Number Of Metastatic Foci Research Articles

Platelet Glycoprotein Ibα Supports Primary Tumor Growth and Experimental Metastasis.

Successful tumor formation and metastatic spread depend on the ability of tumor cells to interact with their host microenvironment. Studies from our groups and others indicate that platelets contribute to tumor host interactions through adhesive and stimulatory functions that may promote tumor growth and dissemination. Here we show that platelet receptor GPIbα plays a critical role in the ability of platelets to support primary tumor development and hematogenous metastasis. We previously developed knockout mice lacking platelet GP Ibα (GPIbαNull, a model of the human Bernard-Soulier syndrome) and mice missing GPIbα, but expressing a fusion protein of cytoplasmic GP Ibα and the extracytoplasmic domain of the interleukin-4 receptor (Ibα/IL-4R). Expression of the Ibα/IL-4R ameliorates macrothrombocytopenia, a characteristic of the Bernard-Soulier syndrome. However, both models result in a severe hemorrhagic state owing to the absence of GPIbα. Congenic mouse strains of both GP Ibα models were generated by 10-generation backcrosses with wild-type (WT) C57BL/6J mice to study the tumorgenic and metastatic activity of syngeneic tumor cells. To analyze primary tumor growth, Lewis lung carcinoma cells (D121) were injected intradermally in the dorsal flank of the hind limb (105 cells/injection) and tumor development analyzed in its initial and advanced stages. Seventeen days after injection, tumors in GPIbαNull and Ibα/IL-4R mice were several-fold smaller than those in WT C57BL/6J mice. Histological examination of early and end-stage tumors revealed three major differences: a severe and immediate onset of tumor necrosis, a lack of new blood vessels large enough to connect to the circulation, and a significant reduction in tumor-associated macrophages. While the proliferation rate of tumor cells in non-necrotic areas of the tumors was unaffected by the lack of GPIbα, the percentage of necrosis in these tumors was 6-fold increased in GPIbαNull mice. Each necrotic area showed increased hemorrhage in the absence of GPIbα. The overall density of microvessels in early primary tumors appeared unchanged, but the number of larger vessels associated with tumors was reduced ~2-fold and the density of tumor-associated macrophages was reduced ~6-fold compared to tumors developing in WT mice. To analyze a contribution of platelet GPIbα to hematogenous tumor metastasis, another syngeneic tumor cell model was used and B16F10.1 melanoma cells were injected into the tail vein (105 cells/mouse) of GPIbαNull, Ibα/IL-4R, or WT mice, to determine the ability of tumor cells to colonize the lungs 14 days later. In the absence of functional GPIbα, the number of metastatic foci at the surface of the lungs was significantly reduced (15-fold, p = 0.0002). These results indicate that successful target organ colonization by tumor cells from the blood stream is severely affected by the lack of extracytoplasmic GPIbα in Ibα/IL-4R animals, and not necessarily by the reduced number of platelets in GPIbαNull animals. The results demonstrate a significant involvement of platelet GP Ibα in primary tumor growth by affecting tumor angiogenesis and the recruitment of tumor-associated macrophages while providing evidence GPIbα directly contributes to target organ colonization by circulating tumor cells. These studies identify platelet GP Ibα as a potential new target for anti cancer therapy.

