Abstract
Purpose :
We have previously shown that uveal melanoma (UM) mutations that are associated with aggressive behavior are not sufficient for metastases to occur. We hypothesize that a UM vascular architecture is necessary for tumor growth and metastases, and that architecture forms first with macrophage infiltration of the UM and later vessel formation.
Methods :
Enucleated human eyes with UM were examined using spatial omic multiplex protein detection for CD31, P4HA1, P4HA2, BAP1, SOX10, and CD68. Antibody based immunofluorescence labeled each target, in cycles of 3 each, followed by bleaching fluorophores with 4.5% peroxide to confirm signal removal between cycles. All images were acquired with Nikon Eclipse TE2000-U widefield microscope. CX3CR1-GFP mice with macrophages labeled with GFP underwent intraocular inoculation of B16F10, Queens, or B16LS9 melanoma cells and at 4 days were imaged using the Heidelberg Spectralis HRT and Phoenix Micron IV OCT. The mice eyes were then enucleated and examined with triple immunohistochemical labeling for CD68/CD31/gp100, and for HIF1α and digitally analyzed for macrophage and endothelial cell location (Scipy, Python).
Results :
Human UM exhibited clusters and empty channels that contained CD68+ macrophages, immature dilated vessels lined by CD31+ cells and some gp100+ UM cells, and well-formed vessels lined by CD31+ cells. Macrophages were concentrated in the UM vs surrounding choroid in the mice imaged by Blue Autofluorescence and OCT. The enucleated mice eyes with gp100+ melanoma exhibited CD68+ macrophages forming tubes/empty spaces, CD31+ endothelial cells in the vicinity of these spaces, lining these vessels. HIF1α was expressed in portions of the tumor that contained many macrophages and few endothelial cells compared with tumor that contained numerous endothelial lined vascular channels. Digital analysis showed macrophages and endothelial cells evenly dispersed in the UM with relative presence and absence of macrophages in avascular and vascular areas, respectively.
Conclusions :
Our study supports the hypothesis that macrophages infiltrate hypoxic areas of UM, create channels, and endothelial cells line these channels forming a vascular supply for the UM.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.