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Exploring tumor heterogeneity
Fessler, E.
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Fessler, E. (2016). Exploring tumor heterogeneity
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Download date: 18 Jul 2017
Annexes
Summary
Nederlandse samenvatting
Curriculum vitae
Portfolio proefschrift
Publication list
Acknowledgements
Summary
Summary
Cancer is a heterogeneous disease, which is reflected both on the cellular as well as the
population level. Tumors have been recognized as ‘abnormal organs’ based on the fact that
transformed cells within one tumor exist in distinct states and intricately crosstalk with nontransformed cells in the tumor microenvironment. The term intra-tumor heterogeneity
conceptualizes this notion, which is nowadays recognized as an important factor determining
response to treatment and development of recurrences. Inter-tumor heterogeneity refers to the
fact that no tumor is like any other, which is illustrated most obviously by the comparison of
tumors arising in different organs. The cells targeted for transformation, the transformationinitiating event, the environmental composition, and many more factors differ between
neoplasms arising for instance in the brain (glioblastomas) and in the colon. Moreover, these
parameters can also differ between tumors arising in the same organ, leading to the formation of
distinct subtypes within a given type of cancer. The drivers of both intra- as well as inter-tumor
heterogeneity are described in Chapter 1, where we also discuss the potential implications of
this diversity on clinical management of patients and how the two forms of heterogeneity are
connected.
Part I – Intra-tumor heterogeneity
In Chapter 2, we describe the cancer stem cell (CSC) concept, which perceives tumors as
hierarchically organized entities. The hierarchy observed in tumors closely parallels the one in
normal organs – (cancer) stem cells at the apex of the hierarchy give rise to transit amplifying
cells, which eventually spawn differentiated progeny. Yet, the hierarchical organization of
tumors might be more flexible with differentiated cells being able to gain CSC characteristics.
We highlight the pathways that control the CSC state and that influence the plasticity within
distinct cellular populations. The cues regulating pathway activity can originate from the tumor
microenvironment and we discuss its role in shaping the CSC state and the impact of CSCs on
tumor progression and metastatic spread to distant organs.
In Chapter 3, we make use of primary cultures derived from glioblastoma specimen: (i)
spheroid cultures, that are enriched for CSCs and (ii) cultures of tumor microvascular endothelial
cells, which constitute the niche for glioblastoma CSCs. We induce differentiation of
glioblastoma cells using multiple stimuli and assess their capacity to dedifferentiate upon
exposure to endothelial cell-derived factors both phenotypically based on marker expression as
well as functionally by determining the clonogenic potential. We purify the differentiated
184
Summary
population using the cell surface marker O4, to assess the dedifferentiation potential of a
population consisting solely of differentiated cells. Soluble factors derived from endothelial cells
are sufficient to inflict CSC features on differentiated glioblastoma cells and we show that basic
fibroblast growth factor alone is able to recapitulate the plasticity instigated by endothelial cells.
Part II – Inter-tumor heterogeneity
In Chapter 4 we describe the existence of subtypes within colorectal cancer (CRC) and how
they can be identified. Historically, CRC has been classified based on genetic and epigenetic
features. Recently, categorization using whole gene expression profiles was propelled into the
spotlight. We highlight the consensus molecular subtype (CMS) classification system, which
unifies several independent gene expression-based classifications of CRC. Moreover, we discuss
model systems that could recapitulate distinct cancer subtypes and might thus proof useful for indepth biological characterization of the CMSs of CRC.
In Chapter 5 we set out to identify the underlying drivers of the mesenchymal, poor prognosis
CRC subtype (CMS4). The data presented indicate that microRNAs (miRs) belonging to the
miR-200 family tune the majority of genes differentially expressed between CMS4 and CMS1-3
tumors. The expression of miR-200 family members is lower in CMS4 compared to the other
subtypes and relates to high levels of methylation of the miR-200 promoter regions. Importantly,
methylation levels of the miR-200 promoter regions predict CMS4 affiliation and prognosis of
stage II colon cancer patients. Besides its usefulness in determining subtype affiliation, this
epigenetic feature also provides an explanation for activity of specific biological programs in
distinct subtypes. The members of the miR-200 family have been implicated in the epithelialmesenchymal transition (EMT) program and their low expression can therefore account for the
mesenchymal appearance of CMS4 tumors. Furthermore, our data suggest that the miR-200
regulatory network is active in the epithelial compartment of mesenchymal colorectal
carcinomas and manipulation of this network in CRC cell lines indicates that the miR-200 family
members indeed determine CMS4-associated gene expression and functional properties.
