The effect of sub-lethal ALA-PDT on the cytoskeleton and adhesion of cultured human cancer cells

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Biochimica et Biophysica A

Regular paper

The effect of sub-lethal ALA-PDT on the cytoskeleton and adhesion of

cultured human cancer cells

Anatoly Uzdenskya,b,*, Elona Kolpakovaa, Asta Juzenienea, Petras Juzenasa, Johan Moana

aInstitute for Cancer Research, 0310 Montebello, Oslo, NorwaybRostov State University, 344090 Rostov-on-Don, Russia

Received 16 July 2004; received in revised form 9 November 2004; accepted 16 November 2004

Available online 15 December 2004

Abstract

5-Aminolevulinic acid (ALA), a precursor of the endogenous photosensitizer protoporphyrin IX, is used in the photodynamic therapy

(PDT) of cancer. Sub-lethal ALA-PDT (1-min irradiation with 370–450 nm blue light, 0.6 mW/cm2 after 2-h incubation with 1 mM ALA)

has been earlier shown to change cell morphology and to inhibit both trypsin-induced detachment of cultured cancer cells from the plastic

substrata and cell attachment to the bottom of the plastic well plates. In the present study, we found that such treatment of human

adenocarcinoma WiDr cells grown in dense colonies stimulated the formation of actin cortex between cells in the colonies and increased the

number of actin stress fibres in some, but not in all, cells. However, ALA-PDT did not change the microtubular cytoskeleton in these cells. A

similar treatment of glioblastoma D54Mg cells, which grow separately and communicate by protrusions, caused loss of fibrillar actin

structures in growth cones, retraction of protrusions, and surface blebbing in some cells. The application of the cytoskeleton inhibitors

cytochalasin D, colchicine or taxol showed that the inhibition of trypsin-induced detachment of photosensitized WiDr cells was related to

ALA-PDT-induced changes in actin and microtubular cytoskeleton. Some signal transduction processes are suggested to be involved in

ALA-PDT-induced changes in cytoskeleton, cell shape, and adhesion.

D 2004 Elsevier B.V. All rights reserved.

Keywords: 5-Aminolevulinic acid-photodynamic therapy; Cell adhesion; Actin; Tubulin

1. Introduction

The photodynamic effect is based on the destruction of

stained cells by highly toxic singlet oxygen that is generated

by photoexcited dye upon light exposure in the presence of

oxygen. This effect is used in cytology as a tool for selective

damage of intracellular organelles [1]. Photodynamic

therapy (PDT) is a promising anticancer treatment based

on light-induced destruction of photosensitized malignant

0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbagen.2004.11.011

Abbreviations: ALA, 5-aminolevulinic acid; CD, cytochalasin D;

EDTA, ethylenediamine tetraacetate; FCS, foetal calf serum; PBS,

phosphate buffered saline; PDT, photodynamic therapy; PpIX, protopor-

phyrin IX; TBS, phosphate buffered saline with milk powder and Triton

X-100

* Corresponding author. Institute for Neurocybernetics, Rostov State

University, 194/1 Stachky ave., NIINK, Rostov-on-Don, 344090, Russia.

Tel.: +7 8632 433577; fax: +7 8632 433588.

E-mail address: [email protected] (A. Uzdensky).

cells and tissues [2,3]. In the last years 5, aminolevulinic

acid (ALA), a biochemical precursor of the potent photo-

sensitizer protoporphyrin IX (PpIX), has been successfully

used for fluorescent diagnosis and photodynamic treatment

of cancer. ALA-derived PpIX accumulates in rapidly

proliferating cancer cells, thus providing selective destruc-

tion of tumours [2,4,5].

PDT has been found to influence cell adhesion [6,7] and

to reduce cancer metastases [8–10]. Cell adhesion is related

to cancer metastases [11–13]. Therefore, some interest has

been focused on the PDT effect on the adhesive properties

of cancer cells. Two aspects of cell adhesion are influenced

by PDT: (a) cell attachment to a plastic substratum

[6,14,15], or to a substratum coated with extracellular

matrix proteins [16,17], or to other cells [6,14]; and (b)

trypsin-induced detachment of cultured cells from a

substratum [6,7,18,19]. Such enzymatic cell detachment,

being a routine cell culture procedure, also characterizes cell

cta 1722 (2005) 43–50

A. Uzdensky et al. / Biochimica et Biophysica Acta 1722 (2005) 43–5044

adhesion. This process may serve as a model of cancer

metastasis because of the involvement of diverse proteolytic

enzymes in the remodelling of extracellular matrix, tumour

invasion, and the formation of metastases.

