Number Of Metastatic Colonies Research Articles

In vitro and in vivo anti-cancer effects of hibernating common carp (Cyprinus carpio) plasma on metastatic triple-negative breast cancer

Uncontrollable proliferation is a hallmark of cancer cells. Cell proliferation and migration are significantly depressed during hibernation state. Many studies believe some factors in the plasma of hibernating animals cause these effects. This study aimed to assess the anti-cancer effects of hibernating common carp (Cyprinus carpio) plasma on 4T1 cancer cells in vitro and in vivo. The effect of hibernating plasma on cell viability, morphology, migration, apoptosis rate, and cell cycle distribution of 4T1 cells was investigated in vitro and in vivo. Hibernating plasma at a concentration of 16 mg/ml significantly reduced the viability of 4T1 cancer cells, without any toxicity on L929 normal fibroblast cells. It could change the morphology of cancer cells, induced apoptosis and cell cycle arrest at the G2/M phase, and inhibited migration. Furthermore, intratumoral injection of hibernating plasma (200 µl, 16 mg/ml) in the tumor-bearing mice caused a significant inhibition of 4T1 breast tumors volume (46.9%) and weight (58.8%) compared with controls. A significant decrease in the number of metastatic colonies at the lungs (80%) and liver (52.8%) of hibernating plasma-treated animals was detected which increased the survival time (21.9%) compared to the control groups. Immunohistochemical analysis revealed a considerable reduction in the Ki-67-positive cells in the tumor section of the hibernating plasma-treated animals compared with controls. Taken together, the SDS-PAGE and mass spectrometry analysis indicated the alpha-2-macroglobulin level in the hibernating fish plasma was significantly increased. It could exert an anti-cancer effect on breast cancer cells and suggested as a novel cancer treatment strategy.

A reverse route into metastasis

Cancer Signaling proteins in the semaphorin family guide cell migration. Transmembrane semaphorins can act both as ligands (the so-called “forward” signaling pathway) and as receptors (the “reverse” signaling pathway). Gurrapu et al. found that semaphorin 4C (Sema4C) acted as a receptor to promote metastasis by activating transforming growth factor–β signaling in invasive breast cancer cell lines. Overexpression of Sema4C in breast or prostate cancer cells resulted in increased numbers of metastatic colonies in mice. High levels of Sema4C in primary breast tumors correlated with increased metastasis in patients. Sci. Signal. 12 , eaav2041 (2019).

Abstract LB-055: Discoidin domain receptor 1 (DDR1) ablation promotes tissue fibrosis and hypoxia to induce aggressive, basal-like breast cancer

Abstract Breast cancer studies implicate the discoidin domain receptor 1 (DDR1) in both pro- and anti-tumor roles. DDR1 is often overexpressed in breast tumors and its activation by collagen promotes tumor cell invasion and potentiates metastatic colonization by cell lines. However, DDR1 transfection into low DDR1 expressing tumor cells hinders cell migration. Moreover, low expression of DDR1 correlates with higher grade human tumors and associates with worse prognosis. To better understand the role of DDR1 in breast tumor progression and metastasis, we established a DDR1 knockout and crossed it into the MMTV-PyMT mouse. DDR1 loss compromised luminal cell adhesion and expanded the basal/myoepithelial population in adult mice. PyMT DDR1-/- tumors grew faster and had increased lung metastasis both in size and number of metastatic colonies. The DDR1-/- tumors broadly exhibited a more basal-like phenotype with enhanced fibrosis and were more hypoxic. Furthermore, these tumors contained more CD90+ CD24+ tumor cells and more K8+ K14+ cells surrounding necrotic regions. Taken together these data suggest DDR1 loss confers a growth advantage favoring basal cell expansion via impaired luminal adhesion and enhanced basal differentiation leading to more aggressive, basal-like disease. Citation Format: Allison P. Drain, Ken Takai, Devon A. Lawson, Laurie E. Littlepage, Marcela Karpuj, Kai Kessenbrock, Annie Le, Kenichi Inoue, Valerie M. Weaver, Zena Werb. Discoidin domain receptor 1 (DDR1) ablation promotes tissue fibrosis and hypoxia to induce aggressive, basal-like breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-055.