The CMSs of CRC present distinct entities, raising the question at what point during
tumorigenesis their development diverges. It has previously been suggested that different colon
cancer subtypes originate from distinct precursor lesions, thus implying that instead of diverging
during tumor development, they follow specific adenoma-carcinoma sequences from the very
beginning. The data presented in Chapter 6 indeed highlight the similarity between sessile
185
Summary
serrated adenomas (SSAs) and the mesenchymal CMS4 of CRC, with respect to gene expression,
expression of selected proteins, and epigenetic features. Additionally, the data suggest that EMT,
a program so far associated with late stage disease, can already be active at the premalignant
stage and might equip SSAs with aggressive features early on.
Next to the straightforward development of one specific precursor lesion to one distinct CRC
subtype, the possibility exists that one type of precursor lesion can spawn carcinomas belonging
to multiple subtypes. In fact, molecular markers such as the BRAFV600E mutation and DNA
hypermethylation suggest that SSAs can develop to the best and the worst prognosis CRC
subtypes, CMS1 and CMS4, respectively. In Chapter 7, we set out to determine developmental
drivers of CRC subtypes and elucidate the effect of the cytokine transforming growth factor-E
(TGFE) on precursor lesions of CRC. Gene expression-based predictions indicate that SSAs
could indeed give rise to CMS1 and CMS4 malignancies and that high levels of TGFE pathway
activity direct SSAs to the mesenchymal, poor prognosis CRC subtype.
In Chapter 8, we discuss the findings of this thesis work in the light of recent publications and
integrate the drivers of tumor heterogeneity identified herein with additional parameters
impacting on tumor (cell) diversity.
186
Nederlandse samenvatting
Nederlandse samenvatting
Kanker is een heterogene ziekte, wat zowel tot uiting komt op cel- als op populatie niveau. De
term intra-tumor heterogeniteit beschrijft het verschil in eigenschappen tussen cellen binnen een
tumor. Door deze verschillen zullen sommige cellen wel, maar andere cellen niet gevoelig zijn
voor therapie waardoor de kanker na eerdere succesvolle behandeling toch uiteindelijk kan
terugkomen. Inter-tumor heterogeniteit verwijst naar het feit dat geen twee tumoren hetzelfde
zijn. Dit verschil is duidelijk waarneembaar tussen tumoren die in verschillende organen
ontstaan; een tumor in de hersenen (glioblastoom) is erg verschillend van een tumor in de darm.
Het celtype, de verandering in de cel, de omgeving van de cel en nog vele andere factoren leiden
uiteindelijk tot deze verschillen. Maar ook binnen de tumoren van hetzelfde orgaan, kunnen veel
van elkaar verschillen, waardoor zogenaamde “subtypes” binnen een tumorsoort worden
gevormd. De factoren die een rol spelen bij zowel intra-tumor als inter-tumor heterogeniteit en
hun relatie tot elkaar worden beschreven in Hoofdstuk 1. Hierin beschrijven we ook de invloed
van heterogeniteit op klinische beslisvorming.
Deel I – Intra-tumor heterogeniteit
In Hoofdstuk 2 bespreken we het concept kankerstamcellen, die zorgen voor een hiërarchisch
georganiseerd systeem in een tumor. De hiërarchie in tumoren lijkt sterk op die in normale
organen – (kanker) stamcellen staan bovenaan in de hiërarchie en ontwikkelen zich tot meer
gedifferentieerde dochtercellen. In kanker is deze hiërarchie wat minder strikt waardoor
gedifferentieerde cellen in staat zijn opnieuw kankerstamcel-eigenschappen te verkrijgen.
We belichten de signaalroutes die belangrijk zijn voor het in stand houden van de kankerstamcel
eigenschappen in tumorcellen. Deze signalen kunnen ook ontstaan in de omgeving van de tumor
en ook de rol hiervan op de stamcel activiteit wordt besproken. Tevens bespreken we de rol van
kankerstamcellen op de ontwikkeling van de tumor en het vormen van uitzaaiingen.
