UNIVERSITATIS OULUENSIS MEDICA ACTA D D 1452 ACTA Janne Capra OULU 2018 D 1452 Janne Capra DIFFERENTIATION AND MALIGNANT TRANSFORMATION OF EPITHELIAL CELLS 3D CELL CULTURE MODELS UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF MEDICINE; BIOCENTER OULU
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Differentiation and malignant transformation of epithelial cells
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UNIVERSITY OF OULU P .O. Box 8000 F I -90014 UNIVERSITY OF OULU FINLAND
A C T A U N I V E R S I T A T I S O U L U E N S I S
University Lecturer Tuomo Glumoff
University Lecturer Santeri Palviainen
Postdoctoral research fellow Sanna Taskila
Professor Olli Vuolteenaho
University Lecturer Veli-Matti Ulvinen
Planning Director Pertti Tikkanen
Professor Jari Juga
University Lecturer Anu Soikkeli
Professor Olli Vuolteenaho
Publications Editor Kirsti Nurkkala
ISBN 978-952-62-1822-9 (Paperback)ISBN 978-952-62-1823-6 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)
U N I V E R S I TAT I S O U L U E N S I S
MEDICA
ACTAD
D 1452
AC
TAJanne C
apra
OULU 2018
D 1452
Janne Capra
DIFFERENTIATION AND MALIGNANT TRANSFORMATION OF EPITHELIAL CELLS
3D CELL CULTURE MODELS
UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF MEDICINE;BIOCENTER OULU
ACTA UNIVERS ITAT I S OULUENS I SD M e d i c a 1 4 5 2
JANNE CAPRA
DIFFERENTIATION AND MALIGNANT TRANSFORMATION OF EPITHELIAL CELLS3D cell culture models
Academic dissertation to be presented with the assent ofthe Doctoral Training Committee of Health andBiosciences of the University of Oulu for public defence inAuditorium F202 of the Faculty of Medicine (Aapistie 5 B),on 16 March 2018, at 12 noon
Reviewed byDocent Varpu MarjomäkiDocent Satu Kuure
ISBN 978-952-62-1822-9 (Paperback)ISBN 978-952-62-1823-6 (PDF)
ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)
Cover DesignRaimo Ahonen
JUVENES PRINTTAMPERE 2018
OpponentDocent Juha Klefström
Capra, Janne, Differentiation and malignant transformation of epithelial cells.3D cell culture modelsUniversity of Oulu Graduate School; University of Oulu, Faculty of Medicine; Biocenter OuluActa Univ. Oul. D 1452, 2018University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland
Abstract
The epithelial cells form barriers that compartmentalize the organs. Carcinomas are cancersstemming from epithelial cells and are the most common cancer type. The aim of this study wasto understand the differentiation and malignant transformation of epithelial Madin-Darby caninekidney (MDCK) cells and to analyse the electrophysiological parameters which regulate theirtransport capacity. Emphasis was placed on comparing different culture environments, both in 2Dand 3D. First, the effects of drugs or basal extracellular fluid composition on MDCK cell, cyst andlumen volumes were analysed using time-lapse microscopy. The results showed that MDCK cellswere capable of both water secretion and reabsorption. The cells were able to perform thesefunctions in a hyperpolarizing or depolarizing environment; change in osmolality of basal fluidwas not required. Taken together, these results validate MDCK cells as a good basic model forstudying kidney function. Next, the aim was to analyse the effect of 2D and 3D cultureenvironments on the gene expression of untransformed MDCK and temperature sensitive ts-Src -transformed MDCK cells and the changes a single oncogene can induce. Microarray analysisrevealed a decrease in the expression of survivin, an inhibitor of apoptosis protein, when switchingthe untransformed cells from 2D environment to 3D. This downregulation of survivin occurs inadult tissues as well, indicating that the cells grown in 3D are closer to the in vivo state than 2Dcells. Src oncogene induced disintegration of cell junctions, but did not downregulate E-cadherinexpression. The last part was to study further the factors controlling survivin expression and itssignificance to cell survival. MDCK cells grown in 3D did not suffer apoptosis if the cellsremained in contact with the extracellular matrix. If MDCK cells were denied of ECM contactsthey were more susceptible to apoptosis than survivin-expressing ts-Src MDCK cells. Finally, ifcells were denied of cell-cell junctions, cells lacking survivin suffered apoptosis even though theyhad proper cell-matrix contacts. Taken together, these results highlighted the importance ofcellular contacts to the cells: MDCK cells needed ECM contacts to differentiate and cell-cellcontacts to avoid apoptosis.
