CpG-ODN-based immunotherapy is effective in controlling the growth of metastasized tumor cells

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG-ODN) act as potent immune stimulators by activating innate immunity through toll-like receptor 9. These immunomodulatory effects of CpG-ODN have been reported to be associated with anti-tumor immunity. In this study, we used a murine B16F10 melanoma model and a CT26 colon cancer model to assess whether CpG-ODN-based immunotherapy was effective in inhibiting tumor cells that have already metastasized to distant organs. Systemic administration of CpG-ODN after melanoma cell injection resulted in a significant inhibition of pulmonary colonization. When CpG-ODN was administered after tumor cell injection, it also inhibited pulmonary metastasis of the tumor cells, albeit to a lesser degree in the latter case. Systemic administration of CpG-ODN after subcutaneous inoculation of CT26 colon cancer cells diminished pulmonary metastasis from the primary tumor sites. Additionally, CpG-ODN also inhibited the growth of pulmonary colonization of the colon tumor cells when CpG-ODN was administered after the primary tumors had been surgically removed. These data indicate that CpG-ODN was effective in inhibiting pulmonary metastasis of the B16F10 melanoma and CT26 colon cancer cells, as well as the growth of metastasized tumor cells. Our results suggest that CpG-ODN-based immunotherapy may be beneficial in controlling micrometastasis after surgery in clinical settings.     

Expression of CXC chemokine receptor-4 enhances the pulmonary metastatic potential of murine B16 melanoma cells

The chemokine receptors CC chemokine receptor (CCR) 7 and CXC chemokine receptor (CXCR) 4 have been implicated in cancer metastasis. To evaluate whether CXCR4 is sufficient to increase tumor metastasis in an organ-specific manner, we transduced murine B16 melanoma cells with CXCR4 (CXCR4-B16) and followed the metastatic fate of the transduced cells in both i.v. and s.c. inoculation models of metastasis. CXCR4-B16 cells demonstrated marked increases (>10-fold) in pulmonary metastasis compared with vector (pLNCX2)-B16 after i.v. and s.c. inoculation of tumor cells. The increase in metastasis could be completely inhibited by T22, a small peptide antagonist of CXCR4. As early as 24 and 48 h after i.v. injection, CXCR4-B16 cells were significantly increased in the lung compared with control B16 cells by 5- and 10-fold (P < 0.05), respectively. CXCR4-B16 cells adhered better to both dermal and pulmonary microvascular endothelial cells relative to control B16 cells. Moreover, CXCL12 promoted the growth of CXCR4-B16 cells in vitro. Whereas expression of CXCR4 in B16 cells dramatically enhanced pulmonary metastasis, metastasis to the lymph nodes, liver, and kidney was rare. Immunohistochemical staining of both primary human cutaneous melanoma and pulmonary metastases revealed CXCR4 expression. Thus, CXCR4 plays a potentially important role in promoting organ-selective metastasis, possibly by stimulating tumor adhesion to microvascular endothelial cells and by enhancing the growth of tumor cells under stress.     

A new mechanism for tumor-induced platelet aggregation. Comparison with mechanisms shared by other tumors with possible pharmacologic strategy toward prevention of metastases

