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

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.