genetically fluorescent melanoma bone and organ...

Genetically Fluorescent Melanoma Bone and Organ MetastasisModels1

Meng Yang, Ping Jiang, Zili An, Eugene Baranov,Lingna Li, Satoshi Hasegawa,Maraya Al-Tuwaijri, Takashi Chishima,Hiroshi Shimada, A. R. Moossa, andRobert M. Hoffman 2

AntiCancer, Inc., San Diego, California 92111 [M. Y., P. J., Z. A.,E. B., L. L., S. H., M. A-T., R. M. H.]; Department of Surgery,University of California, San Diego, California 92103-8220 [M. Y.,S. H., M. A-T., A. R. M., R. M. H.]; and Department of Surgery,Yokohama City University School of Medicine, Yokohama, Japan236 [M. Y., S. H., T. C., H. S.]

ABSTRACTWe report here the establishment and metastatic prop-

erties of bright, highly stable, green fluorescent protein(GFP) expression transductants of the B16 mouse malignantmelanoma cell line and the LOX human melanoma line. Thehighly fluorescent malignant melanoma cell lines allowedthe visualization of skeletal and multiorgan metastases afteri.v. injection of B16 cells in C57BL/6 mice and intradermalinjection of LOX cells in nude mice. The melanoma cell lineswere transduced with the pLEIN expression retroviral vec-tor containing the GFP and neomycin resistance genes. Sta-ble B16F0 and LOX clones expressing high levels of GFPwere selected stepwisein vitro in levels of G418 of up to 800mg/ml. Extensive bone and bone marrow metastases ofB16F0 were visualized by GFP expression when the animalswere sacrificed 3 weeks after cell implantation. Metastasesfor both cell lines were visualized in many organs, includingthe brain, lung, pleural membrane, liver, kidney, adrenalgland, lymph nodes, skeleton, muscle, and skin by GFPfluorescence. This is the first observation of experimentalskeletal metastases of melanoma, which was made possibleby GFP expression. These models should facilitate futurestudies of the mechanism and therapy of bone and multior-gan metastasis of melanoma.

INTRODUCTIONCutaneous melanoma is increasing in incidence. This dis-

ease is largely confined to whites, in whom the age-adjusted

incidence rate in the United States is about 12 per 100,000persons and is 3-fold higher (30 per 100,000) in some geo-graphic areas (1–3). In 1935, only 1 in 1,500 persons developedmelanoma. The incidence has increased dramatically to 1 in 250persons in 1980 and to 1 in 135 persons in 1987. Assumingpresent trends, the incidence will be 1 in 90 persons by the year2000 (4). Therefore, the development of more effective methodsfor the prevention and treatment of this disease is urgent andwould be highly facilitated by a useful animal model that closelymimics the clinical situation.

During the past 15 years, experimental models of thehuman malignant melanoma LOX have been established inathymic nude mice and rats (5–8). Studies with these modelshave shown that the LOX cells have a preference for growth inthe lungs of the host.

In man, the lymph nodes, lung, brain, liver, and bone (9)are the most common sites of melanoma metastasis (10).

To develop effective therapeutics for this pathologicalprocess, a simple, highly reproducible, and easily used animalmodel is needed.

Although injection of B16 melanoma cells into the leftcardiac ventricle resulted in tumor colonies in the skeletal sys-tem and most organs of the mouse (11), this procedure iscomplicated and is not convenient enough to be used in large-scale investigation. There have not been any reports demonstrat-ing bone metastasis from B16 melanoma cells injected into thetail vein. In previous studies, i.v. injection of 105 melanomacells resulted in observable lung tumor colonies (11).

We have developed a new model of cancer by highly stabletransfer and expression of theAequorea victoriajellyfish GFP3

gene in tumor cells. The GFP-expressing tumor cells enablevisualization of tumor growth, invasion, micrometastasis, andmetastasis in fresh viable tissue after transplantation to mice(12–14). In the present investigation, GFP expression in anorthotopic intradermal injection model of LOX cells in nudemice and in an experimental metastasis model of B16 inC57BL/6 mice has revealed the multiorgan and very extensivebone metastatic potential of melanoma. These new metastasismodels should be very useful for studying the biology of met-astatic melanoma and for the development of therapy of thisdisease.

