Inhibition of metastasis of lewis lung carcinoma by a synthetic peptide within growth factor-like domain of urokinase in the experimental and spontaneous metastasis model

Int. J. Cancer: 57, 727-733 (1994) Publication ofthe International Union Against Cancer Publication de I Union Internationale Contre le Cancer 0 1994 Wiley-Liss, Inc.

INHIBITION OF METASTASIS OF LEWIS LUNG CARCINOMA BY A

UROKINASE IN THE EXPERIMENTAL AND SPONTANEOUS METASTASIS MODEL

SYNTHETIC PEPTIDE WITHIN GROWTH FACTOR-LIKE DOMAIN OF

Hiroshi KOBAYASHI'.3, Junko GOTOH', Michio FUJIE~, Hiromitsu SHINOHAM', Nobuhiko MONIWA' and Toshihiko TERAO~ LDeoartmerzt of Obstetrics and Gvnecolom and 2Eaiiiumerit Center, Harnamatsu University School of Medicine, Handacho 3600,

Y 1 1

Hamamatsu, Shizuoka, 431-31, Japan.

Four synthetic peptides (residues 20-30 and 17-34) within the growth factor-like domain (GFD) of murine and human urokinase-type plasminogen activator (uPA) were examined to determine whether they inhibit production of experimental and spontaneous lung metastasis by murine Lewis lung carcinoma (3LL) cells. In an in vivo experimental metastasis assay, which determines mainly the later steps of the metastatic migration process (extravasation from the bloodstream and then growth into pulmonary tumor), none of the peptides introduced by i.v. single co-injection into syngeneic C57B1/6 mice inhibited pulmonary metastasis, when 3LL cells were pre-incubated with the peptides followed by i.v. co-injection of the peptide and cells. In addition, none of the peptides, when injected i.p. daily for 7 days after i.v. tumor cell inoculation, reduced the number of lung tumor colonies. In a second in vivo assay that measures metastasis from a primary tumor (spontaneous metastasis model), multiple i.p. injections of the mouse peptide 17-34 for 7 days after S.C. tumor cell inoculation significantly inhibited metastatic lung tumor colonization in a dose-dependent manner, whereas human peptide 17-34 had no effect. Mouse and human peptide 20-30 had no effect either. The inhibition of lung metastasis was not due to direct antitumor effects of mouse peptide 17-34. Our results indicate that occupation of uPA receptors on 3LL cells by the enzymatically inactive mouse peptide 17-34 or prevention of rebinding of uPA synthesized by tumor cells to their receptor specifically reduced tumor cell invasion and formation of metastasis and that uPA may regulate more efficiently the mechanism involved in the entry of tumor cells into vascular circulation than extravasation during the metastatic process. o 1994 Wilq-Liss, Inc.

The invasion and metastasis of tumor cells from a primary lesion to distant sites in target organ are multistep dynamic processes. Of these, the invasiveness of tumor cells into extracellular matrices represents one of several properties necessary for the formation of metastasis (Dan0 et al., 1985; Liotta and Stetler-Stevenson, 1991). The tumor cells secrete various specific proteolytic enzymes such as serine-proteinase (urokinase-type plasminogen activator (uPA)], metalloprotein- ase (collagenases), cysteine-proteinases (cathepsins B and L) and aspartic-proteinase (cathepsin D) (Schmitt et al., 1991). Results obtained with various systems devised to investigate the mechanisms of tumor invasion and inhibition of experimental metastasis in vifro have suggested that proteinase inhibitors and specific antibodies for uPA, plasmin and collagenase prevent cancer cell invasion to chick chorioallantoic membrane (Ossowski and Reich, 1983; Ossowski, 1988), human amnion (Mignatti et al., 1986) and reconstituted basement membranes (Albini et id., 1987). Evidence for a role of cathepsin B-uPA-plasmin-collagenase activation cascade in cancer cells has been provided (Mignatti et al., 1986; Ossowski, 1988; Kobayashi et al., 1991). The increased invasive potential of tumor cells correlates well with cell surface-associated proteolytic activity. The constitutive saturation of their uPA receptor with uPA molecule synthesized by tumor cells may be a property of tumor cells, providing a mechanism for the invasiveness (Stoppelli et al., 1986). The receptor-binding domain within uPA has been localized to the growth factor-

like domain (GFD) of the amino-terminal fragment (ATF) of uPA (Appella ef al., 1987). If all membrane receptor sites of tumor cells are occupied by enzymatically inactive uPA fragments such as GFD or ATF, pro-uPAs synthesized by tumor cells would not be able to bind to their own receptors. Proteolytic activity attributable to uPA on the tumor cell surface would decrease as the number of free binding sites decreases.