Keywords: apoptosis, live cell microscopy, MDCK cells, polarity, Src kinase, survivin,transepithelial transport
Capra, Janne, Epiteelisolujen erilaistuminen ja pahanlaatuistuminen. Kolmi-ulotteiset soluviljelymallitOulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; Biocenter OuluActa Univ. Oul. D 1452, 2018Oulun yliopisto, PL 8000, 90014 Oulun yliopisto
Tiivistelmä
Epiteelisolut ovat erikoistuneet toimimaan rajapintana elimen ja ympäristön välillä. Ihmistenyleisin syöpä on epiteelisoluista alkunsa saanut karsinooma. Tämän tutkimuksen tarkoituksenaoli ymmärtää Madin-Darby-koiran munuaisen solujen (MDCK) erilaistumista ja pahanlaatuistu-mista sekä analysoida sähköfysiologisia tekijöitä, jotka säätelevät näiden solujen kuljetustoimin-taa. Erityisenä kiinnostuksen kohteena oli erilaisten kasvuympäristöjen vertailu. Farmakologis-ten aineiden tai basaalisen, solunulkopuolisen nesteen koostumuksen vaikutusta MDCK-solu-jen, -kystan sekä luumenin kokoon tutkittiin valomikroskooppisten aikasarjojen avulla. Tuloksetosoittivat MDCK-solujen olevan kykeneviä sekä veden eritykseen että absorptioon, niin hyper-polarisoivassa kuin depolarisoivassakin ympäristössä. Basaalisen nesteen osmolaliteetin muutos-ta ei tarvittu. Nämä tulokset osoittavat MDCK-solujen olevan hyvä munuaisen tutkimuksenperusmalli. Seuraavaksi analysoitiin kaksi- ja kolmiulotteisten (2D ja 3D) viljely-ympäristöjenvaikutusta ei-transformoitujen MDCK-solujen ja lämpötilaherkkien ts-Src-transformoitujenMDCK-solujen geenien ilmentymiseen sekä yhden onkogeenin aktivoimisen aikaansaamia muu-toksia. Microarray-analyysi osoitti apoptoosin estäjän, surviviinin, ilmentymisen vähenemisen,kun kasvuympäristö vaihdettiin 2D-ympäristöstä 3D-ympäristöön. Koska surviviinin vähenemi-nen on normaali tapahtuma aikuisissa kudoksissa, voitiin todeta, että 3D-ympäristössä kasvatetutsolut ovat lähempänä luonnonmukaista olotilaa kuin 2D-ympäristössä kasvaneet. Src-onkogeenisai aikaan soluliitosten hajoamisen, mutta ei vähentänyt E-kadheriinin ilmentymistä. Tutkimuk-sen viimeinen osa keskittyi surviviinin ilmentymistä säätelevien tekijöiden analysoimiseen jasurviviinin merkitykseen solujen eloonjäämiselle. 3D-ympäristössä kasvaneet MDCK-soluteivät kärsineet apoptoosista edellyttäen, että solut pysyivät kosketuksissa soluväliaineeseen. Jossolut irtautuivat soluväliaineesta, ne päätyivät herkemmin apoptoosiin kuin surviviinia ilmentä-vät ts-Src MDCK-solut. Mikäli solujen väliset liitokset pakotettiin avautumaan, solut joutuivatapoptoosiin, vaikka ne olivat kosketuksissa soluväliaineeseen. Yhteenvetona nämä tuloksetkorostavat solujen kontaktien merkitystä: MDCK-solut tarvitsevat soluväliainekontakteja erilais-tumiseen ja solujen välisiä kontakteja välttyäkseen apoptoosilta.
This thesis is based on the following publications, which are referred to throughout
the text by their Roman numerals:
I Capra JP, Eskelinen SM (2017) MDCK cells are capable of water secretion and reabsorption in response to changes in the ionic environment. Can J Physiol Pharmacol 95(1): 72-83.
II Töyli M, Rosberg-Kulha L, Capra J, Vuoristo J, Eskelinen S (2010) Different responses in transformation of MDCK cells in 2D and 3D culture by v-Src as revealed by microarray techniques, RT-PCR and functional assays. Lab Invest 90(6):915-928.
III Capra JP, Eskelinen SM (2017) Correlation between E-cadherin interactions, survivin expression, and apoptosis in MDCK and ts-Src MDCK cell culture models. Lab Invest 97(12):1453-1470.
In this study, sensitive 4D imaging of living cells and quantitative analysis was used
to validate the use of MDCK cysts grown in Matrigel as a fitting model for
functional analysis of cell behaviour, especially when conducting research on
processes occurring within minutes or hours, rather than days. Modern microscopy
enables reliable and sensitive monitoring of such processes, producing supremely
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valuable data for testing the response of the cell cysts to drugs, toxins or
physiological changes, as well as in processes such as malignant transformation.
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6 Discussion
Epithelial cells have a multitude of roles in basically all of the functions of the body
of a multicellular organism. Their ability to attach to each other and form barriers
that compartmentalize organs and separate them from each other, together with the
plasticity to enable alterations in their functions, make them indispensable for a
functioning organism. Due to their importance, the understanding of their structure
and functions is pivotal, yet much is still unclear.
