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.

Background Liver metastases severely reduce the long term survival of colorectal cancer patients. Long non-coding RNAs (lncRNAs) CCAT1 and CCAT2 have previously been found to be associated with impaired patient outcomes in primary colorectal cancer. We aimed to elucidate the role of CCAT1 and CCAT2 in colorectal liver metastases. Methods Total RNA was isolated from 97 human tissue samples of colorectal liver metastases and adjacent normal liver tissue. Gene expression analysis was performed by RT-qPCR and Multiplex ELISA and correlated with patient characteristics and survival. Gene expression, cancer cell migration, invasion, and proliferation were studied after siRNA-mediated knockdown of CCAT1, CCAT2, and MYC in metastatic colorectal cancer cell lines Colo205 and HROC277Met2. Results Elevated expression levels of lncRNAs CCAT1 and CCAT2, and their common target MYC in colorectal liver metastases were associated with prolonged progression-free survival after liver resection. High expression of CCAT1 was likewise associated with prolonged overall survival. Knockdown of CCAT1, CCAT2, and MYC resulted in increased migratory and invasive potential in metastatic colorectal cancer cell lines. Gene expression analysis revealed alterations in constituents of Wnt signaling following knockdown. Conclusion Our findings demonstrate tumor-suppressive functions of lncRNAs CCAT1 and CCAT2 in colorectal liver metastases. They suppress Wnt signaling directly and indirectly through target gene MYC and might prevent further metastatic spread from colorectal liver metastases.

<p>Figure S1. MAMs are more angiogenic and express more Flt1 than TAMs. Figure S2. Efficient depletion of Flt1 in CD11b+ cells. Figure S3. Macrophage infiltration, vessel density and VEGFR1+ cells have no impact on overall survival in patients with primary tumor and liver metastasis of colorectal cancer. Figure S4. Vessel density and VEGFR1 expression influence progression-free survival in patients with synchronous liver metastasis of colorectal cancer. Figure S5. No differences between pTU and LM in the total number of stained cells.</p>

<p>Supplementary methods, tables and supplementary figure legends Table S1. Patient characteristics. Table S2. Kind of neo-adjuvant therapy from LM patients included in the analysis of blood samples by flow cytometry. Table S3. Primer list used for qRT-PCR. Table S4. Association of circulating CD11b+ CD68+ VEGFR1+ cells with clinicopathologic variables in patients with liver metastasis of colorectal cancer. Table S5. Univariate analysis of factors associated with progression-free survival in patients with liver metastasis of CRC. Table S6. Multivariate analysis of factors associated with circulating VEGFR1+ macrophages and progression-free survival in LM patients.</p>

<p>Figure S1. MAMs are more angiogenic and express more Flt1 than TAMs. Figure S2. Efficient depletion of Flt1 in CD11b+ cells. Figure S3. Macrophage infiltration, vessel density and VEGFR1+ cells have no impact on overall survival in patients with primary tumor and liver metastasis of colorectal cancer. Figure S4. Vessel density and VEGFR1 expression influence progression-free survival in patients with synchronous liver metastasis of colorectal cancer. Figure S5. No differences between pTU and LM in the total number of stained cells.</p>

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<p>Supplementary methods, tables and supplementary figure legends Table S1. Patient characteristics. Table S2. Kind of neo-adjuvant therapy from LM patients included in the analysis of blood samples by flow cytometry. Table S3. Primer list used for qRT-PCR. Table S4. Association of circulating CD11b+ CD68+ VEGFR1+ cells with clinicopathologic variables in patients with liver metastasis of colorectal cancer. Table S5. Univariate analysis of factors associated with progression-free survival in patients with liver metastasis of CRC. Table S6. Multivariate analysis of factors associated with circulating VEGFR1+ macrophages and progression-free survival in LM patients.</p>

<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>