Abstract 3644: Combination of OX40L fusion protein with an anti-tumor vaccine reduces lung metastasis and prolongs survival in a murine model of metastatic triple negative breast cancer

Abstract OX40 is a costimulatory receptor on activated T-cells that potentiates the proliferation, survival, and memory formation of the CD4+ and CD8+ immune populations upon its interaction with OX40L on antigen presenting cells. The engagement of OX40 on regulatory T-cells (Tregs) with its ligand has also been shown to inhibit their suppressive capabilities. The use of exogenous OX40L agonists for cancer immunotherapy is particularly attractive in combination with cancer vaccines in order to boost the immune activity of antigen specific T-cells and overcome self-tolerance and immunosuppressive mechanisms of the tumor. We investigated the efficacy of this strategy in the 4T1 murine mammary tumor model of metastatic triple-negative breast cancer by combining an OX40L fusion protein (OX40L-FP) with a poxvirus-based cancer vaccine (MVA-Twist-TRICOM) that induces T-cell responses specific to the tumor antigen Twist, a transcription factor highly expressed in lung metastases. OX40L-FP was administered 3 and 6 days after the prime and boost vaccinations to maximize its interaction with OX40 on activated T-cells. As single agents, OX40L-FP and MVA-Twist-TRICOM had minimal effect on the formation of lung metastases in 4T1 tumor-bearing mice. However, when combined, the two immunotherapies significantly decreased the number of metastatic colonies per lung, and prolonged survival after surgical resection of the primary tumor. Antibody depletion studies demonstrated that the observed anti-tumor effects were mediated by both CD4+ and CD8+ T-cells. Immune subset analysis revealed that the combination therapy increased the total number of CD4+Foxp3- and CD4+ effector memory T-cells in both the lung and periphery compared to the monotherapy controls. More importantly, the two agents synergistically induced greater CD4+ Twist-specific T-cell responses, as determined by ex vivo proliferation assays using T-cells isolated from the lung and spleen that were stimulated with Twist protein. Although the combination had no added effect on the number of total CD8+ and CD8+ effector memory T-cells in relation to the vaccine monotherapy, tetramer staining revealed a significant increase in CD8+ T-cells that were specific for Twist. We also observed a greater number of CD8+ T-cell memory precursors, as well as a greater number of IFNγ-producing CD8+ T-cells in both the lung and periphery. Tregs isolated from the lung and splenic tissue were found to be significantly less suppressive following the combination therapy, compared to those isolated from mice that received the OX40L-FP and MVA-Twist-TRICOM single agent treatments. This study provides the rationale for pairing OX40 agonists with cancer vaccines to stimulate CD4+ and CD8+ T-cell responses, while inhibiting Treg function, as a way to induce specific immunity and clearance of metastatic tumors. Citation Format: Anthony S. Malamas, Scott A. Hammond, Jeffrey Schlom, James W. Hodge. Combination of OX40L fusion protein with an anti-tumor vaccine reduces lung metastasis and prolongs survival in a murine model of metastatic triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3644. doi:10.1158/1538-7445.AM2017-3644

MiR-106b-5p promotes renal cell carcinoma aggressiveness and stem-cell-like phenotype by activating Wnt/β-catenin signalling.

PurposeTo examine the role of miR-106b-5p in regulating the cancer stem-cell-like phenotype in clear cell renal cell carcinomas (ccRCC).Experimental DesignReal-time PCR was performed to evaluate miR-106b-5p levels in ccRCC cell lines and patients specimens. A series of in vivo and in vitro assays were performed to confirm the effect of miR-106b-5p on ccRCC stemness phenotype.ResultsccRCC cells and tissues expressed more miR-106b-5p than normal controls. Gain- and loss-of-function studies demonstrated that overexpression of miR-106b-5p in ccRCC cells increased the spheres formation ability and the proportion of side population cells. Ectopic expression of miR-106b-5p in ccRCC cells increased tumour growth rates and the number of metastatic colonies in the lungs by using an orthotopic kidney cancer model and a tail vein injection model, respectively. Mechanistic studies revealed that, miR-106b-5p has an activating effect on Wnt/β-catenin signalling. miR-106p-5p overexpression simultaneously targets multiple negative regulators of the Wnt/β-catenin pathway, namely, LZTFL1, SFRP1 and DKK2. In addition, we also confirmed that miR-106b-5p and its targets expression correlates with the overall-survival of ccRCC patients from TCGA.ConclusionsThese findings suggest that miR-106b-5p mediates the constitutive activation of Wnt/β-catenin signalling, likely serving as a potential therapeutic target for ccRCC.

