Augmentation of B16 melanoma lung colony formation in C57BL/6 mice having marked granulocytosis

The effect that polymorphonuclear leukocytes (PMN) may have on the metastatic colonization of tumor cells is controversial: some laboratories have reported that PMN can inhibit metastasis whereas others have shown an augmentation effect. We have exploited our finding that a particular fibrosarcoma (BMT-11) transplanted subcutaneously into syngeneic C57BL/6 mice induces a progressive increase in the number of circulating PMN, to re-examine this question. We used such "granulocytosis-positive" mice as recipients of B16 melanoma cells to examine the influence of significant granulocytosis on the level of lung tumor colonies. The number of B16 melanoma lung colonies detected after intravenous (i.v.) injection was significantly higher in BMT-11 tumor-bearing mice with granulocytosis than in control (non-tumor-bearing) mice. Retention of 125IUdR-labelled B16 cells 24 hr after the i.v. injection was 3 to 10 times greater in mice with granulocytosis than in controls. Either simultaneous injection, or preinjection of PMN with B16 cells, increased the lung-colonizing capacity of B16 melanoma cells. These results suggest that abnormally increased numbers of PMN in the peripheral blood, particularly in the lung circulation, can enhance the ability of tumor cells to colonize or metastasize.     

Inhibition of B16 melanoma growth and metastasis in C57BL mice by vaccination with a syngeneic endothelial cell line

Background

Key role of angiogenesis in tumor growth and metastasis based on accumulating evidence and recent progress of immunotherapy have led us to investigate vaccine therapy targeting tumor angiogenesis.

Methods

C57BL/6J mice were vaccinated with a syngeneic endothelial cell line Tpit/E by subcutaneous injection once a week. Prior to ninth vaccination, the mice were challenged with B16/F10 melanoma cells by subcutaneous inoculation on the back for the tumor growth model or by tail venous injection for the lung metastasis model. Development of subcutaneous tumor and lung metastasis was monitored by computed tomography scanning, which enabled accurate evaluation with the minimized sacrifice of mice.

Results

Vaccination with Tpit/E cells inhibited subcutaneous tumor growth and appearance of lung metastasis compared to control. Survival period was elongated in the Tpit/E vaccination in both of the two models. We also obtained hybridomas secreting specific antibodies to Tpit/E cells from a mouse vaccinated with the cells, indicating that specific immune response to the syngeneic endothelial cells was elicited.

Conclusion

These results suggest that vaccination with an autologous endothelial cell line may be effective against melanoma.     

组胺对C57BL/6小鼠B16黑色素瘤转移行为的影响

背景与目的:肿瘤生物治疗中微血管壁通透性是大分子抗体偶联药物进入瘤体的主要屏障.组胺可通过增加微血管壁通透性和组织液的生成促进抗体偶联物在肿瘤局部的富集.本研究观察组胺所致微血管壁通透性的增加和组织液的生成增多是否会增加肿瘤血行及/或淋巴的转移.方法:采用瘤细胞悬液接种法将小鼠黑色素瘤细胞B16注入小鼠左上肢腋部皮下,建立C57BL/6小鼠荷瘤模型.实验组接种第6天起,背部皮下注射300 mg/kg组胺,隔日一次,共5次,对照组注射等体积生理盐水.组织化学方法检测肿瘤细胞在各脏器的转移情况.分别用Student t-test和四格表资料确切概率法分析组胺对肿瘤细胞增殖和转移的作用.结果:接种成瘤率100%.300 mg/kg组胺隔日一次皮下注射能明显抑制肿瘤的生长,实验组与对照组瘤重分别为(5.26 1.55)g和(6.96±1.31)g,二者间差异有统计学意义(P＜0.01).实验组与对照组淋巴结转移率分别为33.3%和75.0%,血行转移率分别为25.0%和75.0%,差异均有统计学意义(P＜0.05).结论:组胺能够抑制C57BL/6小鼠B16黑色素瘤的血行和淋巴转移,该抑制作用部分与其抑制瘤细胞的增殖有关.     

