Factor XIII transglutaminase supports hematogenous tumor cell metastasis through a mechanism dependent on natural killer cell function

Summary.  Background:  Multiple studies suggest that the hemostatic and innate immune systems functionally cooperate in establishing the fraction of tumor cells that successfully form metastases. In particular, platelets and fibrinogen have been shown to support metastatic potential through a mechanism coupled to natural killer (NK) cell function. As the transglutaminase that ultimately stabilizes platelet/fibrin thrombi through the covalent crosslinking of fibrin, factor (F) XIII is another thrombin substrate that is likely to support hematogenous metastasis. Objective:  Directly define the role of FXIII in tumor growth, tumor stroma formation, and metastasis. Methods:  Tumor growth and metastatic potential were quantitatively and qualitatively evaluated in wild-type mice and gene-targeted mice lacking the catalytic FXIII-A subunit. Results:  Loss of FXIIIa function significantly diminished hematogenous metastatic potential in both experimental and spontaneous metastasis assays in immunocompetent mice. However, FXIII was not required for the growth of established tumors or tumor stroma formation. Rather, detailed analyses of the early fate of circulating tumor cells revealed that FXIII supports the early survival of micrometastases by a mechanism linked to NK cell function. Conclusions:  Factor XIII is a significant determinant of metastatic potential and supports metastasis by impeding NK cell-mediated clearance of tumor cells. Given that these findings parallel previous observations in fibrinogen-deficient mice, an attractive hypothesis is that FXIII-mediated stabilization of fibrin/platelet thrombi associated with newly formed micrometastases increases the fraction of tumor cells capable of evading NK cell-mediated lysis.     

Immune-mediated modulation of breast cancer growth and metastasis by the chemokine Mig (CXCL9) in a murine model

Current immunotherapies are limited by several factors, including the failure to recruit sufficient numbers of immune effector cells to tumors. The chemokine monokine induced by gamma-interferon (Mig; CXCL9) attracts activated T cells and natural killer (NK) cells bearing the chemokine receptor CXCR3. We investigated Mig as an immunotherapeutic agent in a syngeneic murine model of metastatic breast cancer. We transfected the highly malignant murine mammary tumor cell line 66.1 to stably express murine Mig cDNA. Immune-competent mice injected with Mig-expressing tumor cells developed smaller local tumors and fewer lung metastases, and they survived longer than mice injected with vector-control tumor cells. Mig-mediated inhibition of local tumor growth was lost in the absence of host T cells. Mig-transduced tumors had increased numbers of CD4 T cells compared with vector-control tumors, consistent with the T-cell chemoattractant property of Mig, and many tumor-infiltrating host cells expressed CXCR3. NK cells had not been examined previously as a possible effector cell in Mig-based therapies. Our studies now show that NK cells are critical to the mechanism by which Mig limits metastasis. Inhibition of angiogenesis was not implicated as a mechanism of Mig-mediated therapy in this model. These studies support the hypothesis that by manipulating the Mig-CXCR3 gradient, it is possible to direct host immune effector cells to tumors, curtailing both local tumor growth and metastasis. These studies also implicate host NK cells as an additional effector cell critical for Mig-mediated control of metastasis.     

Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) have been identified in humans and mice as a population of immature myeloid cells with the ability to suppress T cell activation. They accumulate in tumor-bearing mice and humans and have been shown to contribute to cancer development. Here, we have isolated tumor-derived exosomes (TDEs) from mouse cell lines and shown that an interaction between TDE-associated Hsp72 and MDSCs determines the suppressive activity of the MDSCs via activation of Stat3. In addition, tumor-derived soluble factors triggered MDSC expansion via activation of Erk. TDE-associated Hsp72 triggered Stat3 activation in MDSCs in a TLR2/MyD88-dependent manner through autocrine production of IL-6. Importantly, decreasing exosome production using dimethyl amiloride enhanced the in vivo antitumor efficacy of the chemotherapeutic drug cyclophosphamide in 3 different mouse tumor models. We also demonstrated that this mechanism is relevant in cancer patients, as TDEs from a human tumor cell line activated human MDSCs and triggered their suppressive function in an Hsp72/TLR2-dependent manner. Further, MDSCs from cancer patients treated with amiloride, a drug used to treat high blood pressure that also inhibits exosome formation, exhibited reduced suppressor functions. Collectively, our findings show in both mice and humans that Hsp72 expressed at the surface of TDEs restrains tumor immune surveillance by promoting MDSC suppressive functions.     

