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Functional switching of leukemic cells by stromal contact
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Figure 7. Dependency on the mesenchymal progenitor cells for generation of stem-like leukemic subsets. (A) Identification of VCAM-1-expressing mesenchymal stromal cells in the bone marrow (BM) of mice. % of VCAM-1(+) cells among murine mesenchymal cells (CD45-31-Ter119-) in fresh BM were compared for CD44(+) and CD44(-) cells. Shown are the representative flow cytometry plots (left) and quantification (right) for frequency of VCAM-1(+) cells among the indicated murine mesenchymal stromal subsets in fresh BM. (B) Comparisons of frequency of VCAM-1(+) cells in mice BM between the mesenchymal progenitor and non-progenitor subsets of murine mesenchymal stromal cells. Mesenchymal progenitor subsets in fresh mice BM were defined by PDGFRa(+)/Sca-1(+) or PDGFRa(+)CD51(+) based on published reports.41-43,50 (C) Leukemogenesis in the Bis knockout (KO) mouse model. MN1 leukemic cells were transplanted into Bis KO mice, where mesenchymal progenitor populations are selectively decreased. Two weeks after transplantation into neonates of each mice model, engraftment of leukemic cells in BM and % LSK among engrafted leukemic cells were analyzed. Shown are the experimental design (upper) and % LSK leukemic subsets among engrafted leukemic cells for each indicated mice recipient (lower, left) and % engraftment of leukemic cells (GFP (+)) in BM (lower, right) (mean ± standard error of the mean [SEM], n=6 for wild- type [WT], n=27 for hetero, n=10 for KO). (D) Comparisons of mesenchymal progenitor cell numbers in BM of acute myeloid leukemia (AML) patients with respect to the clinical course. (upper) Experimental design. Fresh uncultured BM of AML patients without prior treatment were analyzed for cytogenetic abnormalities of leukemic blasts and content of mesenchymal progenitor cells (MPC; CD45-31-235a-146+166-) in fresh BM. Five years after the initial analysis, MPC numbers in patients’ fresh BM were compared with subsequent clinical courses (maintenance of complete remission or relapse) with respect to the karyotype of leukemic blasts. (lower) Mean numbers of MPC (CD146+166-) in fresh BM of AML patients for each indicated clinical course and karyotype (mean ± SEM, n=14 for normal karyotype, n=5 for mixed-lineage leukemia [MLL], n=10 for others).
quiescence in cell cycle (Online Supplementary Figure S8)
In order to examine these findings in primary leukemic cells, we examined the response of primary AML blasts from five to seven individual patients to mesenchymal stro- ma. Primary AML blasts exhibited a significant induction of CD90(+) cells upon stromal contact, which was further increased by IL-4 treatment during co-culture (Figure 6C and D). Sort-purified subsets of CD90(+) and CD90(-) leukemic cells exhibited similar switching of phenotypes to maintain constant ratios in CD90(+) subsets in total leukemic cells, as observed for murine leukemic cells (Figure 6E). Moreover, the CD90 (+) subset generated during stro- mal contact exhibited higher resistance to Ara-C (Figure 6F) than the remaining CD90(-) cell population in the same co- culture, demonstrating a similar drug-resistance of newly emerging leukemic subsets in primary human leukemic
cells.
Gene expression study on two independent patients
showed that CD90(+) subsets of primary human AML cells are significantly enriched with gene sets specific for LSC47
than the remaining CD90(-) cells (Figure 6G), and enriched with gene sets involved in the interaction with the extracel- lular matrix (ECM) or focal adhesion (Online Supplementary Figure S9).
Thus, subsets of human leukemic cells in contact with stroma exhibit a stem-like properties to acquire drug-resis- tance through interaction with stroma.
Stromal heterogeneity for generation of stem cell-like leukemic subsets
Extensive heterogeneity has been documented among mesenchymal populations in BM stroma.48,49 Therefore, we investigated the mesenchymal subpopulations responsible for generation of Sca-1(+) cells. Given that VCAM-1 expressing MSC played a role in the generation of drug- resistant subsets, we examined VCAM-1 expression among stromal cell populations in the BM. VCAM-1(+) mesenchy- mal cells were predominantly enriched by a CD44(-) popu- lation, where colony-forming mesenchymal progenitor cells (MPC) are exclusively localized41-43,50 (Figure 7A).
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