the impact of microenvironment and dissertation.pdf · chapter 5 experimental antibody therapy of...

The impact of microenvironment and surgery on colon carcinoma metastasis

new concepts in development and therapy

The work described in this thesis was performed within a collaboration of the departments

of Surgical Oncology and Molecular Cell Biology and Immunology, VU University Medical

Center, Amsterdam, The Netherlands.

The publication of this thesis was financially supported by:

ERBE Benelux B.V.

Harlan Netherlands B.V.

Cover: In vitro cultured CC531s tumor cells. The target of the CC531s specific antibody

(MG4-γ1a) is stained red, cytokeratin is stained green and nuclei are stained blue.

Cover Design & Layout: G.J. van der Bij

Printed by: Wöhrmann Print Service

Copyright © G.J. van der Bij, Amsterdam, 2008

All rights reserved. No part of this book may be reproduced, stored in a retrieval system,

or transmitted or by any means, electronic, mechanical, photocopying, or otherwise

without the prior permission of the holder of the copyright.

VRIJE UNIVERSITEIT

The impact of microenvironment and surgery on colon carcinoma metastasis

new concepts in development and therapy

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad Doctor aande Vrije Universiteit Amsterdam,

op gezag van de rector magnificusprof.dr. L.M. Bouter,

in het openbaar te verdedigenten overstaan van de promotiecommissie

van de faculteit der Geneeskundeop vrijdag 7 november 2008 om 15.45 uur

in het auditorium van de universiteit,De Boelelaan 1105

door

Gerben Joost van der Bij

geboren te Alkmaar

promotoren: prof.dr. S. Meijer prof.dr. R.H.J. Beelen

copromotor: dr. M. van Egmond

8

11

2231

33

TABLE OF CONTENTS

General outline of the thesis

Chapter 1 The perioperative period is an underutilized window of therapeutic opportunity in patients with colorectal cancer

Submitted

Chapter 2 Anti-β1-integrin antibody reduces surgery-induced adhesion of colon carcinoma cells to traumatized peritoneal surfaces

Annals of Surgery 2008 Jan;247(1):85-94

Chapter 3 Blocking α2-integrins on rat CC531s colon carcinoma cells prevents operation-induced augmentation of liver metastases outgrowth

Hepatology 2008 Feb;47(2):532-43

Chapter 4 Therapeutic potential of Kupffer cells in prevention of liver metastases outgrowth

Immunobiology 2005;210(2-4):259-65

Chapter 5 Experimental antibody therapy of liver metastases reveals functional redundancy between FcγRI and FcγRIV

The Journal of Immunology, accepted for publication

Chapter 6 Successful prevention of surgery-induced liver metastases development after anti-tumor monoclonal antibody therapy is mediated by the innate mononuclear phagocyte network

Submitted

Chapter 7 The role of macrophages in tumor development Cellular Oncology 2005;27(4):203-13

Chapter 8 Macrophages direct tumor histology and clinical outcome in a colon cancer model

Journal of Pathology 2005 Oct;207(2):147-55

Chapter 9 Tumor infiltrating macrophages reduce development of peritoneal colorectal carcinoma metastases

Cancer Letters, in press

Chapter 10 General discussion and recommendations

Appendices A. Dutch summary / Nederlandse samenvatting B. List of publications C. Dankwoord D. Curriculum Vitae

ADCC antibody-dependent cellular cytotoxicityAPC adenomatous polyposis colib-FGF basic-fibroblast growth factorBSA bovine serum albuminCAR Committee for Animal ResearchCEA carcinoembryonic antigenCI confidence intervalCR3 complement receptor 3CRC colorectal cancer CSF-1 colony stimulating factor-1DHR dihydrorhodamineDiI 1,1’-dioctadecyl-3,3,3’,3’- tetramethylindocarbocyanine perchlorateDiO 3,3’-dioctadecyloxacarbocyanine perchlorateDMSO dimethyl sulfoxideECM extracellular matrixEGF epidermal growth factorFcγR Fc gamma receptorFCS fetal calf serumFOV fields of visionGM-CSF granulocyte macrophage- colony stimulating factorGS glucocorticosteroidsHBSS hanks’ balanced salt solutionHGF hepatocyte growth factorHIF hypoxia inducible factorHLA-DR human leukocyte antigen DRHPLC high performance liquid chromatographyICAM intercellular adhesion moleculeIFN interferonIL interleukinIg immunoglobulini.p. intraperitoneali.v. intravenousKC Kupffer cellKO knock-outLSEC liver sinusoidal endothelial cellLPS lipopolysaccharidemφ macrophagemAb monoclonal antibody

MCP monocyte chemotactic proteinM-CSF macrophage-colony stimulating factorMDP muramyl dipeptideMMP matrix metalloproteinaseMTP-PE muramyl tripeptide phosphatidylethanomalineNK cell natural killer cellNO nitric oxidePBS phosphate buffered salinePDGF platelet derived growth factorPC peritoneal carcinomatosisPi3k phosphoinositide-3 kinasePMA phorbol myristate acetatePMN polymorphonuclear neutrophilPO peroxidaseROS reactive oxygen speciesSEM scanning electron microscopysLex sialyl Lewis XTEM transmission electron microscopyTGF transforming growth factorTh T helperTNF tumor necrosis factorVCAM vascular cell adhesion molecule VEGF vascular endothelial growth factorvWf von Willebrand factorZO-1 zona occludens 1

ABBREVIATIONS

General introduction and outline of the thesis

General introduction and outline of the thesis | 9

Colorectal carcinoma (CRC) is one of the most common forms of solid cancer and especially

the development of CRC metastases is associated with high mortality and morbidity.

Better understanding of the mechanisms that contribute to metastases formation will

set the stage for innovative new therapeutic strategies that improve patient outcome. As

such, several aspects of metastases development were investigated in this thesis, which

has led to several novel insights. Moreover, new experimental treatment strategies have

been explored, which were based on the gained knowledge and might benefit patients

with CRC in the future.

It is becoming clear that surgery itself contributes to metastases development. In

Chapter 1 the relationship between surgical resection of colorectal carcinoma and tumor

recurrence is reviewed. Potential mechanisms by which surgery can promote tumor

recurrence are addressed, and current and future therapeutic options that exploit the ‘peri-

operative window of opportunity’ are discussed. In Chapter 2 and 3 a novel mechanism

for metastases development is identified, demonstrating that surgery leads to enhanced

tumor cell adhesion. This route of operation-associated metastasis bypasses several

steps that are essential in classical metastases development. In this way, surgery greatly

facilitates tumor recurrence. Moreover, by interfering with the key feature of surgery-

associated metastases development, namely tumor cell binding to exposed extracellular

matrix components, we were able to attenuate metastases development.

