(CANCER RESEARCH 50, 5526-5530. September I. 1990|

Establishment and Characterization of a Tumorigenic Murine Vascular EndothelialCell Line (F-2)Ken-Ichi Toda,1 Kaoru Tsujioka, Yukiya Maruguchi, Kazuhiro Ishii, Yoshiki Miyachi, Kagemasa Kuribayashi, and

Sadao ImamuraDepartment of Dermatology (K-I. T., K. T., Y. Ma., Y. M., S. I.], Institute for Immunology, Faculty of Medicine [K. K.], and Institute for Virus Research ¡K.l.], KyotoUniversity, Kyoto 606, Japan

ABSTRACT

A new murine cell line, designated F-2, was established from anultraviolet light-induced tumor which developed on the back skin of aBALB/c x C57BL/6 F¡-nu/nunude mouse. More than 1 x IO5F-2 cellsinjected into nude mouse skin produced rapidly developing hemangiom-atous lesions. F-2 had a 14-h doubling time under 10% fetal calf serum-containing Dulbecco's modified Eagle's medium culture conditions without any cell growth factor supplementation, and it showed "cobblestone"'

appearance at confluency. F-2 was able to rapidly differentiate on Matri-gel with resultant fine network structure and tubule formation. Ultrastructural observations of the tubule demonstrated that the F-2 cells wereconnected to each other by intermediate junctions and arranged to formspaces, but that no Weibel-Palade bodies were found in the cytoplasm.F-2 also showed the active uptake of fluorescent-labeled acetylated low-density lipoprotein at 37°Cbut not at 4 < , and it showed prominent

binding to the lectin, Griffonia simplicifolia I agglutinin. The addition offibroblast growth factor did not facilitate the growth of F-2 cells. Thesefindings strongly suggest that F-2 is a transformed cell line with tumori-genicity and vascular endothelial cell properties, and it may be useful inthe study of vascular tumor biology.

INTRODUCTION

Malignancy of vascular tissues is usually fatal and hardlycontrolled by medication (1). Since vascular tissue tumors arerelatively rare in the clinical field and few tumorigenic cell linespreserving vascular endothelial cell properties are available inbasic research, information about the regulatory mechanism(s)of endothelial cell tumorigenesis is quite obscure. To learnmore about tumor biology of vascular endothelial cells, it isimportant to develop tumorigenic vascular endothelial celllines.

Recently, we investigated mid-wave length UVB2 tumor induction (2) in BALB/c x C57BL/6 F,-nu/nu (hereafter calledCB6F|-/i«/Hw) mice, initiated tissue culture from the inducedtumors, and established several cell lines (3). Although theinduced tumors were found to be fibrosarcomas or squamouscell carcinomas (2) and the most established cell lines from theinduced tumors generated fibrosarcomas in vivo, we were alsoable to obtain a unique cell line, designated F-2, which showedcutaneous cyst formation after its inoculation into allogenic orsyngenic nude mouse skin. In this paper, we describe themorphological and cell biological characteristics of F-2, providing strong evidence that F-2 was originated from vascularendothelial cells.

Received 11/28/89; revised 5/1/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed, at Department ofDermatology, Faculty of Medicine, Shogoin, Sakyo-ku, Kyoto, Japan 606.

2The abbreviations used are: UVB. ultraviolet B; FN, fibronectin: DMEM,Dulbecco's modified Eagle's medium: FBS. fetal bovine serum: vWF, von Wille-brand factor; Di-I-Ac-LDL, acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl-l-3-,3,3',3'-tetramethylindocarbocyanine percholate; GSA-I, Griffonia simplicifolia I agglutinin; UEA-I, Ulex europaus I agglutinin; FGF. fibroblast growth factor: HUVEC, human umbilical vein endothelial cells.

MATERIALS AND METHODS

Mice. Breeding pairs of nude mice (nu/nu) and their heterozygouslittermates (nu/+) with BALB/c and C57BL/6 backgrounds were originally purchased from Clea Japan Inc., Tokyo, Japan. CB6F,-nu/numice were raised and maintained under pathogen-free conditions inlaminar flow hoods in the Facility of Experimental Animals, Facultyof Medicine, Kyoto University.

