Self-Doping Surface Oxygen Vacancy-Induced Lattice Strains for Enhancing Visible Light-Driven Photocatalytic H2 Evolution over Black TiO2.

The synergistic effect of surface oxygen vacancy with induced lattice strains on visible light-driven photocatalytic H2 evolution over black TiO2 was investigated. Experimental measurements and theoretical calculations on the lattice parameters of black TiO2 show that surface oxygen vacancies induce internal lattice strain during two-step aluminothermic reduction, which regulates the band structure and optimizes the photoinduced charge behavior of black TiO2. The hydrogen evolution rate of black TiO2 with strain modification shows a 12-fold increase to 1.882 mmol/g· h (equal to 4.705 µmol/cm2·h) under visible light illumination. The metastable state caused by the surface oxygen vacancies leads to the formation of a high-energy surface, which enhances visible light absorption and improves the photoinduced charge separation efficiency. Furthermore, the internal lattice strain provides the driving force and channel for the directional movement of photoinduced electrons from the bulk to the high-energy surface for photocatalytic H2 evolution. This strategy provides a new method for designing a high-performance photocatalyst for H2 production.

A meta-analysis of the efficacy of ureteroscopic lithotripsy and extracorporeal shock wave lithotripsy on ureteral calculi.

PURPOSE: To re-evaluated the clinic efficacy of ureteroscopic lithotripsy (URS) and extracorporeal shock wave lithotripsy (ESWL) on ureteral calculi with Cochrane systematic reviews in this paper. METHODS: We searched clinical randomized controlled trials and prospective controlled trials in databases such as Cochrane library, Medline, Springer, Elsevier Science Direct, PubMed. Pooled estimate of risk ratios (RRs), standard mean difference (SMD) with 95% confidence intervals (CIs) were used as measure of effect sizes. Summary effect estimates were also stratified by sample size, study design and study region. The overall effect sizes were derived using a random-effects model or fixed-effects model when appreciated, and meta-analysis were conducted with software RewMan 5.0. RESULTS: The meta-analysis suggested that there were significant differences of post-treatment stone free rate, repeat treatment rate, patients' satisfaction, incidence of postoperative complications, operation time and hospital stays between ESWL treatment cases and URS treatment cases. But in the sample sizes analysis, there were no significant differences of the post-treatment stone free rate and repeat treatment rate when the sample sizes were less than 100. CONCLUSIONS: Compared to the ureteroscopic lithotripsy treatment, extracorporeal shock wave lithotripsy treatment provided a significantly lower post-treatment stone free rate, but it also obviously brought out less postoperative complications, shorter operation time and hospital stays.

A meta-analysis of the efficacy of ureteroscopic lithotripsy and extracorporeal shock wave lithotripsy on ureteral calculi

PURPOSE: To re-evaluated the clinic efficacy of ureteroscopic lithotripsy (URS) and extracorporeal shock wave lithotripsy (ESWL) on ureteral calculi with Cochrane systematic reviews in this paper. METHODS: We searched clinical randomized controlled trials and prospective controlled trials in databases such as Cochrane library, Medline, Springer, Elsevier Science Direct, PubMed. Pooled estimate of risk ratios (RRs), standard mean difference (SMD) with 95% confidence intervals (CIs) were used as measure of effect sizes. Summary effect estimates were also stratified by sample size, study design and study region. The overall effect sizes were derived using a random-effects model or fixed-effects model when appreciated, and meta-analysis were conducted with software RewMan 5.0. RESULTS: The meta-analysis suggested that there were significant differences of post-treatment stone free rate, repeat treatment rate, patients' satisfaction, incidence of postoperative complications, operation time and hospital stays between ESWL treatment cases and URS treatment cases. But in the sample sizes analysis, there were no significant differences of the post-treatment stone free rate and repeat treatment rate when the sample sizes were less than 100. CONCLUSIONS: Compared to the ureteroscopic lithotripsy treatment, extracorporeal shock wave lithotripsy treatment provided a significantly lower post-treatment stone free rate, but it also obviously brought out less postoperative complications, shorter operation time and hospital stays. .

VEGF165 is necessary to the metastatic potential of Fas(-) osteosarcoma cells but will not rescue the Fas(+) cells.