In Chapter 4, 5 and 6 various strategies are discussed, which may benefit patients that

undergo surgery for CRC. In Chapter 4 the pivotal role of Kupffer cells (liver macrophages,

KC) in the protection against liver metastases development is described. This review

furthermore discusses how stimulation of KC function by mediators such as granulocyte

macrophage-colony stimulating factor or tumor specific monoclonal antibodies (mAb)

can prevent liver metastases outgrowth. The concept of prevention of colon carcinoma

metastases after surgery, using tumor-specific mAb, is further explored in Chapter 5

and 6. We showed that administration of such antibodies very efficiently prevents liver

metastases development. Moreover, we were able to identify both Kupffer cells and

monocytes are the cells responsible for the therapeutic effect, which was mediated via

the IgG Fc receptors FcγRI and FcγRIV.

Thus, we have shown in chapter 5 and 6 that macrophages have cytotoxic properties and

can contribute to defense against tumors. However evidence is now accumulating that also

demonstrate a tumor-promoting role of macrophages. Therefore the role of macrophages

in CRC metastases outgrowth is studied in Chapter 7, 8 and 9 of the thesis. Chapter 7

reviews the current knowledge of the role macrophages play in tumor development. The

opposing functions of classically activated (M1) ‘tumor inhibiting’ macrophages versus

alternatively activated (M2) ‘tumor promoting’ macrophages in malignant development

10 | General introduction and outline of the thesis

are discussed. In Chapter 8 the role of macrophages in CRC metastases development

is investigated in more detail. This study showed that accumulation of macrophages in

tumors was associated with poor differentiation, hereby supporting that macrophages

can have a tumor-promoting role. Importantly however, macrophages also decreased

the number of metastases and increased survival demonstrating that macrophages have

potent cytotoxic ability as well. As two different macrophage populations were detected

in tumors, the contribution of the resident versus newly infiltrated macrophages was

investigated in Chapter 9. Although it is currently hypothesized that newly recruited

monocytes differentiate into tumor promoting M2 macrophages, we showed that reducing

migration of monocytes accelerated tumor development. This indicates that in CRC newly

infiltrated monocytes are crucial in controlling metastatic disease. Finally, Chapter 10

addresses the clinical implications of the experimental data from this thesis, and new

therapeutic strategies are recommended.

General discussion and recommendations10

12 | Chapter 10

The aim of the work presented in this thesis was to obtain novel insights in the development

and therapy of colorectal cancer (CRC) metastases. Accordingly, the relation between

surgery and CRC metastases development, as well as the complex role of macrophages

in tumor development has been investigated. This has led to new concepts for preventing

metastases in patients with CRC, which should be implemented in the clinic as soon as

possible to fully utilize the peri-operative window of opportunities.

New insights in surgery-induced metastases development

CRC is one of the most prevalent solid organ cancers in both males and females.

Approximately one million cases are recorded every year worldwide, and over half a

million patients die from this disease yearly.1 Currently, surgical removal of the primary

colorectal carcinoma is the preferred treatment, which provide the best chance for cure.1

Unfortunately, post-surgical development of metastases is a frequent complication, which

is accompanied by high morbidity and mortality. Approximately 25 to 33% of all patients

already have metastatic disease at the time of diagnosis. Moreover, another 25 to 30%

of patients who do not have visible evidence of metastases at the time of diagnosis and

who are therefore eligible for surgery with curative intent, will develop metastases within

5 years.2,3

The secondary malignancies in CRC patients originate from tumor cells that have

disseminated from the primary tumor, and either spread via the venous circulation,

lymphatics or directly via peritoneal cavity. Under physiological circumstances, the process

of metastases formation is highly inefficient, as disseminated tumor cells have a limited

life span and are rapidly eliminated by the immune system.4 However, several studies

showed that surgery itself enhances the risk of metastases development.5-7 (reviewed in

chapter 1) Moreover, we showed that animals that underwent abdominal surgery, which is

required for removal of primary colon carcinoma, had substantially increased metastases

outgrowth in the liver and peritoneal cavity.8,9 (chapter 2 and 3)

Circulating tumor cells can be detected in the majority of CRC patients prior to surgery.10

Additionally, various reports suggest that handling of the tumor during resection can result

in spilling of tumor cells, as increased numbers of tumor cells have been observed after

surgery in the peritoneum, circulation and liver.11-14 Additionally, it is generally accepted

that surgery induces a transient immune suppression and it has been proposed that

this phenomenon might impair anti-tumor responses. However, we were unable to find

any effect of surgery on number or function of immune cells in the liver. Additionally,

previous in vivo studies with CC531s cells showed that either local or systemic trauma

did not enhance growth of established peritoneal metastases. In contrast, enhanced

tumor development was only observed when a tumor cell suspension was injected during

General discussion and recommendations | 13

surgery.5,15 These results suggested that increased implantation of tumor cells accounts

for increased tumor outgrowth after surgery, rather than an enhancement of tumor cell

proliferation or impairment of anti-tumor responses. Peritoneal imprints of operated rats

confirmed that directly damaging of the peritoneum resulted in enhanced adhesion of

rat CC531 colon carcinoma cells to submesothelial extracellular matrix (ECM) proteins

in vivo, which was also observed by electron microscopy. The inflammatory reaction of

the peritoneal cavity led to retraction of mesothelial cells, hereby also exposing ECM at

peritoneal surfaces that had not been traumatized directly. Adhesion of cells to ECM is

predominantly facilitated by integrins, which are widely expressed on most cells, including

practically all tumor cells. We demonstrated that β1 integrin subunits represented the

primary mediators involved in adherence to either isolated ECM components or excised

traumatized rat and human peritoneum. Furthermore, incubation of CC531s cells with

anti-β1 integrin antibodies resulted in a significant decrease of tumor cell adhesion in

vivo (chapter 2).

In analogy, abdominal surgery induced rapid impairment of tight junction integrity and

deposition of vWf in the liver sinusoids. Both these findings are indicative of endothelial

stress, which can result in retraction of endothelial cells and in increased exposure of

subendothelial ECM. Electron microscopy showed adhesion of tumor cells to subendothelial

ECM in the liver sinusoids after surgery. These observations, together with the notion

that blocking α2 integrins on CC531s cells completely reverted increased tumor cell

adhesion and metastases outgrowth after surgery, supports that surgical trauma results

in increased binding of tumor cells to exposed ECM (chapter 3).

As such, we have proposed a new model of metastasis development in the context of

surgery that differs from classical metastasis in several ways. First, surgical resection of

the primary tumor may result in tumor cell spillage, which overcomes the need of complex

cellular changes, such as loss of E-cadherin and β-catenin expression, which are normally

required for tumor cell detachment.11,13,14,16 Second, in classical metastasis disseminated

tumor cells need to express specific adhesion molecules in order to adhere to endothelial

or cells or peritoneal surfaces.3 By contrast, surgical trauma induces exposure of ECM

and thereby facilitates high affinity binding through commonly expressed integrins, which

contributes to tumor recurrence and metastases outgrowth after resection.

