Supplementary figures 1-8. Supplementary Figure 1. The amino acid sequence of scFv GC33 in the construct of anti-GPC3 CAR. Supplementary Figure 2. The expression of CD64 and CD86 in the artificial antigen-presenting cell aK562-64/86. Supplementary Figure 3. Tumor outgrowth of Huh-7 cells was completely abolished by GPC3-targted CAR T cells in vivo. Supplementary Figure 4. Growth suppression on established s.c. HCC xenografts by GPC3-targeted CAR T cells. Supplementary Figure 5. Mice bearing orthotopic Huh-7 tumors. Supplementary Figure 6. The up-regulation of Bcl-XL protein in αGPC3-28BBZ CAR T cells was stimulated by GPC3-specific antigen. Supplementary Figure 7. The GPC3 expression in the normal kidney and gastric glands. Supplementary Figure 8. The homogeneity of GPC3 expression was analyzed in the human primary HCC tissues (n=75).

PDF file - 734K, Figure S1. EGFRvA is widely expressed in various cancer cell lines and tissues. Figure S2. The upregulation of EGFRvA in glioma tissues compared with paired adjacent non-neoplastic brain tissues and a poor prognosis in patients with high-grade gliomas. Figure S3. EGFRvA promotes cell migration and invasion in vitro and in vivo. Figure S4. EGFRvA caused the activation of STAT3 and increased expression of HB-EGF. Figure S5. The positive feedback regulation between HB-EGF and p-STAT3 in EGFRvA-expressing cells. Figure S6. Cell viability and cell adhesion of U87MG EGFRvA cells treated with STAT3 inhibitor AG490, and siRNA-mediated knockdown of STAT3 in U87MG transfectants. Table S1. Clinical characteristics of patients with low or high expression of EGFR or EGFRvA in 52 glioma patients. Table S2. Higher expressed genes in U87MG EGFRvA cells versus U87MG EGFR cells. Table S3. Lower expressed genes in U87MG EGFRvA cells versus U87MG EGFR cells. Table S4. Primers used in this study.

Objective: To investigate the effect of novel epidermal growth factor receptor variant type A (EGFRvA) on migration of human glioma U87MG cells, and to explore its possible mechanism.Methods: The lentiviral expression vector pWPT-GFP [recombined with green fluorescent protein (GFP) gene, as the blank control], pWPT-EGFRwt (recombined with EGFR wild-type gene, as the negative control) and pWPT-EGFRvA (recombined with EGFRvA gene) was respectively co-transfected with packaging vector psPAX2 and pMD2.G into 293T cells to produce lentiviral particles. Subsequently, the lentiviral particles were separately infected into U87MG cells to establish U87MG GFP, U87MG EGFRwt and U87MG EGFRvA cell lines. The overexpression levels of EGFRwt and EGFRvA in U87MG EGFRwt and U87MG EGFRvA cells were detected by flow cyctometry (FCM) and Western blotting. The migration capability of U87MG cells after transfection was investigated by Transwell migration test. The real-time fluorescent quantitative-PCR (RFQ-PCR) was performed to validate the varied mRNA expression levels of 52 genes which had been screened out in three types of cells by microarray. Then the protein expression levels of 4 tumor metastasis-related genes which had significantly varied mRNA expression levels in RFQ-PCR results were confirmed by Western blotting.Results: After U87MG cells were stably infected with lentivirus particles, U87MG EGFRwt and U87MG EGFRvA cells overexpressed EGFRwt and EGFRvA, respectively. The migration capability of U87MG EGFRvA cells was stronger than that of U87MG EGFRwt cells (P < 0.001). The expression levels of FBLN2 and ROBO1 mRNAs were higher in U87MG EGFRvA cells than those in U87MG EGFRwt cells (both P < 0.05), whereas the expression levels of CDH13 and SOX2 mRNAs were the opposite (both P < 0.05). U87MG EGFRvA cells had a significantly lower expression levels of CDH13 and SOX2 proteins which were related to tumor metastasis (both P < 0.05).Conclusion: Overexpression of EGFRvA can promote the migration of U87MG cells through regulating the expressions of FBLN2, ROBO1, CDH13 and SOX2 genes in tumor cells. DOI:10.3781/j.issn.1000-7431.2015.11.874