Fig 2: Overexpression of CFD inhibits proliferation, viability, and invasion of CESC cells. (A) RT-qPCR analysis confirming the successful overexpression of CFD in C33A and SiHa cells. (B) CCK-8 assay showing reduced cell viability in CFD-overexpressing C33A and SiHa cells compared to control cells. (C, D) EdU assay demonstrating decreased proliferation in CFD-overexpressing C33A and SiHa cells. Representative images (C) and quantitative analysis (D) are shown. (E-H) Transwell assays showing impaired invasion and migration in CFD-overexpressing C33A and SiHa cells. Representative images and quantitative analysis are presented. Data are presented as mean ± SD from three independent biological replicates (n = 3). Statistical significance was determined using two-way ANOVA with Bonferroni’s post-hoc test. P < 0.001.

Fig 3: Overexpression of CFD inhibits CD8 + T cell exhaustion. (A, B) Representative protein images (A) and quantitative analysis (B) showing increased secreted CFD protein levels in co-culture supernatants when CESC cells were transfected with control vector or overexpressed CFD vector. (C) CCK-8 assay was performed on CD8 + T cell to evaluate cytotoxicity. (D) Flow cytometry was used to evaluate the CD8 + T cell number. (E-G) Flow cytometry analysis of exhaustion marker expression on CD8 + T cells: PD-1 (E), TIM-3 (F), and LAG3 (G). (H) Flow cytometry analysis of tumor-intrinsic PD-L1 expression on adherent CESC cells. (I-J) Representative protein images (I) and quantitative analysis (J) of PD-L1 protein expression in CESC cell lysates following direct co-culture with CD8⁺ T cells. Data are presented as mean ± SD from three independent biological replicates (n = 3). Statistical significance was determined using two-way ANOVA with Bonferroni’s post-hoc test. P < 0.001.

Fig 4: Downregulation of CFD expression in CESC tissues correlates with reduced CD8 + T cell infiltration and diagnostic potential. (A) Volcano map of DEGs analyzed from GSE39001 dataset. (B) Quantitative PCR analysis of CFD mRNA expression in paired tumor and adjacent normal tissues from 43 CESC patients. Data are presented as mean ± SD from three independent biological replicates (n = 43 patient samples). Statistical significance was determined using Student’s two-tailed t-test. P < 0.001. (C) ROC curve analysis (data from 43 paired samples) evaluating the diagnostic potential of CFD mRNA levels in distinguishing CESC tissues from normal tissues. (D, E) Representative images (D) of Western blotting analysis and quantitative analysis (E) showing reduced CFD protein levels in tumor tissues compared to adjacent normal tissues, n = 4. Data are presented as mean ± SD from three independent biological replicates (n = 4 patient samples). Statistical significance was determined using Wilcoxon rank-sum test. P < 0.001. (F) Immune cell infiltration analysis using the TIMER2.0 database revealed a significant positive correlation between CFD expression and CD8 + T cell infiltration in CESC.

Fig 5: Enhanced tumor growth inhibition by combination therapy of autologous TILs and CFD overexpression in a PDX model. (A) Representative images of tumors harvested from mice from (1) Control, (2) LV-NC (empty vector without TIL), (3) LV-CFD (CFD overexpression without TIL), (4) TIL, (5) TIL + lv-NC, and (6) TIL + lv-CFD groups. (B) Tumor weight measurements showing significant reduction in tumor growth in mice treated with TIL therapy alone and further inhibition in the combination therapy group. (C) Tumor volume measurements over time demonstrating enhanced suppression of tumor growth in the combination therapy group compared to TIL therapy alone. (D, E) Representative IF images (D) and quantitative analysis (E) of CD8 expression in tumor tissues of mice treated with TIL therapy, CFD-overexpressing lentivirus, or their combination. (F, G) Representative IHC images (F) and quantitative analysis (G) of PD-L1 expression in tumor tissues of mice treated with TIL therapy, CFD-overexpressing lentivirus, or their combination. Data are presented as mean ± SD from three independent biological replicates (n = 5). Statistical significance was determined using one-way ANOVA with Tukey’s post-hoc test. P < 0.001.