Cancer and Inflammation

Cancer and Inflammation
Role of Macrophages and Monocytes
Alexander Hedbrant
Faculty of Health, Science and Technology

Biomedical Sciences
DISSERTATION | Karlstad University Studies | 2015:36

Role of Macrophages and Monocytes
Alexander Hedbrant
Cancer and Inflammation - Role of Macrophages and Monocytes

Alexander Hedbrant
DISSERTATION
Karlstad University Studies | 2015:36
URI: urn:nbn:se:kau:diva-37086
ISSN 1403-8099
ISBN 978-91-7063-654-7
The author
Distribution:
Karlstad University
Faculty of Health, Science and Technology
Department of Health Sciences
SE-651 88 Karlstad, Sweden
+46 54 700 10 00
Print: Universitetstryckeriet, Karlstad 2015
WWW.KAU.SE
It doesn't stop being magic just because you know how it works.
Terry Pratchett, The Wee Free Men
Abstract
Macrophages are cells of the innate immune system that can be found
in large quantities in cancer tumors and affect cancer progression by
regulating growth and invasiveness of cancer cells. There are two
main phenotypes of macrophages denoted M1 and M2. In this thesis,
the M1 and M2 phenotype of human macrophages were characterized,
and effects of the macrophages on the growth and invasiveness of colon and lung cancer cells were studied.
Macrophages of the M1 phenotype, but not the M2 phenotype, inhibited growth of both colon and lung cancer cells, and the inhibition for
some of the cancer cell lines was induced by cell cycle arrest in the
G1/G0 and/or G2/M cell cycle phases. In the colon cancer cell line,
the macrophage induced cell cycle arrest was found to attenuate the
cytotoxic effect of the chemotherapeutic drug 5-FU. Macrophages
were also shown to express high levels of proteases (matrix metalloproteinase-2 and 9) and high levels of proteins of the urokinase-type
plasminogen activator (uPA) system, in comparison to the lung cancer
cell lines studied. Expression of these has been found to predict poor
outcome in lung cancer, and the results suggest macrophages to be
important contributors of these in lung tumors. Furthermore, the M1
phenotype was found to express higher levels of the uPA receptor
than the M2 phenotype.
Prostaglandin E2 (PGE2) is a potent inflammatory molecule expressed by e.g. macrophages and monocytes, and inhibition of its expression has been shown to reduce the risk of colon cancer. Green tea
and black tea was found to be potent inhibitors of PGE2 formation in
human monocytes, and the inhibitory effects of green tea was likely
due to its content of the polyphenol epigallocatechin gallate. Rooibos
tea also inhibited PGE2 formation, but was less potent than green and
black tea. The primary mechanism for the inhibition was via inhibition of expression of enzymes in the PGE2 formation pathway, and
primarily microsomal prostaglandin E synthase-1.
List of Content
ABSTRACT ........................................................................................................................... 2
LIST OF CONTENT .............................................................................................................. 3
LIST OF PAPERS ................................................................................................................. 5
THE AUTHORS CONTRIBUTION TO THE PAPERS ........................................................ 6
COMMONLY USED ABBREVIATIONS ............................................................................... 7
INTRODUCTION ................................................................................................................... 8
Cell cycle regulation ........................................................................... 8

Colorectal cancer ............................................................................... 10
Treatment of CRC...............................................................................11
5-fluorouracil ......................................................................................11
Lung cancer ....................................................................................... 13
Risk factors for lung cancer .............................................................. 14
The tumor microenvironment .......................................................... 15
Macrophages ..................................................................................... 15
The M1 phenotypes of macrophages ................................................ 17
The M2 phenotypes of macrophages ................................................ 18
Tumor associated macrophages ....................................................... 19
Monocytes ......................................................................................... 19
The extra cellular matrix .................................................................. 20
The urokinase-type plasminogen activator system......................... 20
The urokinase-type plasminogen activator system in cancer ..........23
Polyphenol content of green, black and rooibos tea ........................23
Prostanoids ........................................................................................ 25
Prostaglandin E2 ...............................................................................26
Prostaglandin E2 in cancer ............................................................... 27
BACKGROUND AND AIM.................................................................................................. 29
METHODS .......................................................................................................................... 30
Culture of cancer cell lines ............................................................... 30

Isolation of human peripheral blood monocytes and differentiation
into macrophages .............................................................................. 31
Differentiation of THP-1 monocytes into macrophages ..................32
Cell counting ......................................................................................32
Detection of proteins and PGE2 .......................................................33
Enzyme linked immunoassays .............................................................................................. 33
Western blot .......................................................................................................................... 33
Immunostaining ..................................................................................................................... 34
Protein siRNA knockdown ................................................................ 35

RNA extraction and cDNA synthesis ................................................ 35
Quantitative real-time PCR...............................................................36
Cell cycle analysis ..............................................................................36
Apoptosis ........................................................................................... 37
Preparation of tea extracts ................................................................ 37
Cell free assay for the combined enzymatic activity of COX-2 and
mPGES-1............................................................................................ 37
Gelatin zymography ......................................................................... 38
RESULTS AND DISCUSSION ........................................................................................... 39
Characteristics of cultured human macrophages of different

phenotypes ........................................................................................39
Macrophages and cancer cell growth ...............................................44
Mechanisms of growth inhibition .......................................................................................... 46
Implication of macrophages during 5-FU treatment of colon cancer

cells ................................................................................................... 49
Anti-inflammatory effects of green, black and rooibos tea extracts 52
Macrophage expression of proteases or protease activity-related
genes and implications for lung cancer cells .................................... 54
CONCLUDING REMARKS ................................................................................................. 57
ACKNOWLEDGEMENTS ................................................................................................... 59
REFERENCES .................................................................................................................... 61
List of papers
I.
Engstrm A, Erlandsson A, Delbro D and Wijkander J:
Conditioned media from macrophages of M1, but not M2 phenotype,
inhibit the proliferation of the colon cancer cell lines HT-29 and
CACO-2. Int J Oncol 44: 385-392, 2014.
II.
Hedbrant A, Erlandsson A, Delbro D, Wijkander J: Conditioned media from human macrophages of M1 phenotype attenuate
the cytotoxic effect of 5fluorouracil on the HT29 colon cancer cell
line. Int J Oncol 46: 37-46, 2014.
III.
Hedbrant A, Wijkander J, Seidal T, Delbro D, Erlandsson A: Macrophages of M1 phenotype have properties that influence
lung cancer cell progression. Tumor Biol. 2015
IV.
Hedbrant A, Nathalie Oberschmid, Bart van Aerde, Ingrid Persson, Ann Erlandsson, Wijkander J: Green, black and rooibos
tea inhibit prostaglandin E2 formation in human monocytes by inhibiting expression of enzymes in the prostaglandin E2 pathway. Manuscript
*Name change august 2014 from Engstrm to Hedbrant
The authors contribution to the papers

Paper I A major part of experimental work, evaluating results,
planning and writing
Paper II A major part of experimental work, evaluating results,
Paper III A major part of evaluating results and writing, part of experimental work and planning
Paper IV A major part of experimental work, evaluating results,
Commonly used abbreviations

5-FU
CCL
CD
CDK
CM
COX
cPLA2
CRC
CXCL
DPD
ECM
EGCG
FCS
GM-CSF
IFN
IL
iNOS
LPS
M-CSF
MMP
MTHFR
mPGES
(N)SCLC
PAI
PG(E2)
SCC
siRNA
TAM
TLR
TNF-
TP
TS
UMPS
uPA(R)
5-fluorouridine
C-C motif ligand chemokine
cluster of differentiation
cyclin-dependent kinase
conditioned media
cyclooxygenase
cytosolic phospholipase A2
colorectal cancer
C-X-C motif ligand chemokine
dihydropyrimidine dehydrogenase
extracellular matrix
epigallocatechin gallate
foetal calf serum
granulocyte-macrophage colony stimulating factor
interferon
interleukin
inducible nitric oxide synthase
lipopolysaccharide
macrophage colony stimulating factor
matrix metalloproteinase
methylene tetrahydrofolate reductase
microsomal prostaglandin E synthase
(non)-small cell lung cancer
plasminogen activator inhibitor
prostaglandin (E2)
squamous cell carcinoma
small interfering RNA
tumor associated macrophage
toll-like receptor
tumor necrosis factor alpha
thymidine phosphorylase
thymidylate synthetase
uridine monophosphate synthetase
urokinase-type plasminogen activator (receptor)
Introduction
Cancer is the name for diseases in which cells acquire an uncontrolled
cell division and are able to invade nearby tissues and spread to other
organs via the blood and lymph systems. As stated, cancer is not one
disease, but instead represents hundreds of diseases originating from
different tissues of the body and from different cell types. Cancer is a
leading cause of death worldwide, second only to cardiovascular diseases [1]. The development of cancer is a slow process that requires
multiple genomic alterations. The alterations can either be direct
changes to the DNA sequence, i.e. mutations, or alterations of the
DNA that affects the expression of genes without changing the DNA
sequence, so called epigenetic alterations. Genetic alterations commonly found in cancer are those that affect genes which regulate functions such as cell division, apoptosis or DNA repair. During the progression from normal cells to malignant ones, the cells will have to
acquire multiple features necessary to become cancer cells. In year
2000, six hallmarks was proposed as necessary for cells to become
malignant, which included: evading growth suppressors, resisting cell
death, inducing angiogenesis, sustaining proliferative signaling, enabling replicative immortality and activating invasion and metastasis
[2]. Further hallmarks of cancer have been proposed since then that
includes evading immune destruction and deregulating cellular energetics [3]. Although genetic alterations are vital for cancer progression, an accumulating amount of data stresses the importance of the
inflammatory cells within the tumor stroma which creates a tumor
microenvironment in which cancer cells can thrive and progress [3,
4].
Cell cycle regulation

In order to maintain the structure and function of tissues and organs,
regulation of the amount of cells present is necessary. The formation
of new cells through cell division and the removal of old ones through
programmed cell death (apoptosis) are tightly controlled during normal conditions. Cells can be in four different phases of a cell cycle,
termed Gap (G)1, synthesis (S), G2 and mitosis (M)-phase. A cell
which has not initiated cell division is in the G1 phase and exerts its
8
biological functions and grows in size until it is ready to proceed

through the cell cycle. Once the cell has passed a checkpoint in G1
called the restriction point, it will be dedicated to go through each
phase of the cell cycle, even in the absence of growth signals [5]. In Sphase it synthesizes a double setup of its genome. G2 is an intermediate control-phase before the initiation of mitosis in which the chromosomes segregate and the cell divides (cytokinesis). After cell division the two daughter cells are once again in G1. Cells in G1 may also
exit the cell cycle and enter a quiescent stage called G0 [6].
The progression through each cell cycle phase is facilitated by the action of cyclin-dependent kinases (CDKs) which are activated by proteins called cyclins, named so because the amount of these proteins
cycles throughout the different cell cycles. Thus, certain cyclin-CDK
complexes are important for the progression through different phases
of the cell cycle. For instance, cyclin D type cyclins are needed for G1S transition [7]. They bind to and activate CDK4 or CDK6 which in
turn inactivates the retinoblastoma protein (Rb) by phosphorylation.
Rb normally blocks the E2F transcription factors; therefore inactivation of Rb enables E2F mediated expression of other cyclins and proteins needed for progression into S-phase.
For the discovery of CDKs, cyclins and cell cycle checkpoints, the researchers Paul Nurse, Timothy Hunt and Leland Hartwell were
awarded the Nobel Prize in 2001 [8].
The amount of active cyclin-CDK complexes available is modulated by
several mechanisms. The amount of cyclins available is controlled by
transcriptional regulation and/or by ubiquitin-dependent degradation
by the proteasome [9]. Furthermore, the cyclin-CDK complexes can
be inactivated by cyclin kinase inhibitors and also by inhibitory phosphorylation by Wee1 family of kinases (Wee1 and Myt1) [10, 11]. The
inhibitory phosphate groups can in turn be removed by active CDC25
phosphatases (CDC25 A-C). The expression and activation of cell cycle regulatory genes can be induced e.g. if the integrity of the DNA is
disturbed or if the cell is deprived of essential nutrients. If the damage
to the cell is beyond repair, it can instead be triggered to enter apoptosis, a controlled destruction of the cell.
Colorectal cancer
Cancer originating from the colon or rectum (colorectal cancer, CRC)
is the third most common cancer for both men and women, and one
of the leading causes of cancer death, with 700 000 deaths worldwide
in 2012 [12, 13]. About 95% of all CRC is adenocarcinomas, meaning
that they originate from the epithelial cells of the colon. Hereditary
CRC syndromes, including Familial Adenomatous Polyposis (FAP)
and Hereditary Non Polyposis CRC (HNPCC) accounts for approximately 5% of all CRC. These hereditary diseases are due to inherited
mutations in the tumor suppressor gene Adenomatous Polyposis Coli
(APC) for FAP or in genes of the DNA mismatch repair system, such
as MutL homolog 1 (MLH1) or MutS homolog 2 (MSH2) for HNPCC
[14]. Inheritable susceptibility has been estimated to account for
about 35% of all CRC, and conversely, about 65% of all CRC is due to
environmental factors [15]. For sporadic CRC, known non-lifestyle
risk factors include age and inflammatory bowel diseases i.e. Crohns
disease and ulcerative colitis [16]. Lifestyle factors associated with an
increased risk of CRC include physical inactivity and obesity, high
consumption of red or processed meat or alcohol, and a low consumption of fibers, while aspirin use reduces the risk of CRC [17-20]. CRC
is much more common in developed regions such as Europe and
North America with incidence rates up to 10 times higher compared
to developing regions, due to many of the risk factors being strongly
associated with a western world lifestyle [21].
The progression of CRC from benign adenomas into adenocarcinomas
is a slow process suggested to take 10-15 years in which multiple genetic alterations are acquired. There are three known molecular
pathways for the progression of CRC, mediated either by chromosomal instability (CIN), microsatellite instability (MSI) or the CpG island methylator phenotype (CIMP) [22]. The CIN pathway which accounts for approximately 70% of all sporadic CRC was first described
by Faeron & Vogelstein in 1990 [23]. It involves a multistep progression with inactivation of tumor suppressors e.g. APC, and at a later
stage p53, and also activation of oncogenes, e.g. k-RAS; mutations
primarily acquired through loss or gain of large chromosomal regions
due to chromosome instability. Chromosome instability has been suggested to arise from mutations in genes that control chromosome segregation during cell division [24]. MSI, the second molecular pathway
10
which accounts for approximately 15% of all CRC, is due to somatic

