Increased experimental pulmonary metastasis in pregnant mice

The effect of pregnancy on experimental pulmonary metastasis was studied. Compared to the incidence of pulmonary metastasis induced by G6 cells in non-pregnant mice, the incidence of such metastasis was found to be greatly enhanced when the cells were injected i.v. in the latter half of pregnancy. The maximum enhancement was seen on the 15th day of pregnancy. The incidence of pulmonary metastasis returned to the level observed in non-pregnant mice when the cells were injected 4 days after parturition. Pregnancy also significantly increased the incidence of pulmonary metastasis of 2 other cell lines (3LL and Colon 26). Injection of G6 cells after hysterectomy performed on the 15th day of pregnancy resulted in decreased lung colonization, similar to that seen after parturition. Quantificative analysis of the arrest of G6 cells labeled with [¹²⁵1]-5-iodo-2′-deoxyuridine in the lungs showed that the tumor-cell clearance from the lungs during the 24–72 hr after tumor-cell injection was much slower in pregnant than in non-pregnant mice. The continuous administration of β-estradiol and/or progesterone, which maintained serum levels of the hormones equivalent to those prevailing on the 15th day of pregnancy, did not affect the lung colonization of G6 cells. Tumor-cell-platelet aggregation was more extensive with platelets obtained from mice at the 15th day of pregnancy than with those from non-pregnant mice. When platelets isolated from pregnant mice were injected into normal mice 5 min before G6 injection, lung metastasis was also enhanced. These findings suggest that a pregnant host is handicapped with regard to pulmonary metastasis, this being partly due to increased platelet-aggregating activity in response to tumor cells. © 1995 Wiley-Liss Inc.     

Inhibition of experimental pulmonary metastasis by controlling biodistribution of catalase in mice

In a previous study, we showed that targeted delivery of bovine liver catalase to hepatocytes by direct galactosylation augmented the inhibitory effect of the enzyme on experimental hepatic metastasis of colon carcinoma cells (unpublished data). Here, we examined the ability of catalase to inhibit tumor metastasis to the lung by controlling its biodistribution. Four types of catalase derivative, Gal-CAT, Man-CAT, Suc-CAT and PEG-CAT, were synthesized. Experimental pulmonary metastasis was induced in mice by i.v. injection of 1 x 10(5) colon 26 tumor cells. An i.v. injection of catalase (35,000 units/kg) partially, but significantly, decreased the number of colonies in the lung 2 weeks after tumor injection, from 93 +/- 29 (saline injection) to 63 +/- 23 (p < 0.01). Suc-CAT, Man-CAT and Gal-CAT showed effects similar to those of catalase on the number of colonies. However, PEG-CAT greatly inhibited pulmonary metastasis to 22 +/- 11 (p < 0.001). Furthermore, s.c. injection of catalase also greatly inhibited metastasis (11 +/- 6, p < 0.001). Neither inactivated catalase nor BSA showed any effects on the number of metastatic colonies, indicating that the enzymatic activity of catalase to detoxify H(2)O(2) is the critical factor inhibiting metastasis. (111)In-PEG-CAT showed a sustained concentration in plasma, whereas s.c.-injected (111)In-catalase was slowly absorbed from the injection site. These results suggest that retention of catalase activity in the circulation is a promising approach to inhibit pulmonary metastasis.     

Effects of neutrophil-mediated pulmonary endothelial injury on the localization and metastasis of circulating Walker carcinosarcoma cells

The lung, a frequent site of cancer metastases, is also a susceptible target in several models of endothelial injury. In previous studies we have demonstrated that such injury, induced by bleomycin or by exposure to high concentrations of atmospheric oxygen, can facilitate the localization and metastasis of circulating tumor cells. Here we have tested the hypothesis that neutrophil-mediated injury to pulmonary endothelium has a similar effect. In Sprague-Dawley rats, intravenous injection of cobra venom factor resulted in complement activation, rapid sequestration of neutrophils in the lung, and endothelial damage, demonstrated by morphology, and by increased protein content and leakage of intravenous 125I-albumin into bronchoalveolar lavage fluids. When 125I-iododeoxyuridine-labeled Walker carcinosarcoma cells were injected intravenously during the period of endothelial injury, the pulmonary capillaries contained aggregates of neutrophils and tumor cells 2 h later, and there was a 3-fold increase in pulmonary tumor cell localization after 24 h in treated animals, compared to controls. Enhancement of tumor cell localization was prevented by pretreatment of the rats with catalase or by antineutrophil antisera. When animals were examined 2 weeks after cell injection, treatment groups had significantly more metastatic tumor nodules and a greater area of lung tissue involved by metastatic tumors. We conclude that neutrophil-mediated damage to the pulmonary endothelium can significantly increase the trapping of circulating tumor cells, and is likely to be clinically important since the large vascular bed of the lung is susceptible to host-mediated injury.     

