Cancer and InflammationRole of Macrophages and Monocytes

Alexander Hedbrant

Alexander H

edbrant | Cancer and Inflam

mation | 2015:36

Cancer and Inflammation

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.

DISSERTATION | Karlstad University Studies | 2015:36 DISSERTATION | Karlstad University Studies | 2015:36

ISSN 1403-8099

Faculty of Health, Science and TechnologyISBN 978-91-7063-654-7

Biomedical Sciences

DISSERTATION | Karlstad University Studies | 2015:36

Cancer and InflammationRole of Macrophages and Monocytes

Alexander Hedbrant

Print: Universitetstryckeriet, Karlstad 2015

Distribution:Karlstad University Faculty of Health, Science and TechnologyDepartment of Health SciencesSE-651 88 Karlstad, Sweden+46 54 700 10 00

© The author

ISBN 978-91-7063-654-7

ISSN 1403-8099

URI: urn:nbn:se:kau:diva-37086

Karlstad University Studies | 2015:36

DISSERTATION

Alexander Hedbrant

Cancer and Inflammation - Role of Macrophages and Monocytes

WWW.KAU.SE

1

“It doesn't stop being magic just because you know how it works.”

– Terry Pratchett, The Wee Free Men

2

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 co-

lon and lung cancer cells were studied.

Macrophages of the M1 phenotype, but not the M2 phenotype, inhib-

ited 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 metallo-

proteinase-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 ex-

pressed by e.g. macrophages and monocytes, and inhibition of its ex-

pression 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 inhibi-

tion of expression of enzymes in the PGE2 formation pathway, and

primarily microsomal prostaglandin E synthase-1.

3

List of Content

ABSTRACT ........................................................................................................................... 2

LIST OF CONTENT .............................................................................................................. 3

LIST OF PAPERS ................................................................................................................. 5

THE AUTHOR’S 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

4

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

5

List of papers

I. Engström 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: Con-

ditioned media from human macrophages of M1 phenotype attenuate

the cytotoxic effect of 5‑fluorouracil on the HT‑29 colon cancer cell

line. Int J Oncol 46: 37-46, 2014.

III. Hedbrant A, Wijkander J, Seidal T, Delbro D, Erlands-

son A: Macrophages of M1 phenotype have properties that influence

lung cancer cell progression. Tumor Biol. 2015

IV. Hedbrant A, Nathalie Oberschmid, Bart van Aerde, In-

grid Persson, Ann Erlandsson, Wijkander J: Green, black and rooibos

tea inhibit prostaglandin E2 formation in human monocytes by inhib-

iting expression of enzymes in the prostaglandin E2 pathway. Manu-

script

*Name change august 2014 from Engström to Hedbrant

6

The author’s 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,

planning and writing

Paper III A major part of evaluating results and writing, part of ex-

perimental work and planning

Paper IV A major part of experimental work, evaluating results,

planning and writing

7

Commonly used abbreviations

5-FU 5-fluorouridine

CCL C-C motif ligand chemokine

CD cluster of differentiation

CDK cyclin-dependent kinase

CM conditioned media

COX cyclooxygenase

cPLA2 cytosolic phospholipase A2

CRC colorectal cancer

CXCL C-X-C motif ligand chemokine

DPD dihydropyrimidine dehydrogenase

ECM extracellular matrix

EGCG epigallocatechin gallate

FCS foetal calf serum

GM-CSF granulocyte-macrophage colony stimulating factor

IFN interferon

IL interleukin

iNOS inducible nitric oxide synthase

LPS lipopolysaccharide

M-CSF macrophage colony stimulating factor

MMP matrix metalloproteinase

MTHFR methylene tetrahydrofolate reductase

mPGES microsomal prostaglandin E synthase

(N)SCLC (non)-small cell lung cancer

PAI plasminogen activator inhibitor

PG(E2) prostaglandin (E2)

SCC squamous cell carcinoma

siRNA small interfering RNA

TAM tumor associated macrophage

TLR toll-like receptor

TNF- tumor necrosis factor alpha

TP thymidine phosphorylase

TS thymidylate synthetase

UMPS uridine monophosphate synthetase

uPA(R) urokinase-type plasminogen activator (receptor)

8

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 dis-

eases [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 com-

monly found in cancer are those that affect genes which regulate func-

tions such as cell division, apoptosis or DNA repair. During the pro-

gression 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, ena-

bling 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 ener-

getics [3]. Although genetic alterations are vital for cancer progres-

sion, 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 nor-

mal 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

9

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 S-

phase it synthesizes a double setup of its genome. G2 is an intermedi-

ate control-phase before the initiation of mitosis in which the chro-

mosomes segregate and the cell divides (cytokinesis). After cell divi-

sion 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 ac-

tion of cyclin-dependent kinases (CDKs) which are activated by pro-

teins 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 G1-

S 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 inactiva-

tion of Rb enables E2F mediated expression of other cyclins and pro-

teins needed for progression into S-phase.