LIPOSOMAL DELIVERY OF EPHA2 SIRNA IN COMBINATION WITH GEMCITABINE REDUCES PANCREATIC TUMOR GROWTH AND INVASION

Background: New therapeutic targets are needed for pancreatic adenocarcinoma. EphA2 has been found to be over-expressed in pancreatic cancer. EphA2 is a tyrosine kinase receptor from ephrin family, which was reported to be associated with different types of cancer. In this study, we examined the effectiveness of in vivo delivery of a liposomal preparation of siRNA against EphA2 in combination with gemcitabine on pancreatic tumor growth and invasion in mice. Methods: Mpanc-96 cells were stably transfected with the luciferase gene and 25 × 104 cells were injected orthotopically into the pancreas of nude mice. A week later, tumor burden of each mouse was measured by bioluminescence system (IVIS Xenogen). The mice were then divided into 4 groups having similar average tumor load. The groups were then assigned to treatment with vehicle, EphA2 siRNA alone (5μg/per animal/twice a week), gemcitabine alone (100mg/kg.b.wt/twice a week) or the combination of both EphA2 siRNA and gemcitabine. SiRNA was coupled with neutral liposomes as previously reported (Landen CN et. al. Cancer Res. 65:6910-8, 2005) and delivered one day prior to gemcitabine treatment. Imaging assessed tumor development weekly. After six weeks of treatment, the animals were sacrificed, tumors were excised and weighed and metastasis to liver and lung were analyzed. Tumor tissue was used for EphA2 and PCNA staining. Results: In vitro studies indicated that the EphA2 siRNA significantly silenced EphA2 expression in pancreatic cancer cell lines by ~80%. Liposomal siRNA delivery did not have any visible adverse effects on mouse well-being or body weight. Treatments with the EphA2 siRNA alone reduced the numbers of PCNA positive tumor cells (p < 0.05), the number of animals with lung metastasis (20% reduction), and the numbers of metastatic foci (liver 53%, lung 32% reduction) but did not significantly reduce tumor mass compared to controls. Gemcitabine alone reduced the number of animals with lung metastasis (25% reduction), and the numbers of metastatic foci (liver 71%, lung 62% reduction), but did not significantly reduce the numbers of PCNA positive tumor cells, or tumor mass. The combination of EphA2 siRNA and gemcitabine reduced the number of the PCNA positive tumor cells (p < 0.01), the number of metastatic foci (liver 80%, lung 85% reduction), the number of animals with metastasis (liver 25%, lung 50% reduction), and the tumor mass (p < 0.01) compared to each of the other three groups. Thus, for all parameters, the effects of the EphA2 siRNA and gemcitabine in combination were greater than either treatment alone. Conclusion: Liposomal delivery of siRNA is a safe and effective delivery system. EphA2 silencing combined with gemcitabine treatment significantly inhibits pancreatic tumor progression and invasion in an experimental model.

Inhibition of growth and metastasis of hepatocellular carcinoma by rapamycin: experiment with mice

To investigate the effects of rapamycin (RPM) in inhibiting the growth and metastasis of hepatocellular carcinoma (HCC). Human HCC cells of the line MHCC97H with a high potential of metastasis were divided into 3 groups to be cultured with cyclosporine A (CsA) 100 ng/ml, RPM 10 ng/ml, or CsA + RPM for 48 hours. Flow cytometry was used to examine the apoptosis and cell cycle MTT method was used to examine the effect of RMP on the proliferation of the MHCC97H cells. RT-PCR was used to detect the mRNA expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hypoxia-inducible factor-1alpha (HIF-1alpha), and transforming growth factor b (TGFb). Another MHCC97H cells were cultured in complete medium without RPM for 48 hours, then the protein expression of VEGF in the supernatant was detected by ELISA. Twenty-eight nude LCI-D20 mice were inoculated with human HCC cells and then divided into 4 groups to be fed with CsA (25 mg/kg), RPM (2 mg/kg), CsA + RPM, and normal saline (0.2 ml, as control group) for 35 days. Then the mice were killed to take the weight of inoculated tumor, measure the blood drug concentration, calculate the lung metastasis rate and number of metastatic foci, and observe pathology of the lung. CsA showed no effect on the cycle of the MHCC97H cells. The MHCC97H cells of the RPM and CsA + RPM groups arrested at the stage G(0)/G(1) (both P = 0.000). MMT method also showed that the proliferation of the MHCC97H cells in the RPM and CsA + RPM groups were blocked (P = 0.003 and P = 0.002). However, CsA did not influence the proliferation of the MHCC97H cells. Flow cytometry showed that RPM did not promote the apoptosis of the MHCC97H cells. RT-PCR showed that RPM down-regulated the mRNA expression of VEGF and HIF-1alpha (both P < 0.05), however, did not influence the mRNA expression of bFGF, TGFb, and TGFb. The VEGF protein level in the supernatant of the culture fluid of MHCC97H cells of the RPM group was (890.3 +/- 25.1) pg/ml, significantly lower than that of the control group, (1583.7 +/- 17.3) pg/ml (P = 0.000). The tumor inhibiting rate of the RPM group was 63.7%, not significantly different from that of the RPM + CsA group (80.9%, P = 1.000). The metastatic rate of the CsA and control groups were both 100% with a higher number of metastatic tumors in the CsA group (P = 0.046). RPM significantly inhibits the growth and metastasis of HCC. RPM-based immunosuppressive regimen may be of value in HCC patients receiving liver transplantation.

Effect of interleukin-18 on metastasis of mouse osteosarcoma cells.

