Persistence of immunogenic pulmonary metastases in the presence of protective anti-melanoma immunity

We have developed a murine melanoma model that allows us to investigate the mechanisms by which spontaneous, immunogenic melanoma metastases escape immunological destruction in syngeneic mice. In the current study, we tested the hypothesis that loss of immunogenicity is an obligatory step in the persistence of pulmonary metastases. Fragments of syngeneic K1735-M2 tumor were implanted in the outer edge of one pinna per C3H/HeN mouse, and the growing tumors were removed 2-3 weeks later. Two weeks after removal of the tumors, the mice demonstrated effective T-cell-mediated immunity to s.c. challenge with K1735-M2 cells. However, lung metastases appeared in 23% of the immunized mice within 9-12 weeks after the initial tumor implantation. The expression of protective immunity to s.c. tumors required the presence of both CD4+ and CD8+ T cells. The immunized mice had specific CTLs capable of killing both K1735-M2 melanoma cells and the cells of nine independently derived melanoma metastases. Furthermore, K1735-M2 immunization protected these mice from s.c. tumor challenge with all nine metastatic cell lines. Our results demonstrate that the persistence of these metastases within the lung was not attributable to emergence of antigen-loss variants in immunized hosts. Our model provides an approach to investigate other mechanisms by which spontaneous metastases escape from immunological control and an opportunity to improve immunotherapy of melanoma metastases.     

Tumor cells expressing anti-CD137 scFv induce a tumor-destructive environment

For immunotherapy to become more effective, there is a need to maximize the antitumor response at the tumor site as well as to eliminate tumor cell variants that lack a given tumor antigen or the ability to present it. We have previously shown that wild-type (WT) cells from the K1735 melanoma (K1735-WT) are rejected following vaccination with cells (K1735-1D8) transfected to express scFv from the anti-CD137 monoclonal antibody 1D8, and that CD4(+) T cells and natural killer (NK) cells are needed for this rejection. We now show that tumors harvested 4 to 10 days after mice had been transplanted with K1735-1D8 cells or a mixture of K1735-1D8 and K1735-WT cells contained more NK cells and that they had an increased percentage of CD4(+) T lymphocytes producing IFNgamma or tumor necrosis factor-alpha. We further show that the percentage of NK cells was higher in B16-1D8 melanomas expressing anti-CD137 scFv than in the WT tumors and that the percentage of FoxP3(+) cells was lower. Admixture of 10% K1735-1D8 cells prevented the progressive growth of transplanted K1735-WT cells in syngeneic mice and also of cells from the antigenically different sarcoma Ag104. Inhibition of WT tumor cells by tumor cells transfected to express anti-CD137 scFv was shown also with the TC1 carcinoma and B16 melanoma. Furthermore, injection of an adenovirus vector, Ad-1D8, which encodes anti-CD137 scFv into established B16 melanomas, significantly prolonged the survival of tumor-bearing mice and could induce regression. Our data suggest that targeting of anti-CD137 scFv to tumors should be explored for therapy for some human cancers.     

Endostatin对结肠癌生长和转移抑制作用的实验研究

背景与目的:结肠癌的生长、转移是血管生成依赖性的,血管生成抑制剂有望通过抑制肿瘤血管生成,诱导结肠癌细胞凋亡,有效地抑制结直肠癌的生长和转移。肿瘤的抗血管生成治疗对选择手术时机和方式、制定综合治疗方案,提高结肠癌患者5年生存率都具有重要意义。本实验研究血管生成抑制因子Endostatin对结肠癌生长和转移的抑制作用,并探讨其对结肠癌细胞凋亡的影响。方法:采用人结肠腺癌细胞株SW1116完整组织块裸鼠原位种植,建立类似于临床的结肠癌转移裸鼠模型。种植后第1周开始皮下注射Endostatin,每天一次,剂量为0mg/kg(对照组)、5mg/kg、10mg/kg、20mg/kg(治疗组),共用6周。种植后第7周处死动物,测量原位肿瘤瘤重、抑瘤率、肿瘤微血管密度(intratumoralmicrovesseldensity,MVD)、肿瘤细胞凋亡指数(apoptoticindex,AI),观察肿瘤细胞腹膜、肝、其他脏器转移及腹水情况。结果:Endostatin剂量为0mg/kg、5mg/kg、10mg/kg、20mg/kg时,原位肿瘤瘤重分别为(1.31±0.36)g、(0.42±0.17)g、(0.21±0.09)g、(0.13±0.05)g;抑瘤率分别为0%、67.9%、84.0%、90.1%;MVD分别为(12.8±4.1)、(5.9±2.5)、(2.2±1.4)、(0.74±O.3);AI分别为(3.87±2.61)%、(6.89±5.18)%、(13.24±4.76)%、(20.97±9.04)%;腹膜转移率分别为9     