Axl signaling induces development of natural killer cells in vitro and in vivo.

Natural killer (NK) cells have been well known to play a critical role in innate immunity, but they are also capable of regulating adaptive immunity through the induction of T cell-mediated memory response and B cell-mediated autoimmune response. NK cells are differentiated from hematopoietic stem cells (HSCs) in the bone marrow (BM), and a series of surface molecules are expressed on NK cells in a differentiation stage-specific manner. Axl receptor tyrosine kinase is originally identified as homeostatic regulators for antigen-presenting cells, and its ligand, growth-arrest-specific gene 6 (Gas6), has been reported to promote cell survival, proliferation, and migration, but their regulatory role in the development and effector function of NK cells is not yet fully understood. In this study, to investigate whether Axl is required for the regulation of NK cell development, the expression of mature NK (mNK) cell-specific receptors and NK cell-associated genes was analyzed in the differentiated HSCs-derived NK cells in vitro and the NK cells harvested from Axl-/- mice. We found that agonistic anti-Axlantibody or recombinant Gas6 specifically upregulated the expression of mNK cell-specific receptors, such as LY49A, Ly49G2, Ly49C/F/I, NKG2A/C/E (1.5- to 3.5-fold increase), and NK cell-associated genes, such as IL-2Rβ (2.3- or 2.4-fold increase), Perforin (4.1- or 2.1-fold increase), IL-15Rα (2.14- or 2.04-fold increase), and IFN-γ (3.3- or 2.8-fold increase) compared to each isotype control, whereas it was abrogated by treatment ofAxl-Ig. Anti-Axlantibody or rGas6 also induced a 2.5- or 1.9-fold increase in the proliferation of developing NK cells compared to each control, respectively. mNK cell populations expressing mNK cell-specific receptors were reduced about twofold in NK cells differentiated from HSCs of Axl-/- mice compared with those of wild-type mice. Furthermore, the triggering of Axl signaling by agonistic anti-Axlantibody promoted the cytolytic activity (1.5- to 1.9-fold increase) against target tumor cells. In B16F10 melanoma-bearing mice, the number of metastatic colonies was decreased by 83% by the administration of mNK cells treated with anti-Axlantibody compared to control Ig. These data suggest that Axl plays an essential role in the regulation of NK cell development as well as NK effector function.

Abstract 5244: Store-operated Ca2+ entry (SOCE) regulates melanoma progression.

