Abstract
Purpose :
Uveal melanoma (UM) is the most common primary intraocular tumor in adults, and half cases develop incurable liver metastases. As the liver is the first and often the only metastatic site of UM, this suggests the existence of a favorable microenvironment for UM cells. Our hypothesis is that extracellular vesicles (EVs) are released by cancer cells from the ocular tumor precondition the hepatic stroma by activating hepatic stellate cells (HSteCs) to promote the colonization by metastatic cells. Our study aims to determine the role of melanomic EVs in the mechanisms governing the remodeling of the liver microenvironment, including the stromal stiffness.
Methods :
EVs were isolated from UM cells and choroidal melanocytes by differential centrifugation. The impact of melanomic EVs on the viability, metabolism and gene expression profile of targeted cells was studied using MTS assay, LIVE/DEAD assay, and RNA-Sequencing. Activation of the mechanotransduction signaling pathways were studied by immunofluorescence and Western blotting (FAK phosphorylation and YAP nuclear translocation). HSteCs were seeded on hydrogels of varying stiffness (0.8/2.5/8 kPa; which mimic the stiffness of healthy to highly fibrotic liver), and then exposed to EVs to study their level of activation/contractility by confocal microscopy (αSMA+) and traction force microscopy. The preconditioning of the liver by melanomic EVs were studied by in vivo fluorescence imaging in immunodeficient mice. The EV-driven activation of HSteCs and their remodeling of the hepatic stroma were analyzed by confocal and polarization microscopy.
Results :
Melanomic EVs increased the proliferation and energy metabolism of HSteCs, and activated pro-fibrotic signaling pathways. The increased rigidity of hydrogels, which mimic advanced pathological stages, favored the internalization of melanomic EVs by HSteCs. The preconditioning of the liver by melanomic EVs promoted the development of metastases and the production of a stiffer extracellular matrix in the animal model.
Conclusions :
Melanomic EVs preconditioned the liver by establishing a pre-metastatic niche through the activation of stellate cells. A better understanding of the bidirectional crosstalk between metastatic UM cells and stellate cells at the premetastatic and micro-metastatic stages in the liver will allow to identify new molecular mechanisms or biophysical features that could be therapeutically targeted.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.