MATERIALS AND METHODSGFP Expression Vector. The pLEIN retroviral vector

(CLONTECH, Palo Alto, CA) expresses enhanced GFP and theneomycin resistance gene on the same bicistronic message,which contains an internal ribosome entry site (IRES) (14).

Received 6/7/99; revised 8/23/99; accepted 8/25/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported in part by United States National Cancer Institute Grant R44CA 53963.2 To whom requests for reprints should be addressed, at AntiCancer,Inc., 7917 Ostrow Street, San Diego, CA 92111. Phone: (858) 654-2555; Fax: (858) 268-4175; E-mail: [email protected]. 3 The abbreviation used is: GFP, green fluorescent protein.

3549Vol. 5, 3549–3559, November 1999 Clinical Cancer Research

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Fig. 1 Stable high-level GFPexpression LOX human mela-noma transductantsin vitro andin vivo.a, the human malignantmelanoma cell line LOX wastransduced with retroviral vec-tor pLEIN that expresses GFPand the neomycin resistancegene on the same bicistronicmessage. Stable clones express-ing high levels of GFP wereselected in 800mg/ml G418.Bar, 80 mm. b, LOX-GFP or-thotopic tumor with intense ex-pression of GFP in the nudemouse. The tumor is growingthrough the whole layer of theskin. The epidermis (EP) anddermis (D) are indicated.Bar,400 mm.

Table 1 Multi-organ, brain and skeletal metastases of GFP-expressing LOX human melanoma cells after intradermal implantation in nude miceTen nude mice were each injected intradermally with 13 106 cells. The mice were sacrificed at 6–8 weeks, at the time of significant decline

in performance status.

Mouse no.

Sites of tumor spread

Brain Lung Pleural membrane Liver Spleen Kidney Adrenal gland Lymph node Bone

1 2 1 1 1 2 2 2 1 22 1 1 1 1 2 1 1 1 23 2 1 1 2 2 2 2 2 24 1 1 2 2 2 2 2 1 25 1 1 2 2 2 2 2 1 26 2 1 1 2 2 2 2 2 27 1 1 2 2 2 2 2 2 28 1 1 1 2 2 2 2 1 19 2 1 2 1 2 2 1 1 2

10 2 1 2 2 2 2 1 1 2

3550Melanoma Bone and Organ Metastasis Imaged by GFP



Packaging Cell Culture, Vector Production, Transfec-tion, and Subcloning. PT67, an NIH 3T3-derived packagingcell line expressing the 10 Al viral envelope, was purchasedfrom CLONTECH. PT67 cells were cultured in DMEM (IrvineScientific, Santa Ana, CA) supplemented with 10% heat-inac-tivated fetal bovine serum (Gemini Bioproducts, Calabasas,CA). For vector production, packaging cells (PT67) at 70%confluence were incubated with a precipitated mixture ofN-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethylsulfate reagent (Boehringer Mannheim) and saturating amounts

of pLEIN plasmid for 18 h. Fresh medium was replenished atthis time. The cells were examined by fluorescence microscopy48 h after transfection. For selection, the cells were cultured inthe presence of 500-2000mg/ml G418 (Life Technologies, Inc.,Grand Island, NY) for 7 days.

Retroviral GFP Transduction of LOX and B16 Cells.For GFP gene transduction, LOX cells (National Cancer Insti-tute, Bethesda, MD) and B16 cells (American Type CultureCollection, Manassas, VA) at;25% confluence were incubatedwith a 1:1 precipitated mixture of retroviral supernatants of

Fig. 2 Lung metastasis ofLOX cells visualized by GFP.a, the surface of the lung of anude mouse. No metastasis wasdetected under bright-field mi-croscopy. Bar, 1280 mm. b,same field as ina. Numerousmicrometastases and metasta-ses are visualized by GFP ex-pression in the lung under flu-orescence microscopy.Bar,1280mm.

3551Clinical Cancer Research



PT67 cells and RPMI 1640 (Life Technologies, Inc.) containing10% fetal bovine serum (Gemini Bioproducts) for 72 h. Freshmedium was replenished at this time. Cells were harvested bytrypsin-EDTA 72 h after transduction and subcultured at a ratioof 1:15 into selective medium that contained 200mg/ml G418.The level of G418 was increased stepwise to 800mg/ml for bothLOX and B16 cells. Clones expressing GFP were isolated withcloning cylinders (Bel-Art Products, Pequannock, NJ) usingtrypsin-EDTA and then amplified and transferred by conven-tional culture methods.