For the discovery of CDKs, cyclins and cell cycle checkpoints, the re-

searchers 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 phos-

phorylation 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 cy-

cle 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 apop-

tosis, a controlled destruction of the cell.

10

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 approxi-

mately 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. Crohn’s

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 consump-

tion 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 ge-

netic alterations are acquired. There are three known molecular

pathways for the progression of CRC, mediated either by chromoso-

mal instability (CIN), microsatellite instability (MSI) or the CpG is-

land methylator phenotype (CIMP) [22]. The CIN pathway which ac-

counts for approximately 70% of all sporadic CRC was first described

by Faeron & Vogelstein in 1990 [23]. It involves a multistep progres-

sion 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 sug-

gested to arise from mutations in genes that control chromosome seg-

regation during cell division [24]. MSI, the second molecular pathway

11

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 microsatel-

lite repeats, i.e. simple DNA sequence repeats of 1-6 base-pair. The

third molecular pathway for CRC, CIMP, is characteristic for epige-

netic silencing of tumor suppressor genes or DNA mismatch repair

genes through methylation of promoter regions rich in 5’-cytocine-

guanine-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 tu-

mor, which can be combined with adjuvant chemotherapy for colon

cancer or radiation therapy (alone or in combination with pre-

operative chemotherapy) for rectal cancer. The chemotherapy primar-

ily involve a combination therapy that include the cytostatic drug 5-

fluorouracil (5-FU), folinic acid (also known as leucovorin) which en-

hances 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 monoclo-

nal antibody therapy against the biological targets vascular endotheli-

al growth factor or epidermal growth factor receptor [29].

5-fluorouracil

5-FU was discovered already in 1957, and is still a highly used cyto-

static drug for the treatment of CRC and other cancers, although there

are now also modified versions of it in use, e.g. the orally adminis-

tered 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 mono-

phosphate (dTMP), therefore, inhibition of TS leads to a shortage of

12

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 con-

verted 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 5-

FU because it elevates the intracellular levels of 5, 10-MTHF [32]. 5-

FU may also be converted into fluorouridine triphosphate (FUTP) and

cause RNA damage, starting with conversion of 5-FU into fluorouri-

dine monophosphate (FUMP) by uridine monophosphate synthetase

(UMPS) [33]. 5-FU is catabolized into the non-toxic substance dihy-

drofluorouracil 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

13

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, squa-

mous 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 differ-

ences between the subgroups of NSCLC [37].

Treatment of NSCLC involves surgical resection of the tumor if possi-

ble, 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 be-

fore/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 be-

tween 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 echino-

derm 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 muta-

tion, more common in the Asian population) and 5% (EML4-ALK) of

the NSCLC patients, and primarily in non-smokers with adenocarci-

noma [42, 43].

SCLC is a neuroendocrine cancer, originating from the neuroendo-

crine Kulchitsky cells found in the bronchial epithelium. Neuroendo-

crine cells release biologically active substances such as hormones or

paracrine signaling substances, upon nerve stimulation. As such,

14

SCLCs sometimes produce hormones including antidiuretic hormone

and adrenocorticotrophic hormone [44]. SCLC is a fast growing, ag-

gressive 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 region-

al lymph nodes at the time of diagnosis [45]. Treatment of SCLC in-

volves 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]. Alt-

hough 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 consti-

tuted 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]. Fur-

thermore, 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 carci-

noma, 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 in-

clude 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].

15

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 sys-

tem 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 malig-

nant 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 in-

flammation also as a driving force for cancer progression.

Already in year 1863, Rudolf Virchow [54] observed leukocyte infiltra-

tion in cancer tumors and made a connection between chronic in-

flammation and cancer. A number of chronic inflammatory diseases

are now known to greatly increase the risk to develop cancer, e.g. in-

flammatory bowel diseases that increase the risk of colon cancer, hep-

atitis induced chronic liver inflammation that increases the risk of

hepatocellular carcinoma and Helicobacter pylori infections that in-

crease the risk of stomach cancer [55-57]. Also, the use of anti-

inflammatory 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 pro-

mote cancer progression in a number of ways. These include produc-

tion of angiogenic or mitogenic factors, production of proteases that

degrade extracellular matrix components allowing tumor cells to in-

vade 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 devel-

opment, immune functions including immune surveillance, killing of

pathogens and cancer cells, antigen presentation and pro- and anti-

16

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

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 ex-

plained 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 granulocyte-

macrophage 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 adult-

hood 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 macro-

phages, 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. mi-

croglia 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 func-

tions 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 activa-

tion 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 recogni-

tion 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], scav-

17

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 pro-

inflammatory 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 in-

flammation, 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 macro-

phages. 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 interferon-

IFN-)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 differenti-

ate macrophages to an alternatively activated M2 phenotype [87-89].

