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© 2019 Pinto, Rios, Durães, Ribeiro, Machado, Mantovani, Barbosa, Carneiro and Oliveira. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., Macrophages are one of the immune populations frequently found in colorectal tumors and high macrophage infiltration has been associated with both better and worst prognosis. Importantly, according to microenvironment stimuli, macrophages may adopt different polarization profiles, specifically the pro-inflammatory or M1 and the anti-inflammatory or M2, which display distinct functions. Therefore, concomitantly with the number of tumor-associated macrophages (TAMs), their characterization is fundamental to unravel their relevance in cancer. Here, we profiled macrophages in a series of 150 colorectal cancer (CRC) cases by immunohistochemistry, using CD68 as a macrophage lineage marker, CD80 as a marker of pro-inflammatory macrophages, and CD163 as a marker of anti-inflammatory macrophages. Quantifications were performed by computer-assisted analysis in the intratumoral region, tumor invasive front, and matched tumor adjacent normal mucosa (ANM). Macrophages, specifically the CD163+ ones, were predominantly found at the tumor invasive front, whereas CD80+ macrophages were almost exclusively located in the ANM, which suggests a predominant anti-inflammatory polarization of TAMs. Stratification according to tumor stage revealed that macrophages, specifically the CD163+ ones, are more prevalent in stage II tumors, whereas CD80+ macrophages are predominant in less invasive T1 tumors. Specifically in stage III tumors, higher CD68, and lower CD80/CD163 ratio associated with decreased overall survival. Importantly, despite the low infiltration of CD80+ cells in colorectal tumors, multivariate logistic regression revealed a protective role of these cells regarding the risk for relapse. Overall, this work supports the involvement of distinct microenvironments, present at the intra-tumor, invasive front and ANM regions, on macrophage modulation, and uncovers their prognostic value, further supporting the relevance of including macrophage profiling in clinical settings., This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT/MCTES in the framework of the project MAGICIAM: a MAcrophaGe Immunomodulatory-delivery system to prevent Cancer Invasion and Metastasis (POCI-01-0145-FEDER-031859). FCT further supported this work under MP PhD grant (PD/BD/81103/2011), CD post-doctoral grant (SFRH/BPD/99442/2014), and MO FCT Investigator grant (IF/01066/2012).

We aimed to refine the value of CDX2 as an independent prognostic and predictive biomarker in colorectal cancer (CRC) according to disease stage and chemotherapy sensitivity in preclinical models. CDX2 expression was evaluated in 1045 stage I–IV primary CRCs by gene expression (n = 403) or immunohistochemistry (n = 642) and in relation to 5-year relapse-free survival (RFS), overall survival (OS), and chemotherapy. Pharmacogenomic associations between CDX2 expression and 69 chemotherapeutics were assessed by drug screening of 35 CRC cell lines. CDX2 expression was lost in 11.6% of cases and showed independent poor prognostic value in multivariable models. For individual stages, CDX2 was prognostic only in stage IV, independent of chemotherapy. Among stage I–III patients not treated in an adjuvant setting, CDX2 loss was associated with a particularly poor survival in the BRAF-mutated subgroup, but prognostic value was independent of microsatellite instability status and the consensus molecular subtypes. In stage III, the 5-year RFS rate was higher among patients with loss of CDX2 who received adjuvant chemotherapy than among patients who did not. The CDX2-negative cell lines were significantly more sensitive to chemotherapeutics than CDX2-positive cells, and the multidrug resistance genes MDR1 and CFTR were significantly downregulated both in CDX2-negative cells and in patient tumors. Loss of CDX2 in CRC is an adverse prognostic biomarker only in stage IV disease and appears to be associated with benefit from adjuvant chemotherapy in stage III. Early-stage patients not qualifying for chemotherapy might be reconsidered for such treatment if their tumor has loss of CDX2 and mutated BRAF. This work was financially supported by the Cancer Clinic, Oslo University Hospital (Grant No.: 2017-19 supporting JB with researcher fellowship), the Norwegian Cancer Society (Grant No.: 182759-2016, to RAL; and Grant No.: 6824048-2016, to AS), the foundation K. G. Jebsen Colorectal Cancer Research Centre, Stiftelsen Kristian Gerhard Jebsen, the South-Eastern Health Regional Authorities of Norway, and the Research Council of Norway, in cooperation with the University of Oslo, through the ?Toppforsk? grant (RAL, Project Number 250993/F20). This work was financially supported by the projects NORTE-01-0145-FEDER-000029 and NORTE-01-0145-FEDER-000003, supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was also supported by FEDER?Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020?Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT?Funda??o para a Ci?ncia e a Tecnologia/Minist?rio da Ci?ncia, Tecnologia e Inova??o in the framework of the project ?Institute for Research and Innovation in Health Sciences? (POCI-01-0145-FEDER-007274). RB acknowledges FCT for financial support (SFRH/BPD/68276/2010).