The aforementioned plasticity is one of the most fascinating aspects of
epithelial cells. As barriers, they must work together and form strong enough bonds
to withstand the physical forces affecting them when the organism moves, and keep
up homeostasis by disallowing free entry of extracellular constituents. Yet, they
cannot be completely impermeable and can rarely maintain constant permeability
to the same molecules, thus requiring a carefully regulated signalling network that
allows changes in plasma membrane permeability according to the needs of the
organism.
The carefully regulated functions of epithelial cells, together with their
abundance, make them also susceptible to mutations leading to malignant
transformation. Against this background, it is not surprising that 90% of all cancers
are epithelia-derived carcinomas. Epithelial cells, by nature, are not exceedingly
migratory or invasive, and thus, for malign carcinomas to develop, they need to
adopt aspects of cells that are more capable in these functions, such as
mesenchymal cells.
Complex signalling networks are needed for regulation of cellular
differentiation and formation of epithelial tissue junctions. Failure in the regulatory
processes can lead to severe metabolic diseases and/or carcinomas. In the present
work, the aim was to develop a simple in vitro model system which enabled
sensitive and detailed analysis of kidney epithelial cell phenotype and behaviour
and the consequences of the activation of an oncogene.
6.1 Plasticity of epithelial cells can be seen in the rapid response
of MDCK cells to changes in ionic environment
Secretory and absorptive organs, kidney included, rely on the asymmetrical
distribution of transporters and channel proteins on their apical and basal
membranes. The expression of these types of proteins varies from tissue to tissue,
and even in different parts of the same organ, such as kidney proximal and distal
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tubule. In addition, the localization and the activity of these transport proteins vary
depending on the circumstances. Indeed, it seems likely that different parts of the
kidney are able to function in different roles when the preferred method of kidney
function, glomerular filtration, is compromised, i.e. proximal tubule cells are able
to secrete as well as absorb. This redundancy becomes advantageous, when facing
acute or chronic renal failure or hypertonic dehydration.
Cell cultures have been a tremendously successful tool when studying
epithelial secretory and absorptive properties. In these functions, ion transport has
a pivotal role. Traditionally, cellular ion transport properties have been studied
using Ussing chamber technique, where a monolayer of cells growing on a
permeable support covers it fully, forming a barrier between the two halves of the
chamber, thus blocking free transport of molecules through the filter membrane
underneath (Ussing 1947, Hoffmann 2001). When studying ion transport, the
potential difference or electrolyte current across the two halves of the chamber is
measured. This method is excellent when conducting studies regarding ion currents
or cell permeability, and factors affecting these functions, such as drugs, nutrients
or physical elements. Also, ex vivo epithelial samples from the tissue of interest can
be used, if available (Li et al. 2004, Li et al. 2012). A limitation of the system,
however, is that it cannot be utilized to study movement of water or generation of
hydrostatic pressure.
Culturing cells in ECM allows building highly organized, differentiated cysts
with a well-developed apico-basal axis. These cysts resemble epithelial structures
in vivo, and, due to the sealed luminal space, it can be used to study water
movement through epithelial cells. Using activators and inhibitors, different
channel proteins contributing to water movement can be analysed, and the
composition of the buffer on basal side of the cells can easily be changed (Li et al.
2004, Li et al. 2012). As a downside, changing the composition of the apical buffer
is not possible. In most previous works, quantitative analysis was carried out using
low-resolution light microscopy and only the enlargement of the cyst was measured.
Hence, the information concerning lumen and cell volumes is missing (Tanner et
al. 1992, Yang et al. 2008b, Yuajit et al. 2013, Buchholz et al. 2014).
In more detail, the present study shows that, depending on the ionic
environment of the basal fluid and potential difference across the cell membrane,
chloride ions and water are able to flow through MDCK cell cysts into the apical
lumen or out of it. Accumulation of Cl- into the luminal space creates luminal
negative potential which induces water flow into the lumen, as was seen with
TMACl and (direct or indirect) chloride channel activators. On the other hand,
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decreasing the electrical potential difference between the basal fluid and the cells,
i.e. depolarizing it, using KCl or, for a more profound effect, making the plasma
membrane freely permeable to K+ with nigericin, results in water reabsorption from
the lumen and luminal shrinkage.
The method depicted here works also when examining the various plasma
membrane transporter proteins. Here, the roles of chloride channel activators
forskolin and lubiprostone as well as CFTR- and ANO1-inhibitors in MDCK cyst
lumen expansion were investigated. With highly specific inhibitors, other channel
proteins and their effects on lumen, cyst and cell size could also be studied,
potentially giving valuable information about the influence of drugs on cellular ion
and water transport. The cell swelling could be separated from cyst enlargement,
enabling the identification of the Donnan effect on the epithelium.
