FZD7 regulates BMSCs-mediated protection of CML cells

Inspite of effective treatment with imatinib (IM), chronic myeloid leukemia (CML) is still an incurable disease. Some patients became refractory because of IM resistance. Bone marrow mesenchymal stem cells (BMSCs) have been implicated a role in promoting CML cells' resistance against IM treatment. The detailed molecular mechanisms, however, remain largely unknown. In this study, we found that BMSCs increased the expression of FZD7 and activated Wnt/β-catenin signaling pathway in CML cells. BMSCs from CML patients showed increased efficiency to accelerate CML cell proliferation, enhance the drug resistance of K562 cells and up-regulate the expression of FZD7. Antagonism of FZD7 expression by shRNA significantly suppressed proliferation and increased IM sensitivity of CML cells co-cultured with BMSCs cells. Our findings suggest that FZD7, involved in canonical Wnt signaling pathway, plays a critical role in mediating BMSCs-dependent protection of CML cells, and potentially provide a novel therapeutic target for CML.


MTT assay
Briefly, cells were cultured in 96-well plates and incubated with 10 μL MTT (5 mg/mL) at 37°C for 4 hrs, and then 100 μL 10% SDS (PH 4.0) was added to each well and incubated over night. The absorbance was measured at 570 nm. Each sample was performed in triplicates.

Cell cycle analysis
Cell cycle analysis was performed using the cell cycle kit (BestBio, Shanghai, China), according to the manufacturer's instructions. Samples were analyzed by FACS (Beckman, CA).

Apoptosis assay
The apoptosis assay was performed using Annexin V-APC/PI apoptosis detection kit (Ebioscience, San Diego, CA), according to the manufacturer's instructions. Fluorescence of at least 10,000 cells was determined on a FACS Calibur flow cytometer to determine the percentage of apoptotic cells.

Isolation of CD34 +
CD34 + hematopoietic cells were isolated from bone marrow samples using the CD34 MicroBead Kit (Miltenyi Biotec) according to the manufacturer's instructions as following: 1. Determine cell number. 2. Centrifuge cell suspension at 300 × g for 10 minutes.
Aspiratesupernatant completely. 3. Resuspend cell pellet in 300 μL of buffer for up to 10 8 totalcells. 4. Add 100 μL of FcR Blocking Reagent for up to 10 8 total cells. 5. Add 100 μL of CD34 MicroBeads for up to 108 total cells. 6. Mix well and incubate for 30 minutes in the refrigerator(2−8°C). 7. Wash cells by adding 5-10 mL of buffer for up to 108 cellsand centrifuge at 300 × g for 10 minutes. Aspirate supernatantcompletely. 8. Resuspend up to 10 8 cells in 500 μL of buffer. 9. Proceed to magnetic separation: (1) Place column in the magnetic field of a suitable MACSSeparator. For details refer to the respective MACS Columndata sheet.
(5) Remove column from the separator and place it on a suitablecollection tube.
(6) Pipette 1 mL buffer onto the column.Immediately flush out the magnetically labeled cellsby firmly pushing the plunger into the column.
(7) To increase the purity of CD34 + cells, the elutedfraction can be enriched over a second MS Column. Repeat the magnetic separation procedure as described insteps 1 to 6 by using a new column.

Luciferase assays
The TOPFlash construct contains a combinationof TCF binding sites placed upstream of luciferasecDNA. The negative control FOPFlash construct containsmutated TCF binding sites. Renilla luciferase pRL-TK was cotransfectedas an internal control for transfection efficiency.Transfections were performed using a Nucleofector (Amaxa) according to the manufacturer's instructionswith minor modifications. Briefly, 1 × 106 K562 cellstransduced with short hairpin RNAs that target FZD7 or control were transfected with 2.5 μg of either TOPFLASH or FOPFLASH along with 0.25 μg of Renilla cytomegalovirus (CMV) construct. After 24 hours, cell lysates were prepared and reporter activity was measured using the Dual-LuciferaseReporter Assay System (Promega).V