INTRODUCTION

Intermediate filaments (IFs) are one of the three majorcytoskeletal systems in eukaryotic cells. They constitute amultigenic family of proteins whose expression is highlytissue-specific (reviewed by Fuchs and Weber, 1994).Vimentin, a class III IF predominantly expressed in cells ofmesenchymal origin, has several intriguing features. Duringmouse embryonic development, vimentin is expressed veryearly in motile cells like parietal endoderm cells (Lane et al.,1983), primary mesenchymal cells that delaminate from theembryonic ectoderm (Franke et al., 1982) and neural crest cells(Houle and Fedoroff, 1983; Cochard and Paulin, 1984).Vimentin is also expressed in immature cells before beingreplaced later by more specialized networks. Thus, stem cellsof neuroepithelium at around 9 days of gestation expressvimentin, whereas their differentiated derivatives express eitherglial fibrillary acidic protein (GFAP) in astroglial cells orneurofilament in neurons (Cochard and Paulin, 1984).Similarly, around day 8 and 9 of mouse gestation, presumptivemyotome cells and myoblasts express vimentin that is laterreplaced by desmin in myotubes and muscles (Fürst et al.,

1989). In adult mice, in addition to being expressed inmesoderm-derived tissues (connective and adipose tissue,endothelial and blood cells, glomerular renal cells, etc),vimentin is also expressed in the epithelial cells of the lens andcertain types of mature astrocytes. Thus, in contrast to other IFwhich exhibit a narrow pattern of expression, vimentin is fairlybroadly expressed. However, in spite of its remarkableexpression profile in embryonic and adult mice, the biologicalfunction of vimentin has remained elusive. Functional deletionof vimentin in mice has unexpectedly not revealed any obviousdifferences with respect to overall development, breeding,structural or functional properties of distinct tissues or organs(Colucci-Guyon et al., 1994). The first evidence for anabnormal phenotype in these mutant animals was documentedfor a subset of astrocytes, wherein the lack of vimentin networkprecluded the formation of an organized GFAP network (Galouet al., 1996). More recently, a cerebellar defect and an impairedmotor coordination has been observed in mice lackingvimentin (Colucci-Guyon et al., 1999). Finally, deficiencies inthe modulation of vascular tuning (Terzi et al., 1997) and themechanotransduction of shear stress (Henrion et al., 1997)have also been reported in Vim−/− mutant mice.

3463Journal of Cell Science 113, 3463-3472 (2000)Printed in Great Britain © The Company of Biologists Limited 2000JCS1642

Vimentin is a class III intermediate filament protein widelyexpressed in the developing embryo and in cells ofmesenchymal origin in the adult. Vimentin knock-out micedevelop and reproduce without any obvious defect. Thisis an unexpected finding in view of the high degree ofconservation of the vimentin gene among vertebrates.However, it does not exclude the possibility of a role forvimentin in pathological conditions, like tumorigenesis.To address this question directly, we have used ateratocarcinoma model involving the injection of ES cellsinto syngeneic mice. ES cells lacking vimentin weregenerated from 129/Sv Vim−/− mice with high efficiency.The absence of vimentin did not affect ES cell morphology,viability or growth rate in vitro. Tumours were induced bysubcutaneous injection of either Vim−/− or Vim+/+ ES cells

into Vim+/+ and Vim−/− mice, in order to analyse the effectof the absence of vimentin in either the tumorigenic cellsor the host mice. No significant differences were foundin either tumour incidence, size or vascularization ofteratocarcinomas obtained with all possible combinations.Vim−/− ES-derived tumours showed the same cellularcomposition typical of teratocarcinomas induced by wild-type ES cells together with a very similar apoptotic pattern.Taken together, these results demonstrate that in this modelvimentin is not essential for efficient tumour growth anddifferentiation in vivo.

Key words: Teratocarcinoma, Vimentin, Embryonic stem cell,Knock-out mice

SUMMARY

Teratocarcinomas induced by embryonic stem (ES) cells lacking vimentin: an

approach to study the role of vimentin in tumorigenesis

Francina Langa 1,*, Chantal Kress 1, Emma Colucci-Guyon 1, Huot Khun 2, Sandrine Vandormael-Pournin 1,Michel Huerre 2 and Charles Babinet 1,‡

1Unité de Biologie du Développement, URA C.N.R.S. Institut Pasteur, 25 rue Dr Roux, Paris, France2Unité d’Histopathologie, Institut Pasteur, 25 rue Dr Roux, Paris, France*Present address: Departamento de Biologia Molecular y Celular, Centro Nacional de Biotecnologia (CNB-CSIC), Carretera de Colmenar Viejo km 15,5, 28049 Madrid,Spain‡Author for correspondence (e-mail: chbabi@pasteur.fr)

Accepted 20 July; published on WWW 13 September 2000

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Thus, despite the fact that mice lacking vimentin seem todevelop normally, several phenotypic differences have beenuncovered, under certain stress and/or pathological conditions(Eckes et al., 1998; Henrion et al., 1997; Terzi et al., 1997). Inthis context, we felt that it would be interesting to look at theeffect of the absence of vimentin on tumorigenesis. Vimentinhas been implicated in tumoral processes by severalobservations. Classically, it was considered to be amesenchymal marker helping to distinguish between sarcomasand carcinomas (Osborn and Weber, 1982, 1983; Leader et al.,1987). However, it appeared that vimentin can be found insome tumours of epithelial origin including breast, renal,thyroid, ovarian, pulmonary or prostatic carcinomas (McNuttet al., 1985; Azumi and Battifora, 1987; Buley et al., 1987;Viale et al., 1988; Kartenbeck, 1989), raising some doubtsconcerning its differential diagnostic value. Furthermore,vimentin has been shown to be coexpressed unusually withkeratins in various tumour cells during their progression fromthe primary to the metastatic tumour stage (Ramaekers et al.,1983; Thompson et al., 1992; Chu et al., 1996). In breastcancer, vimentin expression is preferentially found in highlyproliferative carcinomas with low levels of estrogen receptors,being associated to poor prognosis (Raymond and Leong,1989; Domagala et al., 1990, 1994). Overexpression ofvimentin in breast carcinoma models leads to increasedmotility and invasiveness in vitro, which can be transientlydown-regulated by treatment with antisense oligonucleotidesto vimentin (Hendrix et al., 1996, 1997). However, vimentinexpression could not clearly discriminate between benign andinvasive breast lesions, even though it was correlated with hightumour grade and decreased survival in ductal carcinomas(Heatley et al., 1993; Holck et al., 1993).