M1 macrophages act pro-inflammatory through the release of pro-

inflammatory 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, pos-

sess 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

18

many proteins have been reported to be up-regulated in M1 macro-

phages, 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 re-

ceptor as IL-4 [99]. Further studies of the M2 phenotype have since

then revealed a wide array of soluble factors or membrane bound pro-

teins differentially expressed in the M2-phenotype, which generally

include an anti-inflammatory, wound-healing phenotype that pro-

motes 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, respective-

ly [100, 101].

Following the IL-4/IL-13 pathway of inducing the M2 phenotype, sev-

eral 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 in-

duced by FcR stimulation together with TLR agonists; and the M2c

phenotype, induced by glucocorticoids or IL-10 [102-104]. Others

19

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 com-

monly used [105]. Other markers include macrophage scavenger re-

ceptor 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 associat-

ed 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 can-

cers, 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 deple-

tion of macrophages, inhibition of macrophage recruitment to tu-

mors, 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 ag-

onist, anti-IL4 antibodies, or IFN- plus LPS has been demonstrated

to convert macrophages from an M2 phenotype into the M1 pheno-

type [117, 119, 120].

Monocytes

Monocytes are not merely precursors of macrophages, but can them-

selves 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 phago-

cytes, and can present antigens. There are two major populations of

20

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+CD16-

monocytes 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 re-

ceptor needed for CCL2 induced chemotaxis, which is one of the pri-

mary 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 prom-

inently by TGF- [125]. Proteases on the other hand, facilitate degra-

dation of the ECM, which among others include plasmin, matrix met-

alloproteinases (MMPs), cathepsins, and the a disintegrin and metal-

loproteinase domain (ADAM) family of proteases. Production of ECM

is very important during wound healing, but excessive ECM produc-

tion 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 µM [127]. Activation of

plasmin is mediated by two serine proteases, namely tissue-type

plasminogen activator (tPA) and urokinase-type plasminogen activa-

tor (uPA). One major role of plasmin is fibrinolysis, the process of dis-

21

solving blood clots. tPA is the primary activator of plasminogen in fi-

brinolysis, which is stored in endothelial cells, and upon release, can

bind to fibrin. This binding increases the catalytic efficacy of tPA re-

garding plasminogen activation over 100-fold [128, 129]. Plasmin can

also be activated within tissues to facilitate ECM degradation; a pro-

cess 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 metasta-

size [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 tis-

sues is regulated by a series of proteins, including binding of plasmin-

ogen 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 recep-

tors drastically increases its proteolytic activation by uPAR-bound

uPA; therefore, cellular expression of uPAR and plasminogen recep-

tors 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 pro-

uPA 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 intracellu-

lar domain, but is instead bound to the cell membrane via a glyco-

sylphosphatidylinositol anchor. Even so, uPAR can act as a signal

transducer through interaction with other trans-membrane cell sur-

face 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 3-

kinase/AKT-pathway [139-141]. Furthermore, uPAR binds vitron-

22

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 metallo-

proteinases to cleave off domain DI, leaving domain DII-III still an-

chored 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: In-

ternational Journal of Molecular Sciences [135])

23

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 can-

cer [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], col-

orectal [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 pro-

teins, 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 de-

pending 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 mecha-

nisms that inhibits inflammation, promotes vascular health, acts anti-

microbial and anti-tumorigenic [169-173]. For example, anti-

inflammatory effects of polyphenols include inhibition of NF-kB sig-

naling, 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 addi-

tion to their anti-inflammatory effects be attributed to reactive oxygen

scavenging and anti-angiogenic effects as well as anti-

24

proliferative/pro-apoptotic effects of certain polyphenols toward can-

cer 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 rooi-

bos 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 pre-

serving the catechins [169]. The catechins, which belong to the fla-

vanol subgroup of the flavonoids, constitute approximately 30-40% of

the water soluble dry materials in green tea, and 80-90 % of the fla-

vonoid content [169, 176].