We demonstrate the presence of cell-surface-localized uPA on murine Lewis lung carcinoma (3LL) cells (Whur et al., 1980). To detect and to determine the characteristics of cell-associated uPA, several means including cell ELISA, flow cytometric analysis, and immunoadsorbent-amidolytic assay were employed.

Since the uPA-binding site resides in the NH2-terminal region in view of the capacity of uPA to bind, peptide 20-30 (residue 20-30 of the amino-terminal region of pro-uPA) and peptide 17-34 (residue 17-34), which represent sequences within the GFD of human and mouse uPA respectively, were chemically synthesized. These synthetic peptides were examined to determine whether they inhibit production of experimental and spontaneous pulmonary metastasis by murine 3LL Lewis lung carcinoma cells.

Our results indicate that saturation of the uPA receptor on 3LL cells by mouse peptide 17-34 leads to a decrease in their metastatic potential in an in vivo spontaneous metastasis model but not in an in vivo experimental metastasis model, and this inhibition is dose-dependent and non-toxic. Moreover, we conclude that the mode of action of this peptide is not the inhibition of tumor cell attachment to Matrigel and cell chemotactic migration in an in vitro assay. Our results suggest that uPA may act in an initial step (intravasation) of the metastatic process.

MATERIAL AND METHODS Animals

Specific-pathogen-free female C57B1/6 mice, 6-8 weeks-old, were purchased from Charles River (Kanagawa, Japan). The care and use of the animals were in accordance with our institution's guidelines.

Cells and culture A murine 3LL xenograft, selected for its high lung coloniza-

tion potential, was kindly provided by Chugai Pharmaceuticals (Dr. M. Saitoh, Tokyo). The tumor was maintained by serial subcutaneous transplantation in C57BU6 mice. The 3LL cells were maintained as monolayers in plastic dishes in Eagle's minimal essential medium supplemented with 10% FBS,

3To whom correspondence should be addressed, at Department of Obstetrics and Gynecology, Hamamatsu University School of Medi- cine, Handacho 3600, Hamamatsu, Shizuoka, 431-31, Japan. Fax: 81-53-435-1626.

Received: October 29,1993 and in revised form December 14,1993.

728 KOBAYASHI ET AL.

sodium pyruvate, non-essential amino acids, L-glutamine and vitamins (GIBCO, Grand Island, NY) at 37°C in a humidified incubator with 5% COz in air (Whur et al., 1980).

The promyeloid cell line U937, which binds pro-uPA/uPA via GFD to specific cell-surface receptors, was cultured at 5 x los cells/ml in RPMI 1640 supplemented with 10% FCS. U937 cells were differentiated to macrophage-like cells by incubation in the above medium containing 1 pM phorbol-12- myristate-13-acetate (PMA). A 3-day incubation period re- sulted in 60% adherent cells. Adherent cells detached by 1 mM EDTA in PBS ( 5 min, 23°C) were harvested by centrifugation, resuspended in PBS and then used for the binding experiments.

Thioglycolate-elicited peritoneal macrophages were obtained from C57B1/6 mice by i.p. injection of 1 mi of thioglycolate broth 4 days before sacrifice, cells were collected, selected by overnight adherence and cultured in DMEM (GIBCO) supplemented with 5% heat-inactivated FBS. The cultures were washed 24 hr later and placed in DMEM in the presence of Concanavalin A (0.1 pM). The concentrated medium was used as a source of mouse pro-uPA (Vassalli et al., 1977; Wohlwend et al., 1987; Estreicher et al., 1989).

GFD-related synthetic peptide The amino acid sequences of human and mouse uPA differ

with regard to several single residues in the amino-terminal domain (Blasi et al., 1986). The amino acid sequences within the GFD of human and mouse uPA (residues 20-30 and 17-34; numbering refers to the sequence of the human enzyme; murine uPA has one extra N-terminal residue) were synthesized employing an automated peptide synthesizer (Ap- plied Biosystems 430A Peptide synthesizer, Tokyo) using the chemistry recommended by the manufacturer and purified by reversed-phase HPLC (Senshu Pack, ODs-H-5251, 20 x 250 mm, number 05059); 5 mg of each peptide were dissolved in 100 pl of acetic acid and 900 p1 of Hepes buffer. The pH was maintained at 6.5-7.0 by adding sodium bicarbonate when necessary. The peptide solutions were centrifuged before use. Fresh peptide solutions were prepared for each experiment.

