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

Carlos O. Rodriguez, Jr., D.V.M., Ph.D., Torrie A. Crabbs, D.V.M., Dennis W. Wilson, D.V.M., Ph.D., Virginia A. Cannan, B.S., Katherine A. Skorupski, D.V.M., Nancy Gordon, M.D., Nadya Koshkina, Ph.D., Eugenie Kleinerman, M.D., and Peter M. Anderson, M.D., Ph.D "Aerosol Gemcitabine: Preclinical Safety and In Vivo Antitumor Activity in Osteosarcoma-Bearing Dogs", 2010 Aug;23(4):197-206.

                                                                                  doi: 10.1089/jamp.2009.0773



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.



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.



Osteosarcoma is the most common malignancy of bone in the dog(1) and in young humans.(2) It is estimated that 10,000 dogs and 1,000 children per year in the United States develop osteosarcoma. In children, the 5-year survival time following surgery and intravenous chemotherapy ranges from 60–70% in the nonmetastatic setting(3) to 10–30% in cases of pulmonary metastsis.(4–6) As is the case in children, the addition of anthracycline and platinum-based chemotherapy after aggressive local tumor control increases both time to the development of lung and/or bone metastases and overall survival time,(1,7–16) which ranges in canine patients from 8 months to 14 months.(1,7–16) Despite the doubling of the survival time with the addition of chemotherapy, death continues to be attributed to the development of metastases.(1,7–16) Therefore, because metastatic disease is the impediment to cure in osteosarcoma, treatment strategies that improve the control of pulmonary metastasis should be explored.

Gemcitabine (2′,2′-difluorodeoxcycytidine, Gemzar, dFdC) is a deoxycytidine analogue,(17) which has demonstrated clinical utility in the management of solid(18–24) and hematological(25–27) human malignancies, but whose activity has only recently been explored in veterinary oncology.(28–33) In contrast to the large body of preclinical and clinical literature devoted to gemcitabine in the management of carcinomas, relatively fewer studies have been performed in osteosarcoma in humans,(23,24,34–40) and to date, none have been reported in canine osteosarcoma. Gemcitabine is routinely administered intravenously as either a 30-min or longer infusion.(18–24,26,35,38–40) A major limitation in the control of pulmonary metastasis with the use of the systemic administration of drugs, however, is the reduced drug concentration that is delivered to the lungs due to dilution in the blood volume. Aerosol delivery can bypass this limitation and is a strategy that may improve the control of pulmonary metastases.

In mouse models, aerosol delivery of gemcitabine has been demonstrated to significantly inhibit the growth of primary osteosarcoma tumors and of established lung metastases in a Fas-dependent manner.(41,42) Inhaled gemcitabine also prevented metastatic spread, with no evidence of toxicity to normal tissues. In a relatively larger animal model, aerosolization of gemcitabine was shown to deposit in a moderate, but significant, quantity in the peripheral lung compartment of the baboon with no evidence of pulmonary or systemic toxicity.(43) The clinical feasibility of aerosol delivery in the dog has been demonstrated with other chemotherapeutic and immunomodulatory agents.(44–46) The purposes of this study were to assess the pulmonary versus systemic toxicity of this regional approach to chemotherapy delivery, to examine the histopathological effects against gross osteosarcoma pulmonary metastasis, and to assess the role of the Fas/FasL pathway in this relevant large-animal tumor model.


Materials and Methods

Patients and clinical monitoring

Dogs evaluated at the William R. Prichard Veterinary Medical Teaching Hospital at the University of California at Davis between January 1, 2006 and December 31, 2007, which had a histological diagnosis of osteosarcoma, which harbored radiological evidence of gross pulmonary metastasis, and which were in otherwise good health were eligible for enrollment on this trial. Signed owner consent, IACUC, and internal CTRB approval were obtained. Dogs were custom-fitted with a polyethylene hood and sent home for desensitization to the wearing of the hood and to the noise of the nebulizer. The dog was returned for baseline complete blood count (CBC), serum biochemical analysis (SBA), and arterial blood gas (ABG) analysis and thoracic radiographs once desensitization had occurred (median 3 days). Routine clinicopathological (CBC, SBA, ABG) monitoring occurred at 2 weeks, 4 weeks, and monthly thereafter; serial radiographs were obtained monthly.

