Liposomal encapsulation enhances the antitumour efficacy of the vascular disrupting agent ZD6126 in murine B16.F10 melanoma

Vascular disrupting agents (VDAs) are able to affect selectively tumour endothelial cell morphology resulting in vessel occlusion and widespread tumour cell necrosis. However, single-agent antitumour activity of VDAs is typically limited, as tumour regrowth occurs rapidly following drug treatment. To improve the therapeutic effectiveness of VDAs, we investigated liposomal targeting using ZD6126 as a model VDA. ZD6126 is a phosphate-prodrug of the tubulin-binding vascular disrupting agent ZD6126 phenol. ZD6126 was encapsulated into long circulating PEG-liposomes for passive targeting and PEG-liposomes conjugated with peptide ligands containing the RGD-motif for active targeting to alpha(v)-integrins on tumour endothelial cells. ZD6126 could be stably encapsulated, and liposomes displayed minimal leakage in vitro (<10% in 3 weeks). In vivo, upon intravenous injection, free ZD6126 was rapidly converted into ZD6126 phenol, which was cleared from the circulation within minutes. In contrast, ZD6126 encapsulated into either RGD-targeted or PEG liposomes showed prolonged blood circulation times (t(1/2)=10 h), and ZD6126 phenol exposure was also prolonged (t(1/2)=8 h). Both liposomal formulations displayed tumour accumulation plus hepatosplenic uptake by local macrophages. The altered pharmacokinetics and tissue distribution profiles of both liposomal ZD6126 formulations resulted both in single-dose and multiple-dose regimes, in improved therapeutic efficacy in established murine B16.F10 melanomas compared with free ZD6126. The passively and actively targeted liposomes showed equal antitumour efficacy, indicating that delivery of ZD6126 to the tumour tissue may suffice to disrupt tumour blood vessels without the need for specific targeting to the tumour endothelium.     

Antimetastatic efficacy of niosomal pentoxifylline and its combination with activated macrophages in murine B16F10 melanoma model

The aims of the present study were a) to enhance the effectiveness of antimetastatic agent, Pentoxifylline (PTX) by encapsulation in niosomes and b) to investigate the anticancer activity by combination therapy involving activated macrophages and PTX solution/PTX niosomes. Niosomes were prepared by lipid film hydration method. Particle size distribution revealed bimodal distribution with median vesicle size of 462 nm. The entrapment efficacy of PTX niosomes was found to be 9.64%. A cumulative release of 82.43% from niosomal suspension was observed at the end of 21 hours. Intravenous administration of niosomal PTX (6 mg/kg and 10 mg/kg) resulted in significant reduction in lung nodules in an experimental metastatic B16F10 model suggesting accumulation of PTX in a distant target organ-lung. Light microscopic observations of histologic sections showed a decrease in number of tumor islands in the lung. Macrophages activated by intraperitoneal injection of Iscove's Modified Dulbecco's Medium (IMDM) containing 20% fetal calf serum (FCS) followed by in vitro incubation with muramyl dipeptide (MDP) were more effective in controlling tumor spread than those activated by FCS alone. Combination therapy of activated macrophages and PTX solution/niosomal PTX showed no additive or synergistic effect in controlling tumor spread. Carbon clearance studies revealed that PTX inhibits the phagocytic ability of activated macrophages, thereby resulting in the failure of combination therapy.     

In vitro assay demonstrates similar invasion profiles for B16F1 and B16F10 murine melanoma cells

The variants of the B16 murine melanoma cell line were assayed for their invasive characteristics in the membrane invasion culture system (MICS) and concomitantly tested for their ability to form lung metastases in vivo. Specifically, the B16F1 (low metastatic variant) and the B16F10 (high metastatic variant) murine melanoma cell lines were examined for their ability to invade human amniotic basement membranes (BMs) in vitro and simultaneously examined for lung colony formation in vivo. The B16F1 and B16F10 cell lines both demonstrated similar invasion profiles over 72 h with the total percent invasion through the BMs for both cell lines not exceeding 5.0%. In vivo observations reconfirmed the significant difference in the metastatic capabilities of the 2 variants. These data suggest that tumor cells with differing metastatic propensities can invade an amniotic BM at similar rates, but their survival and metastatic lesion forming capabilities in vivo may vary considerably.     

Neoadjuvant immunotherapy with interferon of the spontaneously metastasizing murine B16F10L melanoma.