Abstract Background: Melanoma is the worst poor-prognosis skin cancer in the U.S. Although Ca2+ is well recognized as a major second messenger, the role of Ca2+ remains poorly understood in the cancer research field including melanoma. Store-operated calcium entry (SOCE) is the mechanism of Ca2+ entry from the extracellular space in response to Ca2+ depletion in the endoplasmic reticulum (ER). SOCE is regulated by interaction between STIM1 (stromal interaction molecule 1) in the ER and Orai (ORAI calcium release-activated calcium modulator) in the plasma membrane. Our previous studies have demonstrated the presence of SOCE is observed in melanoma cells, and potentially regulates cell migration and cell cycle. In the present study, we extensively examined the effects of SOCE on migration and proliferation in multiple melanoma and melanocyte cell lines as well as on metastasis in mice. Method: Changes in intracellular Ca2+ level were measured by Fluo4-AM, a Ca2+-sensing fluorescent dye. In order to ablate STIM-1 or Orai-1, shRNA for each protein was induced with lentiviral infection in SK-Mel-2, SK-Mel-24 and C8161 cells. Proliferation was measured by the MTT assay. Migration was examined with the Boyden chambers and the scratch assay. Immunocytochemistry was performed to count the number of lamellipodia with F-actin staining. The experimental metastasis assay was performed as follows. Three weeks after the intravenous injection of SK-Mel-2 cells with knockdown of Orai1 or STIM1 in Balb/c nu/nu mice, the lungs were removed and fixed with picric acid. The number of metastatic colonies in the lung surface was counted. Results: SOCE, as demonstrated by enhancement of Ca2+ entry from extracellular space after Ca2+ depletion in the ER, was observed in 5 primary and 3 metastatic human melanoma cell lines, a mouse melanoma cell line, human and mouse melanocyte cell lines. In addition, metastatic, but not primary, melanoma cell lines showed significantly greater SOCE compared to melanocytes, suggesting that SOCE may positively correlate with malignancy of melanoma. Knockdown of STIM1 inhibited proliferation in metastatic melanoma cells (SK-Mel-2, SK-Mel-24 and C8161) (p<0.01), suggesting that SOCE activates proliferation of melanoma. Inhibition of SOCE with knockdown of Orai1 or STIM1 suppressed migration in cell lines tested (SK-Mel-2 and SK-Mel-24) (p<0.01) and the SOCE inhibitors suppressed it in cell lines (SK-Mel-2 and C8161) (p<0.01 for both cell lines), indicating that SOCE regulates melanoma migration. Indeed, the number of lamellipodia, which reflects the activity of migration, was decreased by deletion of STIM1 or Orai1 (p<0.01). The experimental metastasis assay demonstrated that the number of lung colonies was reduced by knockdown of STIM1 or Orai1 (p<0.01). Conclusion: Our results demonstrated that inhibition of SOCE suppressed proliferation, migration, and metastasis in melanoma, thus SOCE could be a new target for melanoma therapy. Citation Format: Masanari Umemura, Erdene Balijinnyam, Stefan Feske, Mariana S. De Lorenzo, Lai-Hua Xie, Yoshihiro Ishikawa, Kousaku Iwatsubo. Store-operated Ca2+ entry (SOCE) regulates melanoma progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5244. doi:10.1158/1538-7445.AM2013-5244

A dynamic model for tumour growth and metastasis formation

A simple and fast computational model to describe the dynamics of tumour growth and metastasis formation is presented. The model is based on the calculation of successive generations of tumour cells and enables one to describe biologically important entities like tumour volume, time point of 1st metastatic growth or number of metastatic colonies at a given time. The model entirely relies on the chronology of these successive events of the metastatic cascade. The simulation calculations were performed for two embedded growth models to describe the Gompertzian like growth behaviour of tumours. The initial training of the models was carried out using an analytical solution for the size distribution of metastases of a hepatocellular carcinoma. We then show the applicability of our models to clinical data from the Munich Cancer Registry. Growth and dissemination characteristics of metastatic cells originating from cells in the primary breast cancer can be modelled thus showing its ability to perform systematic analyses relevant for clinical breast cancer research and treatment. In particular, our calculations show that generally metastases formation has already been initiated before the primary can be detected clinically.

Abstract 1483: Metastatic characteristics and radiosensitivity of thyroid carcinoma cells depends on HIF-1 and PI3K signaling in vitro and in vivo