In addition to the overexpression of vimentin in the above-mentioned cancers, vimentin expression has also beenassociated with reversion of the transformed phenotype. Thus,an intriguing observation is that the expression of vimentin iscapable of suppressing the transformed phenotype of BHKcells (Eiden et al., 1991). Furthermore, loss of the malignantphenotype of transformed CHO-K1 cells induced by cAMPderivatives involves vimentin phosphorylation as one of theprimary steps implicated in the reverse transformation reaction(Chan et al., 1989).

In this work, we took advantage of our recently createdvimentin knock-out mice as a potent and clear-cut tool tore-examine the possible involvement of vimentin intumorigenesis. We chose the model of experimentalteratocarcinomas initially obtained by ectopic injection of earlyembryos or embryonal carcinoma cells into syngeneic mice(Stevens, 1970). These tumours are particularly interestingbecause they normally contain cell types derivatives of all threegerm layers (Stevens, 1970; Martin, 1980; Damjanov, 1993);furthermore, they may be obtained by ectopic injection of EScells (Martin, 1981; Hilberg and Wagner, 1992). Thus, in thisstudy, both vimentin null and wild-type ES cells were used toinduce teratocarcinomas by subcutaneous injection either inthe wild-type or the mutant mice. The different combinationsof ES cells and host mice allowed us to monitor the effect ofthe presence/absence of vimentin either in the tumour cells orin the tissues of the host animal.

Our results demonstrate that the absence of vimentin in EScells and/or host mice did not affect either the efficiency of

teratocarcinoma formation nor the normal development ofthese experimental tumours. Therefore, we can conclude thatin this model vimentin expression is dispensable for cellproliferation and differentiation.

MATERIALS AND METHODS

AnimalsVimentin-null mice were obtained by targeted inactivation of thevimentin gene in mice (Colucci-Guyon et al., 1994). All experimentswere performed on Vim1/Vim1 knockout mice, in which theendogenous vimentin gene has been disrupted by an in-frameinsertion of Escherichia coliβ-galactosidase coding sequences intoexon 1 of vimentin gene. These mice are of 129/Sv pure geneticbackground.

ES cellsIsolation of Vim−/− ES cellsVim−/− ES cell lines were established as described by Abbondanzo etal. (1993). Three to five-week-old 129/Sv Vim−/− females weresuperovulated by standard procedures and mated with Vim−/− 129/Svmales. Embryos were collected by flushing the uteri of females 3.5days post-coitum (dpc) (plug=0.5 day) with DMEM (Gibco) culturemedium containing 10% fetal calf serum, 10−4 M 2-mercaptoethanol,and antibiotics. Late morula to blastocyst stage embryos were thentransferred onto culture dishes coated with mitomycin C-treatedprimary embryonic mouse fibroblasts and cultured in ES cell medium(DMEM high glucose, 2 mM glutamine, 1 mM Na-pyruvate, 1×nonessential aminoacids, 10−4 2-mercaptoethanol, 1000 i.u./ml LIF(Esgro), 50 IU penicillin/ml, 50 mg streptomycin/ml, all productsfrom Life Technologies), and 15% fetal bovine serum tested for EScell culture (Seromed). Blastocysts were cultured without mediumchange. After 4-6 days, individual inner cell masses were picked,trypsinized, mechanically dissociated and replated onto fresh feedercells in 1.5 cm wells. Within 6-10 days, colonies having acharacteristic undifferentiated morphology (ES cell-like) wereobserved. They were trypsinized, dissociated, and reseeded in 1.5 cmwells with fresh feeder cells. Four to six days later, ES colonies couldbe clearly identified, which were trypsinized and transferredprogressively to larger feeder-containing culture dishes foramplification. Thereafter all cell lines were routinely maintainedaccording to the protocol of Robertson (1987).

Vim+/+ ES cells129/Sv CK35 ES cells (Kress et al., 1998) were used as wild-typecontrol.

ES sex determinationThe sex of ES cell lines obtained was determined by PCR using Smcgene primers (Smc-1 and Smc-2; Mroz et al., 1999). PCR reactionswere cycled at 95°C for 30 seconds, 50°C for 30 seconds and 72°Cfor 30 seconds for 35 cycles.The presence of a Y chromosome wasindicated by a double band under 350 bp, whereas genomic DNA offemale cells showed only a single band.

Karyotype analysisKaryotype analysis was performed according to the method of Trimanet al. (1975). ES cells were passaged once onto gelatin-precoateddishes to reduce feeder cells. One day after passage, exponentiallydividing population of cells, which had been re-fed with medium 3hours previously, were incubated with 0.02 mg/ml colcemid for 1 hourat 37°C. Cells were then trypsinized, rinsed with phosphate bufferedsaline (PBS) and centrifuged. After complete aspiration of thesupernatant, 6 ml of hypotonic (0.56% w/v) KCl solution were addeddropwise and the cell suspension incubated for 10 minutes at room

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3465Teratocarcinomas induced by vimentin-lacking ES cells

temperature. Cells were then pelleted and 2 ml of ice-cold fixative(methanol:glacial acetic acid, 3:1 freshly prepared) were addeddropwise with gentle agitation and incubated for 5 minutes at roomtemperature. Three changes of fixative were then performed byspinning out the cells and finally fixed cells were spread by releasinga single drop of suspension from a Pasteur pipette positioned 10 cmabove the centre of a pre-cleaned glass microscope slide. After thefixative was evaporated, individual slides were carefully examinedunder phase contrast (×200) to check the number and quality of thechromosome spreads. At least 40 metaphases were analysed.

Production of chimeras and germline transmissionChimeras were generated essentially as described (Bradley, 1987). 3.5dpc blastocyst stage embryos were obtained from C57BL/6 femalesnaturally mated with males of the same strain. The blastocysts wereinjected with 6-10 ES cells and injected embryos were transferred touterine horns of pseudopregnant 2.5 dpc B6D2F1 females. Pups born17 days after injection were identified as chimeric few days later onthe basis of the agouti pigmentation in their coat. Six-week-old malechimeras were mated with C57BL/6 mice and germ line transmissionwas scored by the presence of agouti offspring.

Induction of teratocarcinomas and histological analysis oftumoursVim−/− and Vim+/+ ES cells (5×106 in 0.3 ml of PBS) were injectedsubcutaneously into isogenic 8-week-old 129/Sv wild-type or Vim−/−male mice. After three or six weeks, tumours were excised, fixed inbuffered formalin and subsequently embedded in paraffin wax, usingstandard histological techniques. Sections were then cut from themidplane of the tumours, stained with hematoxylin and eosin (H&E),and observed and photographed in a Leica DIAPLAN microscope.

