Lymphatic vessels (LVs), lined by lymphatic endothelial cells (LECs), are indispensable for life. However, the role of metabolism in LECs has been incompletely elucidated. In the present study, we reported that LEC-specific loss of OXCT1, a key enzyme of ketone body oxidation (KBO), reduces LEC proliferation, migration and vessel sprouting in vitro and impairs lymphangiogenesis in Prox1 ΔOXCT1 mice. Mechanistically, OXCT1 silencing lowers acetyl-CoA levels, TCA metabolite pools, nucleotide precursor and dNTP levels required for LEC proliferation. Ketone body supplementation to LECs induces the opposite effects. Notably, elevation of lymph ketone body levels by a high-fat, low-carbohydrate ketogenic diet increases lymphangiogenesis after corneal injury and improves LV function and growth in a mouse model of microsurgical ablation of LVs in the tail, which recapitulates the features of acquired lymphedema in humans.✓ Ketogenic diet and β-OHB enhances lymphangiogenesis after cornea injury ✓ Ketogenic diet decreases tail lymphedema and improves the functionality of lymphatic vessels

Since a detailed inventory of endothelial cell (EC) heterogeneity in breast cancer (BC) is lacking, here we perform single cell RNA-sequencing of 26,515 cells (including 8433 ECs) from 9 BC patients and compare them to published EC taxonomies from lung tumors. Angiogenic ECs are phenotypically similar, while other EC subtypes are different. Predictive interactome analysis reveals known but also previously unreported receptor-ligand interactions between ECs and immune cells, suggesting an involvement of breast EC subtypes in immune responses. We also identify a capillary EC subtype (LIPEC (Lipid Processing EC)), which expresses genes involved in lipid processing that are regulated by PPAR-γ and is more abundant in peri-tumoral breast tissue. Retrospective analysis of 4648 BC patients reveals that treatment with metformin (an indirect PPAR-γ signaling activator) provides long-lasting clinical benefit and is positively associated with LIPEC abundance. Our findings warrant further exploration of this LIPEC/PPAR-γ link for BC treatment.

Tumor vessel co-option (VCO) is a non-angiogenic vascularization mechanism that is a possible cause of resistance to anti-angiogenic therapy (AAT). Multiple tumors are hypothesized to primarily rely on growth factor signaling-induced sprouting angiogenesis, which is often inhibited during AAT. During VCO however, tumors invade healthy tissues by hijacking pre-existing blood vessels of the host organ to secure their blood and nutrient supply. Although VCO has been described in the context of AAT resistance, the molecular mechanisms underlying this process and the profile and characteristics of co-opted vascular cell types (endothelial cells (ECs) and pericytes) remain poorly understood, resulting in the lack of therapeutic strategies to inhibit VCO (and to overcome AAT resistance). In the past few years, novel next-generation technologies (such as single-cell RNA sequencing) have emerged and revolutionized the way of analyzing and understanding cancer biology. While most studies utilizing single-cell RNA sequencing with focus on cancer vascularization have centered around ECs during sprouting angiogenesis, we propose that this and other novel technologies can be used in future investigations to shed light on tumor EC biology during VCO. In this review, we summarize the molecular mechanisms driving VCO known to date and introduce the models used to study this phenomenon to date. We highlight VCO studies that recently emerged using sequencing approaches and propose how these and other novel state-of-the-art methods can be used in the future to further explore ECs and other cell types in the VCO process and to identify potential vulnerabilities in tumors relying on VCO. A better understanding of VCO by using novel approaches could provide new answers to the many open questions, and thus pave the way to develop new strategies to control and target tumor vascularization.