Our previous studies showed that Fas expression correlates inversely with the metastatic potential of osteosarcoma (OS) cells and that the manipulation of Fas expression alters the lung metastatic phenotype. However, the role of VEGF in the growth and metastases of OS is unclear. The purpose of this study was to determine whether altering VEGF expression affects lung metastatic potential. LM7 metastatic OS cells were transfected with a small interfering RNA targeting human VEGF165 (LM7/siVEGF165) or a pcDNA4 plasmid expressing human VEGF165 (LM7/VEGF). We confirmed that VEGF165 expression was decreased in LM7/siVEGF165 cells and was increased in LM7/VEGF clones compared with control transfected clones. Fas expression was not altered in these transfected clones. We also transfected LM7 cells with Fas (LM7/Fas) or Fas together with VEGF165 (LM7/Fas/VEGF) to determine whether the overexpression of VEGF165 could enhance the metastatic potential of LM7 OS cells with high Fas expression (Fas(+)). LM7/siVEGF165 and LM7/Fas cells showed decreased lung metastatic potential. In addition, the overexpression of VEGF had no effect on the ability of LM7/Fas cells to form lung metastases. We therefore concluded that VEGF165 is critical to lung metastatic potential but is not sufficient to allow Fas(+) OS cells to survive in the Fas ligand lung microenvironment.

Targeting Lyn inhibits tumor growth and metastasis in Ewing's sarcoma.

Src family tyrosine kinases (SFK) play an important role in growth and metastasis of many types of human malignancies. However, their significance in Ewing's sarcoma remains to be elucidated. The purpose of this study was to evaluate the role of Lyn, one member of the SFK, in Ewing's sarcoma growth and metastasis and to determine whether a SFK inhibitor can induce Ewing's tumor regression. Lyn was expressed and activated in TC71, A4573, and SK-ES human Ewing's sarcoma cells. Lyn expression was seen in 13 of 15 patient tumor samples, 6 of which showed Lyn activation. Specific inhibition of Lyn using small interfering RNA significantly decreased primary tumor growth and lytic activity, and also reduced lung metastases in vivo. Down-regulation of Lyn resulted in decreased invasive capacity of tumor cells in vitro. AP23994, a small-molecule SFK inhibitor, decreased Lyn kinase activity and suppressed TC71 cell growth in vitro in a dose-dependent manner. Furthermore, treatment of mice bearing s.c. TC71 tumors with AP23994 or with polyethylenimine/Lyn-small interfering RNA gene therapy resulted in reduced Lyn kinase activity and significant tumor growth suppression. EWS/FLI-1, which is translocation fusion protein associated with Ewing's sarcoma, regulated Lyn gene expression and kinase activity. These data suggest that targeting Lyn may be a new therapeutic approach in treatment of Ewing's sarcoma.

Bone marrow subsets differentiate into endothelial cells and pericytes contributing to Ewing's tumor vessels.

Hematopoietic progenitor cells arising from bone marrow (BM) are known to contribute to the formation and expansion of tumor vasculature. However, whether different subsets of these cells have different roles in this process is unclear. To investigate the roles of BM-derived progenitor cell subpopulations in the formation of tumor vasculature in a Ewing's sarcoma model, we used a functional assay based on endothelial cell and pericyte differentiation in vivo. Fluorescence-activated cell sorting of human cord blood/BM or mouse BM from green fluorescent protein transgenic mice was used to isolate human CD34+/CD38(-), CD34+/CD45+, and CD34(-)/CD45+ cells and mouse Sca1+/Gr1+, Sca1(-)/Gr1+, VEGFR1+, and VEGFR2+ cells. Each of these progenitor subpopulations was separately injected intravenously into nude mice bearing Ewing's sarcoma tumors. Tumors were resected 1 week later and analyzed using immunohistochemistry and confocal microscopy for the presence of migrated progenitor cells expressing endothelial, pericyte, or inflammatory cell surface markers. We showed two distinct patterns of stem cell infiltration. Human CD34+/CD45+ and CD34+/CD38(-) and murine VEGFR2+ and Sca1+/Gr1+ cells migrated to Ewing's tumors, colocalized with the tumor vascular network, and differentiated into cells expressing either endothelial markers (mouse CD31 or human vascular endothelial cadherin) or the pericyte markers desmin and alpha-smooth muscle actin. By contrast, human CD34(-)/CD45+ and mouse Sca1(-)/Gr1+ cells migrated predominantly to sites outside of the tumor vasculature and differentiated into monocytes/macrophages expressing F4/80 or CD14. Our data indicate that only specific BM stem/progenitor subpopulations participate in Ewing's sarcoma tumor vasculogenesis.

