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Cancer Biology and Signal Transduction

The Tyrosine Kinase Inhibitor Imatinib AugmentsExtracellular Fluid Exchange and ReducesAverage Collagen Fibril Diameter in ExperimentalCarcinomaP. Olof Olsson1, Renata Gustafsson1, Ren�e in 't Zandt2,Tomas Friman3, Marco Maccarana4,Emil Tykesson4, Åke Oldberg4, Kristofer Rubin1, and Sebastian Kalamajski5

Abstract

A typical obstacle to cancer therapy is the limited distribution oflow molecular weight anticancer drugs within the carcinomatissue. In experimental carcinoma, imatinib (STI571) increasesefficacy of synchronized chemotherapy, reduces tumor interstitialfluid pressure, and increases interstitial fluid volume. STI571 alsoincreases the water-perfusable fraction inmetastases from humancolorectal adenocarcinomas. Because the mechanism(s) behindthese effects have not been fully elucidated, we investigated thehypothesis that STI571 alters specific properties of the stromalextracellular matrix. We analyzed STI571-treated human colorec-tal KAT-4/HT-29 experimental carcinomas, known to have a well-developed stromal compartment, for solute exchange and gly-cosaminoglycan content, as well as collagen content, structure,and synthesis. MRI of STI571-treated KAT-4/HT-29 experimental

carcinomas showed a significantly increased efficacy in dynamicexchanges of solutes between tumor interstitium and blood. Thiseffect was paralleled by a distinct change of the stromal collagennetwork architecture, manifested by a decreased average collagenfibril diameter, and increased collagen turnover. The glycosami-noglycan content was unchanged. Furthermore, the apparenteffects on the stromal cellular composition were limited to areduction in anNG2-positive stromal cell population. The currentdata support thehypothesis that the collagennetwork architectureinfluences the dynamic exchanges of solutes between blood andcarcinoma tissue. It is conceivable that STI571 reprograms distinctnonvascular stromal cells to produce a looser extracellular matrix,ultimately improving transport characteristics for traditional che-motherapeutic agents. Mol Cancer Ther; 1–10. �2016 AACR.

IntroductionCarcinomas are characterized by a deranged vasculature with

aberrant blood flow and leaky blood vessels, leucocyte infiltrates,and a dense and often a fibrotic stromal extracellular matrix(ECM). In addition, a pathologically elevated interstitial fluidpressure (IFP) characterizes both human and experimental car-cinomas. These stromal properties contribute to the limiteddistribution of blood-borne drugs within carcinoma tissue (1–3).

Several agents that lower IFP in experimental carcinomasincrease the interstitial fluid volume in parallel to an increase ofthe uptake and efficacy of small molecular weight chemothera-peutic agents (1, 4–6). Previous studies have revealed a correla-

tion between IFP and the properties of the collagen network instroma such as collagen fibril density, fibril structure, or networkarchitecture in experimental carcinomas (7–11). In normal looseconnective tissues, IFP is actively controlled by connective tissuecells that exert a tension on the collagen/microfibrillar network,thereby restraining the swelling of the underhydrated glycosami-noglycan (GAG)/proteoglycan ground substance (12). Increasesin GAG/proteoglycan ground substance will increase IFP provid-ed there is a collagen network that resists the tendency towardincreased swelling. Such an example was demonstrated in pan-creatic ductal adenocarcinomas (PDAC) where high concentra-tions of both collagen type I and the GAG hyaluronan correlatedwith a particularly high IFP and solid stress in the stroma (13). In atherapeutic experiment, enzymatic degradation of hyaluronanlowered IFP, restored blood flow, and improved efficacy ofgemcitabine (13). Another study with an experimental model ofPDAC showed that inhibition of the Hedgehog cellular signalingpathway reduced stromal fibrosis, reduced collagen content, andincreased blood flow and efficacy of gemcitabine (14).

STI571 is a low-molecular weight tyrosine kinase inhibitororiginally developed for the treatment of chronic myeloid leuke-mia by blocking the kinase activity of the pathogenic Bcr-Ablfusion protein (15, 16). STI571 also selectively inhibits theintrinsic receptor tyrosine kinases in discoidin domain receptors,c-Kit, colony-stimulating factor-1 receptor (CSF-1R), and PDGFreceptor-a and -b (16, 17). STI571 lowers tumor IFP in severalmodels of experimental carcinoma (including the KAT-4/HT-29model) without affecting tumor growth (18). Furthermore,

1Department of Laboratory Medicine, Translational Cancer Research,Lund University, Lund, Sweden. 2Bioimaging Center, Lund University,Lund, Sweden. 3Department of Pharmaceutical Biosciences, UppsalaUniversity, Uppsala, Sweden. 4Department of Experimental MedicalSciences, Matrixbiology, LundUniversity, Lund, Sweden. 5Departmentof Medical Biochemistry and Microbiology, SciLife Laboratories,Uppsala University, Uppsala, Sweden.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Kristofer Rubin, Lund University, Medicon Village, 406,Scheelev€agen 2, Lund SE-223 63, Sweden. Phone: 467-0425-0364; Fax: 461-8471-4673; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-16-0026

�2016 American Association for Cancer Research.

