The Relationship between Motility Factor - Cancer Research

(CANCER RESEARCH 51, 2699-2705, May 15, 1991]

The Relationship between Motility Factor Receptor Internalizaron and the LungColonization Capacity of Murine Melanoma Cells1

Hideomi Watanabe,2 Ivan R. Nabi,3 and Avraham Raz4

Cancer Metastasis Program, Michigan Cancer Foundation, Detroit, Michigan 48201

ABSTRACT

The in vitro motility of B16-F1 melanoma cells is enhanced byincubation with a monoclonal antibody against gp78, previously characterized as a motility factor receptor. This antibody was used to study therelationship between motility stimulation in vitro and metastatic abilityin vivo in the B16-F1 and K-1735 murine melanoma systems. While bothhigh- and low-metastatic variants exhibited enhanced in vitro motility inresponse to the anti-gp78 monoclonal antibody, only the high-metastaticcells exhibited an increased metastatic ability. Surface immunofluores-cence of low-metastatic cells was distributed more diffusely compared toa highly localized patching of gp78 on high-metastatic cells, suggestingthat the directed endocytosis of gp78 to form a single leading edge isrelated to the metastatic ability of a cell, while fluorescence-activatedcell sorter analysis revealed decreased gp78 surface expression in high-metastatic clones. Priming of cells by preventing internalization of gp78-antibody complexes by pertussis toxin resulted in a marked enhancementof pulmonary mÃ©tastasesby the treated cells which was directly correlated with decreased surface expression of gp78 following washout ofpertussis toxin. These results suggest that cell motility induced bymotility factor receptor occupancy may play a role in the process ofmetastasis and that the ligand-receptor complex internalization from thecell surface is involved in control of cell kinesis during metastasis.

INTRODUCTION

Tumor cell motility has been suggested as a key event of themetastatic cascade, and correlations have been demonstratedbetween cell motility and spontaneous and experimental metastatic potential of various tumor cells (for a review see Refs. 1and 2). Several factors have been implicated as modulatingagents for cell motility (3). Recently two types of motilityfactors, designated scatter factor (4, 5) and AMF5 (6), have

been identified. It has also been suggested that growth factorsincluding fibroblast growth factor (7) and insulin-like growthfactor (8) may affect cell motility. A monoclonal antibody,SLOW-1, was reported to inhibit the locomotion of cells by anindirect signaling mechanism, through a specific cell surfacereceptor involved in motile control (9). We have recently demonstrated that a mAb against a M, 78,000 cell surface glycopro-

tein (gp78) stimulates cell motility in vitro (10) which leads tothe identification of gp78 as a receptor for motility factor(s)(10,11).

The involvement of gp78 in the metastatic process is basedon the initial findings showing that the expression of 0-linked

Received 7/16/90; accepted 3/5/91.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.

' This work was supported by a grant to A. R. from the Paul Zuckerman

Support Foundation for Cancer Research.2Present address: Department of Orthopaedic Surgery. Gunma University

School of Medicine, Maebashi, Gunma 371. Japan.3 Present address: Department of Cell Biology and Anatomy. Cornell Univer

sity Medical College, New York, NY 10021.4 To whom requests for reprints should be addressed, at Michigan Cancer

Foundation. Meyer L. Prentis Cancer Center, 110 E. Warren Avenue, Detroit,Ml 48201.

5The abbreviations used are: AMF, autocrine motility factor: mAb, monoclonal antibody; PBS, phosphate-buffered saline (pH 7.2); PT, pertussis toxin; FACS,fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate.

oligosaccharides on gp78 was directly correlated with the metastatic ability of the cells and with the ability of anti-gp78polyclonal antibodies to enhance the metastatic ability oftreated cells (12, 13). Here, we have utilized the motility-stimulating ability of the anti-gp78 mAb to define the role ofgp78 in metastatic dissemination.