Immunocyto(histo)chemistry assaysCultured ES cells fixed in ethanol-acetic acid (95:5) (5 minutes,–20°C) or deparaffined formalin-fixed tumour sections were firstsaturated with 1% normal goat serum to minimize unspecific binding.Specific primary antibodies (rabbit antiserum against vimentin(Dupouey et al., 1985), rabbit antiserum against nestin (Lendahl et al.,1990), or mouse anti-Factor VIII monoclonal antibodies (Dako,Germany) were then incubated for 1 hour followed, after thoroughlyrinsing in PBS, by a 30 minute incubation of FITC-conjugated goatanti-rabbit (mouse) IgG (Boehringer Mannheim, Germany). Afterwashing with PBS for 20 minutes and mounting in an aqueousmedium, photographs were taken in a Leica DIAPLAN fluorescencemicroscopy.

Apoptosis assayTo detect apoptotic cells, tumour sections were treated afterdeparaffination with the In Situ Cell Death Detection kit (BoehringerMannheim, Germany). Briefly, deparaffined tumour sections wereincubated with proteinase K (20 µg/ml in 10 mM Tris-HCl, pH 7.4)for 15 minutes at 25°C. Endogenous peroxidases were theninactivated with 0.3% H2O2 in methanol for 30 minutes at roomtemperature. After permeabilization with 0.1% Triton X-100 in 0.1%sodium citrate (2 minutes, 4°C), tumour sections were incubated inTUNEL reaction mixture for 60 minutes at 37°C. After the final wash,nuclei with fragmented DNA were visualized by treatment with asolution of 0.25 mg/ml of diaminobenzidine (DAB), 3 mg/ml of nickel

sulfate and 0.003% H2O2. The substrate reaction was stopped after 6-10 minutes by rinsing the slides in H2O for 5 minutes. Slides weremounted and observed in a Leica DIAPLAN microscope.

RESULTS

Isolation and characterization of ES cells lackingvimentinTo isolate ES cells homozygous for a null mutation in thevimentin gene, we collected blastocysts from a cross betweenVim1/Vim1 129/Sv mice and generated ES cell lines (seeMaterials and Methods). From a total of 126 Vim1/Vim1

blastocysts or late morula stage embryos, 83 attached inner cellmasses (ICMs) were picked, and 15 ES cell lines were isolated.Thus, the efficiency of recovery of ES cell lines from 129/Svvimentin null ICM (18%) was in the expected range(Robertson, 1987; Nagy et al., 1993). PCR analysis using sex-specific primers revealed that 12 of the ES cell lines were XYversus 3 XX, which was in good agreement with a preferentialestablishment of XY ES cell lines (Robertson, 1987; Hooper,1992).

We performed karyotype analysis on the ES cell lines. Wechose two male cell lines exhibiting a normal diploid karyotype(Vim−/− 52 and Vim−/− 70). Both cell lines were shown to becompetent for germ-line colonization (see Table 1). Theabsence of vimentin protein in both clones was confirmed byimmunocytochemistry (Fig. 1C,E). Positive controls werecolonies of wild-type 129/Sv CK35 XY ES cells (Kress et al,1998) which display strong vimentin staining (Fig. 1A).Nestin, an intermediate filament present in neuroepithelialprecursors (Lendahl et al., 1990) was present in both Vim+/+and Vim−/− ES cells, although staining was somewhat weakerin Vim−/− cells (Fig. 1B,D,F).

Thus, the absence of vimentin did not prevent the isolationof ES cells at an expected rate. Moreover, Vim−/− ES cellsexhibit the same undifferentiated morphology and growth rateas wild-type CK35 ES cells (data not shown). Finally, theefficiency of germ line colonization appeared to be particularlyhigh: indeed, all of the 4 male chimeras tested for each clonetransmitted the ES genotype to 100% of their progeny(Table 1).

Tumour development in wild-type and vimentin nullanimalsA major feature of ES cells is their capacity to formteratocarcinomas containing cells of all three germinal layersfollowing subcutaneous injection into syngeneic mice (Martin,1981). The availability of both ES cells and mice lackingvimentin allowed us to test all the possible cross combinations.First, to determine whether the absence of vimentin had anyeffect on the formation of teratocarcinomas in vivo we injectedboth Vim−/− 52 et Vim−/− 70 ES clones into syngeneic 129/Sv

Table 1. Efficiency of producing germline chimeras with Vim−/− ES cell linesNo. of blastocysts Pups born Chimeras tested No. of germ line

ES cell line Passage* injected (chimeras) (% of chimerism) chimeras‡

Vim−/−52 P4-P6 30 13m+4f (14) 4m (90-100%) 4 (100% transmission)Vim−/−70 P4-P6 32 9m+2f (10) 4m (90-100%) 4 (100% transmission)

*Number of trypsinizations after initial blastocyst culture. m: male, f: female. ‡Thirty to 60 offspring were surveyed for each chimeric mouse.

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wild-type mice. Moreover, to determine whether the absenceof vimentin in the host plays a role in the teratocarcinomadevelopment, control Vim+/+ CK35 ES cells were injected inwild-type and mutant mice. Finally, the effect of the totalabsence of vimentin in the system was investigated by injectingVim−/− ES cells into Vim−/− mice.

Preliminary experiments performed with control ES cellsand mice indicated that 5×106 cells injected subcutaneouslywere sufficient to give rise to well-analysable tumours. Thus,palpable masses developed at the site of injection within 10days of inoculation and tumours were excised in twocomparable series, three or six weeks after injection. Asummary of the results obtained with the differentcombinations of host and ES cell genotypes is presented inTable 2. Notably, all but one of the sixty injected micedeveloped tumours. Thus, teratocarcinoma formation appearsto be independent on the presence of vimentin, in thetumorigenic ES cells or in the host cells.

An important aspect related to tumorigenesis is the possibledevelopment of metastases. None of the 60 mice injected(control or mutant) sacrified three or six weeks after

inoculation of the Vim−/− or Vim+/+ ES cells showedmetastatic nodules, as evidenced by macroscopic inspectionduring organ excision (See Table 2). These observations werelater confirmed by anatomopathological analysis; liver andlung were fixed and sectionned and their microscopicexamination revealed no evidence of metastases.

