During metastasis, malignant cells escape the primary tumor, intravasate lymphatic vessels, and reach draining sentinel lymph nodes before they colonize distant organs via the blood circulation. Although lymph node metastasis in cancer patients correlates with poor prognosis, evidence is lacking as to whether and how tumor cells enter the bloodstream via lymph nodes. To investigate this question, we delivered carcinoma cells into the lymph nodes of mice by microinfusing the cells into afferent lymphatic vessels. We found that tumor cells rapidly infiltrated the lymph node parenchyma, invaded blood vessels, and seeded lung metastases without involvement of the thoracic duct. These results suggest that the lymph node blood vessels can serve as an exit route for systemic dissemination of cancer cells in experimental mouse models. Whether this form of tumor cell spreading occurs in cancer patients remains to be determined.

In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.

Inflammation stimulates lymphatic endothelial cells to release exosomes, which accumulate in the perivascular stroma. Brown et al. show that these exosomes promote the directional migration of dendritic cells along guidance cues in complex environments by enhancing dynamic cellular protrusions in a CX3CL1-dependent manner.

Type 2 diabetes is associated with microvascular damage that causes frequent infections in the skin and chronic ulcers as a result of impaired wound healing. To trace the pathological changes, we performed a comprehensive analysis of lymphatic vessels in the skin of type 2 diabetic versus nondiabetic patients. The dermis revealed enhanced lymphatic vessel density, and transcriptional profiling of ex vivo isolated lymphatic endothelial cells (LECs) identified 160 genes differentially expressed between type 2 diabetic and nondiabetic LECs. Bioinformatic analysis of deregulated genes uncovered sets functionally related to inflammation, lymphatic vessel remodeling, lymphangiogenesis, and lipid and small molecule transport. Furthermore, we traced CD68+ macrophage accumulation and concomitant upregulation of tumor necrosis factor-α (TNF-α) levels in type 2 diabetic skin. TNF-α treatment of LECs and its specific blockade in vitro reproduced differential regulation of a gene set that led to enhanced LEC mobility and macrophage attachment, which was mediated by the LEC-derived chemokine CXCL10. This study identifies lymph vessel gene signatures directly correlated with type 2 diabetes skin manifestations. In addition, we provide evidence for paracrine cross-talk fostering macrophage recruitment to LECs as one pathophysiological process that might contribute to aberrant lymphangiogenesis and persistent inflammation in the skin.

Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca2+ levels were measured and pharmacological- or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca2+, Ca2+-calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca2+ release. Thus, Ca2+ signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour–stroma interaction.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-016-2441-5) contains supplementary material, which is available to authorized users.