Abstract The purpose of this study was to determine the importance of the HIF-1 and PI3K pathways in the development of a) a metastatic phenotype and b) desensitization to radiotherapy, in thyroid carcinomas. In vitro scratch wound migration assays and cell spreading assays were performed under various oxygen tensions in the presence or absence of two PI3K inhibitors PI-103 and GDC-0941 (GDC-0941 is currently in clinical trials). The role of HIF-1α and PI3K on these processes was also assessed via genetic inhibition of HIF-1α and PI3K by use of a dominant negative variant (dnHIF) and by over expressing WT PTEN in PTEN-null FTC133 cells. For in vivo studies, follicular (FTC133) and anaplastic (8505c) thyroid carcinoma cells were manufactured to stably express eGFP and implanted sub-cutaneously. Mice were treated with PI-103 and GDC-0941 by intra-peritoneal injection and oral gavage. Spontaneous metastatic colonization to the lungs was confirmed by viewing eGFP postive colonies cultured from digested lung tissue and further by immuno-blotting for eGFP. For irradiation studies, cells were exposed to 0,2,4,6 GY external beam radiation. Both PI3K and hypoxia increased cell migration, cell adhesion and cell spreading on extra-cellular matrix molecules. These processes are key in the development of a metastatic phenotype. Genetic and pharmacological inhibition of both PI3K and HIF-1α reduced the migratory, adhesive and cell spreading potential of thyroid carcinoma cells under varying oxygen tensions. Additionally, pharmacological and genetic inhibition of PI3K and HIF-1α reduced both PI3K and HIF-1 activity resulting in reduced activation of PI3K target proteins (pAKT, pGSK-3β) and reduced expression of HIF-1 target genes (CA-9, VEGF, LDH-A, GLUT-1). In vivo, PI-103 and GDC-0941 reduced PI3K and HIF-1α activity as well as PI3K and HIF-1 target genes in primary follicular and anaplastic tumors. Importantly, mice bearing dnHIF-FTC133 tumors and those treated with GDC-0941 had reduced number of metastatic colonies in the lungs of follicular thyroid tumor bearing mice. Radiation induced HIF-1α activity and expression. GDC-0941 combined with radiation blocked HIF-1 induction, prolonged DNA double-strand breaks and reduced clonogenic survival suggesting a radio-sensitising effect. These data link PI3K, HIF-1 activation and aggressive disease in thyroid carcinoma and suggest PI3K may be an important therapeutic target. One attractive approach would be combination therapy with external beam radiation. The latter is the first line treatment in patients suffering from anaplastic thyroid carcinoma but is seldom successful. With the known desensitizing effects of HIF-1 and PI3K activity on radiation treatment, a combined approach involving both radiation and a PI3K inhibitor may improve both the therapeutic response within the local tumor and reduce metastatic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1483. doi:10.1158/1538-7445.AM2011-1483

Enhanced Experimental Tumor Metastasis with Age in Senescence-Accelerated Mouse

Tumor metastasis is affected by the host immune surveillance system. Since aging may attenuate the host immune potential, the experimental tumor metastasis may be enhanced with age. In the present study, we investigated this alteration of experimental tumor metastasis with age. We used senescence-accelerated mice prone 10 (SAMP10) as a model of aged animals. Natural killer cell (NK) activity, as an indicator of immune surveillance potential, in 8-month-old (aged) SAMP10 mice was observed to be much lower than that in 2-month-old (young) mice. When we examined the in vivo trafficking of lung-metastatic K1735M2 melanoma cells in SAMP10 with positron emission tomography (PET), K1735M2 cells labeled with [2-18F]2-deoxy-2-fluoro-D-glucose ([18F]FDG) were observed in both young and aged SAMP10 just after injection of the cells, whereas the clearance of 18F from the lungs was retarded in aged animals. The accumulation of 5-[125 I]iodo-2'-deoxyuridine ([125 I]IUdR)-labeled K1735M2 cells in the lungs of SAMP10 at 24 h after injection was significantly higher in aged mice. Corresponding to these results, the number of metastatic colonies in the lung was larger in the aged SAMP10 of the experimental tumor metastasis model. The present study demonstrated that the aging process produced a susceptible environment allowing the tumor cells to metastasize due to decrease in the host immune surveillance potential with age.

Chemoprevention of Tumor Metastasis by Liposomal .BETA.-Sitosterol Intake

To investigate chemopreventive effect of liposomal beta-sitosterol on tumor metastasis, we prepared liposomal beta-sitosterol composed of egg yolk phosphatidylcholine for oral delivery. Although orally administered beta-sitosterol (4 micromol as beta-sitosterol/mouse) was not absorbed into plasma, the amount of immune response cytokines such as IL-12 and IL-18 was increased in the small intestine after the liposome intake. Moreover, after daily oral administration of the liposome for 7 d, natural killer (NK) cell activity in the mice was increased, suggesting that the immune surveillance activity of mice was enhanced by the liposomal beta-sitosterol intake. Thus, we examined metastatic potential of B16BL6 melanoma cells, which were intravenously injected into mice after sequential administration of liposomal beta-sitosterol for 7 d. The number of metastatic colonies in the lungs was significantly less than that of control group two weeks after the injections of the cells. These results suggest that daily liposomal beta-sitosterol intake prevents tumor metastasis may be due to enhancement of gut immune surveillance systems.