MATERIALS AND METHODS

Cells and Cell Culture. Cell variants derived from two differentmurine tumor systems were used in these studies. The B16-F1 cell linewas derived from pulmonary mÃ©tastasesproduced by i.v. injection ofB16 melanoma cells (14). Since the 3F3A mAb was originally raisedagainst gp78 expressed in B16-F1 cells (10), this cell variant was usedas a positive control. B16-F10 and B16-F10Lr6 cell lines were derivedfrom B16-F1 melanoma. B16-F10 is a high-metastatic line and wasselected in vivo (14, 15). B16-F10Lr6 is a low-metastatic line and wasselected in vitro for resistance to lysis by lymphocytes (15). The K-1735melanoma was developed in a C3H~ mouse that has been treated with

a short course of exposure to UV radiation followed by chronic paintingof the skin with croton oil (16). K-1735-M1 and K-1735-C1-11 cellswere derived from the parent melanoma tumor as high- and low-metastatic variants, respectively. The Cl-ll cloned cell line was obtained by cloning of the fifth in vitro passage of the parent cells (17).The Ml cell line was derived from pulmonary mÃ©tastasesafter i.v.inoculation of the parent tumor cells (18).

The cells were grown in monolayer on plastic in Dulbecco's modifiedEagle's minimal essential medium supplemented with glutamine, non-

essential amino acids, vitamins, antibiotics, and 10% heat-inactivatedfetal bovine serum. To ensure reproducibility, all experiments weredone with the same batch of fetal bovine serum (control number44N3085; GIBCO). The cells were maintained at 37Â°Cin a humidified

atmosphere of 5% CO2/95% air. To ensure reproducibility, all experiments were performed with cultures grown for no longer than 6 weeksafter recovery from frozen stocks. Cell monolayers that had attainedsemiconfluency were used for preparing single-cell suspensions. Suchcultures were obtained by plating 2 x IO5cells/90-mm dish and cultur-ing for 3 days at 37Â°C.Cells were harvested by overlaying with a thinlayer of 2 mivi EDTA for 2 min at 37Â°Cfollowed by gentle pipeting to

form single-cell suspensions. Viability was recorded following a trypanblue exclusion test.

Phagokinetic Tracks. Uniform carpets of gold particles were preparedon coverslips coated with bovine serum albumin, as described previously(19, 20). Colloidal gold-coated coverslips were placed in 35-mm tissue

culture dishes containing 2 ml complete minimal essential mediumsupplemented with glutamine, nonessential amino acids, vitamins, antibiotics, and 10% heat-inactivated fetal bovine serum, with or without3F3A mAb ascites fluid at a concentration of 25 /il/ml, and then 2000cells were added to each plate. After 24 h, phagokinetic tracks werevisualized using dark-field illumination in a Nikon inverted microscopeat x200. The area cleared of gold particles by at least 30 cells wasmeasured, and the standard error was calculated.

Experimental Pulmonary Metastasis. Cells grown as a monolayerwere incubated for 18 h at 37Â°Cin the presence or absence of 25 p\/m\

3F3A mAb. In the experiments with PT, cells were treated with 500ng/ml PT for 60 min prior to mAb exposure. The cells were washed 3times with cold PBS to wash away nonbound mAb prior to injectioninto mice. Syngeneic mice (C57BL/6J mice for B16 melanoma, C3H/HeJ mice for K-1735 melanoma; The Jackson Laboratory) were inoculated in the tail vein with 5 x 10" cells in 0.2 ml of PBS. After 17 days

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MOTILITY FACTOR RECEPTOR ENDOCYTOSIS AND METASTASIS

for B16 melanoma and 31 days for K-1735 melanoma, the mice weresacrificed and autopsied. Lungs from B16 melanoma were fixed with5% formaldehyde in PBS. and those from K-1735 were fixed withBouin's solution to facilitate observation of tumor colonies. The number

of tumor colonies in each lung was determined under a dissectingmicroscope.

FACS Analysis. The 3F3A mAb ascites was diluted 1:10 in PBScontaining 0.02% sodium azide, and 100 Â¿ilwere added to 10* cells.