Malignant potential of cells isolated from lymph node or brain metastases of melanoma patients and implications for prognosis

We studied the correlation between the formation of brain metastasis and the malignant growth potential of seven human melanoma cell lines, isolated from lymph node metastases (A375-SM, TXM-1, DM-4) or from brain metastases (TXM-13, TXM-18, TXM-34, TXM-40), and the potential of three variants of the mouse K-1735 melanoma. Growth rates in different concentrations of fetal bovine serum and colony-forming efficiency in semisolid agarose were measured, and the tumorigenicity and metastatic ability were determined in nude mice (for the human melanoma cell lines) or in C3H/HeN mice (for the K-1735 variants). The ability to form brain metastasis was tested by injection of cells into the carotid artery. A high colony-forming efficiency in agarose, especially at concentrations of agarose greater than 0.6%, corresponded with high tumor take rates, rapid tumor growth rates, and metastatic colonization of the lungs of the recipient mice. For the human melanomas, the lymph node metastasis-derived cells were more tumorigenic and metastatic than the brain metastasis-derived cells. In the K-1735 mouse melanoma, the tumorigenic and metastatic behavior of the cells after i.v. and s.c. injection corresponded with growth in agarose cultures. However, for growth in the brain after intracarotid injection, the different melanoma cell lines showed similar frequencies of tumor take, regardless of tumorigenicity in other sites of the recipient mice, although mice given injections of brain metastasis-derived cells survived longer than mice given injections of lymph node metastasis (human melanoma) or lung metastasis (K-1735 M-2)-derived cell lines. The results from the human and mouse melanoma cell lines show that the brain metastasis-derived cell lines were not more malignant than the lymph node or lung metastasis-derived cells. These data imply that the production of brain metastasis is not always the final stage of a metastatic cascade.     

Intratumoral administration of endostatin plasmid inhibits vascular growth and perfusion in MCa-4 murine mammary carcinomas

Endostatin, a fragment of the COOH-terminal domain of mouse collagen XVIII is a recently demonstrated endogenous inhibitor of tumor angiogenesis and endothelial cell growth. Antiangiogenic therapy with endostatin in animals requires multiple and prolonged administration of the protein. Gene therapy could provide an alternative approach to continuous local delivery of this antiangiogenic factor in vivo. Established MCa-4 murine mammary carcinomas, grown in immunodeficient mice, were treated with intratumoral injection of endostatin plasmid at 7-day intervals. At the time of sacrifice, 14 days after the first injection, endostatin-treated tumor weights were 51% of controls (P < 0.01). Tumor growth inhibition was accompanied by a marked reduction in total vascular density. Specifically, computerized image analysis showed a 18-21% increase in the median distances between tumor cells and both the nearest anatomical (CD31-stained) vessel [48.1 +/- 3.8 versus 38.3 +/- 1.6 microm (P < 0.05)] and the nearest tumor-specific (CD105-stained) vessel [48.5 +/- 1.5 versus 39.8 +/- 1.5 microm (P < 0.01)]. An increased apoptotic index of tumor cells in endostatin-treated tumors [3.2 +/- 0.5% versus 1.9 +/- 0.3% (P < 0.05)] was observed in conjunction with a significant decrease in tumor perfused vessels (DiOC7 staining), and an increase in tumor cell hypoxia (EF5 staining). Hypoxia resulting from endostatin therapy most likely caused a compensatory increase of in situ vascular endothelial growth factor (VEGF) and VEGF receptor mRNA expression. Increased immunoreactivity of endostatin staining in endostatin-treated tumors was also associated with an increased thrombospondin-1 staining [1.12 +/- 0.16 versus 2.44 +/- 0.35]. Our data suggest that intratumoral delivery of the endostatin gene efficiently suppresses murine mammary carcinoma growth and support the potential utility of the endostatin gene for cancer therapy.     

