Molecular detection of circulating cancer cells with cancer stem cell or mesenchymal characteristics in esophageal squamous cell carcinoma (ESCC)

Abstract

Esophageal squamous cell carcinoma (ESCC) is a clinically challenging disease that requires a multidisciplinary approach. However, the scarcity of genomic data on ESCC hampers our understanding of its biology, disease progression, and rational therapy design. Circulating tumor cells (CTCs), which are shed from primary and metastatic tumors and circulate in the peripheral vasculature, reflect the existing tumor heterogeneity. In this study, a non-biased size-based CTC enrichment strategy in combination with quantitative reverse transcription polymerase chain reaction (RT-qPCR) was utilized to molecularly characterize CTCs with cancer stem cell (CSC) or mesenchymal properties in ESCC. CTCs surviving in the circulation may possess unique epithelial-to-mesenchymal transition (EMT) properties, facilitating the detachment from the primary tumor and the invasion into the bloodstream. Based on this hypothesis, a total of 224 human EMT and metastasis-associated genes were first screened using bioinformatics analysis of next- generation sequencing (NGS) data from ESCC cells and human peripheral blood mononuclear cells (PBMCs) at the single-cell level. Genes that were enriched in ESCC cells, but had extremely low expression in PBMCs, were further validated using RT-qPCR as potential ESCC CTC markers. Marker sensitivity and specificity were assessed using RT-qPCR spike-in assays with varying ratios of ESCC cells and PBMCs, followed by RT-qPCR detection of CTCs enriched from cardiac blood of ESCC patient-derived organoid xenograft (PDOX) mice and clinical ESCC peripheral blood samples. A 10-gene panel—comprising CCND1, ECT2, EpCAM, FSCN1, KRT5, KRT18, MET, TFRC, TWIST1, and VEGFC—for CTC RT-qPCR detection with high sensitivity was evaluated in ESCC. Comprehensive transcriptomic data from ESCC cell lines, mouse ESCC PDOX models, and clinical ESCC peripheral blood samples highlight the significant role of TWIST1 (Twist Family BHLH Transcription Factor 1) in ESCC progression. Gain-of-function (GOF) and loss-of-function (LOF) analyses demonstrate that TWIST1 promotes in vitro cell migration, invasion, and clonogenicity, as evidenced by wound healing assay, transwell assay, and colony formation assay, respectively. Moreover, TWIST1 was also found to enhance the in vivo metastatic potential of ESCC cells, as identified using bioluminescence-based tail vein mouse models. Furthermore, positive TWIST1 expression is associated with chemoresistance to cisplatin in ESCC cells and correlates with progression of disease and poor overall survival in ESCC patients. In addition, our findings demonstrate that TWIST1 promotes malignant potential, including tumor growth, invasion, and chemoresistance, through the TWIST1-TGFBI-ZEB1 signaling pathway in ESCC. Bulk RNA sequencing upon TWIST1 overexpression reveals TGFBI-ZEB1 as downstream signaling targets of TWIST1. GOF analyses of both TGFBI and ZEB1 further emphasize their pro-tumorigenic roles in promoting in vitro cell migration, invasion, and colony formation, as well as cisplatin chemoresistance in ESCC. Moreover, the TWIST1-TGFBI-ZEB1 signaling pathway induces an immunosuppressive tumor microenvironment by upregulating the expression of cytokines and chemokines that attract immunosuppressive cells, thereby contributing to EMT and promoting tumor progression. Collectively, these findings highlight a novel role for TWIST1 in ESCC carcinogenesis, regulating tumorigenicity and CSC properties. These findings suggest that TWIST1 may serve as a prognostic monitoring biomarker and a promising therapeutic target in clinical ESCC treatment.