Stromal cell-derived factor-1 stimulates vasculogenesis and enhances Ewing's sarcoma tumor growth in the absence of vascular endothelial growth factor.

Stromal cell-derived Factor-1alpha (SDF-1alpha) stimulates the migration of bone marrow (BM) cells, similar to vascular endothelial growth factor (VEGF). We previously demonstrated that inhibition of VEGF(165) by small interfering RNA inhibited Ewing's sarcoma tumor growth, tumor vessel formation and recruitment of BM cells to the tumor. To determine the importance of BM cells in tumor vessel development, we investigated the effects of SDF-1alpha on VEGF-inhibited TC/siVEGF(7-1) Ewing's tumor neovasculature formation and growth. The effect of SDF-1alpha on CD34(+) progenitor cell chemotaxis was determined in vivo. Using a BM transplantation model with GFP(+) transgenic mice as BM donors and nude mice as recipients, we evaluated the effect of SDF-1alpha on the recruitment of BM-derived cells to VEGF(165)-inhibited TC/siVEGF(7-1) tumors, as well as its effect on neovasculature development, vessel morphology and tumor growth. SDF-1alpha stimulated the migration of CD34(+) progenitor cells to Matrigel plugs in vivo and promoted the retainment of BM-derived pericytes in close association with perfused, functional tumor vessels. Intratumor inoculation of Ad-SDF-1alpha into TC/siVEGF(7-1) tumors resulted in increased SDF-1 and PDGF-BB expression, augmented tumor growth, an increase in the number of large, lumen-bearing vascular structures, and enhanced vessel pericyte coverage, with no change in VEGF(165). SDF-1alpha stimulates BM cell chemotaxis and the association of these cells with functional tumor vessels. Furthermore, SDF-1alpha enhances tumor neovascularization and growth with no alteration in VEGF(165). Our work suggests that SDF-1-mediated vasculogenesis may represent an alternate pathway that could potentially be utilized by tumors to sustain growth and neovasculature expansion after anti-VEGF therapy.

VEGF165 expression in the tumor microenvironment influences the differentiation of bone marrow-derived pericytes that contribute to the Ewing's sarcoma vasculature.

We previously demonstrated that bone marrow (BM) cells migrate to Ewing's tumors and differentiate into endothelial cells within the tumor vasculature. Recent evidence suggests that the roles of BM cells in tumors are more diverse. We investigated whether non-endothelial cell types critical for tumor vessel development are also derived from migrated BM cells. We utilized BM transplantation with GFP(+) transgenic mice as BM donors and nude mice as recipients to track the fate of migrated BM cells. After engraftment, we injected recipient mice either subcutaneously or intramuscularly with Ewing's sarcoma cells. We labeled functional tumor vessels using intravascular perfusion staining with tomato lectin. We assessed BM cell recruitment/differentiation within the tumor microenvironment using immunohistochemistry. Ewing's tumors contained BM-derived cells that had differentiated into endothelial cells lining perfused tumor vessels. A substantial fraction of recruited BM cells also resided in the vessel vicinity and expressed desmin and PDGFR-beta, indicating smooth muscle cell differentiation. In order to further characterize the role of stem/progenitor cells in Ewing's sarcoma, we sorted Tie2(-) BM cells from Tie2-GFP transgenic mice and then injected them intravenously into Ewing's tumor-bearing mice. Tie2(-) BM progenitors migrated to Ewing's tumors and differentiated into Tie2(+) cells occupying a perivascular residence and expressing alpha-smooth muscle actin, desmin and PDGFR-beta, as well as VEGFR-2. We did not observe differentiation of Tie2(-) cells into Tie2(+) perivascular cells in VEGF(165)-inhibited TC/siVEGF(7-1) tumors. The differentiation of Tie2(-) BM cells into Tie2(+) cells in parental but not VEGF(165)-inhibited tumors indicates that the tumor microenvironment may influence the differentiation pathway.

The osterix transcription factor down-regulates interleukin-1 alpha expression in mouse osteosarcoma cells.