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on March 7, 2020. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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STI571 treatment of the ECM-richKAT-4/HT-29 carcinomamodelincreases the extracellular fluid (ECV) volume from 34% to 42%of total tissue water (TTW), without significantly altering plasmavolume (about 0.9% of the TTW; ref. 19). TTW is the sum ofintracellular, ECV, and plasma. Because the ECV is largely con-tained within the interstitial (stromal) ECM-compartment (12),the data suggest that treatment with STI571 increases the volumeof the ECM in the carcinoma and that this compartment amountsto about 25% of the carcinoma volume. These preclinical dataagree with the findings that treatment of patients with STI571increases the water-perfusable tumor fraction in metastases fromcolorectal adenocarcinoma (20). In the current study, we usedMRI to investigate how STI571 affects the uptake of low-molec-ular weight contrast agents. We also analyzed the stromal ECM toinvestigate potential ECM-related mechanisms that could influ-ence the solute exchange between blood and tumor tissue.

Materials and MethodsTumor model

KAT-4 (ATCC) carcinoma cells were cultured in DMEM orRPMI1640 (Invitrogen) supplemented with 10 % FBS (Saveen-Werner) and penicillin þ streptomycin (SVA). Cells were har-vested with trypsin (SVA) and washed two times in PBS (SVA)beforemaking the final cell suspension in PBS. Fiftymicroliters ofcell suspension containing 2 � 106 KAT-4 was injected subcuta-neously into the left flank of 8-week-old female Fox Chase SCIDmice (CB.17, M&B). Themice were housed at the animal facilitiesof Uppsala and Lund Biomedical Centers. All manipulations ofmice were performed under isoflurane (Abbot Scandinavia) anes-thesia and the experiments were approved by the Ethical com-mittees for animal experiments in Lund and in Uppsala, Sweden.The origin of KAT-4 has been debated—first described to beoriginating from human thyroid carcinoma (21), KAT-4 was latershown to resemble HT-29 human colon carcinoma (22). KAT-4cells, whose identity was investigated by short tandem repeat locianalyses (IdentiCell), were used. KAT-4, as expected (22),matched with HT-29 although alleles D13S317:12 and TH01:9were absent. The cell line has not undergone epithelial-to-mes-enchymal transition (23).

Treatment of animalsTumor sizewasmeasured externally with a caliper. Tumors that

had reached volumes between 0.2 and 0.4 mL were randomizedinto two groups. A treatment group received STI571 (kindlydonated by Novartis) 100mg/kg/d dissolved in PBS and a vehiclegroup received only PBS. In both groups the substances wereadministered orally via gavage for 4 consecutive days.

MRIAnimals were anesthetized with 3.5% isoflurane in mixture of

200 mL/minute oxygen and 200 mL/minute nitrous oxide andmaintained at 1.5% to 2% isoflurane. Inside the magnet, therespiratory rate of the animal was monitored and the bodytemperature was maintained at 37�C using warm air (SA Instru-ments Inc.). Contrast-enhanced MR imaging was performed witha 9.4 T MR scanner (Agilent Inc.) equipped with a 6-cm innerdiameter gradient system having amaximum gradient strength of1,000 mT/m. Reference scans were acquired to ensure the correctanimal position inside the magnet and a total of 23 slices weredefinedwith a gapof 0.25mmto image thewhole tumor area. The

animalwas takenout of themagnet and100mLofOmniscan (Gd-DTPA-BMA; GE Healthcare) was injected intraperitoneally withcare takennot tomove thepositionof the animal.With the animalplaced back in the magnet, contrast-enhanced MR images wereacquired using T1-weighted gradient echo sequence (TR: 100 ms,TE: 2ms, number of averages: 8, resolution inplane 117mm�117mm, slice thickness 0.5 mm). Total scan time was 3 minutes andscans were started with 10-minute interval. Animals were scanneduntil a clear decrease of the contrast enhancement could beobserved in the connective tissue. Typically, the animals werescanned 60 to 80 minutes after injection. Mice were euthanizedafter the measurements.

MRI data analysisAnalysis was carried out using in-house written Matlab scripts

(MathWorks). The first images after injection when no contrastenhancement could be detected were used as reference. Thecarcinoma tissue was manually delineated on all slices andmarked as region of interest (ROI). The tumor volume wascalculated by multiplying the total amount of pixels in the ROIin all slices. For all pixels originating from the tumor tissue, thechange of signal was plotted as a function of time. To reduceprocessing time, images were down-sampled with a factor of 2before curve fitting. The data were filtered using principal com-ponent analysis (PCA; ref. 24) in which the 2 first components,representingmore than95%of the variationof thedata,wereusedto reconstruct the data. Quantitative model fitting of this kind ofdata exists (25), but in this study amore qualitative description ofthe data was chosen. We considered only the pixels with a signalchange greater than three SDs of the noise in the image after PCA.Thus, around 10% of the total number of pixels was not consid-ereddue to lack of any significant signal enhancement. The changein the signal of a pixel inside the tumor was fitted to three modelline shapes; a linear enhancement, a monoexponential enhance-ment (wash-in), and a biexponential enhancement (wash-in/wash-out). The most appropriate curve describing the pixelenhancement was chosen from the best correlation coefficientR2 calculated after the fit.Maximumenhancement, time constantsfor wash-in/wash-out, tumor volume and the distribution of thetype of signal enhancement are reported after the analyses. Resultsare reported in histograms for all pixels in the STI571-treated andthe control group. To compare the distribution for every graphweperformed a Mann–Whitney test (nonparametric, unknowndistribution).