The effect of interleukin-18 (IL-18) on metastasis of highly metastatic LM8 mouse osteosarcoma cells was investigated using nude mice treated with anti-asialo GM1 serum to exclude anti-tumor actions of IL-18 through activation of T and natural killer cells. Injection of LM8 cells which do not express IL-18 receptor beta into a tail vain resulted in the formation of pulmonary and hepatic metastatic foci. Daily injection of mice with IL-18 starting the fifth day from the cell injection had no significant effect on the number of metastatic foci, while five daily injections of IL-18 before and after the cell injection resulted in marked decreases. Culture of LM8 cells with IL-18 for 5 days before the injection into mice produced no significant effect on the number of pulmonary and hepatic metastatic foci. In contrast, pretreatment of mice with IL-18 for 5 days before the cell injection markedly decreased metastatic foci. The retention of LM8 cells in the lung 24 h after their injection was also reduced by the pretreatment of mice with IL-18. Serum obtained from mice pretreated with IL-18 for 5 days suppressed mobility of LM8 cells but IL-18 itself did not. These results suggest that IL-18 inhibits metastasis of LM8 cells partly by inducing a factor(s) in the host which suppresses cell mobility.

Duffy antigen receptor for chemokines attenuates breast cancer growth and metastasis:an experiment with nude mice

To study the effects of Duffy antigen receptor for chemokines (DARC) on the tumorigenesis and metastasis of breast cancer. Human breast cancer cells of the line MDA-MB-435HM with a great potentiality of metastasis were cultured and then divided into 3 groups: MDA-MB-435HM-DARC cell group, to be transfected with pcDNA3/DARC FyB containing human DARC cDNA; MDA-MB-435HM-vect cell group, to be transfected with blank plasmid pcDNA3; and MDA-MB-435HM cell group without transfection. Female BALB/c nude mice were implanted with MDA-MB-435HM cells into the nipple. The size of the implanted cancer was measured once a week. The mice were killed 4, 6, and 8 weeks after the implantation respectively and their lungs were taken out to observe the number of metastatic tumor. Immunohistochemical staining was performed to calculate the microvascular density (MVD). Western blotting was used to detect the matrix metalloproteinase (MMP)-9 protein expression of the lung tissues. ELISA was used to detect the concentrations of the 2 DARC ligands: CNCL8 and CCL2 in the supernatant of culture fluid of the 3 kinds of cells and the metastatic tumors. The expression levels of DARC mRNA and protein of the MDA-MB-435HM cells were 38% those of the MDA-MB-435 cells. The expression levels of CXCL8 and CCL2 of the MDA-MB-435HM-DARC cells were significantly lower than those of the MDA-MB-435HM-vect cells and MDA-MB-435HM cells (both P < 0.05). All mice developed tumor with a tumorigenesis rate of 100%. However, the tumor occurred 17 days after implantation in those mice implanted with MDA-MB-435HM-DARC cells, significantly later then in those implanted with MDA-MB-435HM-vect cells and MDA-MB-435HM cells (9 and 10 days after implantation) the size of tumor in the former group being significantly smaller than those of the 2 latter groups (both P < 0.01). The number of metastatic foci in the lung of the MDA-MB-435HM-DARC group was significantly less than those of the other 2 groups (both P < 0.01). The level of CCI2 of the implanted tumor in the MDA-MB-435HM-DARC group was significantly lower than those of the other 2 groups (both P < 0.01). The MVD of the MDA-MB-435HM-DARC was significantly less than those of the other 2 groups (both P < 0.01) and most of the vessels of the MDA-MB-435HM-DARC group were not newly formed vessels with a diameter < 8 red blood cells. The MMP-9 protein expression levels of the MDA-MB-435HM-vect cells and MDA-MB-435HM-cells were 3.1 and 3.4 times that of the MDA-MB-435HM-DARC group (both P < 0.05). DARC, a chemokine decoy receptor, inhibits the chemokine-induced angiogenesis, thus inhibiting the growth and lung metastasis of breast cancer.

Reduction of liver metastasis of intraocular melanoma by interferon-beta gene transfer.