K7M2 mouse osteosarcoma cells form lytic tumors and are deficient in osterix (Osx), a zinc finger-containing transcription factor required for osteoblast differentiation and bone formation. Our previous studies showed that replacement of Osx suppresses lytic bone destruction. Cytokines, including interleukin (IL)-1alpha, IL-6, IL-11, and prostaglandin E2, have been shown to stimulate osteoclast activity. We showed that IL-1alpha production by K7M2 cells was significantly suppressed following Osx transfection through a transcription-mediated mechanism. Osx had no effect on IL-6, IL-11, or prostaglandin E2. Site-directed mutagenesis and chromatin immunoprecipitation indicated that Osx down-regulated IL-1alpha through an Sp1-binding site on the IL-1alpha promoter. Inhibiting Osx by small interfering RNA in two cell lines (Dunn and DLM8) that expressed high levels of Osx led to enhanced IL-1alpha promoter activity and protein production and altered the phenotype from blastic to lytic. These data suggest that Osx down-regulates IL-1alpha expression in mouse osteosarcoma cells via transcriptional repression of IL-1alpha and this may in turn affect the lytic activity of the tumor cells.

Corruption of the Fas pathway delays the pulmonary clearance of murine osteosarcoma cells, enhances their metastatic potential, and reduces the effect of aerosol gemcitabine.

PURPOSE: Pulmonary metastases continue to be a significant problem in osteosarcoma. Apoptosis dysfunction is known to influence tumor development. Fas (CD95, APO-1)/FasL is one of the most extensively studied apoptotic pathways. Because FasL is constitutively expressed in the lung, cells that express Fas should be eliminated by lung endothelium. Cells with low or no cell surface Fas expression may be able to evade this innate defense mechanism. The purpose of these studies was to evaluate Fas expression in osteosarcoma lung metastases and the effect of gemcitabine on Fas expression and tumor growth. EXPERIMENTAL DESIGN AND RESULTS: Using the K7M2 murine osteosarcoma model, Fas expression was quantified using immunohistochemistry. High levels of Fas were present in primary tumors, but no Fas expression was present in actively growing lung metastases. Blocking the Fas pathway using Fas-associated death domain dominant-negative delayed tumor cell clearance from the lung and increased metastatic potential. Treatment of mice with aerosol gemcitabine resulted in increased Fas expression and subsequent tumor regression. CONCLUSIONS: We conclude that corruption of the Fas pathway is critical to the ability of osteosarcoma cells to grow in the lung. Agents such as gemcitabine that up-regulate cell surface Fas expression may therefore be effective in treating osteosarcoma lung metastases. These data also suggest that an additional mechanism by which gemcitabine induces regression of osteosarcoma lung metastases is mediated by enhancing the sensitivity of the tumor cells to the constitutive FasL in the lung.

Intranasal interleukin-12 gene therapy enhanced the activity of ifosfamide against osteosarcoma lung metastases.

BACKGROUND: Cyclophosphamide (CTX) and ifosfamide (IFX) are alkylating agents used to treat osteosarcoma (OS). It was previously demonstrated that the sensitivity of OS cells to 4-hydroperoxycyclophosphamide (4-HC, the active metabolite of CTX) is augmented by interleukin (IL)-12 in vitro through a mechanism involving the Fas/FasL pathway. The purpose of these studies was to determine whether this synergistic effect is operational in vivo. METHODS: Mice were injected intravenously with human LM7 osteosarcoma cells. Treatment was initiated with IFX (2.5 mg/kg intraperitoneally) with or without intranasal polyethylenimine (PEI):IL-12 gene therapy given twice weekly for 6 weeks. RESULTS: Expression of IL-12 protein in the lung was demonstrated in all mice receiving intranasal PEI:IL-12 but not in control mice or those treated with IFX alone. Increased expression of FasL was detected in lungs of all mice receiving IFX. Both IFX and PEI:IL-12 alone significantly inhibited lung metastasis when compared with control groups (P < 0.05). However, the most significant tumor effect was observed in mice receiving IFX+PEI:IL-12 (P < 0.01). Immunohistochemical staining for CD31 and basic fibroblast growth factor (bFGF) and the number of proliferating cells as quantified by proliferating cell nuclear antigen (PCNA) staining were also most significantly decreased in mice receiving combination therapy. CONCLUSIONS: These data indicate that combining IFX and IL-12 may have therapeutic potential and that this increased efficacy may be mediated through a mechanism involving the Fas/FasL pathway.