The control cells were incubated identically without the 3F3A mAb.After 30 min at 0"C, the cells were washed twice with PBS. and then

FITC-conjugated anti-rat antibody (Zymed: 1:10) was used as thesecondary antibody. After 30 min at 0Â°C.the cells were washed twice

in PBS, and cell surface fluorescence was analyzed using a FACS(FACStar; Becton Dickinson, Mountain View, Ã‡A).A scatter windowwas set to eliminate dead cells and cell debris. The frequency andfluorescence profiles of the stained cells were determined using a laseroutput of 125 mV.

Immunofluorescence. The surface immunofluorescence of gp78 wasperformed by fixing cells with 3.5% paraformaldehyde in PBS for 8min at 2.VC. The cells were washed 3 times with PBS and incubatedfor 30 min at 23Â°Cwith a 1:10 dilution of the 3F3A mAb ascites inPBS. The cells were then washed with PBS and labeled at 23Â°Cwith

FITC-conjugated anti-rat antibody (Zymed). After 30 min the cellswere washed extensively with PBS.

Double labeling of gp78 with a major lysosomal associated membrane glycoprotein. LAMP-1 (21), was performed as described (11).Briefly, cells were fixed and permeabilized by immersion of the coverslide in precooled (â€”80Â°C)methanol for 30 min at -20Â°C. Double

labeling of gp78 with LAMP-1 was performed by the sequential incubation of permeabilized cells with anti-P2B/LAMP-l rabbit antiserum(a gift from Dr. Jim Dennis) and F1TC anti-rabbit IgG (Zymed)followed by the 3F3A mAb and tetramethylrhodamine isothiocyanateanti-rat IgG (Zymed). The coverslips bearing the stained cells weremounted in 90% glycerol in PBS and observed under a Nikon photo-microscope. The cells were photographed using Kodak Tri-X 400 ASAfilm.

Protein Gel Electrophoresis and Blotting. Immunoblots were performed as previously described (10). Briefly, cells grown in a monolayerwere harvested, washed twice in PBS, suspended at 5 x IO*1cells/ml in0.5% Nonidet P-40 in PBS containing I mM EDTA and 1 ITIMphenyl-methylsulfonyl fluoride for 30 min on ice. and clarified by centrifuga-tion. Proteins were analyzed by sodium dodecyl sulfate-polyacrylamidegel electrophoresis in 8% polyacrylamide slab gels according to themethod of Laemmli (22) and electrotransferred to nitrocellulose filters.Nitrocellulose filters were quenched overnight in PBS containing 15%skim milk and 0.2% NaN3 (quench solution). The filters were thenincubated with the 3F3A mAb ascites diluted 1:400 in the quenchsolution for 1 h. The filters were washed 5 times for 10 min in thequench solution and then incubated for l h in the quench solution withi:5I-sheep anti-rat antibody (1 jjCi/10 ml; Amersham). The filters were

then washed twice for 15 min in the quench solution and then twicemore in the quench solution containing 0.1% Tween 20. The filterswere dried and autoradiographed.

RESULTS

Locomotor Activity Stimulated by the Anti-gp78 mAb of High-and Low-Metastatic Cell Variants. The binding of the anti-gp78mAb to gp78 mimics the effect of AMF by enhancing thelocomotor activity of the treated cells (10). This observationraised the question of whether motility stimulation by the anti-gp78 mAb may be related to the metastatic potential of the cell.High- and low-metastatic cell variants from two murine melanoma cell systems were plated on gold particle-coated substrateand were examined in dark-field optics 24 h later (Fig. 1). Theaverage area of the particle-clear zone in the low-metastaticB16-F10Lr6 cells was 5.7 ^nr/h, while the area of the tracksformed by the high-metastatic B16-F10 cells was 10.2 Â¡Â¿nr/h.

