Glutamate Metabotropic Receptor 4 in Breast Cancer: A Potential and Specific Target for Chimeric Antigen Receptor Therapy

Yien Xu^1,2Ayidana Hayierhan^2,3Zexiao Chen^2,4Yumei Ma^2,5Wenjun Zhang^2,5Chaoliang Xu^2,5Ruyi Mei^2,5Xiaoling Zhou^2,5Yuhao Zhu^2,6Pingnan Sun^5,7*Jundong Wu^2,6*

• ¹Department of Radiation Oncology, Shantou Central Hospital, Shantou, 515000, Guangdong, China
• ²Shantou University Medical College, Shantou, China
• ³Stem Cell Research Center, Shantou University Medical College, Shantou, 515041, Guangdong Province,, China
• ⁴The Breast Center, Cancer Hospital of Shantou University Medical College, Shantou, Guangdong 515041,, China
• ⁵Stem Cell Research Center, Shantou University Medical College, Shantou, 515041, Guangdong Province, China
• ⁶The Breast Center, Cancer Hospital of Shantou University Medical College, Shantou, Guangdong 515041, China
• ⁷Department of Gynecology of the First Affiliated Hospital,Shantou University Medical College, Shantou, Guangdong, China

Background: Despite the revolutionary success of chimeric antigen receptor (CAR) therapy in hematologic malignancies, its application in solid tumors is hindered by the scarcity of tumor-specific membrane antigens rigorously validated in clinical specimens. Here, we identified glutamate metabotropic receptor 4 (GRM4) as a novel target with dual advantages: breast cancer (BC)-predominant membrane expression and restricted normal tissue distribution, potentially circumventing on-target off-tumor toxicity. Methods: Through integrative multi-database analysis (DESeq2/edgeR/limma differential screening, CellMarker filtration, the Human Protein Atlas database validation), GRM4 was prioritized. Its expression was validated in non-malignant organs (immunohistochemistry (IHC)), BC cell lines (western blot (WB)/quantitative polymerase chain reaction (qPCR)/immunofluorescence (IF)), 158 BC clinical samples with paired para-cancerous tissues (IHC). Subcellular localization, tumor proportion score, and subtype-specific distribution were analyzed. Clinical correlations and survival outcomes were evaluated using chi-square tests and Kaplan-Meier analysis. Results: Membrane expression was confirmed by IHC in 35.44% of clinical cases, and its presence in breast cancer cell lines was validated by WB, qPCR, and IF. GRM4 exhibited tumor-specific membrane/cytoplasmic expression in 80.38% of BC patients (127/158) across all subtypes (≥70% positivity), with 51.27% showing >50% tumor cell positivity. Critically, GRM4 was absent in normal breast/para-cancerous tissues and confined to the brain in non-malignant organs. While GRM4 correlated with advanced clinical stage (p=0.025) and age (p=0.026), it was independent of overall survival (p=0.449). Conclusions: GRM4 emerges as a novel CAR-associated target for breast cancer, demonstrating tumor-specific overexpression, brain-restricted normal expression, and pan-subtype applicability with potential on-target off-tumor effect.