Related Experiment Video
Updated: May 24, 2025
Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles
Published on: May 14, 2021
Frizzled-7-targeting antibody-derived bifunctional protein retargets NK cells against triple-negative breast cancer
Chenyue Wang1,2, Lisha Wu1,2, Xingxing Li1,2
1Shanghai Key Laboratory of Molecular Imaging, Jiading District Central Hospital Affiliated Shanghai University of Medicine & Health Sciences, Shanghai, China.
Background:
Hypoxia is associated with the evasion of triple-negative breast cancer (TNBC) from immune surveillance. Hypoxia increases the subpopulation of putative TNBC stem-like cells (TNBCSCs) through activating Wnt/β-Catenin signaling. The shedding of MHC class I-related chain A (MICA) is particularly noteworthy in cancer stem cells (CSCs), promoting the resistance of CSCs to natural killer (NK) cell cytotoxicity. To reestablish MICA/NKG2D-mediated immunosurveillance, we proposed the design of a fusion protein (SHH002-hu1-MICA) which consists of Frizzled-7 (Fzd7)-targeting antibody and MICA, serving as an engager retargeting NK cells against TNBCs, especially TNBCSCs.
Methods:
Opal multicolor immunohistochemistry staining was used to validate the expression of membrane MICA (mMICA) and existence of NK cells in TNBC tumors; flow cytometry (FCM) assay was used to detect the expression of Fzd7/mMICA on TNBCs. Biolayer interferometry (BLI) and surface plasmon resonance (SPR) assays were executed to assess the affinity of SHH002-hu1-MICA towards rhFzd7/rhNKG2D; near-infrared imaging assay was used to evaluate the targeting capability. A cytotoxicity assay was conducted to assess the effects of SHH002-hu1-MICA on NK cell-mediated killing of TNBCs, and FCM assay to analyze the effects of SHH002-hu1-MICA on the degranulation of NK cells. Finally, TNBC cell-line-derived xenografts were established to evaluate the anti-tumor activities of SHH002-hu1-MICA in vivo.
Results:
The expression of mMICA is significantly downregulated in hypoxic TNBCs and TNBCSCs, leading to the evasion of immune surveillance exerted by NK cells. The expression of Fzd7 is significantly upregulated in TNBCSCs and exhibits a negative correlation with the expression of mMICA and infiltration level of NK cells. On accurate assembly, SHH002-hu1-MICA shows a strong affinity for rhFzd7/rhNKG2D, specifically targets TNBC tumor tissues, and disrupts Wnt/β-Catenin signaling. SHH002-hu1-MICA significantly enhances the cytotoxicity of NK cells against hypoxic TNBCs and TNBCSCs by inducing the degranulation of NK cells and promotes the infiltration of NK cells in CD44high regions within TNBC xenograft tumors, exhibiting superior anti-tumor activities than SHH002-hu1.
Conclusions:
SHH002-hu1-MICA maintains the targeting property of SHH002-hu1, successfully activates and retargets NK cells against TNBCs, especially TNBCSCs, exhibiting superior antitumor activities than SHH002-hu1. SHH002-hu1-MICA represents a promising new engager for NK cell-based immunotherapy for TNBC.
Insights
A novel fusion protein, SHH002-hu1-MICA, effectively targets and eliminates triple-negative breast cancer (TNBC) stem cells by engaging natural killer (NK) cells. This immunotherapy approach shows promise for treating TNBC by overcoming immune evasion mechanisms.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Hypoxia in triple-negative breast cancer (TNBC) promotes immune evasion and increases cancer stem-like cells (TNBCSCs).
- TNBCSCs shed MHC class I-related chain A (MICA), reducing natural killer (NK) cell cytotoxicity.
- Frizzled-7 (Fzd7) is upregulated in TNBCSCs, correlating with decreased MICA and NK cell infiltration.
Purpose of the Study:
- To develop a fusion protein (SHH002-hu1-MICA) to reestablish MICA/NKG2D-mediated immunosurveillance.
- To target Fzd7-expressing TNBCSCs and retarget NK cells for enhanced cancer cell killing.
Main Methods:
- Immunohistochemistry and flow cytometry to assess MICA, Fzd7, and NK cell expression in TNBC.
- Biolayer interferometry and surface plasmon resonance to evaluate fusion protein affinity.
- In vitro cytotoxicity and degranulation assays with NK cells and TNBCs.
- In vivo studies using TNBC xenografts to assess anti-tumor activity.
Main Results:
- SHH002-hu1-MICA demonstrated strong affinity for Fzd7 and NKG2D, targeting TNBC tissues.
- The fusion protein disrupted Wnt/β-Catenin signaling and enhanced NK cell-mediated cytotoxicity against TNBCs and TNBCSCs.
- SHH002-hu1-MICA promoted NK cell degranulation and infiltration into tumors, showing superior anti-tumor activity.
Conclusions:
- SHH002-hu1-MICA successfully activates and retargets NK cells against TNBCs, particularly TNBCSCs.
- The fusion protein exhibits enhanced anti-tumor activities compared to its precursor, SHH002-hu1.
- SHH002-hu1-MICA represents a promising candidate for NK cell-based immunotherapy in TNBC treatment.