Anti-tumor mAb therapy for the prevention of liver metastases development

After surgical resection of the primary tumor, only minimal residual disease will be present

post-surgically, which renders patients with CRC exceptionally suitable for adjuvant

therapy. This is illustrated by various clinical trials, which show benefit of peri-operative

chemotherapy after tumor resection, including peri-operative chemotherapy in stage III

14 | Chapter 10

CRC patients.17,18 However the effect on stage II patients is limited and as such novel

therapeutic strategies need to be developed.

Currently, mAb therapy is successfully being used to treat several forms of cancer.19,20

Although the value of mAb therapy in the peri-operative setting is not yet determined,

it represents a promising new peri-operative treatment modality for patients that have

CRC. Using two experimental models, we investigated the efficacy of anti-tumor mAb to

prevent liver metastases development after a surgical procedure.

We showed in chapter 5 and 6 that liver metastases can be efficiently be prevented

by post-operative treatment with tumor specific mAb. Administration of anti-gp75 mAb

(TA99, IgG2a) tumor specific mAb in a mouse melanoma model was highly effective in

preventing liver metastases. In accordance with our data, it was previously demonstrated

that IgG2a mAb have the highest therapeutic efficacy in commonly used mouse models

for investigation of anti-tumor therapies.21,22 Interestingly, MG4-γ1 (IgG1) but not MG4-

γ2a (IgG2a) mAb directed against the CC531s tumor cell line displayed high therapeutic

efficacy in rats. This resembles the human situation, in which IgG1 mAb also have the

highest therapeutic efficacy and are commonly used in the clinic.23 Although detailed

knowledge of the rat FcγR system is not yet available, these data suggest that the rat

FcγR system is more alike the human system in comparison with mice. Since animals

studies are a critical component in the development of new mAb based therapies, the

similarities we describe here between the rat and human FcγR system suggest that (nude)

rat models are more suitable for mAb based preclinical research than mouse models.

Although never demonstrated directly in vivo, several studies support that ADCC is

an important effector mechanism of therapy. Clynes et al. showed that effectiveness of

mAb therapy depends on FcγR, which is crucial for ADCC.24 Additional studies give insight

in the subtypes of FcγR involved. Ravetch and Bevaart respectively showed that mAb

therapy for lung metastases was dependent on FcγRIV or FcγRI.21,25 Although somewhat

contradictory, both studies suggest that mAb therapy is mediated by cells of the myeloid

lineage, as these are the only cells that express either FcγRI or FcγRIV.26 Moreover,

we showed in a mouse melanoma liver metastases model that FcγRI and FcγRIV were

functionally redundant, as only one of both receptors was necessary for therapeutic

efficacy (chapter 5).

Further research in our rat model revealed that efficacy of mAb therapy for the

prevention of liver metastases depends on KC mediated tumor cell phagocytosis.

Monocytes are able to compensate partly in the absence of KC, but for this to happen high

doses of mAb are required. When low doses of mAb are administered, monocytes cannot

replace KC and mAb therapy is completely ineffective. This indicates that, although both

KC and monocytes are capable of mediating ADCC, KC are the most important effector


cell population (chapter 6).

In conclusion, we have shown that treatment with anti-tumor mAb efficiently prevents

liver metastases development after a surgical procedure in two experimental animal

models. Moreover, we show that mAb therapy depends on either FcγRI or FcγRIV and that

tumor cells are eradicated by ADCC in vivo. This is mediated by the innate mononuclear

phagocyte network, which represents the most important effector mechanism in our

model.

The role of the innate mononuclear phagocyte network in CRC metastases

development

Besides representing an important effector cell population for ADCC in mAb therapy for

the prevention of CRC metastases, it is becoming increasingly clear that macrophages

and monocytes significantly influence tumor development in general. A large fraction of

the tumor stroma is comprised of macrophages, which have a complicated dual role in

tumor development.27,28 Conventionally, macrophages have been regarded as cells that

produce pro-inflammatory mediators and exert cellular cytotoxicity. Macrophages with

these characteristics are referred to as classically activated (or M1) macrophages and

are commonly found during infections and inflammation.29 In the context of cancer, M1

macrophages may inhibit tumor growth, as they can eradicate tumor cells and stimulate

immune responses.28 By contrast, a different subset of macrophages, called alternatively

activated (or M2) macrophages, is reported to contribute to tumor progression by

producing pro-angiogenic and anti-inflammatory mediators, as well as growth factors

and proteases.29-31 Macrophages frequently accumulate into malignant tissue since a

large number of tumors produce mediators, such as colony-stimulating factor 1 and

macrophage chemotactic protein 1, which attract monocytes.30,32 Local alternative

activation of macrophages may be induced by mediators like interleukin (IL) 4, IL-10 and

IL-13, which are reported to be present in tumors.33 In line with these findings it has even

been proposed that infiltrating monocytes are instructed by the local tumor environment

to differentiate into alternative macrophages and as such assist tumor progression.30

Conflicting reports exist on the exact role of macrophages in tumor progression. In

various types of cancer, including breast, bladder and prostate carcinomas, macrophage

presence is associated with unfavorable prognosis, supporting a tumor-promoting role

for macrophages.34-36 However, macrophage infiltration in colorectal carcinomas seems to

benefit patient outcome.37-39 We show in chapter 8 that selective depletion of peritoneal

or liver macrophages prior to tumor cell inoculation resulted in highly differentiated

tumors. In contrast, tumors of control rats showed a desmoplastic stroma reaction with

hallmark features of malignancy, such as neovascularization, matrix remodeling and poor

16 | Chapter 10

differentiation, supporting that macrophages promote malignant tumor progression.

Remarkably, macrophage-depleted rats, bearing highly differentiated tumors, had worse

prognosis as these rats displayed higher tumor load and poorer survival. Thus, while

macrophages direct tumors towards malignant phenotype, their role in anti-tumor defense

is prevalent.

Immunohistochemical analyses demonstrated the presence of two macrophage

populations, namely ED2+ tissue macrophages at the border of tumor nodules and newly

recruited ED1+ macrophages that were scattered throughout the tumors. This suggested

that residential macrophages are classically activated (M1) and aim to contain tumor

growth, whereas newly recruited macrophages are alternatively activated (M2) and may

produce (angiogenic) factors that stimulate tumor progression.