To evaluate the effect of aerosol treatment on gross pulmonary metastasis, a series of sections of affected lungs from contemporary (aerosol naïve controls) animals, which had received similar treatment for their primary tumors (amputation and intravenous chemotherapy with carboplatin with or without adriamycin), was compiled using a computerized search of the database of necropsy tissues. Criteria for inclusion as controls consisted of a histological diagnosis of pulmonary metastatic osteosarcoma, no prior treatment with gemcitabine chemotherapy, and no intravenous chemotherapeutic treatments within 2 months prior to the animal's death. Thirteen aerosol-naive cases for which tissues from both the primary tumor and metastatic lesions were available for immunohistochemical analysis and that met the inclusion criteria were identified. (Table 2) The degree of necrosis within the metastasis was determined by estimating the overall percentage of necrotic tumor area relative to the total area of the tumor metastases and comparing the results between aerosol-treated and aerosol-naïve lesions.

Table 2.
Patient Characteristics of Dogs With Spontaneously Arising Osteosarcoma that Received no Therapy for Metastatic Disease

Aerosol gemcitabine treatment, dosage, and schedule

The caregiver was educated about personal safety (respirator, mask, gloves, and gowns) and trained to administer the therapy to their dog using a Minimate compressor with nebulizer (Precision Medical, Inc., Northhampton, PA). Utilizing this system, aerosol particles containing gemcitabine were delivered that had a mass median aerodynamic diameter of 0.8 mm with GSD 2.1 as measured with the Andersen Cascade Impactor as previously described.(47) To prevent occupational exposure to ambient air levels of gemcitabine that may have been propagated during therapy, nebulization was performed outdoors.

The gemcitabine was reconstituted per manufacturer's instructions, and metered doses were sent home with the owner for administration on a M/W or T/Th schedule. The initial intent was to escalate dogs by 5 mg per week until 25 mg/dog was achieved or when clinicopathological or radiological evidence of toxicity was identified. When no toxicity was found in the first five dogs, subsequent dogs initiated the therapy at 25 mg/dog on the first treatment. Five additional dogs were started at 50 mg total dose twice weekly.


When the owner or attending veterinarian deemed the quality of life of the dog to be diminished to an unacceptable level the dog was euthanized by intravenous Phenobarbital overdose and submitted for necropsy. Particular attention was paid to collect sections of tissue from the proximal and distal trachea, main stem bronchi, and lung. Lungs were inflated with and fixed in 10% formalin buffer and then embedded in paraffin. Samples of metastatic foci as well as all other organs were collected as per standard necropsy protocol. Particular attention was paid to the collection of smaller (1–2 cm) metastatic nodules to minimize bias in the selection of spontaneously necrotic foci. All of the collected/submitted tissues were immersed in 10% buffered neutral formalin and embedded in paraffin. The paraffin blocks were sectioned at 5 μm, mounted on positively charged glass slides, and stained with hematoxylin and eosin (H&E). A single pair of veterinary pathologists (T.C., D.W.W.) reviewed all of the tissues included in this study.


Prior to staining, 5 μm-thick tissue sections from bone were placed at 60°C for 24 h while similar sections of lung were placed on a hot plate for 20 min. Tissues were deparaffinized in xylene and rehydrated. Incubation with 3% hydrogen peroxide for 12 min was used to block exogenous peroxidase. To reduce nonspecific binding, slides were subsequently incubated with PBS containing 10% normal horse serum and 1% normal goat serum. The primary antibody, polyclonal rabbit anti-Fas [(C-20) AB sc-715 antibody, Santa Cruz Biotechnology, Santa Cruz, CA], diluted at 1:200, was applied to the sections and left overnight at 4°C. The secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) labeled with horseradish peroxidase was then applied for 1 h at room temperature. The slides were then developed with 3′3-diaminobenzidine as a substrate and lightly counterstained with hematoxylin. Sections not exposed to the primary antibody served as negative controls. Normal canine liver was used as the positive control for Fas. Simple PCI software (Olympus, Center Valley, Pa) was used to quantify the data. Three slides per group were selected. Three different tumor areas per slide were quantified and then averaged and the MEAN ratio of positive nuclei to total counted nuclei was plotted; at least 200 nuclei were counted.

Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. After samples were deparaffinized as described above, tissues were incubated with 20 μg/mL proteinase K for 15 min at room temperature. After two 5-min washes with double-distilled water, tissues were incubated with hydrogen peroxide as described previously, washed again with double-distilled water thrice (3 min each), and incubated with terminal deoxynucleotidyl transferase buffer [30 mmol/L Trizma (pH 7.2), 140 mmol/L sodium cacodylate, 1 mmol/L cobalt chloride in double-distilled water] for 10 min at room temperature. Avoiding light, the reaction buffer was added to the tissue sections, and the slides were incubated with terminal transferase (1:400 diluted in terminal deoxynucleotidyl transferase buffer; Boehringer-Mannheim Corp., Mannheim, Germany) and biotin-160 dUTP (1:200 diluted in terminal deoxynucleotidyl transferase buffer; Roche, Indianapolis, IN) in a humid atmosphere at 37°C for 1 h. The enzymatic reaction was terminated by rinsing the sections twice with TB buffer (300 mmol/L NaCl, 30 mmol/L sodium citrate in double-distilled water). The tissues were washed twice (5 min each) with double-distilled water and then PBS and stained with 3,3′-diaminobenzidine as described above. One to two tumor areas per slide for a total of five different slides were analyzed and the MEAN ratio of positive nuclei to total counted nuclei was reported; at least 200 nuclei were counted.

Statistical analysis

A repeated-measures ANOVA was used to determine significance (significance set at p  = ≤ 0.05) of changes in hematological, serum biochemical, or pulmonary function parameters. Significance was confirmed with a Dunnet b post hoc (significance set at p = ≤ 0.01). Statistical analysis for the Fas and TUNEL staining was done using an unpaired, two-tailed Student's t-test (significance set at p = ≤ 0.05).



Pet dogs (patients) were exclusively used in this study. The characteristics of aerosol gemcitabine-treated dogs (n = 20) are described in Table 1. The median age was 9 years (range: 4–12). The median weight was 39 kg (range: 21–57). A similar number of male (9 castrated) and female (11 spayed) were treated. Breeds treated included Great Pyrenees (n = 3), Golden Retriever (n = 3), Rottweiler (n  = 4), and one each, St. Bernard, Bull Mastiff, Old English Sheep Dog, Belgian Tervuren, Great Dane, Portuguese Water Dog, and Bernese Mountain Dog. Osteosarcoma of the skeleton (n = 18) and osteosarcoma of soft parts (n = 2) were treated with amputation or radical excision, respectively. Either carboplatin alone (n = 5) or doxorubicin and carboplatin (n  = 11) was administered in the adjuvant setting to the dogs prior to the detection of metastatic disease. In five dogs, surgery alone was the sole means of control of the primary tumor without additional chemotherapy. Two patients (cases 1 and 8) received intravenous chemotherapy (4 and 6 weeks prior to enrollment) for the treatment of their pulmonary metastasis prior to receiving aerosol gemcitabine. All but one patient (case 19) underwent complete necropsy.

Table 1.
Patient Characteristics of Dogs with Spontaneously Arising Osteosarcoma Treated Twice Weekly with Aerosol Gemcitabine

Aerosol gemcitabine is well tolerated both systemically and locally

Six hundred seventy-two 1-h doses of aerosol gemcitabine were delivered (mean: 28; range: 2–80) twice per week on a M/W or Tu/Th schedule. Median duration of treatment was 60 min (range: 45–75). One dog received only 5 mg twice. One dog received only 10 mg twice. In both of these cases, the disease progressed rapidly and the quality of life fell precipitously and the owner elected euthanasia before the dose escalation could be instituted. Eleven dogs received 25 mg and seven dogs received 50 mg on schedule. There were no delays in treatments reported.

CBCs were normal in all dogs at every visit except for a single grade I neutropenia (1,500) episode (p  > 0.5; data not shown). Neither thrombocytopenia nor anemia occurred. SBA parameters were within reference range in every patient at every visit (p > 0.5; data not shown). Arterial blood gas and alveolar-arterial gradients did not vary from base line in any dog at any time point (p > 0.5; data not shown). No gastrointestinal toxicity was reported by any owner. Body weights did not vary from baseline (p > 0.5; data not shown).

