The Role of SDCBP in Immunotherapy of Colorectal Cancer

Rheumatology and Immunology Life Sciences Immunology Oncology Medicine Cell Biology
Colorectal cancer Immunotherapy Tumor microenvironment Anti-PD1 therapy SDCBP Zinc pyrithione

Role of Syndecan Binding Protein (SDCBP) in Regulating Tumor Microenvironment, Tumor Progression, and Anti-PD1 Efficacy in Colorectal Cancer

Background

In recent years, immunotherapy has brought revolutionary advancements in cancer treatment. However, for colorectal cancer (CRC), anti-programmed cell death 1 (APD1) therapy shows relatively limited efficacy in patients. Only a specific subset of patients with high microsatellite instability (MSI-H) benefits from immune checkpoint inhibitors (ICIs), but these patients account for only 15% of total CRC cases, and even less, 4-5%, in advanced cancer cases. Therefore, there is an urgent need to explore new targets to enhance the efficacy of ICIs in CRC.

The tumor microenvironment (TME) plays a crucial role in regulating resistance to cancer immunotherapy. Tumor-associated macrophages (TAMs) are the most abundant immune cells in the TME. TAMs are categorized into classically activated (M1) macrophages, which trigger immune activation, and alternatively activated (M2) macrophages, which promote tumor progression and influence the efficacy of anti-PD1 therapy by overexpressing immunosuppressive factors TREM2 and CCL2. Furthermore, M2-TAMs directly affect the migration and infiltration of CD8+ T cells in tumors. Hence, reducing M2-TAMs infiltration or promoting their repolarization is an important strategy to enhance immunotherapy efficacy.

Syndecan Binding Protein (SDCBP), also known as Syntenin1 or melanoma differentiation-associated gene-9 (MDA-9), is a member of the protein family containing PDZ domains. It is involved in cell adhesion, migration, and signal transduction processes. Previous studies show that SDCBP is closely related to cancer invasion and metastasis and promotes chemoresistance in CRC by regulating cancer stem cells (CSCs) expansion and migration. High expression of SDCBP in CRC patients is significantly associated with poor overall survival and disease-free survival. However, its role in the complex TME and its impact on immunotherapy remain to be fully elucidated.

Source of Research

This study was conducted by scholars Jiahua Yu, Shijun Yu, Jin Bai, Zhe Zhu, Yong Gao, and Yandong Li from the Oncology and Colorectal Surgery Departments, Shanghai East Hospital, Tongji University School of Medicine. The research results were published in the journal Cancer Gene Therapy in 2024.

Research Process

a) Research Workflow

  1. Cell Culture and Treatment

    • Human CRC cell line RKO, murine CRC cell line CT26, and murine macrophage cell line RAW264.7 were used.
    • Different cell lines were cultured in media containing 10% fetal bovine serum.
    • ZnPT treatment: cells were treated with ZnPT (0.25, 0.5, 1, 1.5, 2.0 µM) for 24 hours.

  2. RNA Interference and Establishment of Stable Cell Lines

    • Synthesized small interfering RNA (siRNA) targeting SDCBP and performed transfection, with puromycin selection for stable transfected cells.

  3. Cell Proliferation Assays

    • Conducted CCK-8 and colony formation assays to assess cell proliferation.

  4. Cell Migration Assays

    • Employed Transwell assays to evaluate the migration ability of CRC cells.

  5. Western Blot Analysis

    • Extracted cellular proteins using RIPA lysis buffer and analyzed them via SDS-PAGE and membrane transfer.

  6. Apoptosis Detection

    • Used flow cytometry to detect the proportion of apoptotic cells.

  7. Macrophage M1 and M2 Polarization

    • Treated RAW264.7 cells with LPS and IL-4 for M1 and M2 polarization respectively, followed by co-culture with CRC cells.

  8. Quantitative Real-Time PCR

    • Extracted total RNA, reverse transcribed to cDNA, and performed quantitative real-time PCR.

  9. Immunofluorescence Staining

    • Used immunofluorescence to detect the expression of surface markers.

  10. Flow Cytometry

    • Analyzed the expression of CD86 and CD206 proteins in macrophage subpopulations.

  11. Animal Experiments

    • Employed BALB/c mouse models to study the effect of ZNPT on tumor growth, metastasis, and its combined efficacy with APD1.

  12. Immunohistochemistry

    • Conducted immunohistochemical staining analysis of tumor samples.

  13. CyTOF Analysis

    • Analyzed immune cell infiltration in mouse tumor samples using Cytometry by Time-Of-Flight (CyTOF).

  14. Bioinformatics Analysis

    • Analyzed SDCBP expression in CRC patients and its relationship with immune cells using BEST and SangerBox platforms.

  15. Statistical Analysis

    • Performed statistical analysis using GraphPad Prism.

b) Research Results

  1. Expression and Clinical Significance of SDCBP in Cancer

    • SDCBP is upregulated in CRC patients and is associated with poor prognosis.
    • High SDCBP expression is significantly related to non-responders to immunotherapy.

  2. In Vitro and In Vivo Validation of SDCBP in CRC

    • SDCBP knockdown significantly inhibited CRC cell proliferation and migration.
    • In mouse xenograft models, SDCBP knockdown combined therapy (SDCBP knockdown and APD1) significantly inhibited tumor growth.

  3. Efficacy of Combination Therapy of ZNPT and APD1

    • ZNPT significantly reduced the expression of SDCBP in CRC cells, inhibiting cell proliferation and promoting apoptosis.
    • In liver metastasis models, the ZNPT and APD1 combination therapy group significantly reduced tumor metastasis.

  4. Immune Cell Changes in the TME

    • CyTOF analysis showed an increase in the proportion of M1 macrophages and a decrease in M2 macrophages after ZNPT treatment.

  5. Effect of SDCBP on Macrophage Polarization

    • Co-culture experiments showed that SDCBP knockdown promoted the repolarization of macrophages from M2 to M1.

c) Research Conclusion

SDCBP promotes CRC progression and immune resistance by regulating macrophage polarization in the TME. ZNPT, as an effective inhibitor of SDCBP, can significantly enhance the efficacy of APD1 therapy by reducing M2 macrophage infiltration and increasing the proportion of M1 macrophages.

d) Research Highlights

  • Significant Findings: High SDCBP expression in CRC is associated with poor response to anti-PD1 therapy.
  • Novel Approach: Enhancing immunotherapy efficacy by inhibiting SDCBP and repolarizing macrophages.
  • Broad Applications: ZNPT, as an SDCBP inhibitor, shows great potential in combination immunotherapy.

Significance and Value of the Research

This study reveals the key role of SDCBP in CRC immunotherapy, providing new insights for enhancing immunotherapy efficacy. ZNPT, by inhibiting SDCBP and reshaping macrophage polarization in the TME, exhibits significant therapeutic potential, offering a new treatment option for CRC patients.