A clinically relevant, syngeneic model of spontaneous, highly metastatic B16 mouse melanoma

We report a syngeneic model of spontaneous metastatic B16-F10 mouse melanoma in C57/BL6 mice with a very high metastatic frequency that mimics clinical metastatic melanoma. The B16 melanoma cells were injected between the skin and cartilage on the dorsal side of the ear. The model generated lymphatic and visceral metastases in all of the tested animals. In mice with large primary tumors, tumor weight correlated with the tumor growth time and also with the number of metastases in lymph nodes and organs. The dorsal ear space between the skin and cartilage enables both lymphatic and hematogenous metastatic spread. The model should be useful to study the mechanism of melanoma metastasis and to develop therapy for this currently untreatable disease.     

Endostatin expression by MDA-MB-435 breast cancer cells effectively inhibits tumor growth

Tumors must induce the formation of new blood vessels in order to grow and metastasize. Endostatin, a cleaved product of collagen XVIII, inhibits endothelial cell proliferation and suppresses tumor growth and metastases. Several recent reports have questioned the efficacy of endostatin as a tumor suppressor in experimental animals. Our objective was to determine whether endostatin expression in breast cancer cells inhibits neovascularization and tumor growth in nude mice. MDA-MB-435 cells were transfected with an endostatin expression vector while control cells were transfected with an empty vector. Endostatin expression and secretion were confirmed by RT-PCR and a dot blot assay. No differences were observed in the growth rates of the endostatin-expressing and control clones in vitro. When injected into male and female nude mice, tumors from the control clones increased in size 10-15 fold over 8-10 weeks. In contrast, the endostatin clones formed small tumors which did not increase in size after the first 3 weeks. The endostatinderived tumors had a significantly higher apoptotic index (5.6%) compared to controls (2.0%) and showed a marked reduction in vascularization. In conclusion, expression of endostatin in MDA-MB-435 breast cancer cells effectively suppressed breast tumor growth by inhibiting angiogenesis and increasing apoptosis.     

Antitumor effect of endostatin overexpressed in C6 glioma cells is associated with the down-regulation of VEGF

Endostatin is an anti-angiogenic agent that blocks endothelial cell proliferation, tumor growth, and metastasis. Several lines of direct evidence show that gliomas express high levels of endostatin. However, the anti-angiogenic activity and cellular mechanisms of endostatin from tumor cells have not been completely elucidated. In this study, we established C6 glioblastoma (GBM) xenografts in nude mice by subcutaneously injecting glioma cell lines, C6-null cells, and stable transfected-C6 cells overexpressing mock vector (C6-mock) and endostatin (C6-endo). We found that overexpression of endostatin in C6-endo cells significantly suppressed the expression of VEGF in tumor cells in vivo as well as in vitro. Furthermore, the tumor growth derived from C6-endo cells was inhibited. Our data demonstrate that endostatin could play a direct role in inhibiting tumor cells, and suggest that enhancing endostatin expression in gliomas could be an effective treatment strategy.     

99mTcOAADT]-(CH2)2-NEt2: a potential small-molecule single-photon emission computed tomography probe for imaging metastatic melanoma