Modern live cell imaging techniques enable the collection of high-resolution
3D stacks and calculating the volumes of the cyst, lumen and cells, separately. This
gives valuable information about directions into which water and ions flow through
the epithelial layers. In the present study, a combination of techniques was used to
study living MDCK cysts over short periods of time after a change in their ionic
environment and the results were used to draw conclusions about how the changes
in transepithelial potential directly affects the volume of the cyst, lumen and cells,
without changes in cell numbers due to proliferation or apoptosis hindering the
interpretation. The results from this study show that the method used can be utilized
when studying the effects of chloride channel manipulation, hyperpolarization or
depolarization on cell cyst and lumen volume. These experiments gave support to
the earlier findings that ClC-2 and VSORCC are actively involved in chloride
secretion in MDCK cells (Cuppoletti et al. 2004, Melis et al. 2014).
6.2 Transformation of MDCK cells by v-Src causes changes in
gene expression, cell phenotype and behaviour
Tumour cells differ from healthy cells in several ways: 1) independence from
growth factors via autocrine excretion or altered activity of growth hormone
receptors; 2) anchorage-independent growth; 3) lack of contact inhibition; 4)
reduced adhesion, and 5) continued proliferation without regard to cell density
(Macdonald et al. 2004).
Kinases are prominently involved in the birth and progression of cancer. Src is
a multifunctional tyrosine kinase that has a role in all of the important aspects of
cancer, but interestingly, is rarely mutated in cancer cells. It contributes to the
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disease by being abnormally activated by other oncogenes (Guarino 2010). In
studies of tumour progression in vivo or in vitro, it is difficult to distinguish the
critical factors affecting cell behaviour with respect to proliferation, migration or
resistance to apoptosis. Thus, in the present study, ts-Src MDCK cell line was used
due to the advantages conferred by the cells’ ability to induce transformation by a
simple temperature change, after which the activation of v-Src occurs within an
hour. This model enables the separate analysis of the migratory and proliferative
capacities of the cells, as well as the occurrence of apoptosis, within a short time
period.
The first aspect of Src function in this study was to analyse the changes
induced by prolonged v-Src activation. In MDCK cells, the temperature-induced
activation of v-Src caused a change in expression of 350 genes when cultivated in
2D, and 1570 genes when cultivated in 3D. The relative small number of changes
in 2D is a sign of malignant behaviour exhibited already by inactive v-Src in ts-Src
MDCK, probably due to the temperature-inactivation of v-Src not being complete,
and thus exhibiting a “leaky” phenotype. Inhibition of Src with pp2 caused
significantly more changes, 3,912 and 6,483, respectively. The sizable difference
between the amount of genes changed in temperature-induced inactivation when
compared to inhibition of Src might be due to pp2 inhibiting other members of the
Src-family as well. When comparing MDCK cells with active v-Src to
untransformed MDCK cells, 2,810 genes changed their expression in 2D, whilst
the expression of 4,315 genes was changed in 3D. All in all, the activation of v-Src
has very different outcomes depending on what kind of environment the cells are
growing in.
When the changes induced by v-Src in 3D were analysed, the expression of
two interesting transcription factors was noticed to be elevated: Kaiso and Nanos.
Both of these transcription factors are linked to p120 catenin, a substrate of v-Src,
and these changes in the expression levels of Kaiso and Nanos might thus play a
role in the transformation process of v-Src transformed epithelial cells. Kaiso is a
transcriptional repressor that has a potential role in tumorigenesis, as it represses
the expression of genes regulated by the TCF/LEF pathway. If p120 catenin is
relocated to the nucleus, it binds to Kaiso and releases the expression of these genes
(van Roy & McCrea 2005). Nanos has an evolutionary conserved function in
embryonic patterning and germ line development and is down-regulated by E-
cadherin. Nanos also induces the cytoplasmic translocation of p120 catenin from
the adherens junctions, impairs cell-cell adhesion and promotes cell motility and
invasion (Strumane et al. 2006).
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E-cadherin is a pivotal player in cell attachment and in multiple different
signalling pathways, and the localization of E-cadherin to the plasma membrane is
a marker of cell polarization. The downregulation of E-cadherin, on the other hand,
is a hallmark of multiple advantage-stage tumour cells, although the loss of E-
cadherin alone is not sufficient to induce tumorigenesis (Jeanes et al. 2008, Lim &
Thiery 2012). Microarray analysis did not reveal any changes in the expression of
cadherins or catenins when v-Src was activated, but significant changes were seen
in the cell morphology: activation of v-Src caused a complete reorganization of
junctional complexes, E-cadherin was localized to the cytoplasm in endocytic
vesicles together with β-catenin and, in some cases, p120-catenin. In 3D, the cyst
had no lumen or apoptosis and the cells were undifferentiated as the cell cluster
was lacking a lumen, and actin and E-cadherin were not localized as they should in
cells that have a proper apico-basal axis. E-cadherin recycling was also disturbed
when v-Src was activated. Microarray analysis showed a decrease in the expression
levels of rab proteins that are important in vesicle trafficking, and this decrease
could explain the observed lack of recycling at permissive temperatures. However,
the activation of Src caused impairments in transportation machinery also by
disintegrating the cytoskeletal structures. Thus, Src activation can affect E-cadherin
recycling in multiple ways.
