Fig 2: Inhibition of F-actin polymerization enhances cisplatin sensitivity and suppresses tumor growth in an orthotopic HNSCC DTP mouse model. (A) Representative tumor growth curves in NOD/SCID mice bearing orthotopic tongue tumors derived from HNSCC DTP cells. Mice were randomized into four groups: vehicle control, cisplatin (10 mg/kg, i.p.), F-actin polymerization inhibitor (2 mg/kg, i.p.), and combination therapy. (B) Combination treatment resulted in a significant reduction in tumor volume compared to monotherapies. (C) Kaplan–Meier survival analysis of mice over the 4-week treatment period. The combination group exhibited significantly prolonged survival (log-rank test, p < 0.05). (D) Representative immunohistochemistry (IHC) staining of excised tumor tissues for Ki-67 (proliferation), cleaved caspase-3 (apoptosis), IPO9 (angiogenesis), and RHOJ. Decreased Ki-67, IPO9, and RHOJ expression, and increased cleaved caspase-3 staining were observed in the combination group. Quantitative IHC scoring (Q-score) of indicated markers. Data are presented as mean ± SD (n = 15 per group). Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. p < 0.05; p < 0.01.

Fig 3: Association of High RHOJ Expression in Cisplatin-Resistant HNSCC with M2 Polarization, Cytoskeletal Remodeling, and Stress Adaptation. (A) IHC staining revealed significantly elevated RHOJ expression in recurrent, cisplatin-resistant HNSCC tissues compared with nonrecurrent, cisplatin-sensitive samples. Quantification using the Quick score method revealed increased staining in tumor cell membranes and endothelial regions (p < 0.001). Parallel pAKT (Ser473) IHC staining in the same cohort showed that pAKT Q-scores positively correlated with RHOJ expression, supporting a link between RHOJ upregulation and AKT pathway activation in resistant tumors. (B–C) immunofluorescence analysis demonstrated colocalization of RHOJ with CD31⁺ endothelial cells and CD206⁺ M2 macrophages, suggesting its role in vascular remodeling and immune suppression. (D) Transcriptomic correlation analysis revealed the positive association of RHOJ expression with AKT (phosphorylation signaling), RAC1 (cytoskeletal remodeling), STAT6 (M2 polarization), and SOD2 (oxidative stress response).

Fig 4: Single-Cell and Molecular Analysis of RHOJ in HNSCC TME. (A) t-SNE plot of single-cell RNA sequencing data, with distinct cell clusters observable in HNSCC TME. (B) Principal component analysis results. Tumor-specific clustering reflects RHOJ-associated transcriptomic differences in tumor progression and TME remodeling. (C) Protein–protein interaction network, in which RHOJ is a central node that interacts with key signaling regulators.

Fig 5: Effect of RHOJ on Tumor Cell Migration and Chemosensitivity in SCC9-P Cells. (A) shRNA-mediated knockdown of RHOJ in SCC9-P cells significantly downregulated IPO9, MSN, and EpCAM expression. (B) Migration assays revealed that RHOJ inhibition reduces the migratory capability of SCC9-P cells. (C) Treatment with F-actin inhibitor Latrunculin B (HY-101,848) disrupted actin polymerization, downregulating tumor-related network genes in SCC9-P cells. (D) Combined treatment with Latrunculin B and cisplatin increased chemosensitivity in a RHOJ-dependent manner, effectively targeting DTP cells. (E) Direct and indirect coculture system setup illustrating interaction between M2 macrophages and HNSCC cells. (F) Indirect coculture indicating that RHOJ silencing reduces SCC9-P and HSC3-P cell viability, whereas RHOJ overexpression increases cell growth. (G) Direct coculture indicating that RHOJ silencing inhibits M2 macrophage activation, whereas RHOJ overexpression significantly promotes M2 cell proliferation.