Doubling Time of Stable GFP Clones. GFP or non-transduced cells were seeded at 1.53 104 in 35-mm culturedishes. The cells were harvested and counted every 24 h usinga hemocytometer (Reichert Scientific Instruments, Buffalo,NY). The doubling time was calculated from the cell growthcurve over 6 days.

Intradermal Injection of LOX. Ten 6-week-oldBALB/c nu/nu male mice were injected intradermally with asingle dose of 13 106 LOX-GFP cells. Cells were first har-vested by trypsinization and washed three times with cold

serum-free medium and then injected in a total volume of 0.1 mlwithin 30 min of harvesting. Cells were inoculated into dorsalskin using a 30 G1/2 precision glide needle (Becton Dickinson)and a 1-ml latex-free syringe (Becton Dickinson).

Tail Vein Injection of B16. Female 6-week-oldC57BL/6 mice (Harlan, Indianapolis, IN) were injected with asingle dose of 53 106 B16F0-GFP C1 cells in the lateral tailvein. Cells were first harvested by trypsinization and washedthree times with cold serum-free medium and then injected in atotal volume of 0.2 ml within 30 min of harvesting.

Analysis of Metastases. Tumor progression occurred inthe tumor-bearing animals along with decreased performancestatus. When performance status was poor, as defined by theonset of cachexia, the animals were sacrificed and autopsied.The primary tumor and all major organs as well as the wholeskeleton were explored. The fresh samples were sliced atapproximately 1 mm in thickness by using disposable mic-rotome blades (Model 818; Leica Instruments GmbH, Nuss-loch, Germany) and observed directly under a fluorescencemicroscope.

Fig. 3 Multiorgan metastasis of LOX cells visualized by GFP.a, numerous micrometastases and metastases visualized by GFP expression in thepleural membrane under fluorescence microscopy.Bar, 400mm. b, metastases are visualized in the liver by GFP expression.Bar, 200mm. c, tumormetastasized to the adrenal gland (small arrows) and kidney (large arrows) visualized by GFP expression.Bar, 400mm. d, tumor metastasized toa lymph node as visualized by GFP expression.Bar, 200 mm.




Microscopy. Light microscopy and fluorescence micros-copy were carried out using a Nikon microscope equipped witha Xenon lamp power supply. A Leica stereo fluorescence mi-croscope model LZ12 equipped with a mercury lamp powersupply was also used. Both microscopes had a GFP filter set(Chroma Technology, Brattleboro, VT).

Animal Care. All animal studies were conducted in ac-cordance with the principles and procedures outlined in the NIHGuide for the Care and Use of Laboratory Animals underassurance number A3873-1.

RESULTS AND DISCUSSIONIsolation of Stable Transductants of LOX Cells Ex-

pressing High Levels of GFP. The GFP-transduced cellswere able to grow in levels of G418 of up to 800mg/ml. Theselected G418-resistant LOX cells had a strikingly bright GFPfluorescence (Fig. 1a). There was no difference in the cellproliferation rates of parental cells and selected transductantsdetermined by comparing their doubling times in monolayerculture.

Stable High-Level Expression of GFP in LOX TumorsGrowing Intradermally in Nude Mice. The mice were sac-rificed 6–8 weeks after intradermal injection of LOX-GFPcells. Ten of 10 mice had very large intradermal and s.c. tumors.The tumor tissue was strongly fluorescent, thereby demonstrat-ing stable high-level GFP expressionin vivo during tumorgrowth (Fig. 1b).

Organ and Bone Metastases of GFP LOX Tumors.Tumor-bearing nude mice were sacrificed at 6 – 8 weekswhen performance status was poor. As summarized in Table1, tumors metastasized to the lung (10 of 10 mice; Fig. 2,aandb); pleural membrane (5 of 10 mice; Fig. 3a); liver (3 of10 mice; Fig. 3b); kidney (1 of 10 mice; Fig. 3c, largearrows); adrenal gland (3 of 10 mice; Fig. 3c, small arrows);lymph nodes (7 of 10 mice; Fig. 3d), and skeletal system (1of 10 mice; Fig. 4a). In five mice, single cancer cells or smallcolonies could be visualized in the brain by GFP fluorescence(Fig. 4, b– d).

