Journal: Cell Reports Medicine

Article Title: SOX4-ZIP14-zinc metabolism mediates oncogenesis and suppresses T cell immunity in nasopharyngeal carcinoma

doi: 10.1016/j.xcrm.2025.102300

Figure Lengend Snippet: Clinical samples demonstrate a significant expression of SOX4 in NPC (A) Uniform manifold approximation and projection (UMAP) plot of 355,955 single cells was generated, including 12 subclusters with each color representing one type of cell. (B) Dotplot showed SOX4 as a signature gene in tumor cluster. (C) The t-SNE algorithm was applied to the binary regulon activity matrix. (D) The rank of regulons in NPC cells was determined based on the regulon specificity score (RSS). (E) SCENIC analysis matched the master regulators with the cell types controlled. (F) Normalized SOX4 expression in two NPC RNA-seq cohorts (two-sided unpaired t test). From left to right: GSE118719 (tumor n = 7, normal n = 4), GSE53819 (tumor n = 18, normal n = 18). (G) The progression-free survival for patients with NPC divided by SOX4 expression from GSE102349 (high n = 26, low n = 62, two-sided log rank test). (H) Multi-IF staining of a representative NPC section (bottom) and a nasopharyngeal lymphatic inflammation (NLI) tissue (top) showed co-expression of LMP-1 (yellow), CK 5/6 (green), and SOX4 (red), with nuclei counterstained with DAPI (blue). Scale bars: 100 μm, 20 μm.

Article Snippet: The lentiviruses of overexpressing of SOX4 (LV-SOX4) and control (LV-CON) from Genechem Co., Ltd. (Shanghai, China).

Techniques: Expressing, Generated, Activity Assay, RNA Sequencing, Staining

Journal: Cell Reports Medicine

Article Title: SOX4-ZIP14-zinc metabolism mediates oncogenesis and suppresses T cell immunity in nasopharyngeal carcinoma

doi: 10.1016/j.xcrm.2025.102300

Figure Lengend Snippet: SOX4 regulates intrinsic phenotypes of NPC (A) A UMAP plot was generated for 7,359 cells, which included 7,266 tumor cells exhibiting high and low expression levels of SOX4 and 93 normal epithelial cells, extracted from patients with NPC and NLI, respectively. (B) Major signaling pathways and biological activities of the different subtypes were calculated in gene set variation analysis (GSVA) using the “ Z score” method. (C) Western blot analysis of SOX4 and EMT markers in NP69 and C666-1 cells transfected with SOX4 and their control group. (D) Stably edited SOX4 affected the migration and invasion ability of NP69 or C666-1 cells in vitro by transwell assay ( n = 3, 24 h), scale bar: 100 μm. (E) The NOD/SCID mice were injected with cells suspensions via tail vein for pulmonary metastasis assay ( n = 6). (F) Effect of SOX4 on tumor formation of cells in the subcutaneous implantation NOD/SCID mouse model ( n = 6). (G) Stemness scores were calculated based on single-cell transcriptomes (tumor cells n = 7,266, normal cells n = 93, Kruskal-Wallis test). (H) The expression levels of β-catenin and c-Myc were quantified by immunoblotting in SOX4-transfected NP69 cells. (I) Cell immunofluorescence staining presented the activation of the Wnt pathway that β-catenin translocated from the cytoplasm to the nucleus following an increase in SOX4, scale bar: 10 μm. (J) Overexpression of SOX4 led to an increase of stemness characteristics in NP69 cells observed by western blot. (K) Representative images demonstrated the capacity for sphere formation in floating cultures, as observed through sphere formation assays ( n = 3), scale bar: 100 μm. (L) In vivo extreme limiting dilution assay (ELDA) using NOD/SCID mice ( n = 6); limiting dilution plots and stem cell frequencies were calculated using ELDA software ( http://bioinf.wehi.edu.au/software/elda/ ). (C, H, and J) Western blots were quantified using ImageJ. (D–J) The “ n ” value represents the number of biologically independent samples in each group. The data were presented as mean ± SD and analyzed using a two-sided unpaired t test, unless otherwise specified.

Article Snippet: The lentiviruses of overexpressing of SOX4 (LV-SOX4) and control (LV-CON) from Genechem Co., Ltd. (Shanghai, China).