In Hoofdstuk 3, gebruiken we twee soorten kweken van glioblastomen, één waarin zowel
kankerstamcellen als gedifferentieerde cellen aanwezig zijn en één waarin de endotheelcellen uit
de omgeving van de tumorcellen gekweekt worden. We forceren kankerstamcellen tot
differentiatie naar meer gedifferentieerde cellen en kijken welke factoren deze cellen nodig
hebben om hun kankerstamcel kenmerken terug te krijgen. We zien dat fibroblast growth factor
uitgescheiden door endotheelcellen voldoende is om dit te bewerkstelligen.
187
Nederlandse samenvatting
Deel II – Inter-tumor heterogeniteit
In Hoofdstuk 4 beschrijven we de verschillende subtypes in darmkanker die gebaseerd zijn op
(moleculaire)
genexpressie.
Verschillende
onderzoeksgroepen
hebben
deze
subtypes
geïdentificeerd en zijn uiteindelijk gezamenlijk tot een consensus classificatie gekomen die moet
leiden tot uniformiteit. Daarnaast beschrijven we modellen die gebruikt kunnen worden bij
experimenten in cellen en muizen om deze subtypes beter te kunnen onderzoeken.
In Hoofdstuk 5 identificeren we factoren die een rol spelen in de ontwikkelen van één van de
subtypes, namelijk het mesenchymale subtype, dat gekenmerkt wordt door een slechte
overlevingskans. We laten zien dat microRNAs (miRs), behorende tot de miR-200 familie, de
genexpressie van deze subgroep tumoren beïnvloedt. De expressie van deze miR familie is lager
in het mesenchymale subtype in vergelijking tot de andere subgroepen, wat komt door
methylatie, een manier om een gen uit te schakelen. We laten ook zien dat deze methylatie van
de miR familie de overleving van darmkanker patiënten kan voorspellen.
Genen van deze familie zijn eerder gelinkt aan een proces dat epithelial-mesenchymal transition
(EMT) heet en lage expressie van deze genen kan dus een verklaring zijn voor het
mesenchymale karakter van deze tumoren.
De subtypes in darmkanker verschillen in grote mate van elkaar en de vraag is wanneer in de
ontwikkeling van tumoren deze verschillen ontstaan. Eerder is gesuggereerd dat verschillende
subtypes uit verschillende premaligne afwijkingen, bijvoorbeeld poliepen ontstaan. Dit
impliceert dat de verschillen al in een vroeg stadium aanwezig zijn en dat deze tumoren
behorende tot een subtype vanaf het begin een andere route bewandelen dan tumoren behorende
tot een ander subtype. In Hoofdstuk 6 laten we de overeenkomsten in genexpressie, eiwitten en
methylatie tussen sessiele (vlakke) poliepen en het mesenchymale subtype zien. Daarnaast laten
we zien dat EMT, een proces dat meestal wordt geassocieerd met vergevorderde ziekte, al actief
is in deze sessiele poliepen waardoor deze poliepen wellicht in een vroeg stadium al agressieve
kenmerken hebben.
Naast de eenvoudige verklaring dat een bepaald subtype uit een bepaalde premaligne afwijking
ontstaat, kan het ook zo zijn dat één type afwijking zich kan ontwikkelen tot tumoren uit
verschillende subtypes. Moleculaire eigenschappen van sessiele poliepen suggereren dat ze zich
kunnen ontwikkelen tot tumoren uit twee subtypes: degene met de beste én degene met de
slechtste prognose. In Hoofdstuk 7 proberen we de factoren die belangrijk zijn voor het
188
Nederlandse samenvatting
ontwikkelen van de tumoren in de verschillende subtypes te identificeren. We bestuderen het
effect van transforming growth factor-E (TGFE) op premaligne afwijkingen. Op basis van
genexpressie zien we dat sessiele poliepen inderdaad tot tumoren uit deze twee subtypes kunnen
leiden en dat verhoogde activiteit van TGFE sessiele poliepen in de richting van het
mesenchymale subtype doet sturen.
In Hoofdstuk 8 zetten we de gevonden resultaten in perspectief van de recente publicaties van
anderen en beschrijven we andere factoren die van invloed zijn op tumor heterogeniteit.
