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Attenuating leukemia chemoresistance in the CNS
data suggest that meningeal-mediated leukemia chemore- sistance is primarily dependent upon cell-cell contact with smaller contributions from a soluble factor(s) secreted by meningeal cells.
Meningeal cells shift the apoptotic balance toward survival in leukemia cells
In order to define the mechanism of meningeal-mediat- ed leukemia chemoresistance we next examined the effects of co-culture on the apoptosis pathway in leukemia cells. In agreement with the annexin-V results (Figure 1B), co-culture of leukemia cells with primary meningeal cells attenuated apoptosis caused by cytarabine or methotrexate treatment as determined by both caspase 3/7 activity and measurement of leukemia cell mitochon- drial potential with the dye TMRE (Figure 2A, B and Online Supplementary Figure S3). Next, we used an apopto- sis antibody array to identify changes in the expression of multiple apoptosis family proteins in leukemia cells co- cultured with meningeal cells relative to suspension. In particular, the levels of several pro-apoptotic proteins (BID, caspase 3 & 8) decreased in leukemia cells co-cul- tured with meningeal cells (Figure 2C). However, assess- ing how the dynamic levels, activities, and complex inter- actions of these and other BCL-2 family of pro-and anti- apoptotic proteins (BH3 proteins) integrate to regulate the overall apoptotic balance is experimentally challenging. To try and capture the overall effect of the meninges on leukemia apoptotic balance, we then utilized BH3 profil- ing, a functional apoptosis assay that uses the response of mitochondria to perturbations by BH3 domain peptides, such as BIM, to predict the degree to which cells are primed to undergo apoptosis by the mitochondrial path- way.27,28 Importantly, decreased mitochondrial priming fol- lowing drug treatment has been shown to be highly pre- dictive of chemotherapy resistance in vitro and in vivo.27,28 Accordingly, we found that leukemia cells co-cultured with meningeal cells exhibited increased cytochrome C retention upon exposure to the BIM peptide compared to leukemia cells in suspension (Figure 2D). These data sug- gest that leukemia cells co-cultured with meningeal cells are significantly less primed to undergo apoptosis through the mitochondrial pathway than the same leukemia cells in suspension.
Meningeal cells increase leukemia quiescence
We also examined the effect of the meninges on leukemia cell cycle and quiescence. As shown in Figure 3A-C, leukemia cells co-cultured with primary meningeal cells are less proliferative, as determined by decreased Ki- 67 staining, significantly decreased S phase, and increased G0/G1 phase. We next used Hoechst-pyronin Y staining to better distinguish between G0 and G1 phases.29 As shown in Figure 3D-F, leukemia cells in co-culture with primary meningeal cells exhibited increased G0 phase, indicative of quiescence, relative to leukemia cells in sus- pension. Glucose uptake also diminished in leukemia cells co-cultured with meningeal cells, further supporting that these leukemia cells are quiescent (Online Supplementary Figure S4).
We then examined cell cycle and quiescence in leukemia cells isolated from the meninges of xenotransplanted mice. As shown in Figure 4A-C, leukemia cells isolated from the meninges of transplanted mice exhibited increased G0/G1 phase and decreased Ki-67 staining rela-
tive to leukemia cells isolated from peripheral blood or bone marrow. In order to assess whether the meninges harbor long-term quiescent leukemia cells, we labeled leukemia cells with a fluorescent membrane dye (DiR or DiD) that is retained in dormant, non-cycling cells but is diluted to undetectable levels within three to five genera- tions in proliferating leukemia cells.30-34 Dye-labeled leukemia cells were then transplanted into immunodefi- cient mice. After having developed systemic leukemia in ~4 weeks, the mice were euthanized and their meninges harvested. Within the meninges we detected low levels of dye-retaining, quiescent leukemia cells by flow cytometry (<1% of total leukemia population) (Figure 4D, E). Further supporting that these dye-retaining leukemia cells are qui- escent, the dye-retaining leukemia cells from the meninges exhibited low expression of the proliferation marker Ki-67 (Figure 4F). We also detected these quiescent leukemia cells within the meninges using confocal microscopy (Figure 4G-I).
We next treated these xenotransplanted mice with cytarabine and measured the percentage of dye-retaining leukemia cells in the meninges by flow cytometry. Cytarabine was given at a dose previously shown to result in plasma levels in the range produced by human high- dose cytarabine regimens that cross the blood-brain barri- er.35,36 Cytarabine, compared to phosphate-buffered saline, also significantly reduced the CNS leukemia burden in xenotransplanted mice, providing functional data that cytarabine is crossing the blood-brain barrier in our murine experiments (Online Supplementary Figure S5). Moreover, the relative increase in dye-retaining leukemia cells after cytarabine treatment is consistent with these cells having increased chemoresistance compared to the dye-negative, proliferating leukemia cells (Figure 4J). Together these data suggest that the meninges harbor qui- escent leukemia cells that are resistant to chemotherapy.
Meningeal-mediated leukemia chemoresistance is a reversible phenotype
We then tested whether removal of leukemia cells from co-culture with meningeal cells restored chemosensitivity. Leukemia cells were dissociated from meningeal cells, purified with CD19 (NALM-6) or CD3 (Jurkat) magnetic beads, and placed back in suspension. As shown in Figure 5A, leukemia cells removed from co-culture exhibited similar sensitivity to methotrexate and cytarabine as leukemia cells in suspension. Likewise, leukemia cells grown in suspension after isolation from the meninges of xenotransplanted mice also exhibited sensitivity to cytara- bine (Figure 5B). Further supporting these results, the cell cycle and apoptosis characteristics of leukemia cells placed back into suspension after co-culture reverted back to baseline (Figure 5C-E). These results suggest that drugs that disrupt adhesion between leukemia and meningeal cells may restore leukemia chemosensitivity in the CNS niche.
Overcoming meningeal-mediated leukemia chemoresistance by disrupting adhesion
Given that meningeal-mediated leukemia chemoresis- tance is a reversible phenotype, we next identified several cell adhesion inhibitors that disrupted leukemia- meningeal adhesion in co-culture (Online Supplementary Figure S6A). We selected Me6TREN for further testing based upon its effectiveness in co-culture, small molecular
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