(A) Legumain expression on TAMs was clearly evident. Tumor-infiltrating macrophages were visualized by H&E staining, as indicated by arrows. Legumain expression is indicated by double staining with anti-legumain Ab (green) combined with anti-CD68⁺ Ab (red). Magnification, ×350. (B) Increased legumain expression on TAMs was confirmed by flow cytometric analyses with double-positive populations of F4/80⁺/CD206⁺ M2 macrophages that were isolated from fresh tumor tissue. (C) Multiple-color flow cytometry demonstrated upregulation of the M2 macrophage marker CD206 on RAW cells after being cultured with IL-4, IL-10, and IL-13 (10 ng/ml). Furthermore, legumain was shown to be highly expressed on F4/80⁺/CD206⁺-positive RAW cells cultured with IL-4, IL-10, and IL-13 (upper panels) compared with wild-type RAW cells (lower panels). (D) Confirmation of legumain expression on RAW cells by Western blotting following stimulation with IL-4, IL-13, and IL-10, either singularly or combined.

(A) RAW macrophage cells, which highly express legumain after culturing with 10 ng/ml IL-4, IL-10, and IL-13, served as target cells in a 4-hour ⁵¹Cr release assay. Splenocytes isolated from mice immunized with the pLegumain vaccine were shown to effectively kill RAW cells treated with these cytokines in vitro at different effector-to-target (E/T) cell ratios but failed to induce cytotoxic killing of unstimulated RAW cells lacking legumain expression. **P < 0.005 compared with control groups. (B) The percentage of TAM populations with specific macrophage markers (CD206 and F4/80) in tumor tissue with or without vaccination was detected by flow cytometry. The percentage of TAM populations among tumor tissue cells isolated from mice treated with our DNA vaccine was shown to be reduced; however, there was no decrease in TAM populations isolated from mice treated with either empty vector or pLegumain following CD8⁺ T cell depletion (**P < 0.005). (C) The results of flow cytometry were confirmed by immunohistochemical staining evaluated by confocal microscopy. The population of TAMs in the tumor stroma was dramatically decreased after vaccination. 4T1 cancer cells are shown in blue and TAMs in red. Magnification, ×50 (H&E) and ×350 (control, empty vector, and pLegumain).

(A) The vaccine decreased the release of growth factors by TAMs. 4T1 breast tumor tissue and mouse serum were harvested 12 days after vaccinations and tumor cell challenge. After 24 or 48 hours culturing, the supernatants of tumor tissue cells were harvested, and the concentrations of TGF-β, TNF-α, and VEGF in serum or supernatants measured by ELISA. There were significant differences between the treatment and control groups. *P < 0.01; **P < 0.005. (B) Immunohistochemical staining was performed to determine expression of these growth factors in the tumor microenvironment. In the vaccine treatment groups, VEGF, TGF-β, and MMP-9 release was decreased after a reduction in TAMs, compared with the empty vector groups. The growth factors are shown in green and 4T1 breast cancer cells in blue. (C) A Transwell migration assay was performed to determine tumor cell migration after vaccination. The number of migrating cells isolated from 4T1 tumor tissue was markedly reduced after vaccination. ^#P < 0.001 compared with the empty vector group. (D) In vivo experiments were performed to determine the ability of mice to form 4T1 tumor metastases. The mice were treated with the vaccine within the therapeutic setting as described above. Tumor metastasis scores and lung weights were measured 25 days after primary tumor excision. The metastasis scores are expressed as the percentage of lung surface covered by fused metastatic foci; 0: none; 1: <5%; 2: 5– 50%; 3: >50%. Scores for n = 8 mice/group were: PBS, 3, 3, 3, 3, 3, 3, 2, 2; empty vector, 3, 3, 3, 3, 3, 3, 3, 2; pLegumain, 2, 2, 1 0, 0, 0, 0, 0. Differences in lung weights between the group of mice treated with vaccine and all control groups were statistically significant (**P < 0.005). Magnification, ×350 (B), ×50 (C).