Cell adhesion to the substrata is mediated by integrins

and other cell adhesion molecules, which link the

extracellular matrix to the intracellular actin cytoskeleton

[20–22]. As recently shown [15,19], non-lethal ALA-PDT,

which inhibits the attachment of cultured cancer cells to

the plastic substratum, as well as trypsin-induced cell

detachment from the plastic surfaces, redistributes aVh3integrin in cultured cancer cells and impairs cell

morphology.

In order to elucidate further the mechanisms underlying

the effect of sub-lethal ALA-PDT treatment on the adhesive

properties of cultured cells [19], we studied the influence of

ALA-PDT on actin and tubulin cytoskeleton in two human

tumour-derived cell lines: adenocarcinoma WiDr and

glioblastoma D54Mg differing in shape and behaviour.

Fig. 1. Cell survival after ALA-PDT (1 mM, 2 h incubation, 1 min

irradiation). (A) WiDr adenocarcinoma cells; (B) D54Mg glioblastoma

cells. Values represent the mean ratio of the number of surviving

photosensitized cells (N) to the number of untreated cells (N0): N/N0FS.E.

2. Materials and methods

2.1. Chemicals and antibodies

Penicillin/streptomycin solution, l-glutamine, 5-amino-

levulinic acid, taxol, cytochalasin D (CD), colchicine, triton

X-100, dimethyl sulfoxide, and mouse anti-a-tubulin were

obtained from Sigma-Aldrich Norway AS (Oslo, Norway).

RPMI-1640 medium and foetal calf serum (FCS) were

obtained from GIBCO BRL, Life Technologies (Roskilde,

Denmark). Trypsin/EDTA solution (0.5 g/l porcine trypsin

and 0.2 g/l EDTAd 4 Na in Hank’s Balanced Salt Solution

with phenol red) was purchased from Sigma-Aldrich Norway

AS (Product number T 3924). It was stored frozen and

warmed to 37 8C prior to use. Rhodamine-labelled phalloidin

was obtained from Molecular Probes (Eugene, OR). As a

secondary antibody, we used Cy3-conjugated goat anti-

mouse IgG obtained from Jackson Immunoresearch Labo-

ratories Inc. (West Grove, PA). Antibodies were dissolved

ex tempera in TBS (20 mM phosphate buffered saline

(PBS) containing 5% (w/v) milk powder and 0.2% (v/v)

Triton X-100). 30 mM stock solution of ALA in medium

was prepared ex tempera before each experiment. It was

further diluted to a final concentration of 1 mM. CD,

colchicine, and taxol were added into the cell cultivation

wells 2 h before irradiation.

2.2. Cell cultivation

The human WiDr cell line was derived from a primary

adenocarcinoma of the rectosigmoid colon [23]. These

cells were maintained in exponential growth in RPMI

1640 medium with 10% FCS, 2 mM l-glutamine, and

100 units/ml penicillin and 100 Ag/ml streptomycin. Cells

were grown in Falcon plastic flasks at 37 8C in a

humidified 5% CO2 atmosphere and were subcultured

twice a week using trypsin/EDTA solution. Glioblastoma

cells D54Mg [24] were grown in Dulbecco’ modified

Eagle medium with 10% FCS, 2 mM l-glutamine, 100

units/ml penicillin, 100 Ag/ml streptomycin, and the

prescribed dosage of non-essential amino acids (NEAA,

Bio Whitacker).