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Fig. 1. Immunostaining of vimentin (A,C,E) and nestin (B,D,F) in wild-type ES cells CK35 (A,B) and vimentin-lacking ES cells clone 52(C,D) and clone 70 (E,F). ES cells were stained after a two-day culture in standard conditions. Arrows indicate ES cell colonies. Some isolatedmurine fibroblasts coming from feeder layer appeared stained with both intermediate filaments.

Fig. 2. Comparative histological analyses of tumours obtained fromwild-type ES cells injected in wild-type mice (left, A-G) and fromvimentin-lacking ES cells injected in vimentin-lacking mice (right, a-g).ES cells (5×106) were injected subcutaneously into isogeneic 129/Svmice and the tumours isolated and fixed 3 weeks later. Sections throughtumours contain a variety of tissue types derived of the three embryoniclayers together with extraembryonic material like parietal endoderm(A,a, arrows). Teratocarcinomas include mainly neural tissue (B,b),keratin whorls (C,c), secretory epithelia (D,d, arrows), glandular tissues(E,e, *), bone and cartilage (F,f) and muscle cells (G,g). Sections arestained with hematoxylin and eosin. The differentiated tissues shown ina-g were seen in several tumours from both clone 52 and clone 70Vim−/− ES cell lines tested. b=bone; ca=cartilage; kw=keratin whorl;m=muscle; Ec=Ectoderm; En=Endoderm; Me=Mesoderm. ×50 (A,a),×250 (D,E,G,b,e,f,g), ×500 (B,C,F,c,d).

3467Teratocarcinomas induced by vimentin-lacking ES cells

Fig. 2

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Table 2 also shows that splenomegaly, often associated withteratocarcinomas (Damjanov and Solter, 1974), is morefrequent in the case of tumours arising from Vim−/− recipients,correlating with a higher weight of tumours, but withoutstatistical significancy.

The weight of tumours obtained in the differentcombinations appeared very heterogenous. The biggestindividual tumours obtained after 6 weeks of inoculation of EScells weighed 10.89 g and 7.95 g and grew from Vim+/+ ES

cells in a Vim+/+ mouse and Vim−/− ES cells in a Vim−/−mouse, respectively. These tumours represented the 30.9% and22.4% of the body weight, but both mice with large tumourburden seemed to be in good health. For comparison andstandarization among animals, the weight of liver and spleen,was used and it appeared very similar between all groups; thus,tumour heterogeneity was most probably due to individualcharacteristics of each animal. As a consequence of thisheterogeneity, the detailed analysis of tumour weight did not

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Fig. 3.Vimentin detection interatocarcinomas obtained fromwild-type ES cells (A,B,E,F) andvimentin-lacking ES cells (C,D) in control wild-type mice(A-D) and vimentin-lackingmice (E,F). (A,C,E) Generalview; (B,D,F) highermagnification showing bloodvessels present into tumours.The antibody used is apolyclonal antiserum againstvimentin. (A) Muscle (m),cartilage (ca), fibroblastic (f) cells and endothelial cells inblood vessels (v) stained inVim+/+ ES cells→Vim+/+ micecombination. (B) Detail ofadipocytes (a) and a blood vessel(v) highly stained with anti-vimentin antibody. (C) Only hostblood vessels were stained withanti-vimentin antibody in Vim−/−ES cells →Vim+/+ micecombination. Inset: ×1000magnification of endothelialcells in a host blood vessel; (D) stained blood vessels fromhost (v) coexist with de novooriginated vessels from Vim−/−ES cells (v-) in Vim+/+ mice. (E) Fibroblastic (f), adipocytic(a) and endothelial cells in bloodvessels (v) stained inVim+/+ EScells→Vim−/− micecombination. (F) Coexistence ofhost unstained vessels (v-) andstained vessels (v, inset)originated from Vim+/+ ES cellsin Vim−/− mice. Vimentinstaining was visualized by greenfluorescence (FITC). Nucleiwere counterstained with EvansBlue. ×200 (A,C,E); ×500(B,D,F).

Table 2. Efficiency of induction of teratocarcinomas by Vim+/+ or Vim−/− ES cells clonesES cell clone No. injected mice No. tumours* Splenomegaly* Metastases*

Vim+/+ Vim−/− Vim+/+ Vim−/− Vim+/+ Vim−/− Vim+/+ Vim−/−Vim−/− 52 10 10 10 10 2 5 – –Vim−/− 70 10 10 9 10 2 8 – –Vim+/+ (CK35) 10 10 10 10 3 2 – –

*Results obtained from all tumours, removed 3 or 6 weeks after ES cell injection.

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show, in general, significative differences between tumoursobtained from all possible combinations (Table 3). Therelatively smaller tumours grown from control wild-type EScells injected in Vim−/− mice might be explained by aimmunological rejection of a tumour arising from Vim+/+ cellsin a host lacking vimentin. This initial effect is progressivelyattenuated and clearly overtaken in 6-week-old tumours (Table 3).

Histological analysis of tumoursTumours obtained from ectopic injection of ES cells inpermissive syngeneic mice are normally defined asteratocarcinomas because they are composed of derivatives ofall three germ layers, distributed in an erratic way (Martin,1981). To determine if the absence of vimentin in ES or in hostmice alters the composition of tumours obtained, tumourswere fixed and sectioned. Histological analysis of all 59teratocarcinomas revealed that both wild-type and mutant ESlacking vimentin can differentiate into a wide range of tissuetypes, including ectodermal, mesodermal and definitiveendodermal derivatives. These various tissue types appearedwith roughly equal frequencies in the wild-type and mutantteratocarcinomas and independently of the presence ofvimentin in the host. Fig. 2 illustrates the major cell types

obtained in the teratocarcinomas derived from representativesamples (Vim+/+ ES cells injected into Vim+/+ mouse andVim−/− ES cells injected inVim−/− mouse). In both we canobserve ectodermal derivatives like neural tissue, neuroglia(compare Fig. 2B and b) and dermal epithelium (Fig. 2C,c);endodermal derivatives like digestive and respiratory epithelia(Fig. 2D,d) and glandular tissues (Fig. 2E,e); and mesodermalderivatives like cartilage and bone, even with hematopoieticcells (Fig. 2F,f) and smooth and striated muscle (Fig. 2G,g).Extraembryonic material was represented by the presence ofparietal endoderm, with its red colour characteristic of hyalin

Fig. 4. In situ apoptosis detectionby TUNEL method interatocarcinomas obtained fromcontrol ES cells injected in wild-type mice (Vim+/+→Vim+/+) andvimentin-lacking ES cells injectedin vimentin-lacking mice(Vim−/−→Vim−/−). (A,B) Generalview of representative tumours;(C,D) fibroblastic cells; (E,F) cartilaginous cells. Nucleifragmentation in apoptotic cells isevidenced by the brown colourwhich corresponds to peroxidasestaining (arrows). ca=cartilaginouscells. Nuclei were counterstainedwith Mayer’s hematoxylin. ×160(A,B); ×400 (C-F).

