Page 150 - Haematologica April 2020

L. Boutin et al.
for mitoxantrone after a 3-day culture without MSC. However, when blasts were co-cultivated in the presence of MSC either from HD (Figure 1C) or AML patients (Figure 1D), the mitoxantrone mean fluorescence intensity (MFI) was lower than that of blasts cultured without MSC. This reduction was observed in most of the patients studied, regardless of the MSC origin (P=0.01, n=6 for HD MSC and n=9 for AML MSC).
Because blasts co-cultured on MSC exhibit a lower intracellular amount of mitoxantrone compared to cells cultivated without MSC, we studied by which mecha- nism the amount of intracellular mitoxantrone was reduced. It is now well known that ATP-binding cassette (ABC) transporters can efflux several molecules including chemotherapy drugs.16 Therefore, we used specific probes such as Dioc2(3), CMFDA and purpurin18 to evaluate the activity of three main pump families such as ABCB1 (MDR1), ABCC1 and ABCG2, respectively. There was a significant increase in the percentage of blasts that efflux the Dioc2(3) probe after a 3-day co-culture on HD MSC [median 16.1% (12.8%) with MSC vs. 6.18 (2%) without MSC; P=0.005, n=9] demonstrating the implication of the ABCB1 pump as part of this process (Figure 2A). The Dioc2,3 MFI was also significantly decreased in AML blasts (P=0.02, n=9) (Figure 2D). We also noticed an increase in ABCC1 and ABCG2 pump activities in a small number of AML patients (Figure 2B and C), but with no significant modulation of CMFDA and purpurin18 MFI (Figure 2E and F). However, when activities of these three ABC transporters were analyzed individually, patient per patient, we observed that their activities were systemati- cally modulated by MSC contact (except for one patient, AML 44) (Online Supplementary Figure S4) and that ABC transporters were differentially active from one patient to another. When blasts were co-cultured on MSC isolated from AML patients, we observed an activation of the three ABC transporters (P=0.01, 0.02 and 0.008, respec- tively; n=3-9) (Online Supplementary Figure S2G-I) associat- ed with a decrease in specific probe MFI (P=0.01, n=3-9) (Figure 2J and L), except for Dioc2,3 MFI which remained high (Figure 2J).
Therefore, our results show that leukemia blasts demonstrate a specific pattern of ABC transporter activity that is modulated by contact with stromal cells in a patient-dependent manner. Moreover, the modulation of ABC transporter activities appears higher when blasts are co-cultivated with MSC isolated from AML patients.
Leukemia blasts adopt a Side Population phenotype after close contact with mesenchymal stromal cells through integrin interactions
ABC transporter activation is known to be involved in SP phenotype acquisition. We therefore analyzed the SP phenotype of blasts before and after co-culture with MSC. Circulating blasts were gated on their SSC CD45low profile in flow cytometry (Figure 3A). In contrast to circulating Lin– HSPC freshly isolated from HD,13 a small proportion of blasts (0.2%; interquartile 0.44%) isolated from approx- imately 50% of AML patients expressed a SP phenotype before any stromal co-culture (Figure 3B). However, after a 3-day co-culture on HD MSC, we observed for all AML patients that a SP population had emerged or was increased within blast cells with a median of around 4%, with an interquartile of 8.65% (P<0.001, n=27). We then analyzed whether co-culture with AML MSC also
induced or increased the percentage of circulating leukemia blasts with an SP phenotype. Similarly to MSC isolated from HD, co-cultures of blasts with AML MSC systematically increased the percentage of SP blasts (P=10-4, n= 8-35) (Figure 3C).
We further studied whether a close contact between blasts and MSC was required to promote the SP pheno- type of blasts. We first addressed this question by analyz- ing the proportion of SP blasts in the adherent fraction of the co-cultures and compared it with that detected in the supernatants (SN). SP-expressing cells were mostly found in the adherent fraction (Figure 3D). We then confirmed the requirement of cell-cell interactions using transwell experiments in which the percentage of SP blasts was strongly reduced when blasts and stromal cells were sep- arated by the transwell insert (n=3) (Figure 3D and E). Therefore, close contacts between leukemia and stromal cells appear to be essential to the promotion of the SP phe- notype in blasts.
We had previously demonstrated that the SP phenotype induced on circulating Lin– HSPC from HD by MSC co- culture was dependent on α4 and β1 integrins as well as CD44 engagement.13 Using blocking antibodies, we tested whether the promotion of the SP phenotype on AML blasts was dependent on MSC interactions through these molecules. Blocking α4 integrin significantly reduced the percentage of SP blasts after MSC co-culture (P=0.04, n=7) while blocking β1 integrin and CD44 interactions had a slight but not significant effect on this proportion (Figure 3F). We then tested the effect of pharmacological inhibitors on the main signaling pathways directly or indi- rectly activated by integrins. To do this, we first assessed the 'efficient dose', corresponding to the highest dose that did not induce toxicity for dasatinib (pSrc inhibitor), LY294002 (pAKT inhibitor), CAS2859863 (pSTAT5 inhibitor) and LY209031 (pGSK3 inhibitor), CPD22 (pILK inhibitor), and simvastatine (HMG coA reductase inhibitor) (data not shown). We then added these inhibitors to co-cultures and quantified the proportion of blasts that have adopted an SP phenotype after a 3-day co-culture on HD MSC. Whereas addition of CPD22 or simvastatine did not affect the percentage of SP blasts (data not shown), a significant decrease in SP blasts was observed when Src (P=0.002, n=9), AKT (P=0.003, n=9), STAT5 (P=0.009, n=9), and GSK3 (P=0.01, n=6) pathways were inhibited (Figure 3G). These data confirm that the stroma-induced SP functionality on AML blasts was partly dependent on integrin interactions, and especially of α4 integrin activa- tion.
Side Population acute myeloid leukemia blasts are quiescent and able to actively efflux chemotherapy through ABCB1 transporters
Side Population phenotype is reported to be linked to quiescence.12–14 Therefore, we used flow cytometry to ana- lyze the cell cycle phases of AML blasts expressing or not expressing the SP phenotype (SP vs. non-SP population/MP for Main Population) after a 3-day co-cul- ture on HD MSC. As expected, SP blasts were in the majority in the G0 phase [median 76% (16.5%)] in con- trast to MP blasts [median 33.45% (18.6%)], which were mainly in the G1-S-G2-M phases (Figure 4A). In agree- ment with this result, gene set enrichment analysis (GSEA) performed with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database on data obtained from
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