Jiufeng Li

CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis

Macrophages, which are abundant in the tumour microenvironment, enhance malignancy. At metastatic sites, a distinct population of metastasis-associated macrophages promotes the extravasation, seeding and persistent growth of tumour cells. Here we define the origin of these macrophages by showing that Gr1-positive inflammatory monocytes are preferentially recruited to pulmonary metastases but not to primary mammary tumours in mice. This process also occurs for human inflammatory monocytes in pulmonary metastases of human breast cancer cells. The recruitment of these inflammatory monocytes, which express CCR2 (the receptor for chemokine CCL2), as well as the subsequent recruitment of metastasis-associated macrophages and their interaction with metastasizing tumour cells, is dependent on CCL2 synthesized by both the tumour and the stroma. Inhibition of CCL2–CCR2 signalling blocks the recruitment of inflammatory monocytes, inhibits metastasis in vivo and prolongs the survival of tumour-bearing mice. Depletion of tumour-cell-derived CCL2 also inhibits metastatic seeding. Inflammatory monocytes promote the extravasation of tumour cells in a process that requires monocyte-derived vascular endothelial growth factor. CCL2 expression and macrophage infiltration are correlated with poor prognosis and metastatic disease in human breast cancer. Our data provide the mechanistic link between these two clinical associations and indicate new therapeutic targets for treating metastatic breast cancer.

A Distinct Macrophage Population Mediates Metastatic Breast Cancer Cell Extravasation, Establishment and Growth

Background The stromal microenvironment and particularly the macrophage component of primary tumors influence their malignant potential. However, at the metastatic site the role of these cells and their mechanism of actions for establishment and growth of metastases remain largely unknown. Methodology/Principal Findings Using animal models of breast cancer metastasis, we show that a population of host macrophages displaying a distinct phenotype is recruited to extravasating pulmonary metastatic cells regardless of species of origin. Ablation of this macrophage population through three independent means (genetic and chemical) showed that these macrophages are required for efficient metastatic seeding and growth. Importantly, even after metastatic growth is established, ablation of this macrophage population inhibited subsequent growth. Furthermore, imaging of intact lungs revealed that macrophages are required for efficient tumor cell extravasation. Conclusion/Significance These data indicate a direct enhancement of metastatic growth by macrophages through their effects on tumor cell extravasation, survival and subsequent growth and identifies these cells as a new therapeutic target for treatment of metastatic disease.

Myeloid WNT7b Mediates the Angiogenic Switch and Metastasis in Breast Cancer

Oncogenic targets acting in both tumor cells and tumor stromal cells may offer special therapeutic appeal. Interrogation of the Oncomine database revealed that 52 of 53 human breast carcinomas showed substantial upregulation of WNT family ligand WNT7B. Immunolabeling of human mammary carcinoma showed that WNT7B immunoreactivity was associated with both tumor cells and with tumor-associated macrophages. In the MMTV-PymT mouse model of mammary carcinoma, we found tumor progression relied upon WNT7B produced by myeloid cells in the microenvironment. Wnt7b deletion in myeloid cells reduced the mass and volume of tumors due to a failure in the angiogenic switch. In the tumor overall, there was no change in expression of Wnt/β-catenin pathway target genes, but in vascular endothelial cells (VEC), expression of these genes was reduced, suggesting that VECs respond to Wnt/β-catenin signaling. Mechanistic investigations revealed that failure of the angiogenic switch could be attributed to reduced Vegfa mRNA and protein expression in VECs, a source of VEGFA mRNA in the tumor that was limiting in the absence of myeloid WNT7B. We also noted a dramatic reduction in lung metastasis associated with decreased macrophage-mediated tumor cell invasion. Together, these results illustrated the critical role of myeloid WNT7B in tumor progression, acting at the levels of angiogenesis, invasion, and metastasis. We suggest that therapeutic suppression of WNT7B signaling might be advantageous due to targeting multiple aspects of tumor progression.

Macrophage-derived extracellular vesicle-packaged WNTs rescue intestinal stem cells and enhance survival after radiation injury.

WNT/β-catenin signalling is crucial for intestinal homoeostasis. The intestinal epithelium and stroma are the major source of WNT ligands but their origin and role in intestinal stem cell (ISC) and epithelial repair remains unknown. Macrophages are a major constituent of the intestinal stroma. Here, we analyse the role of macrophage-derived WNT in intestinal repair in mice by inhibiting their release using a macrophage-restricted ablation of Porcupine, a gene essential for WNT synthesis. Such Porcn-depleted mice have normal intestinal morphology but are hypersensitive to radiation injury in the intestine compared with wild-type (WT) littermates. Porcn-null mice are rescued from radiation lethality by treatment with WT but not Porcn-null bone marrow macrophage-conditioned medium (CM). Depletion of extracellular vesicles (EV) from the macrophage CM removes WNT function and its ability to rescue ISCs from radiation lethality. Therefore macrophage-derived EV-packaged WNTs are essential for regenerative response of intestine against radiation.