(A) Schematic of DNA vaccines constructed with the pCMV/myc/cyto vector backbone where the legumain gene was fused to the C terminal of mutant polyubiquitin. The entire fragment was inserted, and protein expression was demonstrated by Western blotting. Mu-legumain, murine legumain. (B) Prophylactic model: The vaccination schedule was designed for 3 immunizations at 1-week intervals followed by an i.v. challenge with 2 × 10⁵ D121 non–small cell lung cancer cells and 5 × 10⁴ CT26 colon cancer cells and mammary fat pad injection with 7 × 10³ 4T1 breast cancer cells. Lung weights were determined 24 days (D121 or CT26) or 30 days (4T1) after tumor cell challenge and analyzed in each group. Differences between the 2 control groups (PBS and/or empty vector) and the treatment group were statistically significant; **P < 0.005. Pre-challenge lung weight, 0.2 g. (C) Therapeutic model: Groups of BALB/c mice (n = 8) were initially injected in the mammary fat pad with 7 × 10³ 4T1 breast cancer cells and thereafter vaccinated 3 times on days 3, 7, and 11 with PBS, empty vector, or the pLegumain vaccine, respectively, and primary tumors excised on day 12. Survival plots represent results for 8 mice in each of the treatment and control groups. The difference between the empty vector control group and the treatment group was statistically significant; **P < 0.005.

(A) DNA vaccination enhances expression of costimulatory molecules by DCs. Lymphocytes from Peyer’s patches obtained 3 days after vaccination were stained with FITC-labeled anti-CD11cAb in combination with PE-conjugated anti-CD80, anti–MHC class I, or anti-CD40 Abs. *P < 0.05 compared with control groups. (B) Intracytoplasmic IFN-γ release of CD8⁺ T cells was measured by FACS analysis. **P < 0.005 compared with control groups. (C) Production of specific IFN-γ was verified at the single-cell level by ELISPOT. This is indicated for lymphocytes from immunized mice restimulated with either legumain⁺ 4T1 tumor tissue cells or legumain^– 4T1 cells, as indicated by the number of immunospots formed per well. **P < 0.005 compared with treatment group without stimulation; ^##P < 0.005 compared with control groups. (D) Splenocytes isolated from treated mice were effective in killing TAMs according to a ⁵¹Cr release assay (^#P < 0.01 compared with control groups). Inhibition experiments with Abs against H-2K^d/H-2D^d MHC class I antigens showed that T cell–mediated tumor cell lysis was MHC class I antigen restricted. Furthermore, in vivo depletions of CD4⁺ or CD8⁺ T cells indicated that lymphocytes isolated from vaccinated mice, which were thereafter depleted of CD8⁺ T cells, failed to induce cytotoxic killing of target cells. However, depletion of CD4⁺ T cells did not abrogate cytotoxic killing of these same target cells. ^#P < 0.01 compared with PBS or empty vector group.

Suppression of VEGF-induced angiogenesis: BALB/c mice were vaccinated with empty vector, pLegumain, or pLegumain after either CD8⁺ or CD4⁺ T cell depletion in vivo. One week after the last vaccination, Matrigel was implanted s.c. into the midline of the abdomen of mice. Vascularization was induced by VEGF or bFGF. (A) The images were taken by a digital camera 6 days after Matrigel plug implantation. Additionally, the section of Matrigel plugs stained with Masson’s trichrome indicate blood vessel growth in Matrigel plugs, as indicated by arrows. Magnification, ×50. (B) Quantification of vessel growth was performed after in vivo staining of endothelium with FITC-labeled isolectin B4 and evaluation by fluorimetry. There was a decrease in the VEGF-induced neovasculature only after vaccination with the vector encoding legumain but not after vaccination with the empty vector or with pLegumain after depletion of CD8⁺ T cells. **P < 0.005, *P < 0.01 compared with the legumain treatment group. (C) Immunohistochemical staining was performed and evaluated by confocal microscopy. The cross-sections of Matrigel plugs were stained to determine the cell type that grew in or migrated into these plugs. The images indicate that endothelial cells with the CD31 marker or macrophages with the CD68 marker grew in or migrated into Matrigel plugs as indicated (magnification, ×350). H&E staining served as a control (magnification ×50).