Although previous studies have already demonstrated thatpulmonary metastases have been produced with high efficiencyafter i.v., i.p., and s.c. injection of LOX cells, other distantmetastases were not observed (5–8). This situation is similar inother human melanoma models. For example, the s.c. implan-tation MeWo melanoma model can result in numerous largelung metastasis nodules. Transfer of the metastatic lung nodulesto new recipients also led to the appearance of lung metastases.However, other distant metastases were not observed (15). Sub-dermal injection of the MeWo cells resulted in the developmentof highly melanotic and nonencapsulated primary tumors, whichgrew quickly into the dermis and epidermis and metastasized athigh frequency to the draining lymph nodes (16). Althoughthese spontaneous metastasis models have provided useful toolsfor basic and preclinical studies, they still have limitationsbecause they do not fully match the clinical situation of highly

Fig. 4 Skull and brain metas-tasis of LOX cells visualized byGFP. a, tumor metastasized tothe skull as visualized by GFPexpression (arrows).Bar, 80mm. b–d, micrometastases andmetastases (arrows) are visual-ized in the brain of nude miceby GFP expression.Bar, 80mm.




malignant melanoma, perhaps the most aggressively metastaticcancer (17).

The LOX-GFP model revealed by GFP fluorescence theextensive multiorgan metastasizing potential of human ma-lignant melanoma, which also includes the brain and skeletalsystem. These data demonstrate the far-reaching malignancyof this tumor (Table 1). Such a high incidence of multiorganand skeletal metastases of human melanoma could not havebeen visualized before the development of the intradermalGFP model described here, which provided the necessarytools.

Isolation of Stable Transductants of B16F0-GFP CellsExpressing High Levels of GFP. The GFP- and neomycin-containing expression vector-transduced cells were able to growin increasing levels of G418 (up to 800mg/ml). The B16F0-GFPC1 clone resistant to 800mg/ml G418 had a strikingly brightGFP fluorescence (Fig. 5). There was no difference in the cellproliferation rates of parental cells and selected transductants asdetermined by a comparison of proliferation rates in monolayerculture. Both cell lines had a doubling time of approximately24 h (data not shown). The GFP-transduced B16F0 and parental

cells still produced melaninin vitro and in vivo. There are noobvious changes in morphology of the GFP-transduced cellscompared with the parental cells.In vitro properties of transfor-mation such as the serum requirement and anchorage depen-dence of GFP-transformed and parental cells will be comparedin future studies.

Stable High-Level Expression of GFP in B16F0 TumorsGrowing in C57BL/6 Mice. Mice were sacrificed 3 weeksafter i.v. injection of B16F0-GFP C1 cells. Five of five mice hadextensive skeletal and visceral metastases (Table 2). The tumortissue was strongly fluorescent, thereby demonstrating stablehigh-level GFP expressionin vivo during tumor growth (Figs. 6and 7).

GFP-expressing Tumor Colonies in the Skeletal Sys-tem. Tumor colonies were found in the bone and bone marrowof all mice (Table 2). GFP-expressing tumor colonies were mostcommonly found in the vertebral bodies (five of five mice),pelvis (five of five mice), and long bones including the femurand tibia (five of five mice) as well as in the humeris (two offive mice). Metastases were also commonly found in flat bones,

Fig. 5 Stable high-level GFP-expressing B16F0-GFP C1 mu-rine melanoma transductantsinvitro. The murine malignantmelanoma cell line B16F0 wastransduced with the pLEIN vec-tor that expresses enhancedGFP and the neomycin resist-ance gene on the same bicis-tronic message. Stable high ex-pression clones were selected in800 mg/ml G418.Bar, 80mm.

Table 2 Skeletal and multi-organ metastases of GFP-expressing B16F0 cells after i.v. injection in C57BL/6 miceFive C57BL/6 mice were each injected with 53 106 B16F0-GFP C1 cells. Three weeks after the injection of the cells, the mice were sacrificed

and examined.

Mouseno.