Table 3. Tumour weight of teratocarcinomas obtained byinjection of different ES cells clones in Vim+/+ and Vim−/−

miceES cell clone Tumour weight (g)

Vim+/+ host mice Vim−/− host mice 3w* 6w 3w 6w

Vim−/− 52 0.66±0.28 4.22±1.50 0.62±0.31 5.03±2.31Vim−/− 70 0.40±0.28 2.43±1.75 0.98±0.37 3.68±2.40Vim+/+ (CK35) 0.67±0.55 6.26±3.72 0.21±0.16 2.77±1.70

Results are expressed as mean ± s.e.m. ANOVA test revealed absence ofsignificant differences between groups.

*w=weeks of development of tumours in host mice.

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substance, in tumours arising from all combinations of ES andmice (Fig. 2A,a). We have not found other tissues, known toappear less frequently in teratocarcinomas, such as renal orhepatic tissue (Gaillard, 1974).

The heterogeneity in the weight of teratocarcinomas (seeTable 3) appears to be also reflected by variations in thecomplexity in organization and maturity of teratocarcinomasobtained. Indeed, histology of the tumours depended on theirsize, independently of the ES cell type or host animal of origin.Thus, in larger tumours, excised 6 weeks after injection, thedifferent tissues appeared more differentiated, whereas smallertumours contained more undifferentiated or immaturestructures.

Immunohistochemistry and apoptosis interatocarcinomasIn order to visualize vimentin expression in the differenttumours, we performed an immunohistochemical study oftumour sections using a anti-vimentin polyclonal antibody(Dupouey et al., 1985). Vimentin could be visualized inmuscle, cartilage, endothelial and fibroblastic cells interatocarcinomas obtained by injection of Vim+/+ ES cells intoVim+/+ mice (Fig. 3A,B). In tumours obtained from Vim−/− EScells injected in Vim+/+ mice, some vimentin-stained cellswere observed (Fig. 3C). This staining clearly corresponds tothe host cell contribution to the tumour. In fact, vimentin-positive cells in tumours arising from vimentin-lacking EScells consist mainly of endothelial cells of blood vessels andsome fibroblastic areas corresponding to stromal cells whichhave been captured from the vimentin-positive host by thetumour lacking vimentin to allow the tumour to grow. Fig. 3Dillustrate the coexistence of blood vessels coming from the host(positively stained) and those originated from thedifferentiation of the Vim−/− ES cells of origin (negativelystained), indicating that angiogenesis seems to be normallydeveloped from cells lacking vimentin. Fig. 3E illustrates arepresentative tumour grown from Vim+/+ ES cells in a Vim−/−mouse. In this combination, vimentin staining shows a lessuniformity compared to tumours obtained from vimentin-positive cells injected in wild-type animals. This differenceresults from the absence of host contribution to vimentinstaining, and it reveals the contribution of vimentin comingonly from ES cells (Fig. 3E). Fig. 3F shows the presence ofnormal vascularization contributed by the host withoutvimentin, with endothelial cells being negative for vimentinstaining. As expected, no vimentin was detected in tumoursderived from ES cells lacking vimentin injected in Vim−/−animals.

Specific staining of endothelial cells with anti-Factor VIIIantibody (Dako, Germany) confirmed that the host contributionto vimentin staining in the tumours coming from Vim−/− EScells injected in Vim+/+ mice consisted mainly in bloodvessels. Detailed analysis and quantification of stainingobtained with endothelial cell markers revealed no differencesin tumour vascularization in teratocarcinomas arising fromdifferent combinations of ES and mice with or withoutvimentin (data not shown). Thus, the absence of vimentin didnot seem to have any effect in the vascularization of theteratocarcinomas obtained in our system.

To determine if vimentin plays a role in apoptosis in ourteratocarcinoma system, we searched for differences in

apoptotic cells in tumour sections of the teratocarcinomascorresponding to all combinations of ES cells and mice withor without vimentin. By TUNEL immunocytochemistry assay,apoptosis was found in 15-20% of cells in all types of tumours,and no differences were found between all the possiblecombinations. Fig. 4 illustrates similar apoptotic stainingpatterns obtained with combinations containing or lackingvimentin in ES cells and mice (Fig. 4A,B). In both cases,apoptosis was mainly observed in fibroblastic (Fig. 4C,D) andcartilaginous cells (Fig. 4E,F)

DISCUSSION

We have investigated the possible involvement of vimentin intumorigenesis. For this purpose, we chose the model ofteratocarcinomas obtained by ectopic injection of ES cells intosyngeneic mice. We used vimentin knock-out mice recentlygenerated in our laboratory (Colucci-Guyon et al., 1994) asthey represent a source of both animals and ES cells devoid ofvimentin and therefore offer a powerful tool to address the roleof vimentin in tumour progression.

We isolated several ES cell lines from 129/Sv Vim−/− miceusing a standard protocol. The rate of success obtained in thisstudy (12% of explanted blastocysts) was within the samerange reported by other authors (Robertson, 1987; Nagy et al.,1993) and by us (Kress et al., 1998). Thus, the absence ofvimentin did not affect the isolation of ES cell lines from129/Sv blastocysts. In addition, the 15 ES cell lines derived didnot exhibit significant differences neither in morphology or inproliferating potential with respect to other wild-type 129/SvES cells derived in our laboratory (Camus et al., 1996; Kresset al., 1998). Furthermore, these cell lines were highlycompetent for germ-line transmission. Thus, each of four malechimera from two independent clones analysed transmitted ESgenotype to 100% of their progeny. Although these numbersare still low, it is intriguing to consider the possibility of apositive effect of the absence of vimentin on the ability of EScells to colonise a host embryo. Thus, it will be interesting toconfirm these observations and extend them to the other ES cellclones isolated in this study.