Because tumor-induced platelet aggregation appears to play a role in the development of certain experimental tumor metastases, we examined the mechanism(s) of tumor-induced platelet aggregation as well as the effect of various anti-platelets agents. Two mechanisms for tumor-induced platelet aggregation have been previously described: (1) a mechanism which requires complement, a stable plasma factor, divalent cation and a sialo-lipo-pro-tein vesicular component of the tumor membrane for platelet aggregation; and (2) a mechanism which operates via the generation of thrombin and requires a phos-pholipid component of the tumor membrane. We now report a new mechanism of tumor-induced platelet aggregation which is shared by three different tumors: a spontaneously metastatic human melanoma, HM29, a murine melanoma, B16F10, and a carcinogen-induced metastatic murine colon carcinoma, CT26. These tumors do not require cell-surface sialic acid or serum complement as does the first mechanism. They do not require cell-surface phospholipid, as do the tumors representing the other two mechanism. They do not aggregate platelets via the generation of thrombin as do tumors representing the second mechanism. These tumors are unique in that they require a trypsin-sensitive surface protein for activity. The ability of the thrombin-generating tumors to aggregate platelets is uniquely sensitive to two highly specific, synthetic thrombin-competitive inhibitors: DAPA and No. 805. The other two groups of tumors are at least 10 times more sensitive to inhibition of platelet aggregation by elevation of cyclic AMP levels (prostacy-clin, 6-keto-PGE₁, dibutyryl cyclic AMP) and at least 10 times more sensitive to inhibition of prostaglandin synthesis (indomethacin, ibuprofen). Thus, tumor-induced platelet aggregation is heterogeneous with respect to mechanism of action as well as inhibition by anti-platelet pharmacologic agents. Sensitivity to anti-platelet agents correlates with the mechanism by which tumor cells aggregate platelets.     

Long exposure of non-cytotoxic concentrations of methylselenol suppresses the invasive potential of B16F10 melanoma

To assess the inhibitory effects of methylselenol on the invasion of murine B16F10 melanoma cells, we carried out in vivo and in vitro experiments using Se-methylselenocysteine (Se-MSC) and selenomethionine (SeMet), respectively. In an animal experiment, the supplementation of drinking water with Se-MSC (4 ppm Se) led to a significant increase in Se levels in the lung, liver and serum in mice. Mice given a mash diet or water supplemented with Se-MSC (2, 4 and 6 ppm Se in the mash diet, and 2 and 4 ppm Se in the drinking water) displayed an almost completely diminished pulmonary metastasis of B16F10 melanoma cells and an enhanced survival, compared to the control mice which were given a basal diet. Treatment with non-cytotoxic concentrations of SeMet (2.5, 5 and 10 microM plus 0.02 U/ml METase, methioninase) induced a substantial decrease in the expression of integrin alphavbeta3, the FN receptor and adhesion ability to vitronectin (VN) and fibronectin (FN) in B16F10 melanoma cells. Moreover, these compounds suppressed gelatinase activity, invasive ability and wound migration in the culture system. SeMet-METase prevented the conversion of pro-MMP-9 to its active form and decreased pro-MMP-2 activities in a zymogram. The pre-treatment of B16F10 melanoma cells with SeMet-METase led to a decrease in pulmonary metastasis and extended survival in mice injected with tumor cells. Collectively, our results indicate that integrin expression is crucial in promoting the metastatic phenotype in murine B16F10 melanoma cells by supporting specific adhesive and invasive properties, suggesting that Se-MSC effectively reduces the metastasis of B16F10 melanoma cells as a nutritional adjuvant. Methylselenol may also contribute to the suppression of integrin expression.     

Platelets and fibrinogen facilitate each other in protecting tumor cells from natural killer cytotoxicity

The functions of platelets and fibrinogen in protecting tumor cells from natural killer cytotoxicity have been discussed for more than 20 years. However, their exact roles and relationships in the process are still not clear. In this study, we show that tumor cells prefer to adhere to fibrinogen than to platelets, and fibrinogen can enhance the adhesion of tumor cells to platelets. β₃ integrin plays an important role in the adhesion of B16F10 to platelets enhanced by fibrinogen. In the presence of thrombin, fibrinogen forms dense fibrin(ogen) layers around tumor cells. Tumor cells can induce platelets to aggregate and form thrombin. Platelets, as well as thrombin, can help fibrinogen protect tumor cells from lethal contact with natural killer cells and natural killer cytotoxicity. Hirudin, a specific inhibitor of thrombin, can reverse the effect of platelets on fibrinogen in blocking natural killer cytotoxicity. Our results suggest that fibrinogen helps platelets to adhere to tumor cells, and platelets in turn promote more fibrinogen to aggregate around tumor cells by forming thrombin. They facilitate each other in protecting tumor cells from natural killer cytotoxicity. ( Cancer Sci 2009; 100: 859–865)     