One of the very striking changes revealed by the microarray analysis was the
expression of survivin in both untransformed and ts-Src MDCK cells in 2D and ts-
Src MDCK cells in 3D, whereas MDCK cells grown in 3D lack it. The expression
of survivin by untransformed MDCK cells in 2D might explain why they were more
resistant to apoptosis than MDCK cells grown in 3D, and thus less differentiated.
In 3D, low survivin expression levels were accompanied by low mitochondrial
membrane permeability, together with lumen formation and apoptosis, in
untransformed MDCK cells. In ts-Src MDCK cells grown in 3D, upregulated
survivin expression correlated with formation of solid cysts, continued proliferation
and resistance to apoptosis.
6.3 The importance of E-cadherin interactions and survivin
expression on cell fate in untransformed and ts-Src MDCK cells
Similarly to ion transport, also epithelial differentiation and cell polarity has been
studied on cells grown on permeable filters where the cell monolayer forms a
barrier, separating the apical and basal sides. Cells grown on plastic polarize, at
best, partially, since they are unable to take in nutrients from the basal side, whereas
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filter-grown cells that have access to the cell media from the basal side are able to
polarize fully (Balcarova-Ständer et al. 1984). This complete polarization was
utilized to study cell trafficking, endo- and exocytosis or selective manipulation of
apical or basal surfaces, among other things. Growing cells on filters enabled the
study of vesicle transport routes with a technique where only the apical membrane
of highly polarized cells was permeabilized and cells were supplemented with
radiolabelled isotopes or fluorescent probes (Simons & Virta 1987, Bomsel et al.
1989).
Cells grown on filters are still imperfectly differentiated as they continue to
constantly proliferate (or, if their contact-inhibition mechanism is still functioning,
will only cease once the plate is full) and are more resistant to apoptosis. On the
other hand, when grown inside ECM, cells polarize better and differentiate further
than in 2D (Martin-Belmonte & Mostov 2008). The MDCK cell cysts will stop
growing once a certain size is reached and can maintain that size for prolonged
periods of time if no apoptosis signal is received. They are also responsive to
apoptosis signalling (Edmondson et al. 2014). As a downside, studying secretion
and collecting exocytosis vesicles from the apical side is not possible in 3D
environment.
Once a multicellular organism has matured and reached its adult size, it aims
to maintain that size. The cellular homeostasis in an adult organism seeks to balance
the proliferation and cell death and maintain the cell size. Excessive cell division,
hyperproliferation, is needed in wound healing, but can also result in cancer, while
increase in cell size and mass, hypertrophy, is involved in muscular growth due to
exercise, but can lead to cardiac disease and contribute to aging. Hyperactivity, or
abnormally active secretion, occurs normally in antibody production, as part of the
immune response or wound healing, but has also been linked to neurodegenerative
diseases (Alzheimer’s, Parkinson’s and Huntington’s diseases) and aging (Lloyd
2013). For modelling these processes, a 3D system is needed, as they are better
differentiated and can maintain homeostasis, i.e. are closer to how cells behave in
vivo than cells grown on 2D.
The cell and cyst size is regulated by the Hippo pathway, E-cadherin junctions,
inhibition of proliferation signalling and susceptibility to apoptosis. Studying these
regulators separately from each other is extremely difficult. E-cadherin interactions
between two cells are needed for contact inhibition of cells growing in dense
cultures. Moreover, dense cell cultures and mechanical strain, transduced via E-
cadherin and the actin network attached to it, were shown to activate the Hippo
pathway by inactivating Yap or transporting it out of the nucleus (Kim et al. 2011,
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Benham-Pyle et al. 2015). Thus, both plasma membrane localization of E-cadherin
and the Hippo pathway are important for the maintenance of the non-proliferating,
differentiated phenotype of epithelial cells.
In the present study, the role of E-cadherin localization and the expression of
survivin was investigated in the occurrence and regulation of proliferation and
apoptosis. It was shown that untransformed MDCK cells were able to achieve a
high level of differentiation when grown inside ECM, and did not suffer apoptosis
even though they were not expressing survivin. A good way to study the importance
of E-cadherin trans-interactions to apoptosis susceptibility is to expose the cells
grown in different environments to EGTA. The untransformed MDCK cysts had
intact E-cadherin trans-interactions, which are needed for both functioning contact
inhibition of proliferation of cells and maintenance of quiescence without
undergoing apoptosis (Benham-Pyle et al. 2015). In the current work, the E-
cadherin junctions were disturbed by chelation of the calcium ions using EGTA in
2D, 2½D and 3D environments. There was a clear correlation between the
expression of survivin and cell response to EGTA, since the cells with
downregulated survivin quickly underwent apoptosis whereas the cells in 2D were
able to avoid apoptosis. Guo and co-workers (2014) showed that dissociation of E-
cadherin trans-interactions by EGTA resulted in cis-interactions between two E-
cadherin molecules located on the plasma membrane of the same cell. This,
together with the results presented here, emphasized the importance of E-cadherin
trans-interactions for cell survival, as cells were prone to apoptosis even though
they were properly polarized and had contact with the ECM. On the other hand,
while incapable of saving the cells from apoptosis, E-cadherin cis-interactions were
sufficient to downregulate survivin in 3D in the presence of EGTA. The significant
difference in apoptosis susceptibility of cells grown in different environments (2D,
2½D and 3D) was shown in the current work, which clearly demonstrated the
plasticity of the cell behaviour in different environments.
