Related Experiment Video
Updated: Sep 18, 2025
Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Published on: February 8, 2018
Single-cell ligand-receptor profiling reveals an immunotherapy-responsive subtype and prognostic signature in
Chuanzhi Chen1, Jiahui Qi2, Weichao Lin1
1Department of Thyroid Surgery, National Key Clinical Specialty (General Surgery), The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China.
Background:
Triple-negative breast cancer (TNBC) is an aggressive form of cancer that lacks specific targeted therapies. Although ligand-receptor (LR) interactions play a crucial role in intercellular communication and contribute to tumor heterogeneity, their molecular details and potential as prognostic or predictive markers in TNBC have not been thoroughly investigated.
Methods:
We analyzed single-cell RNA sequencing data to categorize TNBC into 12 subgroups and 10 distinct cell types. From this dataset, we identified LR pairs that exhibited significant intercellular crosstalk and evaluated their prognostic relevance in a METABRIC TNBC cohort (n = 298). Through consensus clustering of these LR pairs, two molecular subtypes were defined. Key LR genes were then selected using Lasso regression and stepwise multivariate analysis to build an LR-based prognostic scoring system (LR.score), which was validated using both the METABRIC and GSE58812 datasets (n = 107). Additionally, we performed siRNA-mediated knockdown of the CXCL9/CXCR3 axis in MDA-MB-231 cells, confirming the knockdown via RT-qPCR and Western blot. The functional impact was assessed through proliferation, colony formation, and wound healing assays.
Results:
One subtype (Clust1) demonstrated strong immune cell infiltration, higher immune scores, and enrichment in pathways such as epithelial-mesenchymal transition, angiogenesis, and KRAS signaling-indicative of a basal-like, immune-active phenotype. Among the LR pairs, the CXCL9-CXCR3 axis was identified as a key factor in immune cell recruitment and anti-tumor responses. Functionally, silencing the CXCL9/CXCR3 axis significantly diminished the proliferation, colony formation, and migratory capabilities of MDA-MB-231 cells. Moreover, a higher LR.score was correlated with poorer overall survival (HR = 1.69, 95% CI = 1.12-2.56, P < 0.05) and reduced response to immune checkpoint inhibitors (ICIs), while patients with lower LR.score showed increased sensitivity to ICIs, particularly in anti-PD-L1 cohorts.
Conclusion:
The LR.score serves as an independent prognostic factor and a reliable predictor of immunotherapy response in TNBC. Targeting crucial LR interactions, especially the CXCL9-CXCR3 axis, may enhance immunotherapeutic efficacy and refine prognostic evaluations, paving the way for improved treatment strategies in TNBC.
Insights
This study identifies ligand-receptor interactions in triple-negative breast cancer (TNBC), revealing a prognostic score (LR.score) and the CXCL9-CXCR3 axis as key factors for predicting immunotherapy response and survival outcomes.
Area of Science:
- Oncology
- Immunology
- Genomics
Background:
- Triple-negative breast cancer (TNBC) lacks targeted therapies, and its intercellular communication via ligand-receptor (LR) interactions is poorly understood.
- Investigating LR interactions is crucial for understanding TNBC heterogeneity and identifying prognostic/predictive markers.
Purpose of the Study:
- To categorize TNBC subtypes based on single-cell RNA sequencing data.
- To identify and evaluate the prognostic significance of ligand-receptor pairs in TNBC.
- To develop and validate a prognostic scoring system (LR.score) for TNBC patients.
Main Methods:
- Single-cell RNA sequencing analysis to define TNBC subgroups and cell types.
- Identification and prognostic evaluation of ligand-receptor pairs in TNBC cohorts (METABRIC, GSE58812).
- Development of an LR-based prognostic scoring system (LR.score) using Lasso regression and multivariate analysis.
- Functional validation of the CXCL9/CXCR3 axis via siRNA knockdown in MDA-MB-231 cells.
Main Results:
- Two molecular subtypes of TNBC were identified based on LR pair clustering.
- The CXCL9-CXCR3 axis was identified as critical for immune cell recruitment and anti-tumor responses.
- Silencing the CXCL9/CXCR3 axis reduced cancer cell proliferation and migration.
- A higher LR.score correlated with poorer overall survival and reduced response to immune checkpoint inhibitors (ICIs).
Conclusions:
- The LR.score is an independent prognostic factor and predicts immunotherapy response in TNBC.
- Targeting key LR interactions, like the CXCL9-CXCR3 axis, can potentially improve TNBC treatment strategies and prognostic accuracy.