Hydroxyproline determinationsKAT-4/HT-29 carcinomaswere hydrolyzed in 6mol/LHCl for 4

hours at 120�C at a pressure of 2 atmospheres. Hydroxyprolinecontent in the hydrolysates was determined essentially asdescribed previously(26).

Disaccharide fingerprintKAT-4/HT-29 carcinoma tissues from STI571-treated and con-

trol tumors were lyophilized. GAG preparation, lyase treatment,fluorescence disaccharide labeling and separationwere performedaccording to ref. 27. Briefly, tumors were protease and DNAasedigested, and GAGs were purified on anion-exchange chroma-tography. GAGs were roughly estimated by the carbazol method.Then, 500 ng of GAGs was subjected to chondroitinase ABC(Sigma)degradationor degradationwith amixture of heparinases

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(in house preparation, purified from E. coli, stably singularlytransfected with the pET-15b vector containing heparinase I, orvector pET-19b containing heparinase II or III, as provided byProf. Jian Liu (University of North Carolina, Chapel Hill, NC).Fluorophore-labeling of the resulting disaccharides was per-formed by 2-aminoacridone (AMAC, Sigma). Pre-columnAMAC-labeled disaccharides were analyzed with HPLC-fluores-cence as described previously (27). Quantification was done bycomparison to knownweight ofmock-treated standard disacchar-ides (Iduron).

Electron microscopyFor transmission electron microscopy (TEM) analysis, KAT-4/

HT-29 carcinoma from control and STI571-treated mice werefixed in 0.15 mol/L sodium cacodylate–buffered 2.5% glutaral-dehyde, post-fixed in 0.15mol/L sodium cacodylate-buffered 1%osmium tetraoxide, dehydrated in graded ethanol series, impreg-nated in acetone, and embedded in epoxy resin. Ultra-thin sec-tions were examined in a Philips CM-10 electron microscope(Philips) and the micrographs were quantified with ImageJ soft-ware (NIH, Bethesda, MD). For scanning EM analysis, KAT-4tumors from PBS and STI571-treated mice were processed byalkali maceration as described previously (28) and analyzed in aPhilips 515 electron microscope.

Ex vivo collagen synthesisTumors were excised and cut into 300- to 700-mm slices with a

vibratome (Bannockburn). Samples were weighed (average wetweight 169� 20mg, of all 18 samples) and kept on ice until theywere put in collagen synthesis media (MEM proline-glycine-free(Invitrogen) þ 0.284 mmol/L ascorbic acid (Sigma) þ 0.001mmol/L FeSO4 (Sigma) þ 1 mg/mL BSA (Sigma) þ 0.01mmol/L GM6001 (matrix metalloproteinase inhibitor, Chemi-con) together with 50 mCi/mL of 14C-labeled Proline and Glycine(PerkinElmer) at a Proline to Glycine ratio of 1:6. The slices wereincubated at 37�C in a humidified incubator for 6 hours. There-after, the sampleswere dried in a vacuum centrifuge. Collagenwassolubilized in 0.5mol/LHAc containing 3mg/mLpepsin (Sigma)pH 2.5 for 24 hours at 4�C. The samples were centrifuged 17,000� g for 30 minutes at 4�C and supernatants collected. The super-natants were mixed with 2.8 mol/L NaCl to obtain a finalconcentration of 0.7 mol/L NaCl, in which native triple helicalcollagen was precipitated. Precipitated collagen was washed twotimes in 0.5 mol/L HAc containing 0.7 mol/L NaCl. Dry pelletswere stored frozen, pending further analysis. Each pellet wassolubilized in equal amounts of 2� sample buffer [0.2 mol/LTris-HCl pH 8.8 (Sigma), 18% Glycerol (Merck), 0.01 % brom-phenol blue (Merck), 4% SDS (Merck), 10% 2-Mercaptoethanol(BDH)] and heated at 100�C for 10 minutes. The samples wererun on 6 % SDS-PAGE gels after which the gels were stained byCoomassie blue and dried. The dried gels were exposed to animaging plate (Fuji) and developed in BAS-2500 Bioimaginganalyzer (Fuji). Band intensities were normalized to the individ-ual wet tissue weight.

RT-PCR and real-time qPCRTotal RNA was extracted from tumors (five biological repli-

cates) using TRIzol reagent (Thermo Fisher). Five-hundred nano-grams RNA was used for reversed transcription using SuperscriptVILO (Thermo Fisher). Real-time qPCR was performed with

TaqMan probes listed in Supplementary Table, using an AppliedBiosystems 7300 detection system. Gene expression was normal-ized to Actb transcript.

