Page 151 - Haematologica April 2020

MSC-induced SP phenotype leads to chemoresistance
transcriptomic experiments comparing sorted SP vs. MP blasts showed a repression of cell cycle and DNA replica- tion gene expression in SP than MP cells (Figure 4B). When cultivated on MSC, circulating AML blasts acquire the capability to efflux mitoxantrone through ABC trans- porter activation (Figure 1B and C). We therefore analyzed whether the drug efflux was restricted to SP blasts. To do this, blasts which were co-cultivated on MSC during three
days were incubated with mitoxantrone for the last 30 min of Hoechst incubation. We showed that SP blasts had a lower amount of mitoxantrone compared to MP blasts (Figure 4C). Quantification of mitoxantrone MFI (Figure 4D) in the SP population [median 1,584 (1,545)] showed a 2-fold decrease compared to the MP one [median 3,219 (1,706)] (P=0.0052; n=12) for most of the patients ana- lyzed. We thus evaluated which ABC transporters were
A
B
CDE
Figure 4. The mesenchymal stromal cells (MSC)-induced Side Population (SP) functionality in leukemia blasts is associated with quiescence and chemotherapy efflux through ABC transporter activation. (A) Cytograms and histograms showing gating strategies and percentage of SP and MP blasts in G0 and in G1-S-G2-M after a 3-day co-culture on HD MSC. Cell cycle status of SP versus Main Population (MP) blasts was analyzed adding pyronin Y during Hoechst staining. SP blasts are mostly in G0 [median 76% (16.5%)] compared to MP [median 33.45% (18.6%)] blasts which are in G1-S-G2-M (P=0.0009, n=12, Wilcoxon test). (B) Transcriptomic analysis of SP blasts compared to MP blasts. (C) Cytograms showing gating strategy to evaluate mitoxantrone efflux in SP or MP blasts. Quantification of mitoxantrone MFI (D) in SP [median 1,584 (1,845)] and in MP [median 3219 (1,706)] blasts (P=0.0052, n=12, Wilcoxon test). (E) DioC2,3 MFI evaluating ABCB1 activity in SP [median 239 (4,056)] and MP [median 1,298 (5418)] cells (P=0.023, n=7, Wilcoxon test). **P<0.01; ***P<0.001.
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