JZ\

(M

BIO-FIO B16-F101

K-1735 Ml K-1735CH1Fig. 1. Stimulated motility of high- and low-metastatic cell lines induced by

the 3F3A mAb. Cells Â«ereplated on collodial gold cover slides in culture mediumsupplemented with or without the 3F3A mAb ascites fluid (25 Ml/ml). After 24 hcells were photographed under dark field illumination, and the area of the particlefree area of at least 30 individual cells was measured. The average area was shownwith the unit of fim2/h.

Similarly, the K-1735 high-metastatic variant (Ml) exhibited a4.0-fold higher basal locomotor activity than its low-metastaticcounterpart (Cl-11) (Fig. IB) as has been previously described(20). Treatment of all four cell lines with the anti-gp78 mAbstimulated their motility by approximately 2-fold, as previouslyreported for B16-F1 melanoma cells (10), with no significantdifferences between the high- and low-metastatic clones ofeither melanoma all system (Fig. 1). The effect of the anti-gp78mAb appears to be specific because other mAbs, anti-lectin,anti-H2h, anti-transferrin receptor, and rat IgM have no signif

icant effect on cell migration.Lung Colonizing Response to Anti-gp78 mAb. To examine

whether the lung colonization ability of the melanoma variantscould also be enhanced, cells were incubated with or withoutthe anti-gp78 mAb prior to injection into mice. As shown inTable 1, preincubation with the mAb resulted in a 2.5-foldincrease in the lung colonizing capacity of the high-metastaticcell variants of either tumor system, whereas there was a negligible effect on the colonization of the low-metastatic cells.The stimulation of the lung colonizing ability of the high-metastatic cell variants by the anti-gp78 mAb corroborates theprevious findings in which polyclonal anti-gp78 antibodies weredemonstrated to enhance the metastatic ability of B16-F1 melanoma cells (12).

Lung Colonizing Response to FT. We have previously reportedthat pretreatment of B16-F1 cells with PT prior to addition ofthe anti-gp78 mAb or B16-F1 AMF containing conditionedmedia blocked mainly the stimulated motility without affectingthe basal locomotor activity of the cells (10). To examinewhether PT may affect the enhanced production of pulmonary

2700




Table 1 Effect of the anti-gp78 mAb on lung colonization"

CelllineExperiment

1B16 melanoma

FIOFlOLr6In

vitropretreatment

of3F3A++8,

138,270,1,0,0,16->,->111,

1,No.

of lungcolonies/mouse19,

20,28,29,37,42,6237,63, 64, 78, 79,1032,

3, 4, 4,81,1,2,2,3Median

(range)24

(8-62)63(8-103)2(0-8)1

(0-3)P*<005<0.2

K-1735 melanomaMl

Cl-11

Experiment 2B16 melanoma

FIO

FlOLr6

K-1735 melanomaMl

Cl-11

24, 37, 57, 9059,95, 111, 112, 1320, 0, 0, 0, 0, 00, 0, 0, 0, 0

15,17, 18,21,26,30,31,3632, 44, 50, 52, 66, 80, 88, 96, 1410,1,2,4,5,7,7,9, 140,4,6,7,7,8,10,17

56,66,93,93, 103, 11497, 107, 188, 189, 190,2050, 0, 0, 0, 0, 0, 0, 0, 00, 0, 0, 0, 0, 0, 0, 0, 0, 0

47(24-90)111 (59-132)

0 (0-0)0 (0-0)

23 (15-36)66(32-141)

5 (0-14)7(0-17)

93(56-114)188(97-205)

0 (0-0)0 (0-0)

<0.05

<0.02

<0.02

<0.2

<0.05

<0.2Â°Cells grown in complete minimal essential medium were incubated at 37'C in the presence or absence of the 3F3A mAb at a concentration of 25 Â¿il/mlfor 18 h

prior to i.v. injection of 5 x IO4cells into mice.* Probability of no difference from the group without treatment of the 3F3A mAb (two-tailed Mann-Whitney (/test).