Human peptide 20-30: Val-Ser-Asn-Lys-Tyr-Phe-Ser-Asn-Ile- His-Trp Murine peptide 20-30: Val-Ser-Tyr-Lys-Tyr-Phe-Ser-Arg-Ile-

Human peptide 17-34: Gly-Thr-Cys-Val-Ser-Asn-Lys-Tyr-Phe- Ser- Asn-Ile-His-Trp-Cys-Asn-Cys-Pro Murine peptide 17-34: Gly-Val-Cys-Val-Ser-Tyr-Lys-Tyr-Phe- Ser-Arg-Ile-Arg-Arg-Cys-Ser-Cys-Pro

Quantitative assessment of binding of uPA peptides to the uPA receptor on PMA-stimulated U937 cells by flow cytometry.

PMA-stimulated U937 cells were treated with 50 mM glycine-HCI, 0.5 M NaCI, pH 3.0, first to dissociate receptor- bound uPA. After centrifugation, the cell pellet was adjusted to a density of 1 X lo6 cells/ml in PBS containing 0.1% BSA, pH 7.4. HMW-uPA, pro-uPA, and the synthetic uPA peptides were used as competitors; 1.5 nM of fluorescein isothiocyanate- conjugated pro-uPA (FITC-pro-uPA) in the presence or absence of unlabeled competitors were added to the cells (30 min, 4°C). Cell-associated fluorescence (argon laser excitation at 488 nm) at real-time (no wash) was immediately analyzed by FCM. Non-specific binding was determined in the presence of an excess (0.1 pM) of parent pro-uPA (Kobayashi et al., 1993).

Single-cell proteolytic plaque assay Mouse cells (peritoneal macrophages and the 3LL cells) and

human U937 cells were seeded at a density of lo4 cells/24-well dishes and washed 24 hr later (72 hr later in case of PMA- stimulated U937). After 1 hr pre-incubation with or without a synthetic peptide (murine or human peptides 17-34; 100 pM),

Arg-Arg

the cells were washed twice with cold PBS, 0.1% BSA and further incubated for 1 hr with serum-free culture medium containing 0.1% BSA and in the presence or absence of murine pro-uPA-containing medium or human pro-uPA (approx. 1 pgiml). Again, the cells were washed 4 times with ice-cold PBS, 0.1% BSA and overlaid with a mixture of 1.3% casein, 0.8% agar and 40 pg/ml plasminogen. Cell-associated proteolytic activity was analyzed and photographs were taken after 7 hr of incubation at 37°C (Estreicher et al., 1989).

Cell attachment assay The quantified cell adhesion assay was performed; 96-well

microtiter plates were coated with Matrigel (5 pg/filter, 100 pg/ml; Collaborative Research, Bedford, MA); 100 pl of RPMI 1640 with or without the synthetic peptides were added to each well. After incubation for 1 hr at 37°C 100 pl of the cell suspension (1 x lo6 cellsiml) were added to each well and the plates were incubated further for 2 hr at 37°C. Then the unattached cells were removed by washing the plates twice with medium. Under optimal conditions, usually about 80% of the cells remained attached to the well after washing. The cells were then fixed, stained and counted under an Olympus light microscope ( ~ 2 0 0 ) . The data were expressed as the number of attached cells at randomly chosen areas (each sample was assayed in triplicate and each well was counted in 3 areas) (Kobayashi et al., 1992).

In Vitro cell invasion assay Polycarbonate filters, 8 pm pore size (Costar. Cambridge,

MA), were coated with Matrigel, an extract of basement membrane components (2 hr, 37°C) (Albini et al., 1987; Kobayashi et al., 1992). Wells were pre-treated with the synthetic peptides at various concentrations for 1 hr at 37"C, as described in Table 11. Then 200 pl of cell suspension (1 x lo6 cells/ml) were placed in the upper compartment of the Boyden chamber and incubated for 6 hr at 37°C (Kobayashi et al., 1992). Under these conditions, very few cells died within the first 6 hr, as measured by Trypan blue dye exclusion in preliminary experiments. Each assay was carried out in triplicate and cells from various areas of the lower surface were counted.

Chemotactic assay The chemotactic assay was conducted in a similar fasion

without Matrigel coating (Kobayashi et al., 1992). Invasive cells adhering to the lower surface of the filter were quantitated.