ES lacking vimentin were not compromised in their abilityto form teratocarcinomas in vivo. Thus, the percentage oftumours obtained with ES cells without vimentin injected intowild-type 129/Sv mouse strain, was near 100% (97.5%) of totalinjected mice. 100% efficiency was obtained with controlVim+/+ ES cells injected into wild-type mouse, in goodagreement with the fact that 129/Sv background is highlypermissive for the development of teratocarcinomas (Gardnerand Brook, 1997). Moreover, both normal and mutant cellsgave rise to ectodermal, mesodermal and definitive endodermalderivatives, suggesting that vimentin deficiency did notproduce a generalized effect on lineage commitment in thismodel and confirmed that the absence of vimentin does notprevent cell proliferation and differentiation at specific stagesor sites during development in vivo. This result is also inkeeping with the fact that we observed no significantdifferences in proliferation or doubling time between vimentin-lacking and wild-type ES cells. However, and in contrast, wehave recently observed that the absence of vimentin decreasescell proliferation in primary cultures of fibroblasts and

F. Langa and others

3471Teratocarcinomas induced by vimentin-lacking ES cells

astrocytes, cell types of mesodermal and ectodermal origin,respectively (our unpublished data). Although the reasons forthese differences remain elusive, it could mean that theinfluence of the vimentin network on cell proliferation isdepending on the differentiation state of the cell. Finally, inspite of a striking tumour size heterogeneity, kinetics ofgrowing tumours did not seem to reveal clear-cut differencesexcept for an initial decreased growth of wild-type ES cellsin mutant mice, probably due to a transient immunologicalrejection.

Our mice bearing a vimentin null mutation on a 129/Svbackground have allowed us to address the possibleinvolvement of vimentin in the permisiveness of the hostanimal surrounding tissues for tumour growth. In the processof tumour formation, host animal provides the surroundingstroma (infiltrating cells like macrophages, endothelial cells,lymphocytes and fibroblasts (Grégoire and Lieubeau, 1995),which is responsible for the vascular supply, tumour growthand metastasis (Hanahan and Folkman, 1996). The absence ofvimentin in host mice did not affect the efficiency of tumourprogression, the cell types obtained nor the differentiation stateof teratocarcinomas. In addition, vascularization provided bythe host remained unchanged in absence of vimentin, despitethe high level of vimentin expression in endothelial cells.Indeed, although the exact role of vimentin in vascularresponses remains to be elucidated, Terzi et al. (1997) havedemonstrated that the lack of vimentin affects vascularadaptation to pathological situations such as reduction of renalmass. In other studies, a key role of vimentin in vascularresistance to mechanical stress has also been described(Henrion et al., 1997; Schiffers et al., 2000). In our model ofteratocarcinomas, absence of vimentin in the host mice did notsignificantly alter any of the tumoral parameters analysedincluding vascularization of tumours nor the development ofmetastases. In fact, metastases are infrequent in experimentalteratocarcinomas (Nicolas et al., 1981) except for special casesof testicular injections of EC cells (Stevens, 1970). All tumoursobtained in our model grew progressively and remainedlocalized without metastazing elsewere even after 6 weeks ofinjection and despite the large size of the tumours.

It has been described that male mice of 129 substrainsdevelop spontaneous testicular teratocarcinomas at a relativelyhigh frequency, ranging from 0.26% to 33%, depending on thesubstrain. Normal 129/Sv mice have been reported to havearound 1% incidence of spontaneous teratocarcinomas(Stevens, 1973). In our colony, however, neither wild-type norvimentin-null 129/Sv males have ever developed obviousteratomas or teratocarcinomas. Harvey and co-workers, whoset out to compare spontaneous tumorigenesis between wild-type and p53−/− deficient homozygous 129/Sv mice also founda total absence of spontaneous teratocarcinomas in their colonyof wild-type mice while inactivation of the p53 gene entaileda high incidence (over 50%) of aggressive teratocarcinomas inthe males and the development of various types of tumors(Donehower et al., 1992; Harvey et al., 1993). Our vimentinnull mice, as well as their wild-type counterparts, did notexhibit an obvious susceptibility to tumorigenesis, as nospontaneous tumor could be found in any of the mice undermacroscopic or microscopic inspection. Thus, we can concludethat lack of vimentin does not confer a predisposition to aparticular type of tumour, and this holds true even for old mice

as we have already examined a considerable number of veryold mice of both sexes.

In summary, we have taken advantage of vimentin knock-out mice to address the possible role of vimentin intumorigenesis, using as experimental system the induction ofteratocarcinomas by ES cells. We have performed an extensiveanalysis of the tumours induced under various combinations ofmutant and wild-type cells/animals. Taken together, our resultsshow unambigously that the absence of vimentin did not haveany effect on the various parameters which characterize thesetumours, including rate of tumour formation, tumour size andvascularization, as well as cell types and differentiation.However, our study has not fully addressed the possible roleof vimentin in the metastatic potential of malignant cells.Indeed, vimentin is coexpressed with keratins in varioustumour cells during their migration and their evolution fromthe primary to the metastatic tumour stage (Ramaekers et al.,1983; Thompson et al., 1992; Chu et al., 1996). In addition,vimentin overexpression in breast carcinoma model leads toaugmentation of motility and invasiveness in vitro, which canbe transiently down-regulated by treatment with antisenseoligonucleotides to vimentin (Hendrix et al., 1996, 1997). Apossible role of vimentin in epithelium-mesenchymaltransitions associated with tumours has also been proposed(Gilles and Thompson, 1996). In this context, it will beinteresting to use our Vim−/− mice to determine if thecorrelations observed in the various studies are merelycircumstantial or do have a functional significance in vivo.Therefore, our mice lacking vimentin constitute a selected toolto continue to investigate in vivo and in vitro models oftumorigenesis of specialized cells and the relationship betweenvimentin and tumorigenesis.

We are grateful to Pr. Jean A. Gaillard for his invaluable help withhistological analysis of teratocarcinomas and his warm support.Sabine Maurin, Nicole Wurscher and Patrick Ave are acknowledgedfor technical assistance. We thank Dr Jonnathan Weitzman forthoughtful scientific insight and critical reading of the manuscript. DrPatricia Baldacci and Dr Jacqueline Barra are aknowledged forstimulating discussion. This work was supported by grants fromCNRS and Pasteur Institut. F. L. was a recipient of a EEC and FRMpostdoctoral fellowships.

REFERENCES

Abbondanzo, S. J., Gadi, I. and Stewart, C. L. (1993). Derivation of embryonicstem cell lines. In Guide to Techniques in Mouse Development, vol. 225 (ed. P. M. Wassarman and M. L. DePamphilis), pp. 803-822. Academic Press, Inc.,San Diego, California.

Azumi, N. and Battifora, H. (1987). The distribution of vimentin and keratin inepithelial and nonepithelial neoplasms. Am. J. Clin. Pathol.188, 286-296.

