Cholesterol Promotes Acute Leukemia Progression through Systemic but Selective Endothelial Cell Activation.
Authors: Carvalho Tania , Fragoso Rita , Gomes Ana L , Casalou Cristina , Martins Leila , Barata Joao , Dias Sergio 
Published: April 2020

Abstract
High seric levels of cholesterol are generally associated with endothelial dysfunction. Cholesterol has also been shown to positively affect acute leukemia behavior through direct modulation of leukemia cells proliferation/survival. Yet, the indirect/systemic effects of cholesterol in this setting are not clear. We hypothesized that cholesterol promotes acute leukemia progression also through systemic endothelial cell activation. First, to understand how ALL use cholesterol for their advantage, B-ALL 697 cells were treated with methyl-β-cyclodextrin (MβCD)-cholesterol complexes, after which a modest increase in B-ALL cells proliferation and transwell migration (to VEGF/PlGF, as shown in Fragoso et al 2006) was seen. In contrast B-ALL cells adhesion to endothelium was maximized when the cholesterol content of endothelial cells was raised with methyl-β-cyclodextrin (MβCD)-cholesterol (p ≤ 0.05). Similarly, cholesterol-enriched endothelial cells promoted transendothelial transwell B-ALL migration towards PlGF (p ≤ 0.05). The same effect was not seen with cholesterol-enrichment of B-ALL cells. FACS analysis revealed a significant increase in cellular adhesion molecules (CAMs) expression, specifically P-Selectin and VCAM-1, in cholesterol-enriched endothelial cells (p ≤ 0.05). This raise in CAMs expression is accompanied by enhanced mRNA levels for complement C3 factor and C3a receptor and increased secreted levels of C3a fragment in cholesterol-enriched endothelial cells (p ≤ 0.05). B-ALL cells transendothelial migration assay to PlGF was impaired in the presence of a C3aR antagonism (SB 290157) (p ≤ 0.05). To test these findings in vivo, a 30-day fat food feeding trial in a rodent model of acute lymphoblastic leukemia (ALL) was conducted. One month of fat food (cholesterolenriched) diet consumption resulted in significantly increased serum levels of cholesterol and complement C3a fragment (which is known to be produced by activated endothelium) (p ≤ 0.05). Endothelial cells isolated by FACS sorting (Muc-2 positive) from the BM, spleen, lung and brain of these animals displayed distinct cell adhesion molecules (CAM) P-Selectin and VCAM-1 expression profiles: fat food diet significantly increased P-Selectin expression in BM, spleen and lung endothelial cells but not in the brain (p ≤ 0.05), while VCAM-1 increases in BM, spleen and brain endothelial cells, but not in the lung (p ≤ 0.05). C3aR antagonist intraperitoneal injection in mice on fat diet differentially impairs this CAM activation: P-selectin is downregulated by C3aR antagonism in BM, spleen and lung endothelial cells, VCAM-1 is down regulated in BM and spleen, but no effect is seen for both CAMs in brain endothelial cells. After the 30-day fat food trial, Nod-Scid mice were xenotransplanted with B-ALL (697 cell line) and T-ALL (Molt-4) gfp/luc cells and imaged with Ivis Lumina Imaging System. Fat food diet was associated with enhanced in vivo dissemination and progression in B and T-ALL; marked increase in bioluminescence was seen in both models, with enhanced signal being preferentially located in the cranium, vertebrae, femoral-tibial joint region and sternum for B-ALL, and in the cranium, vertebrae and femoral-tibial joint region for T-ALL. At day 11 post-B-ALL xenotransplantation mice on fat food diet displayed increased leukemia cell numbers in the bone marrow (BM), peripheral blood and spleen. The bioluminescence in the cranium was due to leukemia cells infiltration of the bone marrow of the skull, meninges and hypothalamus. There was a two-fold decrease in survival at day 15 post-B-ALL xenotransplantation in mice on fat food diet (p ≤ 0.05). Importantly, C3aR inhibition partially inhibited leukemia infiltration in peripheral organs. Our data suggests that in a hypercholesterolemic environment, endothelial monolayer permeability, leukemia adhesion to endothelium and leukemia trans-endothelium migration is exerted, at least partially, through C3 pathway activation and C3a signaling, resulting in selective and endothelial-specific CAMs induction. Activation of complement and the production of C3a affects the cross-talk between endothelium and leukemia cells, favoring leukemia progression. Furthermore endothelial cells from different internal organs are distinct in terms of responsiveness to cholesterol and expression of CAM, possibly conditioning leukemia cells spread and invasion.