Techniques: Generated, Expressing, Protein-Protein interactions, Western Blot, Transfection, Control, Stable Transfection, Migration, In Vitro, Transwell Assay, Injection, Immunofluorescence, Staining, Activation Assay, Over Expression, In Vivo, Limiting Dilution Assay, Software

Journal: Cell Reports Medicine

Article Title: SOX4-ZIP14-zinc metabolism mediates oncogenesis and suppresses T cell immunity in nasopharyngeal carcinoma

doi: 10.1016/j.xcrm.2025.102300

Figure Lengend Snippet: SOX4 renders NPC cells resistant to CD8 + T cell-mediated cytotoxicity (A) The effective score of infiltrated CD8 + T cells was computed at the single-cell level (from tumor n = 55,044, from normal n = 1,988, Wilcoxon test). (B) The bulk transcriptomic dataset from the GEO database ( GSE102349 ) containing 88 patients with NPC was used to conduct GSEA analysis to calculate the enrichment of immune-related pathways between the SOX4-high and SOX4-low groups ( n = 44 in each group). (C) The distribution and abundance of infiltrated cytotoxic CD8 + T cells were identified by multicolor immunofluorescence in the NPC and NLI clinical specimens, scale bars: 100 μm, 20 μm. (D–G) In co-culture system of anti-CD19 CAR CD8 + T cells and SOX4-transduced CD19 + cells (E:T = 4:1, duration = 24 h), the apoptosis of target cells was analyzed by flow cytometry (D, n = 3), the concentration of secreted cytokines (IFN-γ and TNF-α) was measured using ELISA (E, n = 3), the release of LDH in the culture medium was quantified with an LDH assay kit (cytotoxicity percentage was calculated followed by the kit manual: percent cytotoxicity = [experimental LDH release − medium background]/[maximum LDH release control − medium background], maximum LDH release control is the maximum LDH release from NPC cell lines without CAR T cells) (F, n = 3), and relative cell viability was assessed through the XTT assay, calculated by cell viability of co-cultured tumor cells normalized to that of tumor cells without CAR T cells (G, n = 3). (H and I) The fraction of cytotoxic CD8 + T cells post co-culture was analyzed by flow cytometry ( n = 3). (J) An in vivo study in subcutaneous implantation of CD19-transfected SOX4-NC and SOX4-KD NPC cells in immunodeficient NSG mice with/without CAR T treatment. Tumors were collected (without CAR T: NC, n = 6; KD, n = 6; with CAR T: NC, n = 6; KD, n = 5). The percentage weight change was calculated by normalizing the tumor weight of the treatment group to that of the corresponding control group. (K and L) Infiltrated cytotoxic CD8 + T cell fractions (K, NC, n = 6; KD, n = 3) and percentage of activation (L, NC, n = 6; KD, n = 3); the tiny tumor in the KD group with rare CD8 + T cell infiltration was excluded from statistical analysis. The relative MFI was calculated by normalizing the MFI of the gene-edited group to that of the corresponding control group. (D–L) The “ n ” value represents the number of biologically independent samples in each group. The data were presented as mean ± SD and analyzed using a two-sided unpaired t test, unless otherwise specified.

Article Snippet: The lentiviruses of overexpressing of SOX4 (LV-SOX4) and control (LV-CON) from Genechem Co., Ltd. (Shanghai, China).

Techniques: Immunofluorescence, Co-Culture Assay, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Control, XTT Assay, Cell Culture, In Vivo, Transfection, Activation Assay

Journal: Cell Reports Medicine

Article Title: SOX4-ZIP14-zinc metabolism mediates oncogenesis and suppresses T cell immunity in nasopharyngeal carcinoma

doi: 10.1016/j.xcrm.2025.102300

Figure Lengend Snippet: ZIP14 is the downstream target of SOX4 in NPC cells (A) Chord diagram showed the correlation between SOX4 and measured solute transporter carriers identified by Pearson correlation analysis in bulk RNA-seq data ( GSE102349 ). (B) Spatial co-localization of SOX4 and ZIP14 ( GSE200310 ). (C) The protein expression of ZIP14 was determined by western blotting in NP69 and C666-1 cells transfected with SOX4 or a control. (D) The top three conservative SOX4-binding sites within the 2,000 bp upstream of the ZIP14 DNA promoter-proximal region were computationally predicted using JASPAR 2024. (E) ChIP-PCR detection of SOX4 binding motif within ZIP14 promoter region. PCR amplification was carried out with DNA fragments that were immunoprecipitated by anti-FLAG M2 antibody (IP), together with total DNA fragment (input) and DNA fragments that were immunoprecipitated by anti-IgG (IgG). (F) The two highest predicted binding sites were subjected to mutation and determined using a dual-luciferase reporter assay. (G) Western blotting was employed to confirm the effective knockdown of the ZIP14 protein in SOX4-overexpressing NP69 cells. (H and I) The fractions of activated (H, n = 3) and cytotoxic (I, n = 3) CD8 + T cells were assessed by flow cytometry, respectively. The “ n ” value represents the number of biologically independent samples in each group. The data were presented as mean ± SD and analyzed using a two-sided unpaired t test, unless otherwise specified.