Bradley, A. (1987). Production and analysis of chimaeric mice. InTeratocarcinomas and Embryonic Stem Cells: a Practical Approach(ed. E. J.Robertson), pp. 131-151. IRL Press, Oxford.

Buley, I. D., Gatter, K. C., Heryet, A. and Masson, D. Y. (1987). Expression ofintermediate filament proteins in normal and diseased thyroid glands. J. Clin.Pathol.40, 136-142.

Camus, A., Kress, C., Babinet, C. and Barra, J. (1996). Unexpected behavior ofa gene trap vector comprising a fusion between the Sh ble and the lacZ genes.Mol. Rep. Dev.45, 255-263.

Chan, D., Goate, A. and Puch, T. T. (1989). Involvement of vimentin in the reversetransformation reaction. Proc. Nat. Acad. Sci. USA86, 2747-2751.

Chu, Y. W., Seftor, E. A., Romer, L. H. and Hendrix, M. J. (1996). Experimentalcoexpression of vimentin and keratin intermediate filaments in human melanomacells augments motility. Am. J. Pathol.148, 63-69.

3472

Cochard, P. and Paulin, D. (1984). Initial expression of neurofilaments andvimentin in the central and peripheral nervous system of the mouse embryo invivo. J. Neurosci.4, 2080-2094.

Colucci-Guyon, E., Portier, M. M., Dunia, I., Paulin, D., Pournin, S. andBabinet, C. (1994). Mice lacking vimentin develop and reproduce without anobvious phenotype. Cell 79, 679-694.

Colucci-Guyon, E., Giménez y Ribotta, M., Maurice, T., Babinet, C. and Privat,A. (1999). Cerebellar defect and impaired motor coordination in mice lackingvimentin. Glia 25, 33-43.

Damjanov, I. and Solter, D. (1974). Embryo-derived teratocarcinoma elicitsplenomegaly in syngeneic hosts. Nature249, 569-571.

Damjanov, I. (1993). Teratocarcinoma: neoplastic lessons about normalembryogenesis. Int. J. Dev. Biol.37, 39-46.

Domagala, W., Lasota, J., Bartowiak, J., Weber, K. and Osborn, M. (1990).Vimentin is preferentially expressed in human breast carcinoma with lowoestrogen receptor and high Ki-67 growth fractionAm. J. Pathol.136, 219-227.

Domagala, W., Striker, G., Szadowska, A., Dukowicz, A., Harezga, B. andOsborn, M. (1994). p53 protein and vimentin in invasive ductal NOS breastcarcinoma-relationship with survival and sites of metastases. Eur. J. Cancer30A,1527-1534.

Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, C. A. J., Butel, J. and Bradley, A. (1992). Mice deficient for p53 aredevelopmentally normal but susceptible to spontaneous tumours. Nature 356,215-221.

Dupouey, P., Benjelloun, S. and Gomès, D. (1985). Immunohistochemicaldemonstration of an organized cytoarchitecture of the radial glia in the CNS ofthe embryonic mouse. Dev. Neurosci.7, 81-93.

Eckes, B., Dogic, D., Colucci-Guyon, E., Wang, N., Maniotis, A., Ingber, D.,Merckling, A., Langa, F., Aumailley, M., Delouvée, A., Koteliansky, V.,Babinet, C. and Krieg, T. (1998). Impaired mechanical stability, migration, andcontractile capacity in vimentin-deficient fibroblasts. J. Cell Sci.111, 1897-1907.

Eiden, M. V., MacArthur, L. and Okayama, H. (1991). Suppression of thechemically transformed phenotype of BHK cells by a human cDNA. Mol. Cell.Biol. 11, 5321-5329.

Franke, W. W., Grund, C., Kuhn, C., Jackson, B. W. and Illmensee, K. (1982).Formation of cytoskeletal elements during mouse embryogenesis. III. Primarymesenchymal cells and the first appearance of vimentin filaments. Differentiation23, 43-59.

Fuchs, E. and Weber, K. (1994). Intermediate filaments: structure, dynamics,function, disease. Annu. Rev. Biochem. 63, 345-382.

Fürst, D. O., Osborn, M. and Weber, K. (1989). Myogenesis in the mouse embryo:Differential onset of expression of myogenic proteins and the involvement of titinin myofibril assembly. J. Cell Biol.109, 517-527.

Gaillard, J. A. (1974). Differentiation and organization in teratomas. Neoplasia andCell Differentiation (ed. G. V. Sherbet), pp. 319-349. S. Karger, Basel,Switzerland.

Galou, M., Colucci-Guyon, E., Ensergueix, D., Ridet, J.-L., Gimenez y Ribotta,M., Privat, A., Babinet, C. and Dupouey, P. (1996). Disrupted glial fibrillaryacidic protein network in astrocytes from vimentin knockout mice. J. Cell Biol.133, 853-863.

Gardner, R. L. and Brook, F. A. (1997). Reflections on the biology of embryonicstem (ES) cells. Int. J. Dev. Biol.41, 235-243.

Gilles, C. and Thompson, E. W. (1996). The epithelial to mesenchymal transitionand metastatic progression in carcinoma. The Breast J.2, 83-96.

Grégoire, M. and Lieubeau, B. (1995). The role of fibroblasts in tumor behavior.Cancer Metast. Rev.14, 339-350.

Hanahan, D. and Folkman, J. (1996). Patterns and emerging mechanisms of theangiogenic switch during tumorigenesis. Cell 86, 353-364.

Harvey, M., McArthur, M. J., Montgomery, J. C., A., Bradley, A. andDonehower, L. A. (1993). Genetic background alters the spectrum of tumors thatdevelop in p53-deficient mice. FASEB J.7, 938-943.

Heatley, M., Whiteside, C., Maxwell, P. and Toner, P. (1993). Vimentin in benignand malignant lesions in the human mammary gland. J. Clin. Pathol.46, 441-445.

Hendrix, M. J., Seftor, E. A., Chu, Y. W., Trevor, K. T. and Seftor, R. E. (1996).Role of intermediate filaments in migration, invasion and metastasis. CancerMetast. Rev.15, 507-525.

Hendrix, M. J. C., Seftor, E. A., Seftor, R. E. B. and Trevor, K. T. (1997).Experimental co-expression of vimentin and keratin intermediate filaments inhuman breast cancer cells results in phenotypic interconversion and increasedinvasive behavior. Am. J. Pathol.150, 483-495.