Direct visualization of the phenotype of hypoxic tumor cells at single cell resolution in vivo using a new hypoxia probe.

ABSTRACT Tumor hypoxia is linked to tumor progression, metastasis, and therapy resistance. However, the underlying mechanisms behind this linkage are not fully understood. Here we present a novel fluorescent mCherry hypoxia-responsive marker that can be used in real time imaging to specifically and sensitively identify hypoxic cells in vivo at single cell resolution. Tumors derived from triple negative tumor cells expressing the hypoxia marker reveal that the hypoxic tumor cells congregate near flowing blood vessels. Using multiphoton microscopy, hypoxic MDA-MB-231 cells were directly visualized and showed a more persistent slow migration phenotype as compared to normoxic cells in the same field in vivo. Hypoxic tumor cells are enriched in the cell population that migrates toward human epithelial growth factor gradients in vivo, and has increased collagen degradation and intravasation activity, characteristics of dissemination and metastasis competent tumor cells. The hypoxia probe introduced in this study provides a specific reporter of hypoxic cell phenotypes in vivo which reveals new insights into the mechanisms by which hypoxia is linked to metastasis.

Abstract A13: Macrophage FLT1 mediated inflammatory response determines breast cancer distal metastasis

Macrophages are abundantly found in the tumor microenvironment and enhance malignancy. At distal metastatic sites, our previous studies identified a distinct population of metastasis associated macrophages (MAMs) that promotes tumor cell extravasation, seeding and persistent growth. These macrophages were derived from circulating inflammatory monocytes recruited by CCL2/CCR2 chemokine signaling and directly promote tumor cell extravasation and metastatic seeding in vivo through VEGF production. Our recent studies identified that MAMs express high levels of cell surface FMS-like tyrosine kinase 1 (FLT1, also known as VEGFR1) after their recruitment. Blockade of FLT1 signaling using specific inhibitory antibodies significantly inhibited the metastatic seeding and persistent growth. Using several genetic models of Flt1 deficiency, we show that macrophage specific FLT1 signaling is critical for breast tumor distal metastatic potential. FLT1 is not expressed by other hematopoietic cells and its inhibition did not affect the recruitment of MAMs, which indicated that specific FLT1 signaling in MAMs are important for their metastasis promoting functions. Indeed, we identified that FLT1 regulates a set of inflammatory response genes including Colony Stimulating Factor 1 (CSF1) a central regulator of macrophage biology. Using a genetic gain-of-function approach we show that CSF1 mediated autocrine signaling in MAMs is downstream of FLT1 and can restore the tumor-promoting activity in MAMs even when FLT1 has been inhibited. Together, our data established a link between inflammation and cancer metastasis and suggested the therapeutic potential of targeting these pathways in treating metastatic disease. Citation Format: Bin-Zhi Qian, Hui Zhang, Jiufeng Li, Eun-Jin Yeo, Neil O. Carragher, Anne R. Bresnick, Richard A. Lang, Jeffrey W. Pollard. Macrophage FLT1 mediated inflammatory response determines breast cancer distal metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A13.

Abstract 2842: CCL2 recruits inflammatory monocytes to facilitate breast tumor metastasis

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Macrophages are abundant in the tumor microenvironment and enhance malignancy through their promotion of angiogenesis and invasion at the primary tumor site as well as extravasation, target organ seeding and persistent growth at distant metastatic sites. To determine the origin and recruitment of tumor associated macrophages, different populations of monocytes were FACS sorted and adoptively transferred into mice bearing MMTV-PyMT induced mouse mammary tumors with or without spontaneous pulmonary metastases. Ly6C+ inflammatory monocytes were preferentially recruited to pulmonary metastases but not primary tumors in a CCL2 (also known as MCP1) dependent manner. Human inflammatory monocytes were also preferentially recruited to pulmonary metastases of human breast cancer cells in immuno-compromised mice in a CCL2 dependent manner. These inflammatory monocytes promote tumor cell trans-endothelial migration in vitro, a process that is abrogated by an anti-CCL2 neutralizing antibody. Neutralizing CCL2 in vivo blocks the recruitment of metastasis associated macrophages and their direct interaction with metastasizing tumor cells, leading to inhibition of tumor cell extravasation and metastatic seeding. Inhibition of tumor cell-derived CCL2 inhibits their metastatic seeding. Secretory factors from inflammatory monocytes were identified to mediate their metastasis-promoting function in vitro and in vivo. Both CCL2 expression and macrophage infiltration are correlated with poor prognosis in human breast cancer. Our data strongly suggest that recruitment of Ly6C+ inflammatory monocytes is the mechanistic link between these two clinical associations and suggests new therapeutic targets for treating metastatic breast disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2842. doi:10.1158/1538-7445.AM2011-2842