Severe pulmonary metastasis in obese and diabetic mice

Although obesity is known as a risk factor for several human cancers, the association of obesity with cancer recurrence and metastasis remains to be characterized. Here, B16-BL6 melanoma and Lewis lung carcinoma cells were intravenously injected into diabetic (db/db) and obese (ob/ob) mice. The number of experimental lung colonies was markedly promoted in these mice when compared with C57BL/6 mice. In contrast, tumor growth at the implanted site was comparable when cells were inoculated orthotopically. The use of B16-BL6 cells stably transfected with the luciferase gene revealed that the increased metastasis reflected a difference mainly within 6 hr after the intravenous inoculation of tumor cells. Administration of recombinant leptin in ob/ob mice abolished the increase in metastasis early on as well as the decrease in the splenic NK cell number. In addition, depletion of NK cells by an anti-asialo-GM1 antibody abrogated the enhanced metastasis in db/db mice. These results demonstrate that metastasis is markedly promoted in diabetic and obese mice mainly because of decreased NK cell function during the early phase of metastasis.     

Intranasal administration of CpG DNA lipoplex prevents pulmonary metastasis in mice

Synthetic oligodeoxynucleotides containing CpG motifs (CpG DNA) can activate immunocompetent cells which offer the potential advantage of antitumor activity. In this study, we used cationic liposomes to complex with CpG DNA (CpG DNA lipoplex) to prevent pulmonary metastasis following intranasal administration in mice. Intranasal administration of CpG DNA lipoplex prior to challenge with both colon26/Luc and B16F10 cells significantly prevented the proliferation of tumor cells, and the survival time of the mice receiving CpG DNA lipoplex was prolonged. After intranasal administration, [³²P] CpG DNA lipoplex mainly distributed in nose and lung and induced higher IFN-γ production in the lung. These results suggest that intranasal administration of CpG DNA lipoplex has a significant effect on preventing pulmonary metastasis in mice.     

Promotion of experimental liver metastasis by tumor necrosis factor

Models for experimental metastasis were established to investigate the influence of rmTNF on tumor-colony formation in the liver. Highly metastatic lymphoma tumor cells were either injected i.v. or inoculated s.c. to form spontaneous metastases. In both systems, administration of rmTNF to the animals led to significant enhancement of the number of liver metastases in comparison with control groups. The number of metastatic tumor-cell colonies at an early stage of metastasis was increased, as well as the number of surface metastases in a late stage. Consequently, TNF-treated animals revealed a higher mortality. The optimal time for TNF to exert this metastasis-enhancing effect was found to be 7 days after tumor inoculation, In vitro adhesion of the lymphoma tumor cells to a mouse endothelioma cell line was strongly inhibited by monoclonal antibodies interfering with the interaction of VCAM-1 with VLA-4. These results support and extend earlier results with a fibrosarcoma lung colonization model. In addition, they show that stimulation of the immune system in tumor-bearing hosts activates tumor-promoting pathways, in addition to having possible beneficial effects.     

抑制Src酪氨酸激酶对小鼠肺转移癌的干预作用

目的:探讨抑制Src酪氨酸激酶对小鼠肺转移癌的干预作用及其机制。方法:采用雄性严重联合免疫缺陷(SCID)小鼠,敲除NK细胞,建立肺腺癌A549细胞诱导的实验性肺转移癌动物模型。每天给予小鼠口服Src酪氨酸激酶抑制剂,研究抑制Src酪氨酸激酶对实验性肺转移癌的影响;采用免疫组化法研究抑制Src酪氨酸激酶对转移瘤中增生指数(Ki67染色)和微血管密度(CD31染色)的影响。结果:A549细胞在肺表面形成弥漫性的转移性损伤,50mg/kg/d Src酪氨酸激酶抑制剂口服治疗组肺表面比对照组光滑,肺重量明显小于对照组和10mg/kg/d治疗组(P<0.05),而10mg/kg/d治疗组的肺重量和对照组相比没有明显差别。A549细胞接种后35天,对照组小鼠体重显著下降(P<0.01),而治疗组未见小鼠体重明显下降。口服Src酪氨酸激酶抑制剂50mg/kg/d,明显减少肺组织内转移性肺损伤的数目和面积,同时显著减少Ki67阳性的增生性肿瘤细胞数(P<0.001)。结论:抑制Src酪氨酸激酶通过抑制局部增殖和浸润抑制A549细胞诱导的肺转移癌。     