Experimental and spontaneous pulmonary metastasis An in vivo experimental metastasis assay determined mainly

the later steps of the metastatic migration process (extravasation from the bloodstream and then growth into pulmonary tumor) and a second in vivo spontaneous metastasis assay measured metastasis from a primary tumor (intravasation, extravasation and growth into pulmonary tumor) (Ostrowski et al., 1986; Yu and Schultz, 1990).

Experimental metastasis. 3LL cells (2.5 x lo5 cells/200 pl RPMI 1640) admixed with various concentrations of the synthetic peptides (5 , 50 and 500 pg/mouse) were given i.v. co-injections into the lateral tail vein of unanesthetized C57B1/6 mice. In addition, each peptide, at doses from 5 to 500 pg/mouse, was injected i.p. daily from day 0 to day 6 (for 7 days, Schedule 1) or from day 7 to day 13 (for 7 days, Schedule 2) after tumor cell inoculation. Controls were injected only with vehicle. Twenty-one days after the inoculation of tumor cells, mice were killed and the lungs were removed, washed in PBS and fixed in Bouin's solution. The lung tumor colonies were counted under a dissecting microscope.

Spontaneous metastasis. 3LL cells (1 x 10h cells/200 pl/ mouse) admixed with various concentrations of the peptides were given S.C. co-injections into the abdominal wall. Then,

UROKINASE AND TUMOR CELL METASTASIS 729

each peptide was administered i.p. for various days after tumor cell inoculation, as described above. Mice were killed 28 days after inoculation of tumor cells. The number of lung tumor colonies was determined.

Statistical analysis

test or by non-parametric test. The data were analyzed for significance by the Student's t

RESULTS

Tumor cell-associated uPA activity and immunocytochemical localization of uPA in murine 3LL cells has been reported by Whur et al. (1980) and Skriver et al. (1984). We confirm the presence of uPA activity on the cell surface by immunological (cell ELISA) and enzymological (amidolytic assay) methods (data not shown). Pro-uPA and HMW-uPA levels in the cell supernatants were measured by immunoadsorbent-amidolytic assay (Kobayashi et al., 1992). A significant increase in the amidolytic activity after activation of uPA by plasmin suggests that most (80%) of the total immunoreactive uPA in the 3LL cell supernatants is enzymatically inactive single-chain uPA (data not shown).

Binding of synthetic uPA peptide to its receptor assessed by flow cytometr): or by single-cell proteolytic plaque assay

Competitive inhibition assays employing flow cytometry were performed to examine the affinity of each uPA peptide responsible for binding to the uPA receptor on human tumor (U937) cells. The results of experiments using PMA-stimulated adherent cells are shown in Figure 1: 50% inhibition of FITC-pro-uPA binding to U937 cells was obtained with 1.5 nM pro-uPA, 3.0 nM HMW-uPA, 100 nM human uPA17-34 and 1,000 nM human uPA20-30 respectively. Murine uPA peptides (17-34 and 20-30) did not bind to the receptor.

The cell-bound uPA activity was analyzed by a single-cell proteolytic plaque assay (Fig. 2). Mouse cells (macrophages and the 3LL cells) and human U937 cells incubated with murine and human pro-uPA, respectively, formed proteolytic plaques. The caseinolytic activity detected around the cells was markedly decreased when mouse cells were incubated with murine synthetic peptide (murine uPA17-34) or human cells

0

L3 L

f w I- -J W [L

L a

" 7-7-- I

0 0.01 0.1 1 .o 10 100 I000

COMPETITOR ( n M )

0, pro-uPA ; 0, HMW-uPA ; A, human UPA,,.~~ ; A, human UPA,,.,, ;

v, mouse uPA,,-,, : v, mouse uPA,,_,,

FIGURE 1 - Inhibition of binding of FITC-pro-uPA to U937 cell receptors by synthetic uPA peptides assessed by flow cytometry. Pro-uPA (0). HMW-uPA (0), human uPA17-34 (A), human uPA20-30 (A), murine uPA 17-34 (V), murine uPA20-30 (V). SD < 10% in all samples.

were incubated with human peptide (human uPA17-34). We confirmed the species specificity of uPA binding to its receptor, as previously reported (Estreicher et al., 1989).

