Migration of Acute Lymphoblastic Leukemia Cells into the Central Nervous System Is Regulated By VEGF
Authors: Muench Vera , Trentin Luca , Herzig Julia , Krauss Johann , Kestler Hans A , te Kronnie Geertruy , Debatin Klaus-Michael , Meyer Lueder Hinrich 
Published: October 2019

Abstract
In acute lymphoblastic leukemia (ALL), central nervous system (CNS) directed therapy is required to achieve long-term remission and survival even in patients without detectable CNS disease, indicating subclinical CNS manifestation in many patients. Hence, prophylactic CNS therapy is indispensable but bears the risk of secondary neoplasms and neurocognitive deficits in ALL survivors. Therefore, a better understanding of mechanisms mediating CNS involvement in ALL are needed in order to identify potential targets for prophylactic and therapeutic intervention.
In this study, we transplanted primary patient B cell precursor (BCP) ALL cells onto NOD/SCID mice and investigated engraftment of human leukemia cells in the recipient's peripheral blood (PB), bone marrow (BM), spleen (S), and meninges by flowcytometry staining for huCD19. Upon disease onset, we identified meningeal infiltration of human ALL cells together with leukemia engraftment in BM, S and PB in a subset of samples (CNSpos) in contrast to absent CNS involvement despite high leukemia cell infiltration of BM, S and PB in other recipients (CNSneg). CNSpos and CNSneg phenotypes were consistently observed in subsequent xenograft passages. In CNSpos animals meningeal leukemia infiltration was also detected by immunohistochemistry on brain sections and meningeal enhancement was detected by small animal magnetic resonance imaging in CNSpos recipients.
We further characterized ALL cells isolated from meningeal and BM infiltrates by gene expression profiling and identified the gene coding for vascular endothelial growth factor A (VEGF) to be highly expressed in ALL cells isolated from the CNS as compared to BM derived cells. Differential expression of VEGF was validated by qPCR and confirmed in independent sample cohorts. Most interestingly, reported functions of VEGF include regulation of cellular growth, vascular permeability, and trans-endothelial cell migration, and elevated VEGF protein levels have previously been described in cerebrospinal fluid specimens collected from ALL and AML patients with CNS leukemia.
VEGF signals through its receptors 1 or 2 (VEGFR1/2). On all primary ALL samples, only expression of VEGFR1 but not VEGFR2 was detected. We used the BCP-ALL cell line Nalm-6, which also expresses VEGF and VEGFR1 and mediates a clear CNSpos phenotype upon engraftment. However, cellular survival, proliferation, apoptosis, and metabolic activity were not affected, neither upon exposure to VEGF or the antagonizing VEGF antibody bavacizumab, nor by stable VEGF overexpression or knock-down, thus indicating absence of autocrine VEGF/VEGFR1 signaling.
We further analyzed whether paracrine VEGF signaling through VEGFR2, which upon activation mediates endothelial cell permeability, is involved in trans-endothelial migration of ALL cells leading to leukemia infiltration of the CNS. Brain endothelial cells (bEND.3) incubated with VEGF showed increased phosphorylation of VEGFR2 downstream signaling molecules (Src, AKT), which indicates activated signaling mediating cellular permeability.
Moreover, we modeled migration of ALL cells through brain endothelial cells in a transwell assay and observed significantly increased migration of Nalm-6 ALL cells through bEND.3 monolayers upon exposure to VEGF or upon overexpression of VEGF as compared to controls. Vice versa, significantly lower numbers of migrated leukemia cells were detected after incubation with bevacizumab or upon VEGF knock down indicating VEGF dependent trans-endothelial migration.
Thus, we identified high expression of VEGF in CNS derived leukemia cells, absent autocrine signaling of VEGF on ALL cells and, most importantly, VEGF dependent trans-endothelial migration of ALL cells indicating VEGF as a possible mediator of CNS leukemia.
Finally, we investigated the impact of VEGF on CNS leukemia manifestation in vivo. Recipient animals transplanted with 3 CNSpos primograft samples (4 experiments, one in repetition) were treated with bevacizumab or control. In all experiments, anti-VEGF treatment significantly reduced the leukemia burden exclusively in the CNS but not in BM, S, and PB compartments, indicating that transmigration of leukemia cells and CNS manifestation in ALL is mediated by VEGF. Thus, targeting of VEGF signaling may serve as a novel strategy to control CNS leukemia in patients.

Disclosures
No relevant conflicts of interest to declare.