An in vivo study of the role of the tumor cell cytoskeleton in tumor cell-platelet-endothelial cell interactions

We recently reported that disruption of tumor cell microfilaments or intermediate filaments resulted in an inhibition of the ability of tumor cells to induce the aggregation of homologous platelets in vitro (H. Chopra et al., Cancer Res., 48: 3787-3800, 1988). Previous investigators demonstrated that disruption of the tumor cell cytoskeleton decreases the ability of these cells to form lung colonies. We proposed that this latter effect is due, in part, to decreased interaction of tumor cells with platelets, following their arrest in the microvasculature. To test this hypothesis, B16 amelanotic melanoma cell microtubules, microfilaments, or vimentin intermediate filaments were disrupted with colchicine (50 microns), cytochalasin D (50 microns), or cycloheximide (50 microns), respectively, and then cells were tail vein injected into syngeneic mice. Both cytochalasin D- and cycloheximide-treated cells formed fewer lung colonies than did control cells. Colchicine, however, failed to inhibit lung colony formation. Neither colchicine nor cycloheximide treatment altered initial pulmonary arrest; however, fewer cycloheximide-treated cells remained in the lungs 8 h postinjection. Greater than 90% of control or colchicine-treated cells were found to be associated with activated platelets, and they also demonstrated typical cell membrane process formation 10 min and 8 h post-tumor cell injection. In contrast, less than 10% of cycloheximide-treated cells were in contact with activated platelets 10 min postinjection. However, by 8 h approximately 90% of cycloheximide-treated cells were in contact with activated platelets. This recovery coincided with the reformation of the B16 amelanotic melanoma vimentin intermediate filament network and the reacquisition of the ability to induce platelet aggregation in vitro. Neither colchicine nor cycloheximide treatment altered initial B16 amelanotic melanoma cell adhesion to murine microvessel-derived endothelial cells. This study provides in vivo evidence in support of our previous findings that disruption of certain cytoskeletal elements (i.e., vimentin intermediate filaments) inhibits the tumor cell ability to activate platelets. This study also suggests that platelet activation may stabilize the initial tumor cell arrest in the microvasculature.     

Inhibition of invasion and experimental metastasis of murine melanoma cells by human soluble thrombomodulin

Thrombomodulin (TM) is an anticoagulant molecule expressed on the endothelial cell surface and soluble TM antigen, which is present in human plasma and urine, represents the products of limited proteolytic cleavage of cell-surface TM. Recently, it was demonstrated that TM is also expressed on the surface of several tumor cells and the expression level of TM negatively correlated with malignancy in cancer. We investigated the effect of soluble TM isolated from human urine (uTM) on the invasion and metastasis of murine melanoma cells (B16F10 cells) through a reconstituted basement membrane (Matrigel) and in a murine model of experimental lung metastasis. Matrigel reconstituted with uTM inhibited the invasion of B16F10 cells in a dose-dependent manner in a range from 10 to 1000 ng/ml uTM as compared with the control Matrigel without uTM. The inhibitory action of uTM was not altered in the presence of an excess amount of hirudin, an inhibitor of thrombin proteolytic activity, but abolished in the presence of anti-human TM IgG. Matrigel reconstituted with thrombin (1 NIH unit/ml) enhanced the invasion level of cells by 1.5-fold relative to the control Matrigel without thrombin. The thrombin-enhanced invasion of B16F10 cells was repressed by addition of hirudin (10 units/ml) or uTM (100 ng/ml) into the Matrigel. Matrigel reconstituted with hirudin (10 units/ml) and uTM (100 ng/ml) additionally accelerated the inhibitory activity of hirudin or uTM on the thrombin-enhanced invasion of B16F10 cells. Moreover, metastatic colonies formed in the lungs of mice injected intravenously with B16F10 cells were significantly reduced by injection of uTM once a day up to 2 days after co-injection of uTM with the cells. These results suggested that Matrigel reconstituted with uTM inhibited the invasion of B16F10 cells in vitro through a thrombin-independent mechanism and the injection of uTM suppressed experimental lung metastasis of the cells in mice.     