Histology shows minimal airway and lung toxicity from aerosol gemcitabine

Mild histological changes were present within the sections of the examined airways that could be directly attributed to aerosol gemcitabine. Four animals exhibited minimal to mild expansion of the submucosa of one or more of their larger conducting airways. The submucosa was thickened by loosely arranged highly vascular connective tissue, which formed short blunt papillary projections that protruded into the airway lumina. No similar lesions were noted among the nontreated animals. Alveoli remained normal in both aerosol-treated and aerosol naïve dogs (Fig. 1A and B). Seven animals, all of which had either pleural effusion or pleural metastases, had marked regionally extensive to generalized chronic proliferative villous pleuritis with prominent, congested vasculature. Six animals had pleural effusion and six animals had pleural metastases; however, only five (of 12) animals possessed both lesions concurrently. In one dog with pleural metastasis, the lungs remained free of pulmonary metastasis. Three dogs with pulmonary and or pleural metastasis developed hypertrophic osteopathy, which was identified antemortem and confirmed postmortem. Alveolar lumina immediately adjacent to metastatic foci were expanded by increased numbers of foamy alveolar macrophages, which frequently possessed abundant intracytoplasmic hemosiderin pigment, indicative of prior hemorrhage. Metastasis was identified in anatomical locations outside of the lungs. These locations included liver (n = 3), spleen (n = 3), kidney (n = 3), adrenal (n = 3), other bones (n = 3), eye (n = 1), and skin (n = 1).

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 ...

Additional histopathological findings in the lungs (data not shown) included multifocal lymphoplasmacytic perivascular inflammation (n = 2), intravascular metastases (n = 3), mild chronic bronchitis (n = 2), peribronchial gland hyperplasia (n = 2), and acute focally extensive neutrophilic interstitial pneumonia (n = 1).

Effect of aerosol gemcitabine on metastatic foci

Extensive, predominately central, intratumoral necrosis was noted within all (100%) aerosol-treated dogs (Fig. 1D). There was marked increase in the incidence and severity of necrosis noted among the treated animals compared to the nontreated cases. The majority (85%) of aerosol-treated metastasis exhibited >25% necrosis. In contrast, only 4 of the 13 (31%) nontreated animals did so and many foci from aerosol-naïve patients had minimal necrosis (Fig. 1C). Forty-six percent of the aerosol-treated animals exhibited >50% necrosis, whereas none of the nontreated animals exhibited >50% necrosis.

Fas expression and apoptosis in spontaneously arising canine osteosarcoma and pulmonary metastases

We examined Fas expression in both the primary tumor (Fig. 2a) and the metastatic foci (Fig. 2b and c) of spontaneously arising canine osteosarcoma. Canine osteosarcoma ranged in the expression of Fas as detected and measured by immunohistochemistry staining intensity (Fig. 2a). Fas staining intensity did not differ between the primary tumors obtained from aerosol gemcitabine-treated and -naïve dogs (p > 0.05; Fig. 2a, compare upper and lower rows). Interestingly, Fas staining intensity was decreased in pulmonary metastases (b) compared to the primary tumor (a), and this difference was statistically significant (p =  0.008; photomicrographs: compare a and b; scatter plot: compare Bone and Untreated). Importantly, the Fas staining intensity in pulmonary metastasis from aerosol gemcitabine-treated animals (c) was greater than that found in both the primary tumor (p = 0.025; photomicrographs: compare a and c; scatter plot: compare Bone and Aerosol Gemcitabine) and in the gemcitabine-naïve metastases (p = 0.0075; Fig. 2, photomicrographs: compare b and c; scatter plot, compare column untreated metastasis and gemcitabine).

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 ...

The FAS/FASL pathway kills metastatic osteosarcoma cells by inducing apoptosis.(41,48–51) To determine if the observed increase in Fas expression resulted in an increase in death of tumor cells by apoptosis, we performed the TUNEL assay (Fig 3). Minimal TUNEL staining was identified in untreated pulmonary metastasis (a). In contrast, TUNEL staining was increased in osteosarcoma cells in the pulmonary metastasis obtained from aerosol gemcitabine treated dogs (b), and this difference was significant (p = 0.028).

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 development of pulmonary metastases is the most common form of treatment failure in both dogs(1) and humans(2) with osteosarcoma. Effective treatment for lung metastases remains a clinical challenge. Therefore, the development of aerosol chemotherapy, which targets delivery to the respiratory system where metastases are most likely to be found, may provide a noninvasive method of therapy for pulmonary metastasis. In this study, we evaluated the novel delivery method of gemcitabine as an aerosol to dogs harboring gross metastasis of osteosarcoma in an effort to determine the preclinical safety and to assess the role of the Fas/FasL pathway in this model system. This is believed to be the first in vivo investigation of the antitumor effects of aerosol gemcitabine in spontaneously arising osteosarcoma in any species.