A small interfering RNA targeting vascular endothelial growth factor inhibits Ewing's sarcoma growth in a xenograft mouse model.

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising therapeutic target for cancer. Vascular endothelial growth factor (VEGF) is a key regulator in vasculogenesis as well as in angiogenesis. TC71 human Ewing's sarcoma cells overexpress VEGF, with a shift in isoform production from membrane-bound VEGF189 to the more soluble VEGF165. Transfection of TC71 cells with a vector-based VEGF targeted small interfering RNA expression system (VEGFsi) inhibited VEGF165 expression by 80% and VEGF165 protein production by 98%, with no alteration in VEGF189 expression. Human microvascular endothelial cell proliferation and migration induced by conditioned medium from VEGFsi-transfected TC71 cells was significantly less than that induced by conditioned medium from TC71 cells and control vector-transfected TC71 cells. Furthermore, after s.c. injection into athymic nu/nu mice, the tumor growth of VEGFsi-expressing TC71 cells was significantly less than that of parental or control vector-transfected cells. Vessel density as assessed by CD31 immunohistochemical analysis and VEGF165 expression as assessed by Northern blotting were also decreased. Intratumor gene therapy with polyethylenimine/VEGFsi also resulted in tumor growth suppression. When inoculated into the tibias of nude mice, VEGFsi-expressing TC71 cells induced osteolytic bone lesions that were less severe than those induced by control groups. These data suggest that targeting VEGF165 may provide a therapeutic option for Ewing's sarcoma.

Herceptin down-regulates HER-2/neu and vascular endothelial growth factor expression and enhances taxol-induced cytotoxicity of human Ewing's sarcoma cells in vitro and in vivo.

We have previously shown that high levels of HER-2/neu protein were overexpressed in human Ewing's sarcoma cells (TC71, SK-ES1) relative to normal human osteoblasts. The purpose of this study was to determine whether herceptin alone or in combination with chemotherapeutic agents could inhibit the growth of Ewing's sarcoma in vitro and in vivo. Western blot analysis showed that the protein levels of HER-2/neu were decreased following herceptin treatment. Cell growth was also inhibited by herceptin in a dose-dependent manner with an IC(50) of 4 mg/mL in TC71 and SK-ES1 cell line, whereas human immunoglobin had no effect. Northern blot and ELISA showed the RNA expression and protein levels of vascular endothelial growth factor were also inhibited by herceptin treatment with no alteration in HIF-1alpha protein and topoisomerase IIalpha expression. Furthermore, Ewing's sarcoma tumor growth was significantly delayed by 100 mg/kg herceptin treatment in our Ewing's sarcoma xenograft mouse model. Combining taxol with herceptin resulted in additive cytotoxicity, whereas herceptin-etoposide, doxorubicin, and 9-nitrocamptothecin combinations did not. Taxol-herceptin enhanced growth inhibition in TC71 cells in vitro compared with either agent alone. Ewing's sarcoma growth was also delayed in vivo and mean tumor size was significantly lower in mice treated with herceptin plus taxol than in those receiving taxol or herceptin alone. These data suggest that herceptin in combination with taxol may be a therapeutic option in the treatment of Ewing's sarcoma.

Osterix, a transcription factor for osteoblast differentiation, mediates antitumor activity in murine osteosarcoma.

Osterix is a novel zinc finger-containing transcription factor that is essential for osteoblast differentiation and bone formation. We hypothesized that osterix might have a role in osteosarcoma tumor growth and metastasis. Northern blot analysis showed that the mRNA level of osterix was decreased in two mouse osteosarcoma cell lines compared with its level in normal mouse osteoblasts. Osterix expression was also decreased in three human osteosarcoma cell lines. Transfection of the osx gene into the mouse osteosarcoma cells inhibited tumor cell growth in vitro and in vivo and significantly reduced tumor incidence, tumor volume, and lung metastasis following intratibial injection. Osterix expression was also associated with decreased osteolysis. Using an in vitro migration assay, osterix suppressed the migration of tumor cells to lung extracts. These results suggest that osterix expression may play a role in osteosarcoma tumor growth and metastasis.

Increased Fas expression reduces the metastatic potential of human osteosarcoma cells.