抑制Src酪氨酸激酶对小鼠肺转移癌的干预作用

目的：探讨抑制Src酪氨酸激酶对小鼠肺转移癌的干预作用及其机制。方法：采用雄性严重联合免疫缺陷（SCID）小鼠,敲除NK细胞,建立肺腺癌A549细胞诱导的实验性肺转移癌动物模型。每天给予小鼠口服Src酪氨酸激酶抑制剂,研究抑制Src酪氨酸激酶对实验性肺转移癌的影响;采用免疫组化法研究抑制Src酪氨酸激酶对转移瘤中增生指数（Ki67染色）和微血管密度（CD31染色）的影响。结果：A549细胞在肺表面形成弥漫性的转移性损伤,50mg/kg/d Src酪氨酸激酶抑制剂口服治疗组肺表面比对照组光滑,肺重量明显小于对照组和10mg/kg/d治疗组（P〈0.05）,而10mg/kg/d治疗组的肺重量和对照组相比没有明显差别。A549细胞接种后35天,对照组小鼠体重显著下降（P〈0.01）,而治疗组未见小鼠体重明显下降。口服Src酪氨酸激酶抑制剂50mg/kg/d,明显减少肺组织内转移性肺损伤的数目和面积,同时显著减少Ki67阳性的增生性肿瘤细胞数（P〈0.001）。结论：抑制Src酪氨酸激酶通过抑制局部增殖和浸润抑制A549细胞诱导的肺转移癌。     

Increase in experimental pulmonary metastasis in mice by L-arginine under inhibition of nitric oxide production by NG-nitro-L-arginine methyl ester

As we have previously reported, intraperitoneal injections of N^G-nitro-L-arginine methyl ester [L-NAME; a competitive inhibitor of nitric oxide (NO) synthase] before and after the injection of B16 melanoma cells through a tail vein increased experimental pulmonary metastasis, while simultaneous injections of L-arginine (a substrate of NO synthase) at a 20-fold higher dose synergistically increased pulmonary metastasis. Our present study was intended to elucidate the mechanisms by which L-NAME alone or together with L-arginine increases metastasis. Injections of L-NAME decreased the serum concentration of nitrite plus nitrate (metabolites of NO) by about 50%, which was not reversed by simultaneous injections of L-arginine. Injections of L-NAME also decreased the diameter of arterioles and venules by 20–30%, while simultaneous injections of L-arginine did not show any significant effect. When collagen- or ADP-induced platelet aggregation was examined using platelet-rich plasma, injections of L-NAME showed little effects on platelet aggregation, while simultaneous injections of L-arginine rather suppressed platelet aggregation. B16 melanoma cells produced NO in culture, and L-NAME (0.2 mM) decreased NO production without effects on viability. Our results suggest that the increased experimental pulmonary metastasis induced by L-NAME can be ascribed partly to the contraction of arterioles and venules, which is induced by the inhibition of endogenous NO production by L-NAME, and that the synergistic effect of L-arginine on metastasis is related to the inhibition of endogenous NO production through unknown mechanisms. Int. J. Cancer 75:140–144, 1998.© 1998 Wiley-Liss, Inc.     

