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H.R. Lee et al.
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Figure 4. Role of stroma-induced interleukin-4 in the generation of the stem cell-like leukemic subset. (A) Effects of interleukin-4 (IL-4) during co-culture of leukemic cells. Left: experimental scheme; middle: % LSK generated during 3-day co-culture of leukemic cells with stroma supplemented with recombinant IL-4 (mean ± stan- dard error of the mean [SEM], n= 6); Right: effects of antibody against IL-4 on generation of LSK during stromal co-culture of leukemic cells. Shown are the mean±SEM for % LSK in leukemic cells (green fluorescent protein positive [GFP+] CD45+) (n=7, *P<0.05). (B) Effects of IL-4 antibody on in vivo generation of LSK. Left: experimental design. Antibody against IL-4 was intraperitoneally administered into recipient mice at each indicated time point before and after transplantation of MN1 leukemic cells. Middle: numbers of LSK leukemic (GFP+) cells in the BM (two femurs and two tibia) of recipient mice. Middle and right: % of MN1 leukemic cell engraftment determined by total leukemic cells (GFP+ cells) (middle) and cells with leukemia-initiating cell properties (LK and LSK) (right), respectively (mean ± SEM, n=6, *P<0.05). (C) Effects of IL-4 antibody on the chemosensitivity of the leukemic subsets. Left: experimental design. After engraftment of MN1 leukemic cells (10 days after transplantation), recipients were injected with IL-4 antibody and chemotherapeutic drug (AraC+doxorubicin) at the indicated times. Right: changes in the chemosensitivity of leukemic subsets by in vivo injected IL-4 antibody. Relative fold decrease in the cell numbers of each leukemic subset compared to the control (phosphate buffered saline) group 3 days after exposure to drug and antibody (mean ± SEM, n=5).
leukemic cells, we established MN1 leukemic cells from hematopoietic progenitors of mice lacking IL-4 receptor a (IL-4Ra knockout [KO]). Co-culture of leukemic cells from IL-4Ra KO or wild-type (WT) with stromal cells led to com- parable frequencies of LSK or LK subsets in each group (Figure 5B). In contrast, when mesenchymal stromal cells from IL-4Ra KO mice were co-cultured with MN1 leukemic cells, significantly lower frequencies of LSK, but not LK sub- sets, were observed compared to the WT stroma group (Figure 5C). Thus, IL-4 signals target mesenchymal stromal cells, rather than leukemic cells, to facilitate stroma-mediat- ed generation of the LSK subset, indicating that IL-4-medi- ated cross-talk promotes the functional evolution of leukemic cells.
Next, to investigate the effects of IL-4 on mesenchymal stroma, we examined whether the mode of cellular interac- tion between MSC and leukemic cells is influenced by IL-4. LSK subsets were predominantly generated among the leukemic cells tightly adherent to the mesenchymal cells, for both MN1 or H9M1 leukemic cells, but seldom among the loosely adherent/suspension leukemic cells (Online Supplementary Figure S5A). Supporting the influence of IL-4 on stromal adherence, the level of vascular cell adhesion
molecules 1 (VCAM-1) in MSC, which mediate stromal adherence of leukemic cells,40 were up-regulated by IL-4 in WT MSC, but not in IL-4Ra KO MSC (Figure 5D). Conversely, in vivo injection of IL-4-neutralizing antibody caused a significant decrease of VCAM-1 expressions in BM mesenchymal cells including subsets enriched for mes- enchymal progenitors (CD44(-)PDGFRa(+))41-43 (Figure 5E).
In order to further examine the influences of stromal VCAM-1 expression level on the generation of LSK subsets, leukemic cells were co-cultured with sort-purified MSC fractions for different levels of VCAM-1. MSC with higher VCAM-1 levels increased LSK generation during co-culture, whereas MSC expressing lower levels of VCAM-1 decreased it, in comparison to LSK cells from unsorted MSC co-cultures (Figure 5F). Similarly, VCAM-1-blocking anti- body significantly decreased stromal adherence of leukemic cells (Online Supplementary Figure S5B), which led to a con- comitant decrease in the generation of the LSK subset (Figure 5G). Moreover, in vivo administration of VCAM-1 antibody caused a significant decrease of LSK numbers in BM (Figure 5H). These data, together with positive expres- sion of VCAM-1 ligands in leukemic cells (Online Supplementary Figure S6) indicates that tight adherence of
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