Effects of the synthetic peptides on cell invasion in vitro We first demonstrated that murine and human peptides did

not increase [3H]thymidine incorporation and proliferation of 3LL cells and did not support the role of these peptides as a growth factor for 3LL cells (data not shown). The mouse peptide 17-34 showed a dose-dependent and statistically significant inhibition of invasion in vitro (Table I). The murine peptide 20-30 and human peptides (20-30 and 17-34, up to a concentration of 500 pg/ml), however, showed no significant inhibitory effects. The chemotactic response of the cells was also tested to determine if the inhibition of the cells to invade basement membranes was due to an inhibition of this response by the murine peptide 17-34 (Table 11). The cells tested showed a good chemotactic migration in the presence of the peptide. In addition, we examined the effects of the murine peptide 17-34 used in the experiments on cell attachment. Under optimal conditions, about 80% of these cells remain attached to the well after washing. No inhibition of attachment to Matrigel was seen with this peptide (Table 11).

Effects of the synthetic peptides on experimental metastasis We examined the effects of murine and human uPA pep-

tides on experimental lung metastasis after i.v. injection of 3LL cells. Each peptide was admixed with the cells and the mixtures were immediately co-injected i.v. into the lateral tail vein of a C57B1/6 mouse. Mice were sacrificed 21 days after co-injection. The results of a representative experiment are shown in Table 111. The number of metastatic foci on the lung surface did not decrease significantly when a single dose of each synthetic peptide (up to 500 pg/mouse) was injected together with the tumor cells into the tail vein. Increasing the number of injections of the murine peptide 17-34 (500 pg/mouse, Schedule 1) led to a slight decrease in the mean number of lung tumor colonies (J > 0.05). Multiple injections of the murine peptide 17-34 at a dose of 500 pglmouseiday on days 7-13 after tumor inoculation did not reduce the number of lung tumor colonies. Furthermore, multiple injections of the murine peptide 20-30 and the human peptides 20-30 and 17-34 did not decrease the number of lung tumor colonies, either.

Effects of the synthetic peptides on spontaneous metastasis We next examined the effects of these peptides on lung

metastasis of 3LL cells using a spontaneous metastasis model. Multiple injections of the synthetic peptides at the dose of 5-500 pg/mouse were carried out on days 0-6 or 7-13 after tumor cell inoculation. Tumor colonies in the lung were monitored 28 days after tumor inoculation. Table IV shows that a single co-injection of each peptide (500 pg/mouse) did not inhibit pulmonary metastasis. However, multiple i.p. injection of the murine peptides (20-30 and 17-34) according to Schedule 1 significantly reduced the number of lung tumor colonies, while the human peptides had essentially no effects. Multiple injections of the murine peptide 20-30 according to Schedule 1 also inhibited the formation of lung metastasis but was less effective than murine peptide 17-34. Multiple injections of the synthetic peptides according to Schedule 2, however, showed no reduction of lung tumor colonies. Even at the dose of 500 pg/mouse, the murine peptide 17-34 showed no significant decrease in the number of lung tumor colonies when administered according to Schedule 2. Thus the extent of the inhibition appears to depend on the dose and the administration schedule. Significant inhibition of 3LL spontaneous lung metastasis was obtained with sequential i.p. injections of the murine peptides during 7 days immediately after tumor cell inoculation. In general, treatment with the uPA peptides

730 KOBAYASHI ETAL.

FIGURE 2 - The relative binding affinities of synthetic uPA peptide vs. pro-uPA for human and mouse cells by single-cell proteolytic plaque assay. After 1 hr pre-incubation with or without a synthetic peptide [murine synthetic peptide uPA17-34 (M-uPA17-34) or human synthetic peptide uPA17-34 (H-uPA17-34); 100 kM] and washing with PBS, 0.1% BSA, the cells [mouse macrophages (A), mouse 3LL cells (B), and human U937 cells (C)] were further incubated for 1 hr with or without murine pro-uPA-containing medium (M-pro-uPA) or human pro-uPA (H-pro-uPA) (approx. 1 pgiml). Cell-associated proteolytic plaque was observed after 7 hr of incubation at 37°C. 1, no peptides; 2, cells were incubated with H-pro-uPA, 3, cells were incubated with M-pro-uPA; 4, after the preincubation with H-uPA17-34 and washing, cells were further incubated with H-pro-uPA (H-uPA17-34 + H-pro-uPA); 5, M-uPA17-34 + H-pro-uPA; 6, H-uPA17-34 + M-pro-uPA; 7, M-uPA17-34 + M-pro-uPA.

TABLE I -EFFECT OF THE SYNTHETIC PEPTIDES ON BASEMENT MEMBRANES INVASION BY 3LL CELLS IN VITRO

TABLE I1 - EFFECT OF THE SYNTHETIC PEPTIDES ON THE CHEMOTACTIC RESPONSE AND CELL ATTACHMENT OF 3LL CELLS

Treatment Invading cellsifield Concentration (LLP i m I \

Control 39 f 8

50 38 k 8 500 31 t 9

Murine peptide 17-34 5 43 f 8 sn 29 2 5’

Murine peptide 20-30 5 45 t 10

..