C57BL/6小鼠B16黑色素瘤细胞脾转移机制

 目的　研究C57BL／6小鼠B16黑色素瘤细胞脾转移与血行转移的相关性。方法　细胞悬液接种法制备C57BL／6小鼠B16黑色素瘤细胞的荷瘤模型，HE染色法观察各组织器官中转移瘤细胞的有无及其生长状态，伊文思蓝尾静脉注射法观察蓝染部位与肿瘤转移阳性部位间的相关性。结果　接种成瘤率100 ％。12只小鼠中有9只出现脾转移，均同时伴有不同程度的局部淋巴结转移， 其中3只尚伴有肺转移。脾转移较肺转移出现早、概率高。大体观脾转移阳性部位位于脾背侧段约全长1／4区域。镜下观脾内转移瘤细胞呈散在分布，生长受抑并向成熟分化。伊文思蓝蓝染部位位于脾背侧段约全长1／4区域，与肿瘤转移阳性部位一致。结论　C57BL／6小鼠的B16黑色素瘤细胞脾转移先于其他脏器转移，其转移途径为血行转移。     

Change in the metastatic mode of B16 malignant melanoma in C57BL/6 mice with ageing and sex

An attempt has been made to determine whether changes in the host environment due to ageing or sex differences can influence the metastatic mode of malignant tumours. B16-F10 melanoma cells with a high pulmonary metastasizing potential were injected into the outer ear of C57BL/6 mice; the growth of primary tumour and metastasis of the cervical node were assessed every two days, and the number of lung compared; and in the second young male and female mice were used. The rate of growth of primary tumours was found to be comparable between young and ole mice. Metastasis in the cervical lymph node occurred earlier in old than in young mice. Lung metastases occurred earlier in old than in young mice, but their growth was slower in old than in young mice. Metastasis in the cervical node occurred earlier in males than in females. More lung metastases were found in male than in female mice.     

Effect of lymphokine-containing sera on adenocarcinoma BW10232 and melanoma B16 in C57BL/6 mice

Lymphokine-containing sera (LKS) inhibited the growth of adenocarcinoma BW10232 and melanoma B16 in C57BL/L mice. Resistance to tumor growth was conferred by daily injections into the challenge site. The injection of LKS did not provide long-term resistance, inasmuch as termination of treatment permitted resumption of tumor growth at the primary tumor site. Whether LKS exerted a direct or indirect effect on the tumor cells to retard their was not certain.     

Ajoene inhibits both primary tumor growth and metastasis of B16/BL6 melanoma cells in C57BL/6 mice

Ajoene is an organosulphur compound derived from garlic with important effects on several membrane-associated processes such as platelet aggregation, as well as being cytotoxic for tumor cell lines in vitro. In the present study, we investigated the effect of ajoene on different cell types in vitro, as well as its inhibitory effects on both primary tumors and metastasis in a mouse model. We found ajoene to inhibit tumor cell growth in vitro, but also to inhibit strongly metastasis to lung in the B16/BL6 melanoma tumor model in C57BL/6 mice. As far as we are aware, this is the first report of the anti-metastatic effect of ajoene. Ajoene also inhibited tumor-endothelial cell adhesion, as well as the in vivo TNF-α response to lipopolysaccharide. Possible mechanisms of its antitumoral activity are discussed in the light of these results.     

B16 melanoma in C57BL-6J mice: kinetics and effects of heterologous serum

B16 melanoma was studied in syngeneic C57BL/6J mice as a therapeutic model of melanoma. The irregular behavior of tumor after trochar passage was eliminated by serial intraperitoneal passage, but abundant melanin production was maintained. Mean viability of dispersed suspensions was 56%. Median survival time after injection of 10⁷ cells intraperitoneally was 11-16 days (average 13.20). In vivo doubling time was estimated at 3.24 days. Tenfold increase time was approximately 10.75 days. Preliminary studies with rabbit antiserum demonstrated possible enhancement after incubation with tumor in vitro, and a possible therapeutic effect after administration in vivo.     