Previously, aerosol gemcitabine chemotherapy has been used in experimental murine models of osteosarcoma(41,42,48) and in vitro in a lung cancer model.(43) As had been the case in murine(41,42,48) and baboon studies,(43) aerosol gemcitabine was well tolerated in our cancer-bearing dogs. Monitoring of subjective (appetite, attitude, activity levels) and objective (body weight, CBC, and SBA) parameters identified only a single episode of Grade I neutropenia, which resolved without intervention and did not recur. Because these dogs were not trained respiratory research animals, we chose to measure arterial blood gas and alveolar–arterial gradients as a surrogate end point for pulmonary function. We identified no decrease in pulmonary function as a result of aerosol gemcitabine. Taken together, these findings confirmed that aerosol gemcitabine is systemically well tolerated, and suggested that local toxicity (manifest as changes in pulmonary function or radiographic appearance) would also be minimal. To more directly assess local toxicity, tissues from aerosol-treated dogs were evaluated by two veterinary pathologists (T.C., D.W.W.). Multiple sections of tissue from all levels of the respiratory tree, including the proximal and distal trachea, main stem bronchi, terminal airways, and alveolar sacs (Fig. 1A and B) were evaluated for evidence of toxicity associated with inhalational exposure to gemcitabine chemotherapy. There was no evidence of a direct toxicological effect associated with aerosol gemcitabine treatment in tumor-free regions of the lung. Although the submucosal expansion present within four of the animals was minimal, no similar lesion was noted among the nontreated animals. There was no apparent association between these findings and total cumulative dose of aerosol gemcitabine (i.e., duration or number of treatments or 25-mg vs. 50-mg group).

The remaining histopathological findings are considered incidental or due to progression of the metastatic process. The most notable adverse lesion was that of moderate to marked intrapulmonary and pleural hemorrhage secondary to necrosis of the metastases; although it is possible that aerosol gemcitabine was responsible for this finding, the progression of the disease was considered a more likely etiology. These clinicopathological and pathological findings confirmed and extended the murine studies(41,42) in the tumor-bearing dog. This study demonstrated that aerosol gemcitabine is well tolerated both by local tissues and by the body as a whole.

The role of the Fas/FasL pathway in the metastatic potential of osteosarcoma in murine models has been investigated.(41,50,51) In the primary tumor cells, cell surface Fas expression was present but was lost in the pulmonary metastases.(41) Studies have demonstrated that Fas-transfected tumor cells express higher levels of Fas on the cell surface and are rendered more sensitive to Fas-induced cell death than parental lines; as a result, these Fas expressing cells produce significantly fewer pulmonary “metastatic” nodules than nontransfected cells.(50) The Fas/FasL system was also noted to be corrupted in pulmonary osteosarcoma metastasis collected from human surgical specimens, where 32% were weakly positive and 60% of the specimens were negative for Fas staining.(48) These data suggested that loss of Fas cell surface expression may aid osteosarcoma in the evasion of local lung resistance and the establishment of metastatic pulmonary nodules. We were interested in determining if this pathway was also involved in our canine model.

As was the case in mouse and human, the canine primary tumors expressed cell surface Fas, which was decreased or lost in the chemotherapy-naive metastatic foci. In contrast, in response to treatment of dogs with aerosol-gemcitabine there was an increase in Fas cell surface staining intensity in the metastatic pulmonary foci (Fig. 2) and an increase in apoptosis as measured by TUNEL (Fig. 3). Other nucleoside analogues such as nelarabine(52) and cytarabine(53) or other anticancer drugs at therapeutic concentrations such as doxorubicin, methotrexate, cisplatin, and etoposide(53,54) have also been shown to increase Fas expression.