ImmunofluorescenceTumors were snap-frozen in isopentane at �80�C, embedded

in OCT (Sakura), and sectioned. Frozen 6-mm sections were fixedin 4% paraformaldehyde (Merck) or acetone (Sigma), blocked in40% serum [20% goat serum (Serotec) and 20% horse serum(SVA, Sweden) or pig serum from Chemicon (Temacula)] andincubated with the following primary antibodies: monoclonal ratanti-mouse CD31 clones Mec 13.3 and 390 (BD Biosciences),monoclonal rat anti-mouse F4/80 (Serotec), polyclonal rabbitanti-mouse NG2 (Chemicon), mouse anti-a-smooth muscleactin (a-SMA) clone 1A4 FITC-conjugated (Sigma), and ratanti-Reticular Fibroblast Marker (RFM; Cederlane Laboratories).The following secondary antibodies were used: FITC-conjugatedgoat anti-rat IgG and Texas Red-conjugated goat anti-rabbit (Vec-tor Laboratories). Exchange of primary and secondary antibodiesfor mouse or rabbit normal IgG or PBS was performed in allcombinations necessary to establish the specificity of the observedstaining. DAPI (40,6-diamidino-2-phenylindole) from Sigma wasused for nuclear staining. Images were retrieved with a NikonEclipse 90i microscope (Nikon Instruments). Image analysis andpixel quantification were performed with Photoshop (Adobe)and ImageJ (NIH, Bethesda,MD) software, respectively. Themeanpercentage of pixels per image was calculated from several imagesper investigated tumor. Colocalization data is presented as thepercentage of CD31-positive pixels that colocalized with NG2,and vice versa.

ResultsSTI571 treatment increased exchange between plasma andinterstitial fluid in KAT-4/HT-29 experimental carcinoma

MRI was used to determine wash-in and wash-out character-istics in KAT-4/HT-29 carcinomas. The average volumes of theinvestigated KAT-4/HT-29 carcinomas as determined byMRIwerenot different between the STI571-treated (0.14� 0.12mL, n¼ 9)and the control group (0.15� 0.08mL, n¼ 8). The distribution ofthe low-molecular weight MRI contrast agent Omniscan withinthe extracellular fluid of experimental carcinomas was followedduring a 60- to 80-minute time course per tumor. Omniscanquickly and freely distributes to the extracellular space where itselimination tends to be rapid and complete (29). The contrastsignal confidently represents extracellular fluid as uptake bycultured cells has been shown to be ineffective and requiresprolonged incubation times (30). Dynamic contrast–enhancedMRI data were recorded and analyzed for each individual pixel.Contrast enhancement in the pixels followed time courses whichfit best either with biexponential, monoexponential, or linearenhancement curves as exemplified in Fig. 1. The total number ofpixels having significant signal enhancement from all STI571-treated (n ¼ 9) and control (n ¼ 8) tumors, respectively, werepooled and compared. Thus, a total of approximately 33,000pixels from the STI571-treated group and approximately 25,000from the control group were examined. Amajority of the pixels inboth groups followed a biexponential enhancement curve (Fig.1A), around 11% to 12% followed a monoexponential (Fig. 1B),and less than7.5% followed a linear enhancement curve (Fig. 1C).In these distributions, no significant differences were recorded

Imatinib Changes Collagen Structure and Increases Transport

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between the treatment and control group. On the other hand,STI571-treated carcinomas displayed significant increases in timeconstants in pixels followingmonoexponential wash-in enhance-ment curves (Fig. 2A) and biexponential wash-in (Fig. 2B) andwash-out (Fig. 2C) enhancement curves (P < 0.001 in Fig. 2A–C).Furthermore, the pixels best described by monoexponentialuptake enhancement curves revealed a significant shift toward

higher signal amplitudes (Fig. 2D, P < 0.001), indicating anincreased uptake after STI571 treatment. When biexponentialwash-out time constants per tumor were compared between thegroups the STI571-treated tumors displayed a significantly higheraverage (Fig. 2F, P ¼ 0.047). Together, the data show that STI571treatment increased the dynamic exchange of solutes betweenblood and carcinoma tissue.

STI571 treatment decreased collagen fibril diameterElectron microscopy was used to assess the overall structure

of the collagen network in STI571-treated and control KAT-4/HT-29 tumors. TEM revealed a difference in collagen fibrildiameter and morphology: Collagen fibrils in STI571-treatedtumors were more heterogeneous in diameter and had dis-torted surface morphology, whereas fibrils in PBS-treatedtumors were more uniform and had a smooth surface (Fig.3A). Quantification revealed a Gaussian distribution of colla-gen fibril diameters in control tumors, whereas a skeweddistribution toward thinner fibril diameters was apparent inSTI571-treated tumors (Fig. 3B). This was also reflected in theaverage collagen fibril diameter, which was 35 nm in STI571-treated mice and 45 nm in control carcinoma (Fig. 3C; P <0.029). These results show that STI571 alters the collagen fibrilthickness and morphology, indicating an effect on cell signal-ing-regulated collagen fibril assembly.

