American Association for Cancer Research
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Supplementary Data from IL-17A Stimulates the Progression of Giant Cell Tumors of Bone

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posted on 2023-03-31, 17:25 authored by Meng Xu, Zhi-Gang Song, Cheng-Xiong Xu, Guang-Hua Rong, Ke-Xing Fan, Ji-Ying Chen, Wei Zhang, Jin-Peng Jia, Gang Han, Wei Wang, Wei Chai, Wen-Tao Liang, Wen-Zhi Bi, Yan Wang

Supplementary Data - PDF file 126K, Supplementary Figure S1. The isolation and culture of MNGCs and SCs from GCTB samples. A. After a quick centrifuge, the raw MNGCs were stained with Hoechst 33342 for 1h and sorted by a flow cytometric cell sorter. The MNGCs were huge and contained high levels of DNA and were thus clearly gated and sorted. A representative TRAP staining of the isolated MNGCs was shown. D. SCs were isolated and a representative microscopic morphology was shown. Supplementary Figure S2. Gene microarray and pathway analysis. A, A heat map of genes that are up-regulated over 1.5-fold by IL-17A in IL-17RA+SCs. The IL-17RA+SCs were treated with DMSO or 10 ng/ml IL-17A. After 48 hrs of IL-17A treatment, RNAs were isolated from cells and then subjected to gene microarray analysis.IL-17A pathway in IL-17RA-positive stromal cells. B, Software-assisted identification of pathways affected by IL-17A exposure, as revealed by Ingenuity Pathway Analysis. Differentially-expressed proteins showing a +1.5-fold change or greater were selected and entered into IPA software to illustrate potential interactions. Supplementary Figure S3. Myc and STAT3 inhibitor treatment suppressed IL-17A stimulated cell proliferation and RANKL expression in IL-17RA+SCs. A, The Myc inhibitor 10058-F4 treatment blocked IL-17A stimulated IL-17RA+ SCs proliferation. B, Treatment with the STAT3 inhibitor AG490 blocked IL-17A-induced RANKL mRNA expression in IL-17RA+SCs. The results are expressed as the mean values plus-minus SD. a,b,c,d data not sharing the same letter are significantly different from one another in each group (P < 0.05) by ANOVA and Duncan's multiple range test



Purpose: Giant cell tumors of bone (GCTB) exhibit aggressive bone lytic behavior. Studies have shown that interleukin 17A (IL-17A) is involved pathologic bone resorption in various skeletal disorders. Thus, we have investigated the role of IL-17A in GCTBs.Experimental Design: We evaluated the progression of GCTBs using Campanacci grading and Enneking staging systems in 74 patients with GCTB. The expression of IL-17A and the IL-17A receptor A (IL-17RA) was assessed in GCTB tissues and in both multinucleated giant cells (MNGC) and stromal cells cultured in vitro using immunostaining and reverse transcription PCR (RT-PCR). The effects of IL-17A on the osteolytic activity of the MNGCs and the proliferation of the stromal cells were investigated using the “pit” formation and MTT assays, respectively. The effects of IL-17A on the expression of proosteolytic factors were examined in primary cultured MNGCs and stromal cells using RT-PCR, Western blotting, and gene expression microarrays.Results: In GCTBs, we detected abundant levels of IL-17A, which were associated with tumor extension and grade. IL-17A is predominantly produced by MNGCs, whereas IL-17RA is expressed by both MNGCs and stromal cells in GCTBs. In the MNGCs, the IL-17A increased the mRNA expression of IL-17A and proosteolytic enzymes, and also enhanced osteolytic ability. In the stromal cells, the IL-17A stimulated cellular proliferation and the expression of proosteolytic factors, including RANKL through myc and STAT3, respectively. In addition, IL-17A stimulated in vivo tumor growth and the extent of angiogenesis in GCTBs.Conclusion: IL-17A stimulates the progression of GCTBs and might represent a useful candidate marker for progression and as a therapeutic target for GCTBs. Clin Cancer Res; 19(17); 4697–705. ©2013 AACR.

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