2.3. Cell survival assay

In order to study ALA phototoxicity, 8�104 cells were

inoculated in 12-well tissue culture plates (Nunc, Roskilde,

Denmark) containing 2 ml of medium. After 2–4 days, cells

were incubated for 2 h with 1 mM ALA at 37 8C in the

darkness. Then, in the darkened room, the dye-containing

medium was replaced by the fresh dye-free medium and cells

were exposed to the light. After light exposure, the cells were

incubated for 24 h at 37 8C. Then, they were rinsed with

0.9% NaCl, fixed for 20 min with 96% ethanol, stained for

20 min with methylene blue, and washed. The absorbance of

the dried wells with the stained cells was measured at 630

nm (methylene blue absorption band) using a Perkin-Elmer

A. Uzdensky et al. / Biochimica et Biophysica Acta 1722 (2005) 43–50 45

LS-50 B spectrofluorimeter equipped with a well plate

reader accessory. 3–4 wells were studied for each light

exposure. Relative survival fractions were calculated from:

N=N0 ¼ logIb � logIð Þ= logIb � logIoð Þ;

where I, Io, and Ib are the light intensities passed through

experimental, non-irradiated control, and blank wells,

respectively.

2.4. Cell detachment assay

In the cell detachment experiment, WiDr cells were

subcultured in plastic 12- or 24-well plates (Nunc, Roskilde,

Denmark, or Costar, Corning Inc., Corning, NY) for 3–4

Fig. 2. Actin distribution in WiDr (A, B) and D54Mg (C–F) cells. In WiDr cells, ac

the stress fibres (A), but after PDT, an actin cortex is also seen at cell surfaces o

concentrated in the cellular cortex and in the growth cones of the cell protrusions (

F). (A–D) confocal images; (E and F) conventional fluorescence microscopy. Ba

days at 37 8C in a humidified 5% CO2 atmosphere until cell

layers were almost confluent. Then, 1 mM ALA was added

for 2 h and the samples were exposed to light. After light

exposure, the medium in the wells was removed and 0.3 ml

of trypsin/EDTA solution (heated to 37 8C) was added to

each well. The well plates were incubated for 5–9 min at 37

8C. Then, 2 ml of FCS-containing medium was added into

each well to stop the proteolytic action of trypsin, and 0.5

ml samples were taken. The number of detached cells was

counted using a Glasstic Slide 10 with grids (Hycor

Biomedical Inc., Garden Grove, CA). The optimal time of

trypsin treatment, at which the biggest difference in cell

detachment between untreated and photosensitized samples

was observed (too few cells were detached after too short

tin localizes in the cellular cortex (mainly on the outer colony border) and in

f cell to cell contact inside a colony (B, arrows). In D54Mg cells, actin is

C and E). ALA-PDT decreases the actin content in the growth cones (D and

rs on Panels (A) and (B): 20 Am; on Panels (C) and (F): 30 Am.

Fig. 3. Modification of trypsin detachment of ALA photosensitized WiDr

cells with cytochalasin D (A), colchicine (B), or taxol (C). The

trypsinization efficiency N/N0 (see the legend for Fig. 1) is plotted. ALA

concentration: 1 mM; time of cells incubation with ALA: 2 h. Values

represent the mean N/N0FS.E.

A. Uzdensky et al. / Biochimica et Biophysica Acta 1722 (2005) 43–5046

trypsin exposure but too long exposure caused detachment

of all cells and no difference was observed), varied between

the experiments. It depended on the experimental condi-

tions: temperature, duration of experimental manipulations

leading to cooling of samples, etc. In order to reduce such

variations, the effect of photosensitization was quantified as

the ratio N/N0, where N and N0 are the numbers of the

detached cells in the photosensitized and control samples,

respectively. The duration of trypsinization was determined

visually in each experiment until the detachment of the

majority of control cells.

2.5. Light exposure

Cells incubated with ALA in well plates were exposed to

light from a bank of fluorescent tubes (Model 3026, Applied

Photophysics, London, UK) with an irradiance of 0.7 mW/

cm2 at the position of the cells. The emission of this lamp

was mainly in the wavelength region 370–450 nm, with a

peak at 405 nm, which is close to the maximum of the Soret

band of PpIX in cells.

2.6. Immunofluorescence

For the microscopic study, approximately 104 cells were

seeded into plastic 6-well plates (Nunc, Roskilde, Denmark,

or Costar, Corning Inc., Corning, NY), in which 2–3 round

glass cover slips (10 mm diameter) were placed and allowed

to grow for 2–3 days before photosensitization and staining.