In chapter 9 we investigated whether newly recruited monocytes contribute to tumor

development. To do so, we studied peritoneal CC531s development in animals that were

treated with flavonoids, which led to reduced monocyte/macrophage migration. Impairment

of macrophage migration resulted in a 2-fold increase in metastases development after

14 days. Immunohistochemical analysis revealed no difference in the number of ED2+

cells, but a strongly reduced presence of newly recruited ED1+ macrophages in tumors

from flavonoid treated groups was observed. Further experiments discarded direct effects

of flavonoids on CC531s tumor cells. This suggests that newly recruited macrophages,

like residential macrophages, play an important role in reducing outgrowth of peritoneal

CRC metastases.

Thus, the current model in which tumor-associated macrophages have a M2 phenotype

does not apply for CRC, as we have demonstrated that M1 and M2 characteristics of

tumor-associated macrophages are present simultaneously. As such, the M1/M2 paradigm

should be seen as a theoretical concept, in which macrophage function is positioned

on a continuum with at one end the M1 macrophage and at the other end the M2

macrophage.

Future perspectives and recommendations

Macrophages and tumor development

It has previously been suggested that prevention of macrophage influx into tumors might

increase patient survival.40 While possibly beneficial for some types of cancer (e.g. breast

cancer, in which high macrophage presence is correlated with poor patient outcome),

we demonstrated that inhibition of macrophage influx into CRC metastases should be

avoided. This is supported by several clinical studies, which showed that macrophage


presence in primary CRC is correlated with better patient outcome.37-39 Although interfering

with macrophage function may therefore represent valuable therapeutic strategies, the

complicated balance between pro- versus anti-tumor characteristics warrants caution. As

such, effort should be made to fully understand the role of macrophages in different types

of cancer before initiating clinical applications.

Peri-operative treatment modalities to prevent CRC metastases

This thesis provides new evidence that surgery can result in enhanced tumor outgrowth

but also demonstrates that the peri-operative period offers an attractive window of

opportunity for therapeutic strategies. However, I feel that these opportunities are still

underappreciated. Optimizing surgical procedures should be strived for as it might lead

to reduced tumor cell spillage during removal of CRC and to decreased surgical trauma

along with the concomitant inflammatory reaction. However, some degree of trauma is

unavoidable and tumor cells may already be present in the circulation prior to surgery.

Detailed knowledge of the underlying mechanisms of surgery-induced metastases

outgrowth is pivotal in the development of new therapies. Effort should be made to

identify the mediators that are released after surgery and result in the systemic changes

that underlie increased tumor cell arrest. Proteomics or multiplex assays of serum from

operated and control patients could be of great value. Blocking of these mediators to

prevent tumor cell arrest and subsequent metastases outgrowth should then be further

investigated. Caution should be taken however regarding modulation of immune responses

in the peri-operative period. Immune suppression by anti-cytokine mAb for example

might impair immune cell function and subsequent impediment of tumor cell eradication

whereas immune stimulation might result in increased tumor cell arrest.

Additionally, we showed that integrins play a pivotal role in the adhesion of tumor

cells in target organs after surgery, and that blocking of integrins using mAb can prevent

surgery-induced metastases outgrowth. As such, additional research should evaluate

safety and capacity of anti-integrin antibodies to prevent tumor recurrence after surgical

resection of CRC.

mAb therapy in patients who are at risk of developing liver metastases, such as

patients undergoing resection of primary tumors, shows great potential for the prevention

of metastases development. Various mAb are available in the clinic and thus can readily

be used in the peri-operative setting. As we have clearly shown the potential of mAb

in preclinical models, clinical trials should be executed to explore the efficacy of mAb

to improve patient outcome. Additionally, knowledge of the mechanisms by which mAb

mediate their effects (ADCC by the innate mononuclear phagocytic network), should be

implemented to optimize mAb therapy. For example, optimizing number and function of KC

18 | Chapter 10

and monocytes by or immunostimulatory mediators like GM-CSF should be evaluated.

Altogether, the work presented in this thesis implies that there are several promising

therapeutic opportunities to optimize outcome of patients suffering from CRC. Because all

resources are currently available to investigate the therapeutic efficacy of peri-operative

anti-tumor mAb therapy and anti-integrin therapy, these promising strategies should be

explored in the clinic without delay.


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20 | Chapter 10

Nimmerjahn, F. and Ravetch, J. V. Divergent immunoglobulin g subclass activity through selective Fc receptor 21. binding. Science, 310: 1510-1512, 2005. Lutterbuese, P., Brischwein, K., Hofmeister, R., Crommer, S., Lorenczewski, G., Petersen, L., Lippold, S., da, S. 22. A., Locher, M., Baeuerle, P. A., and Schlereth, B. Exchanging human Fcgamma1 with murine Fcgamma2a highly potentiates anti-tumor activity of anti-EpCAM antibody adecatumumab in a syngeneic mouse lung metastasis model. Cancer Immunol.Immunother., 56: 459-468, 2007. Adams, G. P. and Weiner, L. M. Monoclonal antibody therapy of cancer. Nat.Biotechnol., 23. 23: 1147-1157, 2005. Clynes, R., Takechi, Y., Moroi, Y., Houghton, A., and Ravetch, J. V. Fc receptors are required in passive and active 24. immunity to melanoma. Proc.Natl.Acad.Sci.U.S.A, 95: 652-656, 1998. Bevaart, L., Jansen, M. J., van Vugt, M. J., Verbeek, J. S., van de Winkel, J. G., and Leusen, J. H. The high-affinity 25. IgG receptor, FcgammaRI, plays a central role in antibody therapy of experimental melanoma. Cancer Res., 66: 1261-1264, 2006. Nimmerjahn, F., Bruhns, P., Horiuchi, K., and Ravetch, J. V. FcgammaRIV: a novel FcR with distinct IgG subclass 26. specificity. Immunity., 23: 41-51, 2005. Mantovani, A., Schioppa, T., Porta, C., Allavena, P., and Sica, A. Role of tumor-associated macrophages in tumor 27. progression and invasion. Cancer Metastasis Rev., 2006. Oosterling, S. J., van der Bij, G. J., Meijer, G. A., Tuk, C. W., van Garderen E., van Rooijen N., Meijer, S., van der 28. Sijp, J. R., Beelen, R. H., and van Egmond, M. Macrophages direct tumour histology and clinical outcome in a colon cancer model. J.Pathol., 207: 147-155, 2005. Gordon, S. Alternative activation of macrophages. Nat.Rev.Immunol., 29. 3: 23-35, 2003. Pollard, J. W. Tumour-educated macrophages promote tumour progression and metastasis. Nat.Rev.Cancer, 30. 4: 71-78, 2004. Mantovani, A., Sozzani, S., Locati, M., Allavena, P., and Sica, A. Macrophage polarization: tumor-associated mac-31. rophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol., 23: 549-555, 2002. van der Bij, G. J., Oosterling, S. J., Meijer, S., Beelen, R. H., and van Egmond, M. The role of macrophages in 32. tumor development. Cell Oncol., 27: 203-213, 2005. Stout, R. D., Jiang, C., Matta, B., Tietzel, I., Watkins, S. K., and Suttles, J. Macrophages sequentially change 33. their functional phenotype in response to changes in microenvironmental influences. J.Immunol., 175: 342-349, 2005. Lissbrant, I. F., Stattin, P., Wikstrom, P., Damber, J. E., Egevad, L., and Bergh, A. Tumor associated macrophag-34. es in human prostate cancer: relation to clinicopathological variables and survival. Int.J.Oncol., 17: 445-451, 2000. Tsutsui, S., Yasuda, K., Suzuki, K., Tahara, K., Higashi, H., and Era, S. Macrophage infiltration and its prognostic 35. implications in breast cancer: the relationship with VEGF expression and microvessel density. Oncol.Rep., 14: 425-431, 2005. Hanada, T., Nakagawa, M., Emoto, A., Nomura, T., Nasu, N., and Nomura, Y. Prognostic value of tumor-associated 36. macrophage count in human bladder cancer. Int.J.Urol., 7: 263-269, 2000. Sickert, D., Aust, D. E., Langer, S., Haupt, I., Baretton, G. B., and Dieter, P. Characterization of macrophage 37. subpopulations in colon cancer using tissue microarrays. Histopathology, 46: 515-521, 2005. Forssell, J., Oberg, A., Henriksson, M. L., Stenling, R., Jung, A., and Palmqvist, R. High macrophage infiltra-38. tion along the tumor front correlates with improved survival in colon cancer. Clin.Cancer Res., 13: 1472-1479, 2007. Lackner, C., Jukic, Z., Tsybrovskyy, O., Jatzko, G., Wette, V., Hoefler, G., Klimpfinger, M., Denk, H., and Zatloukal, 39. K. Prognostic relevance of tumour-associated macrophages and von Willebrand factor-positive microvessels in colorectal cancer. Virchows Arch., 445: 160-167, 2004. Lin, E. Y., Nguyen, A. V., Russell, R. G., and Pollard, J. W. Colony-stimulating factor 1 promotes progression of 40. mammary tumors to malignancy. J.Exp.Med., 193: 727-740, 2001.

