Integrative single-cell and bulk transcriptomic analyses identify timp1 as a candidate regulator of anoikis resistance and metastatic potential in esophageal squamous cell carcinoma

Anoikis is a form of programmed cell death that occurs when cells detach from the extracellular matrix, and resistance to anoikis is considered a key mechanism underlying tumor cell invasion and distant metastasis. However, the molecular mechanisms regulating anoikis in esophageal squamous cell carcinoma (ESCC) remain unclear. This study aimed to identify key anoikis-related genes (ARGs) through integrated multi-omics analysis, construct a prognostic model, and further evaluate the functional relevance of TIMP1 in ESCC. First, integrated analysis of ESCC single-cell RNA sequencing data was performed, and anoikis characteristics across different cell types were assessed by calculating anoikis scores, followed by the identification of related differentially expressed genes (DEGs). Subsequently, weighted gene co-expression network analysis (WGCNA) was performed based on the GSE53625 cohort, and the overlap between bulk transcriptomic DEGs and single-cell DEGs was used to identify key ARGs. A prognostic risk score model was established using univariate Cox regression and least absolute shrinkage and selection operator (LASSO) Cox regression and validated in The Cancer Genome Atlas (TCGA) cohort. A nomogram integrating clinicopathological features was subsequently constructed. Finally, the biological role of TIMP1 in ESCC was validated through in vitro functional assays and in vivo nude mouse models of subcutaneous xenograft growth and lung metastasis. Single-cell analysis revealed heterogeneity in anoikis scores across different cell types. A total of 428 candidate ARGs were identified through the integration of WGCNA and multi-omics analyses. The seven-gene risk model (ATP8B3, KIF18B, TIMP1, BCAT1, SLC39A6, CLEC6A, and TNS3), constructed using LASSO-Cox regression, showed predictive value for patient prognosis in both the GSE53625 and TCGA cohorts. Functional assays indicated that TIMP1 knockdown inhibited ESCC cell proliferation, migration, invasion, and colony formation, while reducing anoikis resistance and promoting apoptosis. Furthermore, in vivo experiments demonstrated that TIMP1 knockdown suppressed subcutaneous tumor growth and reduced experimental pulmonary colonization. In this study, an anoikis-related prognostic model for ESCC was developed through the integration of single-cell and bulk transcriptomic analyses, and TIMP1 was prioritized as a candidate gene associated with anoikis resistance. Functional experiments showed that TIMP1 depletion was accompanied by reduced malignant phenotypes and increased susceptibility to anoikis in ESCC cells. However, the downstream signaling mediators and direct molecular interactions linking TIMP1 to the anoikis-resistant phenotype were not characterized in the present study. These findings support the potential prognostic relevance of TIMP1 and provide a rationale for further mechanistic and translational investigation.