Sites of tumor spread

Bone & bonemarrow Brain Lung

Pleuralmembrane Liver Spleen Kidney

Adrenalgland

Lymphnode

Skeletalmuscle Skin

1 1 1 1 1 1 2 1 1 1 1 12 1 1 1 1 1 2 1 1 1 1 13 1 2 1 1 2 2 1 1 1 1 14 1 1 1 1 1 2 1 1 1 1 15 1 1 1 1 2 2 1 1 1 1 1




Fig. 6 Bone metastasis of B16F0 GFP C1 visualized by GFP.a, skull; top, no metastasis was detected under bright-field microscopy.Bar, 640mm.Bottom, same area as shown at the top, bone metastasis visualized in the skull under fluorescent microscopy.b, vertebral body;top, no metastasiswas detected under bright-field microscopy.Bar, 1280mm. Bottom, same area as shown at the top, bone metastasis visualized in the vertebral bodyunder fluorescent microscopy.c, bone metastases visualized by GFP expression in the humerus and scapula.Bar, 1280mm. d, bone metastasesvisualized by GFP expression in the distal end of the femur.Bar, 1280mm. e, bone metastases visualized by GFP expression in the head of the femur.Bar, 800mm. f, bone metastases visualized by GFP expression in the pelvis.Bar, 1280mm.




Fig. 7 Multiorgan metastasis of B16F0-GFP C1 visualized by GFP expression.a, brain metastasis visualized by GFP expression.Bar, 1280mm. b,extensive metastases visualized in the lung and pericardium by GFP expression (arrows). Bar, 1280mm. c, metastases visualized in the pleural




such as the skull (four of five mice) and scapula (three of fivemice; Fig. 6,a–f).

GFP-expressing Experimental Systemic Organ Metas-tases. Tumor-bearing C57BL/6 mice were sacrificed 3 weeksafter injection of B16F0-GFP C1 cells. Experimental metastaseswere visualized by GFP in the major systemic organs: (a) brain(three of five mice); (b) lung (five of five mice); (c) pleuralmembrane (five of five mice); (d) liver (five of five mice); (e)kidney (five of five mice); (f) adrenal gland (five of five mice);(g) lymph node (five of five mice); and (h) skin and skeletalmuscle (five of five mice; Fig. 7; Table 2).

Previous studies have shown that injection of melanomacells into the left cardiac ventricle resulted in tumor colonies inthe skeletal system and in most organs of the mouse (11). Incontrast, the vast majority of i.v. injected tumor cells arrestrapidly in the lung (11), and arterially injected embryonal car-cinoma cells appear to arrest immediately in the capillary beds(18). In spontaneous metastasis to extrapulmonary organs, somecells must pass through the lung capillary bed into the arterialsupply and lodge in other organs. A major role of the lungcapillaries may be to prevent metastasis by trapping and me-chanically destroying almost all of the tumor cells that enter thevenous circulation (19). In the present study, we increased theamount of i.v. injected tumor cells up to 53 106 and causedextensive skeletal and visceral metastasis as visualized by GFP,indicating that some of the tumor cells could pass through thelung capillary.

In the present study, all five mice injected with tumor cellshad bone metastasis as well as lung metastasis. No mouse hadonly bone metastasis. Despite the injection of 53 106 cells(1003 routine innoculum), the survival of all experimentalanimals was longer than 3 weeks, which was sufficient forseeding of the bone and colonization. The model described inthis report used tail vein injection of tumor cells, which couldsimultaneously seed all organs of appropriate “soil.” Bone seed-ing and colonization would not then necessarily be late events.

Bones with fluorescent tumor colonies were those rich inhematopoietic bone marrow, such as vertebral bodies, femur,tibia, and the pelvis. There were no colonies in the bones thatdid not have bone marrow. The presence of bone marrow alsoappears to be a prerequisite for the establishment of human bonemetastasis (20, 21). Bone marrow appears to support tumorcolonization very extensively. Histopathological evidence ofbone metastases was demonstrated by H&E staining (Fig. 8) andcorrelates to GFP fluorescence in the bone (Fig. 6d). The GFP-fluorescent human tumor cell transductants we have producedcan be visualized as single cells when seeded in various tissues(12, 13). However, thus far, we have not yet found any singlecells seeded in the bone. Future time course experiments willclarify how many GFP-expressing tumor cells must be presentin the bone to be detectable. In the present study, the pattern ofskeletal distribution of tumor colonies closely mimics the pat-tern of metastasis to bone in humans.