The principal and most well studied polyphenol of green tea is epigal-

locatechin gallate (EGCG), which constitute approximately half of the

flavonoid content of green tea, however, more than 30 different poly-

phenols have been identified in green tea extracts, and include flavo-

nols 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 oli-

gomers, named theaflavins and thearubigins, which together consti-

tutes 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 in-

cluding nothofagin and the rooibos unique substance aspalathin [171].

25

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 inflam-

mation and diseases. In 1982 the Nobel Prize was awarded to Sune

Bergström, Bengt Samuelsson and John Vane for their discoveries in

the field of prostaglandins and related substances. Sune Bergström

determined the chemical structure of prostaglandins, Bengt Samuels-

son elucidated the process of their formation and John Vane discov-

ered that the anti-inflammatory properties of aspirin were achieved

through blocking prostanoid synthesis [181].

26

Eicosanoids, which include prostanoids (prostaglandins and throm-

boxane 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 pros-

tanoids, 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 iso-

form that can be induced by various inflammatory stimuli or patho-

logical 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. regula-

tion of blood clotting and vascular tension, bronchoconstriction and

dilation, gastrointestinal motility and secretion, kidney function, my-

ometrial contraction, regulation of inflammation and induction of fe-

ver 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 ei-

ther activates or blocks adenylyl cyclase leading to increased or de-

creased cAMP levels, or activates the diacyl-glycerol/inositol triphos-

phate (DAG/IP3) signaling pathway [184]. Furthermore, non-

enzymatic 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 recep-

tors. PGE2 can be generated by three different PGE synthases, name-

ly, 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 func-

27

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 vasodila-

tion in combination with an increased vascular permeability facilitat-

ing 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 chemoattract-

ants 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 dur-

ing acute inflammation, PGE2 can also act immunosuppressive by

regulating the inflammatory response of other immune cells. It en-

hances expression of the anti-inflammatory cytokine IL-10 and inhib-

its expression of the pro-inflammatory cytokine IL-12 in macrophag-

es, 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 for-

mation is commonly seen in a number of cancers, including CRC and

lung cancer [198-205]. Also, the administration of COX inhibitors re-

duces 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 pro-

gression in a number of cancer models [195, 209-211]. The mecha-

nism for PGE2 cancer promotion has been suggested to be due to a

28

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

system, induction of angiogenesis, inhibition of apoptosis and activa-

tion of mitogenic signaling [212].

29

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 pro-

inflammatory actions including clearance of pathogens and cancer

cells, to wound-healing functions and immune suppression. The

broad functional diversity of macrophages makes them highly inter-

esting to study in the context of a tumor microenvironment because it

gives them the ability to act both pro- and anti-tumorigenic. Elucidat-

ing the interplay between cancer cells and macrophages of different

phenotypes is an important challenge to better understand the pro-

gression 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 macro-

phages 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)

30

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 apop-

tosis, expression of genes important for invasion and metastasis and

resistance of cancer cells to chemotherapy. A cancer cell line of mono-

cytic origin was also cultured and used for differentiation into macro-

phages. The cell lines were routinely sub-cultured twice weekly, cul-

tured in RPMI media supplemented with heat inactivated foetal calf

serum (FCS) and antibiotics (penicillin and streptomycin) and main-

tained at 37˚C 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 Pa-

per

CACO-2 Human colon colorectal adenocarcinoma I, II

HT-29 Human colon colorectal adenocarcinoma I, II

NCI-H69 Human lung carcinoma; small cell lung can-

cer

III

NCI-

H520

Human lung squamous cell carcinoma III

THP-1 Human blood acute monocytic leukemia I

31

Isolation of human peripheral blood monocytes and differentia-

tion 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 adher-

ence method, which utilize the monocytes ability to bind plastic sur-

faces, 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 monocyte-

derived 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 treat-

ment with LPS plus IFN- or IL-4 plus IL-13, respectively. Macro-

phages without any added differentiation stimuli were termed M0

macrophages. Conditioned media (CM) from macrophages were col-

lected 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 macro-

phages 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 com-

monly used methods, e.g. isolation of peritoneal macrophages of mu-

rine 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 prima-

ry healthy cells. The disadvantage of PBMC derived monocyte differ-

entiation is that it is a time-consuming process. There can also be dif-

ferences between donors regarding the amount of monocytes ob-

tained and how they respond to various stimuli.