This study determined whether adenovirus-mediated transfer of the murine interferon-beta (AdCMVIFN-beta) gene protects against liver metastases arising from intraocular melanomas in mice. A replication-deficient adenovirus vector (AdCMVIFN-beta) was used for the in vivo transfer of the murine IFN-beta gene into intraocular melanoma-bearing mice. AdCMVIFN-beta was injected either intravenously or directly into the intraocular melanomas. The effect of gene transfer on liver metastases was ascertained by histopathologic analysis of the livers and by measuring serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), which are two enzymes associated with liver metastases in patients with uveal melanoma. Mice treated with two intratumoral injections of AdCMVIFN-beta had a 68% reduction in metastatic liver lesions (P = 0.016) and a 51% reduction in liver enzyme levels compared with control mice (P = 0.02). However, the antimetastatic effect of AdCMVIFN-beta was not directly attributable to the adenovirus vector or virus-mediated cytolysis of tumor cells. Intravenous treatment with AdCMVIFN-beta resulted in an 86% reduction in the number of metastatic foci in the liver (P = 0.014) and a 61% reduction of serum AST levels compared with mice treated with AdCMVLacZ (P = 0.015). AdCMVIFN-beta treatment produced a sharp increase in the NK cell activity that was demonstrable in vivo and in vivo. In vivo depletion of NK cells by anti-asialo GM1 antibody abrogated the antimetastatic effects of AdCMVIFN-beta. The results support the feasibility of activation of NK cell function through gene transfer as one possible therapeutic strategy for reducing hepatic metastases of uveal melanomas.

Differential response of primary and metastatic melanomas to neutrophils attracted by IL-8.

IL-8 is a strong chemoattractant for neutrophils, and it is constitutively produced by many tumors, including human melanomas. To determine the biologic importance of IL-8 for melanoma cells from primary and metastatic lesions, we transduced selected cell lines constitutively producing low levels of IL-8 with IL-8 cDNA using a replication-deficient adenoviral vector. Nontumorigenic SBcl2 primary melanoma cells formed tumors when transduced with increasing plaque-forming units of IL-8 per cell. However, at high IL-8 transduction levels (100 ng/ml/10(5) cells in 48 hr), tumor growth was impaired due to massive neutrophil infiltration. A similar biphasic response was observed in WM115 primary melanomas, which are tumorigenic but not metastatic. Depletion of neutrophils with an antibody that blocks the accumulation of granulocytes at the site of inflammation enabled transduced primary melanomas secreting high levels of IL-8 to survive and grow. In contrast, highly tumorigenic and metastatic 451Lu cells showed marked increases in tumor growth and number of metastatic foci in the lungs depending on the expression levels of IL-8. Cytotoxicity assays with isolated neutrophils confirmed the preferential killing of primary over metastatic melanoma cells. SBcl2 cells stimulated by IL-8 to form tumors in immunodeficient mice were induced to produce VEGF, suggesting that the angiogenic response is enhanced due to increased growth factor production. Our results demonstrate that nontumorigenic primary melanomas depend on IL-8 stimulation in vivo for growth and that tumor growth depends on the level of neutrophil infiltration. Metastatic melanomas proliferate in vivo independently of infiltrating neutrophils.

Inhibition of lung metastasis of osteosarcoma cell line POS-1 transplanted into mice by thigh ligation

Using a model with external ligation of the thigh, the effect of ischemia-reperfusion injury on tumor growth and the activity of lung metastasis was investigated in mice inoculated a spontaneous murine osteosarcoma cell line (POS-1) in vivo. POS-1 cell suspension was inoculated into the right hind footpad of 70 mice. Four weeks after inoculation, the ipsilateral thigh was ligated for 3 h in 15 mice and the contralateral thigh in 15 mice. Another ten mice were inoculated with POS-1 without ligating the thigh. The number of metastatic foci on the lung surface 6 weeks after inoculation was 2.29±0.98 (mean±SE) foci/lungs in mice with ipsilateral ligation and 6.25±2.41 in mice with contralateral ligation, which were significantly lower than control (13.40±1.42 in mice no ligation) ( P<0.01). The number of metastatic foci on the lung surface in mice with intraperitoneal injection of superoxide dismutase (SOD) and catalase was 3.25±0.65 (mean±SE) foci/lungs in mice with ligation which was significantly greater than that in mice without SOD and catalase injection 1.29±0.97 ( P=0.04). Cell viability was 9.12±4.07% with 100 μM H 2O 2 in 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. It revealed that at concentrations of 100 μM H 2O 2 or higher was cytotoxic to POS-1. In cell invasion assay, the number of invading cells with 10 μM H 2O 2 was 2.80±0.53 cells/field, which was significantly lower than control (5.93±0.18) (mean±SE), indicating that low-dose H 2O 2 suppressed invasion of POS-1. These results suggested that reperfusion injury had selective cytotoxicity to POS-1 through producing reactive oxygen species. Activated oxygen was considered to inhibit the regional growth and the ability of lung metastasis of POS-1 cells.