Table 2 Effect of the anti-gp78 mAb with pertussis toxin on lung colonization"

In vitropretreatmentExperiment

1Control3F3AC3F3A +FT"PTExperiment

2Control3F3AC3F3A +VIaPTExperiment

3Control3F3AC

3F3A +PT*PTNo.

of lungcolonies/mouse0,2,

2,4,9, 13, 13, 13,253,5,9, 15, 17,21,23,248,28,41,43,46,64,69,82,1200,0,2,4,

10, 10,14,380,0,0,0,0,0,

1,20, 1,2,2,3,3,3,4,76,9, 18, 19,20.24,29,32.540,0,0.0,0,

1, 1,3,4

3, 9, 19, 38, 42, 42, 50, 57,7921,29, 30, 36, 38, 42, 60, 61, 74, 81

34,46,51,52,60, 121, 132, 141,188,22027,

27, 28, 42, 46, 48, 72, 74Median

(range)9

(0-25)16(3-24)46(8-120)7

(0-38)0

(0-2)3 (0-7)

20(6-54)0(0-4)

42(3-79)i40(21-81)90(34-220)44

(27-74)ft<0.2

<0.002<0.2<0.01

<0.002<0.2<0.2

<0.02<0.2

" Mice were given i.v. injections of 5 x IO4B16-F1 cells.* Probability of no difference from the group with treatment of neither the

3F3A mAb nor PT (two-tailed Mann-Whitney U test).e Cells grown in complete minimal essential medium were incubated at 37"C

in the presence of the 3F3A mAb at a concentration of 25 Â¿il/mlfor 18 h prior toi.v. injection.

* Cells were treated with 500 ng/ml PT 60 min prior to interaction of the cells

with the 3F3A mAb.

mÃ©tastasesinduced by the anti-gp78 mAb, B16-F1 cells werepretreated with 500 ng/ml PT for 60 min and then treatedovernight with PT and the anti-gp78 antibody. Mice wereinoculated with cells preincubated (18 h) with PT only, withantibody only, or with PT followed by antibody. In threeseparate experiments, PT alone had no effect on pulmonarymetastasis (Table 2). The anti-gp78 mAb alone exhibited varying effects on B16-F1 colonization ability, perhaps reflectingits median metastatic potential between the nonresponsive B16-FlOLr6 and the high-metastatic B16-F1 cell lines. However,

cells pretreated with PT and incubated overnight in the presence

100 -

100- II

Fig. 2. Cell surface staining with the 3F3A mAb by flow cytofluorometry ofB16-F10 cells (/a), B16-F10Lr< cells (Â¡b).Ml cells (Ila), or Cl-11 cells (lib).Cells were labeled with the 3F3A mAb at 4Â°C,washed with fluorescein-labeledrabbit anti-rat antibody, and analyzed in the FACStar (Becton Dickinson). Ascatter window was set to eliminate dead cells and cell debris.

of both PT and the anti-gp78 mAb exhibited significant increases in the level of lung colonization (Table 2).

Cell Surface Distribution of gp78. gp78 is localized to theleading and trailing edges of motile B16-F1 melanoma cellsand BALB/C-3T3/A31 fibroblasts (IO).6 In order to examine

the quantitative expression gp78 on the cell surface, cells wereimmunofluorescently labeled and analyzed by FACS. As seen

11. R. Nabi, H. Watanabe, and A. Raz, unpublished results.

2701




Fig. 3. Indirect immunofluorescent labeling of B16-F10Lr6 (A), B16-F10 (B), Cl-11 (C), or Ml cells (D) with the anti-gp78 mAb. X 2200.

in Fig. 2, in both tumor systems the high-metastatic variantswere labeled less than their low-metastatic counterparts. Itshould be noted that this difference is not due to nonspecificbindings of FITC-conjugated anti-rat antibody, since the binding patterns of anti-rat antibody to both high- and low-metastatic cells without the first mAb were indistinguishable. Theseresults from the B16 and K-1735 melanoma systems werefurther corroborated by direct microscopic visualization. Approximately 80% of B16-F10Lr6 cells and 75% of Cl-11 cells