Evaluation of [99mTc]oxotechnetium(V) complexes of the amine-amide-dithiol (AADT) chelates containing tertiary amine substituents as small-molecule probes for the diagnostic imaging of metastatic melanoma has shown that technetium-99m-labeled AADT-(CH2)2-NEt2 (99mTc-1) has the highest tumor uptake and other favorable biological properties. We have, therefore, assessed this agent in a more realistic metastatic melanoma model in which, after i.v. tail injection, a highly invasive melanoma cell line, B16F10, forms pulmonary tumor nodules in normal C57BL6 mice. Small melanotic lesions develop in the lungs and, on histologic examination, appear as small black melanoma colonies, increasing in size and number with time after tumor cell injection. Groups of mice received tumor cell inocula of 2 x 10(5), 4 x 10(5), or 8 x 10(5) B16F10 cells; 14 days later, 2 hours after 99mTc-1 administration, lung uptake of 2.83 +/- 0.21%, 3.63 +/- 1.07%, and 4.92 +/- 1.61% injected dose per gram of tissue (% ID/g), respectively, was observed, compared with normal lung uptake of 2.13 +/- 0.2% ID/g (P < 0.05). Additionally, a higher level of 99mTc-1 accumulation was seen 17 days after tumor cell inoculation as the lung lesions grew. These in vivo studies coupled with additional in vitro and ex vivo assessment show that 99mTc-1 has high and specific uptake in melanoma metastases in lungs and can potentially follow the temporal growth of these tumors.     

Host-derived MMP-13 exhibits a protective role in lung metastasis of melanoma cells by local endostatin production

Background:

Although matrix metalloproteinases (MMPs) are implicated in tumourigenesis and cancer progression, the role of MMP-13 in melanoma cell metastases is poorly understood.

Methods:

Lung metastases of mouse melanoma B16BL6 cells were analysed in MMP-13 knockout (KO) and wild-type (WT) mice after intravenous injection. The mRNA and protein expression of MMP-13 in lung tissues was analysed by RT–PCR, real-time PCR, immunoblotting and immunohistochemistry. The expression of SDF-1α, CXCR4 and endostatin, and effects of endostatin to cultured melanoma cells and lung metastases were also studied.

Results:

Lung metastases of B16BL6 cells were significantly higher by 2.5–5.7-fold in MMP-13 KO mice than in WT mice. The expression of MMP-13 in WT mouse lung tissue was stimulated on day 1 after intravenous injection of the melanoma cells and MMP-13 was immunolocalised to vascular endothelial cells in the lungs. Endostatin formation, but not degradation of SDF-1α, in the lung tissue was associated with reduced lung metastasis in WT mice. Endostatin significantly inhibited migration of B16BL6 cells in monolayer wounding assay and remarkably suppressed Matrigel invasion and transendothelial invasion of the cells. In addition, lung metastases of melanoma cells in MMP-13 KO mice were reduced by intraperitoneal administration of endostatin.

Conclusion:

Our results suggest that MMP-13 is overproduced by endothelial cells in the lungs with melanoma cells and has a protective role in lung metastasis by local generation of endostatin.     

CT7 (MAGE-C1) antigen expression in normal and neoplastic tissues

Angiogenesis is a vital component of the development and progression of many human solid tumors. Glioblastoma multiforme is one of the most highly vascularised class of solid tumors. Thus, we have investigated the potential antitumourigenic activity of endostatin, an angiogenic inhibitor, in the rat C6 glioma model. We have engineered C6 cells that endogenously express mouse endostatin in order to assess the growth of C6 tumors in vivo when endostatin is constitutively expressed. Endostatin secreted by stably transfected C6 cells is biologically active as shown by its inhibition (26%) of bFGF-stimulated proliferation of BAECs in culture. The subcutaneous implantation of endostatin-C6 cells in athymic (nu/nu) mice resulted in a reduced tumor growth rate (90% inhibition) compared to control cell lines throughout the duration of our experiments. Tumor inhibition was associated with a 50% reduction in the number of vessels, which were also smaller in morphology. However, endostatin-C6 tumors were no more necrotic than control tumors. The implantation of endostatin-C6 cells into immunocompetent Wistar rat brains also resulted in reduced tumor volumes (71% inhibition) compared to controls. Tumor cells were sparsely localised along the injection tract but had not formed discrete tumors. Despite the inhibitory response mediated by endostatin on C6 growth, complete tumor inhibition or dormancy was not observed in either the athymic or immunocompetent tumor models. These findings demonstrate that the endogenous expression of endostatin by C6 glioma cells results in a reduced tumor growth rate in vivo that is associated with an inhibition of tumor angiogenesis. Our data suggest that endostatin should be developed as an adjuvant gene therapy for the effective treatment of gliomas.     