UVB Irradiation. UVB irradiation was performed by the methodpreviously described (4). Briefly, UV irradiation was administered witha bank of 4 sun lamps (FL20SE; Toshiba Electric Co., Tokyo, Japan).These tubes provided a continuous UV spectrum with a peak at 305nm. Tube-to-target distance was 15 cm. UV output measured by a UVradiometer was 0.44 mW/cm2 at 305 nm. The mice were irradiated for

1 h, 3 times/wk for 27 wk.Establishment of Cell Lines. The UV-induced tumors were adapted

to culture from the first transplant generation. Several cell lines, including F-2, which was denoted $-2 in our previous paper (3), and afibrosarcoma cell line, c5-ld, were established as clones by limitingdilution culture immediately after adaptation in vitro and were maintained in DMEM (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% FBS (G1BCO, New York, NY), 100 units/ml ofpenicillin G, and 100 Mg/m' of streptomycin at 37°Cin a humidified

atmosphere of 5% CO2 in air (3). For the doubling time determination,cells were plated in duplicate at 2 x l O4cells/60-mm Petri dish (Corning

Glass Works, Corning, NY). Cell numbers were counted on Days 0, 1,2, 3, 4. 5, 6, and 7, and doubling times were derived from the exponential growth phase. All cells used in this study were tested for Myco-plasma contamination and were found to be negative during the courseof the experiments.

Chromosomal Analysis. Chromosome preparations were achieved byharvesting 3- to 4-day-old subcultures after incubation at 37°Cwith 0.2

MgofColcemidper 10 ml of DMEM with 10% FBS for 4 h. Metaphasepreparations followed the techniques described previously (5) using a20-min hypotonie treatment with 0.075 M KC1. All slides were stainedwith a conventional Giemsa solution.

Tumorigenicity. To determine in vivo tumorigenicity, IO5to IO6viableF-2 cells were injected into the backs of CB6F,-nu//i« mice s.c. Thetumors which developed were dissected and processed for light microscopic observations.

Cell Culture on Extracellular Matrices. F-2 cells (5 x 10') were

cultured on either immobilized FN (Sigma, St. Louis, MO), type Icollagen (Vitrogen 100; Collagen Corp., CA), or basement membranecomponents (Matrigel; Collaborative Research, Inc.. Waltham, MA),which were reconstituted as before by incubating a 35-mm tissue culturedish (Corning) with each substrate (6, 7). After the cells were culturedfor 12 to 24 h on these matrices, the morphology and reorganizationof the cells were monitored under a phase-contrast microscope (Nikon,Tokyo, Japan). In some experiments, F-2 cells cultured on Matrigelwere processed for electron microscopic observations by the previouslydescribed method (8).

Uptake Study with Fluorescent Di-I-Ac-LDL. F-2 or <î-ldcells wereincubated for 4 h at 37°Cwith DMEM containing the 10 pg/m\ of Di-

I-Ac-LDL (BiomédicalTechnologies, Inc., Cambridge, MA) (9). Afterthe medium was removed, the cells were washed 3 times and visualizedunder a fluoresent microscope (Nikon) with filters adjusted for rho-damine fluorescence.

Lectin Stain. F-2 cells were used for lectin histochemical studies,where the lectins, GSA-I or UEA-I, were stained as previously described(10). Briefly, the cells, which were cultured on chamber slides (Lab-

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TUMORIGENIC MURINE VASCULAR ENDOTHELIAL CELL LINE

Tek, Naperville, IL), were air dried, overlayered with biotinylated GSA-I (EY Lab., San Mateo, CA) or biotinylated UEA-I (Vector Lab.,Burlingame, CA) for 30 min at room temperature, rinsed with phosphate-buffered saline, and reacted with fluorescein ¡sothiocyanate-labeled avidin D (Zymed Lab., San Francisco, CA) for 30 min at roomtemperature. All samples were rinsed with phosphate-buffered salineand examined under a Nikon fluorescence microscope.

Effects of FGF on F-2 Cell Growth. F-2 cells or HUVEC were seededat 1 x IO3 cells per 6-cm culture dish (Corning) in DMEM with 5%FBS or in the medium containing 100 ng/ml of bovine pituitary gland-derived FGF (Collaborative Research, Bedford, MA). The medium waschanged every other day. The cell number was counted on Days 1, 2,3, 4, and 5.

