Macrophages induce “budding” in aggressive human colon cancer subtypes by protease-mediated disruption of tight junctions

// Kari Trumpi 1, * , Nicola Frenkel 1, * , Timo Peters 1 , Nicoline M. Korthagen 2, 3 , Jennifer M.J. Jongen 1 , Danielle Raats 1 , Helma van Grevenstein 1 , Yara Backes 4 , Leon M. Moons 4 , Miangela M. Lacle 5 , Jan Koster 6 , Danny Zwijnenburg 6 , Inne H.M. Borel Rinkes 1 and Onno Kranenburg 1 1 UMC Utrecht Cancer Center, University Medical Center Utrecht, Utrecht, The Netherlands 2 Orthopedics, University Medical Center Utrecht, Utrecht, The Netherlands 3 Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands 4 Gastroenterology & Hepatology, University Medical Center Utrecht, Utrecht, The Netherlands 5 Pathology, University Medical Center Utrecht, Utrecht, The Netherlands 6 Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands * These authors have contributed equally to this work Correspondence to: Onno Kranenburg, email: o.kranenburg@umcutrecht.nl Keywords: colorectal; budding; metastasis; consensus molecular subtypes; matrix metalloprotease Received: July 15, 2017      Accepted: February 24, 2018      Published: April 13, 2018 ABSTRACT Primary human colorectal tumors with a high stromal content have an increased capacity to metastasize. Cancer-associated fibroblasts (CAFs) promote metastasis, but the contribution of other stromal cell types is unclear. Here we searched for additional stromal cell types that contribute to aggressive tumor cell behavior. By making use of the ‘immunome compendium’—a collection of gene signatures reflecting the presence of specific immune cell-types—we show that macrophage signatures are most strongly associated with a high CAF content and with poor prognosis in multiple large cohorts of primary tumors and liver metastases. Co-culturing macrophages with patient-derived colonospheres promoted ‘budding’ of small clusters of tumor cells from the bulk. Immunohistochemistry showed that budding tumor clusters in stroma-rich areas of T1 colorectal carcinomas were surrounded by macrophages. In vitro budding was accompanied by reduced levels of the tight junction protein occludin, but OCLN mRNA levels did not change, nor did markers of epithelial mesenchymal transition. Budding was accompanied by nuclear accumulation of β-catenin, which was also observed in budding tumor cell clusters in situ . The NFκB inhibitor Sanguinarine resulted in a decrease in MMP7 protein expression and both NFκB inhibitor Sanguinarine and MMP inhibitor Batimastat prevented occludin degradation and budding. We conclude that macrophages contribute to the aggressive nature of stroma-rich colon tumors by promoting an MMP-dependent pathway that operates in parallel to classical EMT and leads to tight junction disruption.