Dexamethasone modifies the susceptibility to serum cytotoxicity and increases the metastatic efficiency of a colon carcinoma cell line

Metastatic inefficiency is a phenomenon by which a majority of tumor cells is lost in the blood stream during the metastatic process. We investigated the effects of dexamethasone (DEX), a synthesized glucocorticoid, on the serum susceptibility of a colon carcinoma cell line, HT-29, with respect to metastatic inefficiency. The susceptibility to serum cytotoxicity of these carcinoma cells is possibly an important factor in metastatic inefficiency. In this study, we used glucocorticoid because it modifies the function of the plasma membrane and has been shown to enhance the hematogenous metastasis of some tumor cells. Using HT-29 cells that had been treated with DEX in vitro, the following factors were evaluated: the metastasis of intrasplenic injected cells; in vitro and in vivo proliferation; motility; the production of matrix metalloproteinases (MMPs); and the expression of the membrane complement regulatory proteins CD46, CD55, and CD59. The number of viable cells in the liver after an intraportal injection of tumor cells was determined by the expression of human β-globin mRNA that is aberrantly expressed in HT-29 cells. In addition, we investigated 100% serum-induced proliferation, susceptibility, and apoptosis. Treatment with DEX was found to accelerate liver metastasis; here, the number of metastatic colonies and the weight of the liver were both significantly increased in DEX-treated HT-29 (HT-29DEX) cells. In contrast, there was no difference in terms of cell motility; the production of MMPs; or the expression of CD46, CD55, or CD59 between the HT-29 and HT-29DEX cells. The HT-29DEX cells exhibited enhanced proliferation in the serum, as well as resistance to cytotoxicity when exposed to 100% serum. In addition, DEX slightly inhibited serum-induced apoptosis. Finally, the expression of colon cancer-derived β-globin mRNA was detectable 24 h after intravenous injection, but only in samples obtained from the HT-29DEX-, but not in those from the HT-29-inoculated mice. These results indicate that DEX reduced the metastatic inefficiency of the HT-29 cells, resulting in a hematogenous metastasis-prone phenotype. It is thus expected that the acquisition of resistance against serum cytotoxicity is among the mechanisms that contribute to the efficiency of hematogeneous metastasis.

Involvement of reactive nitrogen oxides for acquisition of metastatic properties of benign tumors in a model of inflammation-based tumor progression

The cells of a weakly tumorigenic and non-metastatic murine fibrosarcoma (QR-32) are converted into highly malignant tumors (acquiring metastatic potential) once they have grown in vivo after being co-implanted with gelatin sponge which induces inflammation. In the present study, we examined whether nitric oxide (NO) is involved in the inflammation-based tumor progression by administrating a specific inhibitor to inducible nitric oxide synthase, aminoguanidine (AG). First, we co-implanted 1 x 10(5) QR-32 cells with gelatin sponge (10 x 5 x 3 mm piece) into a subcutaneous space in C57BL6 mice. Administration of AG in drinking water (1%) had started 2 days before the tumor implantation and continued until the termination of the experiment. The incidence of tumor formation and the tumor growth did not differ between AG-treated group and -untreated group. On day 28, we excised the arising tumors to establish culture cell lines for evaluation of their acquisition of metastatic phenotype in other normal mice. Metastasis incidence and the number of metastatic colonies were significantly reduced in the tumor cell lines obtained from AG-treated mice compared to those from non-treated mice (p < 0.05). Immunohistochemical analysis demonstrated that inducible nitric oxide synthase and nitrotyrosine in the inflamed lesion were reduced in the AG-administered mice. However, intensity of 8-hydroxy-2-deoxyguanosine was not different between the groups. These results showed that nitric oxide and its reactive nitrogen oxide species cooperatively play a pivotal role in the progression of benign tumor cells in inflamed lesions.

Z-100, an immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis strain Aoyama B, augments anti-tumor activities of X-ray irradiation against B16 melanoma in association with the improvement of type 1T cell responses.

