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

Abstract

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.

FIG. 1.

Activation of the Fas/FasL apoptotic pathway by interaction of Fas+ cells with FasL. The classical (type I) apoptosis pathway involves caspase 8. Fas (a) binds with Fas ligand (FasL, b), the death-inducing signaling complex (DISC) (c) assembles, procaspase 8 is cleaved, and caspase 8 (c) is activated. Cleaved caspase 8 (d) triggers activation of effector caspases 3, 6, and 7 (e), and apoptosis occurs (f). The mitochondrial pathway involves caspase 9. Procaspase 9 (g) is activated upon the release of cytochrome c (h), which binds apoptotic protease activating factor- 1 (Apaf-1) (i). The activated complex, apoptosome, (j) cleaves and activates effector caspases 3, 6, and 7 (e), with subsequent apoptosis (f). FADD = Fas-associated death domain protein; FLIP = FLICE inhibitory protein; Bcl-2 = B-cell lymphoma protein 2; IAPs = inhibitor of apoptotic proteins.

FIG. 2.

Fas expression in metastatic and non-metastatic OS cells. (A) Northern blot analyses of Fas expression in nonmetastatic parental (P) SAOS cells and the metastatic LM2 and LM6 sublines. GADPH = glyceraldehyde-3-phosphate dehydrogenase. (B) Cell surface Fas in nonmetastatic K7, intermediate metastatic K12, and highly metastatic K7M2 mouse OS cells as quantified using flow cytometry. MFI = mean fluorescence intensity. (C) Immunohistochemistry for Fas expression in K7M2 lung metastases. Normal lung (a). Osteosarcoma lung nodule (b). Tumor necrosis (c). Fas+ cells are depicted in brown and Fas− cells are depicted in blue.

FIG. 3.

Blocking the Fas signaling pathway allows Fas+ cells to induce lung metastases following i.v. injection. Fas+ K7 cells were transfected with FDN, Fas-associated death domain dominant negative or a control (neo) vector and injected into mice. Mice were euthanized 4 weeks later, and lung metastases were quantified.

FIG. 4.

Aerosol Gemcitabine (GCB) induces Fas expression and the regression of established OS lung metastases in BALB/C mice.^(36,38) (A) GCB upregulates Fas expression. Flow cytometry of Fas expression in OS K7M3 cells, showed Fas upregulation in the treated group. MFI = mean fluorescence intensity. (B,C) K7M3 cells (3 × 10⁶) were injected i.v. into Balb/c mice. Mice were treated with aerosol GCB weekly (b) or 3 × /week (c) for 2.5 weeks. Mice were then euthanized. Lung nodules were quantified and analyzed for Fas expression by immunohistochemistry (C). Brown depicts Fas+ cells and blue Fas− cells.

FIG. 5.

Aerosol GCB induces apoptosis of OS lung metastases. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used as a marker for apoptosis. Positive cells are stained brown. Lung tumor (a) was used as a positive control. Untreated OS lung metastasis (b) has low to no TUNEL expression compared to GCB treated (c).