Neutrophils responsive to endogenous IFN-β regulate tumor angiogenesis and growth in a mouse tumor model
Jadwiga Jablonska, … , Stefan Lienenklaus, Siegfried Weiss
Jadwiga Jablonska, … , Stefan Lienenklaus, Siegfried Weiss
Published March 8, 2010
Citation Information: J Clin Invest. 2010;120(4):1151-1164. https://doi.org/10.1172/JCI37223.
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Neutrophils responsive to endogenous IFN-β regulate tumor angiogenesis and growth in a mouse tumor model

Abstract

Angiogenesis is a hallmark of malignant neoplasias, as the formation of new blood vessels is required for tumors to acquire oxygen and nutrients essential for their continued growth and metastasis. However, the signaling pathways leading to tumor vascularization are not fully understood. Here, using a transplantable mouse tumor model, we have demonstrated that endogenous IFN-β inhibits tumor angiogenesis through repression of genes encoding proangiogenic and homing factors in tumor-infiltrating neutrophils. We determined that IFN-β–deficient mice injected with B16F10 melanoma or MCA205 fibrosarcoma cells developed faster-growing tumors with better-developed blood vessels than did syngeneic control mice. These tumors displayed enhanced infiltration by CD11b+Gr1+ neutrophils expressing elevated levels of the genes encoding the proangiogenic factors VEGF and MMP9 and the homing receptor CXCR4. They also expressed higher levels of the transcription factors c-myc and STAT3, known regulators of VEGF, MMP9, and CXCR4. In vitro, treatment of these tumor-infiltrating neutrophils with low levels of IFN-β restored expression of proangiogenic factors to control levels. Moreover, depletion of these neutrophils inhibited tumor growth in both control and IFN-β–deficient mice. We therefore suggest that constitutively produced endogenous IFN-β is an important mediator of innate tumor surveillance. Further, we believe our data help to explain the therapeutic effect of IFN treatment during the early stages of cancer development.

Authors

Jadwiga Jablonska, Sara Leschner, Kathrin Westphal, Stefan Lienenklaus, Siegfried Weiss

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Figure 5

Altered characteristics of CD11b+Gr1+ neutrophils isolated from Ifnb1–/– mice.

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Altered characteristics of CD11b+Gr1+ neutrophils isolated from Ifnb1–/–...
(A) Percentage of CXCR4+ neutrophils in blood of tumor-bearing Ifnb1–/– and C57BL/6 mice. (B) Percentage of CXCR4+ neutrophils isolated from tumors of Ifnb1–/– and C57BL/6 mice. Tumors were removed; single-cell solutions were prepared, stained, and analyzed using the BD LSR II System. Data were analyzed with FACSDiva software. Experiments were done twice with at least 5 animals per group. Data represent mean ± SEM. *P ≤ 0.01. (C) IFN-β treatment downregulates Vegf, Mmp9, and Cxcr4 gene expression. Tumors were removed; single-cell solutions were prepared and stained, and CD11b+Gr1+ neutrophils were isolated. Monolayers of such cells were incubated with 5 U rmIFN-β; after 4 hours, RNA was isolated, cDNA prepared, and gene expression measured using real-time RT-PCR. Cells were derived from 5 pooled tumors. All experiments were repeated at least 2 times. (D) High expression of Cxcr4 correlates with higher expression of c-myc and Stat3, and rmIFN-β downregulates both c-myc and Stat3. CD11b+Gr-1+ neutrophils were sorted from tumors and placed in culture with 5 U rmIFN-β. After 4 hours, cDNA was prepared as described in C. Cells were sorted from 5 pooled tumors. All experiments were repeated at least 2 times. (E) Expression of intracellular pSTAT3 in blood and tumor-infiltrating neutrophils was compared between control and Ifnb1–/– mice. Cell suspensions from blood and tumors were prepared, stained, and analyzed using the BD LSR II System. Data were analyzed with FACSDiva software. Experiments were done twice with at least 5 animals per group.