RESULTS

Morphology in Vivo.. Tumorigenesis studies revealed that aquantity of more than 1 x 10s F-2 cells (1, 5, or 10 x 10s cells,

each in 3 mice) generated soft hemorrhagic tumors in nudemouse skin (Fig. 1). The tumor induced by F-2 cells adhered tothe underlying tissue and was composed of an encapsulated cystfilled with blood. Light microscopic studies demonstrated thatthe tumor was composed of a blood-filled cyst lined by a fewlayers of tumor cells, which occasionally proliferated into thecystic cavity, forming capillary lumina-like spaces. Nuclearatypism and mitotic figures were not remarkable. Capillaryproliferations were noticed around the cystic tumor (Fig. 2).

Morphology in Vitro. The F-2 cells placed on plastic culturedishes showed a "cobblestone" appearance at confluency (Fig.

3). Under the present culture condition, the doubling time ofF-2 cells was 14 h. When cells were cultured on FN or type Icollagen, the cells showed anastomosing reticular structureswithin 24 h (Fig. 4A). The cells cultured on Matrigel formedhoneycomb-like network structures within 12 h (Fig. 4B). Smallislands composed of F-2 cells were connected to each other bystraight cellular cords. An F-2 cell was much more closelyapposed to the adjacent cell on Matrigel than on plastic, FN,or type I collagen. The ultrastructural studies of the cellularcord generated on Matrigel showed that, in its cross-section, F-2 cells were arranged to make spaces among themselves with

Fig. 2. Light microscopic observations of the developed tumor. The tumorwas composed of an encapsulated cyst filled with blood (A), x 40. The cyst waslined by a few layers of tumor cells, which occasionally formed capillary' lumina-like spaces (B), x 400.

Fig. 1. Tumor induction following F-2 cell inoculation. One x IO6 F-2 cellswere inoculated into a BALB/c nude mouse. One wk after the inoculation, a largehemorrhagic and cystic tumor developed at the inoculation site.

Fig. 3. Phase-contrast photomicrograph of cultured F-2 cells. The cells sa characteristic "cobblestone" appearance at confluency. x 200.

resultant tubular structure formation (Fig. 5^4). The cells wereconnected to each other by intermediate junctions and hadpinocytotic vesicles, but no Weibel-Palade body-like organdíeswithin their cytoplasm (Fig. 5B). Di-I-Ac-LDL was taken upby F-2 under serum-free culture conditions at 37°Cbut not at4°C(Fig. 6), whereas it was not taken up by a fibrosarcoma cell

line, d-ld. Furthermore, GSA-I, but not UEA-I, bound to thecell surface (Fig. 7).

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TUMORIGENIC MURINE VASCULAR ENDOTHELIAL CELL LINE

¿:î^%.- -^ Jl

BFig. 4. Culture of F-2 cells on type I collagen or Matrigel. F-2 cells cultured

on collagen gels exhibited an anastomosing reticular structure in 24 h (A), x 100.When the cells were cultured on Matrigel for 12 h. they formed a finely organizedhoneycomb-like network structure (B). x 200. Small islands (stars) composed ofF-2 cells were connected to each other by straight cellular cords (arrows).

Fig. 5. Electron microscopic observations of the cross-section of cellular cord.F-2 ceils were arranged to make spaces (stars) among themselves (A) (x 4,500),and they were connected to each other by intermediate junctions (arrows) to formtubule-like structures. Each F-2 cell contained pinocytotic vesicles but no Weibel-Palade bodies in its cytoplasm (B) (x 14,000).

Chromosomal Analysis. Twenty-nine metaphase cells wereexamined with Giemsa stains. The chromosome number variedfrom 64 to 68, with a peak of 66 (Table 1).

Response of F-2 to FGF. The growth rate of F-2 cells wasmaintained in the culture medium with or without exogenousFGF, whereas that of HUVEC was greatly enhanced by theaddition of FGF to the medium (Fig. 8).

DISCUSSION

Present studies have clearly demonstrated that F-2 is a tu-morigenic cell line preserving vascular endothelial cell properties because (a) inoculation of F-2 into nude mouse skin produced hemangiomatous lesions, (b) F-2 was specifically positivefor the accelerated Di-I-Ac-LDL uptake, and (c) GSA-I wasable to bind on the cell surface of F-2 cells. The uptake of Di-I-Ac-LDL at an accelerated rate at 37°Cis a cytological marker

for vascular endothelial cells or macrophages (9), and GSA-Ibut not UEA-I is reported to selectively bind on the cell surfaceof murine endothelial cells or epithelial cells (11). On the otherhand, the classical criteria for identification of human vascularendothelial cells include "cobblestone" appearance in in vitro

culture, synthesis of vWF by the cells, and existence of Weibel-Palade bodies in the cytoplasm (12). Although F-2 cells exhibita "cobblestone" appearance, they were negative for the im-

munostaining against vWF (data not shown) and lacked Weibel-Palade bodies in their cytoplasm. However, vWF production is