mutations in genes of the DNA mismatch repair system [25]. MSI
primarily cause frameshift mutations in genes containing microsatellite repeats, i.e. simple DNA sequence repeats of 1-6 base-pair. The
third molecular pathway for CRC, CIMP, is characteristic for epigenetic silencing of tumor suppressor genes or DNA mismatch repair
genes through methylation of promoter regions rich in 5-cytocineguanine-3 repeats (CpG islands) via DNA-methyltransferase [26].
The CIN/MSI/CIMP pathways are not mutually exclusive but can all
contribute to various degrees in sporadic CRC progression [27].
Treatment of CRC
Treatment of colorectal cancer includes surgical resection of the tumor, which can be combined with adjuvant chemotherapy for colon
cancer or radiation therapy (alone or in combination with preoperative chemotherapy) for rectal cancer. The chemotherapy primarily involve a combination therapy that include the cytostatic drug 5fluorouracil (5-FU), folinic acid (also known as leucovorin) which enhances 5-FU cytotoxicity, and either the cytostatic drug oxaliplatin
(platinum-based compound) or irinotecan (topoisomerase inhibitor),
which gives rise to the FOLFOX or FOLFIRI chemotherapy regimen,
respectively [28]. Chemotherapy can be supplemented with monoclonal antibody therapy against the biological targets vascular endothelial growth factor or epidermal growth factor receptor [29].
5-fluorouracil
5-FU was discovered already in 1957, and is still a highly used cytostatic drug for the treatment of CRC and other cancers, although there
are now also modified versions of it in use, e.g. the orally administered capecitabine which is a prodrug that is enzymatically converted
into 5-FU in the body [30, 31]. 5-FU is classified as an anti-metabolite
and its primary mode of action is through inactivation of the enzyme
thymidylate synthetase (TS). Using 5-10 methylenetetrahydrofolate
(5, 10-MTHF) as cofactor, TS catalyzes the conversion of deoxyuracil
monophosphate (dUMP) into the nucleotide deoxythymidine monophosphate (dTMP), therefore, inhibition of TS leads to a shortage of
11
dTMP and subsequent failure for the cell to proceed through S-phase
of the cell cycle. This will lead to cell death or cell cycle arrest.
In order for 5-FU to become active, it needs to be enzymatically converted inside the cell to fluorodeoxyuridine monophosphate
(FdUMP). This is a multistep process starting with conversion of 5-FU
into 5-fluorodeoxyuridine by thymidine phosphorylase (TP). FdUMP
inhibits TS through a ternary complex which includes FdUMP, TS and
also its cofactor 5, 10-MTHF. Folinic acid increases the efficacy of 5FU because it elevates the intracellular levels of 5, 10-MTHF [32]. 5FU may also be converted into fluorouridine triphosphate (FUTP) and
cause RNA damage, starting with conversion of 5-FU into fluorouridine monophosphate (FUMP) by uridine monophosphate synthetase
(UMPS) [33]. 5-FU is catabolized into the non-toxic substance dihydrofluorouracil by dihydropyrimidine dehydrogenase (DPD). DPD
which is highly expressed in the liver, rapidly degrade most of the
administered 5-FU, giving it a short half-life of about 10 min in vivo
[34]. DPD deficiency is found in about 3-5% of the population which
can cause severe side-effects of 5-FU treatment, or even death [35]. A
simplified model of 5-FU metabolism can be seen in figure 1.
Figure 1. Metabolism and effects of 5-fluorouracil (5-FU). Enzyme abbreviations;

DPD: dihydropyrimidine dehydrogenase, TP: thymidine phosphorylase,
UMPS: uridine monophosphate synthetase, TS: thymidylate synthetase,
MTHFR: methylene tetrahydrofolate reductase
12
Lung cancer
Lung cancer is the leading cause of cancer death, with an annual 1.5
million deaths worldwide [13]. Cancers originating from the lungs are
commonly divided into non-small cell lung cancer (NSCLC) or small
cell lung cancer (SCLC), with approximately 15% of the lung cancers
being SCLC and 85% NSCLC [12]. NSCLCs, which are of bronchial
epithelial origin, can be further divided into adenocarcinoma, squamous cell carcinoma (SCC) or large cell carcinoma (LCC), depending
on their histological appearance, with adenocarcinoma and SCC being
the most common subtypes. The prognosis for NSCLC is poor, and
although somewhat better than for SCLC, the median survival of
NSCLC patients is still less than one year [36], with no major differences between the subgroups of NSCLC [37].
Treatment of NSCLC involves surgical resection of the tumor if possible, but this group includes only about 15% of all NSCLC patients
[36]. Treatment otherwise usually includes a combination of two
chemotherapeutic drugs [38]. Radiation therapy can also be included
e.g. as an alternative to surgery of non-resectable tumors, or before/after surgery to shrink the tumor or remove residual cancer cells,
respectively. Historically, the choice of chemotherapy for the different
subtypes of NSCLC has not been separate, however, a distinction between the efficacy of certain chemotherapies for the SCC and non-SCC
histology has now been acknowledged, with the anti-mitotic agent
Docetaxel being superior for SCC and the folate antimetabolite
Pemetrexed being superior for non-SCC NSCLC [39]. Also, targeted
treatment for patients with an activating mutation in the epidermal
growth factor receptor (EGFR) or for patients expressing the echinoderm microtubule-associated protein-like 4 - anaplastic lymphoma
kinase (EML4-ALK) fusion protein using specific kinase inhibitors for
these proteins have been proven superior to chemotherapy [40, 41].
These mutations, however, are only found in about 15% (EGFR mutation, more common in the Asian population) and 5% (EML4-ALK) of
the NSCLC patients, and primarily in non-smokers with adenocarcinoma [42, 43].
SCLC is a neuroendocrine cancer, originating from the neuroendocrine Kulchitsky cells found in the bronchial epithelium. Neuroendocrine cells release biologically active substances such as hormones or
paracrine signaling substances, upon nerve stimulation. As such,
13
SCLCs sometimes produce hormones including antidiuretic hormone

and adrenocorticotrophic hormone [44]. SCLC is a fast growing, aggressive cancer which forms metastases early. Due to the lack of early
detection methods for SCLC and its high propensity to metastasize,
60-70% of the SCLC patients will have metastases beyond the regional lymph nodes at the time of diagnosis [45]. Treatment of SCLC involves chemotherapy, which can be combined with radiation therapy
if the disease is not widespread, while the possibility to use surgery is
uncommon and any benefit from surgery is doubtful [45, 46]. Although SCLC initially responds very well to chemotherapy or radiation
therapy, most patients soon relapse, and by then the tumors will have
acquired resistance to these treatments. Consequently, the diagnosis
for SCLC is poor, with a median survival of less than a year [36].
Risk factors for lung cancer

In the early 1900s, lung cancer was a very rare disease, which constituted only about 1% of all cancers [47]. Since then, it has become the
most common cause of cancer death in the world, which primarily can
be attributable to a large increase in tobacco smoking, as tobacco
smoking accounts for approximately 87% of the lung cancer deaths in
men and 70% of the lung cancer deaths in women today [47]. Furthermore, 17% of lung cancers in non-smokers have been estimated to
be due to second hand smoke [48]. SCLC has the strongest correlation
with smoking, followed by squamous cell carcinoma, large cell carcinoma, and finally adenocarcinoma [49]. Smoking can induce chronic
inflammation in the lungs, including chronic obstructive pulmonary
disease, and the inflammatory processes are greatly contributing to
the development of cancer [50]. Radon gas exposure is the second
most common cause of lung cancer [51]. Other known risk factors include asbestos or other air pollutants, e.g. from combustion emissions
[52]. There is also an individual inherited susceptibility, as a family
history of lung cancer increases the risk of acquiring this disease, both
in smokers and non-smokers [53].
14
The tumor microenvironment

A cancer tumor does not merely consist of cancer cells; it is instead a
heterologous mixture of cell types, including cells of the immune system and other non-malignant cells, e.g. fibroblasts, endothelial cells
and epithelial cells. The progression of cancer does not solely depend
on the genomic alterations that accumulate within the cancer cells,
but is also highly dependent on the non-malignant cells of a tumor
(known as stromal cells). The stromal cells together with the malignant cells create a milieu, the so called tumor microenvironment,
which may either promote or inhibit cancer progression. Although
inflammatory cells have the potential to kill cancer cells, there is an
accumulating amount of evidence that proves the importance of inflammation also as a driving force for cancer progression.
Already in year 1863, Rudolf Virchow [54] observed leukocyte infiltration in cancer tumors and made a connection between chronic inflammation and cancer. A number of chronic inflammatory diseases
are now known to greatly increase the risk to develop cancer, e.g. inflammatory bowel diseases that increase the risk of colon cancer, hepatitis induced chronic liver inflammation that increases the risk of
hepatocellular carcinoma and Helicobacter pylori infections that increase the risk of stomach cancer [55-57]. Also, the use of antiinflammatory drugs (non-steroid anti-inflammatory drugs) reduces
the risk to develop colorectal cancer [20]. The inflammatory cells may
initiate cancer progression through DNA-damaging reactive oxygen
and nitrogen species leading to mutations, and they may further promote cancer progression in a number of ways. These include production of angiogenic or mitogenic factors, production of proteases that
degrade extracellular matrix components allowing tumor cells to invade and metastasize, and expression of anti-inflammatory mediators
allowing tumor cells to escape immune surveillance [54, 58, 59].
Macrophages
Macrophages are tissue resident cells of the innate immune system
which are found in all human organs. They are important for many
functions including tissue homeostasis, tissue remodeling and development, immune functions including immune surveillance, killing of
pathogens and cancer cells, antigen presentation and pro- and anti15
inflammatory regulation [60]. They were first discovered by lie

