C3 + cancer-associated fibroblasts promote tumor growth and therapeutic resistance in gastric cancer via activation of the NF-κB signaling pathway

Gastric cancer (GC) remains one of the most lethal malignancies globally, with limited therapeutic options. Cancer-associated fibroblasts (CAFs), a diverse population of stromal cells within the tumor microenvironment (TME), play a central role in tumor progression and therapeutic resistance. However, the specific markers identifying tumor-promoting CAF subsets in GC have yet to be fully characterized. Through animal studies and RNA sequencing, complement C3 (C3) emerged as a key marker linked to tumor-promoting CAF subsets. Single-cell sequencing and multiplex immunofluorescence staining confirmed that C3 expression is predominantly localized within CAFs. Independent cohort analyses demonstrated a strong association between elevated levels of C3 CAFs and poor clinical outcomes in GC patients. To further investigate, small interfering RNA (siRNA)-mediated knockdown of C3 in CAFs was employed in vitro, with subsequent experiments, including cell migration assays, cell viability assays, and immunofluorescence, revealing significant functional impacts. C3 secreted by CAFs promoted Epithelial-mesenchymal transition (EMT) and accelerated cancer cell migration. Patients with minimal C3 CAF infiltration exhibited a higher probability of deriving therapeutic benefit from adjuvant treatments. Furthermore, C3 CAFs were associated with immunosuppressive effects and an immune-evasive microenvironment marked by CD8 + T cell dysfunction. A lower prevalence of C3 CAFs correlated with improved responsiveness to immunotherapy in GC patients. Enrichment analysis highlighted pronounced activation of the NF-κB signaling pathway in C3 CAFs relative to their C3 counterparts, supported by elevated phosphorylation levels of IKK, IκBα, and p65 in C3 CAFs compared to both C3 CAFs and normal fibroblasts (NFs). Silencing p65 nuclear translocation in CAFs through siRNA significantly suppressed C3 secretion. The study suggests that NF-κB pathway-mediated CAF activation enhances C3 secretion, driving EMT, migration, chemoresistance, and immune evasion in GC progression. Targeting the NF-κB/C3 signaling axis in CAFs may offer a viable therapeutic strategy for GC management.