Nadezhda V. Koshkina

Aerosol gemcitabine inhibits the growth of primary osteosarcoma and osteosarcoma lung metastases.

Osteosacarcoma (OS) lung metastases are often resistant to chemotherapy. Most anticancer drugs are administered systemically. In many cases this is followed by dose‐dependent toxicity, which may not allow the achievement of therapeutic levels in lungs to eradicate metastases. We determined the efficacy of gemcitabine (GCB) by administering it directly to the lungs via aerosol and studied the role of the Fas pathway in response to the therapy. We used 2 osteosarcoma lung metastases animal models: human LM7 cells that form lung metastases in mice following intravenous injection and murine LM8 cells, which grows subcutaneously in mice and spontaneously metastasize to the lung. Treatment was initiated when the presence of lung metastases had been established. Aerosol GCB inhibited the growth of lung metastases in mice. Intraperitoneal GCB administration at similar dosage had no effect on lung metastases. Besides its direct effect on lung metastases, aerosol GCB suppressed the growth of subcutaneous LM8 tumor. Histopathological examination of mice receiving aerosol GCB showed no evidence of toxicity. Lungs are distinguished from other tissues by the constitutive expression of FasL. Since exposure of tumor cells to GCB upregulated Fas expression, we hypothesized that the susceptibility of the tumor cells to ligand‐induced cell death by resident lung cells may be increased. Therefore, the Fas pathway may contribute to the therapeutic effect of aerosol GCB.

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.

Fas-negative osteosarcoma tumor cells are selected during metastasis to the lungs: the role of the Fas pathway in the metastatic process of osteosarcoma.

Low expression of Fas by different tumors including osteosarcoma, correlates with poor prognosis. We found that osteosarcoma lung metastases from patients expressed negligible amounts of Fas, but primary tumors often expressed high Fas levels. The reason for this discrepancy is unknown. We hypothesized that because FasL is constitutively expressed in the lungs, Fas-positive (Fas+) tumor cells entering the lungs would bind with FasL and die from Fas-induced apoptosis, resulting in the “selection” of Fas-negative (Fas−) cells, which would eventually form metastases. To test this hypothesis, we injected K7 osteosarcoma cells, which express functional Fas in vitro, into mice and confirmed that its bone tumors were Fas+, but lung metastases were Fas−. Next, to inhibit Fas signaling without affecting Fas expression, we transfected these cells with a FADD-dominant negative (FDN) plasmid and developed K7/FDN cells. Metastases formed by K7/FDN cells contained Fas+ tumor cells. Moreover, K7/FDN cells were retained in the lungs longer and formed more lung metastases than K7 cells. In addition, the incidence of lung metastases in FasL-deficient mice injected with K7 cells was higher than that in wild-type mice. Metastases from FasL-deficient mice but not from wild-type mice contained Fas+ tumor cells. Based on that, we conclude that Fas− osteosarcoma cells are selected during lung metastases formation and that inhibition of Fas signaling in tumors or lack of FasL in the host environment allows the proliferation of Fas+ osteosarcoma cells in the lungs and promotes metastases growth. Therefore, Fas may be considered as a new therapeutic target for osteosarcoma treatment. (Mol Cancer Res 2007;5(10):991–9)

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.

Paclitaxel (taxol) and taxoid derivates for lung cancer treatment: potential for aerosol delivery.

Paclitaxel (PTX, Taxol) has revolutionized cancer treatment in the past decade and is recognized as one of the biggest advances in oncology medicine. In spite of the good clinical efficacy shown by PTX, there is still a growing need to achieve better safety and pharmacokinetic profile of PTX in patients. The standard delivery modalities of intravenous infusion result in multiple side effects, and targeting of the drug to specific areas within the body can result in better efficacy and lower toxicity. Aerosol delivery of therapeutic agents has the potential of localizing the drugs specifically to the lung tissue, with a comparable or better pharmacokinetics as compared to intravenous, oral or intraperitoneal delivery. Aerosol delivery of PTX has not been studied extensively, however, it holds immense potential for improving the efficacy against lung tumors. Early pre-clinical studies in mice and dogs have shown good promise, both for pharmacokinetics of PTX, safety and efficacy in lung cancer models. This review looks at the still developing approach of aerosol delivery of PTX for lung cancer, documents the progress so far and the future directions that can bring this approach to clinical reality.

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

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.

