Targeting tumor-associated macrophages as a novel strategy against breast cancer
Yunping Luo, He Zhou, Jörg Krueger, Charles Kaplan, Sung-Hyung Lee, Carrie Dolman, Dorothy Markowitz, Wenyuan Wu, Cheng Liu, Ralph A. Reisfeld, Rong Xiang
Yunping Luo, He Zhou, Jörg Krueger, Charles Kaplan, Sung-Hyung Lee, Carrie Dolman, Dorothy Markowitz, Wenyuan Wu, Cheng Liu, Ralph A. Reisfeld, Rong Xiang
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Research Article Oncology

Targeting tumor-associated macrophages as a novel strategy against breast cancer

Abstract

Tumor-associated macrophages (TAMs) are associated with tumor progression and metastasis. Here, we demonstrate for the first time that legumain, a member of the asparaginyl endopeptidase family functioning as a stress protein, overexpressed by TAMs, provides an ideal target molecule. In fact, a legumain-based DNA vaccine served as a tool to prove this point, as it induced a robust CD8+ T cell response against TAMs, which dramatically reduced their density in tumor tissues and resulted in a marked decrease in proangiogenic factors released by TAMs such as TGF-β, TNF-α, MMP-9, and VEGF. This, in turn, led to a suppression of both tumor angiogenesis and tumor growth and metastasis. Importantly, the success of this strategy was demonstrated in murine models of metastatic breast, colon, and non–small cell lung cancers, where 75% of vaccinated mice survived lethal tumor cell challenges and 62% were completely free of metastases. In conclusion, decreasing the number of TAMs in the tumor stroma effectively altered the tumor microenvironment involved in tumor angiogenesis and progression to markedly suppress tumor growth and metastasis. Gaining better insights into the mechanisms required for an effective intervention in tumor growth and metastasis may ultimately lead to new therapeutic targets and better anticancer strategies.

Authors

Yunping Luo, He Zhou, Jörg Krueger, Charles Kaplan, Sung-Hyung Lee, Carrie Dolman, Dorothy Markowitz, Wenyuan Wu, Cheng Liu, Ralph A. Reisfeld, Rong Xiang

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Figure 5

Abrogation of TAMs results in decreases of growth factor release, tumor cell migration, and metastases.

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Abrogation of TAMs results in decreases of growth factor release, tumor ...
(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).