Transfection of macrophage inflammatory protein 1 alpha into B16 F10 melanoma cells inhibits growth of pulmonary metastases but not subcutaneous tumors.

Macrophage inflammatory protein 1 alpha (MIP-1 alpha), a CC chemokine, is a chemoattractant for T cells and immature dendritic cells. Plasmacytoma cells expressing MIP-1 alpha generate a cytotoxic T cell response without affecting tumor growth. To understand this discrepancy, we compared a local tumor model with a metastatic one using MIP-1 alpha-transfected B16 F10 melanoma cells. Clonal idiosyncrasies were controlled by selecting three lipotransfected tumor clones and two pcDNA vector transfected control clones with equivalent in vitro proliferative capacities. No significant differences were seen between the MIP-1 alpha-producing and control melanoma cells after s.c. injection in the hind leg. All animals had a leg diameter of 10 cm in 18.5-21.5 days. However, after i.v. injection the number of pulmonary foci was significantly reduced in the MIP-1 alpha-producing clones. Injection of 10(6) control transfected cells resulted in a median of 98.5 tumor foci in 2 wk, whereas the injection of the MIP-1 alpha-producing clones resulted in 89.5, 26.5, and 0 foci. The number of metastatic foci was inversely proportional to the amount of MIP-1 alpha produced by the clone in vitro. Flow cytometry showed a significant increase in CD8(+) cells in lungs of mice with MIP-1 alpha-transfected tumors 3 days after injection. This increase was not maintained 10 days later despite continued production of MIP-1 alpha. The protection offered by transfection with MIP-1 alpha was significantly impaired in beta(2)-microglobulin(-/-) mice. Our findings suggest that MIP-1 alpha is effective in preventing the initiation of metastasis, but not at sustaining an effective antitumor response.

Results of treatment for metastatic osteosarcoma with neoadjuvant chemotherapy and surgery.

The purpose of the current study was to define the survival outcome variables for the 85 patients with Stage IIB osteosarcoma treated with neoadjuvant chemotherapy at the authors' institution from 1982 to 1997. A minimum 4-year followup or death was a requisite for inclusion. Forty-three patients were relapse-free survivors and 14 had no evidence of disease at followup for an overall survival of 67%. Twenty-nine patients had thoracotomy and nine have no evidence of disease with a minimum 4-year followup from last thoracotomy. The mean time to metastasis after diagnosis for patients presenting with Stage IIB disease was 12.8 months. There was no difference in the survival for any of the three chemotherapy protocols, used during the 15 years included in this analysis. There was a significant relation between length of time to relapse and survival. For each additional year without relapse, there is an 18% increase in chance of survival. In patients who were treated with thoracotomy, the number of metastatic nodules was a significant predictor of survival; specifically, each nodule increased the risk of death by 43%. A favorable outcome in this cohort of patients is related to the length of time between initiation of therapy and diagnosis of metastasis, and the number of metastatic foci.

Acceleration of lung metastasis by up-regulation of CD44 expression in osteosarcoma-derived cell transplanted mice

The effect of CD44-phenotypic expression on metastasis to the lung was studied using a spontaneous murine osteosarcoma-derived cell line, POS-1, stimulated with lipopolysaccharide (LPS). POS-1 cells were inoculated into the hind paws of 20 C3H/HeJ mice and produced a visible mass in all mice in 5 weeks, and these transplanted tumors resulted in lung metastasis in all mice. The number of metastatic foci in the lungs was 12.0±2.1 (mean±SD) with LPS-stimulated cells, which was significantly higher than that of unstimulated cells (5.8±1.4; N=10 for each; P<0.05). Hyaluronate (HA), a ligand of CD44, inhibited a number of lung metastases in a dose-dependent manner (0.5% HA, 3.0±1.1; 0.005% HA, 5.1±1.5; without HA, 8.6±1.7; N=10 for each; P<0.05, each group with HA versus the group without HA). Adhesion assay by coculturing POS-1 cells and lung microvascular endothelial cells on culture plate showed that the adhesion was significantly lower in HA treated POS-1 than those without HA (1.18±0.12 and 2.74±0.17, respectively, P<0.05). These results suggest that lung metastasis was accelerated by up-regulation of CD44.

