Breast Cancer Research and Treatment 57: 271–275, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.

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Antimetastatic effect of desmopressin in a mouse mammary tumor model Daniel F. Alonso1, Guillermo Skilton1, Eduardo F. Far´ıas2 , Elisa Bal de Kier Joff´e2 , and Daniel E. Gomez1 1 Laboratory 2 Research

of Molecular Oncology, Department of Science and Technology, Quilmes National University; Area, Institute of Oncology Angel H. Roffo, Buenos Aires, Argentina

Key words: breast cancer, desmopressin, metastasis, tumor cell aggregation

Summary We have investigated the effects of desmopressin (DDAVP), a synthetic analog of the natural hormone vasopressin, on experimental lung colonization of mammary tumor cells using a syngeneic BALB/c mouse model. Coinjection of DDAVP (1–2 µg/kg body weight) at the time of i.v. inoculation of F3II carcinoma cells or LM3 adenocarcinoma cells significantly inhibited the formation of experimental lung metastases. In both cases, the number of pulmonary nodules was reduced about 70%. Inhibition of metastasis was also obtained with i.v. administration of DDAVP 24 h after tumor cell inoculation. Interestingly, the inhibition of lung metastasis was not due to direct cytotoxic effects of DDAVP on mammary tumor cells. The in vitro formation of multicellular aggregates in the presence of citrated plasma from control and DDAVP-treated mice was also examined. Control plasma rapidly induced a significant tumor cell aggregation. In contrast, in the presence of plasma from DDAVP-treated mice, tumor cells remained as a single cell suspension. DDAVP may help to dissolve the protective fibrin shield of circulating tumor cells. Our data suggest, for the first time, that adjuvant DDAVP therapy may impair successful implantation of circulating mammary tumor cells.

Introduction Cancer spread is a complex process that requires a series of sequential steps. During hematogenous metastasis, tumor emboli must survive transport in the circulation, adhere to blood vessels, and invade the vessel wall [1]. The vast majority of circulating tumor cells are eliminated rapidly, although aggregation with each other and with platelets or coating of emboli with a fibrin network enhances tumor cell survival. Fibrin clots also occur at sites of tumor cell arrest in the microcirculation [2, 3]. Recently, we have examined the effects of neuropeptide hormones on our mouse mammary carcinoma model F3II [4]. We reported that vasopressin and its synthetic derivative desmopressin (DDAVP; 1desamino-8-D-arginine vasopressin) can modulate tumor cell growth in vitro and the secretion of urokinase, a profibrinolytic enzyme involved in hematogenous metastasis [5]. In this regard, enhancement of pericel-

lular fibrinolysis may prevent coating of tumor emboli with fibrin, therefore decreasing the survival of tumor cells in the circulation. In vivo, DDAVP induces an increase of intravascular fibrinolysis. It has a prolonged duration of action compared to the natural hormone vasopressin, having a time to peak levels of 30–60 min after intravenous injection and a plasma half-life of 5–10 h [6]. In the present study, our goal was to investigate the effects of DDAVP on experimental lung colonization of metastatic mammary tumor cells. Materials and methods Synthetic peptide DDAVP acetate was purchased from Ferring Pharmaceuticals (Malmö, Sweden). For some in vitro experiments, DDAVP was also obtained from Sigma (St. Louis, MO).

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Cell lines and culture conditions

Tumor cell aggregation

The mouse mammary tumor cell lines F3II (sarcomatoid carcinoma) and LM3 (poorly differentiated adenocarcinoma) were grown as monolayers in minimal essential medium (MEM, Gibco, Grand Island, NY) supplemented with 5% heat-inactivated fetal bovine serum, 2 mM glutamine, and 80 µg/ml gentamycin [7, 8]. For harvesting, cells were trypsinized using standard procedures.

The formation of multicellular aggregates in the presence of plasma from control and DDAVP-treated mice was examined. Animals were injected i.v. with DDAVP or saline, as indicated above. After 30 min, arterial blood was drawn in the presence of 0.38% sodium citrate and platelet-poor plasma was prepared by centrifugation. A single-cell suspension (1 × 106 cells per ml in MEM) was mixed with mouse plasma at a final dilution of 1:10. Aliquots containing 0.5 ml of the cell suspension were placed in glass tubes and incubated in agitation at 37◦ C for 15–60 min. The aggregation was stopped by fixing the cells with 4% formaldehyde. The number of single cells in suspension was determined and the extent of aggregation was calculated [10].