Metchnikoff [61] who was awarded the Nobel prize in year 1908 for
his findings on these phagocytes in immune responses. Macrophages
are according to the mononuclear phagocytic system classically explained to be derived from circulating monocytes originating from
hematopoietic stem cells in the bone marrow. Once a monocyte enters
a tissue it starts to differentiate into macrophages if in the presence of
either macrophage colony-stimulating factor (M-CSF) or granulocytemacrophage colony stimulating factor (GM-CSF) [62]. However, it is
now clear that some tissue macrophages are instead derived from
cells of the yolk sac or fetal liver which can sustain throughout adulthood via self-renewal [63]. Tissue resident macrophages derived from
cells of the yolk sac or fetal liver which are not replenished through
circulating monocytes during homeostasis include microglia in the
central nervous system, Kupffer cells in the liver and lung macrophages, while e.g. intestinal macrophages originate from monocytes
[64, 65].
Macrophages are a heterogeneous group of cells, which varies greatly
in function depending on the tissue they reside in [66] and can further
be polarized into different phenotypes within the tissue [67]. E.g. microglia in the central nervous system supports nerve cell development
and osteoclasts in the bone marrow are needed to form the cavities
within bone in which hematopoiesis takes place [68]. General functions of macrophages includes engulfing (phagocytosis/endocytosis)
of debris and dying cells, to support tissue repair and remodeling
through stimulation of ECM synthesis [69] and release of, or activation of proteases [70], and to support cell growth and angiogenesis
e.g. by the expression of heparin-binding epidermal growth factor-like
growth factor (HB-EGF) and vascular endothelial growth factor
(VEGF) [71, 72]. Meanwhile, as the macrophages carry out functions
during homeostasis, they also act as sentinels as a first line of defense
against pathogens or injury. They express a variety of pattern recognition receptors that recognize intra- or extra-cellular pathogens, which
upon stimulation promotes the clearance of these pathogens, via
phagocytosis and via release of inflammatory mediators. Pattern
recognition receptors includes the Toll-like receptors (TLRs) [73], the
NOD-like (nucleotide-binding oligomerization domain) receptors
[74], RIG-I-like (retinoic acid-inducible gene 1) receptors [75], scav16
enger receptors [76], C-type lectins [77] and receptors that recognize
targets opsonized by antibodies (Fc receptors) [78] or factors of the
complement system [79]. Recognition of a pathogen commonly leads
to phagocytosis, antigen presenting, and expression of proinflammatory mediators e.g. IL-1 IL-6, TNF- and nitric oxide [80],
leading to recruitment and activation of immune cells as well as direct
killing of the pathogen. Following initiation and development of inflammation, macrophages also play an important role in resolution of
inflammation through release of potent anti-inflammatory cytokines,
such as IL-10 and transforming growth factor (TGF)- [81-83]. Many
of the studies on macrophages has been undertaken on macrophages
from other species, primarily from mice, due to the ease of extracting
e.g. peritoneal macrophages from these animals, but some caution has
to be acknowledged regarding inter-species differences of macrophages. One important difference is that mouse macrophages produce
high amounts of nitric oxide, whereas human macrophages express
this molecule to a much lower degree [84].
The M1 phenotypes of macrophages

The idea of (classical) macrophage activation was first described by
Mackaness [85] in the 1960s as he discovered that macrophages upon
a secondary exposure to bacteria possessed increased antimicrobial
activity. This phenotype was later found to be induced by interferonIFN-)a cytokine released primarily from T-helper 1 cells [86].
Macrophages activated by IFN- or bacterial stimuli such as LPS, are
nowadays referred to as classically activated macrophages or M1
macrophages. Others have proposed GM-CSF to also induce an M1
phenotype, in contrast to M-CSF that has been proposed to differentiate macrophages to an alternatively activated M2 phenotype [87-89].
M1 macrophages act pro-inflammatory through the release of proinflammatory cytokines, e.g. IL-1, IL-6, IL-12, IL-23 and TNF- and
through production of reactive oxygen- and nitrogen species [86, 90,
91]. They also express higher levels of cyclooxygenase (COX)-2, possess elevated antigen presenting abilities and express low amounts of
the anti-inflammatory cytokine IL-10 [89, 92]. Markers exclusively
expressed by M1 macrophages have been proven difficult to find, but
17
many proteins have been reported to be up-regulated in M1 macrophages, which therefore might serve as markers in conjunction with a
pan-macrophage marker such as CD68. These include inducible nitric
oxide (iNOS), the T-cell costimulatory receptor CD80, the FcR1B
(CD64) and major histocompatibility complex, class II, DR (HLA-DR)
[93-97].
The M2 phenotypes of macrophages

The concept of an alternatively activated macrophage phenotype,
which were later also termed M2 macrophages, or the M2 phenotype,
was first proposed by Gordon and colleagues in 1992 [98], which they
argued to be induced by the T helper 2 cell produced cytokine IL-4.
They reported IL-4 to up-regulate the macrophage mannose receptor,
to induce morphological differences and to inhibit the expression of
pro-inflammatory cytokines, including IL-1, IL-8 and TNF-and to
inhibit nitric oxide production. Later, IL-13 was discovered to mediate
similar effects as IL-4, due to, in part, signaling through the same receptor as IL-4 [99]. Further studies of the M2 phenotype have since
then revealed a wide array of soluble factors or membrane bound proteins differentially expressed in the M2-phenotype, which generally
include an anti-inflammatory, wound-healing phenotype that promotes a T-helper 2 type immune response. They have been reported
to express high levels of the anti-inflammatory cytokines IL-10 and
TGF-, low levels of pro-inflammatory cytokines e.g. IL-12, and high
levels of arginase which converts metabolites away from nitric oxide
production and over to production of polyamines and L-proline,
which promotes cell proliferation and collagen production, respectively [100, 101].
Following the IL-4/IL-13 pathway of inducing the M2 phenotype, several other pathways to induce alternative phenotypes of macrophages
have been proposed. Ever since, the classification of M2 macrophages
has been somewhat confusing, where some researchers choose to
classify all alternatively activated macrophages as M2 macrophages,
while others categorize the M2 phenotypes into different subsets i.e.;
the M2a phenotype, induced by IL-4/IL-13; the M2b phenotype induced by FcR stimulation together with TLR agonists; and the M2c
phenotype, induced by glucocorticoids or IL-10 [102-104]. Others
18
view the M1/M2 phenotypes as the extreme endpoints of activation

states, where the macrophages in vivo can be polarized more or less
towards the M1 or M2 phenotypes depending on the inflammatory
milieu present [67]. Several markers for M2 macrophages have been
proposed, with the scavenger receptor CD163 being the most commonly used [105]. Other markers include macrophage scavenger receptor 1 (CD204), mannose receptor C type 1 (CD206), and for mouse
macrophages also chitinase-like 3 (YM-1), and resistin like alpha
(RELMa, FIZZ-1) [97, 106-108].
Tumor associated macrophages

Macrophages are commonly found in cancer tumors (tumor associated macrophages, TAMs) and have been proposed to both inhibit and
promote the progression of cancer [109]. This discrepancy is in large
due to the high functional diversity of macrophages. M1-like TAMs
have been correlated with an improved prognosis in a number of cancers, including colorectal cancer [95, 110] and lung cancer [93, 94].
Conversely, M2-like TAMs have been associated poor prognosis in the
same cancers [111-114]. In general, however, TAMs seem to primarily
be of an M2-like phenotype that actively promotes cancer progression
[115]. In support of this notion, in experiments on mice, either depletion of macrophages, inhibition of macrophage recruitment to tumors, or the use of a macrophage deficient mouse model, all reduces
tumor progression [116-118]. The phenotype of TAMs, however, are
not terminal, but can be changed upon external stimuli. For instance,
either anti-IL-10 receptor treatment in combination with a TLR-9 agonist, anti-IL4 antibodies, or IFN- plus LPS has been demonstrated
to convert macrophages from an M2 phenotype into the M1 phenotype [117, 119, 120].
Monocytes
Monocytes are not merely precursors of macrophages, but can themselves perform many of the inflammatory functions of macrophages.
They too, express pattern recognition receptors and can respond to
pathogens in a similar fashion as macrophages do, they are phagocytes, and can present antigens. There are two major populations of
19
monocytes found in the blood, CD14+/CD16- monocytes and

CD14+(low)/CD16+ monocytes [121]. The CD14+/CD16+ monocytes
constitute a minor population of a more pro-inflammatory phenotype
and with higher antigen presenting abilities. However, CD14+CD16monocytes have been proposed to be the primary subset recruited to
inflamed tissue to differentiate into macrophages [122]. This is based
on the observation that CD14+CD16+ monocytes lack the CCR2 receptor needed for CCL2 induced chemotaxis, which is one of the primary chemokines for monocyte recruitment during inflammation
[123].
The extra cellular matrix

The extracellular space within tissues contains a net of polymers such
as collagens and elastic fibers which are contained in a viscoelastic gel
of e.g. hyaluronan, proteoglycans and various glycoproteins, all of
which constitutes the extra cellular matrix (ECM) [124]. The ECM
creates a scaffold for the cells to adhere to and is needed to maintain
the function and shape of the tissue. The principal ECM producing
cell is the fibroblast, which can be activated by cytokines, most prominently by TGF- [125]. Proteases on the other hand, facilitate degradation of the ECM, which among others include plasmin, matrix metalloproteinases (MMPs), cathepsins, and the a disintegrin and metalloproteinase domain (ADAM) family of proteases. Production of ECM
is very important during wound healing, but excessive ECM production in tissues can cause fibrosis, which may impair normal tissue
function. The degradation of ECM is needed for angiogenesis and for
late stages of wound healing; however, proteases may also facilitate
tumor invasion and metastasis [126].
The urokinase-type plasminogen activator system

Plasminogen is the inactive zymogen of the serine protease plasmin
which is released into the systemic circulation by the liver and kept at
a plasma concentration of approximately 2 M [127]. Activation of
plasmin is mediated by two serine proteases, namely tissue-type
plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). One major role of plasmin is fibrinolysis, the process of dis20
solving blood clots. tPA is the primary activator of plasminogen in fibrinolysis, which is stored in endothelial cells, and upon release, can
bind to fibrin. This binding increases the catalytic efficacy of tPA regarding plasminogen activation over 100-fold [128, 129]. Plasmin can
also be activated within tissues to facilitate ECM degradation; a process that is needed for non-pathological processes such as wound
healing [130] and angiogenesis [131], but also for the progression of
cancer, enabling cancer cells to invade nearby tissue and to metastasize [132].
The ECM degradation is partly mediated directly by plasmin, but also
through its proteolytic activation of other proteases, including matrix
metalloproteinases (MMPs) [133, 134]. Activation of plasmin in tissues is regulated by a series of proteins, including binding of plasminogen to plasminogen receptors, and activation of plasminogen by uPA
bound to its receptor (uPAR). Furthermore, the activation can be
blocked by inhibitors directed towards plasminogen (2-antiplasmin)
or uPA (plasminogen activator inhibitor (PAI)-1 and PAI-2) [135]. See
figure 2 for an overview of plasmin activation.
There has been many plasminogen receptors identified, expressed on
a variety of cells, such as endothelial cells, inflammatory cells and
cancer cells [136]. Cellular binding of plasminogen to one of its receptors drastically increases its proteolytic activation by uPAR-bound
uPA; therefore, cellular expression of uPAR and plasminogen receptors results in locally high plasmin activation at the cell surface of
these cells [137]. Furthermore, the plasmin inhibitor 2-antiplasmin
is unable to inactivate receptor-bound plasmin [137].
uPA is produced as an inactive pro-uPA zymogen, but binding to
uPAR allows for serine proteases, including plasmin to cleave prouPA into its active form and binding of plasminogen and pro-uPA to
their respective receptors can facilitate reciprocal activation of their
zymogen forms [138]. The uPAR protein does not have an intracellular domain, but is instead bound to the cell membrane via a glycosylphosphatidylinositol anchor. Even so, uPAR can act as a signal
transducer through interaction with other trans-membrane cell surface proteins e.g. integrins or G-protein coupled receptors, to initiate
intracellular signaling including extracellular-signal-regulated kinase
(ERK) activation or activation of the phosphatidylinositol 3kinase/AKT-pathway [139-141]. Furthermore, uPAR binds vitron21
ectin, a protein of the ECM, and uPAR-vitronectin binding is thought

to mediate cell attachment and migration [142]. uPAR can be cleaved
from the cell surface i.e. by glycosylphosphatidylinositol specific
phospholipase D1 or cathepsin G to generate soluble uPAR (suPAR)
[143, 144]. suPAR, in contrast to its cell-bound counterpart, does not
increase the ability of bound uPA to activate plasminogen [137]. uPAR
contains three homologous domains (DI-III), which all contributes to
the binding of uPA, however, upon binding to uPA, conformational
changes of uPAR exposes a linker peptide between domains DI and
DII which enables proteases such as uPA, plasmin or matrix metalloproteinases to cleave off domain DI, leaving domain DII-III still anchored to the cell membrane, but unable to bind uPA or vitronectin
[145, 146]. The inhibitors PAI-1 and PAI-2 are both able to inhibit
both tPA and uPA, but PAI-1 is considered the primary physiological
inhibitor [147, 148].
Figure 2. Plasmin activation by the uPA/uPAR system. (The figure is adapted

with permission from Multidisciplinary Digital Publishing Institute: International Journal of Molecular Sciences [135])
22
The urokinase-type plasminogen activator system in cancer

uPA and uPAR expression in cancer has been extensively investigated
and high levels of these proteins in tumors have been correlated with
a worse outcome in numerous cancers including colorectal [149, 150],
lung [151, 152], breast [153-155], gastric [156, 157], and prostate cancer [158, 159], with both tumor cells and stromal cells as possible
sources of these proteins. In addition, suPAR (DI-III, DI or DII-III)
can be found at elevated levels in the blood of cancer patients, and
high plasma/serum levels has been suggested as possible biomarkers
for a poor prognosis in various cancers, including lung [160, 161], colorectal [150, 162] and breast cancer [163]. Seemingly paradoxical,
high levels of the uPA inhibitor PAI-1 have also been correlated with a
worse prognosis, while high PAI-2 expression has been correlated
with a favorable outcome [155, 164, 165]. The negative outcome of
PAI-1 has been suggested to be due to its ability to bind other proteins, e.g. lipoprotein receptors or vitronectin to activate cell signaling
or disrupt cell adhesion to facilitate metastasis, respectively [166,
167].
Polyphenol content of green, black and rooibos tea

