Editor’s Note: A dynamic understanding of the tumor microenvironment (TME) is essential for uncovering the mechanisms behind treatment response and disease progression. At the 2025 ASCO Annual Meeting, Professor Jieqiong Liu from Sun Yat-sen Memorial Hospital, Sun Yat-sen University, presented an innovative translational study (Abstract #1022) exploring the efficacy and safety of a novel combination therapy in patients with HER2-positive metastatic breast cancer who had failed second-line or later treatments. The team employed single-cell sequencing to analyze TME changes before and after treatment with KN046, a bispecific anti-CTLA-4/PD-L1 antibody, and KN026, a bispecific anti-HER2 antibody. The results revealed distinct immunological differences between responders and non-responders and identified the baseline macrophage-to-lymphatic endothelial cell (Mϕ/LEC) ratio as a predictive biomarker of treatment response. This study offers new insights into individualized care strategies for breast cancer.Study Overview
Title: Longitudinal tissue analysis and correlation of microenvironmental changes with combined immunotherapy and targeted therapy response in metastatic breast cancer.
Background: This study is part of a multicenter phase II clinical trial (NCT04521179) evaluating the efficacy and safety of a novel bispecific antibody targeting CTLA-4 and PD-L1 (KN046) in combination with KN026, a bispecific antibody targeting HER2, in patients with advanced HER2-positive breast cancer who had progressed after second-line or later therapies. The combination therapy demonstrated an objective response rate (ORR) of 47.2%, with grade 3/4 toxicities observed in 27.8% of patients and no grade 5 adverse events. Understanding changes in the tumor microenvironment before and after treatment is critical for identifying patient subgroups that derive benefit and for uncovering mechanisms of resistance. Thus, this study employed single-cell sequencing to analyze pre- and post-treatment tumor samples.
Methods: Paired tumor tissue samples were collected from patients enrolled at Sun Yat-sen Memorial Hospital and Fujian Medical University Union Hospital. Single-cell RNA sequencing (scRNA-seq) and single-cell T-cell receptor sequencing (scTCR-seq) were performed on the matched samples.
Results: A total of 30 tumor samples were collected from 17 patients. Thirteen patients had both pre- and post-treatment samples available, while the remaining four had either baseline or post-treatment biopsies. After rigorous quality control, 334,183 cells were analyzed through scRNA-seq.
TME analysis revealed that in the treatment-sensitive group, there was clonal expansion of CD8⁺ T cells, particularly in progenitor exhausted T cells (TEXP). Immune checkpoint molecules such as PD-1 were upregulated. The abundance of CD16⁺ natural killer (NK) cells and conventional dendritic cells (cDC1 and cDC2) increased, and the IFN-γ signaling pathway was activated. Macrophages (Mϕ) shifted toward a pro-inflammatory (M1-like) phenotype with enhanced antigen presentation capabilities.
In contrast, the treatment-resistant group showed significantly higher baseline Mϕ infiltration (P < 0.05). After treatment, key immune populations including effector T cells, NK cells, and cDC1/cDC2 were depleted, with activation of apoptotic pathways. Regulatory T cells (TREG) were activated, and tumor epithelial cells exhibited upregulation of immune evasion pathways such as JAK/STAT and PI3K/AKT, indicating a heightened immunosuppressive state.
Importantly, the baseline Mϕ/LEC ratio emerged as a powerful predictor of treatment response, with an area under the curve (AUC) of 0.89.
Conclusion: This study revealed that patients who responded to combined targeted and immunotherapy experienced robust immune activation, while non-responders exhibited a suppressed immune environment. The baseline Mϕ/LEC ratio was identified as an effective predictor of clinical response. These findings underscore the value of longitudinal single-cell multi-omics in capturing dynamic TME changes and highlight its clinical potential in predicting treatment outcomes and guiding personalized therapy for HER2-positive metastatic breast cancer.
Expert Commentary
Professor Jieqiong Liu:
The combination of anti-HER2 targeted therapy and chemotherapy has significantly improved survival for patients with metastatic HER2-positive breast cancer. However, treatment options remain limited for patients who experience disease progression after multiple lines of therapy, especially those involving small molecule tyrosine kinase inhibitors (TKIs) or antibody-drug conjugates (ADCs). To address this unmet need, we initiated a multicenter, single-arm phase II clinical trial to evaluate the efficacy and safety of KN046 (a bispecific antibody targeting CTLA-4 and PD-L1) in combination with KN026 (a bispecific HER2-targeting antibody) in patients with advanced HER2-positive breast cancer who had progressed following prior HER2-targeted therapy or ADCs.
The study results, published in Clinical Cancer Research in April this year, demonstrated an objective response rate (ORR) of 47.2%, with a low incidence of grade 3/4 toxicities. This chemotherapy-free, dual-targeted immunotherapy approach offers a promising new treatment option for patients with advanced HER2-positive disease.
Nonetheless, resistance remains a significant challenge, with at least half of the patients in the study showing limited or no response to the treatment. To identify patients who are more likely to benefit and to better understand the underlying mechanisms of treatment efficacy and resistance, we performed single-cell RNA sequencing and T cell receptor sequencing on paired tumor samples collected before and after therapy from a subset of patients.
Our analysis revealed dynamic and time-dependent shifts in multiple immune cell populations and phenotypes within the tumor microenvironment (TME), which correlated with treatment sensitivity. These findings provide novel insights into the immunologic remodeling induced by dual-targeted immunotherapy, as well as the molecular underpinnings of resistance. Notably, we found that the baseline macrophage-to-lymphatic endothelial cell (Mϕ/LEC) ratio effectively distinguished between responders and non-responders, offering a valuable biomarker to support personalized treatment decisions.
Finally, I would like to express my sincere gratitude to Professor Jianyou Liao from Sun Yat-sen Memorial Hospital and Professor Chuangui Song from Fujian Cancer Hospital for their invaluable support and contributions to this study.
Professor Jieqiong Liu
Department of Breast Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University