ZD1839, a selective epidermal growth factor receptor tyrosine kinase inhibitor,shows antimetastatic activity using a hepatocellular carcinoma model

The epidermal growth factor receptor (EGFR) is highly expressed in many human tumors and provides a new target for anticancer drug development. EGFR-targeted agents have shown promising antitumor activity in preclinical and clinical trials. However, little is yet known about the effect of these new agents on tumor metastasis. Here, we investigate the effects of ZD1839 (Iressa), a selective EGFR tyrosine kinase inhibitor, on the metastatic properties of murine hepatocellular carcinoma CBO140C12. ZD1839 inhibited not only cell growth but also epidermal growth factor-induced chemotactic migration and production of active matrix metalloproteinase-9 in vitro. In mice, orthotopic implantation of a fragment of CBO140C12 tumor into the liver resulted in the formation of a solitary tumor nodule and intrahepatic metastasis. ZD1839, given p.o., inhibited growth of the implanted tumor and intrahepatic metastasis by approximately 50%. These results indicate that EGFR signaling plays an important role in tumor metastasis and that ZD1839 is effective at inhibiting intrahepatic metastasis.     

A novel bystander effect involving tumor cell-derived Fas and FasL interactions following Ad.HSV-tk and Ad.mIL-12 gene therapies in experimental prostate cancer

To enhance the NK population induced by Herpes Simplex virus thymidine kinase (HSV-tk) gene transduction and ganciclovir (GCV) treatment, adenovirus-mediated (Ad) expression of IL-12 was added to Ad.HSV-tk + GCV as combination gene therapy. This approach resulted in improved local and systemic growth suppression in a metastatic model of mouse prostate cancer (RM-1). In vitro assay of tumor infiltrating lymphocytes noted superior lysis of both RM-1 and Yac-1 targets with combination therapy, but in vivo depletion of NK cells only negatively impacted on systemic growth inhibition. TUNEL assay of primary tumors noted induction of apoptosis between two and four times higher than controls lasting for 6-8 days post-vector injection. After demonstrating that Ad.HSV-tk/GCV and Ad.mIL-12-induced IFN-gamma independently up-regulated expression of FasL and Fas, respectively, studies examined tumor cell-mediated death through Fas/FasL-induced apoptosis as a mechanism of primary tumor growth suppression. In vitro, combination therapy at low vector doses resulted in synergistic growth suppression, which could be negated by the addition of anti-FasL antibody. In vivo co-inoculation of an adenovirus expressing soluble Fas resulted in combination therapy-treated tumors, which were three times larger than expected, and a reduction in apoptosis to baseline levels. In FasL knockout mice, combination therapy maintained the superior results experienced in wild-type mice, indicating that tumor cell, not host cell FasL, was responsible for Fas transactivation. Therefore, the combination of Ad.HSV-tk/GCV + Ad.mIL-12 results in enhanced local growth control via apoptosis due to tumor cell expression of Fas and FasL and improved anti-metastatic activity secondary to a strong NK response.     

