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The lymphatic system is composed of a hierarchical network of fluid absorbing lymphatic capillaries and transporting collecting vessels. Despite distinct functions and morphologies, molecular mechanisms that regulate the identity of the different vessel types are poorly understood. Through transcriptional analysis of murine dermal lymphatic endothelial cells (LECs), we identified Foxp2, a member of the FOXP family of transcription factors implicated in speech development, as a collecting vessel signature gene. FOXP2 expression was induced after initiation of lymph flow in vivo and upon shear stress on primary LECs in vitro. Loss of FOXC2, the major flow‐responsive transcriptional regulator of lymphatic valve formation, abolished FOXP2 induction in vitro and in vivo. Genetic deletion of Foxp2 in mice using the endothelial‐specific Tie2‐Cre or the tamoxifen‐inducible LEC‐specific Prox1‐CreERT2 line resulted in enlarged collecting vessels and defective valves characterized by loss of NFATc1 activity. Our results identify FOXP2 as a new flow‐induced transcriptional regulator of collecting lymphatic vessel morphogenesis and highlight the existence of unique transcription factor codes in the establishment of vessel‐type‐specific endothelial cell identities. SYNOPSIS: Molecular mechanisms that regulate the identity of functionally specialized lymphatic vessel types are incompletely understood. The current study identifies transcription factor FOXP2 as a signature gene and a critical transcriptional regulator of collecting lymphatic vessels. Transcriptome analysis of murine endothelial cells identifies specific expression of Foxp2 in collecting lymphatic vessels.FOXP2 expression is induced after initiation of lymph flow and regulated by fluid shear stress.Genetic deletion of Foxp2 in mice leads to collecting lymphatic vessel and valve defects.FOXP2 acts downstream of the flow‐responsive transcription factor FOXC2 to regulate NFATc1 activity. [ABSTRACT FROM AUTHOR]

Simple Summary: Tumor draining lymph nodes (TDLNs) are the most common metastatic sites in human cancer but are also essential sites for induction of tumor immunity. How different types of primary tumors affect the anti-tumor immune response in the LNs is not fully understood. By analyzing biobank tissue from breast cancer patients, we demonstrate that invasive breast cancer induce dramatic pre-metastatic LN changes affecting the structure and function of the specialized LN vasculature and associated stromal cells, required for recruitment of T-lymphocytes into the LNs. These changes could not be seen in patients with non-invasive breast cancer and provide new insights of how invasive tumors can disrupt essential functions within the immune system. The data also shows promise of LN stromal and vascular changes as possible future biomarkers for prediction of disease progression in human cancer. The tumor-draining lymph nodes (TDLNs) are primary sites for induction of tumor immunity. They are also common sites of metastasis, suggesting that tumor-induced mechanisms can subvert anti-tumor immune responses and promote metastatic seeding. The high endothelial venules (HEVs) together with CCL21-expressing fibroblastic reticular cells (FRCs) are essential for lymphocyte recruitment into the LNs. We established multicolor antibody panels for evaluation of HEVs and FRCs in TDLNs from breast cancer (BC) patients. Our data show that patients with invasive BC display extensive structural and molecular remodeling of the HEVs, including vessel dilation, thinning of the endothelium and discontinuous expression of the HEV-marker PNAd. Remodeling of the HEVs was associated with dysregulation of CCL21 in perivascular FRCs and with accumulation of CCL21-saturated lymphocytes, which we link to loss of CCL21-binding heparan sulfate in FRCs. These changes were rare or absent in LNs from patients with non-invasive BC and cancer-free organ donors and were observed independent of nodal metastasis. Thus, pre-metastatic dysregulation of core stromal and vascular functions within TDLNs reflect the primary tumor invasiveness in BC. This adds to the understanding of cancer-induced perturbation of the immune response and opens for prospects of vascular and stromal changes in TDLNs as potential biomarkers. [ABSTRACT FROM AUTHOR]