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

Abstract

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.

Mechanisms of Tumor-Induced Lymphovascular Niche Formation in Draining Lymph Nodes

“…For instance, mice overexpressing vascular endothelial growth factor (VEGF)-A or VEGF-C in a model of chemically induced skin carcinogenesis showed prominently enhanced LN lymphangiogenesis, concomitant with a significant increase of LN and distant organ metastasis ( Hirakawa et al., 2005 , Hirakawa et al., 2007 ). Importantly, these and other studies using experimental models of nasopharyngeal carcinoma ( Qian et al., 2006 ) and malignant melanoma ( García-Caballero et al., 2017 , Harrell et al., 2007 ) have identified the onset of LN lymphatic remodeling prior to the colonization of the LN by metastatic cells. These data suggest that, even in the absence of metastatic tumor cells, tumor-associated lymphangiogenic factors can be drained from the primary tumor or released by locally activated cells to initiate changes in preparation of a pre-metastatic “lymphovascular niche” ( Hirakawa et al., 2005 , Ogawa et al., 2014 , Olmeda et al., 2017 ).…”

Consensus guidelines for the use and interpretation of angiogenesis assays

“…In this model, sprouting lymphangiogenesis is reproduced in a complex in vivo microenvironment. This application is based on the use of a biomaterial, gelatin sponge that is inserted in the mouse ear to develop a large lymphatic network [ 145 ]. The gelatin sponge is cut into pieces of about 3 mm 3 , which are transferred in a 96-well plate (one sponge piece per well) for pre-treatment ( Fig.…”

Spatial architecture of the immune microenvironment orchestrates tumor immunity and therapeutic response

“…The immunoregulatory components and their functions within niches have been thoroughly studied and recently reviewed ( 100 , 142 ). Several studies have progressively revealed the spatial distribution of immune and host stromal components within niches ( 143 – 145 ).…”