No effect of STI571 on the amount or structure of GAGs intumors.

GAGs were extracted and purified from tumors. Chondroitinsulfate (CS)/dermatan sulfate (DS), hyaluronan (HA), orheparan sulfate (HS) fluorescent-labeled disaccharides wereobtained after chondroitinase ABC or a mixture of heparinasestreatment, respectively, and quantified after HPLC separation.No differences were seen in the amounts of CS/DS, HA, or HSbetween STI571-treated or control tumors (coefficient of varia-tions ranged from 9% to 19%; Fig. 4A). In KAT-4/HT-29carcinomas, the high level of HS, representing 33% of totalGAGs, most likely reflects a high number of HS-expressingcarcinoma cells (31). No differences in the structure of theCS/DS or HS chains between STI571-treated or control tumorscould be detected (Table 1).

STI571 treatment did not significantly affect total collagencontent.

The observed decrease in fibril diameter in KAT-4/HT-29tumors from STI571-treated mice could reflect a decreasedcollagen content. Hydroxyproline measurements indicated atendency to this effect in STI571-treated tumors; the decrease,however, was not statistically significant (Fig. 4B). A similartrend but no significant difference in hydroxyproline contentwas observed in carcinomas grown in animals treated withSTI571 for 8 days compared with vehicle-treated tumors(1.13 � 0.52 mg/g wet weight, n ¼ 4, P > 0.2 vs. pooledcontrols).

STI571 treatment significantly increased collagen type Isynthesis, but did not affect collagen gene expression

The KAT-4/HT-29 carcinoma is rich in ECM and has a well-developed stromal compartment (8, 19, 32). Treatment withSTI571 had no discernable effect on the gross appearance ofthe collagenous matrix as judged from stainings with Sirius red

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Examples of the three different types of time courses for uptake of contrastagent. A, biexponential uptake. B, monoexponential uptake. C, linear uptake;y-axes, contrast enhancement that is proportional to contrast concentration(a.u., arbitrary units); x-axes, time (minutes). Data are obtained from threeindividual pixels. Points are acquired data points and lines showcalculated best-fit curves.

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(Fig. 5A). Gene expression of collagens, small leucine-rich pro-teoglycans, and enzymes involved in posttranslational modifica-tions of collagens varied largely in KAT-4/HT-29 carcinomas(Supplementary Table S1). STI571 treatment had no significanteffect on expression levels of most transcripts, except for a signif-icantly increased expression of the Lox and Loxl2 genes. Next, weinvestigated whether STI571-treatment affected collagen synthe-sis. Metabolic labeling ex vivo for 6 hours of freshly isolated thicksections from STI571- and vehicle-treated carcinomas, revealed asignificantly increased de novo synthesis of pepsin-resistant colla-gen type I in STI571-treated carcinoma (Fig. 4C and Supplemen-tary Fig. S1). The amounts of pepsin-resistant collagen from thesections did not differ between STI571- and vehicle-treated car-cinomas when normalized to tissue wet weights (SupplementaryFig. S1).

STI571 treatment did not affect endothelial staining andpericyte coverage, but reduced an NG2-positive extravascularcell population in the tumor stroma

Sections from KAT-4/HT-29 carcinomas grown in STI571-trea-ted and control mice were stained for the endothelial markerCD31, a-SMA, RFM, macrophages (F4/80) and the pericytemarker NG2 (Fig. 5A). STI571 treatment had no apparenteffect on the number of cells expressing RFM and a-SMA(Fig. 5B), that is, cells that could be both mural pericytes (33)and stromalmyofibroblasts (34, 35). STI571 induced a decreasednumber of F4/80-positive macrophages (Fig. 5B); this reduction,however, was not statistically significant. Total NG2 staining wassignificantly decreased (P < 0.05) by STI571 treatment (Fig. 5Aand B). Quantification of pixels representing blood vessels(CD31) and NG2-positive pericytes showed that the ratio

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STI571 increases dynamic exchange between the blood and the tumor interstitium as measured by MRI. Histograms in A–E show number of pixels in STI571-treated(red) and control tumors (blue) distributed according to their time constants calculated from the equations of the fitted curves. A, monoexponentialwash-in. B, biexponential wash-in. C, biexponential wash-out. D, distribution of maximum amplitudes in individual pixels taking up contrast according themonoexponential kinetics. E, distribution of maximum amplitudes in individual pixels taking up contrast according the biexponential kinetics. F, average timeconstants in STI571-treated tumors (n¼9) and control tumors (n¼ 8); error bars, SEM. � ,P <0.001 by the Student t test.G, heatmap illustratingwash-out dynamics inPBS- and STI571-treated tumors, scale goes from blue (low time constant) to yellow (high time constant).






between colocalized CD31 and NG2 versus CD31 alone wasunaltered in STI571-treated tumors, that is, no difference invascular NG2 positive pericyte-coverage was detected (Fig. 5Aand C). Thus, it can be inferred that the reduction in NG2 stainingreflects a decrease of extravascular NG2-positive cells or a reducedNG2 expression by such cells.