189
Curriculum vitae
Curriculum vitae
Evelyn Fessler was born on March 25th 1987 in Laupheim, Germany to her parents Marlene and
Günther Fessler. After having lived in a tiny village for 19 years and having completed her
primary education, she moved to Erlangen in 2006, where she studied Molecular Medicine at the
Friedrich Alexander University. She completed her studies with her undergraduate thesis work
performed in the laboratory of Prof. Dr. Robert A. Weinberg (Whitehead Institute for
Biomedical Research/MIT, Cambridge, USA) and graduated with a Diplom (equivalent to MSc)
in 2011. Her undergraduate thesis work focused on the role of immune cells in cancer cell
metastasis under the supervision of Dr. Asaf Spiegel. In 2011, she was awarded a PhD
scholarship from the AMC Graduate School for Medical Sciences, which allowed her to join the
laboratory of Prof. Dr. Jan Paul Medema at the Academic Medical Center in Amsterdam (The
Netherlands). She continued to investigate the role of the tumor microenvironment and
additional causes of tumor heterogeneity during her doctoral work, which is presented in this
thesis.
190
Portfolio proefschrift
Portfolio proefschrift
PhD training
Year
ECTS
The Amsterdam international medical summer school:
molecular pathways in cancer, initiation, maintenance and therapy
2011
1
Laboratory animal science, article 9 (Utrecht, NL)
2011
3.9
Practical biostatistics
2015
1.1
Department seminars (CEMM)
2011-2016
5
AMC oncology seminars (OASIS)
2011-2016
5
Workshop R2: analysis of tumor genomics data
2014
0.1
Cancer Genomics meeting (Amsterdam, NL)
2012/14/15
2.2
Annual OOA PhD retreat (Ermelo, NL, poster presentation)
2012
1
22nd biennial congress of the European Association for
Cancer Research (EACR, Barcelona, ES)
2012
1
Gordon Research Seminar: stem cells & cancer
(Ventura, CA, USA, oral and poster presentation)
2015
1
Gordon Research Conference: stem cells & cancer
(Ventura, CA, USA, poster presentation)
2015
1
AMC oncology seminar (OASIS, Amsterdam, NL, oral presentation)
2015
1
Cancer Genomics meeting (Amsterdam, NL, poster presentation)
2015
1
Keuzeonderwijs
2012
0.5
Master student (University of Amsterdam)
2013-2014
1.5
Courses
Seminars and workshops
Presentations and conferences
Supervision
Awards
PhD scholarship from the AMC Graduate School for Medical Sciences
2011
191
Publication list
Publication list
Fessler E, Medema JP. Colorectal cancer subtypes: developmental origin and microenvironmental regulation.
Submitted.
Fessler E, Jansen M, De Sousa E Melo F, Zhao L, Prasetyanti PR, Rodermond H, Kandimalla R, Linnekamp JF,
Franitza M, van Hooff SR, de Jong JH, Oppeneer SC, van Noesel CJM, Dekker E, Stassi G, Wang X, Medema JP,
Vermeulen L. A multidimensional network approach reveals miRNAs as determinants of the mesenchymal
colorectal cancer subtype. Oncogene, in press (2016).
Fessler E, Drost J, van Hooff SR, Linnekamp JF, Wang X, Jansen M, De Sousa E Melo F, Prasetyanti PR, IJspeert
JEG, Franitza M, Nürnberg P, van Noesel CJM, Dekker E, Vermeulen L, Clevers H, Medema JP. TGFE signaling
directs serrated adenomas to the mesenchymal colorectal cancer subtype. EMBO Molecular Medicine 8(7), 745-760
(2016).
Spiegel A, Brooks MW, Houshyar S, Reinhardt F, Ardolino M, Fessler E, Chen MB, Krall JA, DeCock J,
Zervantonakis IK, Iannello A, Iwamoto Y, Cortez-Retamozo V, Kamm RD, Pittet MJ, Raulet DH, Weinberg RA.
Neutrophils suppress intraluminal NK-mediated tumor cell clearance and enhance extravasation of disseminated
carcinoma cells. Cancer Discovery 6(6), 630-649 (2016).
Guinney J, Dienstmann R, Wang X, de Reyniès A, Schlicker A, Soneson C, Marisa L, Roepman P, Nyamundanda
G, Angelino P, Bot BM, Morris JS, Simon IM, Gerster S, Fessler E, De Sousa E Melo F, Missiaglia E, Ramay H,
Barras D, Homicsko K, Maru D, Manyam GC, Broom B, Boige V, Perez-Villamil B, Laderas T, Salazar R, Gray
JW, Hanahan D, Tabernero J, Bernards R, Friend SH, Laurent-Puig P, Medema JP, Sadanandam A, Wessels L,
Delorenzi M, Kopetz S, Vermeulen L, Tejpar S. The consensus molecular subtypes of colorectal cancer. Nature
Medicine 21(11), 1350-1356 (2015).