6.4 Ts-Src MDCK cells as a model for malignant transformation of
epithelial cells
Cancer cells have traditionally been studied in 2D environment, and analysis has
focused on signal transduction from cell surface receptors into nucleus,
characterization of mutations or measurements of invasion potential of cells grown
on collagen or agarose (Edmondson et al. 2014). 2D environment makes mass-
culture of cells possible for high-throughput analyses and genetic screening, but
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does not allow studying 3D cysts and the changes that occur in them. In addition,
cancer cells grown in 2D are even more dedifferentiated than cancer cells in 3D,
which can make them more resistant to apoptosis and thus reduce the usefulness of
data gathered from these experiments when considering cancer pathology in vivo
(Edmondson et al. 2014, Ravi et al. 2015, Ravi et al. 2017). Temperature-sensitive
Src MDCK cells allow the study of very early steps in malignant transformation
via activation of Src-oncogene, both in 2D and 3D, with a very simple way to
initiate the transformation process. Obviously, the benefits of this cell line for
studying other oncogenes are more limited.
In the present study, the early phases of cellular transformation were monitored.
In ts-Src MDCK cells grown at permissive temperature, E-cadherin localization
varied from lateral membranes to the cytoplasm, but since survivin expression was
upregulated as soon as v-Src was activated, the cells avoided apoptosis. When
grown on rigid cell culture plates, the survivin expression was upregulated
regardless of the Src activation status. In this environment the untransformed
MDCK cells seemed to have a “semi-differentiated” phenotype. The expression of
survivin might even function as an indicator of cellular differentiation.
Lumen filling is usually depicted to happen as transformed cells proliferate into
the lumen. In this study, ts-Src MDCK cell cysts in Matrigel were cultured first at
non-permissive temperature to form cysts with only a single layer of cells and a
lumen in the middle. Then, the cells were shifted to the permissive temperature and
the consequences of v-Src activation were monitored using a spinning disc confocal
microscope. The time-lapse experiment revealed that the lumen collapsed due to
the loss of tight junctions and subsequent loss of intraluminal hydrostatic pressure.
This occurred before any cell proliferation into the lumen could take place. The
current study shows that v-Src is unable to induce cell migration or downregulation
of E-cadherin and thus is not a very strong oncogene when compared to Ras or Myc.
This is supported by the earlier discovery that Src is unable to induce
transformation without STAT3, which is a signal transducer and a Src-substrate that
is also capable of activating genes regulating cell division and survival, including
survivin. Furthermore, cells where Src is activated without STAT3 suffer apoptosis
(Bromberg et al. 1998, Turkson et al. 1998, Geletu et al. 2013).
Behrens and co-workers (1993) noted that activation of v-Src induces
migratory behaviour in ts-Src MDCK cells when cultivated on top of a layer of
collagen at the permissive temperature. Throughout the current study, no invasive
behaviour or tubulogenesis was detected in ts-Src MDCK cells grown inside
Matrigel or the mixture of collagen I and Matrigel, possibly due to the stronger
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polarization cues and growth factors provided by Matrigel. Tubulogenesis has been
induced in untransformed MDCK cells grown inside collagen I by adding
hepatocyte growth factor (HGF; also known as scatter factor) to the cells. HGF
launches the EMT process of the cells, and cells take on a mesenchymal phenotype
and start scattering from the cyst and initiate tubulogenesis by sending long
extensions into the surrounding matrix (Montesano et al. 1991; O’Brien et al. 2004).
Since Src alone is not able to induce tubulogenesis, and thus EMT, in MDCK cells,
the ts-Src MDCK is not perfect for modelling the processes involved in EMT, but
does provide a good model for monitoring early events in malignant transformation.
Cancer cells typically produce more ROS than healthy cells, and are thus more
reliant on their antioxidant and free-radical scavenging capabilities (Prasad et al.
