Spatial Transcriptome Profiling Identifies DTX3L and BST2 as Key Biomarkers in Esophageal Squamous Cell Carcinoma Tumorigenesis

Oncology Biochemistry Medicine Immunology Genetics Life Sciences Cell Biology
Esophageal squamous cell carcinoma Tumorigenesis Spatial transcriptome Single-cell transcriptome Tumor-associated macrophage Polarization Spatial biomarker Functional studies

Digital Spatial Transcriptomics Highlights DTX3L and BST2 as Crucial Biomarkers in Esophageal Squamous Cell Carcinoma Tumorigenesis

Background and Research Question

Esophageal cancer (EC) remains a globally prevalent malignant disease with high incidence and mortality rates. Esophageal squamous cell carcinoma (ESCC), the most common histological subtype in Asian populations, carries extremely poor prognosis due to its rapid progression, treatment resistance, and high metastatic potential. Early detection and treatment are critical to reducing the mortality rates of ESCC patients. However, the cellular and molecular mechanisms underlying ESCC initiation and early tumorigenesis remain largely unclear, particularly regarding the progression from normal tissue to low-grade dysplasia, high-grade dysplasia, and ultimately invasive cancer.

To date, most research has focused on tumor cell behavior in ESCC while neglecting the interactions between tumor cells and the tumor microenvironment (TME). Recent studies have revealed that cancer evolution is majorly influenced by the overall potency of the cellular ecosystem rather than solely by tumor cells. Additionally, traditional high-throughput RNA sequencing (bulk RNA-seq) and single-cell RNA sequencing (scRNA-seq) have limitations in exploring TME-related mechanisms due to the loss of spatial localization and intercellular interaction information. Therefore, utilizing advanced spatial transcriptomic tools to uncover molecular remodeling and immune environment changes across histologically distinct regions is crucial for elucidating oncogenic mechanisms and identifying therapeutic targets.

Research Context

This study was conducted by Rutao Li and colleagues, with contributors from organizations including the First Affiliated Hospital of Soochow University, the Fourth Affiliated Hospital, and the Shenzhen Engineering Center. The paper was published in Genome Medicine (2024) under the title: “Spatial transcriptome profiling identifies DTX3L and BST2 as key biomarkers in esophageal squamous cell carcinoma tumorigenesis.”

Objectives and Methodology

This study utilized digital spatial profiling (Nanostring GeoMx DSP) and single-cell spatial transcriptomics (CosmX SMI) to investigate molecular and immunological changes during the progression from normal esophageal tissues to cancer. The aim was to identify key molecular features in ESCC and propose potential biomarkers or therapeutic targets for this disease.

Key Methodological Steps:

  1. Sample Collection: Multi-stage tissue samples from 11 patients with PT1 ESCC were analyzed, including 4 distinct lesion regions: normal tissue, low-grade dysplasia (LGD), high-grade dysplasia (HGD), and carcinoma.
  2. Molecular Compartmental Analysis: Spatially defined transcriptional alterations in epithelial cells, immune cells, and non-immune stromal cells were assessed.
  3. Validation Approaches: Single-cell spatial molecular imaging (CosmX SMI), immunohistochemistry (IHC), RNA-seq datasets, and scRNA-seq data were integrated to confirm candidate genes and immune alterations.
  4. Functional Investigations: Genes—DTX3L and BST2—were experimentally validated in vitro and in vivo for their impact on tumor progression and modulation of the immunosuppressive microenvironment.

Findings and Results

Mapping Molecular and Immune Changes

This study comprehensively documented distinct molecular and immunological transitions across cancer initiation and progression. Key discoveries include:

  1. Molecular Alterations:

    • Significant gene expression changes in epithelial cells and stromal compartments were observed during the transition from normal to malignancy.
    • DTX3L was notably elevated in epithelial cells, while BST2 was highly expressed in stromal cells along with increased immune-suppressive signals.