TRAIL-R deficiency in mice enhances lymph node metastasis without affecting primary tumor development

TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in established tumor cell lines but not nontransformed cells. Herein, we demonstrate a role for the apoptosis-inducing TRAIL receptor (TRAIL-R) as a metastasis suppressor. Although mouse models employing tumor transplantation have shown that TRAIL can reduce tumor growth, autochthonous tumor models have generated conflicting results with respect to the physiological role of the TRAIL system during tumorigenesis. We used a multistage model of squamous cell carcinoma to examine the role of TRAIL-R throughout all steps of tumor development. DMBA/TPA-treated TRAIL-R-deficient mice showed neither an increase in number or growth rate of benign papillomas nor an increase in the rate of progression to squamous cell carcinoma. However, metastasis to lymph nodes was significantly enhanced, indicating a role for TRAIL-R specifically in the suppression of metastasis. We also found that adherent TRAIL-R-expressing skin carcinoma cells were TRAIL resistant in vitro but were sensitized to TRAIL upon detachment by inactivation of the ERK signaling pathway. As detachment from the primary tumor is an obligatory step in metastasis, this provides a possible mechanism by which TRAIL-R could inhibit metastasis. Hence, treatment of cancer patients with agonists of the apoptosis-inducing receptors for TRAIL may prove useful in reducing the incidence of metastasis.     

DNA vaccination with CD44 variant isoform reduces mammary tumor local growth and lung metastasis

We have shown recently that cDNA vaccination, using a virtual lymph node, ameliorates experimental allergic encephalomyelitis. Successful cure from mammary tumor requires resolution of local tumor growth and metastases. We have examined whether targeting of CD44 cell surface adhesion molecule by cDNA vaccination plays a role in resolving mammary tumor development. We show here that CD44 cDNA vaccination decreases the tumor mass and metastatic potential in experimental mammary tumor of BALB/c mice. Vaccination of mice, inoculated with the mammary tumors, by cDNA of CD44 variant (CD44v) but not by cDNA of standard CD44, markedly reduced local tumor development and lung metastasis. Concomitantly, transfection of CD44 antisense into a highly metastatic mammary tumor cell line disrupted the CD44 expression of the cells and reduced their ability to establish local tumors as well as metastatic colonies in the lung. Moreover, when CD44v, but not standard CD44 sense cDNA, was transfected into the poorly metastatic cell line, tumor development was markedly enhanced. It is possible therefore that DNA vaccination with a specific CD44v construct could induce an immune resistance to mammary tumor progression.     

Suppression of tumor formation in lymph nodes by L-selectin-mediated natural killer cell recruitment

Natural killer (NK) cells are known to reject certain tumors in vivo; however, the ability of NK cells to prevent metastasis of tumors into secondary lymphoid organs has not been addressed. Here, we report that in tumor-bearing hosts, NK cells are recruited to regional lymph nodes in wild-type mice, but not in mice deficient for L-selectin or L-selectin ligands. By adoptive transfer and complete Freund's adjuvant stimulation experiments, we demonstrated that L-selectin on NK cells and L-selectin ligands on endothelial cells are essential for NK cell recruitment to lymph nodes. Furthermore, freshly isolated resident lymph node NK cells lysed tumors efficiently, and metastasis of B16 melanoma cells to draining lymph nodes was suppressed in wild-type or Rag-1-deficient mice, but not when NK cells were depleted. Although L-selectin-deficient NK cells efficiently lysed tumor cells in vitro, NK cell-dependent suppression of tumor metastasis was diminished in mice deficient for L-selectin or L-selectin ligands because of insufficient NK cell recruitment to lymph nodes. Moreover, tumor metastasis was substantially inhibited in L-selectin-deficient mice reconstituted with wild-type NK cells. These findings indicate that L-selectin-mediated NK cell recruitment plays a crucial role in the control of tumor metastasis into secondary lymphoid organs.     