Henrion, D., Terzi, F., Matrougui, K., Duriez, M., Boulanger, C. M., Colucci-Guyon, E., Babinet, C., Briand, P., Friedlander, G., Poitevin, P. and Lévy, B. I. (1997). Impaired flow-induced dilation in mesenteric resistance arteriesfrom mice lacking vimentin. J. Clin. Invest.100, 2909-2914.

Hilberg, E. and Wagner, E. F. (1992). Embryonic stem (ES) cells lackingfunctional c-jun: consequences for growth and differentiation, AP-1 activity andtumorigenicity. Oncogene 7, 2371-2380.

Holck, S., Pedersen, L., Schiodt, T., Zedeler, K., Mouridsen, H. and Schidt, T.(1993). Vimentin expression in 98 breast cancers with medullary features and itsprognostic significance. Virchows Arch.,422, 475-479.

Hooper, M. L. (1992). Embryonic carcinoma and embryonal stem cells. InEmbryonal Stem Cells. Introducing Planned Changes into the Animal Germline,Vol. 1: Modern Genetics(ed. H. J. Evans), pp. 5-23. Harwood Academic GmbH:Chur, Switzerland.

Houle, J. and Fedoroff, S. (1983). Temporal relationship between the appearanceof vimentin and neural tube development. Dev. Brain Res.9, 189-195.

Kartenbeck, J. (1989). Intermediate filament proteins. Diagnostic markers intumour pathology. Interdiscip. Sci. Rev.14, 278-283.

Kress, C., Vandormael-Pournin, S., Baldacci, P., Cohen-Tannoudji, M. andBabinet, C. (1998). Nonpermissiveness for mouse embryonic stem (ES) cellderivation circumvented by a single backcross to 129/Sv strain: establishment ofES cell lines bearing the Omd conditional lethal mutation. Mamm. Genome9,998-1001.

Lane, E. B., Hogan, L. M., Kurkinen, M. and Garrels, J. I. (1983). Co-expressionof vimentin and cytokeratin in parietal endoderm cells of early mouse embryo.Nature303, 701-704.

Leader, M., Collins, M., Patel, J. and Henry, K. (1987). Vimentin: an evaluationof its role as a tumour marker. Histopathology11, 63-72.

Lendahl, U., Zimmerman, L. B. and McKay, R. D. G. (1990). CNS stem cellsexpress a new class of intermediate filament protein. Cell 60, 585-595.

Martin, G. R. (1980). Teratocarcinomas and mammalian embryogenesis. Science209, 668-676.

Martin, G. R. (1981). Isolation of a pluripotent cell line from early mouse embryoscultured in medium conditioned by teratocarcinoma stem cells. Proc. Nat. Acad.Sci. USA 78, 7634-7638.

McNutt, M. A., Bolen, J. W., Gown, A. M., Hammar, S. P. and Vogel, A. M.(1985). Co-expression of intermediate filaments in human epithelial neoplasms.Ultrastruct. Pathol. 9, 31-43.

Mroz, K., Carrel, L. and Hunt, P. A. (1999). Germ cell development in the XXYmouse: evidence that X chromosome reactivation is independent of sexualdifferentiation. Dev. Biol.207, 229-238.

Nagy, A., Rossant, J., Abramow-Newerly, W. and Roder, J. C. (1993). Derivationof completely cell culture-derived mice from early passage embryonic stem cells.Proc. Nat. Acad. Sci. USA90, 8424-8428.

Nicolas, J. F., Jakob, H. and Jacob, F. (1981). Teratocarcinoma-derived cell linesand their use in the study of differentiation. In Functionally Differentiated CellLines(ed. G. Satol), pp. 185-210. A. R. Liss, New York.

Osborn, M. and Weber, K. (1982). Intermediate filaments: Cell-type-specificmarkers in differentiation and pathology. Cell 31, 303-306.

Osborn, M. and Weber, K. (1983). Biology of disease: Tumor diagnosis byintermediate filament typing-A new tool for surgical pathology. Lab. Invest.48,372-394.

Ramaekers, F. C. S., Haag, D., Kant, A., Moesker, O., Jap, P. H. K. and Vooijs,G. P. (1983). Coexpression of keratin- and vimentin-type intermediate filamentsin human metastatic carcinoma cells. Proc. Nat. Acad. Sci. USA80, 2618-2622.

Raymond, W. A. and Leong, A. S. Y. (1989). Vimentin-a new prognosticparameter in breast carcinoma? J. Pathol. 158, 107-114.

Robertson, E. J. (1987). Embryo-derived stem cell lines. In Teratocarcinomas andEmbryonic Stem Cells: a Practical Approach(ed. E. J. Robertson), pp. 71-112.IRL Press: Oxford.

Schiffers, P. M. H., Henrion, D., Boulanger, C. M., Colucci-Guyon, E., Langa,F., van Essen, H., Fazzi, G. E., Levy, B. I. and De Mey, J. G. R. (2000). Alteredflow-induced arterial remodeling in vimentin-deficient mice. Arterioscler.Thromb. Vasc. Biol. 20, 611-616.

Stevens, L. C. (1970). The development of transplantable teratocarcinomas fromintratesticular grafts of pre-and post-implantation mouse embryos. Dev. Biol.21,364-370.

Stevens, L. C. (1973). A new inbred strain in mice (129/ter Sv) with a highincidence of spontaneous congenital testicular teratomas. J. Nat. Cancer Inst.50,235-242.

Terzi, F., Henrion, D., Colucci-Guyon, E., Federici, P., Babinet, C., Levy, B. I.,Briand, P. and Friedlander, G. (1997). Reduction of renal mass is lethal in micelacking vimentin. J. Clin. Invest.100, 1520-1528.

Thompson, E. W., Soonmyoung, P., Brünner, N., Sommers, C. L., Zugmaier,G., Clarke, R., Shima, T. B., Torri, J., Donahue, S., Lippman, M. E., Martin,G. R. and Dickinson, R. B. (1992). Association of increased basementmembrane invasiveness with absence of estrogen receptor and expression ofvimentin in human breast cancer cell lines. J. Cell Phys. 150, 534-544.

Triman, K. L., Davisson, M. T. and Roderick, T. H. (1975). A method forpreparing chromosomes from peripheral blood in the mouse. Cytogenet. CellGenet.15, 166-176.

Viale, G., Gambacorta, M., Dell’Orto, P. and Coggi, G. (1988). Coexpression ofcytokeratins and vimentin in common epithelial tumours of the ovary: animmunocytochemical study of eighty-three cases. Virchows Arch. A. Pathol. Anat.Histopathol.413, 91-101.

F. Langa and others