Page 127 - Haematologica Vol. 110 - January 2025

ARTICLE - CITE-seq analysis of HU effects on CML cells
expressed genes and proteins were assessed by the Wil- coxon rank sum test in R using Seurat’s FindMarkers and FindAllMarkers functions.
Results
Hydroxyurea alters the proportions of stem and progenitor cells in blood and bone marrow from patients with chronic myeloid leukemia
Peripheral blood and BM samples obtained from 17 newly diagnosed CML patients before and after HU treatment were analyzed for SPC frequencies using flow cytometry (Figure 1A). The analysis revealed a non-significant reduc- tion of the frequency of CD34+ cells following HU treatment (Figure 1B), possibly reflecting reduced proliferation of CML SPC. In addition, the frequency of immature CD34+CD38- cells among CD34+ SPC in BM was significantly lower after treatment, with a similar but non-significant trend in blood (Figure 1C). Results from paired samples obtained before
A
H. Komic et al. and after HU treatment from three patients supported
these findings (Online Supplementary Figure S1).
Proteo-transcriptomic CITE-sequencing analysis of chronic myeloid leukemia stem and progenitor cells
To further elucidate the effects of HU on the CD34+ CML SPC compartment, paired blood and BM samples from two patients (obtained before and 7 or 9 days after HU treatment) were subjected to proteo-transcriptomic CITE-sequencing analysis of 596 genes and 51 proteins (Figure 2). Twen- ty-six thousand single CD14-CD34+ cells were successfully sequenced with sequencing saturations of 93% and 97% for mRNA and AbSeq libraries, respectively. After quality control, 21,044 cells remained for analysis.
Hydroxyurea treatment induces erythroid maturation among chronic myeloid leukemia stem and progenitor cells
Following integration of mRNA expression data by patient, dimensionality reduction and clustering of cells from all six
BC
Figure 1. Hydroxyurea alters chronic myeloid leukemia stem and progenitor cell frequencies in blood and bone marrow. (A) Sam- ples and representative gating strategy used for flow cytometry analysis of the effects of hydroxyurea (HU) on stem and progen- itor cells in blood and bone marrow from patients with chronic phase chronic myeloid leukemia (CML). (B, C) Effect of HU treat- ment on the frequency of (B) CD34+ and (C) CD34+CD38- cells in CML patients’ blood and bone marrow samples obtained before (N=7) or a median of 9 days (range, 4-19) after HU treatment (N=10). Statistics by the Mann-Whitney test. Error bars represent the standard error of mean. BM: bone marrow; CP-CML: chronic phase chronic myeloid leukemia; SSC: side scatter; FSC: forward scatter; MNC: mononuclear cells; PBMC: peripheral blood mononuclear cells.
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