Reduction of pulmonary metastases of B16 melanoma by human recombinant interleukin 2 and lymphokine-activated killer cells in immunosuppressed C57BL/6 mice receiving anticancer agent

The immunosuppressive effect of a water-soluble nitrosourea derivative, 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), was evaluated in terms of the cytotoxicity of spleen lymphocytes, and the restoring effect of lymphokine-activated killer (LAK) cells and/or human recombinant interleukin-2 (rIL-2) on the cytotoxicities of spleen lymphocytes was examined in ACNU-treated C57BL/6 mice. In addition, we tested whether the administration of LAK cells and/or rIL-2 could reduce the increased numbers of pulmonary metastases in ACNU-treated mice. The maximum effective dose of ACNU suppressed the cytotoxicity of spleen lymphocytes and pretreatment with ACNU enhanced the induction of artificial pulmonary metastases. The administration of LAK cells and/or human rIL-2 restored the cytotoxicity of spleen lymphocytes against YAC-1 and syngeneic B-16 melanoma cells in ACNU-treated mice, and these treatments partially suppressed the increased numbers of artificial pulmonary metastases of B-16 melanoma cells in ACNU-treated mice. These results are extremely important in providing a rationale for the introduction of adoptive immunotherapy using LAK cells and rIL-2 in patients with advanced cancer who are being treated with anticancer agent(s).     

IFN-α1 gene transfection completely abolishes the tumorigenicity of murine B16 melanoma cells in allogeneic DBA/2 mice and decreases their tumorigenicity in syngeneic C57BL/6 mice

The murine B16 melanoma (H-2^b) was transfected with a retroviral vector containing the mouse IFN-α₁ gene. IFN-α₁-transfected cells produced IFN-α in vitro and exhibited an altered phenotype characterized by a decreased rate of multiplication, enhanced expression of H-2 antigens, an antiviral state to VSV, and decreased pigmentation. Control and IFN-α₁-transfected cells were tested for their ability to grow in syngeneic (H-2^b) CS7B1/6 and allogeneic (H-2^d) DBA/2 mice. IFN-α₁producing B16 clones were less tumorigenic after s.c, i.p., and i.v. routes of injection than IFN-non-producer BI6 clones in syngeneic CS7B1/6 mice. IFN-α₁-producing B16 cells were, however, totally rejected by allogeneic DBA/2 mice regardless of the routes and inocula tested, while control B16 cells grew in and killed DBA/2 mice. The total rejection of IFN-α₁-transfected B16 cells in allogeneic mice appeared to be dependent on T cells as these cells grew in DBA/2 nude mice. Incubation of IFN-α-producing clones with anti-mouse IFN-α/β prior to injection into C57BI/6 mice did not enhance their tumorigenicity. Likewise, injection of C57BI/6 and DBA/2 mice with antibody to IFN-α/β did not enhance the tumorigenicity of IFN-α₁-transfected cells. C57B1/6 mice immunized with irradiated IFN-α₁ cells were only slightly protected against a subsequent challenge with parental B16 cells. In contrast, DBA/2 mice immunized with irradiated IFN-α₁ cells exhibited tumor-specific, long-lasting immunity to subsequent challenge with parental B16 cells. © 1995 Wiley-Liss, Inc.     

Efficacy of acetylsalicylic acid (aspirin) in skin B16-F0 melanoma tumor-bearing C57BL/6 mice

Several epidemiological studies show that aspirin can act as a chemopreventive agent and decrease the incidences of various cancers including melanoma. In this work, we investigated the in vitro and in vivo efficacy of acetylsalicylic acid (ASA) as an antimelanoma agent in B16-F0 cells and skin B16-F0 melanoma tumor mouse model. Our findings indicate that the IC₅₀ (48 h) for ASA in B16-F0 melanoma cells was 100 μM and that ASA caused a dose- and time-dependent GSH depletion and increase in reactive oxygen species (ROS) formation in B16-F0 melanoma cells. Male C57BL/6 mice were inoculated s.c. with 1?×?10⁶ B16-F0 melanoma cells. ASA (80, 100, and 150 mg/kg) was initiated on day 1 or day 7, or day 9 after cell inoculation and continued daily for 13, 7, and 5 days, respectively. Animals were weighed daily and sacrificed on day 13. The tumors were excised and weighed. The animals receiving 13 days of ASA therapy at 80, 100, and 150 mg/kg demonstrated tumor growth inhibition by 1?±?12 %, 19?±?22 %, and 50?±?29 %, respectively. Animals receiving 7 days of therapy at 80, 100, and 150 mg/kg demonstrated tumor growth inhibition by 12?±?14 %, 27?±?14 %, and 40?±?14 %, respectively. No significant tumor growth inhibition was observed with 5 days of therapy. ASA at 100 and 150 mg/kg caused significant tumor growth inhibition in C57BL/6 mice when administered for 13 and 7 days, respectively. The results obtained in this study are consistent with the recent epidemiologically based report that aspirin is associated with lower melanoma risk in humans.     

