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Role of lymphatic endothelial cell-associated autophagy in melanoma dissemination

Lymphatic vessels are a crucial component of the circulatory system that maintains homeostasis by facilitating tissue fluid and dietary fat absorption and aid in inflammatory and immune responses. Disruptions in their function, driving either excessive lymphatic growth or contraction, have been linked to various diseases, including inflammation, cancer metastasis, and lymphedema. Understanding the mechanisms that regulate lymphatic vessel function and the process of lymphangiogenesis, the formation of new lymphatic vessels is therefore of great importance. Previous studies have highlighted the essential role of autophagy, a lysosomal pathway responsible for degrading macromolecules and organelles, in endothelial cell biology, with a particular focus on blood vessels. However, the potential impact of autophagy on lymphatic endothelial cells (LECs) remained unclear. Our research has shown that in a model of injury-driven corneal lymphangiogenesis, LECs deficient in autophagy lose their responsiveness to lymphangiogenic stimuli, both in vitro and in vivo. We found that loss of Atg5 in LECs caused an impairment in the PROX1-VEGFR3 axis that sustains LEC identity. We revealed that the underpinning mechanism relies on impaired degradation of lipid droplets (LDs) by lipophagy in Atg5-depleted LECs, which compromises mitochondrial fatty acid oxidation (FAO) and reduced levels of acetyl-CoA, which is required to support epigenetic regulation of PROX1-targeted genes, including VEGFR3 and CPT1A. Importantly, restoring the ability of mitochondria to use FAO, by supplementing acetate to the mice, rescued corneal lymphangiogenesis in LEC-Atg5 deficient conditions, thus providing further support to our mechanistic data. Lymph nodes (LNs) are specialized structures that serve as immune centers, where lymphocytes encounter antigens and differentiate into effector cells, ultimately leading to the development of effective adaptive immune responses. These complex processes rely on the efficient transport of antigens and immune cells to and from the LNs. Having established a role for autophagy in LEC homeostasis, we wondered whether it could also locally regulate immune responses, more specifically anti-cancer immune responses elicited by immunotherapy with immune checkpoint blockade (ICB). We show that in LEC-specific autophagy-deficient mice, lymphocyte egress from (Td)LNs is impaired, both in steady state and tumor conditions. Genetic deletion of Atg5 in LECs led to increased secretion of S1P by LN LECs, which disturbed the intranodal availability of sphingosine-1-phosphate (S1P), acting as the main exit signal for lymphocytes in secondary lymphoid organs (SLOs). Spatial analysis, via multiplex immunohistochemistry staining of the TdLN, showed a defect in lymphocyte intranodal localization. Single-cell RNA-sequencing of TdLNs from WT and ATG5LEC-KO mice unraveled that loss of Atg5 remodeled niche-specific LEC phenotypes, in particular the so-called ceiling LECs of the subcapsular sinus, and changed expression of genes involved in proliferation, sphingolipid metabolism, lymphocyte trafficking and LEC-T cell interactions. The entrapment of lymphocytes in TdLNs abrogated the effects of ICB, as primed lymphocytes failed to reach the tumor parenchyma. In conclusion, these studies provide evidence for a context-dependent, crucial role of autophagy in inflammation-induced LEC responses, lymph node immune functions, and systemic lymphocyte trafficking, with relevant implications for lymphangiogenesis and anti-cancer immune responses following immunotherapy.

Date:11 Sep 2017 →  28 Sep 2023
Keywords:Endothelial- specific autophagy
Disciplines:Morphological sciences, Oncology
Project type:PhD project