Experimental metastasis assay BALB/c inbred mice with an age of 12–16 weeks and an average weight of 25 g were employed. Tumor cells at a concentration of 2×105 cells per 0.3 ml MEM per mouse were injected into the lateral tail vein of unanesthetized mice. Lung weights were measured 3 weeks later and the number of surface lung nodules was determined under a dissecting microscope, as described in [9]. DDAVP treatment Tumor cells were resuspended in serum-free MEM with or without DDAVP at a final dose of 1– 2 µg/kg body weight (25–50 ng per 0.3 ml MEM per mouse). To analyze whether DDAVP was mediating a direct antitumor effect, tumor cell suspensions were pretreated for 30–60 min at 37◦C in the presence of equivalent concentrations of DDAVP (80–160 ng/ml MEM). After DDAVP washout, tumor cells were pelleted, resuspended in serumfree MEM, and then injected i.v. in the absence of DDAVP. In complementary experiments, to study the extent of DDAVP effect on experimental metastasis, DDAVP was injected i.v. in 0.1 ml of normal saline 24 h after inoculation of tumor cells. Cell survival and in vitro cytotoxicity Tumor cell suspensions were incubated at 37◦ C in serum-free MEM with appropriate concentrations of DDAVP. After 1–5 h, cell viability was assessed by trypan blue exclusion technique. DDAVP effect was also assessed for 24–48 h on semiconfluent tumor cell monolayers in serum-free MEM. Monolayers were then washed, fixed, stained with toluidine blue, and solubilized with 1% SDS. The number of cells was estimated by measuring the absorbance at 595 nm.

Results Inhibition of experimental lung colonization of mammary tumor cells by DDAVP Coinjection of DDAVP at the time of i.v. inoculation of both F3II or LM3 cells remarkably inhibited the formation of experimental lung metastases. The number of pulmonary nodules was reduced about 70% in DDAVP-treated mice (Table 1). In vitro pretreatment of tumor cells with comparable concentrations Table 1. Effect of DDAVP on lung colonization by mouse mammary tumor cells Treatment

Number of lung nodulesc , median (range) F3II cells LM3 cells

Control DDAVP coinjectiona DDAVP pretreatmentb

14 (5–36) 5 (0–12)∗ 19 (3–66)

10 (1–33) 3 (1–18)∗ 12 (8–37)

a Tumor cells were resuspended in serum-free MEM and coinjected i.v. with DDAVP, at a final dose of 1 µg (F3II cells) or 2 µg (LM3 cells) per kg of mouse weight. b Tumor cell suspensions were pretreated in serum-free MEM for 30–60 min in the presence of equivalent concentrations of DDAVP and then washed, pelleted, resuspended, and injected i.v. in the absence of DDAVP. c The number of lung metastases was determined 3 weeks after injection of tumor cells (2 × 105 cells per 0.3 ml MEM per mouse). Each treatment group consisted of at least eight inbred mice. Values are representative of three independent experiments, using female or male mice. No sex-dependent metastatic properties were observed for either cell line. ∗ p < 0.01, Kruskal–Wallis test.

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Figure 1. Effect of administration of DDAVP 24 h after tumor cell injection on lung metastases. DDAVP, at a dose of 1 µg/kg, was administered i.v. 24 h after inoculation of F3II mammary tumor cells. Lung weights (open bars) and the number of experimental lung metastases (filled bars) were determined 3 weeks later. Data are expressed as mean ± SEM and each group consisted of 10 mice. In control animals, lung weights averaged 0.32 g and the number of metastatic lung nodules ranged from 4 to 60. ∗ p < 0.05 and ∗∗ p < 0.01, Mann–Whitney test.

of DDAVP followed by peptide washout did not reduce the incidence of lung colonies, ruling out the possibility that DDAVP was mediating its antimetastatic activity through a direct effect on tumor cells. As shown in Figure 1, inhibition of metastasis was also obtained with i.v. administration of DDAVP 24 h after tumor cell inoculation. Extrapulmonary tumor colonies were not found in any of the control mice or mice treated with DDAVP. Lack of in vitro cytotoxicity of DDAVP DDAVP did not reduce cell viability of tumor cell suspensions at the doses employed (Figure 2A). Similarly, semiconfluent monolayers were not affected by incubation for 24–48 h in the presence of DDAVP (Figure 2B). Inhibition of tumor cell aggregation by DDAVP After 15 min in the presence of diluted plasma from DDAVP-treated mice, most of the mammary tumor cells remained as a single cell suspension. In contrast, control plasma induced a significant tumor cell aggregation during the same time (Table 2). Furthermore, a clot was formed in control tubes and tumor cell clumps were trapped in a fibrin gel matrix. Later in

