Nancy Gordon

Treatment of pregnant breast cancer patients and outcomes of children exposed to chemotherapy in utero

As women in the US delay childbearing, it has been hypothesized that the incidence of breast cancer diagnosed during pregnancy will increase. There are very little prospective data on the treatment of pregnant women with breast cancer with chemotherapy and even less data on the outcomes of their children who were exposed to chemotherapy in utero.

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.

Aerosol Gemcitabine: Preclinical Safety and In Vivo Antitumor Activity in Osteosarcoma-Bearing Dogs

BACKGROUND Osteosarcoma is the most common skeletal malignancy in the dog and in young humans. Although chemotherapy improves survival time, death continues to be attributed to metastases. Aerosol delivery can provide a strategy with which to improve the lung drug delivery while reducing systemic toxicity. The purpose of this study is to assess the safety of a regional aerosol approach to chemotherapy delivery in osteosarcoma-bearing dogs, and second, to evaluate the effect of gemcitabine on Fas expression in the pulmonary metastasis. METHODS We examined the systemic and local effects of aerosol gemcitabine on lung and pulmonary metastasis in this relevant large-animal tumor model using serial laboratory and arterial blood gas analysis and histopathology and immunohistochemistry, respectively. RESULTS AND CONCLUSIONS Six hundred seventy-two 1-h doses of aerosol gemcitabine were delivered. The treatment was well tolerated by these subjects with osteosarcoma (n = 20). Aerosol-treated subjects had metastatic foci that demonstrated extensive, predominately central, intratumoral necrosis. Fas expression was decreased in pulmonary metastases compared to the primary tumor (p = 0.008). After aerosol gemcitabine Fas expression in the metastatic foci was increased compared to lung metastases before treatment (p = 0.0075), and even was higher than the primary tumor (p = 0.025). Increased apoptosis (TUNEL) staining was also detected in aerosol gemcitabine treated metastasis compared to untreated controls (p = 0.028). The results from this pivotal translational study support the concept that aerosol gemcitabine may be useful against pulmonary metastases of osteosarcoma. Additional studies that evaluate the aerosol route of administration of gemcitabine in humans should be safe and are warranted.

Natural killer cell therapy and aerosol interleukin-2 for the treatment of osteosarcoma lung metastasis

Survival of patients with osteosarcoma lung metastases has not improved in 20 years. We evaluated the efficacy of combining natural killer (NK) cells with aerosol interleukin‐2 (IL‐2) to achieve organ‐specific NK cell migration and expansion in the metastatic organ, and to decrease toxicity associated with systemic IL‐2.

The Role of Fas/FasL in the Metastatic Potential of Osteosarcoma and Targeting this Pathway for the Treatment of Osteosarcoma Lung Metastases

Pulmonary metastases remain the main cause of death in patients with Osteosarcoma (OS). In order to identify new targets for treatment, our laboratory has focused on understanding the biological properties of the tumor microenvironment that contribute to or interfere with metastasis. Dysfunction of the Fas/FasL signaling pathway has been implicated in tumor development, and progression. Here we describe the status of Fas expression in murine nonmetastatic K7 and metastatic K7M2 cells and human nonmetastatic SAOS and LM2 and metastatic LM6 OS cells. We demonstrated that Fas expression correlates inversely with metastatic potential. Pulmonary metastases from patients were uniformly Fas- supporting the importance of Fas expression to the metastatic potential. Since FasL is constitutively expressed in the lung, our data suggests that Fas+ tumor cells undergo apoptosis and are cleared from the lung. By contrast, Fas- tumor cells evade this host defense mechanism and form lung metastases. We confirmed these findings by blocking the Fas pathway using Fas Associated Death Domain Dominant-Negative (FDN). Fas+ cells transfected with FDN were not sensitive to FasL, showed delayed clearance and formed lung metastases. Fas+ cells were also able to form lung metastases in FasL-deficient mice. Using our mouse model systems, we demonstrated that aerosol treatment with liposomal 9-Nitrocamptothecin and Gemcitabine (chemotherapeutic agents known to upregulate Fas expression) increased Fas expression and induced tumor regression in wild type mice. Lung metastases in FasL deficient mice did not respond to the treatment. We conclude that Fas is an early defense mechanism responsible for clearing invading Fas+ tumor cells from the lung. Fas- cells or cells with a nonfunctional Fas pathway evade this defense mechanism and form lung metastases. Therapy that induces Fas expression may therefore be effective in patients with established OS lung metastases. Aerosol delivery of these agents is an ideal way to target treatment to the lung.

Aerosol therapy for the treatment of osteosarcoma lung metastases: targeting the Fas/FasL pathway and rationale for the use of gemcitabine.

Lung metastases are the main cause of death in patients with osteosarcoma (OS). Salvage chemotherapy has been largely unsuccessful in improving the long-term survival of these patients. Understanding the mechanisms that play a role in the metastatic process may identify new therapeutic strategies. We have demonstrated that the cell surface Fas expression, the Fas/FasL signaling pathway, and the constitutive expression of FasL in the lung microenvironment play a critical role in the metastatic potential of OS cells. Here we review the status of Fas expression in two sets of OS cells, human SAOS and LM7 and murine K7 and K7M2, which differ in their ability to metastasize to the lungs. We demonstrated that Fas expression inversely correlated with metastatic potential. Evaluation of Fas expression in a set of lung metastases from patients demonstrated low or no Fas expression consistent with our hypothesis that Fas+ osteosarcoma cells cannot form metastases. The absence of FasL in the lung allows Fas+ osteosarcoma cells to form metastases indicating that the microenvironment is an important contributor to the metastatic potential of osteosarcoma cells. Disruption of the signal transduction pathway using Fas-associated death domain dominant negative (FDN) also allowed Fas+ cells to form lung metastases. Aerosol Gemcitabine (GCB) upregulated Fas expression and induced tumor regression in wild-type Balb/c mice but not Fas L-deficient mice. In conclusion, Fas constitutes an early defense mechanism that allows Fas+ tumor cells to undergo apoptosis when in contact with constitutive FasL in the lung. Fas- cells or cells with a corrupted Fas pathway evade this defense mechanism and form lung metastases. The aerosol delivery of chemotherapeutic agents that upregulate Fas expression may benefit patients with established pulmonary metastases.

