Immune checkpoint inhibitors (ICI) have made progress in the field of anticancer treatment, but a certain number of PD-L1 negative OSCC patients still have limited benefits from ICI immuno-therapy because of primary immune evasion due to immunodeficiency. However, in existing human OSCC cell lines, cell models that can be used to study immunodeficiency have not been reported. The objective of this study was to establish a PD-L1 negative OSCC cell line, profile whether the presence of mutated genes is associated with immune deficiency, and explore its influence on the immune recognition of CD8⁺ T cells in vitro. Here, we established a novel tongue SCC cell line (WU-TSC-1), which escapes from immune recognition by antigen presentation defects. This cell line was from a female patient who lacked typical causative factors. The expression of PD-L1 was negative in the WU-TSC-1 primary tumor, transplanted tumor, cultured cells and lipopolysaccharide stimulation. Whole exome sequencing (WES) revealed that WU-TSC-1 harbored missense mutations, loss of copy number and structural variations in human leukocyte antigen (HLA) class I/II genes. The tumor mutation burden (TMB) score was high at 292.28. In addition, loss of heterozygosity at beta-2-microglobulin (B2M)—a component of all HLA class I complex allotypes—was detected. Compared with the commonly used OSCC cell lines, genetic alterations in HLA class I and B2M impeded the proteins’ translation and inhibited the activation and killing effect of CD8⁺ T cells. In all, the WU-TSC-1 cell line is characterized by genetic variations and functional defects of the HLA class I complex, leading to escape from recognition by CD8⁺ T cells.

RNA-binding proteins (RBPs) critically regulate post-transcriptional gene networks, yet their roles and mechanisms in oral squamous cell carcinoma (OSCC) remain underexplored. Dysregulated RBPs were identified through integrated analysis of RNA-seq and single-cell RNA-seq. The oncogenic functions of IGF2BP2 were evaluated through tissue microarrays, CCK-8, transwell assays, mouse xenografts, and Igf2bp2-deficient mouse models of tongue SCC (TSCC). Subsequently, we utilized RNA-seq, RIP-seq, RIP/MeRIP-qPCR, and dual-luciferase reporter assays to investigate IGF2BP2-target genes. Furthermore, cell co-culture system and mouse TSCC models were used to validate the therapeutic effect of the IGF2BP2 inhibitor. IGF2BP2 was the most markedly upregulated RBP in OSCC cells and cancer-associated fibroblasts (CAFs), correlating with unfavorable prognosis. IGF2BP2 deprivation significantly impaired human OSCC proliferation and metastasis, and delayed mouse TSCC onset. Mechanistically, IGF2BP2 stabilized EGFR and PIK3R1 mRNA via m6A-dependent interactions, thereby sustaining activation of the EGFR/PI3K/AKT oncogenic axis. Pharmacological inhibition of IGF2BP2 exhibited anti-OSCC efficacy in vivo and in vitro by concurrently suppressing EGFR and PI3K/AKT pathway activity, overcoming anti-EGFR resistance resulting from cell-intrinsic PI3K/AKT hyperactivation and CAF-secreted factors. Our findings identified IGF2BP2 as a master regulator of OSCC progression and a promising therapeutic target, offering an alternative strategy for OSCC patients suffering anti-EGFR resistance.

The interaction between programmed cell death 1 ligand 1 (PD-L1) and programmed cell death protein 1 (PD-1) protects tumor cells from immune surveillance. PD-L1 exon 3 is a potential alternative exon and encodes an Ig variable (IgV) domain. Here, we found that a lack of exon 3 leads to the significant loss of cellular membrane locations and the dramatically reduced protein expression of PD-L1, indicating that PD-L1 exon 3 is essential for its protein expression and translocation to the cell membrane. Notably, oral cancer cells show almost no exon 3 skipping to ensure the expression of the full-length, functional PD-L1 protein. We discovered two key exonic splicing enhancers (ESEs) for exon 3 inclusion. Two efficient antisense oligonucleotides (ASOs) were identified to block these two ESEs, which can significantly trigger exon 3 skipping and decrease the production of full-length, functional PD-L1 on the surface of cancer cells. Treatment of oral cancer cells with these ASOs significantly enhanced immune cells’ suppression of cancer cell proliferation. Surprisingly, these two ASOs also significantly inhibited cell growth and induced cell pyroptosis in oral cancer cells. Altogether, the results of our study demonstrate the pivotal roles of exon 3 in PD-L1 expression and provide a novel anti-PD-L1 method.