Thrombin increases the metastatic potential of tumor cells

Initial arrest of tumor cells in the microvasculature and their attachment to the endothelium and subendothelial matrix (SEM) are essential prerequisites for metastasis to occur. Factors mediating these interactions are viewed as important determinants of the tumor-cell metastatic phenotype. In this work we have studied the effects of thrombin, its analogs and its precursors on the adhesive properties and metastatic potential of tumor cells. We show that α-thrombin, the native form of the key coagulation enzyme, is capable of enhancing tumor-cell adhesion to both the endothelium and SEM components represented by fibronectin. Subclotting, physiological concentrations of α-thrombin produced a 2- to 5-fold increase in tumor-cell adhesion. A bell-shaped dose-response curve was observed, with maximal effect at 0.1 U/ml. Maximum effect occurred when cells were exposed to the agonist for 15 min and exposure for up to 4 hr resulted in enhanced tumor-cell adhesion. Prolonged incubation with thrombin resulted in a decline in the thrombin-enhanced adhesion which reached unstimulated control levels by 24 hr. Thrombin precursors and active-site-inhibited thrombin analogs only had minimal adhesion-enhancing activity; nitro- and exosite-α-thrombin, which retain a functional active site, mimicked, although to a lesser degree, the action of α-thrombin. Tumor-cell incubation with thrombin resulted in an upregulated cell-surface expression of the α_llbβ₃ integrin, a receptor mediating interactions between tumor cells and endothelial cells, and between tumor cells and SEM. Antibodies against α_llbβ₃ integrin effectively inhibited thrombin-enhanced tumor-cell adhesion. Thrombin effects on tumor cells involved the PKC signal transduction pathway as thrombin-enhanced adhesion was inhibited by pre-incubation with PKC inhibitors and a transient PKC translocation from cytosol to membrane was observed following thrombin challenge. In vivo, thrombin-treated tumor cells demonstrated a 2-fold increase in their lung-colonizing ability. In contrast to the adhesion results, the metastasis-enhancing effects of α-thrombin were mimicked by a thrombin precursor (prothrombin) and thrombin analogs.     

Expression of a Mr 41,000 glycoprotein associated with thrombin-independent platelet aggregation in high metastatic variants of murine B16 melanoma

In the previous study, we generated a monoclonal antibody, 8F11, against NL-17, a high metastatic clone derived from a metastatic variant of murine colon adenocarcinoma 26. 8F11 inhibited platelet aggregation induced by NL-17 and recognized a Mr 44,000 membrane protein as antigen. In the present study, the reactivity of 8F11 to murine B16 melanoma and its metastatic variants was examined, and the antigen recognized by 8F11 on the cell surface was characterized. 8F11 was found to strongly react with 3 metastatic variants of B16 melanoma. In contrast, only slight reactivity was observed with parent B16 melanoma. The reactivity of the antibody to these cells was in the order B16F10 greater than B16BL-6 greater than B16F1 much greater than B16. Western blot analysis showed a Mr 41,000 protein as the antigen recognized by 8F11 on the cell surface of B16F10 cells. The Mr 41,000 antigen appeared to be a glycoprotein that bound to wheat germ agglutinin as has been observed for the Mr 44,000 antigen of NL-17. To elucidate the functional role of the Mr 41,000 antigen in B16 melanoma, platelet aggregation induced by B16 and B16F10 was compared. B16 was reported to stimulate platelet aggregation by the generation of thrombin, whereas B16F10 was found to activate platelet by at least 2 mechanisms: one dependent on thrombin and the other independent on thrombin. The activity of B16 and its metastatic variants to induce platelet aggregation in the presence of MD805, a synthetic antagonist of thrombin, well correlated with the reactivity of 8F11 to these cells. 8F11 blocked platelet activation by B16F10 under conditions preventing thrombin activity such as enzymatic formation of lysolecithin through the treatment of the cell surface with phospholipase A2 or in the presence of MD805. These data indicate that Mr 41,000 glycoprotein recognized by 8F11 on metastatic variants of B16 melanoma is involved in the thrombin-independent platelet aggregation. A positive correlation was observed between the levels of Mr 41,000 glycoprotein expression of B16 and its metastatic variants and their pulmonary metastasis after i.v. injection, suggesting Mr 41,000 glycoprotein, as well as other factors reported previously, may play an important role in the hematogenous spread of B16 melanoma.     