Figure 2. Tumor cell survival after DDAVP exposure: (A), cell viability of F3II carcinoma cells in suspension in the absence (squares) and presence of DDAVP, at a concentration of 80 ng/ml (triangles) or 160 ng/ml (circles); (B), DDAVP effect on cultured monolayers in vitro for 24–48 h, at a concentration of 80 ng/ml (open bars) or 160 ng/ml (filled bars). In all cases, SD was less than 10%. Similar results were obtained using LM3 adenocarcinoma cells. Table 2. Aggregation of F3II mammary tumor cells in the presence of control and DDAVP-treated mouse plasma Treatmenta

Percentage of aggregationb (mean ± SD) 15 min 30 min 60 min

Control (saline) DDAVP

73 ± 13 9 ± 3∗

78 ± 32 61 ± 28

84 ± 36 78 ± 11

a Animals were injected i.v. with DDAVP (1 µg/kg) or saline. After

30 min, arterial platelet-poor plasma was obtained. b The extent of aggregation was calculated from triplicate determina-

tions according to the following equation: (1−Nt /Nc )×100, where Nt (treated) represents the number of single cells after incubation in the presence of diluted mouse plasma and Nc (control) represents the original number of single cells in suspension. ∗ p < 0.001, t-test.

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the experiment, however, tumor cell aggregation also tended to increase in the presence of DDVAP-treated mouse plasma.

Discussion To the best of our knowledge, this is the first report of inhibition of lung colonization of mammary tumor cells by DDAVP, the synthetic derivative of the peptide hormone vasopressin. Further investigations will determine the precise mechanism by which DDAVP exerts antimetastatic effect in mice. Nevertheless, our results suggest that the effect of DDAVP is exerted in the early stages of metastasis, possibly limiting the formation of tumor cell emboli as well as altering the interaction of cancer cells with endothelium at the target organ. The possibility that the antimetastatic properties of DDAVP are associated with a direct cytotoxic effect was ruled out by the fact that in vitro pretreatment of tumor cells with the peptide did not modify their capacity to produce lung tumor colonies. Moreover, DDAVP did not reduce the viability of either tumor cell suspensions or semiconfluent monolayers. Metastatic tumor cells entering into the blood stream interact with components of the hemostatic system. This interaction results in fibrin deposition around tumor cells, determining the formation of microthrombi that increase the efficiency of the metastatic cascade [2, 11]. Fibrin deposition may determine an enhanced tumor cell aggregation and trapping in the target organ, and also protects tumor cells from destruction by host immunity [12]. In this regard, we have reported an enhancement of lung colonization by F3II cells administering a synthetic inhibitor of the profibrinolytic enzyme urokinase during the first stages of metastasis formation [13]. Conversely, it is known that DDAVP induces a rapid and marked increase in plasma levels of tissuetype plasminogen activator, the major effector of endogenous fibrinolysis [14, 15]. Hence, DDAVP could help to dissolve the protective fibrin shield of circulating tumor cells, as indicated by the reduction of cell aggregation in the presence of plasma from DDAVP-treated animals. Besides, DDAVP may modulate tumor-associated urokinase activity and enhance pericellular fibrinolysis in tumor emboli [4]. However, we cannot exclude that other actions of DDAVP could be mediating the described results. For instance, DDAVP may modify tumor cell attachment

by altering P-selectin expression on endothelial cells [16] or platelets [17]. DDAVP may also alter hemodynamics of blood flow or induce lysis of metastatic tumor cells through the production of nitric oxide from the vasculature [18, 19]. DDAVP has been previously used in patients with diabetes insipidus and in a variety of bleeding disorders [6, 20]. DDAVP appears to be a safe and effective hemostatic agent for use during surgery in patients with hemophilia or Von Willebrand disease [21]. Our data demonstrated antimetastatic properties of DDAVP in a mouse mammary tumor model, administering a dosage within the range that other authors have previously used and proved enhanced antidiuretic and hemostatic effects (0.3–4 µg/kg). These doses have the advantage of being well characterized from a pharmacological point of view [20–22]. The present observations suggest a potential clinical application of DDAVP during cancer resection. As the tumor is mobilized by the surgeon, a large number of viable tumor cells are liberated into the circulation. This fact has been confirmed by reverse transcription polymerase reaction in breast cancer surgery [23]. Whichever the mechanism of action involved, it seems that a safe pharmacological agent, such as DDAVP, may have a new clinical use: administered at the time of surgery, it may decrease or even prevent the metastatic implantation of tumor cells released during the surgical manipulation.

Acknowledgements The authors thank Dr Raúl Altman for useful discussions. This work was supported by grants from the Third World Academy of Sciences and Quilmes National University to D.F.A. and D.E.G.

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Address for offprints and correspondence: Daniel F. Alonso, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, R. Sáenz Pena 180, Bernal (1876) Buenos Aires, Argentina; Fax: (54-11) 4365-7132; E-mail: [email protected]