Appendices

A. Dutch summary / Nederlandse samenvattingB. List of publicationsC. DankwoordD. Curriculum Vitae

22 | Appendix A - Dutch summary / Nederlanse samenvatting

NEDERLANDSE SAMENVATTING

Operatieve verwijdering van darmkanker en de ontwikkeling van metastasen

Kanker van de dikke darm (colon carcinoom) is de op één na vaakst voorkomende soort

kanker. Jaarlijks wordt bij ongeveer een miljoen mensen de diagnose darmkanker gesteld

en sterven er wereldwijd ongeveer 500.000 mensen aan deze ziekte per jaar. Momenteel

is operatief verwijderen van het kankergezwel de beste behandeling. Alhoewel deze

behandeling de prognose van de patiënt sterk verbetert, blijft het terugkomen van de

kanker in de darm (recidieven) en uitzaaiingen (metastasen) naar andere plekken in

het lichaam gevreesde complicaties die een slechte prognose met zich meebrengen. Dit

wordt geïllustreerd door de bevinding dat na operatieve verwijdering van darmkanker, de

kanker zal terugkomen in 25 tot 30 % van alle patiënten. Bij de meerderheid van deze

patiënten zaait de kanker uit naar de lever. Kankercellen kunnen in de lever terechtkomen

via de bloedbaan nadat ze zich hebben afgescheiden van het primaire gezwel (het colon

carcinoom). Onder normale omstandigheden zullen de meeste kankercellen niet uitgroeien

tot metastasen, maar worden opgeruimd door het immuunsysteem. In hoofdstuk 1, 2 en

3 van dit proefschrift laten wij echter zien dat een operatie, welke altijd noodzakelijk is

voor de verwijdering van een colon carcinoom, de kans op recidieven en metastasen in

de lever aanzienlijk verhoogt. In deze hoofdstukken laten wij tevens zien dat dit komt

doordat de aanhechting van kankercellen in de buikholte en lever vlak na de operatie

sterk verhoogd is. De verhoogde aanhechting van kankercellen wordt veroorzaakt doordat

steungevend bindweefsel (de extracellulaire matrix) bloot komt te liggen na een operatie.

Extracellulaire matrix komt in bijna elk weefsel voor, onder andere rondom bloedvaten

en onder het buikvlies. Kankercellen kunnen makkelijk binden aan deze blootliggende

extracellulaire matrix met behulp van een bepaald type adhesiemoleculen, namelijk

integrines. Nadat kankercellen via deze integrines zijn gehecht aan de extracellulaire

matrix, kunnen ze uitgroeien tot metastasen. Door de functie van integrines op kanker-

cellen te blokkeren, waren wij in staat kankercelaanhechting en metastasevorming na

operatie tegen te gaan (figuur 1). Uit hoofdstuk 1, 2 en 3 kan geconcludeerd worden dat

integrine-gemedieerde kankercelaanhechting aan blootliggende extracellulaire matrix de

oorzaak is van verhoogde uitgroei van metastasen na een operatie.

Antilichaamtherapie voor de preventie van levermetastasen

Nadat het primaire gezwel is verwijderd, blijft er hoogstens een klein restant van het

kankerweefsel in de patiënt achter (minimal residual disease). Het tijdstip na de operatie

is daarom zeer geschikt voor therapie (adjuvante therapie), dat als doel heeft dit laatste

beetje overgebleven kankercellen te vernietigen (hoofdstuk 4). Alhoewel er veelbelovende

Appendix A - Dutch summary / Nederlanse samenvatting | 23

resultaten zijn behaald met behandelingen waarbij chemotherapie wordt gegeven rondom

de operatie, blijft er behoefte aan betere therapieën. Onder andere wordt er onderzoek

gedaan naar de mogelijkheid het immuunsysteem te stimuleren zodat kankercellen

kunnen worden vernietigd door de immuuncellen van de patiënt. Tumorcellen worden

echter slecht herkend door het immuunsysteem, waardoor deze strategie vaak niet goed

werkt. Daarom hebben wij onderzocht of toediening van tumor-specifieke antilichamen

na de operatie levermetastasen kunnen voorkomen. Deze antilichamen binden aan

kankercellen waardoor deze kankercellen makkelijk herkend en vernietigd kunnen worden

Figuur 1. Kankercelaanhechting na operatie. (A) Onder normale omstandigheden is er geen blootliggende extracellulaire matrix. Hierdoor kunnen kankercellen slecht aanhechten in weefsels en ontwikkelen maar zeer weinig kankercellen zich tot metastasen. (B) Na een operatie komt de extracellulaire matrix echter bloot te liggen, waardoor kankercellen, via integrines, makkelijk aanhechten en kunnen uitgroeien tot metastasen. (C) Door integrines op kankercellen te blokkeren met behulp van anti-integrine antilichamen, kan de aanhechting van kankercellen aan blootliggende extracellulaire matrix na operatie worden voorkomen. Doordat kankercellen niet meer kunnen aanhechten, zullen ze zich ook veel minder snel ontwikkelen tot metastasen.


door het immuunsysteem van de patiënt (figuur 2). Op dit moment wordt antilichaam

therapie al toegepast bij diverse soorten kanker, waaronder bij darmkanker. Echter is de

werkzaamheid van antilichaamtherapie direct na operatieve verwijdering van darmkanker

nog niet onderzocht.