32

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 differen-

tiated 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 Bürker 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 (pa-

per 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 Bürker chambers. To generate a single cell suspension

of H69 cells, the cells were separated using a specialized dissociation

33

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

a hemocytometer such as Bürker 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 sig-

nals which are used to estimate the amount of cells present. Disad-

vantages 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 re-

leased into the culture medium, whereas cell surface proteins or in-

tracellular 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. Sep-

arated proteins were transferred onto a polyvinylidene fluoride mem-

brane using a wet transfer system. The membrane was blocked with

an appropriate blocking buffer and was then incubated with the pri-

mary antibody overnight at 4˚C (See Table II for antibody targets).

Following primary antibody binding, an appropriate horseradish pe-

roxidase linked secondary antibody was applied and signals were de-

34

veloped using an enhanced chemiluminescent (ECL) substrate and

light intensities were detected by exposure to x-ray film. After detec-

tion, the antibodies could be stripped of to allow reuse of the mem-

brane 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 immuno-

histochemistry.

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 stain-

ing. 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 per-

formed using a Dako autostainer and visualized using the standard

method of horseradish peroxidase and 3,3’-diaminobenzidine. The

nuclei were then counterstained with Mayer’s haematoxylin and the

slides dehydrated and mounted. Tonsil tissue was used as control.

35

Table II. Antibodies used

Antibody

target

Application Paper

p21 Western blot II

-actin Western blot II

COX-1 Western blot IV

COX-2 Western blot IV

cPLA2 Western blot IV

mPGES-1 Western blot IV

CD68 IHC, ICC I-III

CD163 IHC, ICC I- III

MMP-2 IHC III

MMP-9 IHC III

uPAR IHC, ICC III

Protein siRNA knockdown

Knockdown of p21 protein expression (paper II) was achieved in HT-

29 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 nega-

tive 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 manufactur-

er’s instructions. The RNA quantity and purity was measured via the

absorbance value at 260 nm and the 260/280 nm ratio, respectively.

36

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 soft-

ware [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 validat-

ed 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 tar-

gets. However, when measuring mRNA it is important to acknowledge

that the level of mRNA may not always reflect the amount of corre-

sponding 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 pro-

pidium 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 pre-

sent in the cell. A signal of 1N=G1-phase (a single setup of chromo-

some 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 apop-

tosis, 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.

37

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 as-

say, the cells were fixed with 1 % paraformaldehyde dissolved in PBS

and subsequently further fixed in ice-cold 70% ethanol. Paraformal-

dehyde 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 apop-

tosis, by fluorescently labeling the exposed 3’-hydroxyl ends of DNA

fragments. This method is a more reliable and sensitive method com-

pared 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 µm sterile filter and frozen

in aliquots at -20°C 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 stimulat-

ed 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 soni-

cation. 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 supple-

mented with L-glutathione and the mixture was pre-incubated with or

without the addition of tea extracts or polyphenols (EGCG or querce-

38

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 esti-

mates from the SDS-PAGE separation.

39

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 num-

ber 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 anti-

inflammatory properties were investigated with regard to their ability

to inhibit PGE2 formation, an inflammatory mediator strongly associ-

ated 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 pheno-

type and the anti-inflammatory M2 phenotype. These phenotypes can

be generated in vitro in a number of ways, and one of the most com-

monly 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 macrophag-

es (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 pan-

macrophage 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 differentiat-

ed cells were also CD163 positive to a large extent (70% and 80% posi-

tive cells, respectively) while the large majority of the M1 differentiat-

ed cells were CD163 negative (Fig 5 a-c). At the mRNA level, M1 dif-

40

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 dif-

ferentiated state within this timeframe. In agreement with these re-

sults, 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 differ-

entiated 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, in-

cluding 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 IL-

10 was found in the M1DIFF CM. However, the same induction of IL-

10 has been observed by others [221] and is likely due to the M-CSF

differentiation which has been reported to yield macrophages that re-

sponds with IL-10 release upon LPS treatment [223].

In general, the M1/M2DIFF CM contained higher amount of the cyto-

kines 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 cyto-

kines detected (by the RayBiotech protein array) M0DIFF CM and

M2DIFF CM was near to identical. Furthermore, M0 and M2 macro-

phages expressed CD163 to a similar extent. These results support

41

findings by others which have suggested M-CSF to differentiate mac-

rophages 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 follow-

ing the 48 h culture post differentiation stimuli (Fig. 5 d-f).

Differentiation into M1 macrophages also resulted in lower cell num-

bers 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 acti-

vate proliferation of tissue resident macrophages in a mouse model

[224].

42

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.

43

Ta

ble

III

. P

hen

oty

pic

mR

NA

an

d p

rote

in e

xp

ress

ion

of

cult

ure

d h

um

an

ma

cro

ph

ag

es o

f th

e M

1, M

2 a

nd

M0

ph

eno

typ

es.