Fig. 6. Di-I-Ac-LDL uptake by F-2 cells. F-2 cells took up Di-I-Ac-LDL at37°Cbut they did not at 4°C.

a marker for differentiated endothelial cells, and transformedcells may lose vWF producibility (13). Furthermore, a recentreport demonstrated that Weibel-Palade bodies were not alwaysreliable ultrastructural markers even for normal endothelialcells in a murine system ( 14). In these contexts, our observationsseem to be sufficient to conclude that F-2 was originated fromvascular endothelial cells. Also supporting our conclusion isevidence that F-2 cells rapidly generate fine honeycomb-like

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TUMORIGENIC MURINE VASCULAR ENDOTHELIAL CELL LINE

Fig. 7. Agglutinin binding to F-2 cells. F-2 cells were positive for GSA-Ibinding (A) but negative for UEA-I (B).

105-

DAYS

Fig. 8. Proliferation of F-2 cells and HUVEC in the culture medium with orwithout exogenous FGF. One thousand F-2 cells or HUVEC were seeded in 6-cm culture dishes in DMEM, supplemented with 5% FBS. After 5 days of culture,F-2 cells in the medium plus 100 ng/ml of FGF (•)grew similarly to those inthe medium only (O), while the number of HUVEC in the medium plus 100 ng/ml of FGF (A) increased about 6-fold compared with that in the medium only(A).

Table I Distribution of chromosome numbers in F-2

Chromosome64No.

of metaphase" 46556611676683

°After the subcultured F-2 cells were treated with Colcemid, metaphasepreparations were achieved by hypotonie treatment. Details are found in "Materials and Methods."

network and tubular structures under culture conditions withthe basement membrane component, Matrigel (7).

At present, two procedures have been reported for obtainingtumorigenic murine vascular endothelial cell lines, (a) Injectionof 1,2-dimethylhydrazine dihydrochloride s.c. induced malignant endothelioma and angiosarcoma in the liver of BALB/cmice, from which two endothelial cell lines, DIO and D14, wereestablished (15). However, it is unclear whether those linesretain endothelial cell properties other than Kaposi's sarcoma-

like tumor formation in vivo, (b) The other technique was tocreate transgenic mice with polyoma virus middle T-oncogeneexpression (16, 17) or to inoculate the in v/'/ra-constructed Rous

sarcoma virus variant encoding the middle T-antigens (18).These mice systemically developed multiple cavernous hemang-iomas, from which several highly tumorigenic endothelial celllines were established. These cell lines were rather differentiated, since they produced vWF, showed contact inhibition,and failed to grow in soft agar. Although F-2 cells are alsohighly tumorigenic, they do not exhibit middle T-oncogeneintegration (data not shown). Furthermore, F-2 cells do notshow contact inhibition, and they can grow in soft agar (datanot shown). These observations indicate that F-2 is a lessdifferentiated endothelial cell line than middle T-oncogene-integrated ones.

Tumors induced by UV irradiations in a murine system aregenerally fibrosarcomas (19) or squamous cell carcinomas (20)and only endotheliomas (21-23). Our previous study demonstrated that 89% of UVB-induced tumors were fibrosarcomas,and 11% were squamous cell carcinomas (2) but that there wereno hemangiomas. Therefore, the establishment of the F-2 cellline might be by chance. F-2 cells can be easily maintainedwithout the usual requirement of growth factors such as FGF(24) or tumor-conditioned media (25), and they do not respondto FGF stimulation, which suggests that they produce somegrowth factors like FGF and proliferate in an autocrine manner.

To our knowledge, F-2 is the first established tumorigenicvascular endothelial cell line derived from a UV-induced tumor.This cell line, which has the characteristics of high tumorigenic-ity in vivo as well as high growth activity without any specificsupplementation in vitro, may be useful for learning vasculartumor biology.

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1990;50:5526-5530. Cancer Res   Ken-Ichi Toda, Kaoru Tsujioka, Yukiya Maruguchi, et al.   Vascular Endothelial Cell Line (F-2)Establishment and Characterization of a Tumorigenic Murine

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