(low-metastatic variants) had multiple surface domains stainedby the anti-gp78 mAb (Fig. 3, A and C) while most of the high-

metastatic cell variants (approximately 90% of FIO and 75%of Ml cells) manifested a single labeled domain along the edgeof the cells (Fig. 3, B and D). Since the coordinate extension ofa single leading edge is crucial to the directed locomotion of acell (23), the multiple surface domains on the low-metastatic

variants may reflect the extension of multiple leading edgesresulting in a random rather than a directional motility inresponse to the anti-gp78 mAb. Such an altered motility patternwould not be detectable in the two-dimensional phagokinetic

track assay but would be evident in the metastatic response ofthe cells (Fig. 1; Table 1).

[Â»traccilular Distribution of gp78. In A31 fibroblasts, gp78 islocalized intracellularly to elongated beaded vesicles which col-ocalize with LAMP-1, a lysosomal glycoprotein aligned withmicrotubules,6 and we have proposed that gp78 is translocated

within such tubular vesicles. To examine whether the colocali-zation of the intracellular gp78 with lysosomes is also presentin melanoma cells, cells were immunofluorescently double-labeled with the anti-gp78 mAb and antibodies to the lysosomalantigens LAMP-1. As seen in Fig. 4, gp78 and LAMP-1 exhibit

partial codistribution identifying gp78 as a lysosomal glycoprotein. There were no differences among the four melanoma celllines in the distribution of intracellular gp78 and LAMP-1.Colocalization of the proteins was detected primarily in thelarger vesicular structures. The anti-gp78 mAb, however, alsolabeled other smaller vesicles which were not labeled by anti-LAMP-1 antibodies and may represent endosomes, as seen infibroblasts.6

Decrease in Surface Expression of gp78 l h after Removal ofPT. The unexpected increase in the lung colonizing ability ofcells treated with both PT and the anti-gp78 antibody leads us

to believe that PT inhibition of G protein may be blocking theinternalization of gp78 required for motility stimulation. Such

2702



MOTILITY FACTOR RECEPTOR ENDOCYTOS1S AND METASTASIS

Fig. 4. Double indirect immunofluorescent labeling for gp78 (A) and LAMP-1 (B). Cl-11 cells were fixed and permeabilized prior to treatment with anti-P2B/LAMP-1 antisera and fluorescein goat anti-rabbit IgG followed by anti-gp78and rhodamine goat anti-rat IgG. No cross-reactivity of the two secondaryantibodies to cells labeled with either of the two primary antibodies was detected.The three other cell lines showed the same distribution and colocalization as theCl-11 cells.

treated cells would not endocytose gp78 in overnight cultureand would be injected into the mice with anti-gp78 antibodystill on the cell surface. Placement of the cells in a PT freeenvironment, either during washing of the cells with cold PBSor following injection, would allow the recruitment to the cellmembrane of newly synthesized G protein which had not beeninhibited by PT. Following inoculation into the mouse, inter-nalization at 37Â°Cof the gp78-antibody complex would occur

with the resultant motility stimulus.To determine whether such a hypothesis could explain the

synergistic effect of the anti-gp78 mAb and PT on lung colonization, we analyzed the surface distribution of gp78 on cellstreated with PT. B16-F1 cells were cultured in the presence of500 ng/ml PT for 18 h and then allowed to recover in PT freemedium for 1 h. PT treatment did not affect the cell surfaceexpression of gp78 compared to that of untreated cells (datanot shown). However, replacement of the media for l h withregular media lacking PT resulted in the decreased cell surfaceexpression of gp78 both quantitatively by FACS analysis (Fig.5A) and qualitatively by surface immunofluorescence (Fig. 5, B