Fessler E, Borovski T, Medema JP. Endothelial cells induce cancer stem cell features in differentiated glioblastoma
cells via bFGF. Molecular Cancer 14(157), (2015).
Büller NV, Rosekrans SL, Metcalfe C, Heijmans J, van Dop WA, Fessler E, Jansen M, Ahn C, Vermeulen JL,
Westendorp BF, Robanus-Maandag EC, Offerhaus GJ, Medema JP, D'Haens GR, Wildenberg ME, de Sauvage FJ,
Muncan V, van den Brink GR. Stromal Indian hedgehog signaling is required for intestinal adenoma formation in
mice. Gastroenterology 148(1), 170-180 (2015).
Colak S, Zimberlin C, Fessler E, Hogdal L, Prasetyanti P, Grandela C, Letai A, Medema JP. Decreased
mitochondrial priming determines chemoresistance of colon cancer stem cells. Cell Death and Differentiation 21,
1170-1177 (2014).
De Sousa E Melo F, Vermeulen L, Fessler E, Medema JP. Cancer heterogeneity - a multifaceted view. EMBO
Reports 14(8), 686-695 (2013).
De Sousa E Melo F, Wang X, Jansen M, Fessler E, Trinh A, de Rooij LP, de Jong JH, de Boer OJ, van Leersum R,
Bijlsma MF, Rodermond H, van der Heijden M, van Noesel CJ, Tuynman JB, Dekker E, Markowetz F, Medema JP,
Vermeulen L. Poor-prognosis colon cancer is defined by a molecularly distinct subtype and develops from serrated
precursor lesions. Nature Medicine 19(5), 614-618 (2013).
Fessler E, Dijkgraaf FE, De Sousa E Melo F, Medema JP. Cancer stem cell dynamics in tumor progression and
metastasis: is the microenvironment to blame? Cancer Letters 341(1), 97-104 (2013).
De Sousa E Melo F, Colak S, Buikhuisen J, Koster J, Cameron K, de Jong JH, Tuynman JB, Prasetyanti PR, Fessler
E, van den Bergh SP, Rodermond H, Dekker E, van der Loos CM, Pals ST, van de Vijver MJ, Versteeg R, Richel
DJ, Vermeulen L, Medema JP. Methylation of cancer-stem-cell-associated Wnt target genes predicts poor prognosis
in colorectal cancer patients. Cell Stem Cell 9(5), 476-485 (2011).
192
Acknowledgements
Acknowledgements
Dear Jan Paul, thank you for giving me the opportunity to be part of your team and for your
support. You personify the fact that there is a lot of fun to be had in science and your enthusiasm
is a great source of inspiration. Thank you for challenging discussions and ‘adventurous’
experiments, and most importantly, thank you for your trust and for believing in me throughout
these years.
It was a pleasure to work with you, Louis. Your ceaseless optimism and ability to see the
positive side of every piece of data and situation is admirable. Thank you for being my copromotor!
I would like to thank my committee members, Gijs van den Brink, Jarno Drost, René Bernards,
Ron van Noorden, Steven Pals, and Tom Würdinger, for evaluating this thesis, for preparing an
opposition, and for participating in the defense.
Dear Bob, thank you for giving me the opportunity to work in your lab and perform exciting
research. It was a great privilege to be part of your team! Thank you, Asaf, for your supervision
and trust. May the force be with you in the years to come! A big thank you to Mary and Annie
for taking care of me during my time in Cambridge, and to Jordan, Wai, Tsukasa, Katharina,
Nora, and Philipp.
I was fortunate to have been working together with great collaborators during my PhD. Evelien,
Joep, and Suzie – thank you for including patients, collecting material, and for your continued
interest in our research. Many thanks to Carel and Marnix for your pathological assistance.
Thank you, Berend, for being a source of infinite wisdom about cell sorters, for teaching me how
to use them, and for your continued support.
A special thank you to Xin and Sander – your bioinformatical skills are priceless. Thank you for
patiently answering all my questions and for performing a plethora of analyses ‘just to see how it
looks’.
I would like to thank everyone in the LEXOR team for always having an open ear and lending a
helping hand. I am very grateful to have spent my PhD working in this unique team.