Polyphenols are compounds that exist naturally in plants, with high
contents found in e.g. fruits, berries, vegetables, nuts and herbs.
These compounds are further categorized into four main groups depending on their structure, i.e. flavonoids, stilbenes, phenolic acids
and lignans [168]. A schematic figure of polyphenol nomenclature can
be seen in figure 3. In general, consumption of these compounds are
thought to have beneficial effects on human health, primarily through
their potent antioxidant capacity, but also through specific mechanisms that inhibits inflammation, promotes vascular health, acts antimicrobial and anti-tumorigenic [169-173]. For example, antiinflammatory effects of polyphenols include inhibition of NF-kB signaling, an important transcription factor for many pro-inflammatory
cytokines, inhibition of nitric oxide, and inhibition of prostaglandin
synthesis [173]. Anti-carcinogenic effects of polyphenols can in addition to their anti-inflammatory effects be attributed to reactive oxygen
scavenging and anti-angiogenic effects as well as anti23
proliferative/pro-apoptotic effects of certain polyphenols toward cancer cells [174, 175].

Flavonoids are the main group of polyphenols found in green, black
and rooibos tea, but the composition of flavonoids differ between
these tea types. Green and black tea is generated through different
processing of the leaves from the plant Camellia sinensis while rooibos tea is produced from the leaves and stems of another plant,
Aspalathus linearis. The tea leaves of black tea are processed in a
manner that allows enzymatic oxidation (sometimes referred to as
fermentation) of catechins, the primary polyphenols found in tea
leaves, via polyphenol oxidase, whereas the leaves of green tea are
heated during manufacturing to inactivate this enzyme, thereby preserving the catechins [169]. The catechins, which belong to the flavanol subgroup of the flavonoids, constitute approximately 30-40% of
the water soluble dry materials in green tea, and 80-90 % of the flavonoid content [169, 176].
The principal and most well studied polyphenol of green tea is epigallocatechin gallate (EGCG), which constitute approximately half of the
flavonoid content of green tea, however, more than 30 different polyphenols have been identified in green tea extracts, and include flavonols e.g. quercetin, kaempferol and myricitin and catechins such as
epigallocatechin, epicatechin gallate and epicatechin [177, 178]. Black
tea have a similar total amount of polyphenols as green tea, however,
the catechins of green tea is to a large extent oxidized into larger oligomers, named theaflavins and thearubigins, which together constitutes 60-70% of the total polyphenols in black tea [179]. Rooibos is
usually fermented to yield red tea, but unfermented green rooibos is
also produced. Rooibos tea does not contain catechins; instead the
flavonoid content of rooibos tea includes flavonols e.g. quercetin and
rutin, flavones such as orientin and vitexin and dihydrochalcones including nothofagin and the rooibos unique substance aspalathin [171].
24
Figure 3. Major classes of polyphenols including example structures. (The figure

is adapted with permission from Cambridge Journals: British Journal of
Nutrition [180])
Prostanoids
Prostaglandins were first discovered in the 1930s as substances in
seminal fluids and vesicles mediating smooth muscle contraction of
the uterus and were later found to be important mediators of inflammation and diseases. In 1982 the Nobel Prize was awarded to Sune
Bergstrm, Bengt Samuelsson and John Vane for their discoveries in
the field of prostaglandins and related substances. Sune Bergstrm
determined the chemical structure of prostaglandins, Bengt Samuelsson elucidated the process of their formation and John Vane discovered that the anti-inflammatory properties of aspirin were achieved
through blocking prostanoid synthesis [181].
25
Eicosanoids, which include prostanoids (prostaglandins and thromboxane A2) and leukotrienes, are signaling molecules derived from
20-carbon fatty acids, predominantly from the fatty acid arachidonic
acid, and exert autocrine and paracrine functions [182]. Arachidonic
acid is released from cell membrane phospholipids by phospholipase
A2 (PLA2) enzymes and can be processed by cyclooxygenase (COX)-1
or COX-2 into prostaglandin (PG) H2, the precursor of all prostanoids, which include PGD2, PGE2, PGF2, PGI2 and thromboxane
A2. COX-1 is a constitutively expressed enzyme responsible for basal
housekeeping expression of PGs, while COX-2 is an inducible isoform that can be induced by various inflammatory stimuli or pathological conditions [183]. The different prostanoids are produced from
the substrate PGH2 by their respective synthases.
The prostanoids are expressed in different tissues and cell types to
modulate various physiological functions and conditions, e.g. regulation of blood clotting and vascular tension, bronchoconstriction and
dilation, gastrointestinal motility and secretion, kidney function, myometrial contraction, regulation of inflammation and induction of fever and pain [182]. The prostanoids exert their biological functions
upon binding to their respective G-protein coupled receptor, IP, FP,
DP1, DP2, TP or EP1-EP4. Depending on receptor, ligand binding either activates or blocks adenylyl cyclase leading to increased or decreased cAMP levels, or activates the diacyl-glycerol/inositol triphosphate (DAG/IP3) signaling pathway [184]. Furthermore, nonenzymatic conversion of PGD2 can form 15 deoxy-PGJ2, which acts
upon nuclear receptor peroxisome proliferative receptor (PPAR) to
mediate anti-inflammatory effects [185].
Prostaglandin E2
Of all prostanoids, PGE2 is the main mediator of inflammation and
has the most diverse functions due to its four different target receptors. PGE2 can be generated by three different PGE synthases, namely, microsomal PGE synthase (mPGES)-1, mPGES-2 and cytosolic
MPGES (cPGES). mPGES-2 and cPGES are constitutively expressed
whereas mPGEs-1 is induced by inflammatory stimuli and considered
the primary PGE synthase for PGE2 formation during inflammation
and cancer [186]. The formed PGE2 can either exert its various func26
tions via receptor ligand binding, or be metabolized into an inactive

metabolite via 15-PG dehydrogenase.
Acute inflammation is characterized by four main features, namely
rubor (red flare), calor (heat), tumor (swelling) and dolor (pain) and
all of these features are induced by PGE2 during inflammation. PGE2
induces vasodilation which causes the heat and red flare due to the
increased local blood flow [187]. The swelling is caused by vasodilation in combination with an increased vascular permeability facilitating leukocyte infiltration. PGE2 can increase vascular permeability
through binding to EP3 receptors on mast cells, stimulating these
cells to release histamine, a known mediator of hyperpermeability
[188]. Furthermore, PGE2 is a chemoattractant for mast cells, and
also induces mast cells and epithelial cells to produce chemoattractants for other immune cells thus further contributing to the leukocyte
infiltration at the site of inflammation [189-191]. The pain induced by
PGE2 is mediated via EP receptors on neuronal nociceptors [192].
Although these pro-inflammatory effects are mediated by PGE2 during acute inflammation, PGE2 can also act immunosuppressive by
regulating the inflammatory response of other immune cells. It enhances expression of the anti-inflammatory cytokine IL-10 and inhibits expression of the pro-inflammatory cytokine IL-12 in macrophages, and also reduces the macrophages phagocytic ability [193, 194].
PGE2 also act immunosuppressive by inhibiting the cytotoxic activity
of natural killer cells and cytotoxic T lymphocytes [195, 196], and
through inhibiting the expression of the pro-inflammatory cytokine
IFN- from T helper 1 cells [197].
Prostaglandin E2 in cancer
Overexpression of PGE2 or the enzymes responsible for PGE2 formation is commonly seen in a number of cancers, including CRC and
lung cancer [198-205]. Also, the administration of COX inhibitors reduces the risk of CRC development and recurrence [206, 207], and
epidemiological studies suggests a reduced cancer risk by the use of
COX inhibitors also for cancers originating from other tissues [208].
In animal models, PGE2 has also been shown to increase cancer progression in a number of cancer models [195, 209-211]. The mechanism for PGE2 cancer promotion has been suggested to be due to a
27
variety of its pleiotropic effects, including its effects on the immune

system, induction of angiogenesis, inhibition of apoptosis and activation of mitogenic signaling [212].
28
Background and aim

Macrophages, which mainly originate from circulating monocytes, are
cells of the innate immune system that often is present within cancer
tumors in large quantities. Macrophages, which can be of different
phenotypes, have a broad variety of functions, ranging from proinflammatory actions including clearance of pathogens and cancer
cells, to wound-healing functions and immune suppression. The
broad functional diversity of macrophages makes them highly interesting to study in the context of a tumor microenvironment because it
gives them the ability to act both pro- and anti-tumorigenic. Elucidating the interplay between cancer cells and macrophages of different
phenotypes is an important challenge to better understand the progression of cancer and to develop more effective cancer therapies. The
aim of this thesis was to study the monocyte-macrophage cell lineage
and possible implications of their presence in cancer tumors.
In more detail, the aims of the different subprojects included:
Elucidate differences of the M1 and M2 phenotype of human macrophages regarding expression of inflammatory mediators and surface
antigens (paper I)
Determine if macrophages of the M1 and M2 phenotype:
- Can affect the cell growth of colon cancer and lung cancer cells
(paper I, II, III)
-
Can affect the efficacy of 5-FU to inhibit colon cancer cell

growth (paper II)
Express different levels of MMPs and genes of the uPA/uPAR

system, and how their expression of these genes compares to
the expression levels of these genes found in lung cancer cells of
SCLC and SCC origin (paper III)
Determine if and how green-, black- and rooibos tea extracts inhibit
PGE2 formation in human monocytes (paper IV)
29
Methods
This section provides an overview of the methods used in the papers
presented in this thesis. A more detailed description of the methods is
presented in the individual papers.
Culture of cancer cell lines

Cancer cell lines originating from colon and lung tissue (Table I) were
used to study how macrophages can influence cancer cells on various
aspects of carcinogenesis including cancer cell proliferation and apoptosis, expression of genes important for invasion and metastasis and
resistance of cancer cells to chemotherapy. A cancer cell line of monocytic origin was also cultured and used for differentiation into macrophages. The cell lines were routinely sub-cultured twice weekly, cultured in RPMI media supplemented with heat inactivated foetal calf
serum (FCS) and antibiotics (penicillin and streptomycin) and maintained at 37C in a humidified atmosphere with 5% CO2.
Table I. List of cell lines used in the papers included in the thesis
Cell line
Species
Tissue Disease
CACO-2
HT-29
NCI-H69
Human
Human
Human
colon
colon
lung
NCIH520
THP-1
Human
lung
Paper
colorectal adenocarcinoma
I, II
colorectal adenocarcinoma
I, II
carcinoma; small cell lung can- III
cer
squamous cell carcinoma
III
Human
blood
acute monocytic leukemia
30
Isolation of human peripheral blood monocytes and differentiation into macrophages