We have previously demonstrated that administration of interferon a/b (IFN) for 4-5 days after challenge with a transplantable Moloney sarcoma virus-induced tumor completely inhibited tumor development. In the present study, we examined the therapeutic effects of IFN on mortality induced by metastatic dissemination of the B16F10L murine melanoma. IFN was administered at various times in relation to the surgical removal of primary tumor: days -5 to -1 prior to tumor excision (neo-adjuvant protocol), or for 5 days after tumor excision, beginning on days 1, 6 or 11 after excision of the primary tumor (adjuvant protocols). The neo-adjuvant protocol was superior to all other protocols, significantly increasing percentage survival (56% vs. 0%) and median survival time (greater than 84 days vs. 33 days) compared to untreated controls, as well as to all adjuvant protocols. In contrast, IFN treatment on days 1 to 5 after excision of the primary tumor decreased median survival time of cases compared to untreated controls (20 days vs. 33 days). Both IFN-induced inhibition and enhancement of metastatic dissemination were dose-dependent, with higher amounts of IFN producing greater inhibition or enhancement. The superior therapeutic efficacy of the neo-adjuvant IFN treatment was associated with increased spleen and lung-derived natural killer cell cytolytic activity (on days -4, 0 and 2) followed by a later (day 13) increase in lung-associated cytolytic T-cell responses.     

In vitro and in vivo effects of polyhaemoglobin-tyrosinase on murine B16F10 melanoma

Melanoma is an increasingly common fatal skin cancer. Many groups are carrying out research on potential treatments for melanoma. One of these approaches has shown that lowering tyrosine can inhibit the growth of melanoma in cell cultures and of B16BL6 melanoma in mice. However, humans cannot tolerate tyrosine-restricted diets for lowering tyrosine because of nausea, vomiting and weight loss. We report here our preparation and characterization of a novel soluble polyhaemoglobin-tyrosinase complex. This preparation prevents native tyrosinase from having adverse effects and from rapid removal after injection. The preparation inhibited murine B16F10 melanoma cell growth in culture and delayed its growth in a mice model. Intravenous injection of the preparation lowers the systemic tyrosine level without causing adverse effects such as vomiting and weight loss in mice. It is therefore possible that this complex could be useful in the treatment of human melanoma.     

Nude mouse model to study passive humoral immunotherapy directed against B16 F10 murine melanoma

A model to study passive humoral immunotherapy of experimental melanoma was generated by subcutaneous injection of B16 F10 murine melanoma cells in the midtail of BALB/C nude (nu/nu) mice. Mice were challenged with melanoma cells pretreated: (1) with complete culture medium, (2) with 10% adjuvant control serum, (3) with 10% anti-fECA (formalinized extracellular antigens) immune serum, or (4) with a monoclonal antibody (mAb H2-3-3) specific for the B700 melanoma-associated antigen. All control mice challenged with melanoma cells pretreated either with culture medium or with medium containing adjuvant control serum (Groups I and II) died during the observation period of 84 days. At day 84, 60% of the mice challenged with melanoma cells pretreated with anti-fECA immune serum (Group III) survived, as did 100% of the mice challenged with cells pretreated with mAb H2-3-3 (Group IV). Injection of melanoma cells pretreated with mAb H2-3-3 was associated with the greatest reduction of subsequent local tumor growth and the lowest number of metastatic lung tumors. The inhibitory effects of immune sera in vivo also correlated with in vitro effects of anti-fECA immune serum and mAb H2-3-3, determined on B16 F10 melanoma target cells using assays for DNA synthesis and antibody dependant cellular cytotoxicity (ADCC). In sum, this nude mouse model for the study of passive humoral immunotherapy of experimental melanoma was utilized to demonstrate significant protective effects against B16 F10 melanoma cell challenge by treatment with anti-fECA immune sera or a melanoma-specific monoclonal antibody. © 1994 Wiley-Liss, Inc.     

Role of natural killer and T-cells in interferon induced inhibition of spontaneous metastases of the B16F10L murine melanoma.

