Page 107 - Haematologica June

Table 1. High leukemia-initiating cell frequencies in TTLshort /poor prognosis acute lymphoblastic leukemia. Estimated leukemia-initiating cell (LIC) frequencies of 2 TTLshort and 2 TTLlong ALL samples. Limiting dilution analysis.
Leukemia initiating cells in ALL
ment of primary cells in a BCP-huALL model.14 These two phenotypes were strongly associated with patients’ out- come and the TTLshort/high-risk phenotype involved increased activation of the mammalian target or rapamycin (mTOR) pathway14,15 and deficient apoptosis signaling.16
In this study, we identified annotations to distinct cell cycle compartments as a biological discriminator identi- fying a cellular subfraction with higher LIC activity capa- ble of driving leukemia reconstitution in a NOD/SCID/huALL mouse model.
Methods
Twenty patient-derived xenograft samples established by trans- plantation of patients’ ALL cells into NOD/SCID mice (NOD.CB17-Prkdcscid/J, Charles River) as previously described14,16 were used in this study. The patients’ samples were obtained with informed consent in accordance with the institution’s (Ulm University) ethical review board; all animal experiments were approved by the appropriate authority (Regierungspräsidium Tübingen) and carried out following institutional and national guidelines on the care and use of laboratory animals. The charac- teristics of the patients and their malignancies are summarized in Online Supplementary Table S1.
For gene expression analysis, RNA was prepared from sorted cells and analyzed using Affymetrix Human Genome-U133 Plus 2.0 arrays. Gene-expression data were deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO series accession #GSE71836). Cell cycle staining was per- formed as previously described17-19 labeling DNA/RNA with Hoechst (Molecular Probes, ThermoFisher Scientific) and pyronin Y (Polysciences, Hirschberg, Germany). Post-sorting analysis con- firmed the positions of the sorted populations within the sorting gates. Viable cells (identified by trypan blue exclusion) were trans- planted into NOD/SCID mice (105 viable cells/recipient); group sizes were chosen based on the availability of cells after sorting.
Secondary recipients were transplanted with unsorted cells (1x105) isolated from mice with full-blown leukemia, which had been transplanted with G1blow or G2/M sorted cells. For analysis of reac- tive oxygen species (ROS), cells were stained with CM- H2DCFDA (Invitrogen/ThermoFisher Scientific) and analyzed by flow cytometry. DNA was labeled with Hoechst. Post-sorting analysis confirmed high/low ROS levels. Viable cells (identified by trypan blue exclusion) were transplanted (105 viable cells/recipi- ent); cells sorted based on FSC-A/SSC-A and FSC-A/FSC-H gates were used as control. Drug sensitivities (cell death upon pred- nisolone or cytarabine) were investigated in sorted cellular sub- fractions.
Statistical analyses were carried out using the Mann-Whitney test, the unpaired t-test with Welch correction (two-tailed), the one sample t-test or the log-rank test (two-tailed) as indicated. P≤0.05 was considered statistically significant.
Additional and detailed information on the methods used can be found in the Online Supplementary Data.
Results
Leukemia-initiating cell potential in B-cell precursor acute lymphoblastic leukemia is associated with cell cycle activity
We previously characterized the engraftment potential of primary BCP-ALL cells transplanted into NOD/SCID mice and found that a rapid engraftment and a short time to leukemia (TTLshort) are associated with poor patients’ outcome.14 The capacity of a cell to give rise to a pheno- typically equal progeny in vivo is considered a stem cell’s hallmark feature.20 We evaluated LIC activities by trans- planting decreasing numbers of four primograft samples (TTLshort, n=2; TTLlong, n=2) in limiting dilutions (105 to 101 cells) assessing leukemia engraftment 25 weeks after transplantation. Interestingly, higher LIC frequencies (ID04, LIC: 1/329, TTL: 9 weeks; ID05, LIC: 1/739, TTL: 10 weeks) were observed in TTLshort/poor prognosis
Sample
ID04
ID05
ID12
ID11
TTL N. cells transplanted
105
104 short 103 102 101
Recipients transplanted Recipients engrafted
88 88 87 84 80
LIC frequency
1/329
1/739
1/2159
1/74028
105 88
104 88 short 10386 102 81 101 80
105 88 104 88
long 103
102 80
83 101 80
105 86
104 81 long 10380 102 80 101 80
TTL: time to leukemia; N: number.
haematologica | 2018; 103(6)
1009