Plu

s si

gn

in

di-

cate

pro

tein

ex

pre

ssio

n/r

elea

se a

nd

min

us

sig

n i

nd

ica

te n

o d

etec

tab

le p

rote

in (

in C

M o

r o

n t

he

cell

s d

etec

ted

by

im

mu

no

cyto

chem

-

istr

y).

+/-

in

dic

ate

po

ssib

le b

ut

low

ex

pre

ssio

n.

Pro

tein

lev

els

wer

e d

etec

ted

sem

i-q

ua

nti

tati

vel

y (

n=

2-4

). F

old

ch

an

ges

are

co

m-

pa

red

to

M0

ma

cro

ph

ag

es a

nd

at

lea

st 4

in

dep

end

ent

exp

erim

ents

wit

h m

acr

op

ha

ges

fro

m d

iffe

ren

t d

on

ors

wer

e u

sed

fo

r th

e

mR

NA

an

aly

sis.

D:

do

wn

-reg

ula

ted

, U

: u

p-r

egu

late

d,

ND

: n

ot

det

ecte

d.

Em

pty

fie

lds

ind

ica

te t

ha

t n

o m

easu

rem

ents

ha

ve

bee

n

do

ne.

Res

ult

s fo

r m

RN

A d

ata

dir

ectl

y f

oll

ow

ing

dif

fere

nti

ati

on

an

d f

or

pro

tein

da

ta 4

8 h

po

st d

iffe

ren

tia

tio

n i

s n

ot

sho

wn

in

an

y

of

the

pa

per

s in

clu

ded

in

th

e th

esis

.

D

iffe

ren

tia

ted

ma

cro

ph

ag

es o

r m

acr

op

ha

ge

DIF

F C

M

ma

cro

ph

ag

es 4

8 h

po

st d

iffe

ren

tia

tio

n o

r m

acr

op

ha

ge

CM

M

1 M

2

M0

M

1 M

2

M0

Ta

rget

m

RN

A

pro

tein

m

RN

A

pro

tein

m

RN

A

pro

tein

m

RN

A

pro

tein

m

RN

A

pro

tein

m

RN

A

pro

tein

CD

68

+

+

+

+

+

+

C

D16

3

D 1

6-f

old

+

/-

D 2

-fo

ld

++

1

++

+

1

D 6

-fo

ld

1

u

PA

R

U 7

-fo

ld

++

1

+

1 +

M

MP

-2

U 1

0-f

old

+

D

3-f

old

-

1

MM

P-9

1

++

1

++

1

IL

-6

U 1

90

-fo

ld

++

+

U 1

0-f

old

-

1 -

ND

-

ND

-

ND

IL-8

U

16

00

-fo

ld

++

+

D 2

-fo

ld

+

1 +

U

16

-fo

ld

++

+

1 +

1

IL

-10

D

2-f

old

+

++

1

+

1 +

U

4-f

old

+

1

+

1

IL-1

2

U 5

-fo

ld

+

1 -

U 3

-fo

ld

1

1

IL

-13

N

D

N

D

N

D

N

D

N

D

N

D

C

CL

2

D 2

-fo

ld

++

1

++

1

++

1

+

1 +

+

1

CC

L5

++

+

+

+

+

C

CL

17

D 6

-fo

ld

+

U 6

-fo

ld

++

+

ND

+

/-

U 1

7-f

old

+

C

CL

18

U 2

4-f

old

U 1

15-f

old

1

U 6

0-f

old

U 6

0-f

old

1

CX

CL

1

+

-

-

-

-

CX

CL

5

+

-

-

+

-

CX

CL

9

U 2

6,0

00

-fo

ld

+

1 -

1 +

/-

U 9

00

-fo

ld

+

1 +

/-

1

TN

F-

U

95

-fo

ld

++

1

+/-

1

- U

13

-fo

ld

- D

2-f

old

-

1

CD

80

U

50

0-f

old

U 6

-fo

ld

1

U

10

-fo

ld

1

1

H

LA

-DR

A

U 3

7-f

old

U 3

-fo

ld

1

U

16

-fo

ld

1

1

44

Macrophages and cancer cell growth

Macrophages have the potential to both inhibit and promote prolifer-

ation of cells, including cancer cells, and both negative and positive

correlation regarding tumor associated macrophages and cancer pro-

gression 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 M1–like macrophages in part appears to

depend on the localization of these macrophages, as M1-like macro-

phages 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 mac-

rophages is one way to study if and how macrophages affect various

steps of cancer cell progression, that may explain the prognostic val-

ues found for tumor associated macrophages. In the papers included

in this thesis, the ability of macrophages of both the M1 and M2 phe-

notypes to affect cancer cell growth was studied. The results demon-

strated that macrophages of the M1, but not M2 phenotype, caused a

reduction in cell numbers of cancer cells originating from colon (pa-

per 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 dif-

ferentiation, 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 sub-

stances 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.