Wij hebben in twee verschillende

diermodellen laten zien dat antilichaam-

therapie de uitgroei van levermetastasen

na een operatieve ingreep kan voorkomen

(hoofdstuk 5 en 6). Tevens hebben we

door middel van experimenten in muizen,

waarin specifieke onderdelen van het

immuunsysteem niet meer werken (knock-

out muizen), kunnen achterhalen welke

onderdelen van het immuunsysteem

belangrijk zijn voor de werkzaamheid van

antilichaamtherapie. Fcγ receptoren, met

name FcγRI en FcγRIV, bleken cruciaal. Deze

receptoren zijn aanwezig op verschillende

cellen van het immuunsysteem en kunnen

antilichamen herkennen (Figuur 2). Als een

immuuncel een antilichaam herkent, welke

gebonden is aan een kankercel, kan de

immuuncel de kankercel vernietigen. Om

vervolgens nauwkeuriger te kijken welke

immuuncellen verantwoordelijk zijn voor het

effect van de antilichaamtherapie en hoe deze

immuun-cellen de kankercellen vernietigen,

hebben we in een rattenmodel verder

onderzoek gedaan. Hieruit bleek met name

dat de macrofagen van de lever (Kupffer

cellen) kankercellen na antilichaamtherapie

vernietigen. Deze Kupffer cellen zijn in

afwezigheid van tumor-specifiek antilichaam

ook al zeer goed in staat kankercellen te

vernietigen, maar doen dit nog beter wanneer

antilichaamtherapie gegeven wordt. Met

behulp van verder microscopisch onderzoek

Figuur 2. Antilichaamtherapie. (A) Onder normale omstandigheden kunnen immuuncellen kankercellen slecht herkennen. (B) Na toediening binden tumor-specifieke antilichamen aan kankercellen. (C) Immuuncellen kunnen nu, met behulp van Fc receptoren, de kankercellen makkelijk herkennen. (D) Nadat de kankercel is herkend, zal deze vernietigd worden door de immuuncel.


is tevens duidelijk geworden dat Kupffer cellen de kankercellen door fagocytose vernietigen

(ze ‘eten’ de kankercellen op). Samenvattend hebben wij in hoofdstuk 4, 5 en 6 laten

zien dat met name Kupffer cellen verantwoordelijk zijn voor het therapeutische effect van

antilichaamtherapie ter preventie van colon carcinoom metastasen.

De rol van macrofagen in metastase ontwikkeling

Macrofagen zijn volop aanwezig in vele soorten kanker, waar ze allerlei functies kunnen

vervullen. Behalve dat macrofagen een belangrijke rol spelen in antilichaamtherapie ter

bestrijding van metastasen, kunnen ze de ontwikkeling van gezwellen in het algemeen

sterk beïnvloeden (hoofdstuk 7). Klassiek wordt de macrofaag gezien als een immuuncel

die ontstekingsmediatoren produceert en ziekteverwekkers zoals bacteriën opruimt.

Dit type macrofaag noemt men de ‘klassiek geactiveerde’ of M1 macrofaag en wordt

normaliter gevonden bij ontstekingen en infecties. Er wordt gedacht dat deze macrofagen

kankercellen kunnen vernietigen en kunnen bijdragen aan een immuunreactie tegen

het gezwel. Macrofagen kunnen ook een aantal andere functies hebben, bijvoorbeeld

het stimuleren van celgroei en de aanleg van bloedvaten. Dit type macrofaag wordt

‘alternatief geactiveerd’ of M2 genoemd. Vanwege deze functies zouden M2 macrofagen

de groei van gezwellen kunnen versnellen (figuur 3). De huidige gedachte is dat de

macrofagen die in gezwellen zitten vooral van het M2 type zijn en bijdragen aan groei van

de kanker. Hierover bestaan echter tegengestelde berichten. In een aantal soorten kanker

(o.a. borst-, blaas- en prostaatkanker) is de aanwezigheid van macrofagen inderdaad

geassocieerd met een slechte prognose wat suggereert dat macrofagen bijdragen aan

de groei van gezwellen. Maar het is ook duidelijk geworden dat de aanwezigheid van

Figuur 3. De effecten van M1 en M2 macrofagen op tumorgroei. M1 macrofagen in en rondom tumoren dragen bij aan remming van tumorgroei doordat zij kankercellen vernietigen en bijdragen aan een goede afweerreactie tegen het gezwel. M2 macrofagen daarentegen bevorderen tumorgroei door productie van groeifactoren, stimulatie van bloedvatvoorziening en onderdrukking van afweerreacties.


macrofagen in andere typen kankers (onder andere in colon carcinoom) juist gunstig is

voor de prognose van de patiënt.

Wij hebben in een diermodel laten zien dat macrofagen een grote rol spelen bij de

ontwikkeling van colon carcinoom metastasen (hoofdstuk 8). De aanwezigheid van

macrofagen leidt tot slecht gedifferentieerde gezwellen (normaliter geassocieerd met een

slechtere prognose), wat duidt op de aanwezigheid van M2 macrofagen. Macrofagen zijn

ook zeer belangrijk in het beperken van de groei van gezwellen, omdat dieren zonder

macrofagen een veel grotere hoeveelheid gezwellen ontwikkelden. Er wordt aangenomen

dat vooral nieuw gerekruteerde macrofagen die in gezwellen terechtkomen uiteindelijk

M2 macrofagen worden en de groei van de kanker bevorderen. Zodoende hebben wij

de toevoer van nieuwe macrofagen in gezwellen geblokkeerd om te onderzoeken of dit

de groei kan remmen (hoofdstuk 9). Het tegenovergestelde bleek waar, want blokkade

van macrofagentoevoer leidde tot een verhoogde groei. Dus ook nieuw gerekruteerde

macrofagen hebben eigenschappen van M1 macrofagen en dragen bij aan beperking van

de groei van colon carcinoom metastasen. Samenvattend kan geconcludeerd worden

dat in colon carcinoom metastases macrofagen aanwezig zijn die zowel M1 als M2

karakteristieken hebben. De goede M1 karakteristieken hebben echter de overhand over

de ongunstige M2 eigenschappen (figuur 4).