Blocked by a KiSS

Cancers can spread to other tissues through a process called metastasis. Ohtaki et al. isolated and characterized a ligand (metastin) from placenta for the orphan G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) hOT7T175. Metastin is believed to be the product of the KiSS-1 , a known metastasis suppressor. Application of metastin or metastin protein fragments promoted an elevation in intracellular Ca 2+ concentration in cells transfected to express the orphan GPCR. Application of metastin to hOT7T175-transfected Chinese hamster ovary or B16-BL6 melanoma cells inhibited their migration and chemotaxis toward fetal calf serum; and in the melanoma cells, metastin induced the formation of stress fibers and phosphorylation of focal adhesion kinase and paxillin. Treatment of animals induced to form metastatic tumors through the injection of the transfected B16-BL6 cells showed that metastin decreased the number of metastatic foci. However, metastin did not affect growth of the primary tumor. These results are intriguing and may point to a new direction for inhibiting tumor metastasis, as well as contribute to the identification of a new GPCR and ligand that regulate cell adhesion. T. Ohtaki, Y. Shintani, S. Honda, H. Matsumoto, A. Hori, K. Kanehashi, Y. Terao, S. Kumano, Y. Takatsu, Y. Masuda, Y. Ishibashi, T. Watanabe, M. Asada, T. Yamada, M. Suenaga, C. Kitada, S. Usuki, T. Kurokawa, H. Onda, O. Nishimura, M. Fujino, Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 411 , 613-617 (2001). [Online Journal]

Contribution of angiogenesis to the progression of colon cancer: possible inhibitory effect of angiogenesis inhibitor TNP-470 on tumor growth and hepatic metastasis.

The contribution of angiogenesis to tumor growth and hepatic metastasis of colorectal cancer was investigated by means of immunohistochemical study and in vitro and in vivo experiments. Colorectal cancer specimens from 30 patients with hepatic metastasis and 39 patients without hepatic metastasis were studied by staining with antibodies against factor VIII-related antigen. Microvessel count in patients with liver metastasis was significantly higher than in those without liver metastasis (p<0.005). The effect of TNP-470 was evaluated with in vitro and in vivo experiments using human colon cancer cell line, LM and the highly hepatic metastasis cell line, LM-H5. The effect of TNP-470 on the proliferation of the cancer cells and human umbilical vein endothelial cells (HUVECs) was examined. TNP-470 inhibited more sensitively the proliferation of HUVECs than cancer cells in vitro. IC50 was approximately 3 pg/ml in HUVECs and approximately 2 microg/ml in cancer cells. The effect of TNP-470 on the growth of xenografts and liver metastases by LM-H5 in nude mice was examined. TNP-470 (30 mg/kg) was administered by subcutaneous injection every third day for 4 weeks. TNP-470 inhibited both the growth of xenograft and the hepatic metastasis. The number of metastatic foci in the liver was 78.2+/-30.1 in the control group and 20.6+/-16.5 in the treated group. These results suggest that TNP-470 is a potent agent to inhibit tumor growth and hepatic metastasis of colon cancer.

Effects of Linomide on growth and metastasis of implanted human gastric cancer in nude mice.

To elucidate the effect of angiogenesis inhibitor, Linomide, on tumor growth and metastasis in nude mice implanted with human gastric cancer. A metastatic model of gastric cancer was established using orthotopic implantation of histologically intact tumor tissues into the gastric wall of nude mice. Linomide (0, 80, 160 mg·kg(-1)) was given p.o. every day after the implantation, and the mice were sacrificed after 10 wk to detect tumor size and metastasis. The microvessel counts were measured by immunohistochemical staining using a monoclonal antibody against Human Factor VIII related antigen. Linomide treatment significantly decreased the size of the implanted tumors (control group: 1.36 ± 0.81 cm(3) vs Linomide treated group: 0.84 ± 0.51 cm(3) and 0.62 ± 0.35 cm(3), P < 0.05 and 0.01, respectively). Additionally, an antimetastatic effect of Linomide was clearly demonstrated in a dose dependent manner: mice given 80 mg·kg(-1) Linomide developed liver metastasis in 4 of 10 cases, mice given 160 mg/kg developed metastasis in only 1 of 10 mice, while it developed in 19 of 28 mice of the control group (P < 0.05 and 0.01, respectively). The number of metastatic foci was also significantly less in the treated group. Furthermore, the microvessel counts in tumors of treated mice was reduced by 33%-42% as compared with the control tumors (P < 0.01). Linomide has a strong inhibitory activity against in vivo tumor growth and metastasis of gastric cancer, effectively suppressing the growth of the primary tumor, preventing liver metastasis, and attenuating the rate of neovascularization.

Effect of medium-chain triglyceride emulsion in total parenteral nutrition on experimental hepatic metastasis in the rat.