PURPOSE: The process of metastasis requires the single tumor cell that seeds the metastatic clone to complete a complex series of steps. Identifying factors responsible for these steps is essential in developing and improving targeted therapy for metastasis. Resistance to receptor-mediated cell death, such as the Fas/Fas ligand pathway, is one mechanism commonly exploited by metastatic cell populations. EXPERIMENTAL DESIGN AND RESULTS: LM7, a subline of the SAOS human osteosarcoma cell line with low Fas expression, was selected for its high metastatic potential in an experimental nude mouse model. When transfected with the full-length Fas gene (LM7-Fas), these cells expressed higher levels of Fas than the parental LM7 cells or LM7-neo control-transfected cells. These cells were also more sensitive to Fas-induced cell death than controls. When injected intravenously into nude mice, the LM7-Fas cell line produced a significantly lower incidence of tumor nodules than control cell lines. Lung weight and tumor nodule size were also decreased in those mice injected with LM7-Fas. Levels of Fas were quantified in osteosarcoma lung nodules from 17 patients. Eight samples were Fas negative, whereas the remaining 9 were only weakly positive compared with normal human liver (positive control). CONCLUSIONS: Our results demonstrate that altering Fas expression can impact the metastatic potential of osteosarcoma cells. We conclude that the increase of Fas on the surface of the LM7 osteosarcoma cells increased their sensitivity to Fas-induced cell death in the microenvironment of the lung, where Fas ligand is constitutively expressed. Thus, loss of Fas expression is one mechanism by which osteosarcoma cells may evade host resistance mechanisms in the lung, increasing metastatic potential. Fas may therefore be a new therapeutic target for osteosarcoma.

Interleukin-12 enhances the sensitivity of human osteosarcoma cells to 4-hydroperoxycyclophosphamide by a mechanism involving the Fas/Fas-ligand pathway.

Cyclophosphamide (CY) and its derivative ifosfamide are alkylating agents used to treat osteosarcoma (OS). The purpose of these studies was to determine whether alkylating agents affect the expression of Fas ligand (FasL) and whether interleukin 12 enhances the sensitivity of human OS cells to alkylating agents. 4-Hydroperoxycyclophosphamide (4-HC), the preactivated CY compound, and 4-hydroperoxydidechlorocloclophosphamide (4-HDC), its nonalkylating analogue, human OS LM6 cells, and a clone of cells derived by transfection with the interleukin 12 gene (LM6-#6) were used for these studies. Incubation of LM6 and LM6-#6 with 10 micro M 4-HC increased the expression of FasL mRNA (2.5- and 3.0-fold, respectively). By contrast, 4-HDC, Adriamycin (ADR), cisplatin (CDP), and methotrexate (MTX) had no effect on FasL mRNA expression. Increased FasL expression after treatment with 4-HC was also demonstrated by immunohistochemistry and flow cytometry. Drug-induced FasL was functional and mediated cell death. We examined the effect of FasL up-regulation by 4-HC on LM6 and LM6-#6 cells. Flow cytometry showed that LM6-#6 cells expressed 2.2-fold more Fas than LM6 cells. Cytotoxicity of 4-HC, 4-HDC, ADR, CDP, and MTX on LM6, LM6-neo, and LM6-#6 were quantified. Colony-forming assay revealed an IC(50) of 2.10 micro M for 4-HC in LM6-neo cells compared with 0.41 micro M in LM6-#6 cells. The IC(50) for 4-HDC, ADR, CDP, and MTX were not significantly different between the two cell lines. We concluded that the increased expression of Fas enhanced LM6-#6 sensitivity to 4-HC. These data indicate that Fas/FasL may be involved in the cytotoxic pathway of CY. Combining biological agents with chemotherapeutic agents that have complementary Fas/FasL pathway actions may offer new therapeutic alternatives.

Association of alphavbeta3 integrin expression with the metastatic potential and migratory and chemotactic ability of human osteosarcoma cells.