2703

Fig. 5. Internalization of gp78 in B16-F1 melanoma cells following PTtreatment and removal. Indirect immunofluorescent labeling of cell surface in thepresence of PT for 18 h (B) and l h after washout of PT (O- Down-regulationwas shown quantitatively in FACS analysis (A). Surface expression of gp78 wasanalyzed by FACS (A) and indirect surface immunofluorescence (B and C)following treatment of cells with PT for 18 h (A and B) and l h after washout ofPT (A and C). Gp78 internalizaron following PT washout was demonstratedboth quantitatively (A) and qualitatively (C), x 1800.




and C). PT treatment of cells incubated with anti-gp78 mAbmay serve to prime the cells, preventing receptor activation andinternalization, so that only following removal of the PT canthe motility stimulus of the anti-gp78 mAb be transmitted tothe cell.

DISCUSSION

Local invasion of the host stroma by cells from the primarytumor is the first step in the metastatic process. Mechanismssuch as generation of mechanical pressure, the release of protease, and an increased motility of tumor cells are believed toplay an important role in this process (24, 25). After dispersalthrough vessels, tumor cells arrest in the capillary bed of distantorgans and extravasate into the organ parenchyma. The attainment of an extravascular position is believed to involve anactive locomotion similar to that responsible for the initialinvasion into the blood vessels (1, 2, 24, 25). Therefore, locomotion of tumor cells and its regulation is thought to be crucialfor metastasis. This was supported by results obtained in aprevious study on the motility of variants derived from K-1735melanoma, in which a direct correlation was found betweenlocomotion in vitro and lung colonization in vivo (20). In adifferent study a Fourier analysis revealed that cell motility,especially pseudopodal extension, correlates with metastaticpotential in several cell lines of Dunning R3327 rat prostaticadenocarcinoma with different metastatic capacities (26). Itshould also be pointed out that several tumor cells were reportedto grow in an intravascular location at the site of arrest andextravasate by breaking the enclosing vessel (28, 29).

The mechanisms by which tumor cells regulate locomotoractivity during metastasis are not clear, and it has been suggested that during locomotion cells either use internal programsor respond to signals from the environment. Several factorsincluding growth factors have been implicated as signal modulating agents for cell motility (8). Human synovial cells producea M, \ 3,000 stimulant for polymorphonuclear locomotion (27).Atnip et al. (30) have shown that both chemotactic and che-mokinetic movements of MTLn3 rat mammary adenocarcinoma cells are stimulated by the MTLn3 derived cytokine (M,53,000) and have suggested that the production of such cyto-kines may represent a phenotypic difference between high- andlow-metastatic variants (30). Previous studies have indicatedthat extracellular proteins, i.e., collagen, laminin, and fibronec-tin, and their degradative products are chemotactic for sometumor cells (31, 32).

We have recently demonstrated that anti-gp78 mAb stimulates in vitro cell migration (10, 11). gp78 was identified as apossible receptor for motility factor because the binding of theanti-gp78 mAb to the cell surface mimics the binding of amotility factor stimulating cell locomotion (10) and shows adirect binding competition between AMF and anti-gp78 togp78.7 In the present study, to determine the relationship

between motility factor receptor activation and metastatic capacity, we have used the anti-gp78 mAb as a motility stimulusand examined the motility response in vitro and metastaticresponse in vivo in cell pairs exhibiting low- or high-metastaticpotential. In both the B16 and K-1735 melanoma systems thehigh metastatic cell variants exhibited a higher basal locomotoractivity in the absence of an external stimulus. Preincubationof cells with the anti-gp78 resulted in a significant increase in

7 Siliciri et al., unpublished observation.

the lung colonizing capacity of high-metastatic variants,whereas there was no significant response to the mAb in thelow-metastatic variants. The in vitro motility assay of area

clearing does not measure directional movement. While wedetected an increased in vitro motility of the low- metastaticvariants, this may reflect the multifaceted extension of manyleading edges and random movement and not the directionalextension of a single leading edge. The finding that low-metastatic variants, which seem to show multidirectional leadingedge extension, did not respond to the anti-gp78 mAb in lungcolonizing capacity indicates that metastasis requires a directional motility stimulation.