Interleukin-18 inhibits osteolytic bone metastasis by human lung cancer cells possibly through suppression of osteoclastic bone-resorption in nude mice

Interleukin (IL)-18 exhibits antitumor as well as antiosteoclastogenic activities. These findings suggest that IL-18 is a potential tool for the treatment of cancers with associated osteolytic bone metastasis. We have previously shown that systemic daily administration of recombinant (r) IL-18 inhibits the development of osteolytic bone metastasis by human breast cancer cells. Here we demonstrate that systemic daily administration of rIL-18 (1 microg/mouse/d) for 21 days significantly inhibited the number and the total area of osteolytic bone metastasis by RWGT2 human lung cancer cells in nude mice. No severe adverse effects were observed. Natural killer (NK) cells did not increase in splenocytes from rIL-18-treated mice, and the in vitro NK activity of splenocytes against RWGT2 cells was only weakly enhanced in the presence of IL-18. The administration of rIL-18 made no difference in the growth of subcutaneous tumors, histologic indices (mitotic index, apoptotic index, and Ki-67-labeling index) of subcutaneous tumors or metastatic bone foci, or in the number of osteoclasts along the bone surface adjacent to tumors. Moreover, serum levels of cytokines including interferon-gamma, IL-1alpha, IL-6, tumor necrosis factor-alpha, and granulocyte/macrophage colony-stimulating factor, which regulate bone-resorbing activity of osteoclasts, were evaluated. Among them, IL-6 was remarkably downregulated in rIL-18-treated mice. These findings suggest that IL-18 inhibits osteolytic bone metastasis possibly through suppression of osteoclastic bone-resorption mediated in part by IL-6.     

Adenovirus gene transfer vectors inhibit growth of lymphatic tumor metastases independent of a therapeutic transgene

Adenovirus (Ad) gene transfer vectors traffic to regional lymph nodes (RLNs) after footpad injections in mice, resulting in localized production of interferon gamma (IFN-gamma). With this background, we evaluated the hypothesis that Ad vector administration may inhibit RLN tumor metastasis independent of the transgene in the expression cassette. Tumors of MM48, a cell line with a propensity toward lymphogenous metastasis, were established in the footpads of syngeneic C3H mice, and E1(-)E3(-) Ad vectors encoding no transgene (AdNull) or encoding an irrelevant transgene (AdCD; Escherichia coli cytosine deaminase with no 5-fluorocytosine administration) were administered (10(10) particles) in a peritumoral location. Both vectors suppressed the growth of tumor in the regional (popliteal) lymph node. This effect was localized to the regional, but not distant, lymph nodes (p < 0.05). Heat inactivation of the vector or decreasing the dose of the vector to 10(9) particles did not suppress RLN growth of the tumor when compared with 10(10) particles of active AdNull (p < 0.05 and p < 0.01, respectively). The ability of an E1(-)E4(-) vector expressing beta-galactosidase (AdRSVbetagal.11) to suppress RLN tumor growth showed that the E4 region of the Ad vector was not responsible for the effect. Blocking either IFN-gamma or natural killer (NK) cells with systemic antibody treatment in immunocompetent mice allowed rapid growth of RLN metastases despite Ad vector administration, and Ad vector injection into the footpads of tumor-free mice induced the accumulation of NK cells in the RLN. These data demonstrate that, in a metastatic murine tumor model, a low dose (10(10) particles) of replication-deficient Ad vectors inhibits RLN metastases independent of a therapeutic transgene, an effect that is mediated, at least in part, by IFN-gamma and NK cells.     

The difference in immunological properties between lymph node metastatic and non-metastatic cell lines of MCA-induced fibrosarcoma of C4W mice.