Effects of cytokine-mediated modulation of nm23 expression on the invasion and metastatic behavior of B16F10 melanoma cells

The molecular mechanisms of tumor invasion and metastasis are yet to be fully elucidated. A potential tumor-metastasis-suppressor gene nm23 has been described in certain rodent and human tumors. In the present study, we examined the potential anti-invasive and anti-metastatic effect of nm23 gene in B16F10 cells, a malignant murine melanoma cell line. Transfection of nm23 gene into B16F10 melanoma ceils resulted in significant suppression of the invasiveness and metastatic ability of melanoma cells and significantly enhanced the survival of tumor-bearing mice. B16F10 melanoma cells transfected with nm23 produced significantly less soluble ICAM-I and were more susceptible to LAK-cell-mediated cytotoxicity. Co-culture of B16F10 melanoma cells with IL-2 had no effect on nm23 expression, whereas treatment with PGE₂, TNF-α and IFN-γ resulted in down-regulation of nm23 expression. Concomitantly, in vivo treatment with TNF-α or IFN-γ in experimental mice increased pulmonary metastases and lowered the overall survival period, as compared with IL-2 treatment alone. These results provide evidence that nm23, in addition to its anti-metastatic function, could also be involved in modulating tumor-target-structure expression, in down-regulating invasive potential and in production of soluble intracellular adhesion molecules. The down-regulation of nm23 by TNF-α, IFN-γ and particularly by PGE₂ warrants re-examination of current immunotherapeu-tic protocols and of the role played by PGE₂ in tumor progression. © 1995 Wiley-Liss, Inc.     

CXCR4 enhances adhesion of B16 tumor cells to endothelial cells in vitro and in vivo via beta(1) integrin

The chemokine receptor, CXCR4, is expressed by human melanomas, and its ligand, CXCL12, is frequently produced at sites of melanoma metastasis. Herein, we examine CXCR4-enhanced binding of B16 murine melanoma cells to endothelial cells (ECs) and recombinant adhesion molecules in vitro to determine the role of tumor- and EC-derived adhesion molecules in tumor metastasis. By flow cytometry, unstimulated primary lung ECs showed constitutive expression of vascular cellular adhesion molecule 1 (VCAM-1), whereas skin-derived ECs did not. All B16 cell lines tested showed constitutive expression of alpha(4) and beta(1) integrin chains but showed no expression of beta(2) integrins. CXCR4-B16 arrest on VCAM-1/immunoglobulin-coated plates and tumor necrosis factor alpha-stimulated ECs under physiological shear stress conditions (1.5 dynes/cm(2)) was rapid, resistant to shear stress of 10 dynes/cm(2), and showed no evidence of rolling before arrest. In vitro, CXCR4-B16 cell binding to ECs was blocked by anti-beta(1) and anti-CXCL12 monoclonal antibodies. In vivo, metastasis of CXCR4-B16 cells to murine lungs was strongly inhibited by anti-CXCL12 and two different anti-beta(1) monoclonal antibodies. Finally, CXCR4-B16 exposed to CXCL12 rapidly increased binding affinity for soluble VCAM-1/immunoglobulin as detected by a flow cytometric assay. Thus, beta(1) integrins play a critical role in CXCR4-mediated B16 tumor cell metastasis in vivo and may be a potential target for inhibition of tumor metastasis, particularly to the lung.     