Conclusie en aanbevelingen

Figuur 4. Het netto effect van alle macrofagen op ontwikkeling van metastasen. Alhoewel M2 macrofagen tumorgroei bevorderen, is het netto effect van alle macrofagen (M1, M2 en nieuw gerekruteerde macrofagen) tumorgroeiremmend.


Macrofagen en de ontwikkeling van metastasen

Er wordt gesuggereerd dat het verminderen van de hoeveelheid macrofagen in gezwellen

de prognose van de patiënt kan verbeteren. Dit lijkt inderdaad een goede aanpak bij

typen kankers waarbij een hoog aantal macrofagen in het gezwel correleert met een

ongunstig ziektebeloop (zoals borstkanker). Ons onderzoek laat echter zien dat deze

aanpak niet wenselijk is betreffende colon carcinoom metastasen, en dus beter kan

worden vermeden. Een andere strategie ter behandeling van kanker is het stimuleren

van de aanwezigheid van M1 macrofagen en het verminderen van M2 macrofagen. De rol

van M1 en M2 macrofagen in gezwellen is echter zeer gecompliceerd en nog niet volledig

begrepen. Daarom is voorzichtigheid geboden bij therapeutische interventies en zal er

meer onderzoek gedaan moeten worden om de rol van de macrofaag in de ontwikkeling

van gezwellen volledig in kaart te brengen.

Peri-operatieve behandelmogelijkheden voor het bestrijden van metastasen

Het onderzoek beschreven in dit proefschrift laat zien dat verwijdering van het primaire

gezwel, alhoewel noodzakelijk en potentieel genezend, paradoxaal genoeg kan bijdragen

aan uitzaaiing van het gezwel en verergering van het ziektebeeld. Tegelijkertijd

hebben we laten zien dat interventies in de periode rondom de operatie verschillende

veelbelovende mogelijkheden bieden deze metastaseontwikkeling tegen te gaan. Ik ben

van mening dat de mogelijkheden die ter beschikking staan, momenteel erg worden

ondergewaardeerd. Een voor de hand liggende strategie ter bestrijding van metastasen

is het optimaliseren van chirurgische procedures waardoor weefselschade, ontsteking

en het losraken van kankercellen worden geminimaliseerd. Elke operatie zal niettemin

leiden tot gunstigere omstandigheden voor de ontwikkeling van metastasen in meer of

mindere mate. Om deze reden is het nodig de exacte mechanismen te begrijpen die ten

grondslag liggen aan de verhoogde uitgroei van metastasen na chirurgisch trauma zodat

nieuwe therapieën ontwikkeld kunnen worden. Wij hebben in een diermodel laten zien

dat na een operatie de extracellulaire matrix in de buikholte en lever vrij komt te liggen

waardoor kankercellen met behulp van integrines makkelijk kunnen aanhechten. Deze

aangehechte kankercellen kunnen hierna uitgroeien tot metastasen. Door de interactie

van integrines en extracellulaire matrix te voorkomen, kan de uitgroei van metastasen

worden tegengegaan. Toekomstig onderzoek in dit veld moet zich richten op de veiligheid

en de doeltreffendheid van het blokkeren van integrines in patiënten die een operatie

ondergaan voor de verwijdering van een colon carcinoom.

Een andere veelbelovende strategie voor het voorkomen van metastasen na

operatieve verwijdering van colon carcinoom is antilichaamtherapie. Verschillende

tumorspecifieke antilichamen zijn reeds beschikbaar. Aangezien wij duidelijk de potentie


van antilichaamtherapie na operatieve verwijdering van colon carcinoom hebben laten

zien, is de logische volgende stap te onderzoeken wat de effectiviteit is in de kliniek.

Daarnaast moet de kennis van de werkingsmechanismen van antilichaamtherapie die er

momenteel is, worden gebruikt om antilichaamtherapie te optimaliseren. Een voorbeeld

hiervan is het stimuleren van de hoeveelheid en activatie van Kupffer cellen in de lever,

aangezien deze cellen een belangrijke rol spelen in antilichaamtherapie ter bestrijding

van levermetastasen.

Er kan geconcludeerd worden uit het werk dat in dit proefschrift beschreven is, dat

er veelbelovende therapeutische strategieën zijn, die de prognose van patiënten met

een colon carcinoom kunnen verbeteren. Een aantal van deze strategieën moet voordat

ze in de kliniek kunnen worden geëvalueerd eerst nader worden onderzocht. Dit geldt

onder andere voor therapieën waarin het veranderen van het aantal en de functie van

macrofagen in tumoren centraal staat. Er is echter geen reden om klinisch onderzoek

naar de veiligheid en effectiviteit van integrine-blokkering en antilichaamtherapie na

operatieve verwijdering van colon carcinoom langer uit te stellen. Klinisch onderzoek naar

deze therapieën moeten dan ook zo snel mogelijk worden gestart.


VERKLARENDE WOORDENLIJST

Adhesiemolecuul Molecuul aan de buitenkant van cellen dat

verantwoordelijk is voor de aanhechting aan andere

cellen of structuren.

Antilichaam (tumor specifiek) Een molecuul (antistof) dat één bepaald ander

molecuul op kankercellen kan herkennen en binden.

Antilichaamtherapie Therapie waarbij tumor specifieke antilichamen

worden gegeven die binden aan kankercellen. Deze

kankercellen kunnen vervolgens makkelijk worden

herkend en vernietigd door het immuunsysteem van

de patiënt.

Chemotherapie Therapie waarbij stoffen worden gegeven die vooral

delende cellen doden. Aangezien kankercellen over het

algemeen snel delen, is deze therapie zeer geschikt

voor het bestrijden van kanker.

Colon carcinoom Kankergezwel uitgaande van de epitheellaag van de

dikke darm.

Extracellulaire matrix Steungevend bindweefsel dat in bijna elk type weefsel

voorkomt.

Fagocytose Het proces waarbij een cel vaste deeltjes, bijvoorbeeld

een andere cel, opneemt om deze vervolgens te

vernietigen.

Immuuncel Cel van het immuunsysteem, bijvoorbeeld een

macrofaag.

Integrines Familie van adhesiemoleculen, veelvoorkomend op

kankercellen.

Knock-out muis Genetisch gemodificeerde muis waarbij een of

meerdere genen zijn uitgezet. Zeer geschikt voor het

onderzoeken van de functie van bepaalde genen.

Kupffer cel Macrofaag van de lever.