2017). This makes them more vulnerable to drugs that elevate the levels of
intracellular ROS, such as PL. MDCK cells transformed by v-Src were also more
susceptible to PL-induced apoptosis than untransformed cells. Pro-oxidants, like
PL, have been shown to inhibit several transcription factors, like STAT3, NF-κB,
Sp1, Sp3 and Sp4, and downregulate expression of genes involved in cell growth,
survival, inflammation and angiogenesis (Pathi et al. 2011, Han et al. 2014, Park
et al. 2014, Bharadwaj et al. 2015). PL-treatment was able to downregulate survivin
expression of the ts-Src MDCK cells as well. NAC, an antioxidant, rescued the
cells from PL-induced apoptosis, but was not able to upregulate survivin expression,
indicating some other mechanism than increased apoptosis resistance via survivin
to be behind the rescue. NAC-treatment caused reappearance of E-cadherin to the
lateral membranes of ts-Src MDCK cells, indicating an improvement in
differentiation state of the cells. Earlier, NAC-treatment has been shown to prevent
gamma-irradiation-induced disruption of TJs and AJs in mouse colon cells and to
improve the differentiation state of normal human epidermal keratinocytes (NHEK)
and Caco-2 colon cancer cells by elevating their E-cadherin expression (Shukla et
al. 2016). Taken together with the results from this study, it seems NAC protects
cancer cells from PL-induced apoptosis by improving their differentiation by
returning E-cadherin to the lateral membranes.
6.5 Comparison of the effects of 2D and 3D growth environment on
cell behaviour
The importance of cell-based assays to the advancement of medical sciences cannot
be understated. Cultured cells provide a simple, fast and cost-effective alternative
to animal testing, and, more importantly, allow the use of human cells without a
94
constant need for patient donors. Established cell lines provide homogenous sample
material and thus improve predictability and reproducibility. The majority of the
experiments made with cultured cells use monolayers of cells grown on hard plastic
in 2D, which differs remarkably as a growth environment from the in vivo
environment where cells are surrounded by other cells and ECM. This difference
between artificial and natural environment can result in data that does not
correspond to the situation in vivo and may thus be misleading or outright false or
artificial. This delays the advancement of science, and, especially in drug
development, causes significant extra costs in terms of both money and time. Some
of these costs could be avoided by using cell-based assays where the cells are closer
to their state in vivo than in 2D cultures. This way, ineffective or toxic compounds
could be eliminated already before clinical trials. 3D culture of cells presents a
method which occupies a space between 2D and living tissue, being still more cost-
effective and less labour-intensive than animal trials, but simultaneously providing
cells an environment more akin to their natural state (Edmondson et al. 2014).
Cells grown in 3D differ from 2D cells in several important ways, both
morphologically and physiologically. 3D environment provides spatial cues for
differentiation and also physically limit the movements of the cells, while cells in
monolayer have increased degrees of freedom for spreading and proliferation.
Physical constraints require cells with invasive tendencies to also be able to break
down the surrounding ECM. Thus, a hallmark of malignant transformation is the
ability to express enzymes that can break down ECM (Partanen et al. 2012).
Monolayer cells also receive homogenous amounts of nutrients and growth factors
from the provided medium, whilst cells in 3D cysts receive nutrients more unevenly
due to the possible penetration problems through the scaffold material. Cells
growing inside the cyst might also receive less nutrients and even less oxygen
compared to the outermost cells of the cyst. This creates a cyst with cells at various
stages, from proliferating to quiescent, apoptotic, hypoxic and even necrotic,
creating heterogeneity perceived in tissues. In addition, cells grown in 3D are able
to execute their internal programming, like form a lumen (Partanen et al. 2007,
Edmondson et al. 2014, Ravi et al. 2015). Importantly, cells grown in 3D are often
less sensitive to drugs than cells in 2D due to this heterogeneity in cell stages and
levels of differentiation (Edmondson et al. 2014, Ravi et al. 2017).
In the present study, the levels of gene expression of untransformed MDCK
cells grown in 2D were compared to cells grown in 3D. The microarray analysis
revealed 6474 gene expression changes when the environment was changed from
2D to 3D. One of the genes that underwent significant changes with the change in
95
environment was survivin, which was expressed in cells grown in 2D, but
downregulated in 2½D and to an even greater extent in 3D. Cells grown in 3D were
also more susceptible to apoptosis than cells grown in 2D when denied of E-
cadherin trans-interactions. These results again show that the 3D ECM environment
correlates better with normal tissue environment than the 2D plastic environment.
The present study also reveals drastic differences in gene expression and phenotype
that activation of a single oncogene, in this case v-Src, can have in 2D when
compared to 3D, effectively highlighting that results obtained from 2D cell-culture
experiments should be analysed with great care and conclusions pertaining to in
vivo situations drawn with utmost vigilance.
6.6 Use of time-lapse imaging of live cells as a tool for monitoring
cellular processes
In highly dynamic and complex structures like cells, a single time-point snapshot,
such as images of fixed cells or Western blots, can give an incomplete picture of
the whole process. Often, continuous observation of the process is needed. For this,
time-lapse imaging of live cells is an invaluable tool. Time-lapse can utilize
transmitted light, or dyes and/or fluorescent probes which stain a specific part of
the cell, or fusion proteins with an attached fluorescent tag produced by the cell
itself. Time-lapse imaging of live cells is always a balancing act between picture
quality and cell well-being, and many aspects have to be optimized for good quality
data. These include temperature, CO2 and O2 levels, pH, availability of nutrients
and minimizing evaporation and phototoxicity (Coutu & Schroeder 2013).
In the present study, time-lapse imaging of live cells has been used extensively.