Human peripheral blood mononuclear cells (PBMC) were isolated
from buffy coats of healthy blood donors via gradient centrifugation.
Monocytes were selectively isolated from other PBMCs by the adherence method, which utilize the monocytes ability to bind plastic surfaces, a characteristic not shared by other PBMCs [213]. Following an
overnight culture, the monocytes were subjected to tea extracts or
other treatments (paper IV), or used for the generation of monocytederived macrophages (paper I-III).
For the differentiation of monocytes into macrophages the monocytes
were cultured for 6 days in RPMI with the addition of macrophage
colony stimulating factor (M-CSF). The generated macrophages were
further differentiated into M1 or M2 macrophages using a 48 h treatment with LPS plus IFN- or IL-4 plus IL-13, respectively. Macrophages without any added differentiation stimuli were termed M0
macrophages. Conditioned media (CM) from macrophages were collected at two time-points, the first time-point being the media used
for differentiation into M1 or M2 macrophages, which consequently
contains the exogenously added differentiation stimuli. These CM
were termed M1DIFF and M2DIFF CM. The differentiated macrophages were washed and cultured for an additional 48 h without any
differentiation stimuli added. These CM were collected and termed
M1 and M2 CM. See figure 4 for an overview of the culture procedure.
The advantage of the method used to generate macrophages is that it
generates macrophages of human origin, as opposed to other commonly used methods, e.g. isolation of peritoneal macrophages of murine origin. There are also monocyte and macrophage cancer cell lines
available which can be used, however, there are always concerns when
using cell lines whether they respond as would be expected by primary healthy cells. The disadvantage of PBMC derived monocyte differentiation is that it is a time-consuming process. There can also be differences between donors regarding the amount of monocytes obtained and how they respond to various stimuli.
31
Figure 4. Flowchart illustrating the method used for generation of macrophages

of different phenotypes
Differentiation of THP-1 monocytes into macrophages

Cells of the THP-1 acute monocytic leukemia cell line can be differentiated into macrophages with phorbol 12-myristate 13-acetate (PMA)
[214, 215]. In this thesis, THP-1 macrophages were generated by
stimulation of THP-1 cells with 160 nM PMA for 24 h (paper I) and
subsequently cultured for 48 h to generate THP-1 macrophage CM.
The advantage of THP-1 monocyte differentiated macrophages is as
discussed above, the ease to generate these macrophages compared to
the generation of monocyte derived macrophages from primary cell
cultures, while a disadvantage is that THP-1 macrophages originate
from cancer cells which makes it difficult to know how they deviate
from normal healthy macrophages.
Cell counting
Cells were counted in Brker chambers using trypan blue to exclude
non-viable cells. Adherent cells were loosened with a trypsin/EDTA
solution prior to counting. If loose cells were observed in the culture
media prior to counting, it was collected and counted separately (paper I). H69 cells grow in suspension in aggregates and are difficult to
get into a single cell solution needed for a reliable assessment of cell
number using Brker chambers. To generate a single cell suspension
of H69 cells, the cells were separated using a specialized dissociation
32
solution (Accumax). The advantage of assessing cell numbers by using

a hemocytometer such as Brker chambers is that it gives a direct
measurement of the amount of cells present, as opposed to other
methods e.g. the XTT and MTT methods that measure secondary signals which are used to estimate the amount of cells present. Disadvantages of using hemocytometers includes that it is time-consuming
and that it requires single cell suspensions.
Detection of proteins and PGE2

Proteins were detected using a number of methods described below.
Enzyme linked immunoassays were performed for soluble factors released into the culture medium, whereas cell surface proteins or intracellular proteins were detected in cell lysates using western blot, or
through immunostaining of fixed cells from cultures or formalin fixed
and paraffin embedded biopsies. The advantage of immunostaining is
that it visualizes the expression of proteins in individual cells, but it is
a less quantitative method as compared to western blot analysis,
which is a semi-quantitative method.
Enzyme linked immunoassays

The concentration of IL-10 (paper I), IL-12 (paper I) and PGE2 (paper
IV) in conditioned media was analyzed using commercially available
ELISA or EIA kits. A protein array utilizing the ELISA principle
(RayBiotech) was used to semi-quantitatively measure the expression
of 42 different cytokines in macrophage conditioned media (paper I).
Western blot
Cells were lysed in Laemmli sample buffer [216] including protease
inhibitors and were subjected to SDS-PAGE to separate proteins. Separated proteins were transferred onto a polyvinylidene fluoride membrane using a wet transfer system. The membrane was blocked with
an appropriate blocking buffer and was then incubated with the primary antibody overnight at 4C (See Table II for antibody targets).
Following primary antibody binding, an appropriate horseradish peroxidase linked secondary antibody was applied and signals were de33
veloped using an enhanced chemiluminescent (ECL) substrate and

light intensities were detected by exposure to x-ray film. After detection, the antibodies could be stripped of to allow reuse of the membrane for the detection of other proteins. Detected -actin (paper II)
or COX-1 (paper IV) signals were used as loading control.
Immunostaining
Formalin fixed and paraffin embedded SCLC and lung SCC biopsies,
which were decoded to remove any possible links to the patients, were
used in paper III for the detection of various antigens using immunohistochemistry.
Immunocytochemistry was performed with cells either cytospun onto
glass slides or cells cultured on 4-well chamber slides. These cells
were dried and fixed in formalin prior to immunocytochemical staining. Chamber slides have the advantage that the cells retain their
morphology as opposed to cytospun samples. However, the chamber
slides used in these studies were of glass which may affect how the
cells adhere and grow in comparison to their growth on plastic cell
culture plates.
Slides for both immunohistochemistry and immunocytochemistry
were immunostained using the same procedures. Epitope retrieval
was performed as suggested by the manufacturer of the antibody (see
Table II for antibodies used) and the staining procedure was performed using a Dako autostainer and visualized using the standard
method of horseradish peroxidase and 3,3-diaminobenzidine. The
nuclei were then counterstained with Mayers haematoxylin and the
slides dehydrated and mounted. Tonsil tissue was used as control.
34
Table II. Antibodies used
Antibody
target
p21
-actin
COX-1
COX-2
cPLA2
mPGES-1
CD68
CD163
MMP-2
MMP-9
uPAR
Application
Paper
Western blot
Western blot
Western blot
Western blot
Western blot
Western blot
IHC, ICC
IHC, ICC
IHC
IHC
IHC, ICC
II
II
IV
IV
IV
IV
I-III
I- III
III
III
III
Protein siRNA knockdown

Knockdown of p21 protein expression (paper II) was achieved in HT29 cells using small interfering RNA (siRNA). Western blot analysis of
cell lysates was performed to control that the knockdown had been
successful. A negative siRNA scramble sequence was used as a negative control. siRNA knockdown is mediated by cleaving mRNAs with
sequences complementary to the siRNA sequence. A disadvantage of
siRNA is that it might only reduce the expression of the target gene
and only for a limited time, in contrast to knock-out methods which
makes the cells completely unable to produce the target protein.
RNA extraction and cDNA synthesis

Extraction of total RNA and reverse transcription to generate cDNA
was done using commercially available kits according to manufacturers instructions. The RNA quantity and purity was measured via the
absorbance value at 260 nm and the 260/280 nm ratio, respectively.
35
Quantitative real-time PCR

mRNA expression levels were measured using real-time PCR and
SYBR green. Fold change of treated sample vs untreated control was
calculated using the ct method (paper III) or the REST2009 software [217] (paper II). GAPDH (paper III) or multiples housekeeping
genes (paper II), were used for calculating fold differences. Primer
pair efficiencies were calculated using the Linreg PCR software, which
calculates the efficiencies from the slope of the logarithmic phase of
the real-time PCR data [218]. The amplified PCR product was validated by melt-curve analysis and the size of the amplified PCR product
was validated by agarose gel electrophoresis for each primer pair. The
method of quantitative mRNA detection using real-time PCR has the
advantage of being very sensitive as it greatly amplifies the original
material, and it also allows for easy detection of a large number of targets. However, when measuring mRNA it is important to acknowledge
that the level of mRNA may not always reflect the amount of corresponding protein produced.
Cell cycle analysis

Cell cycle analysis (paper I-III) was measured with a FACScalibur flow
cytometer on cells fixed with ice-cold ethanol and stained with propidium iodide, a molecule that is fluorescent if bound to nucleic acid.
RNA was degraded by RNAse H prior to analysis, therefore, the
amount of light emitted was proportional to the amount of DNA present in the cell. A signal of 1N=G1-phase (a single setup of chromosome pairs), 2N=G2/M-phase (a double setup of chromosome pairs)
and a signal between 1N-2N= S-phase. The distribution of cells in
each phase was calculated using the ModFit software. The amount of
cells with a signal < 1N (subG1 peak) was used as an estimate of apoptosis, as one of the characteristics of apoptosis is degradation of DNA.
Flow cytometry has the advantage of acquiring data for single cells at
a rapid pace, allowing quantifiable signals from tens of thousands of
individual cells to be used for each sample.
36
Apoptosis
Apoptosis was analyzed on a FACScalibur flow cytometer, either as
described previously (in the cell cycle analysis section), or through the
use of a commercially available terminal deoxynucleotidyl transferase
dUTP nick end labeling (TUNEL) assay (paper I). For the TUNEL assay, the cells were fixed with 1 % paraformaldehyde dissolved in PBS
and subsequently further fixed in ice-cold 70% ethanol. Paraformaldehyde is used to cross-link DNA-fragments so that they do not leak
out of the cells as they are permeabilized with ethanol. The TUNEL
assay measures DNA fragmentation which is a characteristic of apoptosis, by fluorescently labeling the exposed 3-hydroxyl ends of DNA
fragments. This method is a more reliable and sensitive method compared to the previously mentioned sub-G1 method.
Preparation of tea extracts

Green, black, and rooibos tea extracts were prepared by adding 20 ml
of boiling PBS to 1 g dry tea material. After 5 min incubation, the tea
extracts were filtered twice through a 0.22 m sterile filter and frozen
in aliquots at -20C until use. These solutions were referred to as 1:20
dilutions. (i.e. 1 g tea/20 ml PBS). The time of extraction and solvent
used (water-based) for extraction of polyphenols was chosen to mimic
the conditions of tea brewing for normal dietary use.
Cell free assay for the combined enzymatic activity of COX-2 and
mPGES-1
The enzymatic activity of COX-2 and mPGES-1 was measured in a cell
free assay with enzymes originating from cell lysates of LPS stimulated monocytes (paper IV). The cells were scraped off in an assay buffer
referred to as buffer x (80 mM KCl, 10 mM HEPES, 1 mM EDTA, pH
7.4) supplemented with protease inhibitors, and lysed using sonication. The lysates were centrifuged at 1700 x g for 10 min to pellet
the nuclei of the cells. The supernatant was collected and used as an
enzyme source for assessment of the combined COX-2 and mPGES-1
activity. The 1,700 x g supernatant was mixed with buffer x supplemented with L-glutathione and the mixture was pre-incubated with or
without the addition of tea extracts or polyphenols (EGCG or querce37
tin) prior to addition of the substrate arachidonic acid. The reaction

was terminated after 30 min and the amount of PGE2 produced was
measured with a PGE2 enzyme immunoassay kit.
Gelatin zymography
Gelatinase activity of MMP-2 and MMP-9 was measured in M1/M2
CM and in CM from H520 or H69 cells through zymography using
commercially available reagents. Zymography is a sensitive method;
however, it will only give a relative quantification of the activity and
furthermore only allows discrimination of proteins through size estimates from the SDS-PAGE separation.
38
Results and Discussion

Inflammatory cells, including macrophages and monocytes and the
inflammatory mediators they produce are of great significance for the
progression of cancer. In this thesis, mainly cell culture was utilized as
a means to study the potential effects of macrophages towards a number of aspects regarding tumor progression of colon cancer and lung
cancer cells, including cancer cell growth, cell invasion and resistance
to cancer therapy. Furthermore, compounds with suggested antiinflammatory properties were investigated with regard to their ability
to inhibit PGE2 formation, an inflammatory mediator strongly associated with the development of colon cancer.
Characteristics of cultured human macrophages of different

phenotypes
As mentioned previously in the background section, macrophages can
be of different phenotypes including the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype. These phenotypes can
be generated in vitro in a number of ways, and one of the most commonly used methods is to stimulate macrophages with LPS and/or
IFN- for the generation of M1 macrophages, and IL-4 and/or IL-13
for the generation of M2 macrophages.
In this thesis the same protocol were used for all included papers to
generate human monocyte derived, M-CSF differentiated macrophages (termed M0 macrophages), which were further differentiated into
M1 macrophages using LPS plus IFN- or into M2 macrophages using
IL-4 plus IL-13. These macrophages were characterized at the end of
the differentiation by their expression of surface antigens, i.e. the panmacrophage marker CD68 and the M2 macrophage marker CD163,
and also by their mRNA expression of a chosen setup of M1 and M2
characteristic genes analyzed both at the end of the differentiation,
and also 48 h post differentiation. All cells regardless of macrophage
phenotype expressed CD68 at the end of differentiation indicating
that the cells were indeed macrophages. The M2, and M0 differentiated cells were also CD163 positive to a large extent (70% and 80% positive cells, respectively) while the large majority of the M1 differentiated cells were CD163 negative (Fig 5 a-c). At the mRNA level, M1 dif39
ferentiation induced expression of some suggested M1 characteristic