We have previously demonstrated that interferon (IFN) treatment of mice bearing the spontaneously metastasizing B16F10L murine melanoma on days -5 to -1 prior to surgical removal (day 0) of the primary tumor resulted in survival of greater than 50% of treated mice. The antitumor effect was correlated with an early increase of natural killer (NK) cell cytotoxicity followed by a later developing specific cytolytic T-cell response. The purpose of this study was to establish definitively the roles of NK, CD4, and CD8 cells as mediators of the antitumor/antimetastatic effects of IFN treatment by administration of anti-asialo-GM1, anti-L3T4, and/or anti-Lyt-2 antisera. Depletion of NK cells, alone or in combination with T-cells, eliminated the protective effect of IFN treatment. Depletion of both CD4 and CD8 cells, however, did not significantly alter the therapeutic effect of IFN therapy. Collectively, these data conclusively demonstrated the importance of NK cells as mediators of the IFN induced antitumor state. However, in mice depleted of CD4 cells alone, the protective effect of IFN was eliminated, in spite of the presence of intact NK cells. In vitro analysis of NK cytotoxicity on day 1 after surgery demonstrated (a) a lack of IFN induced stimulation of NK activity in CD4 depleted mice and (b) a significant increase in both baseline and IFN induced NK cytotoxicity in CD8 depleted mice. These data suggested a CD8 cell mediated inhibition of NK activity/stimulation in CD4 depleted mice, possibly responsible for the lack of response to IFN therapy in that group. These results demonstrate the importance of not only individual components of the immune system but also the interaction of these components in both the natural and IFN induced control of spontaneous B16F10L metastases.     

Nitric oxide-mediated regulation of hypoxia-induced B16F10 melanoma metastasis

Tumour hypoxia is associated with resistance to therapy and with increased invasion and metastatic potential. Recent studies in our laboratory have shown that the hypoxic up-regulation of tumour cell invasiveness and chemoresistance is in part due to reduced nitric oxide (NO) signaling. Using B16F10 murine melanoma cells, we demonstrate here that the increased metastatic potential associated with exposure to hypoxia is mediated by a reduction in cGMP-dependent NO-signaling. Pre-incubation of B16F10 cells in hypoxia (1% vs. 20% O(2)) for 12 hr increased lung colonization ability by over 4-fold. This effect of hypoxia on metastasis was inhibited by co-incubation with low concentrations of the NO-mimetic drugs glyceryl trinitrate (GTN) and diethylenetriamine NO adduct (DETA/NO). In a manner similar to hypoxia, pharmacological inhibition of NO synthesis resulted in a significant increase in lung nodule formation, an effect that was prevented by co-incubation with GTN. An important NO-signaling pathway involves the activation of soluble guanylyl cyclase and the consequential generation of cGMP. Culture in the presence of a non-hydrolysable cGMP analogue (8-Br-cGMP) abrogated the hypoxia-induced lung nodule formation, suggesting that the effects of NO on metastasis are mediated via a cGMP-dependent pathway. These findings suggest that a novel mechanism whereby hypoxia regulates metastatic potential involves a downstream inhibition of cGMP-dependent NO signaling.     

Suramin augments the antitumor and antimetastatic activity of pentoxifylline in B16F10 melanoma

Rapid tumor growth and metastasis are 2 major problems associated with treatment of malignant melanoma. Therefore, drugs that can intervene these processes are of clinical importance. Pentoxifylline (PTX), a methyl xanthine derivative, has been shown to inhibit B16F10 melanoma tumor growth and metastasis. We hypothesized that suramin when combined with PTX enhances its antineoplastic effects, which we have examined using the B16F10 mouse melanoma model. Suramin in simultaneous or sequential combination potentiated the cytotoxic effects of PTX on B16F10 cells. PTX arrested cells in the G0-G1 phase and suramin augmented the effects. Both the drugs inhibited F10 adhesion to laminin, matrigel and collagen type IV and showed enhanced inhibition in combination The combination also demonstrated significantly higher inhibition in cell motility (p = 0.002) and invasion through matrigel (p = 0.005) as compared to the single agents. Suramin synergized with PTX in its effects on secretion of MMP-9 gelatinase. DBA2/J mice implanted with intradermal B16F10 tumor were used as a model to study tumor growth. Animals were intratumorally treated with 50 mg/kg of PTX, 10 mg/kg of suramin and their combinations. Simultaneous administration of the drugs inhibited tumor growth by 5- to 6-folds. Tumor growth was completely blocked in sequential regimen with regression in some cases. The number and size of metastatic nodules on lung was also reduced significantly by the combination treatment. In conclusion, the novel combination of PTX and suramin has synergistic antitumor and antimetastatic activity in B16F10 melanoma and may be a promising approach in treatment of patients suffering from malignant melanoma.     