In the present investigation, GFP expression has revealed

membrane by GFP expression (arrow). Bar, 800mm. d, metastases visualized in the liver by GFP (arrows).Bar, 400mm. e, kidney (large arrows)and adrenal (small arrow) metastasis visualized by GFP expression.Bar, 1280 mm. f, mesenteric lymph gland metastases visualized by GFPexpression.Bar, 1280mm. g, skin metastases visualized by GFP expression.Bar, 1280mm. h, skeletal muscle metastases visualized by GFPexpression.Bar, 200mm.

Fig. 8 Bone metastasis ofB16F0-GFP C1 melanoma inthe distal end of femur (same asFig. 6d). Note the presence oftumor cells between the spi-cules of cancellous bone (whitearrows), the osteoclastic reac-tion (large black arrows), andthe formation of reactive os-teoid by osteoblastic prolifera-tion (small black arrows). H&Estaining. Light microscopy.3200.




bone and visceral metastasis of human and murine melanoma.The fluorescence models have demonstrated for the first timethe very high metastatic potentials of the B16F0 and LOXmelanomas.

In the present study, the pLEIN vector was used totransfect cells with the GFP gene along with the neo geneconferring resistance to Geneticin (G418). In previous stud-ies, the biscistronic GFP vector was used, which contains thedihydrofolate reductase gene to confer resistance to metho-trexate (12, 13). In recent and present studies, selection wascarried out in stepwise increments of the selective agentG418. After selection in what was determined to be themaximum concentration of the selective agent, the cells werecultured in the absence of the selective agent. Only thebrightest GFP clones capable of stable expression of GFP inthe absence of the selective agent were then selected andtested for stabilityin vitro and in vivo. This procedure hasresulted in the selection of tumor cells capable of stable GFPexpressionin vivo as they form tumors and metastasize. Boththe GFP-transduced melanoma cell lines are very stableinvitro and in vivo. B16F0-GFP C1 cells have been passagedfor over 20 generationsin vitro with no obvious changesobserved in fluorescence. In s.c. B16F0 melanoma models,the survival of the mice is longer than 8 weeks. The tumorsare fluorescent until the time of the animal’s death. LOX-GFP cells have been passaged for six generationsin vitro andgrown in vivo for over 3 months, during which time therewere no obvious changes in fluorescence seenin vitro or invivo.

Previous studies transfected tumor cells with thelacZ geneto detect micrometastases (22). However, detection oflacZrequires extensive histological preparation; therefore, it is im-possible to detect and visualize tumor cells in viable fresh tissueat the microscopic level. The GFP technique has greatly en-hanced the resolution of the visualization of micrometastases infresh tissue, allowing detection down to the single-cell level infresh tissue (12, 13).

GFP is more easily visualized than melanin, due to itsbright fluorescence. The purpose of the present report was todemonstrate the potential power of visualization of geneticallyfluorescent models of melanoma. Fig. 8 demonstrates by stand-ard H&E staining that GFP is reporting histologically provenbone metastases. Future studies will compare the histopathologyof GFP B16 and the parental cells in detail.

B16F0-GFP C1 cells produced melaninin vitro andin vivo,similar to the parental cells. GFP-LOX cells did not producemelanin in vitro and in vivo similar to their parental cells.Transduction controls without GFP will be a subject of futurestudies.

The clonogenicity of GFP-transformed tumor cells hasbeen approached in three different experiments. In a study byChishimaet al. (12), a s.c. growing GFP-expressing Chinesehamster ovary tumor was minced, and the cells were grown inculture with expression of GFP maintainedin vitro. In anotherstudy by Chisimaet al. (23), nude mice were injected in the tailvein with GFP-expressing human lung tumor cells. These cellsseeded the lungs, which were removed and grown in three-dimensional histoculture. Progressive colonization of the lungby the GFP-expressing tumor cells over 52 days in histoculture

was observed. In the present study, we observed comparablebone metastatic cells by both GFP expression (Fig. 6d) andH&E staining (Fig. 8). Thein vitro subcloning of GFP-express-ing cells recovered fromin vivometastasis will be undertaken infuture studies.

The new metastasis models of LOX and B16F0 should bevery useful for the study the biological behavior of metastaticmelanoma and for the development of therapy of this disease.

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1999;5:3549-3559. Clin Cancer Res Meng Yang, Ping Jiang, Zili An, et al. ModelsGenetically Fluorescent Melanoma Bone and Organ Metastasis

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