Modeling pre-metastatic lymphvascular niche in the mouse ear sponge assay.

Lymphangiogenesis, the formation of new lymphatic vessels, occurs in primary tumors and in draining lymph nodes leading to pre-metastatic niche formation. Reliable in vivo models are becoming instrumental for investigating alterations occurring in lymph nodes before tumor cell arrival. In this study, we demonstrate that B16F10 melanoma cell encapsulation in a biomaterial, and implantation in the mouse ear, prevents their rapid lymphatic spread observed when cells are directly injected in the ear. Vascular remodeling in lymph nodes was detected two weeks after sponge implantation, while their colonization by tumor cells occurred two weeks later. In this model, a huge lymphangiogenic response was induced in primary tumors and in pre-metastatic and metastatic lymph nodes. In control lymph nodes, lymphatic vessels were confined to the cortex. In contrast, an enlargement and expansion of lymphatic vessels towards paracortical and medullar areas occurred in pre-metastatic lymph nodes. We designed an original computerized-assisted quantification method to examine the lymphatic vessel structure and the spatial distribution. This new reliable and accurate model is suitable for in vivo studies of lymphangiogenesis, holds promise for unraveling the mechanisms underlying lymphatic metastases and pre-metastatic niche formation in lymph nodes, and will provide new tools for drug testing

Periostin in lymph node pre-metastatic niches governs lymphatic endothelial cell functions and metastatic colonization.

peer reviewedAlthough lymph node (LN) metastasis is an important prognostic parameter in cervical cancer, the tissue remodeling at a pre-metastatic state is poorly documented in LNs. We here identified periostin (POSTN) as a component of non-metastatic LNs by applying proteomic analyses and computerized image quantifications on LNs of patients with cervical cancer. We provide evidence for remarkable modifications of POSTN and lymphatic vessel distributions and densities in non-metastatic sentinel and metastatic human LNs, when compared to distant non-metastatic LNs. POSTN deposition at a pre-metastatic stage was demonstrated in a pre-clinical murine model (the ear sponge assay). Its expression by fibroblastic LN cells was assessed by in situ hybridization and in vitro cultures. In vitro, POSTN promoted lymphatic endothelial cell functions and tumor cell proliferation. Accordingly, the in vivo injection of recombinant POSTN together with VEGF-C boosted the lymphangiogenic response, while the metastatic potential of tumor cells was drastically reduced using a POSTN blocking antibody. This translational study also supports the existence of an unprecedented dialog "in cascade", between the primary tumor and the first pelvic nodal relay in early cervical cancer, and subsequently from pelvic LN to para-aortic LNs in locally advanced cervical cancers. Collectively, this work highlights the association of POSTN deposition with lymphangiogenesis in LNs, and provides evidence for a key contribution of POSTN in promoting VEGF-C driven lymphangiogenesis and the seeding of metastatic cells

Lipid droplet degradation by autophagy connects mitochondria metabolism to Prox1-driven expression of lymphatic genes and lymphangiogenesis.

Autophagy has vasculoprotective roles, but whether and how it regulates lymphatic endothelial cells (LEC) homeostasis and lymphangiogenesis is unknown. Here, we show that genetic deficiency of autophagy in LEC impairs responses to VEGF-C and injury-driven corneal lymphangiogenesis. Autophagy loss in LEC compromises the expression of main effectors of LEC identity, like VEGFR3, affects mitochondrial dynamics and causes an accumulation of lipid droplets (LDs) in vitro and in vivo. When lipophagy is impaired, mitochondrial ATP production, fatty acid oxidation, acetyl-CoA/CoA ratio and expression of lymphangiogenic PROX1 target genes are dwindled. Enforcing mitochondria fusion by silencing dynamin-related-protein 1 (DRP1) in autophagy-deficient LEC fails to restore LDs turnover and lymphatic gene expression, whereas supplementing the fatty acid precursor acetate rescues VEGFR3 levels and signaling, and lymphangiogenesis in LEC-Atg5-/- mice. Our findings reveal that lipophagy in LEC by supporting FAO, preserves a mitochondrial-PROX1 gene expression circuit that safeguards LEC responsiveness to lymphangiogenic mediators and lymphangiogenesis.We thank K. Rillaerts, J. Souffreau, and A. Bouche, for expert technical support and Dr. A. Luttun and Dr. A. Zijsen for sharing tools and advices. P.A. is supported by grants from the Flemish Research Foundation (FWO-Vlaanderen; G076617N, G049817N, G070115N), the EOS MetaNiche consortium N degrees 40007532, Stichting tegen Kanker (FAF-F/2018/1252) and the iBOF/21/053 ATLANTIS consortium with G.B. D.H. is the recipient of an FWO Doctoral Fellowship from the Flemish Research Foundation (FWO-Vlaanderen, 1186019N), Belgium. M.B. is supported by the `Fonds voor Wetenschappelijk Onderzoek' (FWO). K.J. is the recipient of an FWO Postdoctoral Fellowship from the Flemish Research Foundation (FWO-Vlaanderen). P.C. is supported by Methusalem funding by the Flemish government, and by an ERC Advanced Research Grant (EU-ERC269073).S

Ear Sponge Assay: A Method to Investigate Angiogenesis and Lymphangiogenesis in Mice.

peer reviewedAngiogenesis and lymphangiogenesis have become important research areas in the biomedical field. The outgrowth of new blood (angiogenesis) and lymphatic (lymphangiogenesis) vessels from preexisting ones is involved in many pathologies including cancer. In-depth investigations of molecular determinants such as proteases in these complex processes require reliable in vivo models. Here we present the ear sponge assay as an easy, rapid, quantitative and reproducible model of angiogenesis and lymphangiogenesis. In this system, a gelatin sponge soaked with tumor cells, cell-conditioned medium, or a compound to be tested is implanted, for 2-4 weeks, between the two mouse ear skin layers. The two vascular networks are next examined through histological procedures

Nouveaux procédés de microscopie tridimensionnelle et algorithmes de traitement et quantification d'images pour étudier les modèles biologiques associés au cancer

Recent progresses in 3D microscopy technologies revolutionise our way to study fundamental biological processes involved in cancer metastasis. We considered several in vitro and in vivo biological models, used various microscopy techniques and developed adapted processing, characterisation and quantification algorithms to study migration, invasion, angiogenesis lymphangiogenesis processes by analysing cell-to-cell and cell-to-matrix interactions

Tumor exposed-lymphatic endothelial cells promote primary tumor growth via IL6.

Solid tumors are composed of tumor cells and stromal cells including lymphatic endothelial cells (LEC), which are mainly viewed as cells forming lymphatic vessels involved in the transport of metastatic and immune cells. We here reveal a new mechanism by which tumor exposed-LEC (teLEC) exert mitogenic effects on tumor cells. Our conclusions are supported by morphological and molecular changes induced in teLEC that in turn enhance cancer cell invasion in 3D cultures and tumor cell proliferation in vivo. The characterization of teLEC secretome by RNA-Sequencing and cytokine array revealed that interleukine-6 (IL6) is one of the most modulated molecules in teLEC, whose production was negligible in unexposed LEC. Notably, neutralizing anti-human IL6 antibody abrogated teLEC-mediated mitogenic effects in vivo, when LEC were mixed with tumor cells in the ear sponge assay. We here assign a novel function to teLEC that is beyond their role of lymphatic vessel formation. This work highlights a new paradigm, in which teLEC exert "fibroblast-like properties", contribute in a paracrine manner to the control of tumor cell properties and are worth considering as key stromal determinant in future studies

Contralateral vascularized lymph node transfer : an optimized mouse model

peer reviewe