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H.R. Lee et al.
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Figure 6. Generation of stem-like, drug-resistant leukemic cells in human leukemia models. (A) Generation of stem-like (CD90(+)) leukemic subsets from each indi- cated human leukemia cell line during stromal co-culture. Leukemic cell populations (CD45(+))were gate separated from mesenchymal stromal cells (MSC) (CD45(- ) for analysis. Shown are the quantification of the frequency of CD90(+) cells in the co-culture (n=7, † <0.2%, *P<0.05). (B) Comparison of drug sensitivity between each leukemic subset. Each indicated human leukemic cell line was exposed to Ara-C (200 nM) for 2 days. Shown are the numbers of surviving (annexinV- propidium iodide-) cells in the culture (mean ± standard error of the mean [SEM], n=6). (C to G) Generation of stem-like, drug-resistant leukemic subsets in human leukemic cells from acute myeloid leukemia (AML) patients. (C) Human leukemia cells from AML patients were co-cultured in the presence or absence of human MSC. Shown are the % CD90 (+) cells in total leukemic cells after co-culture for 3 days and differences were analyzed by Mann-Whitney U test (n=14 from seven individual patients’ samples, P<0.05). (D) Effects of interleukin-4 (IL-4) on the generation of CD90(+) subsets. Leukemic cells were co-cultured with human MSC in the pres- ence or absence of IL-4 (100 ng/mL). Shown are the relative fold increases in % of CD90(+) subsets in leukemic cells (mean± SEM, n= 10 from five individual patient’s samples, *P<0.05). (E) Reversible switching of subsets of human primary leukemic cells to maintain constant equilibrium. CD90(+) and CD90(-) subsets generated during co-culture were sort-purified and re-plated in the co-culture with human MSC. Shown are the fold changes of each subset after plating each purified subset after 3 days of co-culture (n=6, *P<0.05). (F) Comparisons of drug sensitivity between the CD90(+) subset and the rest of the CD90(-) population in primary AML cells after exposure to Ara-C (200 nM). Shown are the relative fold changes in numbers of surviving cells of each population determined by numbers of annexinV-propidium iodide- (PI) cells and differences were analyzed by Mann-Whitney U test (n=14 from seven individual patients’ samples, P<0.05). (G) Enrichment of stem cell signatures in CD90(+) human leukemic cells. Primary human leukemic cells from two AML patients (#4 and #2) were co-cultured with human MSC for 3 days, and the generated CD90(+) and CD90(-) cells were subjected to RNA sequencing analysis. Differentially expressed genes were analyzed by gene set enrich- ment analysis (GSEA-P) for enrichment of 259 genes specific for leukemia stem cells.47 Shown are the plots of enrichment scores (upper) and ranked list of each gene in the order of log2 folds ratio (CD90+/90-) with position at zero indicated (lower).
leukemic cells to VCAM-1 in MSC facilitates emergence of LSK subsets.
Consistent with these findings, gene expression changes in MSC induced by IL-4 treatment during culture revealed 41 differentially expressed genes (DEG), the most profound changes of which were in the gene ontology group related to the ‘binding’ molecular function, supporting their role in the cellular interaction with leukemic cells (Online Supplementary Figure S7A, B). Thus, IL-4 enhances the cellu- lar interaction of stroma and leukemic cells to facilitate stro- ma-dependent evolution of the Sca-1(+) leukemic subset exhibiting drug resistance.
Stroma-induced changes in human leukemic cell models
In order to investigate whether a similar phenomenon
can be seen in human leukemic cells, we examined human AML cells for acquisition of CD90(+) as a phenotype for stem-like subsets based on findings that a subset of CD90(+) cells amongst CD34(+) cells represent long-term repopulating hematopoietic stem cells (HSC)44 and that CD90 expression in human leukemic cells represents high- risk leukemia with stem cell properties.45,46 We first exam- ined human leukemic cell lines, MOLM-14 and MV4-11 (M5 type FAB), and HL-60 (M3 type FAB). For each leukemic cell line tested, co-culture with human BM- derived MSC resulted in the emergence of leukemic subsets with the CD90(+) phenotype, albeit to variable levels (Figure 6A). Moreover, when chemoresistance was com- pared between leukemic subsets, significant resistance to Ara-C treatment was observed selectively for CD90(+) cells in all tested leukemic cells (Figure 6B) similarly exhibiting
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haematologica | 2022; 107(2)