45

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 var-

ious 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 in-

creased angiogenesis.

Although there is a consensus that macrophages in general, and M1

macrophages in particular, have the potential to kill, or inhibit prolif-

eration 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 prolifera-

tion of breast cancer cells [231] and osteosarcoma cells [232], and M-

CSF differentiated M1 macrophages have been reported to inhibit re-

nal carcinoma cell growth [233].

There are a number of factors that could possibly induce the reduc-

tion in cell numbers, including released cytokines or other proteins

[231, 234, 235], reactive oxygen/nitrogen species [236-238] or deple-

tion of essential nutrients [233].

Due to the unstable nature of reactive nitrogen/oxygen species, we

found it unlikely that these compounds would be preserved through-

out the experimental setup, which includes long term storage of the

CM prior to use. Depletion of nutrients could be a possible explana-

tion; however, a dose-dependent growth inhibitory effect was ob-

served 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 prolifera-

46

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

specific protein responsible for the reduction in cell numbers ob-

served. Of the cytokines detected in the M1/M1DIFF CM, direct addi-

tions 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 reduc-

tion 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 respon-

sible 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 mac-

rophages.

Mechanisms of growth inhibition

A reduction in cell numbers after treatment can be attributed to a re-

duction 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 treat-

ed 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 adher-

ent 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 pro-

liferation following a 48 h culture in fresh culture media, indicating

that the treatment caused prolonged damage to the cells resulting in

47

either a sustained cell cycle arrest or a delayed apoptosis not detecta-

ble at the time-point of the apoptosis measurement. Whether apopto-

sis per se caused the cells to detached or if M1DIFF CM treatment

caused cells to detach, thereby inducing apoptosis (anoikis) is not de-

termined.

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 recov-

ered their cell proliferation when cultured in fresh culture media, in-

dicating that the majority of cells were still viable. In contrast, M1 CM

treated CACO-2 colon cancer cells maintained a greatly reduced pro-

liferation 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 phas-

es) 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 contin-

uation 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 re-

sponded to M1 CM treatment with cell cycle arrest, while this treat-

ment did not affect the cell cycle distribution of H69 cells to any ma-

jor 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

48

treatment induced cell cycle arrest. A schematic overview of the func-

tion 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 ar-

rest, 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 knock-

down experiments against p21 did not affect the cell cycle arrest in-

duced by M1 CM, indicating that proteins other than p21 are respon-

sible 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. How-

ever, this has not been confirmed by studies on the protein level.

49

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 can-

cer cells

5-FU is one of the most commonly used chemotherapeutic drugs for

the treatment of colon cancer; however, although combination thera-

pies 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 in-

hibit 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

50

found that 5-FU dose dependently inhibited proliferation of both cell

lines, reaching maximum inhibition at about 10 µM (24 h treatment).

At the concentration of 20 µM, which was chosen for further experi-

ments, 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 ad-

ditional 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 prolifera-

tion 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 CACO-

2 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 in-

hibition.

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 tox-

icity. 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 activat-

ing pathway, TP, was up-regulated. All these changes would, if any-

thing, suggest an increased susceptibility to 5-FU toxicity in M1 CM

treated HT-29 cells, in contrast to our observed data.

51

Figure 7. Simplified overview of key genes involved in the activation and catabo-

lism 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 S-

phase 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 sus-

tained damage to these cells. However, for HT-29 cells a combined 5-

FU 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 5-

FU 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 syn-

thesizing 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 re-

sults 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

52

based chemotherapies, enabling some of the cancer cells to escape

treatment.