Then, the samples were incubated with 1 mM ALA

dissolved in FCS-containing medium for 2 h. They were

exposed to light for 1 min as described above and fixed for

20 min in 3% paraformaldehyde at room temperature. The

treated cells were washed with PBS, incubated with PBS

containing 30 mM NH4Cl for 10 min and again washed

twice for 10 min in PBS. For actin visualization, the cells

were stained for 20 min with rhodamine-labelled phalloidin

at room temperature. For the visualization of microtubules,

the cells were labelled for 20 min with anti-a-tubulin

dissolved in TBS as described above, washed twice with

PBS for 10 min, and then labelled for 20 min at a room

temperature with secondary goat anti-mouse antibodies

diluted in TBS.

After staining, the cover slips were mounted in Mowiol

(Calbiochem, San Diego, CA). Fluorescence microscopy

was performed using the Zeiss Axioplan microscope (Karl

Zeiss, FRG) equipped with epifluorescence and an oil

immersion objective of 63�. A HBO/100 W mercury lamp

was used for fluorescence excitation. Fluorescence was

observed with an excitation filter 450–490 nm, a dichroic

beam splitter FT 510, and a long pass emission filter N630

nm. Fluorescence images were acquired by a CCD-camera

(Princeton Instruments, Princeton, USA) driven by the

software AnalySis (Soft imaging system GmbH, Munster,

Germany). A Leica TCSNT (Wetzlar, Germany) microscope

was used for confocal microscopy. Images were acquired

with 63� (for glioblastoma cells) or 100� (for adenocarci-

noma cells) objectives and captured with the resolution of

1024�1024 pixels. Montages of images were prepared by

A. Uzdensky et al. / Biochimica et Biophysica Acta 1722 (2005) 43–50 47

using the Adobe PhotoShop 4.0 software (Adobe, Mountain

View, CA).

2.7. Statistics

Standard statistical methods based on Student’s criterion

were used. Results are expressed as meanFS.E.

3. Results

3.1. Phototoxicity of ALA-PDT

The survival curves for the photosensitization of

adenocarcinoma WiDr cells and glioblastoma D54Mg cells

incubated for 2 h with 1 mM ALA are shown in Fig. 1. The

fluences giving 90% survival were about 150 and 110 mJ/

cm2 (light exposures 3.7 and 2.7 min, respectively). 1-min

light exposure killed less than 3% of cells.

3.2. ALA-PDT effect on the intracellular actin cytoskeleton

In tightly packed colonies of untreated WiDr cells,

intracellular actin is manifested as stress fibres and cortex

filaments localized at the cellular periphery (Fig. 2A). In a

colony, the actin cortex was localized mainly at the colony

borders. Almost no cortex-specific staining was observed

inside colonies in the proximity of intercellular contacts

(Fig. 2A). 5–10 min after ALA-PDT (1 min light exposure),

the actin cortex appeared in intercellular regions (Fig. 2B).

Fig. 4. Fluorescence micrographs of the a-tubulin distribution in WiDr (A and B)

tubulin distribution in these cell lines. Arrow shows blebbing in the D54Mg cell.

This may indicate a reduction of cell–cell interactions. At

the same time, the number of actin stress fibres increased in

approximately half of photosensitised cells (Fig. 2B) as

compared to that in untreated controls (Fig. 2A).

Glioblastoma cells grew less densely and approached

each other by long protrusions (Fig. 2C). In untreated

control cells, the growth cones in the protrusions were

especially abundant with fibrillar actin structures. The cells

also contained filopodia, actin spots, a few stress fibres, and

an actin cortex (Fig. 2C,E). After ALA-PDT, the intra-

cellular actin was partly redistributed. Its presence in the

growth cones was significantly reduced and no fibrillar

structures were evident (Fig. 2D,F). Actin appeared to be

present in other surface areas (Fig. 2D,F).

Sub-lethal ALA-PDT (1 mM ALA, 2-h incubation; 1-

min irradiation) has been shown to inhibit the trypsin-

mediated detachment of WiDr and D54Mg cells from a

plastic substratum [19]. The presence of 0.5–5 Ag/ml

cytochalasin D (an amount known to disrupt actin micro-

filaments [25]) inhibited trypsin-mediated detachment of

WiDr cells from the plastic substratum in the same manner

as ALA-PDT did (Fig. 3A). In combination, CD and ALA-

PDT had an additive effect. Therefore, the reorganization of

the actin cytoskeleton may be involved in trypsin-induced

cell detachment.