cAMP metabolism in B16 melanoma clones during the formation of experimental and spontaneous metastases

The ability of B16 melanoma clones to form tumor colonies in the lung after i.v. injection (experimental metastases) correlates positively with their capacity to respond to activators of cyclic adenosine 3-, 5-monophosphate (cAMP) metabolism (such as melanocyte-stimulating hormone and forskolin). To investigate whether this relationship is causal, the cAMP responses of 4 B16 melanoma clones of differing colonizing potential have been examined in freshly established stock cell cultures, in pulmonary colonies in vivo and in cultures established from these lesions. In all cases, the cAMP responsiveness of the excised colonies (and cultures derived from them) mimicked the responsiveness of the cultures from which they arose. B16 clones exhibiting low cAMP responsiveness gave rise to few experimental metastases all of which were poorly responsive to activators of cAMP metabolism. Similarly, clones with high cAMP responsiveness formed multiple lung colonies which displayed a marked sensitivity to agents that stimulated cAMP production. Parallel experiments on the spontaneous metastatic behavior of the same clones revealed that the cAMP responsiveness of cells in the primary (intrafootpad) tumor and spontaneous metastatic lesions in the lung faithfully reflected the response profile of the original tumor-cell inoculum but no correlation was found between cAMP responsiveness and the capacity to form spontaneous metastases. These data suggest that cAMP-dependent events may influence the survival, arrest and organ colony formation by cells injected directly into the circulation but appear to be of little or no importance in determining the early event(s) involved in the evolution of spontaneous metastases prior to the entry of cells into the circulation.     

Sequential chemoimmunotherapy with cisplatin, interferon-beta and interleukin-2 inhibits the growth of B16-F1 melanoma in syngeneic mice

Sequential combinations of chemotherapy with biological response modifiers has recently been evaluated as systemic treatment for patients with advanced melanoma. The response rates of the chemoimmunotherapy were reported to be higher than conventional treatment using chemotherapy or biological agents alone. To investigate the effectiveness of such chemoimmunotherapy, we evaluated the antitumour effect of sequential chemoimmunotherapy in vivo using a B16 mouse melanoma system. In this sequential regimen, administration of cisplatin (CDDP) was followed by interferon-beta (IFNbeta) and interleukin-2 (IL-2). This combination therapy synergistically inhibited the growth of B16-F1 melanoma and prolonged the survival of mice bearing B16-F1. In contrast, this therapy did not show any antitumour effect on B16-F10 melanoma. The exact mechanism of the antitumour effect is not clear, but the following results were noted: no synergistic effect of this therapy was detected in nude mice, neutralizing anti-IFNgamma antibody significantly blocked the antitumour effect of this therapy, and the number of apoptotic melanoma cells was significantly increased in melanoma tissues removed from mice treated with this therapy. These results demonstrated the potent immunological antitumour effect of this sequential chemoimmunotherapy.     

CPI-0004Na, a new doxorubicin prodrug, reduces growth of 3LL-H61 carcinoma lung metastases in C57BI/6 mice

The ETAP concept (Extracellularly Tumor-Activated Prodrug) is a new approach developed to overcome the lack of selectivity and the side effects responsible for the limited efficacy of chemotherapeutic agents. CPI-0004Na, a doxorubicin (Dox) prototype prodrug of this type, is less toxic than free Dox and showed increased efficacy against subcutaneous human tumor xenografts. The aim of this study was to assess the efficacy of the prodrug vs Dox (given ip) at their maximal tolerated dose (MTD) for this administration schedule (129.3 micromol/kg and 12.93 micromol/kg, respectively) against experimentally induced 3LL-H61 carcinoma lung metastases in mice. Our results indicate that, Dox has no effect on the number of lung metastases while CPI-0004Na induces a 38.3% reduction on average. When considering the effect on the proportion of the lungs' surface covered by metastases, Dox induces a 39% reduction while the prodrug CPI-0004Na is about two fold more active with a 71% decrease.     