Article Snippet: The lentiviruses of overexpressing of SOX4 (LV-SOX4) and control (LV-CON) from Genechem Co., Ltd. (Shanghai, China).

Techniques: RNA Sequencing, Expressing, Western Blot, Transfection, Control, Binding Assay, Amplification, Immunoprecipitation, Mutagenesis, Luciferase, Reporter Assay, Knockdown, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: SOX4-ZIP14-zinc metabolism mediates oncogenesis and suppresses T cell immunity in nasopharyngeal carcinoma

doi: 10.1016/j.xcrm.2025.102300

Figure Lengend Snippet: NPC cells compete with CD8 T cells for zinc via ZIP14 (A and B) The GSVA enrichment analysis disclosed an increase in zinc imports in SOX4-high tumor cells (A) and a reduction in zinc import in tumor-derived CD8 + T cells (B). (C) Validation of zinc homeostasis in NPC and non-NPC patients from the GEO dataset ( GSE53819 ). (D) The EC50 of zinc to tumor cells (4.468 μM) and CD8 + T cells (6.549 μM) was evaluated by normalized cell viability cultured in the complete medium containing gradient concentration of zinc (XTT, n = 3). (E and F) Proportion of activated CD8 + T cells (E) and cell viability of C666-1 cells (F) under excess zinc (50 μM) and deficient zinc (5 μM) conditions ( n = 3). (G and H) After 24 h of co-culture with ZIP14-KD cells, extracellular zinc concentration (G) in the culture medium and intracellular zinc concentration (H) of CAR CD8 + T cells were determined using the appropriate zinc assay kits ( n = 3). (I) The ZIP14-NC and ZIP14-KD tumor in immunodeficient NSG mice and anti-CD19 CAR T-engrafted humanized NSG mice ( n = 6). The percentage volume change is calculated by normalizing the tumor weight of the treatment group to that of the corresponding control group. (J) The fractions of tumor-infiltrating activated CD8 + T cells and effector CD8 + T cells were analyzed using flow cytometry. (D–J) The “ n ” value represents the number of biologically independent samples in each group. The data were presented as mean ± SD and analyzed using a two-sided unpaired t test.

Article Snippet: The lentiviruses of overexpressing of SOX4 (LV-SOX4) and control (LV-CON) from Genechem Co., Ltd. (Shanghai, China).

Techniques: Derivative Assay, Biomarker Discovery, Cell Culture, Concentration Assay, Co-Culture Assay, Zinc Assay, Control, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: SOX4-ZIP14-zinc metabolism mediates oncogenesis and suppresses T cell immunity in nasopharyngeal carcinoma

doi: 10.1016/j.xcrm.2025.102300

Figure Lengend Snippet: Estimation of SOX4-targeting therapeutic strategy in NPC treatment (A) Schematic representation of the subcutaneous patient-derived xenograft (PDX) inoculation and chemotherapy strategy employed in nude mice. (B) Tumors collected after a 12-day administration of various chemotherapies and AAV-SOX4-RNAi ( n = 5). (C) Tumor growth curves for GEM, DDP, and AAV-SOX4-RNAi treatments compared to the control group. (D) A schematic illustrating the adoptive transfer of human PBMCs into NSG mice inoculated with PDX, along with the administration of monotherapy or combination therapy, which included anti-PD1 and/or AAV-SOX4-RNAi. (E) PDX-derived tumors were collected at the experiment’s endpoint. (F–H) Tumor growth curves (F) were measured for the different groups ( n = 4), and the proportions of tumor-infiltrating activated CD8 + T cells (G) and cytotoxic CD8 + T cells (H) were compared across each group ( n = 3). (C and F) The data were presented as mean ± SD and analyzed using a two-way ANOVA with Tukey's multiple comparisons test. (G–H) The data were presented as mean ± SD and analyzed using a one-way ANOVA test.

Article Snippet: The lentiviruses of overexpressing of SOX4 (LV-SOX4) and control (LV-CON) from Genechem Co., Ltd. (Shanghai, China).

Techniques: Derivative Assay, Control, Adoptive Transfer Assay