Tie2 signaling in the tumor microenvironment orchestrates breast cancer cell dissemination through TMEM doorways

Duran et al., bioRxiv, 2026 asked which cell at a TMEM doorway needs Tie2 signalling for a breast cancer cell to get into the bloodstream. A TMEM doorway is a stable three cell structure, a Mena expressing tumour cell touching a Tie2 high macrophage touching an endothelial cell, and in breast cancer it is the only place tumour cells cross into the circulation. Its density is a clinically validated marker of distant recurrence. All three cell types can express Tie2, so the question was which one carries the function.

The answer came from removing Tie2 from each cell type separately and then putting the cells back together. Tie2 was deleted from the endothelial and tumour cells with CRISPR-Cas9, and knocked down in RAW/LR5 macrophages with AUMsilence sdASO from AUM BioTech. Two oligonucleotides against Tie2, named TEK-1 and TEK-2, and a non-targeting control were applied at 15 μM for 72 hours, with no transfection reagent named in the methods, and the knockdown was confirmed at both transcript and protein level by quantitative PCR and immunoblot. The three sets of cells were then combined in a transendothelial migration assay that reproduces the conditions at a doorway.

Knocking Tie2 down in the macrophages significantly reduced the number of tumour cells crossing the endothelium. Removing Tie2 from endothelial cells or from tumour cells changed nothing, which is what pins the requirement to the macrophage rather than to Tie2 in general. Blocking Tie2 pharmacologically with 1 nM rebastinib gave the same cell type pattern. The conclusion was then reproduced in animals with a separately built inducible macrophage specific Tie2 knockout, which reduced vascular opening at doorways, lowered VEGF-A in tumour macrophages and cut the number of circulating tumour cells.