Abstract
Targeted monoclonal antibodies inhibiting the epidermal growth factor receptor (EGFR), such as cetuximab and panitumumab, represent a primary therapeutic modality for patients with RAS wild-type colorectal cancer liver metastases (CRLM). However, therapeutic resistance almost universally emerges, mediated by complex interactions between tumor cell populations and the surrounding tumor microenvironment (TME). In this study, we integrated high-resolution 10x Visium spatial transcriptomics with single-nucleus RNA sequencing (snRNA-seq) on paired pre- and post-treatment CRLM tissue specimens to decode the spatial microenvironmental remodeling driving anti-EGFR resistance. Spatial transcriptomic profiling uncovered distinct microenvironmental niches associated with treatment recalcitrance. Specifically, post-treatment non-responsive lesions exhibited a marked spatial enrichment of SPP1+ tumor-associated macrophages (TAMs) at the invasive tumor border, establishing a localized immunosuppressive barrier that restricted CD8+ cytotoxic T cell infiltration. Furthermore, spatial ligand-receptor analysis revealed subregional crosstalk where cancer-associated fibroblast (CAF)-derived hepatocyte growth factor (HGF) and transforming growth factor-beta 1 (TGF-β1) induced focal activation of MET and HER2/HER3 signaling in adjacent tumor cells located within hypoxic stromal microdomains. Functional validation using patient-derived organoid co-culture models confirmed that co-stimulation with stromal HGF and TGF-β1 synergistically restored MAPK and PI3K/AKT phosphorylation, successfully rescuing tumor cells from cetuximab-mediated apoptosis. These findings illuminate spatial microenvironmental compartmentalization as a key driver of non-genetic anti-EGFR resistance and suggest that targeting spatial microenvironmental signaling crosstalk represents a promising strategy to overcome therapeutic failure in metastatic colorectal cancer.
