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Metastatic colorectal carcinoma-associated fibroblasts have immunosuppressive properties related to increased IGFBP2 expression 1 1 Introduction 1 2 Materials and methods 2 2.1 Cell lines 2 2.2 Fibroblast isolation and culture 2 2.3 PBMC and monocyte isolation 2 2.4 T cell isolation, pre-activation and CTV labelling 2 2.5 Collection of fibroblast-conditioned medium (CM) 2 2.6 2D co-cultures 2 2.7 3D Collagen gel cultures 3 2.8 siRNA-mediated knockdown 3 2.9 Neutralizing antibody and inhibitor 3 2.10 Cytokine profiling 3 2.11 Flow cytometry 3 2.12 Immunohistochemistry 3 2.13 Immunofluorescence on FFPE tissues 3 2.14 Immunofluorescence of collagen gels 3 2.15 Analysis of gene expression via RNA-seq 4 2.16 Protein-protein interaction network and functional enrichment analysis 4 2.17 Statistical analysis 4 3 Results 4 3.1 Fibroblasts from metastases present different signals to the tumour microenvironment than fibroblasts form primary tumour 4 3.2 MAFs and CAFs differentially modulate macrophages 6 3.3 MAFs reduce macrophage-mediated T lymphocyte activation 8 3.4 MAF-induced modulation of macrophages is IGFBP2-dependent 9 4 Discussion 9 Author contributions 11 Declaration of competing

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