Tumor-associated macrophages drive spheroid formation during early transcoelomic metastasis of ovarian cancer
Mingzhu Yin, Xia Li, Shu Tan, Huanjiao Jenny Zhou, Weidong Ji, Stefania Bellone, Xiaocao Xu, Haifeng Zhang, Alessandro D. Santin, Ge Lou, Wang Min
Mingzhu Yin, Xia Li, Shu Tan, Huanjiao Jenny Zhou, Weidong Ji, Stefania Bellone, Xiaocao Xu, Haifeng Zhang, Alessandro D. Santin, Ge Lou, Wang Min
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Research Article Inflammation Oncology

Tumor-associated macrophages drive spheroid formation during early transcoelomic metastasis of ovarian cancer

Abstract

Tumor-associated macrophages (TAMs) can influence ovarian cancer growth, migration, and metastasis, but the detailed mechanisms underlying ovarian cancer metastasis remain unclear. Here, we have shown a strong correlation between TAM-associated spheroids and the clinical pathology of ovarian cancer. Further, we have determined that TAMs promote spheroid formation and tumor growth at early stages of transcoelomic metastasis in an established mouse model for epithelial ovarian cancer. M2 macrophage–like TAMs were localized in the center of spheroids and secreted EGF, which upregulated αMβ2 integrin on TAMs and ICAM-1 on tumor cells to promote association between tumor cells and TAM. Moreover, EGF secreted by TAMs activated EGFR on tumor cells, which in turn upregulated VEGF/VEGFR signaling in surrounding tumor cells to support tumor cell proliferation and migration. Pharmacological blockade of EGFR or antibody neutralization of ICAM-1 in TAMs blunted spheroid formation and ovarian cancer progression in mouse models. These findings suggest that EGF secreted from TAMs plays a critical role in promoting early transcoelomic metastasis of ovarian cancer. As transcoelomic metastasis is also associated with many other cancers, such as pancreatic and colon cancers, our findings uncover a mechanism for TAM-mediated spheroid formation and provide a potential target for the treatment of ovarian cancer and other transcoelomic metastatic cancers.

Authors

Mingzhu Yin, Xia Li, Shu Tan, Huanjiao Jenny Zhou, Weidong Ji, Stefania Bellone, Xiaocao Xu, Haifeng Zhang, Alessandro D. Santin, Ge Lou, Wang Min

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Figure 4

Reciprocal upregulation of Egf in TAMs and Egfr expression in tumor cells are critical for OC growth.

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Reciprocal upregulation of Egf in TAMs and Egfr expression in tumor cell...
(A–C) TAMs promote ID8 cell proliferation in vitro. Peritoneal spheroids were harvested from OC-bearing mice at 8 weeks after tumor implantation. TAMs and ID8 tumor cells (PE-ID8) were isolated, and PE-ID8 cells were cultured alone or cocultured with TAMs in a Transwell without direct contacts. Naive ID8 cells were used as controls. (A) Total cell number was counted at times indicated. (B) Cell proliferation was measured by Ki67 staining. Scale bar: 100 μm. (C) Ki67+ tumor cells were quantified. n = 9. (D) Reciprocal upregulation of EGF in TAMs and EGFR expression in PE-ID8 cells. Gene expression of Egf and Egfr in TAMs and PE-ID8 was determined by qRT-PCR. Peripheral blood monocytes and naive ID8 tumor cells were used as controls. Relative gene expression is presented as fold change in relation to monocytes as 1.0. n = 3. (E) Immunofluorescent staining of CD68 with EGF or EGFR in spheroids harvested from ascites of OC mice. Representative images of spheroids from n = 5 mice are shown. Scale bar: 20 μm. (F and G) KD of EGF by siRNAs. TAMs were transfected with control (ctrl) or EGF siRNAs for 48 hours. (F) Egf mRNA levels in TAMs detected by qRT-PCR. (G) EGF protein levels in supernatant of TAMs cocultured with ID8 cells were measured by ELISA. (H and I) ID8 cells were treated with EGF in the absence or presence of EGFR inhibitor (10 or 20 μM) for 12 hours. Phospho- and total EGFR and ERK1/2 were determined by Western blot with respective antibodies. Total EGFR, ERK1/2, and GAPDH were determined. Relative phosphorylation levels were quantified. (J and K) TAMs were pretransfected with control siRNA or EGF siRNA. PE-ID8 cells were cultured alone or cocultured with TAMs in a Transwell in the absence or presence of EGF (20 ng/ml) or EGFR inhibitor (20 μM) for 12 hours. Proliferating PE-ID8 cells were stained by Ki67. Representative images are shown (J) with quantification of Ki67+ cells (K). Scale bars: 100 μm. Three different replicates were performed for all experiments. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (2-sided Student’s t test).