INTRODUCTION: Expression of adhesion molecules such as alphavbeta3 integrin has been associated with the metastatic potential of tumor cells. The purpose of this study was to determine whether alphavbeta3 expression correlated with the metastatic potential of human osteosarcoma cells. MATERIALS AND METHODS: We developed a series of sublines (LM2-LM7) from human osteosarcoma SAOS parental cells, with progressively increasing potential to form lung metastases in nude mice after intravenous injection. SAOS parental and LM2 cells were poorly metastatic, but LM7 cells resulted in visible metastatic lung nodules by 6-8 weeks. We quantified alphavbeta3 integrin expression using flow cytometry. RESULTS: alphavbeta3 expression correlated with the metastatic potential of the cells, with LM7 cells showing the highest expression. LM7 cell adhesion to vitronectin decreased after treatment with echistatin, a RGD-containing peptide antagonist of alphavbeta3. LM7 cells demonstrated higher chemotactic activity than SAOS cells to a homogenate made from lung tissue. This chemotactic activity was also inhibited by echistatin. These data indicated that alphavbeta3 was critical for the migration of LM7 cells to the lung homogenate. Chemotaxis to a liver homogenate was the same for LM7 and SAOS cells. Migration of LM7 cells through lung endothelial cells was higher than that through liver endothelial cells, and echistatin again inhibited this migration. CONCLUSIONS: alphavbeta3 integrin expression may play a role in the metastatic potential of osteosarcoma cells by enhancing the ability of the cells to migrate specifically to the lung. Alphavbeta3 integrin may therefore be a potential new target for osteosarcoma.

Aerosol gene therapy with PEI: IL-12 eradicates osteosarcoma lung metastases.

PURPOSE: We determined whether polyethylenimine (PEI), a polycationic DNA carrier, can be used to deliver the interleukin (IL) 12 gene by aerosol to treat established osteosarcoma (OS) lung metastases in a nude mouse model. EXPERIMENTAL DESIGN: Tumor response was assessed using our OS lung metastases model. Treatment with aerosolized PEI containing the murine IL-12 gene (PEI:IL-12; 600 microl PEI and 2 mg IL-12) was given twice weekly for 5-6 weeks. RESULTS: Aerosol therapy for 2 weeks resulted in high expression of both the p35 and p40 subunits of IL-12 in the lungs but not in the livers of mice. Peak IL-12 mRNA expression was seen 24 h after a single aerosol PEI:IL-12 treatment. This expression gradually decreased with continued detection for up to 7 days. IL-12 protein was not detectable in plasma even after 6 weeks of aerosol therapy. The number of lung metastases in mice treated with aerosol PEI:IL-12 was decreased significantly (median, 0; range, 0-33) compared with mice that received PEI alone (median, 37.5; range, 11-125; P = 0.002). Nodule size was also significantly smaller in the aerosol PEI:IL-12 group with 87% of the nodules measuring 1 mm. Weekly aerosol PEI:IL-12 therapy was as effective as twice weekly therapy. CONCLUSIONS: Aerosol therapy resulted in selective gene expression and protein production in the tumor area. Aerosol PEI:IL-12 may avoid the systemic toxicities described previously in patients treated with i.v. IL-12. Because OS metastasizes almost exclusively to the lung, aerosol PEI:IL-12 therapy may provide a therapeutic option, which may be especially valuable.

Interleukin-1 alpha increases the cytotoxic activity of etoposide against human osteosarcoma cells.

The recurrence of pulmonary metastases resistant to salvage chemotherapy continues to be a major problem in osteosarcoma patients. Our goal is to identify novel combinations of biologic response modifiers plus chemotherapeutic agents that can be translated into clinical trials. Response rates of relapsed osteosarcoma patients to etoposide have been extremely low. The present investigation demonstrated that IL-1 alpha dramatically increased the sensitivity of MG-63, SAOS-2, and TE-85 osteosarcoma cells to etoposide when the two agents were used simultaneously. The cytostatic activity of 1 microM etoposide was increased from 35 to 70%, 30 to 65%, and 4 to 90%, respectively, by 5.0 U/ml IL-1 alpha. Analysis using the colony-forming assay to quantify cytotoxicity showed that the percentage of cell survival following exposure to etoposide decreased from 0.81 to 0.56, 0.55 to 0.2, and 0.4 to 0.05 when the combination treatment was used. Increased sensitivity was not seen when etoposide treatment preceded IL-1 alpha treatment. IL-1 alpha also increased the sensitivity of these cells to doxorubicin but not to cisplatin or topotecan. The mechanism of this enhanced activity is independent of p-glycoprotein, drug-uptake, or effects on topoisomerase II.