吉西他滨增加放射性肺损伤作用的实验研究

目的观察吉西他滨不同给药方式对鼠放射性肺损伤的影响,并对其分子机制进行探讨.方法雄性BALB/C鼠,体重25g左右,随机分为空白对照(C)组、单纯照射(R)组、单纯给药(G)组、先给药后照射(PG+R)组、照射与同期给药(R+CG)组,每组80只.实验观察共8个月.采用6MVX线照射,照射面积2.O cm×2.5 cm,照射剂量25Gy分5次.吉西他滨药量80mg/(kg·d),第1、5天腹腔给药.G组给药后第1个月始,每周镜下计数炎细胞总数及分类.每月取左肺组织进行羟脯氨酸含量与病理学检测,右肺用于TGF-βl、EGF和ICAM-1免疫组织化学分析.每月从处死小鼠心脏取血浆100μl用于TGF-β1、EGF和ICAM-1的检测.结果吉西他滨对肺损伤早期主要是炎性细胞渗出(中性粒细胞＞90%),实验后1、3、6个月分别以渗出、增生和纤维化为主.R+CG组羟脯氨酸含量第5个月开始明显高于其他组(P＜0.01),并持续到最后.R+CG组血浆ICAM-1含量照射后第1个月明显高于其他组(P＜0.01),第2个月以后开始下降.R+CG组血浆EGF含量在照射后第2、3个月明显高于其他组(P＜0.01),第4个月后开始下降;TGF-β1含量在照射后第3个月明显高于其他组(P＜0.01),以后含量相对稳定.R+CG组ICAM-1表达在第1个月时明显高于其他组(P＜0.01),第2个月后开始下调;EGF表达在第2、3个月时明显高于其他组(P＜0.01),第4个月后开始下调;TGF-β1表达第3个月开始明显高于其他组(P＜0.01),以后表达相似.结论经病理组织学及羟脯氨酸、TGF-β1、EGF、ICAM-1含量和免疫组织化学分析,照射与吉西他滨同期给药可明显增加放射性肺损伤的发生.     

原发瘤切除对荷人骨肉瘤裸鼠血管生成和肺转移的影响

目的 研究原发瘤切除对骨肉瘤血管生成和肺转移的影响,并探讨其可能的机制及临床意义.方法 用细胞悬液法构建倚人骨肉瘤裸鼠模型,分为实验组(切除原发瘤)、截肢对照组(不切除原发瘤的截肢手术)和空白对照组.用酶联免疫吸附试验(ELISA)法,检测手术前后裸鼠血清中血管内皮生长因子(VEGF)和内皮抑素的表达水平.用HiCN法检测裸鼠体内Matrigel胶体中血红蛋白浓度.应用膨胀压片计数法和组织切片苏木素-伊红对比染色法,观察肿瘤肺部转移灶的情况.结果 手术后实验组血清中的VEGF和内皮抑素浓度较空白对照组和截肢对照组有明显的下降,且内皮抑素下降幅度更大[VEGF分别为(71.43±9.15)pg/ml、(115.81±4.38)pg/ml和(111.68±12.26)ps/ml(P<0.01);内皮抑素分别为(40.77±5.41)ng/ml、(123.18±5.94)ng/ml和(128.06±4.52)ng/ml(P<0.01)];实验组、空白对照组和截肢对照组Matrigel小体中血红蛋白浓度分别为(36.55±2.35)g/L、(16.84±1.15)g/L和(16.29±1.10)g/L(P<0.01).实验组、空白对照组和截肢对照组的肺转移率分别为80.0%、40.0%和35.0%,差异有统计学意义(P<0.05).结论 原发瘤切除可使荷人骨肉瘤裸鼠体内的VEGF和内皮抑素比例失衡,有利于肿瘤血管生成和肺转移,故原发瘤切除后及时抑制肿瘤血管生成,对降低骨肉瘤肺转移有重要意义.     

Conditional activation of Neu in the mammary epithelium of transgenic mice results in reversible pulmonary metastasis

To determine the impact of tumor progression on the reversibility of Neu-induced tumorigenesis, we have used the tetracycline regulatory system to conditionally express activated Neu in the mammary epithelium of transgenic mice. When induced with doxycycline, bitransgenic MMTV-rtTA/TetO-NeuNT mice develop multiple invasive mammary carcinomas, essentially all of which regress to a clinically undetectable state following transgene deinduction. This demonstrates that Neu-initiated tumorigenesis is reversible. Strikingly, extensive lung metastases arising from Neu-induced mammary tumors also rapidly and fully regress following the abrogation of Neu expression. However, despite the near universal dependence of both primary tumors and metastases on Neu transgene expression, most animals bearing fully regressed Neu-induced tumors ultimately develop recurrent tumors that have progressed to a Neu-independent state.     

Vascular endothelial growth factor C promotes human gastric carcinoma lymph node metastasis in mice.