3.3. ALA-PDT effect on the microtubular cytoskeleton

In control, WiDr adenocarcinoma cells microtubules

labelled with a-tubulin-specific antibodies formed a net-

and D54Mg (C and D) cells. ALA-PDT (B and D) does not change the a-

Bars on Panels (A) and (B): 15 Am; on Panels (C) and (D): 30 Am.

A. Uzdensky et al. / Biochimica et Biophysica Acta 1722 (2005) 43–5048

work around the nucleus (Fig. 4A). ALA-PDT did not

induce any changes in this network (Fig. 4B).

In the flattened D54Mg glioblastoma cells, the micro-

tubule network was well developed (Fig. 4C). After ALA-

PDT, some cells retracted their lamellipodia and formed

extracellular blebs containing tubulin (Fig. 4D). In these

cells, the microtubular cytoskeleton was, to some extent,

disaggregated so that the tubulin was concentrated in the

perinuclear region (Fig. 4D). Such cells seemed to be

loosely attached to the plastic substratum.

Colchicine is known to depolymerise microtubules [26].

In our experiments, 1 AM colchicine did not significantly

influence the trypsinization efficiency by itself. However, 1–

5 AM colchicine increased the effect of ALA-PDT on

trypsin detachment of WiDr cells (Fig. 3B) independently of

concentration in this range. In contrast to colchicine, taxol

stabilizes microtubules [26]. Together with ALA-PDT, 1

AM taxol decreased the trypsinization efficiency in an

additive manner (Fig. 3C). Therefore, despite the absence of

visible morphological changes in tubulin organization in

WiDr cells, the microtubules may be involved in trypsin-

induced cell detachment.

4. Discussion

Our present and previous data [15,19] show that sub-

lethal ALA-PDT inhibits both trypsin-mediated detachment

of WiDr or D54Mg cells from plastic substrata and

attachment of suspended WiDr cells to plastic. It is known

that cell adhesion and growth depend on the chemical nature

of the substratum, which may vary from one manufacturer

to another [27]. Nevertheless, we did not observe any

noticeable difference between ALA-PDT effect on trypsin-

induced detachment of WiDr cells from Nunc or Costar

well-plates. Therefore, the difference in surface chemistry

was not of significant importance.

The presence of serum in medium is necessary for

adhesion and growth of diverse mammalian cells in culture.

Vitronectin and fibronectin are the major serum proteins

involved in cell adhesion. These proteins adsorbed on the

plastic surface are recognized by the surface receptor

proteins such as integrins [20–22]. The major integrin in

focal contacts is aVh3 [21,28]. It binds specifically to

vitronectin [29]. Redistribution of aVh3 integrin in WiDr

cells after sub-lethal ALA-PDT treatment has been recently

demonstrated [15]. Trypsin-mediated cell detachment is

promoted by the cleavage of extracellular integrin fragments

interacting with the adsorbed serum proteins.

Photodynamic inhibition of trypsin-mediated cell detach-

ment has been suggested to be due to protein cross-linking

caused either by direct photosensitization of the cell

membrane [6], or by the photosensitization of tissue

transglutaminase [18]. We suggest that PDT-induced inhib-

ition of trypsin detachment may be due to mutual integrin

cross-linking within the pre-existing adhesion clusters and

cross-linking of integrins with other proteins in the focal

contacts.

ALA-PDT-induced changes in cell adhesion were

accompanied with the remodelling of the actin cytoskeleton.

Actin fibres are formed after integrin binding to extracellular

ligands. The following integrin clustering leads to the

formation of large actin bundles (stress fibres) at the focal

contacts [21,22]. PDT-induced increase of the number of

stress fibres in WiDr cells may indicate a strengthening of

the cell-substratum contacts. This may be related to photo-

inhibition of cell detachment. We also observed the

formation of the actin cortex at the inner borders of WiDr

cells growing in contact with each other in colonies. This

may indicate the weakening of intercellular contacts and

isolation of cells. Such a pronounced reorganization of the

actin cytoskeleton, including the formation of stress fibres

and remodelling of the actin cortex, may be governed by the

cellular signal transduction system rather than by local

processes triggered by direct PDT targeting of the cell

membrane where integrin-mediated adhesion contacts are

formed. GTPases as Rho and Rac, responsible for the

formation of stress fibres and lamellipodia, respectively, as

well as protein kinase C and phosphatydylinositol 3-kinase,

playing key roles in adhesion signalling [30–33], may

participate in these processes. Their role in photoinduced

cytoskeleton remodelling should be studied in the future.