In this study, the effects of combination therapy consisting of X-ray irradiation and Z-100 on the survival time of C57BL/6 mice inoculated with B16F10 melanoma were investigated. Survival time was significantly prolonged in B16F10 melanoma-bearing mice treated with the X-ray irradiation (5 Gy) and Z-100 (10 mg/kg s.c.) combination therapy compared with mice irradiated with X-rays alone. The weight of primary tumors and number of metastatic colonies were also significantly suppressed by the combination therapy compared with that in the X-ray irradiation group. These results indicated that Z-100 could enhance the anti-tumor effects of radiotherapy against B16F10 melanoma. On the other hand, the survival time of CD4 knockout mice bearing the same tumors was not prolonged by the combination therapy compared with mice irradiated with X-rays alone, suggesting that CD4+ cells are partly involved in augmentation of the anti-tumor effect of radiotherapy by Z-100. In addition, type 1 cytokine (IL-2, IFN-gamma) production was significantly increased and type 2 cytokine (IL-4, IL-10) production was significantly suppressed in the tumor-bearing mice treated with the combination therapy compared with the X-ray irradiation group. Moreover, interleukin-12 production by CD11c+ cells was also significantly increased in mice treated with the combination therapy compared with the X-ray irradiation group. These results indicate that Z-100 augmented the anti-tumor effects of X-ray irradiation. Moreover, we demonstrated that the effects of Z-100 were expressed at least in part, by the improvement of the T cell responses from type 2-dominant to type 1-dominant via up-regulation of IL-12 production.

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.

Obstructive jaundice facilitates hepatic metastasis of B16F1 mouse melanoma cells: participation of increased VCAM-1 expression in the liver.

Obstructive jaundice facilitates experimental liver metastasis in the rat model, but the detailed mechanisms of this facilitation remain unclear. This study aimed to evaluate the contribution of vascular cell adhesive molecules-1 (VCAM-1) to augmented hepatic metastasis in cases of obstructive jaundice. Obstructive jaundice was induced in male C57BL/6 mice by common bile duct obstruction for 5 days using a Surgiclip. For the biliary decompression, obstructive jaundice was induced for 5 days, followed by removal of the Surgiclip. Liver specimens and blood samples were obtained from animals 5 days after biliary obstruction (OJ5) or sham operation and 2, 5, 11, 14 days after biliary decompression. The expression of VCAM-1 mRNA was increased in the livers from the OJ5 group. Western blot analysis demonstrated increased expression of VCAM-1 protein in the livers of the OJ5 group, in contrast with low VCAM-1 expression in the sham group. The expression of VCAM-1 protein was sustained at high levels at 2 days and decreased at 5 days after biliary decompression (BD5). For the induction of experimental hepatic metastasis, male C57BL/6 mice were randomized to three groups (sham, OJ5, BD5) of six animals each. B16F1 melanoma cells were introduced into the animals by an intraportal injection. Metastatic colonies in the livers were investigated 13 days after inoculation. The mean number of metastatic colonies was significantly increased in the OJ5 group (70.5+/-51.2) compared to that of the sham group (7.2+/-7.9) (p<0.05). This augmentation of hepatic metastasis was abrogated in the BD5 group (16.8+/-20.3). In conclusion, our results suggest that augmented hepatic metastasis in cases of obstructive jaundice are partly mediated through VCAM-1/VLA-4 interaction.

Expression of the anti-metastatic effect induced by Juzen-taiho-to is based on the content of Shimotsu-to constituents.

We investigated the inhibitory effect of oral administration of Juzen-taiho-to, a Kampo Japanese herbal medicine, and its related formulations on the experimental liver and lung metastasis of tumor cells in vivo. Oral administration of Juzen-taiho-to for 7 d before tumor inoculation significantly reduced the number of liver metastatic colonies of colon 26-L5 carcinoma cells and attenuated the increase of liver weight in a dose-dependent manner ranging from 4 to 40 mg/d. Its oral administration for this same period before tumor inoculation also significantly inhibited lung metastasis of B16-BL6 melanoma cells. Juzen-taiho-to originally consisted of 8 crude drugs derived from Shimotsu-to and Shikunshi-to prescriptions together with two crude drugs (Cinnamomi Cortex and Astragali Radix). Oral administration of Shimotsu-to as well as Juzen-taiho-to for 7 d before tumor inoculation resulted in a significant reduction in the number of metastatic colonies and the liver weight as compared with the control, whereas Shikunshi-to did not exhibit such an inhibitory effect. Unsei-in containing four Shimotsu-to constituents was also active in inhibiting liver metastasis. Toki-shakuyaku-san and Ninjin-yoei-to, which include all Shimotsu-to constituents except Rehmanniae Radix and Cnidii Rhizoma, respectively, did not show a significant anti-metastatic effect. Rikkunshi-to and Ninjin-yoei-to, which contain Shikunshi-to constituents, did not affect the inhibition of liver metastasis. Hochu-ekki-to treatment before tumor inoculation also led to a significant inhibition of liver metastasis, probably through an inhibitory mechanism different from Juzen-taiho-to. These results suggest that the anti-metastatic effect of Juzen-taiho-to is partly associated with its Shimotsu-to-derived constituents.