To pursue the process of lymph node metastasis, i.e, the preferential tumor growth in lymph node, we have established the non-metastatic M2B cell line which was derived from 3-methylcholanthrene-induced fibrosarcoma of C4W mouse and a metastatic cell line, M2BLN-M+ which was obtained from metastatic lymph nodes of irradiated C4W mouse which was subcutaneously implanted with cultured tumor cells, because implanted tumor cells were derived from the spontaneous metastatic lymph node of the parental M2B tumor, but regressed in naive C4W mouse. We examined the characteristics of both tumor cell lines in terms of the immunological cellular interactions. M2BLN-M+ showed unexpectedly to be more susceptible to cytotoxicity of immune effectors (NK cell, macrophage and cytotoxic T lymphocyte) than M2B did. When cultured both tumor cells with these effector cells, the growth inhibition of M2BLN-M+ was greater than that of M2B. The regional lymph node of tumor-bearer, however, showed no effective cytotoxic activity as reported by others. On the contrary, when cultured both tumor cells with non-immune lymph node cells, to be surprised, the proliferation of M2B was markedly suppressed, while that of M2BLN-M+ was slightly inhibited. The lymph node cells of M2B-bearing mice showed stronger cytostatic activity to M2B. The results suggest that the cytostatic activity of lymph node cells will be a pivotal factor, concerning the establishment of lymph node metastasis.     

Suppression of tumor metastasis by NK4 plasmid DNA released from cationized gelatin

NK4, composed of the NH(2)-terminal hairpin and subsequent four-kringle domains of hepatocyte growth factor (HGF), acts as an HGF-antagonist and angiogenesis inhibitor. This study is an investigation to evaluate the feasibility of controlled release formulation of NK4 plasmid DNA in suppressing the tumor growth, and lung metastasis. Biodegradable cationized gelatin microspheres were prepared for the controlled release of an NK4 plasmid DNA. The cationized gelatin microspheres incorporating NK4 plasmid DNA could continuously release plasmid DNA over 28 days as a result of microspheres degradation following the subcutaneous injection. The injection of cationized gelatin microspheres incorporating NK4 plasmid DNA into the subcutaneous tissue significantly prolonged the survival time period of the mice bearing Lewis lung carcinoma tumor. Increases in the tumor volume and the number of lung metastatic nodules of NK4 plasmid DNA release group were suppressed to a significantly greater extent than that of solution-injected group (77.4 and 64.0%, respectively). The number of blood vessels and the apoptosis cells in the tumor tissue were significantly suppressed (80.4%) and increased (127.3%) against free NK4 plasmid DNA-injected group. Thus, the controlled release of NK4 plasmid DNA augmented angiogenesis suppression and apoptosis of tumor cells, which resulted in suppressed tumor growth. We conclude that this controlled release technology is promising to enhance the tumor suppression achieved by gene expression of NK4.     

Heparan sulfate mimetic PG545-mediated antilymphoma effects require TLR9-dependent NK cell activation

Heparan sulfate (HS) is an essential component of the extracellular matrix (ECM), which serves as a barrier to tumor invasion and metastasis. Heparanase promotes tumor growth by cleaving HS chains of proteoglycan and releasing HS-bound angiogenic growth factors and facilitates tumor invasion and metastasis by degrading the ECM. HS mimetics, such as PG545, have been developed as antitumor agents and are designed to suppress angiogenesis and metastasis by inhibiting heparanase and competing for the HS-binding domain of angiogenic growth factors. However, how PG545 exerts its antitumor effect remains incompletely defined. Here, using murine models of lymphoma, we determined that the antitumor effects of PG545 are critically dependent on NK cell activation and that NK cell activation by PG545 requires TLR9. We demonstrate that PG545 does not activate TLR9 directly but instead enhances TLR9 activation through the elevation of the TLR9 ligand CpG in DCs. Specifically, PG545 treatment resulted in CpG accumulation in the lysosomal compartment of DCs, leading to enhanced production of IL-12, which is essential for PG545-mediated NK cell activation. Overall, these results reveal that PG545 activates NK cells and that this activation is critical for the antitumor effect of PG545. Moreover, our findings may have important implications for improving NK cell–based antitumor therapies.     