Macrofaag Grote immuuncel die in staat is ziekteverwekkers te

vernietigen en afweerreacties te induceren. Speelt ook

een belangrijke rol bij de herstelfase na ontsteking en

weefselschade.

Macrofaag, M1 type ‘Klassiek geactiveerde’ macrofaag, voornamelijk

betrokken bij de opruiming van ziekteverwekkers en

lichaamsvreemde stoffen.


Macrofaag, M2 type ‘Alternatief geactiveerde’ macrofaag, onder andere

belangrijk voor de herstelfase na ontstekingen en

weefselschade door stimulering van celgroei en

bloedvoorziening.

Macrofaag, nieuw gerekruteerd Jonge macrofaag (monocyt) die vanuit het bloed in

een weefsel is gemigreerd. De lokale omstandigheden

bepalen welke functie de macrofaag gaat uitoefenen

(bijvoorbeeld M1 of M2 functies).

Metastase Uitzaaiing, kwaadaardig gezwel dat op een andere

plaats optreedt dan de oorspronkelijke plaats van het

eerste (primaire) gezwel.

Minimal residual disease Zeer kleine hoeveelheid tumorweefsel die eventueel

na operatieve verwijdering of andere behandeling van

een gezwel overblijft.

Receptor Molecuul in de wand van cellen, welke na activatie

signalen vanuit de omgeving de cel in stuurt.

Receptor, Fcγ Familie van receptoren die (het niet specifieke deel

van) antilichamen herkennen.

Recidief Het terugkomen van een gezwel na een operatie of

andere behandeling op dezelfde plek waar het eerder

gelokaliseerd was.

Appendix B - List of publications | 31

LIST OF PUBLICATIONS

van der Bij GJ, Oosterling SJ, Beelen RHJ, Meijer S, Coffey JC and van Egmond M.The perioperative period is an underutilised window of therapeutic opportunity in patients with colorectal cancerSubmitted

van der Bij GJ, Bögels M, Otten MA, Oosterling SJ, Kuppen P, Meijer S, Beelen RHJ and van Egmond M.Successful prevention of surgery-induced liver metastases development after anti-tumor monoclonal antibody therapy is mediated by the innate mononuclear phagocyte networkSubmitted

Otten MA, van der Bij GJ, Verbeek SJ, Nimmerjahn F, Ravetch JV, Beelen RHJ, van de Winkel JGJ and van Egmond M.Experimental antibody therapy of liver metastases reveals functional redundancy between FcγRI and FcγRIV The Journal of Immunology, accepted for publication

van der Bij GJ, Bögels M, Oosterling SJ, Kroon J, Schuckmann DT, de Vries HE, Meijer S, Beelen RHJ and van Egmond M.Tumor infiltrating macrophages reduce development of peritoneal colorectal carcinoma metastases.Cancer Letters, in press

Oosterling SJ, van der Bij GJ, Bögels M, ten Raa S, Post JA, Meijer GA, Beelen RHJ and van Egmond M.Anti-beta1 integrin antibody reduces surgery-induced adhesion of colon carcinoma cells to traumatized peritoneal surfaces.Annals of Surgery. 2008 Jan;247(1):85-94.

van der Bij GJ, Oosterling SJ, Bögels M, Bhoelan F, Fluitsma DM, Beelen RHJ, Meijer S and van Egmond M.Blocking alpha2 integrins on rat CC531s colon carcinoma cells prevents operation-induced augmentation of liver metastases outgrowth.Hepatology. 2008 Feb;47(2):532-43.

Oosterling SJ, Mels AK, Geijtenbeek TB, van der Bij GJ, Tuk CW, Vuylsteke RJ, van Leeuwen PA, Meijer GA, Meijer S, Beelen RHJ and van Egmond M.Preoperative granulocyte/macrophage colony-stimulating factor (GM-CSF) increases hepatic dendritic cell numbers and clustering with lymphocytes in colorectal cancer patients.Immunobiology. 2006;211(6-8):641-9.

Oosterling SJ, van der Bij GJ, Mels AK, Beelen RHJ, Meijer S, van Egmond M and van Leeuwen PA.Perioperative IFN-alpha to avoid surgically induced immune suppression in colorectal cancer patients.Histology Histopathology. 2006 Jul;21(7):753-60.

Oosterling SJ, van der Bij GJ, Bögels M, van der Sijp JR, Beelen RHJ, Meijer S and van Egmond M.Insufficient ability of omental milky spots to prevent peritoneal tumor outgrowth supports omentectomy in minimal residual disease.Cancer Immunology Immunotherapy. 2006 Sep;55(9):1043-51.

van der Bij GJ, Oosterling SJ, Meijer S, Beelen RHJ and van Egmond M.The role of macrophages in tumor development.Cellular Oncology. 2005;27(4):203-13.

32 | Appendix B - List of publications

van der Bij GJ, Oosterling SJ, Meijer S, Beelen RHJ and van Egmond M.Therapeutic potential of Kupffer cells in prevention of liver metastases outgrowth.Immunobiology. 2005;210(2-4):259-65.

Oosterling SJ, van der Bij GJ, Meijer GA, Tuk CW, van Garderen E, van Rooijen N, Meijer S, van der Sijp JR, Beelen RHJ and van Egmond M.Macrophages direct tumour histology and clinical outcome in a colon cancer model.Journal of Patholology. 2005 Oct;207(2):147-55.

Oosterling SJ, van der Bij GJ, van Egmond M, van der Sijp JR.Surgical trauma and peritoneal recurrence of colorectal carcinoma.European Journal of Surgical Oncology. 2005 Feb;31(1):29-37.

de Gruijl TD, Luykx-de Bakker SA, Tillman BW, van den Eertwegh AJ, Buter J, Lougheed SM, van der Bij GJ, Safer AM, Haisma HJ, Curiel DT, Scheper RJ, Pinedo HM and Gerritsen WR.Prolonged maturation and enhanced transduction of dendritic cells migrated from human skin explants after in situ delivery of CD40-targeted adenoviral vectors.Journal of Immunology. 2002 Nov 1;169(9):5322-31.

Appendix D - Curriculum Vitae | 33

CURRICULUM VITAE

Gerben Joost van der Bij was born on January the 9th 1978 in Alkmaar, The Netherlands.

He graduated high school (atheneum) in 1996 after which he studied medical biology at

the VU University in Amsterdam. After receiving his master’s degree in 2002 he started

as PhD student within a collaboration between the department of Surgical Oncology (prof.

dr. S. Meijer) and Molecular Cell Biology & Immunology (prof. dr. R.H.J. Beelen) at the VU

University Medical Center. The results of his PhD project are described in this thesis. In

September 2007 he started a 1-year program at the VU university to obtain his bachelor’s

degree in medicine, which he completed in August 2008. In October 2008 he will continue

his medical education to acquire his master’s degree.

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