193
Acknowledgements
Felipe, thank you for introducing me to the wonders of cancer stem cell culture. I very much
appreciate your continuous support. One-and-only Tijana, thank you for keeping in touch and for
sharing stories from inside and outside the lab over many coffees. Catarina, I have never learned
as much from a person in such a short time, thank you for sharing your knowledge and
experience. Helene, you have been a truly inspiring office neighbor, never shy of a smart
question and always enthusiastic about (your) research. Lisette, thank you for always instantly
replying to any question and for your readiness to help.
Many thanks to you, Arlene, for your open personality and diligence. You are one-of-a-kind and
I consider myself lucky to have shared many special moments with you and Johan. I am glad I
got to know you, Eva, and will cherish our conversations and your honesty. Maartje, you are the
calmest PhD student I know, thank you for always being a source of serenity. Bregje, I still
remember meeting you on your very first day and I am glad we shared this last part of our PhD
journey. Joyce, Luigi, and Selcuk – it was a great pleasure to work with you and I will dearly
miss your positive spirit. Prashanthi, you are the sweetest person I have ever met, please never
change! Maarten, Salvatore, and Raju, thank you for many discussions and valuable advice.
Valeria, Simone, Serena, Dita, Klaas, Roos, Veronique, Anne, Remy, Stephanie, Aafke, Aarti,
Cynthia, Sanne, Nicolas, Daniel, Sophie, Kristiaan, Lisanne, and Maria thank you for your
gezelligheid and for making the LEXOR team – in the past and present – a conglomerate of great
minds. Thanks to Kate, Joan, Saskia, Hans, and Gregor for your assistance in any aspect of
scientific life.
A big thank you to our fellow LEXORians from MDL – Ana, Silvia, Danielle, and Carmen – and
to everyone from the CEMM department for your support and for many friendly encounters.
I admire you, Janneke, for your determination and fortitude. Thank you for sincerely caring and
for all you have done for me – listening and organizing just being the top of a very long list.
I highly value your opinion and hold you very dear!
Cheriaan, thank you so much for being the most amazing couple and for your friendship. You
have made our time here exceptional with myriad dinners and trips. Thank you for always
having an open door and for making us feel right at home in Amsterdam. Cheryl, thank you for
always being there, for understanding without words, and for exactly knowing how I would feel
in every situation. You mean a lot to me! Jurriaan, thank you for your sincerity and for handling
the taste with me.
194
Acknowledgements
Dörte, zehn Jahre ist es nun her seit wir unsere erste gemeinsame Wohnung bezogen haben und
unsere Freundschaft hat viele Veränderungen überstanden. Egal wie viele Kilometer zwischen
uns liegen, weiß ich, dass Du immer für mich da bist und jedes Mal wenn wir uns wiedersehen
ist es, als hätten wir auch die letzten sechs Jahre zusammen gewohnt.
Gabi und Ewald, danke für Eure Anteilnahme und dass ich mich bei Euch und mit Euch wie
zuhause fühlen kann. Luise, Doris und Gernot – vielen Dank für Eure Herzlichkeit und für viele
schöne Momente. Vielen Dank, Angi und Nico, dass Ihr mich so liebevoll aufgenommen habt
und ich an Eurer kleinen Familie teilhaben darf. Danke, Hanna, dass Du so ein kleiner
Sonnenschein bist und uns so viel Freude bereitest!
Herzlichen Dank an Irmi, Elli, Bruni und Willi, Elfriede und Gerhard, Leni und Sepp, Carmen,
Julian, Manuel, Elena und Angela, Juliane und die beiden Stefans mit Ihren jungen Familien für
Euren Rückhalt und schöne Erinnerungen.
Vielen Dank, Mama und Papa, für Eure – im wahrsten Sinne des Wortes – grenzenlose
Unterstützung. Danke, dass zuhause immer ein sorgenfreier Ort ist und Ihr jederzeit für mich da
seid. Liebe Christina, mit Deiner einzigartigen Tatkraft und Entschlossenheit bist Du ein ganz
besonders Vorbild und ich bin unglaublich stolz auf Dich. Ich bin dankbar, so eine liebevolle
Familie zu haben und jedes Mal aufs Neue verblüffen mich Eure guten Herzen und Eure
Einzigartigkeit!
Lucas, danke für Deine Unterstützung, Dein Verständnis und Deine Warmherzigkeit. Danke,
dass Du jederzeit für mich da bist und ich mich immer auf Dich verlassen kann. Ich freue mich
auf all die Abenteuer, die wir in Zukunft zusammen erleben werden und bin mir sicher, dass wir
gemeinsam alles schaffen können.
Evelyn
195
Acknowledgements
196