Program: Oral and Poster Abstracts
Type: Oral
Session: 506. Hematopoiesis and Stem Cells: Microenvironment, Cell Adhesion and Stromal Stem Cells: Hematopoietic Stem Cells on Stress
First, we confirmed that BCP-ALL cells exhibited their drug resistant phenotype through adhesion to human BM-MSCs using human BCP-ALL cell line, Nalm6. We isolated human BM-MSCs from normal bone marrow samples (AllCells, Emeryville, CA). When Nalm6 cells were co-cultured with human BM-MSCs, they were prone to adhere to BM-MSCs. Nalm6 adhered to BM-MSCs (Nalm6/ad) were more resistant to the treatment with various anti-cancer drugs including doxorubicin than Nalm6 in suspension (Nalm6/su). Immunoblot analysis showed that expression level of anti-apoptotic protein Bcl-2 and phosphorylation level of pro-survival kinase Akt are higher in Nalm6/ad compared with those in Nalm6/su. In cell cycle analysis using Ki67/PI fluorescence-activated cell sorter (FACS) assay, the percentage of populations in G0 phase and S/G2/M phase was higher in Nalm6/ad compared with that in Nalm6/su, which indicated the increase of MRD and the high proliferation of leukemic cells in adhesive population, respectively. Therefore, we considered that detachment of Nalm6 from BM-MSCs is important to restore chemosensitivity of BCP-ALL cells and to eliminate these cells.
Accordingly, we screened drugs that are capable of disrupting the adhesion of Nalm6 to BM-MSCs, and found that several proteasome inhibitors had a such activity. BM-MSCs were treated with bortezomib (10nM for 24h or 100nM for 1h), carfilzomib (3 nM for 24 h or 100 nM for 1 h) or oprozomib (10 nM for 24 h or 300 nM for 4 h), and then washed with PBS and co-cultured with Nalm6 cells. We confirmed that the number of BM-MSCs is not affected with these drugs irrespective of their concentration and incubation time by MTT assay. Co-cultured cells were divided into suspension cells and adherent cells. Each population was stained with monoclonal antibodies against CD19 and CD90 to detect Nalm6 and BM-MSCs, respectively, and analyzed by FACS. When BM-MSCs were pre-treated with bortezomib, carfilzomib or oprozomib, the number of Nalm6/ad was significantly decreased, and inversely, the number of Nalm6/su was increased, as compared to that of co-cultures with untreated BM-MSCs.
Intriguingly, BM-MSCs treated with bortezomib showed aberrant expression of secreted protein acidic and rich in cysteine (SPARC). Immunoblot analysis showed that SPARC expression of BM-MSCs was transiently increased after bortezomib treatment. In co-cultures with BM-MSCs transfected with siRNA targeting SPARC, the number of Nalm6/ad was increased. In addition, the number of Nalm6 adhered to BM-MSCs pre-treated with recombinant human SPARC was lower than that adhered to control non-treated BM-MSCs. Therefore, SPARC showed anti-adhesive property and was one of the molecules associated with adhesion of BM-MSCs to Nalm6.
Finally, we tested whether bortezomib shows therapeutic effects in vivo. Bortezomib administration supported survival of BCP-ALL xenograft model mice utilizing Nalm6, which was concomitant with the decrease in leukemia cells in the femur and in the size of spleen, as compared to those in non-treated control mice.
In summary, Nalm6 cells that adhered to BM-MSCs contributed to construction of chemoresistant population similar to MRD. Here, we found that this population could be detached by treatment of BM-MSCs with bortezomib through transient increase in their SPARC expression. Our findings suggest that bortezomib or other proteasome inhibitors might be promising drugs to treat BCP-ALL through attenuating the adhesion of leukemic cells to BM-MSCs, and shed new insight into a therapeutic strategy in BCP-ALL by targeting BM-MSCs.
Disclosures: No relevant conflicts of interest to declare.
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