Page 147 - Haematologica April 2020

MSC-induced SP phenotype leads to chemoresistance
Results
Leukemia blasts cultivated on mesenchymal stromal cells actively efflux chemotherapy drugs by activating ABC transporters
We first characterized MSC isolated from the BM of AML patients (AML MSC) at diagnosis and compared them to MSC isolated from BM of healthy donors (HD MSC). The number of cumulative population doubling was slower for AML MSC than for HD MSC (Online Supplementary Figure S2A). AML MSC clonogenicity was also weaker than that of HD MSC (Online Supplementary Figure S2B). Because AML MSC were morphologically dif- ferent from HD MSC, we quantified senescent cells using the β-galactosidase activity test. We observed that AML MSC showed 5-10 times more senescent cells than HD MSC as soon as passage 4 (Online Supplementary Figure S2C). This result can explain the limited expansion capac- ity and clonogenicity of AML MSC. Despite these growth
alterations, no differences were observed regarding their phenotype (CD45–CD90+CD73+CD105+) (Online Supplementary Figure S3A) or differentiation capacities, since they were able to differentiate into adipocytes, osteoblasts, and chondrocytes similarly to HD MSC (Online Supplementary Appendix and Online Supplementary Figure S3B and C). We further analyzed their functional roles on AML blast survival by co-culturing blasts on either AML or HD MSC. After a 3-day co-culture, cells were removed and blast viability was evaluated using Annexin V and 7AAD co-staining. Co-culture with HD or AML MSC significantly increases AML blast survival (n>12; P=0.0004) (Figure 1A, left). This difference was also observed when mitoxantrone, a chemotherapy drug, was added to co-cultures (n>12; P=0.0003) (Figure 1A, right).
To try to explain this increased survival, we evaluated the intracellular amount of mitoxantrone in the blast pop- ulation co-cultivated with or without MSC isolated from HD or AML patients (Figure 1B). All blasts were positive
A
BCD
Figure 1. Mesenchymal stromal cells (MSC) from heathy donors (HD) or from acute myeloid leukemia (AML) patients confer a better survival to leukemia blasts, even in the presence of chemotherapy agents. (A, left) Histogram representing the number of living AML blasts after a 3-day culture without MSC [median 51,655 (56,830)] or with MSC from HD [median 91,815 (94,556)] or AML patients [median 56,896 (102,863)]. (Right) The same conditions but in the presence of 50nM of mitoxantrone. Culture conditions in the presence of the different types of MSC confer a significantly improved survival to AML blasts (left: ***P=0.0004; right: ***P=0.0003, n>10, represents the number of AML blasts/MSC donor combinations). (B) Histogram showing fluorescence intensity of AML blasts stained with mitoxantrone after a 3-day culture with or without MSC from HD (one representative experiment of the 6 performed). Control (CT) represents non-stained blasts. (C) Mitoxantrone mean fluorescence intensity (MFI) in AML blasts cultivated [median 2,191 (2,032)] or not [median 3,676 (3,546)] on HD MSC (**P=0.01, n=6, Wilcoxon test). (D) Mitoxantrone MFI in AML blasts cultivated [median 3,861 (1,147)] or not [median 1,821 (460)] on AML MSC (**P=0.01, n=3-9, Wilcoxon test). n: number. **P<0.01; ***P<0.001.
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