  2. Biological Pathways:

    • Oncogenic processes (e.g., cell cycle, DNA repair) were elevated in epithelial compartments.
    • Cytokine- and immune-related pathways (e.g., Interleukin-6 and IL-10 signaling) were upregulated in stromal compartments.

  3. Immunological Environment:

    • A progressively immunosuppressive microenvironment was identified, characterized by increased tumor-supportive M2 macrophages and regulatory T (Treg) cells.
    • Higher CD68 (macrophage marker) and CD163 (M2 macrophage marker) expression levels were validated across cancer progression stages.

Identifying Biomarkers and Cellular Communication

Key signaling pathways and biomarkers were highlighted: - Intercellular Crosstalk: Enhanced tumor epithelial cell-macrophage communication was observed, primarily through the MIF-(CD74+CXCR4) ligand-receptor axis. - Candidate Biomarkers: 1. DTX3L (in epithelial cells) and BST2 (in stromal cells) consistently increased during ESCC progression. 2. Both biomarkers were shown to modulate M2 macrophage polarization, contributing to an immune-suppressive TME.

Functional and Biological Studies

Functional studies validated the roles of DTX3L and BST2:

  1. DTX3L:

    • Knockdown of DTX3L significantly reduced ESCC cell migration and proliferation in vitro.
    • In vivo, DTX3L knockdown suppressed tumor growth in xenografts, with reduced macrophage infiltration and lower M2 macrophage presence.

  2. BST2:

    • Knockdown of BST2 or treatment with anti-BST2 antibodies decreased M2 macrophage polarization markers (e.g., CD163).
    • Conditioned media from BST2-deficient macrophages impaired ESCC cell migration and proliferation.

Clinical Implications and Comparative Studies

Clinical Potential: - DTX3L: First time identified as highly expressed in ESCC, with potential as a diagnostic biomarker or therapeutic target. - BST2: Elevated expression suggested as a tool for early detection and immunotherapy. Anti-BST2 antibody therapies previously explored in multiple myeloma may promise similar applications in ESCC.

Comparison with Prior Studies: - Unlike prior studies (e.g., Liu et al. exploring ESCC precancerous lesions), this study applied molecular segmentation to specific tissue compartments (epithelial, immune, and stromal layers) using DSP, providing a more nuanced view of ESCC progression. - Single-cell and spatial molecular imaging offered subcellular resolution insights, surpassing traditional techniques such as bulk RNA profiling.

Challenges and Limitations

  • Genomic Influence: The lack of genomic data integration limits insight into regulatory mechanisms underlying transcriptional rewiring.
  • Heterogeneity: Although PT1 ESCC serves as an ideal spatial model with multiple histological regions, key expression levels in one compartment may influence other anatomical layers.

Conclusion

Spatial transcriptomic profiling provided a dynamic atlas of molecular and cellular events during ESCC progression, leading to the identification of DTX3L and BST2 as key spatial biomarkers. This framework offers valuable insight into ESCC pathogenesis, its immune microenvironment, and targeted therapeutic opportunities for this disease.

Abbreviations

ESCC: Esophageal Squamous Cell Carcinoma
DSP: Digital Spatial Profiling
SMI: Spatial Molecular Imaging
IHC: Immunohistochemistry
TME: Tumor Microenvironment
MIF: Migration Inhibitory Factor
scRNA-seq: Single-cell RNA Sequencing

Additional Resources/Support

  • Public RNA-seq datasets (e.g., TCGA, GEO Series GSE213565) were analyzed.
  • Animal experiments conducted at Soochow University were ethically approved.
  • Study funded by the National Natural Science Foundation of China.

Future Directions

  • Further clinical testing of anti-BST2 therapies for ESCC immunotherapy.
  • Exploring genomic datasets to correlate molecular findings with mutations.

Research Impacts: This study lays a foundation for developing early interventions in ESCC and improving outcomes through targeted, spatially-informed strategies.