The leading cause of cancer related mortality is metastatic spread of tumor cells to distant sites. Metastasis is a multi step process consisting of intravasation, survival in the circulation, extravasation, survival in and colonization of distant tissues. The complexity of each step of the metastatic process makes it highly inefficient overall and although large numbers of cells disseminate from the primary tumor, only a small fraction of these cells end up forming distant tumors. However rare, disseminated tumor cells ultimately successfully form metastatic colonies and therefore present a critical therapeutic challenge.
Many tumor types are capable of metastasizing to distant sites however the location of these sites varies considerably among different primary tumors. Several studies have shown that the site of metastasis may be determined by a specific gene expression pattern, or signature, in primary tumor cells that mediates metastasis to specific distant organs (Gupta and Massague, 2006
). In addition, tumor induced changes in the microenvironment of distal organs prior to colonization might make certain tissues more receptive to colonization of migrating tumor cells (Joyce and Pollard, 2009
). Recent studies have suggested that factors secreted from the primary tumor may modulate the future site of metastasis in a directed fashion (Erler et al., 2009
). Bone marrow derived cells are mobilized by the primary tumor and directed to the future site of metastasis (Kaplan et al., 2005
) where they take part in the formation of a functional pre-metastatic niche. All of these processes are likely working in concert to determine the future site of metastasis.
Here we describe the accumulation and activation of neutrophils by the primary tumor during the pre-metastatic stage in mouse cancer models. Induced by the primary tumor, these tumor-entrained neutrophils (TENs) accumulate in the circulation and the pre-metastatic lung. Neutrophils are short-lived leukocytes that are part of the innate immune system. Owing to their highly motile nature, neutrophils are quick to respond and provide the first line of defense against infections through phagocytosis, extracellular degranulation and spreading of extracellular traps (Hickey and Kubes, 2009
). Driven by cytokines and chemotactic factors, neutrophils are also recruited to sites of inflammation where they take part in the inflammatory process. The notion that tumors and sites of inflammation, such as wounds, share many similarities is now commonly accepted. Tumors might also be viewed as wounds that will not heal (Dvorak, 1986
) and as such incite an inflammatory response by recruiting leukocytes through the production of various cytokines and chemokines (Coussens and Werb, 2002
The tumor recruited leukocyte population is heterogeneous and consists, in part, of tumor-associated neutrophils (TANs). TANs were previously shown to have a pro-tumorigenic effect at the primary site by secreting pro-tumorigenic factors, promoting angiogenesis and suppressing immune responses (Pekarek et al., 1995
; Shojaei et al., 2008
; Youn et al., 2008
). Interestingly, the pro-tumorigenic effects of neutrophils were shown to be TGF-β dependent and upon TGF-β blockade, neutrophils were shown to switch from the “N2” pro-tumorigenic phenotype to the “N1” anti-tumorigenic phenotype (Fridlender et al., 2009
). Recent studies have also suggested a pro-metastatic role for neutrophils in the pre-metastatic niche. High numbers Gr-1+
cells were shown to accumulate in the pre-metastatic lungs of mice bearing highly metastatic tumors. The Gr-1 epitope is present on both Ly-6G+
cells which are exclusively neutrophils (Daley et al., 2008
) and Ly-6C+
cells in which the epitope is expressed at higher levels on monocytes than neutrophils (Zhu et al., 2007
). Although not formally demonstrated by depletion of Gr-1+
cells, this study suggests that tumor-mobilized lung associated Gr-1+
cells contribute to the formation of a pro-metastatic niche by generating an immunosuppressive environment with abnormal vasculature (Yan et al., 2010
). In another recent study it was shown that G-CSF stimulated neutrophils and monocytes increase the metastatic seeding of circulating tumor cells in the lungs and that both G-CSF and BV8 contribute to the formation of lung metastases. Furthermore, in one 4T1-cell derivative orthotopic model (66Cl4), Ly-6G-specific depletion of neutrophils was found to reduce the metastatic burden in the lungs (Kowanetz et al., 2010
Through careful depletion and adoptive transfer experiments here we examine the role of TENs in a variety of breast cancer models and relate our findings to their presence in the peripheral blood of breast cancer patients.