500 Human oeotide 20-30 500

20 t 61 50 k 11

Human bebtide 17-34 500 39 k 7

Control experiment was carried out in the absence of peptides. The murine and human synthetic peptides (20-30 and 17-34; at dose of 5, 50 and 500 ygiml) were treated as described in “Materials and Methods.” Each experiment was performed in triplicate. Data are expressed as the mean 2 SD of 3 experiments. Similar results were obtained when synthetic peptides were added to the cell suspension before the cells were seeded into the upper chamber.’p < 0.05 compared to control.

proved to be more effective in the spontaneous metastasis than in the experimental metastasis model.

DISCUSSION

In our previous work, we have shown that uPA polypeptides with intact GFD from proteinase-digested pro-uPA inhibit human ovarian cancer cell (HOC-I) invasion in an in vitro reconstituted basement membrane Matrigel assay, when all membrane receptor sites are saturated by enzymatically inactive uPA fragments, such as intact GFD or ATF (Kobayashi et al., 1993). In order to extend our in vitro observation we attempted to selectively saturate uPA receptor with competitive receptor-bound uPA peptides in an in vivo metastasis model.

Invading Attached cellsifield (9%) cellsifield (76) Treatment

Control 100 f 8 100 * 7 Murine peptide 20-30 Murine peptide 17-34 Human peptide 20-30 Human ueutide 17-34

91 f 12

95 f 8

107 & 13 91 2 13

Chemotactic response (invading cellsifield; %) and cell attachment (attached cellsifield; %). the mean ? SD of 3 experiments is shown.

In the present study, the significance of cell-surface uPA expression regarding the invasive potential of tumor cells was examined by saturating free uPA receptors with synthetic uPA peptides. We report here the possible mechanism by which murine uPA peptides with the amino acid sequences responsible for binding to the receptor inhibit invasion and pulmonary metastasis of 3LL cells. We assumed that if cell- associated uPA were essential to the invasive process, invasion would be inhibited after all membrane receptor sites were occupied by synthetic peptides. That is, pro-uPA synthesized and released by tumor cells would not bind to its own receptors, because tumor cells would not have free uPA- binding sites. We confirmed that receptor-bound uPA peptides can also lead to a decrease in the invasive potential of tumor cells [HOC-I (data not shown) and 3LL cells (Table I)] in the in vitro assays and would be worth testing in vivo. The binding of uPA to the receptor is species-specific (Blasi et al., 1986; Estreicher et al., 1989; Yu and Schultz, 1990). We assessed the relative binding affinities of synthetic uPA peptides vs. pro-uPA for human U937 cells, and similar results were shown for the 3LL cells that are the focus of this report (Fig. 2). Human uPA peptides do not bind to mouse cells (mouse macrophages and the 3LL cells), and murine peptides

UROKINASE AND TUMOR CELL METASTASIS 73 1

TABLE 111 -EFFECTS OF SYNTHETIC PEPTIDES ON EXPERIMENTAL METASTASES

n P Concentration Numher of lung c’o of Peptideb Treatment (Kgimousej nodules (mean k SDj inhibition

Experiment 1 Control a Control b Mouse u P A ~ ( ~ ~ ~ ~ Mouse u P A ~ ~ - ~ ~ Human uPAzo-30 Human u P A ~ ~ - ~ ~

Experiment 2

Experiment 3

Mouse uPA2&3o

Experiment 4

Experiment 5

Experiment 6 Control a Human U P A ~ ( ~ ~ ( ,

Human u P A ~ ~ - ~ ~

Single injection Single injection Single injection Single injection

Schedule 1

Schedule 2

Schedule 1

Schedule 2

Schedule 1 Schedule 2 Schedule 1 Schedule 2

0 500 500 500 500 500

0 5 1 5;:

1 5;:

1 5;:

[ 5;:

0 5

0 5

0 5

0 500 500 500 500

32.6 f 11.7 38.8 * 9.8 37.9 * 15.9 33.0 k 10.4 29.9 +- 12.1 36.8 f 7.9

21.3 +- 12.6 19.6 ? 9.3 24.1 2 10.3 25.1 * 13.4

68.5 f 26.4 59.6 f 20.1 70.3 31.6

21.2 * 9.6 22.6 k 13.2 19.8 2 11.1

67.6 f 30.1

13.2 2 8.8

31.3 f 10.6

37.5 f 18.2 29.9 f 12.8

40.0 +- 18.0

41.0 2 13.9 39.9 k 11.0 40.1 * 12.7 38.8 f 13.4 36.9 9.6

-

0 0 0 8 0

-

8 0 0

- 13 0 1

-

0 7

38

- 0 0 4

-

3 2 5

10

8 7 NS 9 NS 9 NS 8 NS 8 NS

8 8 NS 9 NS 9 NS

8 8 NS 7 NS 7 NS

8 7 NS 8 NS 9 NS

8 8 NS 8 NS 8 NS

9 9 NS 8 NS 7 NS 7 NS

Control a: no addition of any peptides. Control b: Kringle-domain (uPA47-135) purified from human pro-uPA was used as additional control.

show no specific affinity for the surface of human U937 cells or human HOC-I cells (Kobayashi et al., 1992), which were confirmed by single-cell proteolytic plaque assay and by flow cytometric analysis. Of the 4 peptides tested, the murine peptide 17-34 significantly inhibited 3LL cell invasion and metastasis in a dose-dependent manner both in the in vitro and in the in vivo systems, indicating that the ability of these peptides to compete with pro-uPA for its receptor apparently determined their ability to inhibit metastasis. This inhibition correlates with the quality and affinity of uPA peptides to saturate uPA receptor. Furthermore, the mode of action of uPA peptides was not due to inhibition of tumor cell attachment or of cell chemotactic migration. uPA peptides did not inhibit or stimulate the growth of tumor cells (data not shown). Besides, these uPA peptides were capable of blocking invasion not only when they were added together with the cells but also when they were pre-adsorbed to Matrigel (Kobayashi et al., 1992).

When we examined the effect of uPA peptides on the formation of lung metastasis, the murine peptide 17-34 caused substantial reduction of the number of pulmonary metastatic foci in the spontaneous metastasis assay. This assay measures multifactorial metastatic processes in which tumor cells leave a primary tumor, intravasate into the bloodstream, attach to the vascular wall, extravasate from the capillaries and grow to target organ (Yu and Schultz, 1990). The extent of inhibition depended on the dose and time of administration. Significant inhibition of the 3LL lung metastasis was obtained with daily injections of the murine peptide 17-34 for 7 days immediately after tumor inoculation. Human uPA peptides were used t o confirm that this effect was not attributable to physicochemical properties such as high viscosity at high concentration in in vivo studies.

The synthetic uPA peptide did not completely inhibit tumor cell invasion and metastasis, suggesting that in addition to intact peptide sequence 17-34, the conformation of GFD is a prerequisite for binding of uPA molecules and for inhibiting invasion. Furthermore, the incompleteness with which the competitive receptor-bound uPA peptides blocked invasion indicates that uPA is not the only mediator involved, suggesting that 3LL cells possess significant uPA/plasmin-indepen- dent proteinase activity. We did not examine the participation of other proteinases such as metalloproteinases (Liotta and Stetler-Stevenson, 1991). Other explanations for incomplete inhibition of tumor cell invasion by uPA peptides are that exogenous uPA peptides d o not displace endogenous pro-uPA/ HMW-uPA from its receptors and that the affinity of the peptides to uPA receptors may be much lower than that of pro-uPA/HMW-uPA produced by tumor cells. uPA receptors may also be re-occupied by uPA released from tumor cells. Competitive inhibition assays employing flow cytometry carried out to investigate the affinity of human uPA peptides responsible for binding to the uPA receptor on HOC-I and U937 cells demonstrated that human uPA peptides have an affinity for the uPA receptor of 100 times less than HMW-uPA.

Notwithstanding these limitations, our study strongly argues for a role of cell-associated uPAiuPA receptor system that facilitates in vitro invasion and in vivo metastases. However, the uPA peptide exerts essentially a minor effect in the experimental metastasis model. The experimental metastasis assay mainly measures the later steps of the metastatic migration process (attachment, extravasation and growth into the target organ) since tumor cells have been directly injected into the bloodstream. We have utilized these highly selective peptides to inhibit the binding of uPA to the receptor believed to play a role in the invasion and metastatic process. Treatment with

732 KOBAYASHI ETAL.

TABLE N - EFFECTS OF THE SYNTHETIC PEPTIDES ON THE SPONTANEOUS METASTASIS

n P Concentration Number of lung % of

(&mouse) nodules (mean ? SD) inhibition Peptides Treatment

Experiment 1 Control a Control b Mouse uPA20-30 Mouse uPAlM4 Human Human u P A ~ ~ - ~