DiscussionBy taking advantage of dynamic MRI to follow wash-in and

wash-out characteristics, we show that treatment of colorectalKAT-4/HT-29 experimental carcinomas with STI571 (Imatinib/Gleevec) resulted in a significant change of the contrast enhance-ment dynamics—a fasterwash-in anda fasterwash-out of contrastagent was observed. This provides a strong indication that STI571treatment induces an augmented exchange between plasma andthe carcinoma extracellular fluid volume. In our study, all pixelswere analyzed individually. The physical process of contrastenhancement is complicated and the areas close to the vasculatureare likely to show biexponential enhancement in our model, thatis, wash-in and -out dynamics, whereas the pixels further awayfrom the blood vessels should tend to follow amonoexponentialuptake curve, that is, wash-in flow. The contrast agent may diffusefrom one pixel to another over time and a linear uptake is likelydue to diffusion to areas with monoexponential uptake profiles(36) Consequently, in the comparison between the different

uptake curves between control and STI571-treated groups, achange in the biexponential time constants will directly influencethe diffusion into the monoexponential areas. Despite the fasterwash-out of contrast agent in the STI571-treated animals, a fastermonoexponential uptake was evident. This finding strongly indi-cates that STI71 improves transport of solutes through the stromainterstitium, thereby making it more accessible for blood streamsolutes. Our current data, therefore, provide one plausible con-tributing mechanism for the enhanced efficacy of taxol (18) anduptake and efficiency of Epothilone B (37) after STI571 treatmentof KAT-4/HT-29 experimental carcinomas.

STI571 reduces IFP and increases the interstitial fluid volume inKAT-4/HT-29 carcinomas (18, 19). A potential mechanism couldinvolve a reduction of fibroblast tensile forces applied on thecollagen/microfibrillar network (1, 18). This is in analogy withwhat has been described for conditions in normal loose connec-tive tissues in which PDGF normalizes an anaphylaxis-inducedlowering of the IFP (38).Our current data suggest an alternative orcomplementary mechanism that involves an STI571-inducedaltered collagen network structure with decreased collagen fibrildiameters. This potentially results in a more flexible collagennetwork that allows for the expansion of the interstitial fluidvolume and reduction of IFP. Collagenous fibers and the GAG/proteoglycan ground substance constitute the two major macro-molecular complexes in any connective tissue and play a decisiverole in the efficacy of fluid and solute transport through tissues

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Ultrastructure of stromal carcinomacollagen fibers. A, micrographsobtained by TEM of KAT-4/HT-29carcinomas from STI571 and controlmice, depicting morphology ofcollagen fibrils. Note, the higherabundance of smaller fibrils in tumorfrom STI571-treated mice. B,histogram showing distribution ofcollagen fibril diameters in STI571(black bars) and control carcinomas.The two populations differedsignificantly when tested with theKolmogrov–Smirnov test (P <0.0001).C, average fibril diameters fromindividual carcinoma in the STI571-treated (n ¼ 8) and control (n ¼ 8)groups. �� , P < 0.05 by the Studentt test; error bars, SD.

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(12, 39). The finding that the amounts of hyaluronan, chondroi-tin/dermatan sulfate, and heparan sulfate per unit carcinoma dryweight were unaffected by STI571 treatment, together with thedata on an expanded interstitial fluid volume (19), suggest thattheGAG/proteoglycan ground substancewas diluted by taking upfluid. The collagen content, measured as the total amount ofhydroxyproline per unit carcinoma wet weight was not signifi-cantly affected by STI571 but the marginal decrease that wasrecorded fits well with an increase in the interstitial fluid volume.In conclusion, our data suggest that STI571 reprograms stromalconnective tissue cells, altering the collagen network allowing foran under-hydrated GAG/proteoglycan ground substance to swell,which in turn reduces the barrier for fluid and solute transportthrough the carcinoma, as inferred from the present MRI data.

Our data also suggest that the mechanism by which STI571induces a change in the collagen network involves an increasedturnover of collagen type I, indicated by an increased collagensynthesis rate combined with an unchanged total hydroxyprolinecontent. The latter probably is due to an increased degradation ofcollagen that offsets the increased synthesis. Collagen transcrip-

tion was not significantly altered in spite of the increased collagensynthesis. This is in agreement with previous data emphasizingthe importance of posttranscriptional regulation of collagen type Isynthesis (23, 40). An increased turnover of the collagen andimpaired maturation of the collagen fibrils is a plausible mech-anism for the observed decrease in average collagen fibril diam-eter. Inhibition of TGF-b1/b3 in KAT-4/HT-29 downregulates thesmall-leucine rich repeat proteoglycan fibromodulin and theeffects on carcinoma IFP and collagen fibrillar network recordedafter TGF-b1/b3 inhibition is mimicked in KAT-4/HT-29 carci-nomas grown in fibromodulin-deficient mice (8). Fibromodulinregulates collagen fibril assembly in dense connective tissues (41)and is upregulated and plays a pathogenic role in several modelsof fibrosis (8, 42–44). STI571 showed a trend to downregulatefibromodulin mRNA in KAT-4/HT-29 carcinoma but, due to alarge variance, the changes did not reach statistical significance. Itis possible that an inability to mature the collagen fibrils resultsfrom disturbances in the enzyme arrays responsible for cross-linkformation. Recently, we showed that fibromodulin interacts withLox and can direct collagen cross-link formation (45). A decreasedexpression of fibromodulin can result in an impairment of fibrilmaturation even if Lox is increased, as it was observed in thecurrent study in which the expression of Lox and Loxl2 wereincreased.