Theophylline administration markedly reduces hepatic and pulmonary implantation of B16-F10 melanoma cells in mice

Theophylline-treated B16-F10 melanoma cells show a lower experimental metastatic potential in vivo. To identify the possible mechanism(s) involved and on the basis of previous reports, we tested the induction of apoptosis in B16-F10 cells. Fluorescence activated cell sorter (FACS) analysis and p53 overexpression in theophylline-treated B16-F10 melanoma cells appeared to suggest enhanced cell death by apoptosis. The in vivo effects of orally administered theophylline in mice were investigated using different treatment schedules in mice that had undergone hepatic or pulmonary colonization with tumour cells. Mice received theophylline in their drinking water according to different protocols: (i) from 3 days before tumour cell inoculation until animal sacrifice ('early treatment'); (ii) from 3 days before until 3 days after tumour cell inoculation ('short treatment'); or (iii) from 3 days after tumour cell inoculation until animal sacrifice ('late treatment'). In the 'early treatment' group, the number of melanoma foci was reduced by 92.3% in the liver and 81.4% in the lung compared with control animals (P < 0.001). In the 'short treatment' group, there was an 80.2% and 72.2% reduction in liver and lung metastases, respectively (P < 0.001). In the 'late treatment' group, the inhibition of metastasis was 59.7% for liver and 45.3% for lung (P < 0.005). Survival studies showed that 50% of the 'early' theophylline-treated animals died 33.2 +/- 2.0 days after intrasplenic injection (control group: 23.1 1.8 days; P < 0.001) and 33.9 +/- 2.5 days after tail vein injection (control group: 24.1 +/- 1.4 days; P < 0.001). Taken together, these observations provide useful information for the potential clinical application of theophylline as a chemotherapeutic agent against malignant melanoma.     

Radiation and concurrent chemotherapy for the treatment of Lewis lung tumor and B16 melanoma tumor in C57/BL mice

C57/BL mice bearing either Lewis lung tumor or B16 melanoma tumor were treated with radiation and concurrent chemotherapy. The treatment results were determined in vivo by tumor regrowth delay assay. When continuous infusion of either Cyclophosphamide (CYCLO) or 5-Fluorouracil (5-FU) or Adriamycin (ADRIA) or Mitomycin-C (MITO-C) was used in combination with continuous radiation at 1 cGy/min, no increase in tumor regrowth delay was observed over that of radiation alone. When multiple drug chemotherapy, FAM (5-FU, ADRIA, MITO-C) was administered in combination with radiation at 80 cGy/min, no increase in tumor regrowth delay was observed over that of radiation alone. In these two murine tumor models, when clinically relevant concentrations of commonly used chemotherapy agents were combined with radiation, no therapeutic advantage was observed.     

Lipid peroxidation and antioxidant capacity in selected tissues of healthy black C57BL/6J mice and B16 melanoma-bearing mice

During the process of melanogenesis free radicals are generated. The aim of this study was to investigate the effects of melanogenesis in B16 melanoma on lipid peroxidation and antioxidant capacity in selected tissues of black C57BL/6J mice. The study was conducted on 24 mice: 12 healthy controls and 12 with a transplanted B16 melanoma. Two weeks after the melanoma transplant, when the average weight of the tumours was approximately 2.0 g, blood samples were taken from the orbital venous plexus. The mice were killed by dislocation of the spinal cord, and the brain, liver and lungs were removed for analysis. The level of thiobarbituric acid-reactive substances (TBARS) and of 1,1-diphenyl-2-picrylhydrazyl (DPPH) reactive substances were determined in full liver, lung and brain homogenates and in serum. The activity of superoxide dismutase (SOD) was determined only in homogenized tissue. The concentration of TBARS and the SOD activity were statistically significantly higher in all the studied tissues from mice with B16 melanoma than in tissues from healthy mice. The antioxidant capacity, however, was lower in the tissues of melanoma-bearing mice. The results obtained demonstrate an increase in oxidative stress in the tissues of mice bearing a transplanted B16 melanoma.