This hypothesis is supported by studies of the surface expression of gp78 on the low- and high-metastatic variants. FACSanalysis showed that the high-metastatic variants express lessgp78 on their surface than the low-metastatic variants in bothmelanoma systems. Immunofluorescent examination revealedthat there is a distinct pattern of distribution of gp78 on thecell surface; low-metastatic variants had multiple domains thatwere stained diffusely, while most of the high-metastatic cellsexhibited a single polarized densely labeled area (Fig. 3). Sucha distribution of gp78 on the cell surface corresponds closely tothe suggestion by Bretscher (33) and Bergmann et al. (34) thaton a stationary cell the internalized membrane is returned atrandom to the cell surface, whereas on a motile cell it istransported through the cell to the leading edge. The diffusesurface labeling of low-metastatic variants may reflect the extension of multiple leading edges with a resultant decrease innet forward movement. Indeed, we have shown that gp78 maybe translocated from the internalized surface (endocytosis) tothe leading edge (exocytosis) through lysosomal compartments.6 Guirguis et al. (35) reported that the formation of

pseudopodal protrusions is stimulated by an AMF. Partin et al.(26) showed that pseudopodal extension correlates with metastatic potential in several cell lines of Dunning R3327 ratmodel of prostatic cancer with different metastatic propensities.Internalized membrane with gp78 may serve as a main sourceof membrane for extension of the leading edge, enabling themore dynamic phenomena of cell motility such as membraneruffling, pseudopodal extension, and translation to occur actively at the leading edge in high-metastatic variants.

PT irreversibly blocks activity of guanine nucleotide-bindingproteins (G proteins) essential for signal transducing (36, 37)and the action on second messengers (38). A PT sensitivepathway is involved in motility stimulated by both AMF (38)and anti-gp78 mAb (10). We have shown here that PT treatment combined with the 3F3A mAb enhances pulmonary metastasis formation following i.v. inoculation. The enhancementis not due to the direct effect of PT, since PT alone has noeffect on the production of pulmonary metastatic nodules;furthermore, PT at a higher concentration (1 Â¿ig/ml)was reported to inhibit liver metastasis (39). It is more likely that PTexerts an enhancing action on the external motility stimulatorswhich make cells motile and metastatic. The receptor-mediatedmotile response is independent of adenylate cyclase and requiresdirect participation of a G protein (40). In the present study,we show that internalization of gp78 occurs when B16-F1 cellsare placed in PT free media for l h after pretreatment with PTfor 18 h. The increase in pulmonary metastasis following treatment with both anti-gp78 mAb and PT (Table 2) could resultfrom a similar process. In the presence of PT the 3F3A mAbbinds to surface-exposed gp78 but is not internalized to the

extent that the motility induction process of the treated cells is2704




blocked. Subsequent washing and injection of the cells intomice would remove PT from the cells and enable them toexpress newly synthesized functional G protein at the cellmembrane. Following cell injection the ligand occupied gp78molecules would be internalized, as shown in vitro in Fig. 5,resulting in the timely stimulation of cell motility which wouldin turn produce increased pulmonary mÃ©tastases.

It is likely that metastatic cells interact with such motilitystimulating factor(s) during the metastatic cascade, and theexpression of membrane surface mediator(s) of cell motilitylike gp78 would therefore play a role in the development ofmÃ©tastases.

ACKNOWLEDGMENTS

We thank J. Jenkins for typing and N. Squires for editing.

REFERENCES

1. Strauli, P., and Weiss, L. Cell locomotion and tumor penetration. Report ona workshop of the EORTC cell surface project group. Eur. J. Cancer, 13: 1-12, 1977.

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1991;51:2699-2705. Cancer Res Hideomi Watanabe, Ivan R. Nabi and Avraham Raz Melanoma CellsInternalization and the Lung Colonization Capacity of Murine The Relationship between Motility Factor Receptor

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