Estrous cycle influences organ-specific metastasis of B16F10 melanoma cells

Some clinical studies suggest that timing of surgery during specific menstrual phases may influence the chances of survival for premenopausal women with breast cancer, whereas other studies failed to find this effect. Because most breast cancer deaths are attributable to metastases, we hypothesized that aspects of the metastatic process might be sensitive to cyclic hormonal fluctuations. Our goal was to develop a mouse model to assess possible mechanisms for the effect of the menstrual cycle on metastatic ability. To separate the effects of the hormonal milieu on the host versus the cancer cells, we began by using melanoma cells. We report here that the estrous phase at the time of entry of B16F10 melanoma cells into the circulation leads to marked differences in organ-specific metastasis, suggesting that this concept merits additional study.     

Regression of established B16F10 melanoma with a recombinant Listeria monocytogenes vaccine.

We have previously shown that Listeria monocytogenes, a gram-positive, facultative intracellular bacterium, is a potent vector for targeting tumor-specific antigens to the immune system. In the present study, we extend these studies to the highly tumorigenic mouse melanoma B16F10, transduced with a model tumor antigen. We are able to induce the regression of primary tumors and established lung metastases by parenteral immunization with a L. monocytogenes recombinant that expresses the same antigen. Adjunctive therapy with granulocyte macrophage colony-stimulating factor or a vaccinia-based vaccine does not result in an improved cure rate over the L. monocytogenes vaccine alone. Tumor regression is accompanied by the expression of inflammatory cytokines in the tumor.     

Effects of niosomal cisplatin and combination of the same with theophylline and with activated macrophages in murine B16F10 melanoma model

The use of cisplatin is limited due to its certain toxic effects. In the present study niosomes of cisplatin by using span 60 and cholesterol were prepared and investigated for antimetastatic activity in experimental metastatic model of B16F10 melanoma. Theophylline and its combination effect with free cisplatin and niosomal cisplatin were also carried out in the same model. The effect of treatment with activated macrophages alone and in combination with cisplatin, theophylline and niosomal cisplatin was also observed. Treatment with niosomal cisplatin (1 mg/kg) and combination of the same with theophylline (15 mg/kg) showed significant reduction in the number of lung nodules as compared to untreated control as well as with free cisplatin (1 mg/kg). The treatment with activated macrophages (activated by using Muramyl dipeptide) significantly reduced the secondary growth of tumor in lung. Niosomal cisplatin showed a significant protection against weight loss and bone marrow toxicity as compared to free cisplatin. These results suggest that cisplatin encapsulated in niosomes has significant antimetastatic activity and reduced toxicities than that of free cisplatin. However theophylline failed to show antimetastatic effect alone or in combination with cisplatin or with activated macrophages.     

RNAi silencing of the WT1 gene inhibits cell proliferation and induces apoptosis in the B16F10 murine melanoma cell line

The Wilms' tumor protein 1 (WT1) is essential for tumor cell proliferation and is highly expressed in various hematological and solid malignancies including human malignant melanoma. We investigated whether WT1 expression is essential for growth in the B16F10 murine melanoma cell line. Toward this end, we examined WT1 protein expression and WT1 isoforms (17AA+/17AA-, KTS+/KTS-) in this cell line. WT1 was silenced by two RNA interference constructs, designated WT1-1 and WT1-2. RNA interference-mediated reduction of WT1 protein expression significantly inhibited B16F10 cell viability. Loss of WT1 also increased caspase-3 and poly-ADP-ribose polymerase activation, as well as apoptotic body formation, chromatin condensation, and DNA fragmentation. Together, these findings implicate decreased WT1 protein levels in the induction of apoptosis. These results imply that WT1 plays a distinct role in B16F10 melanoma growth.     

The effect of combined expression of interleukin 2 and interleukin 4 on the tumorigenicity and treatment of B16F10 melanoma.

