Leukemic cells in the CNS show adaptation to hypoxia, such as reduced proliferation and oxidative phosphorylation, increased glycolysis, and upregulation of vascular endothelial growth factor A (VEGFA). (A) Representative cell cycle analysis of 4′,6-diamidino-2-phenylindole–stained CNS- and BM-derived leukemic cells from xenograft recipients of ALL9 leukemic cells. The fraction of cells in S/G2/M (8.6% vs 26.0%) is indicated. (B) Mean percentages of CNS- and BM-derived leukemic cells that are in S/G2/M from 5 separate recipients of ALL9 leukemic cells. (C) Immunohistochemical analysis of leukemic cell proliferation in BM and brains from xenograft recipients of ALL9 leukemic cells. The percentage of hCD19⁺Ki67⁺ cells out of hCD19⁺ cells are indicated. n = 3 biological replicates. (D) Mean oxygen consumption rates (RFU/h) in CNS- and BM-derived leukemic cells from recipients of ALL9 leukemic cells. n = 3 technical replicates, and data are representative of 2 independent experiments. Data show mean ± standard error of the mean, analyzed with 2-sided, paired Student t test: *P < .05. (E) Gene set enrichment analysis of RNA-sequencing data illustrating enrichment of hypoxia (normalized enrichment score [NES] = 2.34, false discovery rate [FDR] = 0) and glycolysis (NES = 2.25, FDR = 0) associated genes in CNS-derived leukemic cells from recipients of ALL9 cells. (F) Heatmap indicating levels of glycolytic metabolites in paired CNS- and BM-derived leukemic cells from 4 xenograft recipients (1-4) of ALL9 leukemic cells measured by mass spectrometry. 2PG, 2 phosphoglycerate; 3PG, 3 phosphoglycerate; F6P, fructose 6 phosphate; G1P, glucose 1 phosphate; G6P, glucose 6 phosphate. Red indicates high concentration and green low concentration. (G) Quantification of VEGFA (probe ID A_23_P70398) transcripts in CNS- and BM-derived leukemic cells from xenograft recipients of ALL1-ALL4 leukemic cells with microarray. ALL3 contains data from 2 mice (3-1, 3-2). (H) Quantification of VEGFA in RNA sequencing from primary tissues of ALL6-ALL8. (I) VEGFA expression by RQPCR in CNS- and BM-derived leukemic cells from recipients of ALL9 leukemic cells; n = 4 biological replicates. Data show mean ± standard error of the mean, analyzed with 2-sided, paired Student t test: *P < .05.