Integrative Spatial Transcriptomics and Immunoinformatics for Prognostic Multi-Epitope Vaccine Construct Prediction...

Background/Objectives: Synovial sarcoma (SS) is a rare and aggressive soft-tissue malignancy characterized by complex molecular alterations and poor prognosis, highlighting the need for targeted immunotherapeutic strategies. This study aimed to design a rational multi-epitope vaccine targeting the FKBP10 oncoprotein to elicit effective immune responses against SS. Methods: Transcriptomic data from the GEO dataset GSE144190, comprising 10 tumor and 9 normal tissue samples, were analyzed to identify differentially expressed genes (DEGs). Results: Our findings revealed significantly upregulated FKBP10 with a log2 fold change of 3.55, baseMean expression of 1521.84, and adjusted p-value of 8.37 × 10^-26. Mutational analysis across 7782 sarcoma samples indicated a low alteration frequency of ~1.5%, primarily missense variants. Functional mapping showed FKBP10 as a hub interacting with multiple collagen chains and chaperone proteins, implicating its role in extracellular matrix organization and protein folding. Linear B-cell epitope prediction identified 17 epitopes (6-21 amino acids), while T-cell mapping yielded 10 MHC class I and 9 MHC class II high-affinity epitopes, all antigenic (VaxiJen > 0.5) and non-allergenic. A multi-epitope vaccine was constructed incorporating a 50S ribosomal protein L22 adjuvant, linkers, and a 6× histidine tag. Physicochemical analysis showed a molecular weight of 36.43 kDa, pI 6.97, instability index 31.79, aliphatic index 64.88, and GRAVY -0.509, indicating stability and hydrophilicity. Structural modeling validated 82.5% residues in favored regions. Molecular docking revealed strong binding with TLR4 (-9.7 kcal/mol) and TLR9 (-9.4 kcal/mol), and 200 ns molecular dynamics simulations confirmed stable RMSD trajectories, low RMSF at binding residues (<4 Å), persistent hydrogen bonding, compact radius of gyration (38-42 Å for TLR4; ~20 Å for TLR9), favorable total energy (-1400 to -1500 kcal/mol for TLR4; -650 to -720 kcal/mol for TLR9), and stable SASA (~52,000-54,000 Ų). Conclusions: These findings demonstrate that the FKBP10 multi-epitope vaccine is structurally stable, immunogenic, and capable of engaging key innate immune receptors, supporting its potential as a promising immunotherapeutic candidate for synovial sarcoma.