The recent use of interleukin 2 (IL-2) and interleukin 4 (IL-4) single cytokine modified tumour cells in rodent models has demonstrated a potential use of these cytokines to produce autologous cancer cell vaccines. Here we compare the potential therapeutic benefit of transduction with IL-2 or IL-4 alone, and combined IL-2 + IL-4 in B16F10 cells, a murine malignant melanoma of poor immunogenicity. Transduction of B16F10 cells (MHC class I and II negative) to express either IL-2 or IL-4 alone delays the formation of tumours, IL-4 being more effective than IL-2. However, combined expression of IL-2 + IL-4 reduces tumorigenicity more than either cytokine alone. The eventual formation of tumours may result from loss of gene expression, and preliminary results suggest methylation of the retroviral long terminal repeat (LTR), rather than loss of the transduced DNA sequences. Histological examination of tumours expressing either IL-2 or IL-4 alone shows a non-specific inflammatory reaction with an increased tissue infiltrate of immune effectors (monocytes/macrophages, lymphocytes, granulocytes) localised around the tumour. In comparison, when cells expressing combined IL-2 + IL-4 were injected there were more granulocytes present, and perhaps more importantly, these were mainly localised within the tumour. The benefit of combined IL-2 + IL-4 expression results from a local rather than systemic effect as the growth of tumours from cells expressing IL-2 or IL-4 alone injected at distant sites was comparable with a single inoculation of cells expressing either cytokine alone. However, when cells expressing single cytokines IL-2 or IL-4 were mixed and injected at the same site, in comparison with the clonal population of cells expressing combined IL-2 + IL-4, tumour growth was characteristic of IL-4 alone rather than IL-2 + IL-4. Treatment of established tumours with a single injection of lethally irradiated tumour cells expressing IL-2 + IL-4 was sufficient to either reject tumours, or at least delay further tumour development. Furthermore, treatment stimulated an initial non-specific immune reaction that lead to a systemic immunity. Lethally irradiated wild-type cells were also successful in treating some established tumours, although this did not induce any systemic immunity. However, although successful in treatment studies, neither wild-type nor combined IL-2 + IL-4 expressing cells were able to vaccinate animals against a subsequent challenge with live wild-type tumour. These results indicate a potential therapeutic benefit with the use of combination IL-2 + IL-4 transduction of autologous cancer cells.     

GM-CSF膜修饰B16.F10黑素瘤细胞疫苗对小鼠种植肿瘤的抑制作用

目的: 研究粒细胞巨噬细胞集落刺激因子（granulocytemacrophage colony stimulating factor, GMCSF)膜修饰小鼠黑素瘤B16.F10细胞制备的疫苗对小鼠种植肿瘤的抑制作用。方法：采用生物素链亲合素GMCSF融合蛋白技术制备GMCSF膜修饰B16.F10黑素瘤细胞疫苗,将BALB/c小鼠随机分为GMCSF膜修饰B16.F10细胞疫苗组、GMCSF与B16.F10细胞混合疫苗组、B16.F10细胞疫苗组、GMCSF组、生理盐水组。各组于第1天和第7天分别进行免疫接种,第14天皮下注射0.2 ml B16.F10细胞（1×106个细胞）进行攻击,观察各组小鼠无瘤率和生存期;攻击后24 d 用ELISA法检测小鼠脾细胞INFγ分泌水平。结果：接受B16.F10细胞攻击后第60天和第90天,GMCSF膜修饰B16.F10细胞疫苗组小鼠均未成瘤,存活率为100%;GMCSF与 B16.F10细胞混合组无瘤率分别50%和40%,存活率分别为70%和40%;B16.F10细胞疫苗组无瘤率均为20%,存活率分别为80%和20%;GMCSF组和生理盐水组无瘤率为0,无小鼠存活。GMCSF膜修饰B16.F10细胞疫苗组的IFNγ分泌量比其他组明显升高（P<0.01）。结论：GMCSF膜修饰B16.F10肿瘤细胞疫苗可以激发BALB/c小鼠特异性抗肿瘤免疫反应,有效防止B16.F10肿瘤细胞的攻击。     

小鼠黑色素瘤B16F10-Red细胞株的建立与生物学特性分析

目的:构建表达香菇珊瑚红色荧光蛋白(discosomasp red fluorescent protein,DsRed)的小鼠黑色素瘤B16F10-Red细胞株,并检测其生物学特性.方法:用GenEscortTMⅡ转染试剂将pDsRed质粒导入小鼠黑色素瘤B16F10细胞,G418加压培养联合极限稀释法建立稳定、高水平表达DsRed的单克隆细胞系.FCM检测B16F10和B16F10-Red细胞的细胞周期.比较B16F10-Red和B16F10细胞的克隆球形成能力和小鼠体内致瘤能力.结果:稳定表达DsRed的小鼠黑色素瘤B16F10-Red细胞株基本保持了其亲代细胞的特征,能在C57BL/6小鼠腹部皮下形成肿瘤并继续生长和转移.结论:B16F10-Red细胞株构建成功,其移植瘤模型成瘤率和转移情况同B16F10肿瘤相比无明显差别.