Vascular endothelial growth factor (VEGF)-C, one of several members of the VEGF family, is a relatively specific lymphangiogenic growth factor. VEGF receptor (VEGFR)-3 (or Flt4) is a VEGF-C receptor with expression restricted to lymphatic endothelial cells. Since the mechanisms by which carcinoma cells metastasize to lymph nodes remain unclear, we constructed a VEGF-C transfectant (AZ-VEGF-C) from the AZ521 human gastric carcinoma cell line, which ordinarily shows little nodal metastatic potential and little VEGF-C expression. We orthotopically implanted transfected tumor cells into the stomachs of nude mice. The number of mice developing lymph node metastases and the number of lymph node metastases per mouse with nodal metastases were higher than with implants of mock-transfected control cells. Specifically, percentages of mice with lymph node metastases were 95.5% (21/22) for AZ-VEGF-C and 29.4% (5/17) for controls (P<0.01), while mean numbers of involved lymph nodes were 3.76 for AZ-VEGF-C and 1.00 for controls (P<0.01). No difference was found between AZ-VEGF-C and controls regarding cell growth and chemotactic responses in vitro, or in volumes of tumors arising from implanted cells. When we performed immunohistochemical staining for VEGFR-3 in these tumors to investigate lymphangiogenesis by VEGF-C, the number of vessels stained for VEGFR-3 in tumors and surrounding tissues was higher for AZ-VEGF-C than for controls. VEGFR-3-positive vessels occupied 14.9/1000 of microscopically examined areas for AZ-VEGF-C, but only 1.30/1000 for controls (P<0.001). Our results suggest that VEGF-C is a specific lymphangiogenic growth factor with an important role in lymph node metastasis.     

Promotion by polychlorinated biphenyls of lung and liver tumors in mice

Polychlorinated biphenyls (PCB), which are tumor promoters,have been found in human tissues for decades. Their contributionto cancer risk may only now start to appear, due to long humancancer latency and the nature of tumor promotion. Epidemiologicalassociations have been seen between PCB exposure or tissue contentand cancer at several sites. In rodents, tumor promotion byPCBs has been little studied in tissues other than liver. Previously,in an experiment modeling infant carcinogen exposure followingPCBs received in milk, lung and liver tumors, initiated neonatallyin mice by the environmental nitrosamine N-nitrosodimethylamine(NDMA), were promoted by later treatment with Aroclor 1254.The present study was undertaken to confirm and characterizethe effects of Aroclor 1254 on tumor number, latency, size andmalignancy. Male Swiss mice were given NDMA on postnatal day4 and Aroclor 1254 (250 mg/kg) on day 8, and killed at intervals.Eight PCB congeners were quantified in the carcasses. Incidencesof mice with NDMA-initiated lung tumors at 28 weeks of age wereincreased 2.5-fold by PCBs. Multiplicities of lung tumors wereenhanced four-fold by PCBs at 28 and 52 weeks. By 72 weeks tumornumbers were similar in the NDMA-only and NDMA–PCB groups.Liver tumors first occurred in significant numbers at 52 weeksand only in mice receiving both NDMA and PCBs. As for the lung,at 72 weeks the incidence was high in both the NDMA-only andNDMA–PCB groups. Sizes of tumors and liver carcinoma incidencewere not altered by PCB treatment. Carcass analysis revealeda significant positive association between lung tumor numbersat 28 weeks and relative percentage of 2,2',4,4',5-pentachlorobiphenyl,with no other correlations. The results confirm that PCBs promotelung as well as liver tumors, by triggering the early appearanceof latent initiated tumors otherwise presenting in old age.     

Promotion of hepatocarcinogenesis by phenobarbital in c-myc/TGF-alpha transgenic mice

Previous work has shown that phenobarbital (PB) can promote cell survival in double transgenic c-myc/transforming growth factor (TGF)-alpha mice. This was achieved through a suppression of cell death brought about, at least in part, by a general increase in the level of bcl-2 protein and a decrease in TGF-beta1 in treated versus untreated animals. No changes were found in TGF-beta type II receptor or in bcl-X(L) protein levels. In the present work, we followed these animals for up to 31 wk of age (28 wk of treatment), by which time numerous tumors could be observed. A PB-dependent decrease in tumor latency and a significant increase in multiplicity were seen. No statistically significant changes in the phenotype of foci, nodules, or neoplasms were observed after PB administration, and no effect on median tumor size was detected. Levels of the anti-apoptotic protein bcl-2 did not correlate with tumor formation in PB-treated animals. However, in untreated mice, bcl-2 was higher in tumors than in surrounding tissue in all tumors examined. We believe that the PB-dependent modification of tumorigenesis in the livers of c-myc/TGF-alpha mice was predominantly a result of the ability of this drug to block cell death during the early stages of tumor development. The effect of PB was exerted apparently by a pathway similar to, but separate from, that of TGF-alpha. However, these pathways appear to converge downstream, having common effectors in the form of bcl-2 family proteins. Mol. Carcinog. 28:168-173, 2000.