A method was created for monitoring changes in lumen, cyst and cell volumes,
which can give useful information on the roles of transporter proteins and can be a
future platform for testing drugs affecting those transporters. Time-lapse imaging
was also used to monitor the sequence of events occurring when the v-Src oncogene
was activated in ts-Src MDCK cysts. Previous studies have shown that lumen
filling occurs once MDCK cells in a cyst undergo cellular transformation, and this
has been attributed to proliferation of cells into the luminal space. In the current
study, time-lapse imaging of ts-Src MDKC cysts revealed that, once v-Src is
activated, the lumen quickly collapses as the transformation weakens tight
junctions, and hydrostatic pressure inside the lumen is released. This occurs before
any proliferation due to oncogene activation could happen. These kinds of
sequential events cannot be monitored via “snapshots” taken with fixed cells.
96
97
7 Summary and conclusions
The aim of the current study was to understand the mechanisms behind the
maintenance of a differentiated phenotype and analyse the electrophysiological
parameters which regulate the transport capacity of kidney epithelial cells. Special
emphasis was placed on comparing the effects of different culture environments on
these processes. The first part of the study concentrated on analysing the effects of
drugs or the composition of the basal extracellular fluid on cell, cyst and lumen
volumes of untransformed MDCK cells using time lapse microscopy of living cells.
Lack of monovalent cations caused lumen expansion by inducing transepithelial
water flow towards the lumen, presumably via activation of cAMP signalling
pathway or voltage- and volume-sensitive chloride channels. Evacuation of water
from the lumen could be induced by exposing the cells to extracellular fluid lacking
chloride ions, or by depolarization of the cyst. These experiments showed that
MDCK cells were capable of both water secretion and reabsorption. More
importantly, the cells were able to perform these functions when exposed to
hyperpolarizing or depolarizing environment; a change in osmolality of basal fluid
was not required. Taken together, these results validate the status of MDCK cells
as a good basic model for studying the cell biological characters of secretory
epithelium or any tissue with secretory or absorptive properties. The method
presented here allows the analysis of the effects of external stimuli, be it changes
in ion balance or drugs affecting ion transport, on cell, cyst and lumen volumes
over short periods of time.
The second part of the study aimed to analyse the effects 2D and 3D culture
environments have on gene expression of untransformed MDCK and ts-Src MDCK
cells, and the extent of alterations in genetic profile that activation of a single
oncogene can induce. Microarray analysis revealed thousands of changes between
different conditions, but the most intriguing was the decreased expression of
survivin when switching from 2D environment to 3D. This downregulation of
survivin occurs in adult tissues as well, indicating that the cells grown in 3D are
better differentiated than 2D cells, and thus closer to the in vivo state. This is in line
with mitochondrial activity measurements, as the mitochondrial membrane
permeability of 3D-grown MDCK cells decreased swiftly after the first two days
in Matrigel, whilst the membrane permeability of ts-Src MDCK cells remained
higher for longer. This observation could explain the increased apoptosis in MDCK
cells compared to ts-Src MDCK cells, and indicate pro-survival and anti-apoptotic
function of v-Src.
98
Differences in survivin expression generated interest for further study of
factors controlling survivin expression and its significance to cell survival. MDCK
cells grown in 3D were devoid of survivin, but also did not suffer apoptosis, at least
as long as the cells remained in contact with the ECM, whereas MDCK cells grown
on 2D and ts-Src MDCK cells in all environments expressed survivin. If MDCK
cells were denied ECM contacts by growing them in suspension, they were more
susceptible to apoptosis than survivin-expressing ts-Src MDCK cells. Finally, if
cells were denied cell-cell junctions, cells lacking survivin suffered apoptosis even
though they had a proper ECM environment supporting cell-matrix contacts. Taken
together, these results highlighted the importance of cellular contacts to the cells:
MDCK cells needed ECM contacts to differentiate and cell-cell contacts to avoid
apoptosis. Since v-Src did not downregulate E-cadherin expression, it is unable to
bring about complete EMT. Thus, rather than being the initiator of cancer, v-Src
can be considered to be a mild oncogene that supports cancer cell survival.
99
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Original articles
I Capra JP, Eskelinen SM (2017) MDCK cells are capable of water secretion and reabsorption in response to changes in the ionic environment. Can J Physiol Pharmacol 95(1): 72-83.
II Töyli M, Rosberg-Kulha L, Capra J, Vuoristo J, Eskelinen S (2010) Different responses in transformation of MDCK cells in 2D and 3D culture by v-Src as revealed by microarray techniques, RT-PCR and functional assays. Lab Invest 90(6):915-928.
III Capra JP, Eskelinen SM (2017) Correlation between E-cadherin interactions, survivin expression, and apoptosis in MDCK and ts-Src MDCK cell culture models. Lab Invest 97(12):1453-1470.
Reprinted with permission from Canadian Science Publishing (I) and Springer
Nature (II and III)
Original publications are not included in the electronic version of the dissertation.
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