Evidence that intravenously derived murine pulmonary melanoma metastases can originate from the expansion of a single tumor cell

The purpose of these studies was to determine whether hematogenous clonal pulmonary melanoma metastases originate from the expansion of a single cell and if so, by extrapolation, metastasis can be considered a cloning process. Three different cell lines of murine K-1735 melanoma with different metastatic properties and unique karyotypes were injected i.v. into syngeneic C3H/HeN mice as multicell aggregates of individual cell lines or combinations of cell lines. Resultant solitary lung metastases were isolated in culture as individual lines and then karyotyped. Even when heterogeneous clumps of tumor cells were injected, the individual metastases exhibited a karyotype unique to one metastatic cell type. Furthermore, when cellular aggregates were composed of metastatic cells admixed with cells that were tumorigenic but nonmetastatic, the resultant metastases exhibited only the karyotype of the metastatic cells. This finding suggests that the presence of metastatic cells did not change the inability of nonmetastatic cells to proliferate in a distant organ. Collectively, the results indicate that the resultant metastases were of clonal origin owing to the expansion of a single metastatic tumor cell in the lung parenchyma.     

重组腺相关病毒介导人内皮抑素抑瘤作用的实验研究

背景与目的：研究表明,内皮抑素能抑制肿瘤血管的生成,进而抑制肿瘤的生长和转移,然而有关内皮抑素治疗的研究报道却很少。本研究初步探讨了重组腺相关病毒介导内皮抑素对肿瘤生长和转移的作用。方法：用RT-PCR方法获得人内皮抑素基因,构建内皮抑素可分泌表达的通用型重组腺相关病毒（rAAV）载体,包装获得重组腺相关病毒rAAV-SS-Endostain。以动物实验分析其抗肿瘤作用。结果：C57BL/6小鼠肌肉注射10^11TUrAAC-SS-Endostatin一次,该重组病毒对小鼠黑色瘤B16F10移植瘤生长的抑制率为57.1％,在实验肺转移模型中,对转移灶的抑制率为70.7％。结论：重组腺相关病毒介导的内皮抑素能有效抑制肿瘤的生长和转移。     

腺病毒介导的内皮抑素基因治疗小鼠肺癌

目的 探讨内皮抑素对小鼠肺癌生长和转移的抑制作用及其对肿瘤内部新生血管的影响.方法 在C57BL/6小鼠背部皮下注射2×106 lewis肺癌(LLC)细胞,建立小鼠肺癌种植瘤模型,2周后,瘤内注射2×109 pfu内皮抑素腺病毒载体,观察内皮抑素对肿瘤生长、转移及生存率的影响,检测内皮抑素在肿瘤组织的原位表达和血液循环中的表达水平及持续时间.用免疫组化方法,检测肿瘤内部血管密度,观察治疗对肿瘤血管的影响.用透射电镜观察肿瘤细胞的凋亡情况.结果 免疫组化检测结果显示,内皮抑素蛋白在内皮抑素组的肿瘤组织中呈强阳性表达,而在空载体对照组和阴性对照组中呈阴性表达或很少量表达.用酶联免疫吸附实验(ELISA)法检测内皮抑素组血清内皮抑素浓度,第2周可达1540±560 ng/ml;1个月后,血清内皮抑素浓度降至对照水平.内皮抑素组的肿瘤体积和生存率,与空载体对照和阴性对照组比较,差异有统计学意义(P＜0.05).抗CD31抗体标记的肿瘤内血管密度(MVD)在内皮抑素组、空载体对照组和阴性对照组中,分别为37.5±4.6、65.2±5.8和68.5±4.5个/200倍视野,抗CD105抗体标记的肿瘤内MVD分别为10.5±3.2、39.7±5.6和42.4±4.8个/200倍视野,内皮抑素组与空载体对照组和阴性对照组比较,差异有统计学意义(P＜0.05).内皮抑素组的组织在电镜下呈凋亡相的肿瘤细胞多见.结论 腺病毒介导的内皮抑素基因可在体内高效、较长时间表达内皮抑素蛋白,对小鼠皮下种植瘤有一定的治疗作用,其作用的靶点是抑制新生血管的生成.     

