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H.R. Lee et al.
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Figure 2. Reversible and equipotent nature for generation of LSK leukemic subsets. LSK (Sca-1(+)) or LK (Sca-1(-)) subsets of MN1 leukemic cells generated by co- culture were sort-purified and then replated for 3 days in the absence (SF) or presence of mesenchymal stromal cells (+MSC). (A) Flow cytometry profiles. (B and C) Quantitative analysis for expansion of cell numbers for LSK or LK subsets from input numbers of sorted LSK or LK cell populations during the second round of co- culture with murine MSC. Shown are the fold increases of cell numbers compared with input in the second co-culture (B) and final frequencies for LSK from each set of the second co-culture (C) (mean ± standard error of the mean, n=6, *P<0.05).
chastic), but with similar probability of each cells (equipotent) for conversion among the total leukemic cell populations.
Functional heterogeneity acquired in stem cell-like leukemic subsets
We next determined whether the Sca-1(+) stem cell-like leukemic subset arising by stromal contact was functionally distinct. When MN1 leukemic cells were treated with the chemotherapeutic Ara-C during co-culture with mesenchy- mal cells, Sca-1(-) subsets exhibited significant decrease of cell numbers, but the Sca-1(+) subset exhibited higher resistance compared to the Sca-1(-) subsets, with no signif- icant changes in cell numbers (Figure 3A). Drug resistance in the Sca-1(+) subset was similarly reproduced in other leukemia cell types tested (HoxA9/Meis1-induced leukemic cells or C1498 leukemia cell line) (Figure 3A).
The chemoresistance of the Sca-1(+) (LSK) leukemic pop- ulation compared to the rest of the Sca-1(-) (LK) cells was similarly observed in vivo with mice engrafted with MN1 leukemic cells and treated with chemotherapeutic drug (Ara-C and doxorubicin)29 (Figure 3B). Thus, enhanced drug resistance is a common feature of leukemic subsets acquir- ing a Sca-1(+) phenotype upon stromal contact in a range of leukemic cell models.
In order to further investigate the drug resistance of the Sca-1(+) cells, we analyzed their cell cycling in BM and found that % of quiescent cell population (G0) was higher in LSK cells (Figure 3C). We also compared the frequency of leukemic initiating cells (LIC), a functional assay for
leukemia stem cells30 in the Sca-1(+) leukemic subset in comparison to the other subsets. Thus, subsets of Lin(+) cells, Lin-c-kit-, LK (Lin-c-kit+sca-1-) cells and LSK (Lin-c-kit+ sca-1+) leukemic cells generated in the BM of MN1 trans- planted mice were sort purified and transplanted into sec- ondary recipient mice in a limiting dilution assay. However, the LK and LSK populations exhibited a similar frequency of LIC, while exhibited significantly higher frequencies than the other cell populations (Figure 3D; Online Supplementary Figure S2A). These two populations (LK and LSK) also exhibited comparable levels of in vivo leukemic engraftment or in vitro leukemia colony formation (Online Supplementary Figure S2B and C), indicating that the Sca-1(+) subset developed during in vivo leukemogenesis comprise a subset of LIC that does not display significantly different leukemogenic activity compared to their Sca-1(-) counter- parts.
Thus, the Sca-1(+) leukemia subset generated from leukemic cells represents a distinct leukemic cell population that has acquired drug-resistance without altering their leukemogenic activity.
Interleukin-4 plays a role in the emergence of drug- resistant Sca-1(+) cells
We next sought to identify possible signals from the stro- ma that induce emergence of Sca-1(+) cells. Given that altered production of cytokines and/or growth factors are frequently observed in leukemic cells,10 we examined the cytokine/growth factor gene expression induced by stromal contact of leukemic cells (Online Supplementary Figure S3A).
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haematologica | 2022; 107(2)