Selection-free endogenous tagging of cell lines by bicistronic co-expression of the surface antigen NGFR.

Endogenous protein tagging, in contrast to exogenous overexpression of tagged proteins, allows to characterize specific protein functions under defined physiological or pathophysiological conditions without the influence of non-physiological protein levels. The development of generic and homology-independent tagging strategies, exploiting the CRISPR/spCas9 gene editing system in combination with generic tag donor plasmids, allows targeted and precise gene modification in mammalian cells for almost any desirable gene. So far, fluorescent tags or antibiotic resistance cassettes coupled to the endogenous fusion protein expression have been applied to isolate correctly modified clones. However, both can be challenging, especially when endogenously controlled expression of the tagged protein is weak or regulated by cellular signals. Here, we expand the strategy to selection-free endogenous tagging by exploiting exogenous co-expression of surface antigens. These endogenously regulated, but still easily accessible surface antigens allow simple identification and isolation of clones harboring correctly tagged alleles via common sorting procedures (e.g. FACS/MACS). Using metabolically controlled interaction studies of the endogenously tagged mTORC1-regulating GATOR2 complex protein WDR59, we show that endogenous GFP-labeling does not affect complex association of fusion proteins and downstream signaling via mTORC1. In addition, exogenous co-expression of the NGFR surface antigen does not influence conditional protein-protein interactions.•A method for selection-free, site-specific, homology-independent endogenous genetic tagging.•Production of fusion genes for protein visualization in living cells or determination of protein-protein-interactions.•Expression of a fusion protein mirroring physiological expression in its natural genetic context.

Extravesicular TIMP-1 is a non-invasive independent prognostic marker and potential therapeutic target in colorectal liver metastases.

Molecular reprogramming of stromal microarchitecture by tumour-derived extracellular vesicles (EVs) is proposed to favour pre-metastatic niche formation. We elucidated the role of extravesicular tissue inhibitor of matrix metalloproteinase-1 (TIMP1EV) in pro-invasive extracellular matrix (ECM) remodelling of the liver microenvironment to aid tumour progression in colorectal cancer (CRC). Immunohistochemistry analysis revealed a high expression of stromal TIMP1 in the invasion front that was associated with poor progression-free survival in patients with colorectal liver metastases. Molecular analysis identified TIMP1EV enrichment in CRC-EVs as a major factor in the induction of TIMP1 upregulation in recipient fibroblasts. Mechanistically, we proved that EV-mediated TIMP1 upregulation in recipient fibroblasts induced ECM remodelling. This effect was recapitulated by human serum-derived EVs providing strong evidence that CRC release active EVs into the blood circulation of patients for the horizontal transfer of malignant traits to recipient cells. Moreover, EV-associated TIMP1 binds to HSP90AA, a heat-shock protein, and the inhibition of HSP90AA on human-derived serum EVs attenuates TIMP1EV-mediated ECM remodelling, rendering EV-associated TIMP1 a potential therapeutic target. Eventually, in accordance with REMARK guidelines, we demonstrated in three independent cohorts that EV-bound TIMP1 is a robust circulating biomarker for a non-invasive, preoperative risk stratification in patients with colorectal liver metastases.