This study evaluated the effect of total parenteral nutrition (TPN) regimens containing a medium-chain triglyceride (MCT) emulsion on tumor metastasis. Tumor metastasis development was assessed by the number of metastatic foci on the liver surface in rats inoculated with ACL-15 tumor cells via the portal vein. Rats received one of the following TPN regimens: TPN containing an MCT emulsion (group M), in which tricaprylin emulsion served as the MCT and comprised 50% of nonprotein calories (NPC); TPN containing a long-chain triglyceride (LCT) emulsion (group L), in which soybean oil served as the LCT and comprised 50% of NPC; and TPN without lipid emulsion (group G), in which dextrose comprised 100% of NPC. The number of metastatic foci was greatest in rats receiving TPN containing the MCT emulsion on day 11 after tumor cell inoculation and either 11 days of TPN or 2 days of TPN followed by 9 days standard rat chow. TPN containing MCT emulsion increases liver metastasis early in its administration.

Inhibitory Effect of the Angiogenesis Inhibitor O-(Chloroacetyl-carbamoyl)fumagillol(TNP-470) on Tumor Growth and Metastasis of Murine Mammary Tumor Cell Line (JYG-B)Inoculated in Female Nude Mice.

The effect of the potent angiogenesis inhibitor O-(chloroacetyl-carbamoyl) fumagillol(TNP-470), a semisynthetic analogue of fumagillin on tumor growth and metastasis was studied using murine mammary tumor cells (JYG-B) inoculated into female nude mice. Injection of TNP-470 at 10 and 30 mg/kg doses, 3 times a week, until the end of the experiment (41 days)inhibited the s.c.inoculated primary tumor growth in a dose-dependent manner (45%and 65% in volume, respectively). TNP-470 at 30 and 60 mg/kg doses, 3 times a week, until the end of the experiment (35 days), reduced the weight of the i.p. inoculated total tumor mass of abdominal dissemination in a dose-dependent manner (54% and 76%, respectively). Pulmonary metastasis was found in all mice but TNP-470 significantly reduced the lung weight reflecting the total volume of the metastatic foci, and the numbers of metastatic foci in the liver and kidneys was reduced by TNP-470 treatment. These findings show that the angiogenesis inhibitor (TNP-470)has a strong inhibitory effect on tumor grwoth and metastasis in vivo.

Inhibition of Development of Murine Melanoma Lung Metastases by Systemic Administration of Recombinant Platelet Factor 4

When administered locally, recombinant platelet factor 4 (rPF4), a known angiogenesis inhibitor, has been shown to effectively suppress murine melanoma and human colon carcinoma primary tumor growth in mice. It was tentatively concluded that this effect was due to the inhibition of tumor neovascularization. This study has evaluated the effects of systemically administered rPF4 on the growth and establishment of experimental B16F10 melanoma lung metastases in syngeneic mice. B16F10 cells (0.5-1.0 x 10(5) were administered intravenously to mice; 21 days later, the lungs were removed and the tumor foci were counted. Treatments with rPF4 were given to C57BL/6J mice immediately following tumor inoculation either (a) intravenously as a single dose (0.375, 0.75, 1.5, or 2.0 mg) or as multiple doses (6 mg total) over a 48-hour period, (b) subcutaneously or intramuscularly as multiple doses (6 mg total) over a 72-hour period, or (c) subcutaneously as multiple doses (6 mg total) over a 92- or 96-hour period following a delay of 4, 24, or 48 hours. (BALB/cByJ x C57BL/6)F1 (CByB6F1/J) athymic nude mice received rPF4 subcutaneously over a 72-hour period. The ability of rPF4 to block binding of 51Cr-labeled B16F10 tumor cells to matrix-coated microtiter plates was evaluated in vitro. The in vivo effect of intravenously injected rPF4 on the retention of 51Cr-labeled B16F10 cells was examined by determining the remaining lung-associated radioactivity after 30 minutes or 1, 2, or 4 hours. Intravenous administration of rPF4 significantly inhibited the development of metastatic lung nodules in a dose-dependent fashion as assessed by both lesion number (P < .03) and lung weight (P < .05). When initiation of subcutaneous treatment with rPF4 was delayed 24-48 hours, the number of metastatic foci was significantly reduced (P < .05). The antitumor effect of rPF4 was not dependent on a T-lymphocyte-mediated process, since subcutaneous rPF4 treatment also suppressed the number of lung metastases in T-cell-deficient athymic CByB6F1/J nude mice. In vitro experiments demonstrated modest inhibitory effects (28%) of rPF4 on B16F10 tumor cell binding to purified murine vitronectin. However, lung clearance experiments at early time points (0.5 hour and 1 hour) showed that tumor lodging was not affected by rPF4 treatment. The administration of rPF4 by systemic routes inhibited the development of experimental lung metastases. These findings are consistent with the known angiostatic properties of rPF4, and we conclude that inhibition of metastatic tumor formation may be due to inhibition of tumor-induced neovascularization. These results support the testing of rPF4 as an angiostatic agent in the treatment of metastatic tumors.