surface markers, e.g. HLA-DRA [94, 219] and CD80 [97, 220], and
also of several cytokines/chemokines e.g. TNF-, IL-6, IL-8, IL-12
and CXCL-9 (Table III), of which many were up-regulated also 48 h
post differentiation, indicating that the macrophages remain in a differentiated state within this timeframe. In agreement with these results, another study using a similar differentiation protocol reported
that macrophages of either M1 or M2 phenotype require up to 12 days
to revert back to an undifferentiated M0 phenotype [220].
In line with the mRNA data analyzed from these cells, the M1 differentiated cells released many pro-inflammatory cytokines into the
M1DIFF CM, including TNF-, IL-6, IL-8, IL-12, a number of (C-X-C
motif) ligand (CXCL) and (C-C motif) ligand (CCL) chemokines, including CXCL-1, -5, -9, and CCL-5 (Table III, the entire protein array
can be seen in paper I). Some of these cytokines were also detected in
the M2DIFF CM e.g. IL-8 and CCL-5, but at a lower concentration
compared to the amount detected in M1DIFF CM. In the M2DIFF CM
CCL-17 was the only cytokine found to be expressed substantially
higher compared to M1DIFF CM. These cytokine expression profiles
are in general agreement with the findings of others [92, 220-222].
Unexpectedly, even though the anti-inflammatory cytokine IL-10 is
commonly associated with the M2 phenotype [102], high levels of IL10 was found in the M1DIFF CM. However, the same induction of IL10 has been observed by others [221] and is likely due to the M-CSF
differentiation which has been reported to yield macrophages that responds with IL-10 release upon LPS treatment [223].
In general, the M1/M2DIFF CM contained higher amount of the cytokines measured in comparison to their corresponding M1/M2 CM.
However, differences between the cytokines released into M1 and M2
CM were still present, as indicated from the mRNA data, e.g. IL-8 and
CXCL-9 were still present in M1 CM to a larger extent compared to
the amount present in M2 CM, and CCL-2 was detected to a larger
extent in M2 CM compared to M1 CM (Table III).
Regarding cytokine release, the undifferentiated M0 macrophages
were highly similar to the M2 differentiated macrophages; of all cytokines detected (by the RayBiotech protein array) M0DIFF CM and
M2DIFF CM was near to identical. Furthermore, M0 and M2 macrophages expressed CD163 to a similar extent. These results support
40
findings by others which have suggested M-CSF to differentiate macrophages into an M2-like phenotype and induce CD163 expression, as
opposed to GM-CSF suggested to differentiate macrophages into an
M1-like phenotype [87, 88, 97].
The morphology of generated M1 and M2 macrophages differed to
some extent. M1 macrophages tended to be more elongated compared
to the M2 macrophages. This difference was more pronounced following the 48 h culture post differentiation stimuli (Fig. 5 d-f).
Differentiation into M1 macrophages also resulted in lower cell numbers compared to corresponding M2 differentiation (results not
shown). Visual inspections comparing cell numbers before and after
differentiation suggest that the differentiation into M1 macrophages
results in a reduction in cell numbers, in some cases with detachment
of cells observed; however, we cannot exclude the possibility that M2
macrophages also proliferate as IL-4 has been demonstrated to activate proliferation of tissue resident macrophages in a mouse model
[224].
41
Figure 5. Representative images of CD163 immunoreactivity following M0 (a),

M1 (b) and M2 (c) differentiation, and of the morphology of M0 (d), M1 (e)
and M2 (f) macrophages 48 h post differentiation (d-f). a-c are cytospun
onto glass slides and have therefore lost their original morphology.
The monocytic leukemia cell line THP-1 is a commonly used in vitro

model of human macrophages through differentiation with PMA. In
paper I, THP-1 cells were differentiated into macrophage-like cells
using 160 nM PMA for 24 h which caused the THP-1 cells to adhere to
the surface and acquire a macrophage-like morphology. However, we
did not characterize the phenotype of the THP-1 macrophages any
further.
42
CD68
CD163
D 16-fold
uPAR
MMP-2
MMP-9
IL-6
U 190-fold
IL-8
U 1600-fold
IL-10
D 2-fold
IL-12
U 5-fold
IL-13
ND
CCL2
D 2-fold
CCL5
CCL17
D 6-fold
CCL18
U 24-fold
CXCL1
CXCL5
CXCL9
U 26,000-fold
U 95-fold
TNF-
CD80
U 500-fold
HLA-DRA
U 37-fold
Target
+
+
+
++
++
++
+
+++
+++
+++
+
+
+/-
1
1
U 6-fold
U 3-fold
U 6-fold
U 115-fold
U 10-fold
D 2-fold
1
1
ND
1
D 2-fold
+/-
++
+
++
+
+
-
+
++
1
1
1
1
ND
1
1
1
1
43
+/-
++
+
+
+
+
+
+++
U 900-fold
U 13-fold
U 10-fold
U 16-fold
ND
U 60-fold
1
U 7-fold
U 10-fold
1
ND
U 16-fold
U 4-fold
U 3-fold
ND
1
+
+
-
+
+
+/-
++
+
++
+++
+
1
D 2-fold
1
1
U 17-fold
U 60-fold
D 6-fold
1
D 3-fold
1
ND
1
1
1
ND
1
+/-
++
+
+
+
++
+
+
1
1
1
1
1
1
1
1
ND
1
1
1
ND
1
Differentiated macrophages or macrophage DIFF CM

macrophages 48 h post differentiation or macrophage CM
M1
M2
M0
M1
M2
M0
mRNA
protein
mRNA
protein mRNA protein
mRNA
protein mRNA protein mRNA protein
Table III. Phenotypic mRNA and protein expression of cultured human macrophages of the M1, M2 and M0 phenotypes. Plus sign indicate protein expression/release and minus sign indicate no detectable protein (in CM or on the cells detected by immunocytochemistry). +/- indicate possible but low expression. Protein levels were detected semi-quantitatively (n=2-4). Fold changes are compared to M0 macrophages and at least 4 independent experiments with macrophages from different donors were used for the
mRNA analysis. D: down-regulated, U: up-regulated, ND: not detected. Empty fields indicate that no measurements have been
done. Results for mRNA data directly following differentiation and for protein data 48 h post differentiation is not shown in any
of the papers included in the thesis.
Macrophages and cancer cell growth

Macrophages have the potential to both inhibit and promote proliferation of cells, including cancer cells, and both negative and positive
correlation regarding tumor associated macrophages and cancer progression has been found in vivo, depending on cancer type and also
on the phenotype of the macrophages. High tumor macrophage
counts in general are associated with a better prognosis for colorectal
cancer patients [95, 109, 225-228]; however, if markers for M1 and
M2 macrophages are studied separately, some studies have found
M2-like macrophages to correlate with a worse prognosis [113, 114].
For lung cancer, M2-like macrophages are generally associated with
worse prognosis or clinicopathological factors [111, 112, 229, 230],
while the prognostic value of M1like macrophages in part appears to
depend on the localization of these macrophages, as M1-like macrophages within tumor islets, but not within the surrounding tumor
stroma, have been correlated with improved prognosis [93, 94].
Treatment of cancer cells in vitro with conditioned media from macrophages is one way to study if and how macrophages affect various
steps of cancer cell progression, that may explain the prognostic values found for tumor associated macrophages. In the papers included
in this thesis, the ability of macrophages of both the M1 and M2 phenotypes to affect cancer cell growth was studied. The results demonstrated that macrophages of the M1, but not M2 phenotype, caused a
reduction in cell numbers of cancer cells originating from colon (paper I-II) and lungs (paper III). Treatment of colon cancer cells from
the cell lines HT-29 or CACO-2 with CM from macrophages of the M1
phenotype gave a dose-dependent reduction in cell numbers, starting
with an almost complete inhibition of proliferation at full-dose. The
M1DIFF CM (media used for differentiation, which included LPS and
IFN-) was more potent compared to the M1 CM (collected after differentiation, without LPS and IFN-).
The higher potency of M1DIFF CM compared to M1 CM was not due
to the exogenously added LPS/IFN-as direct additions of these substances did not affect the cell numbers to any major extent. It is more
likely that the difference in potency is due to differences in the
amount of soluble factors released into the CM.
44
A similar reduction in cell numbers was observed for lung SCC (H520
cells) and SCLC (H69 cells) cells treated with M1 CM (M1DIFF CM
treatments was not included in this study).
M2 macrophages are suggested to aid cancer progression through various mechanisms, including an increased proliferation of cancer cells
through release of growth factors. However, in our studies we did not
observe an increase in proliferation of cancer cells from colon or lungs
treated with conditioned media from M2 macrophages (neither with
M2DIFF CM or M2 CM). There is a possibility that the cancer cells at
the culture conditions used already were at a maximal proliferative
speed. Others have found an increased proliferation of cancer cells
treated with CM from M2 macrophages when they replaced the FCS
supplemented media to a chemically defined serum-free media [231].
It is also possible that M2 macrophages primarily support tumor
growth in vivo due to factors not reproducible in vitro, such as increased angiogenesis.
Although there is a consensus that macrophages in general, and M1
macrophages in particular, have the potential to kill, or inhibit proliferation of cancer cells, there are only a limited number of studies that
have utilized conditioned media from human M1 or M2 macrophages
to evaluate their effects on the proliferation of cancer cells. GM-CSF
differentiated M1 macrophages have been found to inhibit proliferation of breast cancer cells [231] and osteosarcoma cells [232], and MCSF differentiated M1 macrophages have been reported to inhibit renal carcinoma cell growth [233].
There are a number of factors that could possibly induce the reduction in cell numbers, including released cytokines or other proteins
[231, 234, 235], reactive oxygen/nitrogen species [236-238] or depletion of essential nutrients [233].
Due to the unstable nature of reactive nitrogen/oxygen species, we
found it unlikely that these compounds would be preserved throughout the experimental setup, which includes long term storage of the
CM prior to use. Depletion of nutrients could be a possible explanation; however, a dose-dependent growth inhibitory effect was observed for dilutions as high as 1/8th of full dose (for M1DIFF CM
treatment of HT-29 cells) indicating that depletion of nutrients is not
a major cause for the reduction in proliferation observed. Cytokines
and other proteins play an important role in regulating the prolifera45
tion of various cells; however, we have not been able to pinpoint a

specific protein responsible for the reduction in cell numbers observed. Of the cytokines detected in the M1/M1DIFF CM, direct additions of TNF- and CXCL-9 were tested, which has previously been
reported to inhibit cancer cell growth or induce apoptosis of colon
cancer cells [239, 240] and also to inhibit the proliferation of colon
intestinal epithelial cells [241], but neither cytokine induced a reduction in cell numbers of HT-29 or CACO-2 cells.
It is plausible that it is not a single factor responsible for the reduction
in cell numbers observed, but rather a combined effect of multiple
proteins/other factors. The question of which factor(s) that is responsible for the growth inhibitory effects of our generated M1/M1DIFF
CM remains unresolved so far.
Treatment of colon cancer cells with CM from THP-1 macrophages
caused a reduction in cell numbers with a similar potency as M1 CM,
indicating a possible pro-inflammatory phenotype of the THP-1 macrophages.
Mechanisms of growth inhibition

