Aerosol gemcitabine: preclinical safety and in vivo antitumor activity in osteosarcoma-bearing dogs

Abstract

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.

FIG. 1.

Representative images evaluating the effect of aerosol gemcitabine on canine pulmonary tissues. Dogs with radiographic evidence of pulmonary metastasis were treated with aerosol gemcitabine twice weekly for a median 7 weeks. Tissues were harvested and processed as described in Materials and Methods. Minimal changes were identified in aerosol gemcitabine-naïve (A) or -treated lung (B). Treated metastatic foci (D) demonstrated increased central necrosis as compared to chemotherapy-naïve (C) historical controls.

FIG. 2.

Fas expression in canine osteosarcoma. Representative sections of primary (a) and metastatic (b, c) osteosarcoma lesions removed from aerosol gemcitabine-naïve (a, top row, b) or -treated (a, bottom row, c) dogs were harvested, processed, stained for Fas by immunohistochemistry, and the staining quantitated (scatter plot) as described in Material and Methods. Canine liver served as negative (Fas antibody omitted; NEG) and positive (POS) controls. The quantitation of Fas staining intensity (scatter plot) was lower in untreated pulmonary metastases (□) than in the originating primary osteosarcoma tumor (○), and this difference was significant (p = 0.008). In contrast, pulmonary metastases obtained from aerosol gemcitabine-treated (▪) dogs demonstrated stronger Fas staining intensity than the in the primary tumor (p = 0.025). The difference between untreated and treated metastases was also different and statistically significant (p = 0.0075). Whisker bars represent SEM.

FIG. 2.

Fas expression in canine osteosarcoma. Representative sections of primary (a) and metastatic (b, c) osteosarcoma lesions removed from aerosol gemcitabine-naïve (a, top row, b) or -treated (a, bottom row, c) dogs were harvested, processed, stained for Fas by immunohistochemistry, and the staining quantitated (scatter plot) as described in Material and Methods. Canine liver served as negative (Fas antibody omitted; NEG) and positive (POS) controls. The quantitation of Fas staining intensity (scatter plot) was lower in untreated pulmonary metastases (□) than in the originating primary osteosarcoma tumor (○), and this difference was significant (p = 0.008). In contrast, pulmonary metastases obtained from aerosol gemcitabine-treated (▪) dogs demonstrated stronger Fas staining intensity than the in the primary tumor (p = 0.025). The difference between untreated and treated metastases was also different and statistically significant (p = 0.0075). Whisker bars represent SEM.

FIG. 3.

Apoptosis in metastatic canine osteosarcoma. Representative sections of metastatic canine osteosarcoma lesions removed from untreated (a) or aerosol gemcitabine-treated (b) dogs were harvested, processed, stained for apoptosis using the TUNEL assay and the staining quantitated (graph) as described in Material and Methods. Human prostate served as negative (primary antibody omitted; NEG) and positive (POS) controls. The level of apoptosis was low in untreated pulmonary metastases (□). After exposure to aerosol gemcitabine (▪), there was a marked increase in apoptotic activity in the pulmonary metastases (p = 0.028). Whisker bars represent SEM. Where not visible, the bars are contained within the symbol.

FIG. 3.

Apoptosis in metastatic canine osteosarcoma. Representative sections of metastatic canine osteosarcoma lesions removed from untreated (a) or aerosol gemcitabine-treated (b) dogs were harvested, processed, stained for apoptosis using the TUNEL assay and the staining quantitated (graph) as described in Material and Methods. Human prostate served as negative (primary antibody omitted; NEG) and positive (POS) controls. The level of apoptosis was low in untreated pulmonary metastases (□). After exposure to aerosol gemcitabine (▪), there was a marked increase in apoptotic activity in the pulmonary metastases (p = 0.028). Whisker bars represent SEM. Where not visible, the bars are contained within the symbol.