Evaluation of prognostic factors in patients with stage 1 renal cell carcinoma

During the past thirty-five years, we treated 221 patients with stage 1 renal cell carcinoma. We classified these patients into two groups: 175 patients who did not show recurrence and 45 patients who showed recurrence (excluding one patient who died of other disease). We investigated factors which affect the recurrence after nephrectomy and the survival after recurrence in these two groups. We found two major factors such as tumor diameter and tumor grade which affected the recurrence of renal cell carcinoma. Of these two factors, the grade of malignancy affected the prognosis of the patients with stage 1. Furthermore, as a result of long-term observation, we found that patients with grade I showed good prognosis compared with these with grade II, III and IV. The patients who showed weight loss constituted the majority of recurrent patients. Anaemia, erythrocyte sedimentation rate and positive reaction of C-reactive protein in addition to weight loss were major factors affecting the prognosis of stage 1 patients. In an effort to analysing the features of recurrent patients, there observed that no significant difference of the non-recurrent rate was detected between the patients who showed cancer death after nephrectomy and the patients who were living after nephrectomy. In analysing metastatic lesions and numbers of metastatic foci, we detected relatively long-term survival especially in patients with less than 3 foci of metastases in the lung, compared to those with multiple foci. Furthermore, we found dimension of the metastasis depended on the grade of the primary lesion.

Growth characteristics of recent sputum isolates of Mycobacterium tuberculosis in guinea pigs infected by the respiratory route.

The consideration of virulence must distinguish between infectivity and the ability to cause progressive disease once the infection is established. Several investigators have reported the presence of naturally occurring isolates which differ in virulence for guinea pigs. Isolates from south India which differed with respect to gross disease and number of bacilli recovered from spleen after an intramuscular infection also differed in their efficiencies to initiate an infection, once inhaled and retained. Also, this difference was correlated with differences in the rate of multiplication at the site of implantation and rate of multiplication at sites of hematogenous seeding, as well as the extent of hematogenous seeding. The number of metastatic foci was identified as a quantitative measure of hematogenous seeding, which was not confounded by the rate of multiplication of bacilli. Even allowing for the fourfold-reduced efficiency of low-virulence tubercle bacilli to produce a lesion, this measure clearly revealed a significantly reduced ability of the low-virulence tubercle bacilli to disseminate via the bloodstream.

Immunotargeting of daunomycin to localized and metastatic human colon adenocarcinoma in athymic mice.

A monoclonal antibody (designated SF25), which recognizes a protein antigen expressed on a large number of human colon carcinomas, was used for drug targeting. Daunomycin-antibody conjugates were prepared by two previously described procedures. In one, the drug was bound to the antibody through a spacer of small molecular mass (cis-aconitic acid), while in the other a dextran bridge served as the link between drug and antibody. High substitution rates of drug to antibody were obtained using the latter binding procedure. Both conjugates were tested in vitro against two human colon carcinoma cell lines, LS180 and KM-12. The efficacy of a daunomycin-dextran-SF25 antibody conjugate was tested against colon carcinoma LS180 tumors transplanted at different sites into athymic mice. The specific conjugate was significantly more inhibitory to a subcutaneous tumor growth than its components or their mixture. SF25 antibody alone showed antitumoral effects against all three forms of transplanted tumor tested, namely, local, metastatic or intrahepatic, whereas daunomycin, on its own, was effective only against the subcutaneous tumor. Binding of daunomycin to dextran partially improved its inhibitory activity against the metastatic tumor. The conjugate, daunomycin-dextran-SF25 antibody reduced the number of metastatic foci, increased the survival rate and delayed death. Yet against lymph node metastases it was not significantly better than a mixture of both constituents. However, results obtained with an intrahepatic tumor, a model that mimics the natural progression of the disease, resembled those described with the subcutaneous tumor. Daunomycin-dextran-SF25 antibody was significantly more effective than all components separately and than a mixture of drug and antibody, provided a highly drug-substituted conjugate was used.