Fig 1.
ROS levels in immunophenotypic stem/progenitor cells from representative normal control, myelodysplastic and AML bone marrows.
Flow cytometry histograms show intracellular ROS levels (using DCF) in HSC/MPP/LMPP and CMP/GMP/MEP of representative control BM (A), umbilical cord blood (UCB) (B), four AML BM samples at diagnosis (C), two MDS-RAEB BM samples (D), and two MDS-no-EB BM samples (E). DCF staining with normalised mean fluorescence intensity of each of these populations is shown (using DCF staining of reference lymphocyte population shown as light grey filled histogram, while for stem/progenitor subsets staining is shown as open histograms with different coloured lines as indicated on each plot). Additional examples of DCF staining of AML and MDS samples are shown in S1F–S1H Fig.
Fig 2.
Summarised ROS levels in control, dysplastic and leukemic CD34+ and CD34− blasts.
Charts show normalised DCF MFI (relative to lymphocytes within each sample) of CD34+CD38low subsets (A) and CD34+CD38high subsets (B) from control BM (n = 24), and AML BM (n = 27), MDS/MPD-no-EB BM (n = 12) and MDS-RAEB BM (n = 14). Flt3ITD+ AML = red filled square. CBF-AMLs = green filled squares (see Key in figure). ROS levels in CD34−CD117+ cells were also compared between control BM and CD34− AML (n = 10) and MDS patient BM samples, using the same colour scheme as above (C). CD34−CD117+ cells were subdivided into CD45RA+ and CD45RA−cells to enrich for myeloid and erythroid precursors respectively. Median expression and interquartile range is shown on each plot. P values are shown for data-sets where significant differences were observed (p<0.05, Mann Whitney test with 95% confidence intervals).
Fig 3.
Influence of ROS levels on lineage fate and viability in normal progenitors.
Sorting scheme of normal BM CD34+CD38high cells separated into CD45RA+ and CD45RA−subsets, followed by gating into 4 different populations based on differential DCF staining (A). Sorted cells were seeded onto methylcellulose media supplemented with cytokines. After 2 week culture at 37°C in vitro, colonies were scored using an inverted light microscope and recorded as a percentage of the total colony yield for FACS-sorted control CD34+CD38high CD45RA−(B) and CD34+CD38high CD45RA+ (C) progenitors with different levels of DCF staining. Total colony yield as percentage of input cell number was also determined (D). Colony forming unit assay data is from three independent experiments. Colonies were scored as erythroid (e), macrophage (m), granulocyte (g), granulocyte-macrophage (gm), or granulocyte-erythroid-macrophage mixed (gemm). Viability was determined by Annexin V and 7-AAD staining of normal BM labelled with HSPC-specific mAb. Initial gating was performed on SSClow CD45intCD117+ cells. The pooled viability data from 7 control BM sample are shown (E). The effects of redox modification on DCF staining and cell viability in CD34+CD38high progenitors, is shown from two independent experiments (F). BM cells were treated overnight with the pro-oxidant BSO (100μM) or left untreated (control).
Fig 4.
ROS levels in blast cells of different AML sub-groups.
DCF levels in AML blasts (gated by CD45/SSC/CD117/CD34). Data is shown for total CD34+ blasts in CD34+AMLs (n = 70) (A) and total CD34− CD117+ blasts in CD34− AMLs (n = 23) (B) compared to the equivalent blast subset of control BMs (n = 24). AML samples, except for CBF-AMLs (CD34+, n = 12; CD34−, n = 1), were grouped according to Flt3ITD+/NPM1 mutational status: ITD−/NPM1wt, (CD34+, n = 37; CD34−, n = 4), ITD+/NPM1wt (all CD34+, n = 6), ITD-–/NPM1mut (CD34+, n = 7; CD34−, n = 4), ITD+/NPM1mut, (all CD34−, n = 9). Unk = represents patient samples lacking mutational data.
Fig 5.
Combined ROS, ki67 and BCL2 staining of control and AML stem/progenitor cell subsets.
Plots show representative immunophenotypic blasts stained with DCF, ki67 and BCL2 in control BM (A) CD34+AML BM (B) and CD34− AML BM (C). Isotype controls for each sample were performed to establish negative gates (shown as green dotted rectangle). An expanded population with pro-survival phenotype, ki67lowBCL2high, is observed in both AML types (B and C) and highlighted by black rectangle.
Fig 6.
Summarised ki67 and BCL2 expression in control, AML and MDS subsets.
CD34+CD38low, CD34+CD38high and CD34− (CD117+) blast subsets were compared for ki67-positivity (A), BCL2 expression (using BCL2-specific MFI defined by fold increase over staining with appropriate fluorescent isotype-control mAb) and the aberrant ki67lowBCL2high phenotype (C), which is defined using isotype control staining (see S3E Fig). Data includes 16 control BM, 29 CD34+ diagnosis AML samples (mixed BM/PB), 6 MDS with no excess blasts (no EB) and 6 MDS-RAEB samples. The CD34− plots include data from 11 CD34− diagnosis AMLs. Median expression and interquartile range is shown on each plot. Data is shown for all AML patients (grey squares, filled for CD34+ and open for CD34− AMLs) and genetic subgroups. F+N- denotes ITD+/NPM1wt patients (red filled squares, all CD34+), F+N+ denotes ITD+/NPM1mut patients (red open squares, all CD34−) and F-N+ denotes ITD−/NPM1mut (blue filled open/blue open squares for CD34+/CD34− respectively). CBF-AMLs are shown as green squares.
Fig 7.
ROS-separated ki67 and BCL2 expression in AML stem/progenitor cells.
AML progenitors stained with mAb for surface markers, DCF, ki67 and BCL2 were separated into ROSlowest and ROShighest cells based on the 20% dimmest and 20% brightest DCF staining populations respectively. Charts show ki67 and BCL2 expression respectively in ROSlowest and ROShighest subsets in (A-B) CD34+ AMLs, (C-D) in CD34− AMLs and (E-F) in CD34+ cells in MDS patients with no excess blasts (no EB) and MDS patients with excess blasts (RAEB-1/RAEB-2). Profiles from 5 representative control/normal BMs are shown for CD34+ and CD34−CD117+ cells in the respective charts (grey triangles). AML patients were subdivided into Flt3ITD+/NPM1wt (simplified to F+/N-), Flt3ITD−/NPM1mut (F-/N+), Flt3ITD+/NPM1mut (F+N+), CBF-AMLs, t(9;11)-AMLs and other cases. AML patients with poor early response (refractory/ early-relapse/ delayed remission) indicated by red squares. Patients achieving stable remission after one course of treatment are shown as black squares. Patients for whom early outcome data was unavailable are shown as grey squares. Thresholds for high BCL2 expression are shown as dashed lines on each BCL2 chart. The threshold was set at >mean+2SD of normal CD34+ (>6.9) or CD34− blasts (>3.3).