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Additional file 4: Supplementary Figure 4. Overexpression of CD147 promotes the malignant transformation of JB6. (A-B) Overexpression of CD147 accelerates the growth of JB6 in vitro. Stable overexpression of CD147 in JB6 cells was generated by lentiviral infection. Whole-cell lysates of JB6 cells were extracted and subjected to immunoblot analysis using antibodies against CD147 as described in the Materials and Methods. GAPDH was used as a control (A). CD147-overexpressing JB6 cells showed an increased growth rate (B). Cells were seeded into 96-well plates, and cell viability was examined by a CCK-8 kit as described in the Materials and Methods. Data from multiple experiments are expressed as the mean ± SD. The significance of differences was evaluated using two-way ANOVA. (C) CD147 increases the colony formation ability of JB6 cells. Cells were seeded into 6-well plates, and the number of foci was counted as described in the Materials and Methods. Data from three independent experiments are expressed as the mean ± SD. The significance of differences was evaluated using Student’s t-test. (D-F) Upregulation of CD147 promotes the migration and invasion abilities of JB6 cells. The scratch assay was performed as described in the Materials and Methods. The bar chart graphs shown are from three independent experiments (D). Data are presented as the mean ± SD (n = 3). The significance of differences was evaluated using two-way ANOVA. Transwell assays were performed as described in the Materials and Methods (E). The number of invasive cells per field was calculated, and the data are presented as the mean ± SD (n = 4) of each group (F). The significance of differences between cells was evaluated by Student’s t-test.

Additional file 5: Supplementary Figure 5. Expression of CD147 related genes in normal skin, actinic keratosis, and cSCC in datasets of GSE42677. The correlation of CD147 with CXCL1 (A), CXCL2 (B), CXCL10 (C) and IL6 (D) were determined using Pearson’s correlation analysis.

Additional file 6: Supplementary Figure 6. Overexpression of CD147 promotes the activation of RSK2 and the interaction of CD147 and RSK2 is mediated by CD147D207-230. (A) Whole-cell lysates of JB6-Mock or JB6-CD147OE cells stimulated with EGF were extracted and subjected to immunoblot analysis using indicated antibodies. GAPDH was used as a control. (B) The CD147D207-230 mediates the interaction between CD147 and RSK2. RSK2 and truncated CD147 were co-transfected into 293 T cells. Co-IP was performed with anti-c-Myc antibodies, followed by immunoblotting with the indicated antibodies.

Additional file 2: Supplementary Figure 2. RT-PCR Analyses of the Effect of CD147 on the Gene Expression Profile of EpiCD147-OE Mice. RNA was extracted from EpiCD147-OE and EpiCD147-WT mice. RT-PCR was then performed with different primers (MMP8, CCL1, IL-1β, CCL2, CCL3 and CCL4) as described in the Materials and Methods. The data from multiple experiments (n = 4) are expressed as the mean ± SD. The significance of differences was evaluated using Student’s t-test.

Additional file 3: Supplementary Figure 3. Overexpression of CD147 in CD147-knockdown HaCaT cells rescued the phenotype. (A) Overexpression of CD147 promote the growth of HaCaT cells with CD147-knockdown. Stable overexpression of CD147 in JB6 cells was generated by lentiviral infection. Cells were seeded into 96-well plates, and cell viability was examined by a CCK-8 kit as described in the Materials and Methods. Data from multiple experiments are expressed as the mean ± SD. The significance of differences was evaluated using two-way ANOVA. (B-E) Overexpression of CD147 promotes the migration and invasion abilities of CD147-knockdown HaCaT cells. The scratch assay was performed as described in the Materials and Methods (B). The bar chart graphs shown are from three independent experiments (C). Data are presented as the mean ± SD (n = 3). The significance of differences was evaluated using two-way ANOVA. Transwell assays were performed as described in the Materials and Methods (D). The number of invasive cells per field was calculated, and the data are presented as the mean ± SD (n = 4) of each group (E). The significance of differences between cells was evaluated by one-way ANOVA.

Additional file 4: Table S1. The demographic and clinical information of participants.

Additional file 2: Figure S2. MAN2C1 is generally down-regulated in cancers. (A) MAN2C1 is generally down-regulated in cancers. The expression profile of MAN2C1 in different cancers was analyzed by GEPIA. MAN2B1 is overexpressed in ACC, BRCA, CESC, COAD, ESCA, GBM, LUAD, LUSC, OV, PRAD, READ, SKCM, STAD, TGCT, THCA, UCEC and UCS, but highly expressed in THYM. (B) The expression level of MAN2B1 in melanoma is moderate among different cancers. Comparison of MAN2C1 expression in different cancers was analysed in ProteinAtlas as described in Methods.

Additional file 1: Figure S1. MAN2B1 is generally highly expressed in cancers. (A) MAN2B1 is generally highly expressed in cancers. The expression profile of MAN2B1 in different cancers was analyzed by GEPIA. MAN2B1 is overexpressed in CESC, DLBC, ESCA, GBM, KIRC, KIRP, LAML, LGG, LIHC, PAAD, SKCM, TGCT, UCEC and UC. (B) The expression level of MAN2B1 in melanoma is higher than that in other cancers. Comparison of MAN2B1 expression in different cancers was analyzed in ProteinAtlas as described in Methods.

BackgroundGamma delta (γδ) T cells are attractive effector cells for cancer immunotherapy. Vδ2 T cells expanded by zoledronic acid (ZOL) are the most commonly used γδ T cells for adoptive cell therapy. However, adoptive transfer of the expanded Vδ2 T cells has limited clinical efficacy.MethodsWe developed a costimulation method for expansion of Vδ2 T cells in PBMCs by activating γδ T-cell receptor (γδTCR) and Toll-like receptor (TLR) 7/8 using isopentenyl pyrophosphate (IPP) and resiquimod, respectively, and tested the functional markers and antitumoral effects in vitro two-dimensional two-dimensional and three-dimensional spheroid models and in vivo models. Single-cell sequencing dataset analysis and reverse-phase protein array were employed for mechanistic studies.ResultsWe find that Vδ2 T cells expanded by IPP plus resiquimod showed significantly increased cytotoxicity to tumor cells with lower programmed cell death protein 1 (PD-1) expression than Vδ2 T cells expanded by IPP or ZOL. Mechanistically, the costimulation enhanced the activation of the phosphatidylinositol 3-kinase (PI3K)–protein kinase B (PKB/Akt)–the mammalian target of rapamycin (mTOR) pathway and the TLR7/8–MyD88 pathway. Resiquimod stimulated Vδ2 T-cell expansion in both antigen presenting cell dependent and independent manners. In addition, resiquimod decreased the number of adherent inhibitory antigen-presenting cells (APCs) and suppressed the inhibitory function of APCs by decreasing PD-L1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression in these cells during in vitro Vδ2 T-cell expansion. Finally, we showed that human Vδ2 T cells can be expanded from PBMCs and spleen of humanized NSG mice using IPP plus resiquimod or ZOL, demonstrating that humanized mice are a promising preclinical model for studying human γδ T-cell development and function.ConclusionsVδ2 T cells expanded by IPP and resiquimod demonstrate improved anti-tumor function and have the potential to increase the efficacy of γδ T cell-based therapies.