Expanding the armamentarium for neutrophil‐mediated angiogenesis
Authors: Brian E. Hsu, Peter M. Siegel
Journal: Hepatology (2017)

Potential conflict of interest: Nothing to report. See Article On page 1920 Neutrophils are emerging as important modulators of cancer progression and metastasis, exhibiting both antimetastatic and prometastatic functions.1 Originally considered to be a terminally differentiated and homogenous cell population that was engaged as a first line of defense against microbial infections, neutrophils are now viewed as a highly "plastic" immune cell type in the context of cancer, which can undergo polarization within the tumor microenvironment.2 Neutrophils present in the metastatic microenvironment can mediate tumor cell killing and impair the establishment of metastases.3 In other contexts, infiltrating neutrophils function to promote cancer progression and metastasis.5 With respect to liver metastases, neutrophils promote the formation and growth of hepatic metastases from multiple solid cancers.7 Neutrophils positively impact the metastatic process through a variety of functions, including their ability to enhance cancer cell entrapment/seeding within neutrophil extracellular traps,7 promote cancer cell extravasation,9 and stimulate angiogenesis.8 In the context of tumor angiogenesis, there are several mechanisms through which neutrophils play a proangiogenic role. First, neutrophils express a variety of proangiogenic factors. The best example is the observation that tumor‐associated neutrophils contain significant pools of intracellular vascular endothelial growth factor (VEGF) that can be released upon stimulation,12 and tumor‐associated neutrophils exhibit increased transcription of VEGF mRNA.13 Impairment of Bv8 (prokineticin‐2), a neutrophil‐derived angiogenic factor, with anti‐Bv8 antibodies has been shown to reduce recruitment of myeloid cells, decrease blood vessel density, and inhibit tumor growth upon inhibition.14 In addition to directly producing angiogenic factors, neutrophils can indirectly mobilize angiogenic factors stored in the local extracellular matrix (ECM) through the release of proteases. Indeed, neutrophil‐derived matrix metalloproteinase 9 (MMP‐9) catalyzes the release of VEGF bound to the ECM by proteolytic cleavage, inducing an angiogenic switch.15 Moreover, there are studies that identify a distinct subpopulation of proangiogenic neutrophils (defined as CD49d+VEGFR1high C‐X‐C chemokine receptor [CXCR]4high neutrophils) that are recruited by VEGF‐A to the sites of tissue hypoxia. These proangiogenic neutrophils release larger amounts of MMP‐9 compared to inflammatory neutrophils recruited to infectious sites.16 Fibroblast growth factor 2 (FGF2/basic FGF) can also be liberated from ECM stores through the action of neutrophil‐derived proteases.17 In the current issue, Gordon‐Weeks et al. expand our understanding of neutrophil‐dependent angiogenesis.8 In this study, the investigators demonstrate the presence of infiltrating neutrophils in liver metastases derived from human xenografts as well as in murine models of liver metastasis. Isolated neutrophils (Ly6G+/CXCR2+/CD45+) from HT29‐derived liver metastases exhibited multilobed nuclear morphology, indicating that these infiltrating cells correspond to mature, segmented neutrophils. Interestingly, no differences were observed in circulating low‐density neutrophils that have recently been described in tumor‐bearing animal models.18 To functionally interrogate the role of neutrophils in the expansion and growth of liver metastases, neutrophil depletion experiments were performed only after the cancer cells had seeded the liver. Depletion of neutrophils after 7 or 14 days post‐tumor‐cell injection reduced the metastatic burden at endpoint, whereas neutrophil depletion after 21 days did not. In addition, tumor cell proliferation and vascular density was diminished in liver metastases that formed in neutrophil‐depleted animals. These data argue that neutrophils act early in the colonization phase to enhance the growth of colorectal cancer (CRC) liver metastases by promoting angiogenesis. To gain a mechanistic understanding of how liver‐metastasis–associated neutrophils were contributing to an angiogenic response, mRNA expression profiles of metastasis‐associated neutrophils were compared to neutrophils isolated from normal livers. From these analyses, FGF2 mRNA levels were found to be elevated in neutrophils isolated from CRC liver metastases, whereas angiogenic factors such as VEGF were not significantly different. Moreover, FGF2 expression was localized to Ly6G+ cells and deposited within ECM of liver metastases in situ, and neutrophil depletion resulted in a significant reduction of FGF2 expression in hepatic lesions. Finally, the investigators demonstrate that an FGF2 neutralizing antibody, when administered 1 week post‐tumor‐cell injection, resulted in a reduction of vascular density, cancer cell proliferation, and liver‐metastatic burden. These results phenocopied the consequences of neutrophil depletion; however, mice treated with the neutralizing FGF2 antibody exhibited the same degree of neutrophil infiltration compared to control cohorts. These data argue that blocking neutrophil‐derived FGF2 resulted in impaired angiogenesis rather than a secondary effect resulting from impaired neutrophil recruitment. Indeed, the critical role of FGF2 for enhancing angiogenesis associated with the establishment of liver metastases was demonstrated by the fact that CRC cells engineered to overexpress FGF2 were capable of efficiently forming liver metastases even in the context where neutrophils were depleted. This study demonstrates that neutrophils recruited into the liver‐metastatic microenvironment produce FGF2, which drives angiogenesis and growth of nascent hepatic metastases. These results support previous work demonstrating that neutrophils can indirectly liberate FGF2 from ECM stores through release of heparanase, which was also observed in the current study to be highly elevated in neutrophils recruited to liver metastases (Fig. 1). They also make the novel observation that infiltrating neutrophils are themselves a significant source of FGF2. The precise mechanism of FGF2 release and activation is still in question. Is FGF‐2 that is produced by neutrophils deposited within the ECM and subsequently released by neutrophil‐derived heparanase to induce angiogenesis, or do cancer cells within the growing metastases secrete their own proteases that release and activate FGF2? Given the growing realization that neutrophils represent a much more heterogeneous cell population in the context of cancer, another interesting question is the relationship between the FGF2‐producing neutrophils described in the current study and previously described proangiogenic neutrophils (CD49d+VEGFR1high CXCR4high)—are they the same or distinct neutrophil subsets? Regardless, the demonstration that neutrophil‐derived FGF2 is important for a robust angiogenic response within liver metastases provides a target for a subset of patients where traditional anti‐VEGF therapies have failed.Figure 1: Neutrophil‐derived FGF2 supports the growth of liver‐metastatic cancer cells. Cancer cells first seed the liver. Metastasis‐associated neutrophils that express FGF2 are subsequently recruited to the growing lesions. FGF2 produced by neutrophils stimulates angiogenesis through direct release of active FGF2 or through the release of FGF2 bound to the ECM. Release of ECM‐bound stores of FGF2 may result from heparanase released by neutrophils or by proteases produced by cancer cells. Author names in bold designate shared co‐first authorship.