Development of a xenograft glioma model in mouse brain

Xenograft intracerebral glioma models have been developed in normal mice by growing the rat C6 glioma in either adult or neonatal mouse brains. Using this tumor line it was possible to grow discrete intracerebral gliomas in either CBA or AKR adult mice or neonatal mice. The size of the tumor mass and length of survival was directly related to the number of tumor cells injected and the time after implantation. To obtain localized intracranial tumor growth cells were suspended in a 1% agarose solution before implantation. Following injection of 10(6) cells into the frontal lobe of adult CBA or AKR mice, discrete tumor masses greater than 4 mm in diameter were obtained in 90% of animals at 14 days, and the largest tumors in adult mice occurred between 21 and 28 days after implantation. The tumor size following implantation of 10(6) cells was significantly greater than with 10(5) cells at 7 days (P less than 0.05) and at 14 and 21 days (P less than 0.01). Less than 60% of mice of BALB/c, RIII, or C57 black strains developed tumors greater than 4 mm diameter at 14 days after intracerebral injection of 10(6) C6 cells. Using neonatal mice it was found that when 10(5) cells were injected intracranially tumors greater than 4 mm in diameter developed in 14 of 15 animals within 2 weeks (CBA mice). Similar results were seen in the RIII, AKR, C57 black, and BALB/c strains. Longer growth periods resulted in larger tumors, up to 8 mm in diameter (6 of 10 animals at 20 days). The tumors in the neonatal animals were not as discrete as in the adult mice, and tumor often spread to the meninges and into the lateral ventricles. The tumor harvested from the brain had a cloning efficiency of 1.2 +/- 0.4% (SD). A panel of monoclonal antibodies was raised to the C6 glioma, and this was used to define clearly the margins of the tumor within the brain. The xenograft mouse models should prove useful for the study of the therapy of gliomas.     

Enhancement of radiation effects by pXLG-mEndo in a lung carcinoma model

PURPOSE: Endostatin is a potent antiangiogenesis protein with little or no toxicity that has potential to enhance radiotherapy. The major goal of this study was to evaluate the combination of radiation and endostatin gene therapy in a preclinical lung cancer model. METHODS: Plasmid pXLG-mEndo, constructed in our laboratory, includes the mouse endostatin gene cloned into the pWS4 vector. The kinetics of endostatin expression and efficacy of the pXLG-mEndo and radiation ((60)Co gamma-rays) combination was evaluated in the C57BL/6 mouse-Lewis lung carcinoma (LLC) model. The LLC cells were implanted s.c. and pXLG-mEndo was injected intratumorally 12-14 days later without any transfection agent; a dose of 10 Gy radiation was applied approximately 16 h thereafter. Some groups received each modality twice. Endostatin, vascular endothelial growth factor (VEGF), and transforming growth factor-beta1 (TGF-beta1) were quantified in plasma and tumors, and tumor vasculature was examined. RESULTS: Endostatin expression within LLC tumors peaked on Day 7 after pXLG-mEndo injection. Addition of radiation to pXLG-mEndo significantly enhanced the level of tumor endostatin compared with plasmid alone (p < 0.05). Tumor growth was significantly delayed in mice receiving pXLG-mEndo plus radiation compared with no treatment (p < 0.005), radiation (p < 0.05), and control plasmid (p < 0.05). The number of LLC tumor vessels was reduced after combined treatment (p < 0.05), and significant treatment-related changes were observed in both VEGF and TGF-beta1. CONCLUSIONS: The data demonstrate that delivery of endostatin by pXLG-mEndo as an adjuvant to radiation can significantly enhance the antitumor efficacy of radiotherapy in the LLC mouse tumor model and support further investigation of this unique combination therapy.     

Correlation between the in vitro interaction of tumor cells with an organ environment and metastatic behavior in vivo

The purpose of this study was to determine whether the in vitro interaction of tumor cells with an organ fragment correlated with organ colonization in vivo. Several clones of the K-1735 melanoma that exhibit different potentials for lung colonization or the heterogeneous M-5076 reticulum cell sarcoma which preferentially colonizes the liver, were used. The simultaneous subcutaneous implantation of a lung fragment with high lung-colonizing K-1735 cells stimulated the production of tumors, but this was not the case with the M-5076 cells, which do not colonize the lungs after intravenous injection. The ability of the tumor cells to survive in vitro in a lung fragment correlated with their capacity to produce lung tumor colonies after intravenous injection. Moreover, lung-conditioned medium stimulated the growth of lung-colonizing K-1735-M2 cells but not liver-colonizing M-5076 cells. Collectively, the data suggest that tissue-mediated growth stimulation may be one of the factors influencing the metastatic pattern and potential of tumor cells.     

