Purpose: To investigate the clinical relevance of macrophages in liver metastasis of colorectal cancer and their influence on angiogenesis and patient survival. Moreover to evaluate specific blood monocytes as markers of disease recurrence.Experimental design: In a mouse model with spontaneous liver metastasis, the angiogenic characteristics of tumor-and metastasis (MAM)-associated macrophages were evaluated. Macrophages and the vasculature from 130 primary tumor (pTU) and 123 patients with liver metastasis were assessed. In vivo and in human samples, the clinical relevance of macrophage VEGFR1 expression was analyzed. Blood samples from patients (n ¼ 157, 80 pTU and 77 liver metastasis) were analyzed for assessing VEGFR1-positive (VEGFR1 þ ) cells as suitable biomarkers of disease recurrence.Results: The number of macrophages positively correlated with vascularization in metastasis. Both in the murine model as well as in primary isolated human cells, a subpopulation of MAMs expressing VEGFR1 were found highly angiogenic. While VEGFR1 expression in pTU patients did not predict prognosis; high percentage of VEGFR1 þ cells in liver metastasis was associated with worse patient outcome. Interestingly, VEGFR1 þ -circulating monocytes in blood samples from patients with liver metastasis not only predicted progression but also site of recurrence.Conclusions: Our findings identify a new subset of proangiogenic VEGFR1 þ MAMs in colorectal cancer that support metastatic growth and may become a liquid biomarker to predict disease recurrence in the liver.

Hypoxia is a common feature of solid tumors. Prolyl hydroxylase enzymes (PHD1-3) are molecular oxygen sensors that regulate hypoxia-inducible factor activity, but their functions in metastatic disease remain unclear. Here, we assessed the significance of PHD enzymes during the metastatic spread of colorectal cancer. PHD expression analysis in 124 colorectal cancer patients revealed that reduced tumoral expression of PHD3 correlated with increased frequency of distant metastases and poor outcome. Tumorigenicity and metastatic potential of colorectal tumor cells over and underexpressing PHD3 were investigated in orthotopic and heterotopic tumor models. PHD3 overexpression in a syngeneic tumor model resulted in fewer liver metastases, whereas PHD3 knockdown induced tumor spread. The migration of PHD3-overexpressing tumor cells was also attenuated in vitro. Conversely, migratory potential and colony formation were enhanced in PHD3-deficient cells, and this phenotype was associated with enhanced mitochondrial ATP production. Furthermore, the effects of PHD3 deficiency were accompanied by increased mitochondrial expression of the BCL-2 family member, member myeloid cell leukemia sequence 1 (MCL-1), and could be reversed by simultaneous inhibition of MCL-1. MCL-1 protein expression was likewise enhanced in human colorectal tumors expressing low levels of PHD3. Therefore, we demonstrate that downregulation of PHD3 augments metastatic spread in human colorectal cancer and identify MCL-1 as a novel downstream effector of oxygen sensing. Importantly, these findings offer new insight into the possible, context-specific deleterious effects of pharmacologic PHD inhibition. Cancer Res; 76(8); 2219-30. Ó2016 AACR.

<div>AbstractPurpose:<p>To investigate the clinical relevance of macrophages in liver metastasis of colorectal cancer and their influence on angiogenesis and patient survival. Moreover to evaluate specific blood monocytes as markers of disease recurrence.</p><p><b>Experimental design:</b> In a mouse model with spontaneous liver metastasis, the angiogenic characteristics of tumor- and metastasis (MAM)-associated macrophages were evaluated. Macrophages and the vasculature from 130 primary tumor (pTU) and 123 patients with liver metastasis were assessed. <i>In vivo</i> and in human samples, the clinical relevance of macrophage VEGFR1 expression was analyzed. Blood samples from patients (<i>n</i> = 157, 80 pTU and 77 liver metastasis) were analyzed for assessing VEGFR1-positive (VEGFR1⁺) cells as suitable biomarkers of disease recurrence.</p>Results:<p>The number of macrophages positively correlated with vascularization in metastasis. Both in the murine model as well as in primary isolated human cells, a subpopulation of MAMs expressing VEGFR1 were found highly angiogenic. While VEGFR1 expression in pTU patients did not predict prognosis; high percentage of VEGFR1⁺ cells in liver metastasis was associated with worse patient outcome. Interestingly, VEGFR1⁺-circulating monocytes in blood samples from patients with liver metastasis not only predicted progression but also site of recurrence.</p>Conclusions:<p>Our findings identify a new subset of proangiogenic VEGFR1⁺ MAMs in colorectal cancer that support metastatic growth and may become a liquid biomarker to predict disease recurrence in the liver.</p></div>