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Functional switching of leukemic cells by stromal contact
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Figure 5. Interleukin-4-mediated cross-talk in mesenchymal stromal cells controlling VCAM-1-mediated generation of leukemic subsets. (A to C) Identification of interleukin-4 (IL-4) target in cross-talk of leukemic cells and mesenchymal stromal cells (MSC). (A) Experimental design. IL-4 receptor (IL-4Ra) knockout (Leukemia- KO) or wild-type (WT) leukemic cells were co-cultured with WT mice-derived murine MSC (MSC WT) (left), or MSC from IL-4Ra KO mice (MSC KO) or WT (right) were co-cultured with leukemic cells from WT mice. (B) Effects of IL-4Ra KO out in leukemic cells on the generation of LSK subsets. Shown are the quantification of % LSK and LK cells during co-culture (mean ± standard error of the mean[SEM], n=9, *P<0.05). (C) Effects of IL-4R KO in MSC on the generation of LSK subsets. Shown are the quantification of % LSK and % LK from the co-culture (mean± SEM, n=15, *P<0.05). (D to H) IL-4 targeting of MSC facilitates generation of leukemic subsets by controlling VCAM-1 expression in MSC. (D) Effects of IL-4 signals on VCAM-1 expression levels of MSC. Murine MSC from WT or IL-4R KO mice were treated with recombinant IL-4 and the fold increase of % VCAM-1(+) were analyzed in comparison to the control group (mean± SEM, n=6, *P<0.05). (E) In vivo changes of VCAM-1(+) cells in the bone marrow (BM) of mice injected with IL-4-neutralizing antibody. Mice were intraperitoneally injected with IL-4 antibody for 4 days and ana- lyzed for % VCAM-1(+) cells in BM stromal cells. Shown are the relative fold differences of % VCAM-1(+) cells in the indicated subsets of BM mesenchymal stromal cells relative to the IgG treated mice group (n=5, *P<0.05). (F) Influence of VCAM-1 expression in MSC for generation of LSK subsets. Murine MSC were sort-purified for differences in VCAM-1 expression levels, and the generation of LSK subsets from MN1 leukemic cells co-cultured with each fraction was analyzed. Shown are the flow cytometry plots for sorting of MSC (left) and relative folds for LSK numbers generated in each co-culture group compared to co-culture with unsorted MSC (right) (n=6). (G) Effects of blocking antibody against VCAM-1 on the in vitro generation of LSK subsets. During co-culture of leukemic cells with stroma, the indicated amount of antibody against VCAM-1 was added and changes in the numbers of LSK generated in the co-culture were analyzed (n= 6, *P<0.05). (H) Effects of block- ing antibody against VCAM-1 on the in vivo generation of LSK subsets. Mice transplanted with MN1 leukemic cells were injected with rat immunoglobulin (Ig) or VCAM-1-blocking antibody (intravenous 10 mg/kg) (7 and 10 days after leukemic cell transplantation). Three days after antibody injection, generation of LSK in recip- ient BM was analyzed (n=3 for IgG, n=4 for anti-VCAM1, *P<0.05).
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