Inhibition of pulmonary metastasis by Z-100, an immunomodulatory lipid-arabinomannan extracted from Mycobacterium tuberculosis, in mice inoculated with B16 melanoma.

The anti-metastatic effect of Z-100, an immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis, was investigated in mice bearing B16 melanoma cells. Treatment of BF10 mice implanted with high metastatic B16F10 melanoma cells with a 10 mg/kg dose of Z-100 resulted in the reduction of experimental pulmonary metastasis as compared with that of BF10 mice treated with saline. The number of pulmonary metastatic colonies in BF1 mice (mice implanted with low metastatic B16F1 melanoma cells) was greatly increased after the inoculation of CD4+ CD11b+ CD281+ TCR alphabeta+ type 2 T cells (F10-Th2 cells) derived from BF10 mice, while only a few metastatic colonies were demonstrated in lungs of BF1 mice inoculated with naive CD4+ T cells. However, the numbers of metastatic colonies in BF1 mice were not increased when they were inoculated with the F10-Th2 cell fraction derived from Z-100-treated BF10 mice and the generation of F10-Th2 cells in BF10 mice was effectively suppressed by the Z-100 treatment. These results suggest that Z-100 inhibits pulmonary metastasis of B16 melanoma through the regulation of tumor-associated Th2 cells, which are a key cell in the acceleration of tumor metastasis.

Differential effect of host microenvironment and systemic humoral factors on the implantation and the growth rate of metastatic tumor in parabiotic mice constructed between young and old mice

B16 melanoma cells were injected into the tail vein of young mice, old mice and parabiotic mice constructed between young and old mice, and the number and shape of pulmonary metastases were compared among three experimental groups. In unpaired mice, the number of metastatic colonies in the lungs was 10-fold larger in young than in old mice. In parabiotic mice, the number in young mice was almost comparable with that of unpaired young mice, but the number in old mice approached the level of young mice. Metastatic colonies on the pulmonary surface of young mice were mostly nodular in shape, while those of old mice were flat in shape. The shape of colonies reflecting the tumor growth rate did not change in parabiotic old mice in spite of colonies reflecting the tumor growth rate did not change in parabiotic old mice in spite of an increase in number. In young parabiotic mice, the large and intermediate colonies decreased with a concomitant increase of small ones as compared with unpaired young mice. These results suggest that the implantation of metastatic colonies in the lung is mainly dependent on systemic humoral factors and their growth is mainly dependent on the host local factors in the microenvironment, and distinct age changes of both factors greatly influence the metastatic mode of tumors, respectively.

Inhibition of experimental metastasis of murine fibrosarcoma cells by oligopeptide analogues to the fibronectin cell‐binding site

We have analyzed the effect of the synthetic oligopeptides Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) and Gly-Arg-Gly-Glu-Ser-Pro (GRGESP), analogues to the fibronectin cell-binding sequence, on the formation of experimental lung metastasis. SR-BALB were injected alone or in conjunction with GRGDSP or GRGESP in the tail vein of BALB/c mice. Co-injection with GRGESP reduced by approximately 70% the number of metastatic colonies per mouse. However, co-injection with the closely related peptide GRGDSP, containing the conservative substitution Glu/Asp, did not affect metastatic behavior. GRGESP peptide anti-metastatic activity was not due to a toxic effect on tumor cells or on mice. In vitro adhesion assays testing for a possible effect of the peptide on cell-matrix interactions indicated that the GRGESP peptide did not affect cell adhesion to the matrix proteins tested.