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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]

BACKGROUNDThe authors attempted to obtain shared proteins among lung carcinoma cells by column chromatographies. A glycoprotein with approximately 500 kDa isolated from QG56 cells showed an identical amino acid sequence to 90K/Mac‐2 binding protein (M2BP). This protein has been reported to be highly expressed and to modulate the expression of surface molecules involved in immune responses on cultured cancer cells. Therefore, it would be beneficial for M2BP to be targeted in cancer immunotherapy.METHODSThe authors analyzed the expression of M2BP in lung carcinoma cells and M2BP's immunogenicity as a tumor antigen. Eight cultured lung carcinoma cell lines and 28 tumor tissues from patients with lung carcinoma were examined for the expression of M2BP mRNA and protein. Sera from cancer patients (n = 23) and healthy donors (n = 19) were studied for their reactivity to M2BP peptides by enzyme–linked immunosorbent assay.RESULTSSeven of the 8 (87.5%) lung carcinoma cell lines and 17 of the 28 (60.7%) tumor tissues expressed high levels of M2BP mRNA. Most of the M2BP mRNA‐positive cancer cell lines and tumors also showed M2BP protein expression. The serum levels of antibodies to M2BP were elevated in 30.4% of the patients. In addition, M2BP‐specific immunoglobulin G was observed in all patients with anti‐M2BP antibodies.CONCLUSIONSM2BP is highly expressed in lung carcinoma cells and is sufficiently immunogenic to elicit specific immunity to this molecule in patients with lung carcinoma. M2BP is expected to be useful as a tumor marker and a target antigen in cancer immunotherapy. Cancer 2002;95:1954–62. © 2002 American Cancer Society.DOI 10.1002/cncr.10899

Context.—Podoplanin is a mucin-type glycoprotein and a lymphatic endothelial marker. Immunohistochemical staining for podoplanin is currently used as a routine pathologic diagnosis tool in Japan to identify lymphatic invasion of cancer cells. Recent reports suggest that podoplanin and other proangiogenic molecules are expressed in stromal fibroblasts and myofibroblasts. Objective.—To analyze the distribution of podoplanin expression in tumor stroma and its clinical and biologic significance. Design.—We performed immunohistochemistry for podoplanin on tissue microarrays from 1350 cases of 14 common cancer types. Results.—Two hundred eighty-seven of 662 cases (43%) showed podoplanin expression in the stromal cells within cancer nests. Stromal podoplanin expression in 14 common cancer types was significantly associated with tumor stage (P < .001), lymph node metastases (P < .001), lymphatic invasion (P  =  .02), and venous invasion (P < .001). The stromal cells positive for podoplanin were also positive for α-smooth muscle actin but negative for desmin, confirming a myofibroblasts phenotype. In contrast, myofibroblasts in inflammatory fibrotic lung diseases were podoplanin negative. Lymphatic vessel density was greater in the stromas with podoplanin expression than in the stroma lacking podoplanin-expressing stromal cells (P  =  .01). Survival data were available for non–small cell lung cancer. Stromal podoplanin expression was associated with poorer prognosis in adenocarcinoma (P < .001) and remains statistically significant after adjustment for sex, age, and stage (P  =  .01). Conclusion.—Our data indicate that podoplanin expression in stromal myofibroblasts may function as a proangiogenic biomarker and may serve as a predictive marker of lymphatic/vascular spread of cancer cells and a prognostic marker of patient survival.

In order to induce specific antitumor immunity in mice, we attempted to immunize C57BL / 6 mice with DNA vaccine encoding MUC1 polypeptide. When the mice immunized with MUC1 DNA were challenged with EL4 -muc, MUC1 -transfected syngeneic lymphoma cells, they completely rejected tumors. When DNA vaccine was given to the EL4 -muc tumor -bearing mice, this vaccination was insufficient to suppress tumor growth in the mice. However, activated, but nonprimed dendritic cells ( DCs ) obtained from syngeneic mice and MUC1 DNA vaccine were given simultaneously to the same site of EL4 -muc tumor -bearing mice, tumor growth was markedly suppressed accompanying prolongation of survival time. MUC1 antigen was detected on the DCs at the vaccination site and in regional nodes in the mice which received MUC1 DNA vaccine and DCs. These mice showed markedly enhanced cellular immune responses specific for MUC1 compared to those in mice vaccinated with MUC1 DNA alone. No significant difference in titers of antibodies to MUC1 between the two groups was observed. These results suggest that nonprimed DCs inoculated at the DNA vaccine site are essential for eliciting strong antitumor cellular immunity to suppress tumor growth efficiently in DNA -vaccinated mice. This animal model is useful for developing DNA vaccine for anti-cancer immunotherapy.