Tumor Selective Hyperthermia Induced by Short-Wave Capacitively-Coupled RF Electric-Fields

There is a renewed interest in developing high-intensity short wave capacitively-coupled radiofrequency (RF) electric-fields for nanoparticle-mediated tumor-targeted hyperthermia. However, the direct thermal effects of such high-intensity electric-fields (13.56 MHZ, 600 W) on normal and tumor tissues are not completely understood. In this study, we investigate the heating behavior and dielectric properties of normal mouse tissues and orthotopically-implanted human hepatocellular and pancreatic carcinoma xenografts. We note tumor-selective hyperthermia (relative to normal mouse tissues) in implanted xenografts that can be explained on the basis of differential dielectric properties. Furthermore, we demonstrate that repeated RF exposure of tumor-bearing mice can result in significant anti-tumor effects compared to control groups without detectable harm to normal mouse tissues.

Exploratory analysis of Fas gene polymorphisms in pediatric osteosarcoma patients

Defective apoptosis signaling by the Fas pathway has carcinogenic implications. We analyzed 123 pediatric patients with osteosarcoma for Fas single nucleotide polymorphisms: 2 of the promoter region (−1377 G>A and −670 A>G) and 2 of the coding region (exon 3 18272 A>G and exon 7 22628 C>T). As a comparison group, we used 510 adults without a history of cancer. We found an increased risk of osteosarcoma associated with the heterozygous genotype Fas exon 3 AG (genotype frequency 19.5% in cases vs. 12.0% in controls, P=0.028; adjusted odds ratio=1.6, 95% confidence interval=0.9-2.7], and this association was more pronounced in non-Hispanic whites (20.6% in cases vs. 10.1% in controls, P=0.014; adjusted odds ratio=2.3, 95% confidence interval=1.2-4.6). Additionally, the frequency of the variant allele (exon 3 G) was significantly higher in cases than in controls for both the entire group and non-Hispanic whites (P=0.046 and P=0.030, respectively). We found no significant association between osteosarcoma risk and the other Fas polymorphisms. This study suggests an association between the Fas exon 3 A>G polymorphism and osteosarcoma risk; however, further study is needed with pediatric controls and a larger sample size.

Expression of granulocyte-colony-stimulating factor and its receptor in human Ewing sarcoma cells and patient tumor specimens: potential consequences of granulocyte-colony-stimulating factor administration.

Ewing sarcoma (ES) is a highly vascular malignancy. It has been demonstrated that both angiogenesis and vasculogenesis contribute to the growth of ES tumors. Granulocyte‐colony‐stimulating factor (G‐CSF), a cytokine known to stimulate bone marrow (BM) stem cell production and angiogenesis, is routinely administered to ES patients after chemotherapy. Whether ES cells and patient tumor samples express G‐CSF and its receptor (G‐CSFR) and whether treatment with this factor enhances tumor growth was examined.

Gold nanoparticles stabilized with MPEG-grafted poly(l-lysine): in vitro and in vivo evaluation of a potential theranostic agent

As the number of diagnostic and therapeutic applications utilizing gold nanoparticles (AuNPs) increases, so does the need for AuNPs that are stable in vivo, biocompatible, and suitable for bioconjugation. We investigated a strategy for AuNP stabilization that uses methoxypolyethylene glycol-graft-poly(l-lysine) copolymer (MPEG-gPLL) bearing free amino groups as a stabilizing molecule. MPEG-gPLL injected into water solutions of HAuCl4 with or without trisodium citrate resulted in spherical (Zav = 36 nm), monodisperse (PDI = 0.27), weakly positively charged nanoparticles (AuNP3) with electron-dense cores (diameter: 10.4 ± 2.5 nm) and surface amino groups that were amenable to covalent modification. The AuNP3 were stable against aggregation in the presence of phosphate and serum proteins and remained dispersed after their uptake into endosomes. MPEG-gPLL-stabilized AuNP3 exhibited high uptake and very low toxicity in human endothelial cells, but showed a high dose-dependent toxicity in epithelioid cancer cells. Highly stable radioactive labeling of AuNP3 with 99mTc allowed imaging of AuNP3 biodistribution and revealed dose-dependent long circulation in the blood. The minor fraction of AuGNP3 was found in major organs and at sites of experimentally induced inflammation. Gold analysis showed evidence of a partial degradation of the MPEG-gPLL layer in AuNP3 particles accumulated in major organs. Radiofrequency-mediated heating of AuNP3 solutions showed that AuNP3 exhibited heating behavior consistent with 10 nm core nanoparticles. We conclude that PEG-pPLL coating of AuNPs confers “stealth” properties that enable these particles to exist in vivo in a nonaggregating, biocompatible state making them suitable for potential use in biomedical applications such as noninvasive radiofrequency cancer therapy.