Anti-inflammatory effects of green, black and rooibos tea ex-

tracts

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 gastroin-

testinal 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 prophylac-

tic drugs against colorectal cancer. Tea extracts, and various polyphe-

nols including the green tea catechin EGCG can inhibit PGE2 for-

mation [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 re-

sponsible 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 in-

duced 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 rooi-

bos 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, re-

spectively (Table IV). EGCG were also found to be highly potent at

53

inhibiting mPGES-1 expression, with inhibition observed at 2.5 µM

(Table IV). Although no studies have been conducted regarding inhi-

bition of mPGES-1 expression with tea extracts or EGCG, several pa-

pers has reported COX-2 inhibition with green and black tea extracts

and with EGCG [250-253]. The inhibition of expression of the en-

zymes was accompanied with inhibition of PGE2 formation. Green

and black tea was equally potent inhibitors of PGE2 formation where-

as rooibos tea was about 10-fold less potent (Table IV). Published lit-

erature suggest that the green tea extract at a dilution of 12,800 con-

tain about 3 µM EGCG [254, 255]. If so, the inhibition of PGE2 for-

mation 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 Black tea Rooibos

tea

EGCG

(µM)

Quercetin

(µM)

Inhibition at dilutions/concentrations of:

mPGES-1

expression ≤ 1:51,200 ≤ 1:25,600 ≤ 1:6,400 ≥ 2.5 ≥ 20

COX-2

expression ≤ 1:12,800 ≤ 1:25,600 ≤ 1:3,200 ≥ 10 ≥ 20

cPLA2

expression ≤ 1:6,400 ≤ 1:6,400 ≤ 1:6,400 ≥ 5 ≥ 20

PGE2

release ≤ 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 for-

mation. With this short term treatment it was assumed that that there

54

would be no effects on the expression of the enzymes and that inhibi-

tion of PGE2 formation instead would be due to inhibition of the ac-

tivity of the enzymes. With this assay, quercetin inhibited PGE2 for-

mation dose-dependently with a 50% reduction at 40 µM. 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 as-

say, all tea extracts, and also EGCG inhibited PGE2, however, at con-

siderably higher doses than required for inhibition of the expression

of mPGES-1. EGCG has previously been reported to inhibit the enzy-

matic activity of mPGES-1, but not COX-2 [248]; however, in our as-

say 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 pro-

teins 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, PAI-

1, 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 ex-

pressed considerably higher levels of uPAR, both at the mRNA (7-

fold) and protein levels compared to the M0 and M2 phenotype. Fur-

thermore, treatment of M1 CM to the lung cancer cell lines H520 and

55

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 prog-

nosis, compared to uPAR positivity in the cancer cells [150]. Our re-

sults 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 ex-

pressed 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 mark-

er CD163, and also uPAR, MMP-2 and MMP-9. Macrophages identi-

fied by the expression of either CD68 or CD163 were found in all bi-

opsies, but in varying amount, from few cells to highly infiltrated tis-

sues. CD163 was mostly found to be expressed at a similar extent to

CD68, or even more widespread than CD68, indicating that the ma-

jority of macrophages were of an M2-like phenotype, if CD163 is a

useful marker for M2-like macrophages in lung SCC and SCLC. Oth-

ers, 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 colo-

rectal 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 includ-

ing macrophages was found to express uPAR, MMP-2 and MMP-9 in

56

both lung SCC and SCLC biopsies. Of the cancer cells, uPAR expres-

sion 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 immuno-

reactivity in the cancer cells was found in the majority of the lung SCC

and SCLC biopsies.

57

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 inflammato-

ry 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 macro-

phages 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 in-

duced by the M1 phenotype could attenuate the efficacy of 5-FU

treatment on a colon cancer cell line. These results suggest that mac-

rophages of M1 phenotype could be beneficial in tumors through in-

hibition of cancer cell growth, but that said inhibition could be un-

wanted 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 in-

cluding 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, MMP-

2 and MMP-9. In vitro, the M1 and M2 phenotypes were found to ex-

press 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 suggest-

ed to predict poor prognosis, both the M1 and M2 phenotype may

58

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 po-

tent of the three teas primarily inhibited PGE2 formation by inhibit-

ing 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 need-

ed for inhibition of the expression of the enzymes or PGE2 formation.

59

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 ide-

as.

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 också tacka några utanför jobbet, vänner och familj. Utan er

hade denna avhandling inte varit möjlig:

Mamma och pappa, tack för att ni alltid funnits där. Tack Christopher

Engström, utan dig hade jag kanske varken varit nördig eller för den

delen skrivit en avhandling!

60

Slutligen vill jag tacka Lisa Hedbrant, tack för din kärlek, uppmuntran

och stöttning. Du har betytt mer än något annat för mig under denna

tid.

61

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Cancer and InflammationRole of Macrophages and Monocytes

Alexander Hedbrant

Alexander H

edbrant | Cancer and Inflam

mation | 2015:36

Cancer and Inflammation

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.

DISSERTATION | Karlstad University Studies | 2015:36 DISSERTATION | Karlstad University Studies | 2015:36

ISSN 1403-8099

Faculty of Health, Science and TechnologyISBN 978-91-7063-654-7

Biomedical Sciences