A reduction in cell numbers after treatment can be attributed to a reduction in proliferation, cell cycle arrest, apoptosis, necrosis, or a
combination of these events. Although necrosis was not studied in
detail, treatment with M1/M1DIFF CM did not increase the amount of
non-viable cells (visualized with trypan blue) in either of the colon or
lung cancer cell lines studied (results not shown) indicating that these
CM did not induce necrosis.
Apoptosis measurements were performed using HT-29 colon cancer
cells. An increased apoptosis was observed in HT-29 cells when treated with M1DIFF CM, but not when treated with M1 CM. The M1DIFF
CM affected some of the HT-29 cells to detach from the cell culture
flasks, and a separate apoptosis measurement of detached and adherent cells revealed that the detached cells were apoptotic whereas no
increased apoptosis was detected for the adherent cells. However, the
M1DIFF CM treated adherent cells were unable to recover their proliferation following a 48 h culture in fresh culture media, indicating
that the treatment caused prolonged damage to the cells resulting in
46
either a sustained cell cycle arrest or a delayed apoptosis not detectable at the time-point of the apoptosis measurement. Whether apoptosis per se caused the cells to detached or if M1DIFF CM treatment
caused cells to detach, thereby inducing apoptosis (anoikis) is not determined.
Although M1 CM treatment caused a reduction in cell numbers, it did
not induce apoptosis of HT-29 cells, and no increased cell detachment
was observed for any of the adherent cell lines (HT-29, CACO-2 and
H520). Furthermore, following M1 CM treatment, HT-29 cells recovered their cell proliferation when cultured in fresh culture media, indicating that the majority of cells were still viable. In contrast, M1 CM
treated CACO-2 colon cancer cells maintained a greatly reduced proliferation post treatment. This difference in proliferative recovery post
M1 CM treatment between HT-29 and CACO-2 cells could possibly be
explained by differences in the cells ability to regulate the cell cycle.
Analysis of the cell cycle distribution showed that HT-29 cells, but not
CACO-2 cells, induced cell cycle arrest (in the G0/G1 and G2/M phases) in response to M1 CM (and also M1DIFF CM) treatment. Cell cycle
arrest is an important mechanism which enable cells to repair cell
damage, e.g. to DNA, or to obtain deprived nutrients, before a continuation of the cell cycle that would otherwise lead to damaged cells or
cell death. The cell cycle inhibition observed in M1 CM treated HT-29
cells could therefore play a protective role in the survival and recovery
of these cells.
Different responses regarding cell cycle inhibition was also observed
for the two different lung cancer cell lines studied. H520 cells responded to M1 CM treatment with cell cycle arrest, while this treatment did not affect the cell cycle distribution of H69 cells to any major extent. However, no studies were conducted on the recovery of
H520 and H69 cells post M1 CM treatment, and therefore it is not
confirmed whether H520 cells which responded with cell cycle arrest
recovers proliferation to a greater extent compared to H69 cells which
in similarity with CACO-2 cells do not respond with an apparent cell
cycle arrest.
The mRNA expression of a number of cell cycle regulatory genes were
studied in the colon cancer cell lines, and a few of these (i.e. p16, p21,
p27 and GADD45A) were studied also in the lung cancer cell lines in
order to get an understanding of the mechanisms of the M1 CM
47
treatment induced cell cycle arrest. A schematic overview of the function of the genes studied, and how the expression of these genes were
affected by M1 CM treatment in the cell lines which responded with a
cell cycle arrest (HT-29 and H520) can be seen in Fig. 6. The mRNA
expression of several genes were regulated in M1 CM treated HT-29
cells in a manner consistent with cell cycle arrest in G1/G0 and/or
G2/M. An up-regulation of the cell cycle inhibitor p21 was observed,
which can induce arrest in all cell cycle phases, although mainly in G1
through inhibition of the G1 associated cyclin-CDK complexes [242,
243]. In addition, both cyclin E and CDK2 were down-regulated in M1
CM treated HT-29 cells. The cyclin E-CDK2 complex is necessary for
G1/S transition, and furthermore, is inhibited by p21. CACO-2 cells
treated with M1 CM, which did not display an apparent cell cycle arrest, did not display any changes in the mRNA expression of either
p21, cyclin E or CDK-2. Since p21 from the mRNA data emerged as a
promising candidate for the M1 CM induced cell cycle arrest observed
in HT-29 cells, protein levels of p21 was studied and also found to be
elevated in the M1 CM treated HT-29 cells. However, siRNA knockdown experiments against p21 did not affect the cell cycle arrest induced by M1 CM, indicating that proteins other than p21 are responsible for the observed cell cycle arrest.
Of the lung cancer cell lines studied, M1 CM treated H520 cells, which
responded with G1/G0 arrest, was found to up-regulate all cell cycle
inhibiting genes studied, i.e. p16, p21, p27 and GADD45A, suggesting
one or several of these genes to be of importance for the arrest. However, this has not been confirmed by studies on the protein level.
48
Figure 6. Overview of the studied cell cycle regulatory genes and their mRNA
expression levels in M1 CM treated HT-29 and H520 cells. Filled arrows
denote effects on H520 and open arrows denote effects on HT-29 cells.
mRNA expression of all genes in the figure was studied for HT-29 while
for H520 only the up-regulated CDK inhibitors were studied.
Implication of macrophages during 5-FU treatment of colon cancer cells

5-FU is one of the most commonly used chemotherapeutic drugs for
the treatment of colon cancer; however, although combination therapies with 5-FU and other drugs (i.e. oxaliplatin and leucovorin) have
improved the efficacy of the chemotherapy, approximately 1/3 of the
patients treated with curative intent will relapse within 6 years [244].
It is possible that the tumor microenvironment in some cases enables
cancer cells to avoid treatment. In paper II the efficacy of 5-FU to inhibit proliferation of the colon cancer cell lines HT-29 and CACO-2
was studied, and whether macrophages of the M1 or M2 phenotype
(utilizing CM) could improve or attenuate the efficacy of 5-FU. It was
49
found that 5-FU dose dependently inhibited proliferation of both cell

lines, reaching maximum inhibition at about 10 M (24 h treatment).
At the concentration of 20 M, which was chosen for further experiments, both cell lines hardly recovered their cell growth at all even if
5-FU was washed away and the cells allowed to recover for up to 7 additional days. Addition of M1 or M2 CM during the 5-FU treatment
did not change the cells initial growth inhibition to 5-FU. However, if
the cells were allowed to recover for up to 7 days following 5-FU and
macrophage CM treatment, a greatly increased cancer cell proliferation was found for HT-29 cells having been treated with 5-FU plus M1
CM but not with 5-FU plus M2 CM. This was not observed for CACO2 cells which still did not recover from the 5-FU treatment; however,
CACO-2 cells recovered poorly also from M1 CM treatment alone,
which could possibly mask an effect on the 5-FU induced growth inhibition.
We argued that the M1 CM induced attenuation of 5-FU treatment in
HT-29 cells could depend either on the cell cycle inhibition observed
in HT-29 cells following M1 CM treatment (described in the previous
section), or through regulation of the metabolism of 5-FU inside the
cell, e.g. shunting 5-FU into the catabolic pathway thus avoiding toxicity. M1 CM treatment of HT-29 cells did affect the mRNA expression
of genes involved in 5-FU metabolism, however not in a manner that
would suggest an attenuation of 5-FU toxicity (Fig. 7). The target for
5-FU, namely TS, and furthermore the 5-FU catabolic enzyme DPD,
were both down-regulated, while the first enzyme in the 5-FU activating pathway, TP, was up-regulated. All these changes would, if anything, suggest an increased susceptibility to 5-FU toxicity in M1 CM
treated HT-29 cells, in contrast to our observed data.
50
Figure 7. Simplified overview of key genes involved in the activation and catabolism of 5-FU and how M1 CM treatment affected the expression of these
genes in HT-29 cells (indicated by open arrows).
Effects on the cell cycle could be of importance since 5-FU mainly is

an S-phase specific drug that target cells undergoing DNA synthesis
by inhibiting formation of the nucleotide dTMP. Cell cycle analysis of
5-FU treated HT-29 and CACO-2 cells revealed a major accumulation
of cells in S-phase following treatment indicating that cells entering Sphase were unable to continue through the cell cycle. Even though
apoptosis measurements did not reveal any increased apoptosis in
HT-29 or CACO-2 cells directly following 5-FU treatment, these cells
did not recover their proliferation post treatment indicating a sustained damage to these cells. However, for HT-29 cells a combined 5FU and M1 CM treatment effectively blocked accumulation of cells in
S-phase, and when both treatments were removed, the cells greatly
recovered proliferation. In contrast, CACO-2 cells treated with both 5FU and M1 CM did not avoid accumulation of cells in S-phase, and
also did not show an increased recovery following treatment. These
results suggest that cells not in S-phase and thereby not actively synthesizing DNA, were able to avoid 5-FU cytotoxicity and supports the
hypothesis that M1 CM induced cell cycle arrest (in G0/G1 and G2/M)
are responsible for the attenuation of 5-FU in HT-29 cells. These results may suggest that the presence of M1-like macrophages could be
beneficial in colon cancer tumors by reducing tumor growth, but the
same properties, could in some tumors attenuate the efficacy of 5-FU
51
based chemotherapies, enabling some of the cancer cells to escape

treatment.
Anti-inflammatory effects of green, black and rooibos tea extracts

NSAIDs which inhibit the enzymatic activities of COX-1/COX-2,
needed for synthesis of prostaglandins, reduces the risk of colorectal
cancer [245]. However, long term use of NSAIDs can cause serious,
even life-threatening gastrointestinal side-effects, including gastrointestinal bleedings [246]. Therefore, finding other drugs that inhibits
prostaglandin formation without serious side-effects accompanied
with long term use would be of great interest to be used as prophylactic drugs against colorectal cancer. Tea extracts, and various polyphenols including the green tea catechin EGCG can inhibit PGE2 formation [247, 248]; however, the mechanism of inhibition is not yet
fully elucidated. In paper IV, effects of green, black, and rooibos tea
extracts and of the polyphenols EGCG and quercetin were studied on
several aspects of prostaglandin formation. The main enzymes responsible for production of PGE2 during inflammation is cPLA2
which mobilize free arachidonic acid from membrane phospholipids,
and COX-2 plus mPGES-1 that converts arachidonic acid into PGH2
and subsequently to PGE2, respectively [249].
Through western blot analysis, it was found that human monocytes
without LPS treatment expressed cPLA2, but no, or very low amount
of COX-2 and mPGES-1. However, LPS treatment for 24 h greatly induced the expression of COX-2 and mPGES-1, and also up-regulated
cPLA2 expression about 2-fold. COX-1 expression was unaffected by
LPS treatment.
The green and black tea extracts and the polyphenols EGCG and
quercetin dose-dependently inhibited the LPS-induced expression of
mPGES-1, COX-2, and to a smaller extent also cPLA2, while the rooibos tea extract inhibited mPGES-1 expression and to a small extent
also COX-2 and cPLA2. Of the three enzymes, mPGES-1 expression
was inhibited with the greatest potency with all tea extracts, and green
tea inhibited mPGES-1 with highest potency, while black tea and
rooibos tea were approximately 2-fold, and 10-fold less potent, respectively (Table IV). EGCG were also found to be highly potent at
52
inhibiting mPGES-1 expression, with inhibition observed at 2.5 M

(Table IV). Although no studies have been conducted regarding inhibition of mPGES-1 expression with tea extracts or EGCG, several papers has reported COX-2 inhibition with green and black tea extracts
and with EGCG [250-253]. The inhibition of expression of the enzymes was accompanied with inhibition of PGE2 formation. Green
and black tea was equally potent inhibitors of PGE2 formation whereas rooibos tea was about 10-fold less potent (Table IV). Published literature suggest that the green tea extract at a dilution of 12,800 contain about 3 M EGCG [254, 255]. If so, the inhibition of PGE2 formation and of mPGES-1 expression with green tea could be due to its
EGCG content, whereas the inhibition observed with black tea must
be due to also other compounds, as the content of EGCG in black tea
is only 1/10 or lower than that of green tea [254, 255].
Table IV. Effects of tea extracts and polyphenols on the expression of enzymes
and release of PGE2 in LPS treated human monocytes.
Green tea
Rooibos
EGCG
tea
(M)
Inhibition at dilutions/concentrations of:
mPGES-1
expression
COX-2
expression
cPLA2
expression
PGE2
release
Black tea
Quercetin
(M)
1:51,200 1:25,600
1:6,400
2.5
20
1:12,800 1:25,600
1:3,200
10
20
1:6,400
1:6,400
1:6,400
20
1:51,200
1:51,200
1:6,400
2.5
20
Further studies were conducted to determine if the tea extracts and

polyphenols had any direct effects on the enzymatic activity of cPLA2,
COX-2 and mPGES-1. In a cellular assay, LPS stimulated monocytes
were washed and treated with tea extracts, EGCG or quercetin for 15
min plus 45 min with a calcium ionophore to stimulate PGE2 formation. With this short term treatment it was assumed that that there
53
would be no effects on the expression of the enzymes and that inhibition of PGE2 formation instead would be due to inhibition of the activity of the enzymes. With this assay, quercetin inhibited PGE2 formation dose-dependently with a 50% reduction at 40 M. However,
the tea extracts and EGCG did not inhibit PGE2 formation.
A cell free assay of the combined enzymatic activity of COX-2 and
mPGES-1 to convert arachidonic acid into PGE2 gave inhibition with
quercetin at similar doses as for the cellular assay. In the cell free assay, all tea extracts, and also EGCG inhibited PGE2, however, at considerably higher doses than required for inhibition of the expression
of mPGES-1. EGCG has previously been reported to inhibit the enzymatic activity of mPGES-1, but not COX-2 [248]; however, in our assay no such discrepancy can be made.
Macrophage expression of proteases or protease activity-related