STI571 had no effect on the density of CD31-positive vessels inKAT-4/HT-29 carcinoma, consistent with our earlier findings(19). Furthermore, our data show that the pericyte coverage ofCD31-positive vessels was not affected by STI571 treatment.Although STI571-treatment does not affect plasma volume inKAT-4/HT-29 carcinomas but has been shown to reduce bloodvessel perimeters (19), it cannot be excluded that STI571 increasesblood flow through the tumor. Available data have indeed shownthat modulation of the ECM-composition, notably the majorECM-constituents collagen and/or glycosaminoglycans, influencesolid stress in carcinoma and thereby compression of bloodvessels (13, 14, 46). A less dense and more flexible collagennetwork would enable the opening of vessels and increase bloodflow. It is, thus, possible that STI571 by altering the collagenultrastructure has a dual function that increases drug distributionwithin carcinoma, by improving transport through the stromaand, potentially, tumor blood flow.

A

B

Dry

wei

ght (

mg/

g)W

et w

eigh

t (m

g/g)

HSHACS/DS 0

500

1,000

1,500

STI571PBS

0

100

200

300

Radiolabeled collagen

C

Rel

ativ

e 14

C-in

corp

orat

ion

(%)

*

Hydroxyproline0

1

2

3

Figure 4.

GAGs, collagen content, and synthesis in KAT-4/HT-29 carcinomas. A, GAGswere extracted from lyophilized STI571-treated carcinomas (n ¼ 5) and fromcontrol carcinomas (n ¼ 4), purified, and treated with chondroitinase ABC or amixture of heparinase I, II, III to quantitatively degrade CS/DS and HA, or HS,respectively. The obtained disaccharides were fluorescence labeled, separatedby HPLC, and quantified using defined disaccharide standards. B,hydroxyproline concentration in tumors from STI571-treated for 4 days (n ¼ 7)and PBS control (n¼ 11); error bars, SD.C,quantification ofmetabolically labeledcollagen a1(I)-chains from autoradiographs. Collagen was extracted bypepsin-treatment from tissue sections incubated ex vivo for 6 hours. Data areobtained from 9 STI571- and 9 PBS-treated carcinomas. Band intensities werenormalized to tissue wet weight at harvest. � , P < 0.03 by the Student t test.

Table 1. Compositional analysis of CS/DS and HS chains

PBS STI571

CS/DSDUA-GalNAc (DO) 16.1 � 1.0 15.7 � 1.4DUA-GalNAc-4S (DA) 80.2 � 1.1 80.4 � 1.4DUA-GalNAc-6S (DC) 1.8 � 0.1 1.9 � 0.2DUA-2S-GalNAc-4S (DB) 1.9 � 0.2 2.0 � 0.2DUA-2S-GalNAc-6S (DD) <0.1 <0.1DUA-GalNAc-4S,6S (DE) 0.0 0.0

HSDUA-GlcNAc 54.3 � 0.2 54.9 � 0.3DUA-GlcNS 21.4 � 0.1 21.6 � 0.1DUA-GlcNAc-6S 7.1 � 0.1 7.0 � 0.3DUA,2S-GlcNAc 2.8 � 0.1 2.7 � 0.1DUA-GlcNS-6S 3.0 � 0.0 2.9 � 0.2DUA-2S-GlcNS 7.7 � 0.2 7.5 � 0.2DUA,2S-GlcNAc,6S 1.3 � 0.2 1.3 � 0.1DUA-2S-GlcNS-6S 2.5 � 0.2 2.2 � 0.0

NOTE: Data are expressed as the mole percentage of the disaccharide units(mean � SD of duplicates).






STI571 inhibits several tyrosine kinases, including PDGF recep-tors, with a proposed pathogenic role in fibrosis (47). Previously,we showed that a PDGFB–specific aptamermimicked someof theeffects of STI571 on IFP and increased sensitivity to taxol in KAT-4/HT-29 experimental carcinomas (18). No reports, however,have previously shown the effects of this agent on collagennetwork structure reported on herein. Another target of STI571,ABL is activated down-stream of activated PDGF receptors (48).ABL has been ascribed a role in fibrotic reactions acting upstreamof early growth response factor 1 (Egr-1; refs. 49, 50). Further-more, STI571 attenuates the severity of disease in animal modelsof fibrosis and is currently under clinical testing for the treatmentof fibrotic diseases (51). Inhibition of TGF-b1/b3 in KAT-4/HT-29experimental carcinomas reduces IFP, fibril diameters of stromalcollagen and expression of a subset of inflammation-related genes(8, 32). TGF-b stimulates ABL by a non–Smad-dependent path-way (50, 52). It is, thus, possible that themechanism(s) by whichSTI571 and TGF-b1/b3 inhibitors exert their effects in KAT-4/HT-29 carcinomas involve inhibition of c-Abl and its downstreamtarget Egr-1.

