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methotrexate resistance.16 Similarly, Akers et al. showed that co-culture of leukemia cells with astrocytes, choroid plexus epithelial cells, or meningeal cells enhanced leukemia chemoresistance.17 Together these observations suggest that the pathophysiology of CNS leukemia extends beyond the role of the blood-brain barrier. We hypothesize that the ability of leukemia cells to persist in the unique CNS niche and escape the effects of chemotherapy and immune surveillance likely also play critical roles in CNS leukemia and relapse.
However, while extensive research has demonstrated a critical role of the bone marrow niche in leukemia biology, the impact of the CNS niche on leukemia biology is less well understood.18,19 Herein, we demonstrate that the meninges exert a unique and critical influence on leukemia biology by enhancing leukemia resistance to the chemotherapy agents currently used in the therapy of CNS leukemia. We then leveraged this new understanding of the mechanisms of meningeal-mediated leukemia chemoresistance to identify a novel drug, Me6TREN (Tris[2-(dimethylamino)ethyl]amine), which overcomes leukemia chemoresistance by disrupting the interaction between leukemia and meningeal cells.
Methods
Cells and tissue culture
Leukemia cells were obtained from the American Type Culture Collection (ATCC) or Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and cultured in RPMI medium (Sigma-Aldrich) supplemented with fetal bovine serum 10% (Seradigm) and penicillin-streptomycin (Sigma-Aldrich). Leukemia cell lines included both B-cell (NALM-6, SEM) and T- cell (Jurkat, SEM, MOLT-13) immunophenotypes. The HCN-2 neuronal cell line was obtained from the ATCC. Leukemia cells expressing green fluorescent protein (GFP) were generated as described elsewhere.20 Murine leukemia cells, generated by BCR/ABL p190 expression in hematopoietic cells from CD45.1 Arf-/- mice,21-23 were provided by Dr. Michael Farrar (University of Minnesota, MN, USA). Primary B-ALL cells for co-culture exper- iments were obtained from the University of Minnesota Hematologic Malignancy Bank (IRB #: 0611M96846; pediatric patient at diagnosis). Primary B-ALL cells for in vivo experiments were obtained from the Public Repository of Xenografts [PRoXe;24 sample CBAB-62871-V1; pediatric patient at diagnosis with a t(4;11) translocation]. Primary meningeal cells were obtained from ScienCell and cultured in meningeal medium sup- plemented with fetal bovine serum 2%, growth supplement, and penicillin-streptomycin. Meningeal cells were isolated from multiple different donor specimens and were typically used between passages 3-5.
Murine experiments
NSG (NOD.Cg-Prkdcscid, Il2rgtm1Wjl/SzJ; Jackson Laboratory) mice were housed under aseptic conditions. Mouse care and experiments were in accordance with a protocol approved by the Institutional Animal Care and Use Committee at the University of Minnesota (IRB#1704-34717A). Mice 6-8 weeks old were injected intravenously via the tail vein with ~1-2x106 human leukemia cells. Experiments were then performed 3-5 weeks after injection. In general, after euthanasia of the mice, the heart was perfused with phosphate-buffered saline and the meninges or other tissues were removed using a dissecting microscope, and dissociated by gently washing through a 0.40
Attenuating leukemia chemoresistance in the CNS
mm filter (Millipore). Cells were then stained with fluorescent antibodies against CD19 (NALM-6; eBioscience) or CD3 (Jurkat, eBioscience) and assessed for apoptosis or cell cycle as described. Alternatively, leukemia cells could be purified from meningeal cells using immunomagnetic separation and either CD19 or CD3 antibodies (Stem Cell Technologies or Miltenyi Biotec) and placed back into suspension. In drug treatment experiments, Me6TREN 10 mg/kg and cytarabine 50 mg/kg were given by subcutaneous and intraperitoneal injection, respectively. Cerebrospinal fluid was obtained from mice as described elsewhere.25 Briefly, mice were euthanized and a scalp incision was made at the midline to expose the dura mater over- lying the cisterna magna. Under a dissection microscope, a tapered, pulled glass capillary tube was then inserted through the dura and into the cisterna magna to obtain clear cere- brospinal fluid. Experiments with murine leukemia cells (BCR/ABL p190 expression in hematopoietic cells from Arf-/- mice; CD45.1 background) used C57BL/6 mice. In these experi- ments, ~3000 leukemia cells/mouse were injected via the tail vein.
Statistical analysis
Results are shown as the mean ± standard error of mean of the results of at least three experiments. The Student t-test or analysis of variance was used for statistical comparisons between groups. The log-rank (Mantel-Cox) test was used to calculate P values comparing the mouse survival curves. P values <0.05 were consid- ered statistically significant. Statistical analyses were conducted using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA,USA).
Results
Leukemia cells reside in the meninges of the mouse central nervous system
In order to identify the anatomic site(s) in the CNS within which the leukemia cells reside, we transplanted multiple human ALL cell lines, including NALM-6, Jurkat, and SEM, into immune-compromised mice (NSG) via tail vein injection (Online Supplementary Figure S1A). Mice were not irradiated or conditioned with busulfan prior to transplantation to avoid perturbing leukemia niches. The mice were then euthanized and the CNS examined by histopathology and immunohistochemistry. We identified both the meninges and, to a lesser extent, the choroid plexus as the predominant CNS sites that harbor leukemia cells both before and after treatment with systemic cytara- bine (Online Supplementary Figure S1B). In contrast, parenchymal involvement by leukemia was a rare, and often late, finding. It is possible that the altered immune system of NSG mice could influence CNS leukemia involvement or anatomic distribution. Accordingly, we also tested a pre-B-ALL mouse leukemia model that uti- lizes BCR/ABL p190 expression in hematopoietic cells from Arf-/- mice transplanted into immunocompetent mice.21,22,26 Similar to the xenotransplantation results, leukemia extensively involved the meninges in these mice (Online Supplementary Figure S1C).
The meninges enhance leukemia chemoresistance
We then developed ex vivo co-culture approaches to focus more specifically on the effects of the meninges on leukemia chemosensitivity. We selected meningeal cells based on our immunohistochemical analyses of brains
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