Comparative studies on the quantitative analysis of experimental metastatic capacity

The purpose of these studies was to establish a procedure for determining the relative experimental metastatic potential of unrelated murine tumors. We used three tumors (the B16-F10 melanoma, which is syngeneic to the C57BL/6N mouse, and the K-1735 melanoma and the UV-2237 fibrosarcoma, which are syngeneic to the C3H/HeN mouse). Various numbers of tumor cells were injected into normal or immunosuppressed syngeneic recipients and into 3-week-old BALB/c nude mice. At appropriate intervals, the recipient mice were killed, and the metastatic burden was determined. The number of experimental metastases was not linearly correlated with cell input. Thus, simply comparing the incidence of metastasis resulting from the injection of one predetermined dose of tumor cells did not allow for determination of their relative metastatic capacities. More reproducible and meaningful results were obtained by introducing increasing numbers of viable tumor cells admixed with a constant number of nontumorigenic (X-irradiated) tumor cells serving as carrier. The incidence of metastasis by few or many injected cells is influenced by host factors such as immune status, and therefore determinations of the true metastatic nature of any given tumor necessitate the choice of an appropriate recipient.     

Local versus systemic interleukin-2: tumor formation by wild-type and B7-1-positive murine melanoma cells

Modification of murine K1735 melanoma cells to express the immune costimulator B7-1 had no effect on tumor formation in syngeneic mice. In contrast, <40% of mice inoculated with K1735 cells modified to secrete murine interleukin-2 (IL-2) formed tumors, and no tumors formed when the K1735 cells coexpressed both murine IL-2 and B7-1. However, administration of systemic recombinant human IL-2 had no detectable effect on the formation of tumors by the B7-1-expressing K1735 cells. By contrast, admixtures of IL-2-secreting and B7-1-expressing K1735 cells formed fewer tumors than either cell type alone. Murine IL-2 was effective only when secreted locally, because the IL-2-secreting cells inoculated into the right flank did not affect the growth of the B7-1-expressing cells inoculated into the opposite flank.     

Inhibition of B16BL6 tumor progression by coadministration of recombinant angiostatin K1-3 and endostatin genes with cationic liposomes

Transfection of the antiangiogenic angiostatin and endostatin genes was shown to be an alternative to high-dose administration of angiostatin or endostatin proteins for cancer therapy. We have systematically investigated whether coadministration of the mouse angiostatin kringle 1-3 gene (pFLAG-AngioK1/3) and the endostatin gene (pFLAG-Endo) complexed with cationic liposomes exhibits enhanced therapeutic efficacy. In vitro, the coexpressed mixture of angiostatin K1-3 and endostatin more effectively reduced angiogenesis in chorioallantoic membranes than either angiostatin K1-3 or endostatin alone. In vivo, subcutaneous co-administration of pFLAG-AngioK1/3 and pFLAG-Endo lipoplexes more effectively inhibited vascularization in Matrigel plugs implanted in mice than either one alone. Additionally, subcutaneous administration of these genes inhibited the growth and formation of pulmonary metastases of B16BL6 melanoma cells in mice. Compared to treatment with an empty vector, treatment with pFLAG-AngioK1/3 plus pFLAG-Endo inhibited 81% of tumor growth, while treatment with pFLAG-AngioK1/3 or pFLAG-Endo inhibited tumor growth 70 and 69%, respectively. Cotreatment with the two plasmids after primary tumor excision induced a 90% inhibition of pulmonary metastases versus 79% for pFLAG-AngioK1/3 or 80% for pFLAG-Endo individually. These results suggest that combined administration of angiostatin K1-3 and endostatin genes complexed with cationic liposomes may be an innovated antiangiogenic strategy for cancer therapy.