保尔佳对黑色素瘤细胞转移能力及相关细胞行为的影响

考察保尔佳对恶性肿瘤转移的影响及其相关机制。采用细胞微管吸吮实验技术单细胞定量观察不同浓度的保尔佳处理小鼠黑色素瘤细胞不同时间后，瘤细胞对培养牛肺动脉内皮细胞粘附性的改变，同时采用Ｂｏｙｄｅｎ小室法检测经同样处理的瘤细胞的侵袭穿膜能力。采用瘤细胞注射法制作肺转移荷瘤小鼠模型，观察经处理瘤细胞肺转移结节形成数目的改变。保尔佳处理可明显降低瘤细胞与内皮细胞的粘附性，同时抑制其侵袭穿膜能力，这种作用在１０－２～１０μｇ／ｍｌ浓度范围呈剂量依赖性，在１２～４８ｈ范围呈时间依赖性，短时间（３０ｍｉｎ）处理无明显作用。经保尔佳处理的癌细胞，其在肺组织中形成转移结节的数目明显减少。保尔佳通过抑制瘤细胞转移相关性细胞行为—粘附、侵袭，从而起抑制肿瘤转移的作用     

Comparative in vivo and in vitro proliferation of murine tumor cells in the bone microenvironment

To examine the effect of bone microenvironmental factors on the growth of metastatic cells, the in vivo proliferative features of three murine cell lines were determined at skeletal metastatic sites and correlated with their ability to grow in vitro in the presence of bone-derived factors. Bones, ovaries, adrenals and the brain were most affected by metastasis, following an intraarterial injection of B16/F1 and B16/F10 melanoma and FS/L10 fibrosarcoma cells into C57BL/6 mice. Melanoma cells showed a marked metastatic preference for bone, while fibrosarcoma cells developed brain metastasis in all animals. Tumor burden in bones was highest (19+/-2%) for B16/F10 cells, compared to B16/F1 (10+/-2%) or FS/L10 (3+/-1%) cells. Autoradiographic studies demonstrated organ- and cell type-specific differences in tumor cell proliferation, with B16/F10 cells displaying the lowest labelling indexes in bone (12+/-2% for B16/F10 vs 28+/-2% and 27+/-4% for B16/F1 and FS/L10 cells, respectively). To test if bone-derived factors differentially affected tumor cell growth in these three cell lines H-3-thymidine uptake by these tumor cells was assessed after in vitro incubation with bone-derived conditioned medium. Under these conditions, we observed stimulation of B16/F10 cell proliferation, but inhibition of uptake in the other two cell lines. Thus, these results demonstrate that, in this in vivo experimental model, growth properties of metastatic cells are organ- and cell type-specific. Additionally, we show that the in vitro proliferative behavior of tumor cells in the presence of bone-derived factors correlates and may predict skeletal tumor growth properties in vivo.     

Antimetastatic and antitumor effects of benzoquinonoid AC7-1 from Ardisia crispa

An antimetastatic and cytostatic substance, termed AC7-1, was isolated from Ardisia crispa and identified as a benzoquinonoid compound, 2-methoxy-6-tridecyl-1,4-benzoquinone. It was originally characterized as the potent PAF (platelet-activating factor) receptor-binding antagonist with nonspecific antiplatelet effects on platelet aggregation induced by various agonists including PAF, ADP, thrombin and collagen. The nonspecific antiaggregatory properties of AC7-1 drew our interest given its possible relationship in integrin receptor-binding antagonistic activity. The integrin receptor plays an important role in metastasis and thrombosis as the cell surface transmembrane protein. Based on the aforementioned facts, the antimetastatic activities of AC7-1 were examined using various in vitro and in vivo metastasis assays. AC7-1 strongly blocked B16-F10 melanoma cell adhesion to extracellular matrix (ECM) and B16-F10 melanoma cell invasion. AC7-1 also remarkably inhibited pulmonary metastasis and tumor growth in vivo. AC7-1 inhibited B16-F10 melanoma cell adhesion to only specific synthetic peptides including RGDS. These findings suggest that antimetastatic activities of AC7-1 can be caused by blocking integrin-mediated adherence. We found AC7-1 to be a potential candidate for the development of a new antimetastatic drug.     