Inhibition of Breast Tumor Progression by Systemic Delivery of the Maspin Gene in a Syngeneic Tumor Model

Maspin has been shown to possess tumor-suppressing activity against breast tumor growth and metastasis. To test the therapeutic value of the maspin gene (SERPINB5) in breast cancer, we established a syngeneic breast tumor metastasis model. This model involved the implantation of mammary tumor cells orthotopically to mammary gland and allowed tumors to grow within the gland and become metastatic to other organs. The mammary tumor cells were initially isolated from MMTV-polyoma virus middle T transgenic mice and were selected in vitro for high invasiveness. Here, we demonstrate that the mammary tumor cells were highly invasive and metastatic. Overall, 100% of tumor-transplanted mice developed lung metastasis. Using nonviral liposome as a carrier, we delivered SERPINB5 to mice bearing mammary tumors. Our data showed that both primary tumor growth and metastasis were significantly inhibited in this syngeneic metastasis model. Such inhibition is mediated by SERPINB5 transgene through increased apoptosis in SERPINB5-treated tumors. Thus, SERPINB5 can be used in gene therapy against breast tumor growth and metastasis.     

Interleukin-10 inhibits tumor metastasis through an NK cell-dependent mechanism

Interleukin-10 (IL-10) is a recently described pleiotropic cytokine secreted mainly by type 2 helper T cells. Previous studies have shown that IL-10 suppresses cytokine expression by natural killer (NK) and type 1 T cells, thus down-regulating cell-mediated immunity and stimulating humoral responses. We here report that injected IL-10 protein is an efficient inhibitor of tumor metastasis in experimental (B16-F10) and spontaneous (M27 and Lox human melanoma) metastasis models in vivo at doses that do not have toxic effects on normal or cancer cells. Histological characterization after IL-10 treatment confirmed the absence of CD8+ and CD4+ T cells and macrophages at the sites of tumor growth, but abundant NK cells were localized at these sites. This unexpected finding was confirmed by showing that IL-10 inhibits most B16-F10 and Lox metastases in mice deficient in T or B cells (SCID and nu/nu mice), but not in those deficient in NK cells (beige mice or NK cell-depleted mice). However, IL-10 downregulation of pro-inflammatory cytokine production and/or recruitment of additional effector cells may also be involved in the anti-tumor effect at higher local concentrations of IL-10, since transfected B16 tumor cells expressing high amounts of IL-10 were rejected by normal, nu/nu, or SCID mice at the primary tumor stage, and there was still a 33% inhibition of tumor metastasis in beige mice.     

Roles of microsomal prostaglandin E synthase-1 in lung metastasis formation in prostate cancer RM9 cells

Cyclooxygenase-2 (COX-2) is known to correlate with a poor prognosis of prostate cancer and contribute to tumor metastasis. However, the precise mechanism of this phenomenon remains unknown. We have previously reported that host stromal microsomal prostaglandin E synthase-1 (mPGES-1) appeared critical for tumor-associated angiogenesis and tumor growth. Here, we tested whether or not mPGES-1 has a critical role in lung metastasis formation of prostate cancer. Murine prostate cancer cells (RM9) were intravenously injected and lung metastasis was estimated by counting colonies in the lungs. Mice treated with a selective COX-2 inhibitor, celocoxib, were suppressed lung metastasis compared to vehicle mice. This lung metastasis formation was also reduced in mPGES-1 knockout (mPGES-1 KO) mice, compared with wild type (WT) mice. This was accompanied with reduced angiogenesis around the metastasized colonies of RM9. Plasma protein levels and metastasized lung tissue mRNA levels of vascular endothelial growth factor (VEGF) and stromal cell derived factor-1 (SDF-1) were significantly suppressed in mPGES-1 KO mice in comparison with WT mice. In addition, the expressions of matrix metalloproteinases (MMP)-9, and metalloproteinases (MMP)-2 were down-regulated in metastatic lungs in mPGES-1 KO mice. These results suggested that host mPGES-1 was essential for MMP-2 and MMP-9 up-regulation that enhances tumor metastasis. mPGES-1 appears to be critical for tumor metastasis in prostate cancers. mPGES-1 inhibitors may be useful to protect against prostate cancer metastasis.     