Experiment 2

Mouse u P A ~ ~ ~ ~

Experiment 3

Mouse uPA2w30

Single injection Single injection Single injection Single injection

Schedule 1

Schedule 2

Experiment 4

Mouse u P A ~ ~ - ~ ~

Experiment 5

Mouse u P A ~ ~ - ~ ~

Experiment 6 Control a Human u P A ~ ~ ~ ~

Human u P A ~ ~ - ~ ~

Schedule 1

Schedule 2

Schedule 1 Schedule 2 Schedule 1 Schedule 2

0 500 500 500 5 00 500

0 5

50 500

0 5

50 500

0 5

50 500

0 5

50 500

0 500 500 500 500

29.8 t 10.6 27.6 t 9.5 31.6 2 10.1 33.9 rt 13.6 29.9 f 15.1 38.6 2 9.8

63.4 t 23.1 57.2 f 20.2 66.2 f 23.8 21.2 t 10.6

32.6 k 11.9

31.9 t 15.1 31.1 f 12.3

31.4 f 12.0 36.6 t 11.7 10.8 f 7.7 6.1 f 1.3

42.3 t 16.3 50.1 t 18.9 40.1 t 23.2 39.5 f 20.0

21.1 t 10.1 23.6 t 11.3 26.8 t 11.4 20.9 f 8.8

38.1 f 9.6

26.6 f 13.2

-

7 0 0 0 0

- 10 0

67

- 0 2 5

_.

0 66 81

- 0 5 7

-

0 0 1 0

I 7 7 8 8 7

8 7 8 8

7 7 7 7

7 7 7 7

8 8 7 7

7 7 7 7 7

NS NS NS NS NS

NS NS

< 0.01

NS NS NS

NS <0.01 < 0.01

NS NS NS

NS NS NS NS

uPA peptides in the spontaneous metastasis model proved to be more effective than in the experimental metastasis model. Although the sensitivity of inhibition of invasion will depend on the tumor cells o r the assay system employed, our results indicate that uPA regulates more efficiently the mechanism involved in the entry of tumor cells into the circulation (intravasation) than extravasation during the metastatic process. Our results may be in agreement with the hypothesis that uPA is essential in the early steps of spontaneous metastasis, especially in which tumor cells must penetrate through extracellular matrices surrounding the primary tumors, but not in the later stages (Ossowski and Reich, 1983; Ostrowski et al., 1986; Mignatti et al., 1986; Ossowski, 1988; Hearing et al., 1988; Kobayashi et al., 1992). The role of proteinases in the later steps was not tested in the model of lung colonization and should be also investigated. Our hypothesis does not agree with 2 reports on the expression of the human uPA gene in transformed murine cells promoting its lung colonization ability in mice (Axelrod et al., 1989; Yu and Schultz, 1990). It may be that other factors are rate-determining in the intravasation process rather than the activity of a receptor-bound uPA. Growth factors such as TGFP may in part function by modifying the proteolytic balance expressed by tumor cells. The ability of tumor cells to produce growth factors suggests that some tumor cells might produce an activity that could also affect the uPA levels. uPA exhibits mitogenic activity in some malignant cells and thereby may act as an autocrine or paracrine growth factor on cells in which it is synthesized. A

pivotal role of uPA in regulating paracrine interactions between metastatic cancer cells and target organ via growth factors, such as insulin-like growth factordtheir binding pro- teins and TGFP, may also be postulated. These data suggest that uPA participates not only in the early events of the metastatic process but also in regulating mitogenic activity of metastatic tumor cells.

The biological assays are very different, and elements other than just extravasation vs. intravasation could easily be involved; this conclusion would need better support using other experimental approaches. We must answer the following questions: Why is intravasation more susceptible? What specific step or steps are being targeted, and why is there a difference in this phase vs. extravasation? Does occupancy of the uPA receptor block cell division? What are the circulating concentrations of peptides? Do the tumor cells make uPA after injection? D o the metastases make uPA? In addition, some peptides can possess unexpected activities, and better in vitro evidence would be needed to show that the different peptides do or d o not inhibit receptor and subsequent cell- surface uPA functions. Further experiments are also needed to examine the effect of exogenously applied uPA peptides on the ligandlreceptor interaction.

A C K N O W L E D G E M E N T S

The authors acknowledge the very capable assistance of Drs. K. Takeuchi and M. Itoh during the course of this study.

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Inhibition of metastasis of lewis lung carcinoma by a synthetic peptide within growth factor-like domain of urokinase in the experimental and spontaneous metastasis model