Inhibition of tumor growth in vivo by hyaluronan oligomers

One of the critical events in tumor growth and metastasis is the interaction between tumor cells and host tissue stroma, mediated by different adhesion receptor repertoires in different tumor cell types. Several lines of evidence indicate that interaction between the hyaluronan receptor CD44, expressed on tumor cells, and host tissue stromal hyaluronan can enhance growth and invasiveness of certain tumors. Disruption of CD44-hyaluronan interaction by soluble recombinant CD44 has been shown to inhibit tumor formation by lymphoma and melanoma cells transfected with CD44. Since hyaluronan is a ubiquitous glycosaminoglycan polymer from which oligosaccharides of defined size can be readily purified, we tested the ability of hyaluronan oligomers to inhibit tumor formation by subcutaneously (s.c.) injected B16F10 melanoma cells. Our results indicate that hyaluronan oligomers injected at concentrations as low as 1 mg/ml can markedly inhibit B16F10 melanoma growth, providing a potentially attractive reagent for the control of local tumor development. Int. J. Cancer 77:396–401, 1998. © 1998 Wiley-Liss, Inc.     

Thrombin promotes platelet-mediated melanoma cell adhesion to endothelial cells under flow conditions: role of platelet glycoproteins P-selectin and GPIIb-IIIA.

We investigated the role of platelets in human melanoma cell (line 397) interaction with vascular endothelial cells (ECs) under flow conditions. The ability of the tumour cells to adhere to the EC monolayer was significantly reduced by application of flow at a shear rate of 250 s(-1). A 2.2-fold increase in tumour cell adhesion to ECs under flow was observed upon addition of thrombin receptor agonist peptide (TRAP)-activated platelets but not resting platelets. A similar increase (2.5-fold) in tumour cell adhesion to ECs under flow was observed when the tumour cells were incubated with resting platelets on thrombin-treated ECs. However, thrombin treatment of the ECs alone had no effect on tumour cell adhesion in the absence of platelets. The enhancement of tumour cell adhesion to ECs by TRAP-activated platelets was virtually abolished by blockade of the platelet glycoproteins P-selectin and GPIIb-IIIa by monoclonal antibodies. Blockade of P-selectin also inhibited the direct adhesion of TRAP-activated platelets to ECs, but did not affect the interaction of the tumour cells with platelets immobilized on subendothelial extracellular matrix (ECM). Blockade of GPIIb-IIIa inhibited both platelet-EC and platelet-tumor cell interactions. Our results indicate that tumour cell adhesion to the endothelium under flow is enhanced by platelets under conditions that allow platelet adhesion to ECs. Inhibition studies suggest that activated platelet adhesion to ECs is mediated by P-selectin and GPIIb-IIIA, and tumour cell adhesion to EC-bound platelets--mainly by GPIIb-IIIa.     

The reversible P2Y12 inhibitor ticagrelor inhibits metastasis and improves survival in mouse models of cancer

Tumor cells use activated platelets to promote their proliferation and metastatic potential. Because platelet activation is largely mediated through ADP engagement of purinergic P2Y12 receptors on platelets, we investigated the potential of the reversible P2Y12 inhibitor ticagrelor, a clinical agent used in the prevention of cardiovascular and cerebrovascular events, to inhibit tumor adhesion and metastasis. In B16‐F10 melanoma intravenous and intrasplenic metastasis models, mice treated with a clinical dose of ticagrelor (10 mg/kg) exhibited marked reductions in lung (84%) and liver (86%) metastases. Furthermore, ticagrelor treatment improved survival compared to saline‐treated animals. A similar effect was observed in a 4T1 breast cancer model, with reductions in lung (55%) and bone marrow (87%) metastases following ticagrelor treatment. In vitro, B16‐F10 cells exhibited decreased interaction with platelets from ticagrelor‐treated mice compared to saline‐treated mice, an effect similar to that observed with blockade of glycoprotein IIbIIIa. Similarly, B16‐F10 cells co‐incubated with platelets from ticagrelor‐treated mice exhibited reduced adhesion to endothelial monolayers compared to those co‐incubated with platelets from saline‐treated animals, an effect also observed in vivo. Interestingly, pretreatment of endothelial monolayers with ticagrelor did not result in reduced tumor cell adhesion. These findings support a role for P2Y12‐mediated platelet activation in promoting metastases, and provide proof‐of‐concept for the clinical use of ticagrelor in the prevention of tumor metastasis.     

