadera andra vävnader. Denna teori (hypotesen om cellfusion) hävdar att
cancerceller, genom heterotypisk fusion med somatiska celler, producerar hybrider med
tillväxtfrämjande egenskaper och metastaseringsförmåga. Cellfusion är en vanlig biologisk
process och förekommer normalt i placenta, vid foster- och skelettmuskelutveckling samt
bildning av osetoklaster i benvävnad. Cellfusionsteorin är fortfarande spekulativ även om
flera rapporter under de senaste 10-15 åren har kunnat bevisa att makrofag-cancer fusion
förekommer både in vitro och in vivo samt producerar hybrider med metastaseringsförmåga.
Den kliniska betydelsen av cellfusion vid cancer är oklar. Syftet med denna avhandling är att
studera förekomsten av cellfusion och dess kliniska betydelse i bröstcancer och
kolorektalcancer, som är två vanligt förekommande typer av solida tumörer.
Med utgångspunkt i hypotesen om cellfusion och antagandet att makrofag-cancer fusion kan
skapa hybrider som uttrycker egenskaper från båda modercellerna, undersöktes via
immunohistokemin paraffin-inbäddade tumör- och normal vävnadsprover från patienter med
bröstcancer (n = 133) och kolorektalcancer (två olika patientmaterial med totalt 240 patienter)
med en makrofagspecifik antigen CD163. Uttrycket av CD163 analyserades i förhållande till
makrofaginfiltration och flera kliniska och patologiska variabler, såsom tumörstadium, tid till
metastaser, överlevnadstid, strålning, DNA ploiditeten samt proliferation och apoptos i
cancerceller.
Makrofagfenotyp, uttryckt som CD163, förekommer i både bröst- och kolorektalcancer och är
signifikant korrelerad till avancerad tumörstadier och kortare överlevnad. CD163 expression
var vanligare efter bestrålning och förknippat med minskad apoptos. Proliferation i cancer
celler är relaterad till både makrofaginfiltration och uttryck av CD163.
53
DAP12 är ett membranprotein som uttrycks av makrofager och är viktig för dess
fusionsförmåga och osteoklastfunktion och- bildning. DAP12-uttryck undersöktes i
bröstcancerprover från 133 patienter. DAP12 uttrycks av bröstcancerceller och är signifikant
relaterad till avancerad tumörstadier, skelett- och levermetastaser samt dålig prognos. CD163
var inte korrelerad till vare sig DAP12-uttryck eller makrofaginfiltration. Multivariat analys
visar att CD163 är en viktig prognostisk faktor för både bröst- och kolorektalcancer.
Slutsatserna i denna avhandling stödjer hypotesen om cellfusion och illustrera dess kliniska
betydelse för tumörprogression och metastatering i bröstcancer och kolorektalcancer.
54
ACKNOWLEDGEMENTS
This thesis was produced at the Department of Surgery, Institution for Clinical and
Experimental Medicine (IKE), Linköping University, Sweden. It was a great privilege to
develop my scientific skills as a PhD student at such an excellent academic environment as
this institution. The path towards this thesis spans over several years, and many people have
been involved in and contributed to the ideas presented and the understanding gained. I would
like to acknowledge my debt to all who have helped me during this exciting and unforgettable
journey.
In particular, I wish to express my sincere gratitude to my supervisor Professor Joar Svanvik
for continuous support of my Ph.D. study and research, for his patience, motivation,
encouragement, enthusiasm, kindness, thoughtful guidance and immense knowledge. I could
not have imagined having a better advisor and mentor for my Ph.D. study.
I would also like to express my profound gratitude to Professor Olle Stål and Professor Xiao-
Feng Sun, not only for providing me with the tissue and patient materials included in thesis
but mainly for their generous support, kindness, encouragement, excellent scientific advice
and fruitful discussions.
I am deeply indebted to my dear friend and co-author, Dr Hans Olsson, for introducing me to
the field of cancer histopathology and immunohistochemistry, for his invaluable support,
generosity, friendship and profound knowledge in tumour biology.
To my co-authors, Dr. Siv Doré and Dr. Rihab Elkarim at the department of pathology for
helping me to evaluate immunohistochemical tissue samples as second examinors.
I am deeply grateful to Birgitta Frånlund for kind and excellent practical support with
immunohistochemistry, for her generosity and valuable advice and for teaching me the
techniques of histopathology. I would like to thank Birgitta Holmlund for introducing me to
the field of cancer cell lines and TMA and for practical help in Paper I and IV.
My sincere thanks to Professor Bertil Lindblom, Professor Anders Rosén, Professor Ingemar
Rundquist, Professor Jon Jonasson and Professor Peter Söderkvist for your support and
stimulating scientific discussions in tumour and cell biology.
55
I would like to show my gratitude to my clinical mentors, Dr. Kalev Teder and Dr. Hans
Krook, and to all my colleagues at the Department of Surgery in both Norrköping and
Linköping for your encouragement, kindness and for being wonderful colleagues.
I would also like to thank Lena Trulsson and all the members of the HPG research group for
your splendid collaboration and exciting scientific discussions.
To Dr. Cecilia Gunnarsson, Professor Olle Stål, Dr. Staffan Haapaniemi, Dr. Ann Cherian and
Dr. Claes Juhlin. Thank you for proofreading this thesis.
Last but not least, I would like to thank my family. This thesis would not have been possible
without you all. First and foremost, I owe my deepest gratitude to my beloved mother, Sabiha
Touma Shabo, for her enormous and invaluable sacrifices, constant inspiration, support and
love. To my beloved Josefin, thank you for all your support, love and patience. I am grateful
to my dear brothers Julian and Sivan for their love, never-ending encouragement and great
friendship. A special thought is devoted to the memory of my beloved father for being a
powerful source of inspiration and energy.
56
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