genes and implications for lung cancer cells
Lung cancers in general and SCLC in particular have poor prognosis
and metastases are often found at the time of diagnosis. Proteases
play a key role in enabling cancer cells to invade nearby tissue and to
metastasize by degrading the surrounding ECM. Proteases and proteins important for protease activity, such as the uPA/uPAR system,
may be expressed by cancer cells or by stromal cells e.g. macrophages,
within the tumor. In paper III, expression of protease activity-related
genes were studied in lung cancer cells of SCLC and lung SCC origin,
and also in macrophages of the M0, M1 and M2 phenotypes, to get an
insight into the possible stromal macrophages contribution of these
proteins in lung cancers. The genes studied included uPA, uPAR, PAI1, MMP-2 and MMP-9, which have all been suggested to correlate
with worse outcome in lung cancers [151, 152, 160, 256-259].
It was found that cultured human macrophages of all phenotypes
(M0, M1 and M2) expressed considerably higher mRNA levels of uPA,
uPAR and PAI-1 (10- 10,000-fold) compared to the lung SCC (H520)
and SCLC (H69) cells studied. All macrophage phenotypes expressed
similar mRNA levels of uPA and PAI-1, but the M1 phenotype expressed considerably higher levels of uPAR, both at the mRNA (7fold) and protein levels compared to the M0 and M2 phenotype. Furthermore, treatment of M1 CM to the lung cancer cell lines H520 and
54
H69 significantly up-regulated PAI-1 mRNA expression in both cell

lines. High uPAR positive macrophage counts in colorectal cancer
tumors have been shown to correlate more strongly with poor prognosis, compared to uPAR positivity in the cancer cells [150]. Our results indicate that macrophages; M1 macrophages in particular, may
contribute significantly to the uPAR expression in some forms of lung
cancer, which may be of relevance for tumor progression.
All macrophages expressed MMP-9 mRNA to a similar extent, and
MMP-9 gelatinase activity was found in the M1 and M2 CM, while
MMP-9 mRNA and MMP-9 gelatinase activity was undetected in both
H520 and H69 cells and CM, respectively. The M1 phenotype expressed the highest mRNA levels of MMP-2, and MMP-2 gelatinase
activity could be observed in M1 CM, but not in M2 CM or CM from
the lung cancer cell lines investigated. These results further imply that
macrophages in lung cancer could contribute greatly to the amount of
proteolytic enzymes present in the tumors, if the in vitro results are
applicable to the in vivo situation.
In addition to the in vitro cell culture studies, 23 lung SCC and 10
SCLC biopsies were immunohistochemically stained with antibodies
targeting the pan-macrophage marker CD68, the suggested M2 marker CD163, and also uPAR, MMP-2 and MMP-9. Macrophages identified by the expression of either CD68 or CD163 were found in all biopsies, but in varying amount, from few cells to highly infiltrated tissues. CD163 was mostly found to be expressed at a similar extent to
CD68, or even more widespread than CD68, indicating that the majority of macrophages were of an M2-like phenotype, if CD163 is a
useful marker for M2-like macrophages in lung SCC and SCLC. Others, which have performed a more extensive characterization using
dual-staining for CD68 and various M1 and M2 phenotype markers
have reported that approximately 20-30 % is of an M1-like phenotype,
and 70-80% of an M2-like phenotype in NSCLC [94, 111].
We could also observe CD163 expression in some of the cancer cells in
10/23 lung SCC biopsies and in 2/10 SCLC biopsies. Others have
shown that expression of this macrophage marker in breast, and colorectal cancer cells correlate with poor survival [260], and so it would
be of interest to study if the same can be found for lung SCC. In
agreement with the in vitro findings, a subset of stromal cells including macrophages was found to express uPAR, MMP-2 and MMP-9 in
55
both lung SCC and SCLC biopsies. Of the cancer cells, uPAR expression was found in some of the cancer cells in 22/23 lung SCC biopsies
but was not found in SCLC cells. Both MMP-2 and MMP-9 immunoreactivity in the cancer cells was found in the majority of the lung SCC
and SCLC biopsies.
56
Concluding remarks
Inflammatory cells in tumors can contribute to cancer progression
e.g. through production of growth factors and proteases, but can also
kill cancer cells or inhibit their growth. Macrophages are inflammatory cells often found in large quantities in tumors and possess both
tumor stimulating and tumor killing abilities. There are two main
phenotypes of macrophages, the M1 and M2 phenotype, and effects of
these macrophages on different aspects of cancer progression were
studied in this thesis.
It was found that human monocyte derived M-CSF differentiated
macrophages differentiated into an M1 phenotype, but not macrophages differentiated into an M2 phenotype, were able to inhibit the
cell growth of both colon and lung cancer cells, and that the inhibition
in some cell lines was due to induction of cell cycle arrest in the
G1/G0 and/or G2/M phases. Furthermore, the cell cycle arrest induced by the M1 phenotype could attenuate the efficacy of 5-FU
treatment on a colon cancer cell line. These results suggest that macrophages of M1 phenotype could be beneficial in tumors through inhibition of cancer cell growth, but that said inhibition could be unwanted during 5-FU based chemotherapy if the cell growth inhibition
is accompanied with cell cycle arrest in the G1/G0 or G2/M phases.
The uPA/uPAR system activates uPA and in turn other proteases including MMPs, which can degrade the ECM allowing cancer cells to
escape the primary tumor site. SCLC and lung SCC biopsies revealed
that macrophages are present often in high amount in both SCLC and
lung SCC, and also that these macrophages can express uPAR, MMP2 and MMP-9. In vitro, the M1 and M2 phenotypes were found to express high mRNA levels of uPA, uPAR and PAI-1 compared to the two
lung cancer cell lines investigated, suggesting that macrophages could
contribute greatly to the expression of these proteins in lung tumors.
The M1 phenotype was found to express higher levels of uPAR and
MMP-2 compared to the M2 phenotype, and was also able to induce
PAI-1 mRNA expression in the two lung cancer cell lines investigated.
As the expression of these proteins in lung tumors have been suggested to predict poor prognosis, both the M1 and M2 phenotype may
57
contribute to tumor progression in these cancers, unless the cytotoxic

effects of the M1 phenotype dominate.
COX inhibitors block formation of prostaglandins (e.g. PGE2) and
have been shown to reduce the risk to develop colon cancer. Green,
black and rooibos tea extracts, and also the polyphenols EGCG and
quercetin, were shown to inhibit PGE2 formation in human LPS
stimulated monocytes. The green and black teas primarily inhibited
PGE2 formation by inhibiting expression of mPGES-1, COX-2 and to
a less extent cPLA2, whereas the rooibos tea which was the least potent of the three teas primarily inhibited PGE2 formation by inhibiting mPGES-1 expression. The inhibition of PGE2 formation with
green tea could likely be due to its EGCG content. A direct effect on
the combined enzymatic activity of COX-2 and mPGES-1 could also be
observed with all three teas; however, higher concentrations were
needed for this direct effect on the activity of the enzymes than needed for inhibition of the expression of the enzymes or PGE2 formation.
58
Acknowledgements
I would like to thank all my colleagues at Karlstad University for the
invaluable help and support that you have provided in order to make
this thesis. There are some in particular who I want to mention:
For a start I would like to thank my supervisors, it has been great
working with you. Thank you for this time, for a good team work in a
friendly and helpful atmosphere.
Jonny Wijkander, thank you for being a great supervisor, for the way
you have led this project into the right track, for all the help in the lab,
with the writing of articles and for giving me this opportunity. It has
been great fun working with you.
Ann Erlandsson, thank you for the supervision. It has been inspiring
to work with you, the way you love research and always have new ideas.
Dick Delbro, thank you for the great help in writing the articles and
for sharing your great experience of science.
I would like to thank my co-authors, Tomas Seidal and Ingrid Persson
for valuable input.
I also want to thank the staff at the oncology department at Karlstad
central Hospital for help with immunostainings.
I would also like to thank the PhD students or former PhD students,
Ola Olsson, Filip Rendel and Christina Fjaeraa Alfredsson for making
the time in the lab, and outside, a lot more fun!
Jag vill ocks tacka ngra utanfr jobbet, vnner och familj. Utan er
hade denna avhandling inte varit mjlig:
Mamma och pappa, tack fr att ni alltid funnits dr. Tack Christopher
Engstrm, utan dig hade jag kanske varken varit nrdig eller fr den
delen skrivit en avhandling!
59
Slutligen vill jag tacka Lisa Hedbrant, tack fr din krlek, uppmuntran
och stttning. Du har betytt mer n ngot annat fr mig under denna
tid.
60
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Macrophages are cells of the immune system often found in large numbers in
cancer tumors. They affect multiple aspects of cancer progression, including
growth and spread of cancer cells, and the efficacy of treatments. There are two
major macrophage phenotypes denoted M1 and M2, that have mainly pro- and
anti-inflammatory properties, respectively.
In this thesis, M1 and M2 macrophages were characterized and effects of them
on different aspects of cancer progression were studied using culture of colon,
and lung cancer cells.
The M1 phenotype inhibited proliferation of cancer cells from both colon and
lung. The growth inhibition was for some cell lines accompanied by cell cycle
arrest.
The macrophage induced cell cycle arrest was found to protect colon cancer cells
from the cytostatic drug 5-fluorouracil.
Prostaglandin E2 (PGE2) contributes to colon cancer development and treatment
of monocytes with tea extracts inhibited PGE2 formation via inhibition of
expression of microsomal prostaglandin E synthase-1.
Proteases can degrade the extracellular matrix of a tumor to facilitate cancer cell
invasion and metastasis. The M1 and M2 phenotypes of macrophages expressed
several protease activity related genes to a greater extent than lung cancer cells,
and M1 more so than the M2 phenotype.
ISBN 978-91-7063-654-7
ISSN 1403-8099

Cancer and Inflammation - Role of Macrophages and Monocytes

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Role of Macrophages and Monocytes

Faculty of Health, Science and Technology

Cancer and Inflammation

DISSERTATION | Karlstad University Studies | 2015:36

Cancer and Inflammation - Role of Macrophages and Monocytes

Cell cycle regulation ........................................................................... 8

Culture of cancer cell lines ............................................................... 30

Protein siRNA knockdown ................................................................ 35

Characteristics of cultured human macrophages of different

Implication of macrophages during 5-FU treatment of colon cancer

The authors contribution to the papers

Commonly used abbreviations

Cell cycle regulation

biological functions and grows in size until it is ready to proceed

which accounts for approximately 15% of all CRC, is due to somatic

Figure 1. Metabolism and effects of 5-fluorouracil (5-FU). Enzyme abbreviations;

SCLCs sometimes produce hormones including antidiuretic hormone

Risk factors for lung cancer

The tumor microenvironment

inflammatory regulation [60]. They were first discovered by lie

The M1 phenotypes of macrophages

The M2 phenotypes of macrophages

view the M1/M2 phenotypes as the extreme endpoints of activation

Tumor associated macrophages

monocytes found in the blood, CD14+/CD16- monocytes and

The extra cellular matrix

The urokinase-type plasminogen activator system

ectin, a protein of the ECM, and uPAR-vitronectin binding is thought

Figure 2. Plasmin activation by the uPA/uPAR system. (The figure is adapted

The urokinase-type plasminogen activator system in cancer

Polyphenol content of green, black and rooibos tea

proliferative/pro-apoptotic effects of certain polyphenols toward cancer cells [174, 175].

Figure 3. Major classes of polyphenols including example structures. (The figure

tions via receptor ligand binding, or be metabolized into an inactive

variety of its pleiotropic effects, including its effects on the immune

Background and aim

Can affect the efficacy of 5-FU to inhibit colon cancer cell

Express different levels of MMPs and genes of the uPA/uPAR

Culture of cancer cell lines

acute monocytic leukemia

Isolation of human peripheral blood monocytes and differentiation into macrophages

Figure 4. Flowchart illustrating the method used for generation of macrophages

Differentiation of THP-1 monocytes into macrophages

solution (Accumax). The advantage of assessing cell numbers by using

Detection of proteins and PGE2

Enzyme linked immunoassays

veloped using an enhanced chemiluminescent (ECL) substrate and

Table II. Antibodies used

Protein siRNA knockdown

RNA extraction and cDNA synthesis

Quantitative real-time PCR

Cell cycle analysis

Preparation of tea extracts

tin) prior to addition of the substrate arachidonic acid. The reaction

Results and Discussion

Characteristics of cultured human macrophages of different

ferentiation induced expression of some suggested M1 characteristic

Figure 5. Representative images of CD163 immunoreactivity following M0 (a),

The monocytic leukemia cell line THP-1 is a commonly used in vitro

Differentiated macrophages or macrophage DIFF CM

Macrophages and cancer cell growth

tion of various cells; however, we have not been able to pinpoint a

Mechanisms of growth inhibition