Aerosol interleukin-2 induces natural killer cell proliferation in the lung and combination therapy improves the survival of mice with osteosarcoma lung metastasis.

We have previously shown that aerosol interleukin‐2 (IL‐2) increased the number of intravenously injected human natural killer (NK) cells in the lungs. In this study we investigated whether this increase was secondary to NK cell proliferation and determined the site of the proliferation.

Knockdown of autophagy-related protein 5, ATG5, decreases oxidative stress and has an opposing effect on camptothecin-induced cytotoxicity in osteosarcoma cells

BackgroundAutophagy induction can increase or decrease anticancer drug efficacy. Anticancer drug-induced autophagy induction is poorly characterized in osteosarcoma (OS). In this study, we investigated the impact of autophagy inhibition on camptothecin (CPT)-induced cytotoxicity in OS.MethodsAutophagy-inhibited DLM8 and K7M3 metastatic murine OS cell lines were generated by infection with lentiviral shRNA directed against the essential autophagy protein ATG5. Knockdown of ATG5 protein expression and inhibition of autophagy was confirmed by immunoblot of ATG5 and LC3II proteins, respectively. Metabolic activity was determined by MTT assay and cell viability was determined by trypan blue exclusion. Acridine orange staining and immunoblotting for LC3II protein expression were used to determine autophagy induction. Oxidative stress was assessed by staining cells with HE and DCFH-DA followed by flow cytometry analysis. Mitochondrial membrane potential was determined by staining cells with TMRE followed by flow cytometry analysis. Immunoblotting was used to detect caspase activation, Parp cleavage and p53 phosphorylation.ResultsAutophagy inhibition caused a greater deficit in metabolic activity and cell growth in K7M3 cells compared to DLM8 cells. K7M3 cells exhibited higher basal autophagy levels than DLM8 cells and non-transformed murine MCT3 osteoblasts. Autophagy inhibition did not affect CPT-induced DNA damage. Autophagy inhibition decreased CPT-induced cell death in DLM8 cells while increasing CPT-induced cell death in K7M3 cells. Autophagy inhibition reduced CPT-induced mitochondrial damage and CPT-induced caspase activation in DLM8 cells. Buthionine sulfoximine (BSO)-induced cell death was greater in autophagy-competent DLM8 cells and was reversed by antioxidant pretreatment. Camptothecin-induced and BSO-induced autophagy induction was also reversed by antioxidant pretreatment. Significantly, autophagy inhibition not only reduced CPT-induced oxidative stress but also reduced basal oxidative stress.ConclusionsThe results of this study indicate that autophagy inhibition can have an opposing effect on CPT-induced cytotoxicity within OS. The cytoprotective mechanism of autophagy inhibition observed in DLM8 cells involves reduced CPT-induced oxidative stress and not reduced DNA damage. Our results also reveal the novel finding that knockdown of ATG5 protein reduces both basal oxidative stress and drug-induced oxidative stress.

Fludarabine and once-daily intravenous busulfan for allogeneic bone marrow transplantation for Chediak-Higashi syndrome.

An HLA-identical sibling bone marrow transplant was done for a patient with Chediak-Higashi syndrome. The preparative regimen included intravenous fludarabine (40 mg/m2/dx4) and busulfan (130 mg/m2/dx4). Busulfan was given once daily. Pharmacokinetic studies showed the area under the concentration-time curve of the once-daily intravenous busulfan was similar to that seen with the total daily dose administered with an every-6-hourly regimen. Toxicity was minimal. Myeloid engraftment occurred on day +17 and donor chimerism was complete. Fludarabine and once-daily intravenous busulfan is well tolerated and is adequate for engraftment of sibling transplant in Chediak-Higashi syndrome.

Autophagy in Osteosarcoma

Osteosarcoma (OS) metastatic disease is resistant to conventional chemotherapy. Tumor resistance to chemotherapy has been one of the major areas of concern to clinicians and the topic of many laboratory investigators. Evaluation of mechanisms implicated in OS lung metastasis resistance to chemotherapy has been the focus of some of our most recent work. We have previously demonstrated the therapeutic efficacy of aerosol gemcitabine (GCB) in OS lung metastases. However, a subset of cells fails to respond to GCB treatment and persists as isolated lung metastases in vivo. Autophagy, a physiological mechanism that supports nutritional deprivation under stressful conditions, has been implicated in tumor resistance to chemotherapy. We demonstrated the induction of autophagy by GCB in LM7 metastatic human OS cells and K7M3 metastatic murine OS cells. Inhibition of autophagy resulted in increased sensitivity to GCB in LM7 cells. By contrast, inhibiting autophagy in K7M3 cells decreased GCB sensitivity. Defining the role autophagy plays in chemotherapy response in different tumor types has become of greater importance in order to identify the best suitable therapeutic approach. In this chapter, we summarize some of the most recent work related to autophagy in OS, identify some of the known mechanisms, and address the different roles autophagy plays in chemotherapy response.