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leukemias as compared to decreasing frequencies along with prolonged engraftment (ID012, LIC: 1/2159, TTL: 19 weeks ID11, LIC: 1/74028, TTL: 22 weeks) in TTLlong leukemias (Table 1). Accordingly, only TTLshort cells led to engraftment upon transplantation of 102 cells.
Next, we analyzed expression of the lineage and stem cell markers CD19, CD10, CD34 and CD38, previously described to be characteristic of cells with stem or initiat- ing cell potential.5,9-12 Altogether, 50 patients’ ALL samples, which had been transplanted and characterized for their engraftment phenotype, were analyzed. No differences in marker expression were observed between the two phe- notypes (Figure 1A); however, a trend of higher propor- tions of CD34+ cells in TTLlong/good prognosis samples was seen, in line with earlier reports.21,22 In order to look for stem cell features, which are different from expression of surface markers, we analyzed our previously obtained gene expression data14 using gene set enrichment analysis. We identified 23 gene sets significantly enriched in the TTLshort/high risk profile (false discovery rate q-value ≤ 0.05), of which 17 were annotated to cell cycle functions, pointing to an association of cell cycle regulation with the TTL phenotype and, therefore, LIC activity in ALL (Figure 1B and Online Supplementary Table S2).
To further investigate these findings functionally, we analyzed the proportions of cells in active mitosis in all 20 samples (n=10 TTLshort and n=10 TTLlong), (Online Supplementary Table S1) by staining for phosphorylated histone H3 (Ser10). Significantly higher proportions of mitotic ALL cells were identified in TTLshort compared to TTLlong leukemias (Figure 2A), in line with our gene expres- sion analysis results. Moreover, we investigated cellular proliferation of leukemia cells in vivo in one leukemia of each TTL phenotype. Dividing cells were marked with bromodeoxyuridine and huCD19/bromodeoxyuridine- positive cells were analyzed after labeling/pulse and dur-
AB
ing follow up/chase. At the end of the labeling (day 0), sig- nificantly higher percentages of huCD19/bromodeoxyuri- dine-positive cells were detected in spleen and bone mar- row of TTLshort mice than in TTLlong mice (Figure 2B). Moreover, a clear reduction of bromodeoxyuridine posi- tivity in human ALL cells was observed during chase in TTLshort in contrast to similar or slowly decreasing levels in TTLlong leukemias (Figure 2C). During the experiment, all animals showed similarly high leukemia loads (Figure 2D).
These findings indicate that the LIC frequency is related to a higher in vivo proliferation capacity. Moreover, despite variation in frequencies between different samples, we did not find that LIC in BCP-ALL are extremely rare, which further supports recent observations suggestive of a stochastic stem cell concept in ALL in which many cells possess leukemia-initiating potential.
Cells in early G1-S transition possess higher leukemia-initiating cell potential
Since we found that differences in LIC frequencies and cell cycle progression are associated with distinct engraft- ment capabilities, we hypothesized that leukemia cells in different cell cycle phases are characterized by a specific repopulating potential. We used a cell cycle “live” staining with simultaneous staining of DNA and RNA17,19 distin- guishing G0/G1, S and G2/M phases. In particular, cells in G0/G1 were further divided based on increasing RNA intensity, reflecting transition from G to S phases.23,24 Early cell cycle annotated cells (G0/G1) were subdivided into G1a, G1blow and G1bhigh fractions according to progressive- ly increasing RNA fluorescence. A fourth gate was placed on cells in G2/M (Figure 3A). Staining of G1a/G1blow-sort- ed cells with the proliferation marker Ki-67 revealed that Ki-67-negative resting G0 cells are part of the G1a fraction in this analysis (Online Supplementary Figure S1).
All sorted subpopulations showed leukemia-initiating
Figure 1. Leukemia engraftment is associated with cell cycle activity. (A) No difference in surface marker expression (CD34, CD10 and CD19 n=50; CD38 n=40) as measured by flow cytometry on primary patients’ ALL cells with either TTLshort or TTLlong phenotype. Single and median values are indicated. Mann-Whitney test; P= statistical significance. (B) Representative gene set enrichment analysis plots of cell cycle annotated gene sets in the TTLshort/high LIC activity profile False Discovery Rate (FDR) (q-value ≤0.05).
haematologica | 2018; 103(6)