Given the close interaction between tumor cells and stromal cells in the tumor microenvironment (TME), TME-targeted strategies would be promising for developing integrated cancer immunotherapy. Cancer-associated fibroblasts (CAFs) are the dominant stromal component, playing critical roles in generation of the pro-tumorigenic TME. We focused on the immunosuppressive trait of CAFs, and systematically explored the alteration of tumor-associated immune responses by CAF-targeted therapy. C57BL/6 mice s.c. bearing syngeneic E.G7 lymphoma, LLC1 Lewis lung cancer, or B16F1 melanoma were treated with an anti-fibrotic agent, tranilast, to inhibit CAF function. The infiltration of immune suppressor cell types, including regulatory T cells and myeloid-derived suppressor cells, in the TME was effectively decreased through reduction of stromal cell-derived factor-1, prostaglandin E2, and transforming growth factor-β. In tumor-draining lymph nodes, these immune suppressor cell types were significantly decreased, leading to activation of tumor-associated antigen-specific CD8+ T cells. In addition, CAF-targeted therapy synergistically enhanced multiple types of systemic antitumor immune responses such as the cytotoxic CD8+ T cell response, natural killer activity, and antitumor humoral immunity in combination with dendritic cell-based vaccines; however, the suppressive effect on tumor growth was not observed in tumor-bearing SCID mice. These data indicate that systemic antitumor immune responses by various immunologic cell types are required to bring out the efficacy of CAF-targeted therapy, and these effects are enhanced when combined with effector-stimulatory immunotherapy such as dendritic cell-based vaccines. Our mouse model provides a novel rationale with TME-targeted strategy for the development of cell-based cancer immunotherapy.

Tumor-draining lymph nodes (DLN) are the most important priming sites for generation of antitumor immune responses. They are also the location where an immunosuppressive cytokine, transforming growth factor-β (TGF-β), plays a critical role in suppressing these antitumor immune responses. We focused on TGF-β-mediated immunosuppression in DLNs and examined whether local inhibition of TGF-β augmented antitumor immune responses systemically in tumor-bearing mice models. For inhibition of TGF-β-mediated immunosuppression in DLNs, C57BL/6 mice subcutaneously bearing E.G7 tumors were administered plasmid DNA encoding the extracellular domain of TGF-β type II receptor fused to the human IgG heavy chain (TGFR DNA) i.m. near the established tumor. In DLNs, inhibition of TGF-β suppressed the proliferation of regulatory T cells and increased the number of tumor antigenspecific CD4 + or CD8 + cells producing IFN-γ. Enhancement of antitumor immune responses in DLNs were associated with augmented tumor antigen-specific cytotoxic and natural killer activity in spleen as well as elevated levels of tumor-specific antibody in sera. The growth of the established metastatic as well as primary tumors was effectively suppressed via augmented antitumor immune responses. Inhibition of TGF-β-mediated immunosuppression in DLNs is significantly associated with augmented antitumor responses by various immunocompetent cell types. This animal model pro-vides a novel rationale for molecular cancer therapeutics targeting TGF-β. [Cancer Res 2009;69(12):5142-50]

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Galectin-3 plays crucial roles in tumor progression. However, in non-small cell lung cancer (NSCLC), it remains unclear whether the hypoxic tumor microenvironment enhances galectin-3-induced cell motility. We investigated galectin-3 expression in NSCLC cells under hypoxia, and the possible molecular mechanisms by which galectin-3 influences tumor aggressiveness. Galectin-3 levels in NSCLC cell lines under hypoxia were assessed using reverse transcription PCR and western blotting. To clarify the role of endogenous galectin-3, the effect of galectin-3 knockdown in NSCLC cells was investigated using scratch and invasion assays. The expression and clinicopathological significance of galectin-3 in 57 patients with pN0M0 invasive pulmonary adenocarcinoma were investigated by immunohistochemistry. Both mRNA and protein levels of galectin-3 in the NSCLC cell lines A549 and LK-2 were upregulated by hypoxia. As revealed by scratch and invasion assays, the cell migratory and invasive activities were significantly increased under hypoxia, but were reduced by galectin-3 knockdown. Notably, addition of galectin-3 to the media did not improve the cell motility impaired by galectin-3 knockdown. To clarify the role of endogenous galectin-3 in the enhancement of tumor cell motility under hypoxia, we focused on the function of RhoA. RhoA level in the plasma membrane, but not in the cytoplasm, was increased under hypoxia and decreased by galectin-3 knockdown. RhoA activity was significantly enhanced under hypoxia and effectively inhibited by galectin-3 knockdown. In patients with pN0M0 invasive pulmonary adenocarcinoma, higher galectin-3 expression on tumor cells was significantly associated with tumor cell invasion into microvessels and tumor recurrence after surgery. These data demonstrate that in NSCLC cells under hypoxia, upregulated galectin-3 levels increase the localization of RhoA to the plasma membrane, thus enhancing RhoA activity, which is associated with aggressive cell motility. In pN0M0 invasive pulmonary adenocarcinoma, galectin-3 is a potential biomarker for predicting tumor recurrence after radical surgery.

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