Platelet-aggregating activities of metastasizing tumor cells. III. Platelet aggregation as resulting from thrombin generation by tumor cells

Thrombin generated in the process of platelet aggregation induced by three metastasizing murine tumors was measured using a chromogenic substrate specific for thrombin. Addition of B16 cells or 3LL cells to the platelet-rich plasma induced the generation of a significant amount of thrombin during the lag period preceding aggregation, while that of MH134 cells did not. Thrombin generation was observed in both the presence and the absence of platelets, indicating that platelets are not necessarily required for thrombin generation by these tumor cells. This suggests that the adherence of platelets to tumor cells is not an essential step for the initiation of thrombin-mediated aggregation induced by B16 and 3LL cells. Furthermore, the results of a one-stage clotting assay using plasma deficient in coagulation factors indicated that B16 and 3LL cells generated thrombin through direct activation of factor X.     

苦参碱衍生物M19对于肿瘤细胞转移的抑制作用

目的:探讨苦参碱衍生物M19对于肝细胞癌和黑色素瘤细胞转移的抑制作用。方法:肝癌细胞Hep3B、MHCC-LM3和黑色素瘤细胞B16F10经M19处理后,检测肿瘤细胞体外迁移能力。建立C57BL/6小鼠黑色素瘤B16F10和裸鼠肝细胞癌MHCC-LM3转移模型,经M19体内给药后,观察肿瘤细胞肝、肺转移情况。结果:M19能浓度依赖性地抑制肝癌细胞和黑色素瘤细胞迁移。M19体内给药使B16F10细胞在小鼠肺的转移减少,且还能抑制MHCC-LM3细胞在裸鼠肺和肝的转移。结论:M19能在体内外抑制肝细胞癌和黑色素瘤细胞的转移。     

Modified heparins inhibit integrin αIIbβ3 mediated adhesion of melanoma cells to platelets in vitro and in vivo

The adhesion of tumor cells with platelets is important in the process of tumor metastasis. A huge work has indicated that anti‐adhesion is an effective strategy for metastasis inhibition. In this article, we assess the role of platelet integrin α_IIbβ₃ in adhesion of melanoma cells to platelets and the effects of heparin and modified heparins on the adhesion in vitro and in vivo. We show that platelet integrin α_IIbβ₃ is involved in the interaction of human melanoma A375 cells with platelets, and the high affinity epitope resides on the α_IIb subunit rather than β₃ subunit. Heparin sulfate‐like proteoglycans on tumor cell surface are implicated in the adhesion of A375 cells to integrin α_IIbβ₃. We also show that RO‐heparin, CR‐heparin, N‐2,3‐DS‐heparin and 2,3‐O‐DS‐heparin can significantly inhibit A375 cells binding to the CHO cells expressing integrin α_IIbβ₃ under static and flow conditions, and remarkably inhibit the adhesion of A375 cells to the immobilized platelet layers under flow conditions. We find that A375 cells and B16F10 cells are arrested in the pulmonary vessels and adhered to platelets, and the initial interaction of tumor cells with platelets in lung vessel and long‐term establishment of metastatic foci can be inhibited by heparin as well as CR‐heparin and N‐2,3‐DS‐heparin. These data suggest that modified heparins can inhibit tumor cell‐platelet interaction mediated by platelet integrin α_IIbβ₃ and modified heparins may be a potential substitute for heparin in inhibiting tumor metastasis. © 2009 UICC