Carbonic anhydrase XII mediates the survival and prometastatic functions of macrophages in human hepatocellular carcinoma

Macrophages constitute a major immune component in tumor tissues, but how these cells adapt to and survive in the nutrient-depleted and lactic acid–induced acidic tumor microenvironments is not yet fully understood. Here, we found that levels of carbonic anhydrase XII (CA12) expression were significantly and selectively upregulated on macrophages in human hepatocellular carcinoma (HCC). Transient glycolytic activation of peritumoral monocytes induced sustained expression of CA12 on tumor-infiltrating macrophages via autocrine cytokines and HIF1α pathways. On the one hand, CA12 mediated the survival of macrophages in relatively acidic tumor microenvironments, while on the other hand, it induced macrophage production of large amounts of C-C motif chemokine ligand 8 (CCL8), which enhanced cancer cell epithelial-mesenchymal transition (EMT) and facilitated tumor metastasis. Consistently, the accumulation of CA12⁺ macrophages in tumor tissues was associated with increased tumor metastatic potential and reduced survival of patients with HCC. Selective targeting of tumor-infiltrating macrophages with a CA12 inhibitor reduced tumor growth in mice and was sufficient to synergistically enhance the therapeutic efficacy of immune-checkpoint blockade. We suggest that CA12 activity is a previously unappreciated mechanism regulating the accumulation and functions of macrophages in tumor microenvironments and therefore represents a selective vulnerability that could be exploited in future designs for antitumor immunotherapeutic strategies.     

Investigation of the biological effects of anti-cell adhesive synthetic peptides that inhibit experimental metastasis of B16-F10 murine melanoma cells.

The experimental metastasis of B16-F10 murine melanoma cells is blocked by the anti-cell adhesive pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) derived from the central cell-binding domain of fibronectin. In this report, we show that peptide treatment substantially extends the survival time for mice injected intravenously with B16-F10 cells (8/8 vs. 0/8 mice alive at 150 d), thereby demonstrating the potential efficacy of GRGDS treatment in protection against metastatic colonization. We have also examined the specificity of GRGDS activity by testing a series of related homologues for their effects on experimental metastasis. The overall profile of the relative inhibitory activities of these peptides closely matched their previously established capacity to disrupt adhesion in vitro. Lung retention studies with radiolabeled B16-F10 cells revealed an accelerated rate of cell loss from the lung 0-6 h after coinjection with the active peptide GRGDS. This early effect of GRGDS was consistent with its short circulatory half-life, which was found to be 8 min. Taken together, these results suggest that peptide-mediated inhibition of pulmonary colonization is due to interference with B16-F10 cell adhesion to structures in the target organ. Possible peptide interference in tumor cell-blood cell interactions was examined in order to assess (a) possible biological side-effects of peptide treatment and (b) whether such interactions might be an alternative mechanism for GRGDS-mediated inhibition of pulmonary colonization. GRGDS was found to retain full inhibitory activity when coinjected with B16-F10 cells into mice in which platelet function was impaired by acetylsalicylic acid treatment or into thrombocytopenic mice treated with antiplatelet serum (76-93% inhibition of colony formation). These data suggest that platelet involvement in the effects of the peptide is minimal. Similarly, GRGDS was also found to be a potent inhibitor of experimental metastasis in natural killer (NK) cell-deficient beige mice (86% inhibition), thereby discounting the possibility that GRGDS artifactually enhanced NK cell activity. We conclude as a result of these studies that cell-binding fibronectin peptides are specific inhibitors of experimental metastasis that prolong survival, that they appear to function by blocking the adhesion of B16-F10 cells to structures in the target organ, and that they do not appear to act through side effects on certain metastasis-related blood cell functions. In the future, derivatives of fibronectin peptides may be potentially useful prophylactic agents for interfering with the process of metastasis.