There was no reduction in the stromal cell populations thatstained positive for RFM and a-SMA, or both. The a-SMA–

expressing cells in the tumor stroma are often referred to asmyofibroblasts, and are regarded as the major collagen type I-synthesizing cells in tumors (34). Our findings that STI571had no major effect on total hydroxyproline (collagen) con-tent or the number of a-SMA–expressing in STI571-treatedtumors are in agreement with that a-SMA–expressing cells arethe major collagen-producing cells. It is, however, possiblethat STI571 altered the expression of collagen assembly reg-ulators by a-SMA–expressing cells resulting in the productionof a less dense collagen network. In contrast, STI571 treatmentreduced the number of NG2-expressing cells. The amount ofthese cells colocalizing with CD31-positive cells was, however,unchanged, inferring a decrease in extravascular NG2-expres-sing cells. Our previous work has shown that overexpressionof PDGF-B in murine skin induces the expansion of NG2-positive stromal cells and that pericytes are the likely pre-cursors for these cells (35). It is possible that treatment withSTI571, which inhibits PDGF receptors, hampers the expan-sion of the NG2-positive stromal cell population and thatthese cells could have a role in modifying the ECM.

Together, our data suggest that STI571 (Imatinib/Gleevec)improves the distribution of low-molecular weight compounds

PBSSTI571A

C

*

Are

a co

vera

ge (%

)

CD31NG2F4/80RFMaSMA0

2

4

6

NG2 to CD31

CD31 to NG2

0

5

10

15

20STI571PBS

Col

ocal

ized

pix

el (%

)

B

Figure 5.

Effects of STI571 on stroma characteristics inKAT-4/HT-29 carcinoma. A, gross morphologyshowing Sirius red staining of thick sections (30mm) showing an abundant collagen matrix inthe stroma of KAT-4/HT-29 with no discernableeffect by STI571 treatment. B, epifluorescenceimages of tumors from STI571-treated andcontrol mice, depicting CD31-positiveendothelium (red) and NG2-positive (green)mural pericytes and stromal cells. C, each of theinvestigated markers were quantified anddisplayed as a percentage of the whole imagearea containing positive staining. C,quantification of vascular coverage byNG2-positive areas. Data are shown as thepercentage of NG2- or CD31-positive pixels thatcolocalized with the other marker, CD31 or NG2,respectively, in tumors from STI571- and PBS-treated tumors. � , P < 0.05 by theMann–Whitney test; error bars, SEM; scale bars,50 mm in A and B.

Olsson et al.





into carcinoma interstitiumbyaltering the collagenultrastructure,but not the overall content of collagen or glycosaminoglycans.Drugs that specifically alter collagen ultrastructure should beimportant tools in improving the uptake, and thereby efficacy,of commonly used chemotherapeutics.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: P.O. Olsson, R. in 't Zandt, T. Friman, M. Maccarana,Å. Oldberg, K. Rubin, S. KalamajskiDevelopment of methodology: P.O. Olsson, R. Gustafsson, R. in't Zandt,T. Friman, M. Maccarana, K. RubinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P.O. Olsson, R. Gustafsson, R. in't Zandt,M. Maccarana, E. Tykesson, K. Rubin, S. KalamajskiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P.O. Olsson, R. Gustafsson, R. in 't Zandt, T. Friman,M. Maccarana, K. Rubin, S. KalamajskiWriting, review, and/or revision of the manuscript: P.O. Olsson, R. Gustafs-son, R. in 't Zandt, T. Friman, M.Maccarana, Å. Oldberg, K. Rubin, S. Kalamajski

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R. Gustafsson, K. RubinStudy supervision: R. Gustafsson, K. Rubin

AcknowledgmentsLund University Bioimaging Center (LBIC) at Lund University is gratefully

acknowledged for providing experimental resources.

Grant SupportThis study was supported by funds from the Swedish Cancer Society (to K.

Rubin and M. Maccarana), the Swedish Research Council (to K. Rubin), the

Alfred €Osterlund Foundation (to K. Rubin, Å. Oldberg, and S. Kalamajski), theKochFoundation (toK. Rubin), theCrafoord Foundation (to S. Kalamajski), theMagnus Bergvall Foundation (to S. Kalamajski), the ÅkeWiberg Foundation (toS. Kalamajski), and Uppsala and Lund Universities.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 20, 2016; revised June 6, 2016; accepted June 23, 2016;published OnlineFirst July 29, 2016.

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Olsson et al.




Published OnlineFirst July 29, 2016.Mol Cancer Ther P. Olof Olsson, Renata Gustafsson, René in 't Zandt, et al. Fibril Diameter in Experimental Carcinoma

CollagenExtracellular Fluid Exchange and Reduces Average The Tyrosine Kinase Inhibitor Imatinib Augments

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