Necrosis, however, is not a hallmark of Fas-mediated cell killing. The observed central necrosis with viable peripheral tumor cells and no inflammatory response has been observed in lesions resected following salvage chemotherapy or tumor necrosis factor (TNF) treatment.(55,56) In contrast to those findings, we found an increased number of inflammatory cells immediately adjacent to the metastatic foci. This inflammatory response could be secondary to the increased incidence and severity of intratumoral necrosis noted within the treated animals. Previous studies in mice have evaluated the histopathological changes in metastases after only 4–12 treatments (2–6 weeks of twice weekly treatment).(41,42) In the human studies pulmonary inflammation was either not evaluated(48) or the metastatic tissues were removed after a single dose of salvage chemotherapy.(56) In contrast, our patient dogs received a median of 28 treatments (14 weeks of twice weekly treatment). Therefore, it is possible that with prolonged therapy the initial Fas-mediated apoptosis of tumor cells in the metastatic foci is eclipsed by progressive destruction of the metastatic nodule so that the predominant pathological aspect within the foci would be necrotic with attendant inflammation, as was seen in our dogs. As was the case in the local tissues, there was no apparent association between these findings and total cumulative dose of aerosol gemcitabine (i.e., duration or number of treatments or 25-mg vs. 50-mg group). The study was not powered to identify an association between dose and degree of response.

It is also possible that both/either the gemcitabine chemotherapy itself and/or the gemcitabine-induced upregulation of Fas in the FasL-rich environment of the lung resulted in the marked necrosis of the metastasis. The central necrosis could also be a direct effect of gemcitabine on the tumor neovasculature.(57) Previous studies in the baboon have demonstrated marked variability in gemcitabine deposition in the lung both between therapy sessions and between subjects.(43) We did not measure plasma levels or deposition of gemcitabine or the accumulation of gemcitabine triphosphate (the active metabolite) in the various areas of the lung in these patients because all had last received therapy more than 2 days prior to euthanasia and because the levels would likely be extremely low. Variability in pulmonary deposition could also account for the marked variability in both the observed, FAS expression, TUNEL staining, and necrosis. Objective measure of the deposition of aerosolized gemcitabine in the dog lung in normal dogs is being considered in an effort to be able correlate degree of biological response with any local toxicity or tumor response.

Metastatic osteosarcoma carries a poor long-term prognosis in the dog. The median time (days) to metastasis [disease-free interval (DFI); 223] and the overall survival (OS; 289) of approximately 700 dogs with osteosarcoma in relapse for which no treatment was performed can be estimated (66 days ± 44) from the literature. Our cohort of 21 dogs had a median DFI (139), survival in relapse (79), and OS (262), which were similar. Although aerosol gemcitabine demonstrates FAS-mediated antitumor properties against metastatic canine osteosarcoma, none of these dogs had radiographic resolution of their disease and these survival numbers underscore our realization that this novel delivery will need to be combined with other modes of therapy to maximize survival both in this relevant large animal model and in children with metastatic osteosarcoma.

The metastatic cascade is a complex process requiring multiple steps that necessitate the perfect union of host, tumor, and local microenvironmental factors. We acknowledge the important and necessary complementary roll played by local FasL in the eradication of metastatic osteosarcoma. In gld mice (that lack normal FasL function), aerosol gemcitabine had no effect in eradicating metastases from the lung. In the case of our dogs, we identified metastatic foci outside of the lung in the relatively FasL poor locations such as liver, kidneys, and skin. Although Fas was not specifically evaluated in these extrapulmonary foci, their presence speaks to the need for additional treatment strategies in combination with aerosol gemcitabine in the management of metastatic osteosarcoma.

In conclusion, the aerosol method of gemcitabine administration was very well tolerated regionally and systemically by our canine patients. The results from this pivotal translational study in a highly relevant immune competent large-animal tumor model of human osteosarcoma support the notion that aerosol gemcitabine may be useful against pulmonary metastasis of osteosarcoma. Although there were no cures, because there was a demonstrated histological tumor response, additional studies that evaluate the aerosol route of administration of gemcitabine as a form of therapy in metastatic pediatric osteosarcoma combined with other metastasis-effective, FasL-inducing treatments such as IL-12 or ifosfamide; or immunity modulators, such liposome encapsulated muramyl tripeptide, are warranted.


This work was funded by a Center for Companion Animal Health Grant 06-07 (C.O.R.) and National Institute of Health Grant CA42992 (E.S.K.). C.O.R. would like to thank Charlotte Marra for her generous support, and is indebted to the guardians of these beloved companions for their tireless effort; without their dedication, this work would not have been possible. P.M.A. acknowledges Research Support from the University of Texas M.D. Anderson Cancer Center, and is grateful for additional research support from the Thorrison, Rosen, Belvo, Haynie, Meares, Silberblatt, Brodie, Jager, and Lash families.

Author Disclosure Statement

No conflicts of interest exist.


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