The microtubules are other cytoskeleton components

responsible for the cell shape. They do not directly link

integrins to actin fibres. Their role in cell adhesion is

assumed to be auxiliary and regulatory [32,33]. For

instance, microtubules growing towards focal adhesions

may deliver the brelaxing factorQ that stimulates the

dissolving of the focal adhesions at the cell periphery

[34]. The disruption of microtubules stimulates the for-

mation of stress fibres and the assembly of adhesion

plaques and triggers the integrin-dependent signalling

cascade [33,35,36]. Microtubules have shown sensitivity

to PDT [37,38]. However, in our experiments, sub-lethal

ALA-PDT did not visibly change the microtubular cyto-

skeleton in WiDr cells. Nevertheless, the experiments with

microtubule modulators indicated their involvement in

ALA-PDT-induced inhibition of trypsin-mediated cell

detachment. It was of interest that both colchicine

(preventing microtubule assembly [26]) and taxol (stabiliz-

ing microtubules [26]) enhanced the ALA-PDT-induced

increase of cell resistance to trypsinization.

Different morphological changes and reorganization of

actin cytoskeleton were observed in ALA-PDT-treated

glioblastoma cells D54Mg. These cells do not form dense

colonies as WiDr cells do and establish intercellular contacts

by cytoplasmic protrusions. After sub-lethal ALA-PDT,

glioblastoma cells lost the fibrillar actin structures in the

growth cones of the protrusions, indicating adhesion

weakening at these sites. Simultaneously, the actin cortex

enlarged in the central cellular regions (Fig. 2D,F). A certain

fraction of the photosensitized D54Mg cells demonstrated

A. Uzdensky et al. / Biochimica et Biophysica Acta 1722 (2005) 43–50 49

surface blebbing and segregation of tubulin-containing

blebs. Some blebs were formed from the retracted protru-

sions in the course of cytoskeleton reorganization and cell

rounding. Blebbing of the cell surface is often accompanied

by the disruption of microtubules [37,38] and the reorgan-

ization of actin microfilaments [39]. Sub-lethal ALA-PDT,

which induced cell blebbing in our experiments, inhibited

trypsin detachment of the D54Mg cells. The hallmarks of

adhesion reduction (loss of fibrillar actin structures in distal

ends of cell protrusions and surface blebbing) and adhesion

strengthening (inhibition of detachment) are not in agree-

ment, and a more detailed study is necessary to resolve this

contradiction.

The observed photodynamic inhibition of cell attachment

and enzymatic detachment are in agreement with the data on

the correlation between attachment rate and organ-coloniz-

ing capacity of cancer cells [40], with the published

observations that PDT decreases the metastatic potential of

surviving cancer cells [10,41], and with reported reduction

of tumour metastases after PDT compared to that after

surgery [8,9]. This gives PDT an advantage compared to

other treatment modalities.

In conclusion, sub-lethal ALA-PDT (1-min blue light

exposure after 2 h cells incubation with 1 mM ALA),

which inhibits the attachment of WiDr cells to plastic and

trypsin-induced cell detachment from plastic substrata,

caused the reorganization of the actin cytoskeleton. The

effects differed in the two cell lines. In WiDr adenocarci-

noma cells, which form densely packed colonies, ALA-

PDT-induced formation of an actin cortex inside the

colonies and increased the number of actin stress fibres in

some cells. The microtubule cytoskeleton appeared to be

unchanged. Similar treatment of glioblastoma D54Mg

cells, which do not form colonies and communicate via

protrusions, caused the loss of fibrillar actin structures in

the growth cones, retraction of protrusions, and surface

blebbing in some, but not in all cells. Cytochalasin D,

colchicine, and taxol influenced ALA-PDT effect on

trypsin-induced detachment of WiDr cells showing the

involvement of actin and microtubule cytoskeleton in this

process. Some signal transduction processes are suggested

to be involved in ALA-PDT-induced changes in cytoske-

leton, cell shape, and adhesion.

Acknowledgements

The present work was supported by the Research

Foundation of the Norwegian Radium Hospital (RF) and

by the Norwegian Cancer Society (DNK).

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