C3+ cancer-associated fibroblast-derived GAS6 promotes cervical cancer metastasis via activation of AXL signaling pathway in a syngeneic orthotopic mouse model

Metastatic cervical cancer remains a leading cause of cancer-related death in women, although several mouse models of metastatic cervical cancer exist, many fail to preserve an immunocompetent primary tumor microenvironment, limiting their ability to adequately model metastatic progression. This study aimed to establish a syngeneic orthotopic cervical cancer model that faithfully reproduces the metastatic cascade and defines cellular interactions associated with metastasis in tumor microenvironment. U14 cells derived from mouse cervical cancer were engineered to stably express luciferase (U14-Luc) for the detection of micrometastases via bioluminescence imaging. The syngeneic orthotopic mouse model was established by intra-cervically injecting U14-Luc into immunocompetent C57BL/6 mice under direct visualization. Primary and metastatic tumor burden were detected by in vivo or ex vivo organ bioluminescence imaging. Histological analysis of tumor tissues was performed using Hematoxylin-Eosin staining. Single-cell RNA sequencing was used to identify cellular interactions and molecular pathways in primary tumors (PTs) and paired lymph node metastasis tumors (LNMTs). In this mouse model, primary tumors consistently developed near the external cervical os and vaginal fornix. By day 14 post-injection, cancer cells had invaded into the pelvic viscera, particularly the rectum, and disseminated to regional lymph nodes. By day 28, distant metastases, predominantly in the lungs, were observed. Single-cell analysis revealed that crosstalk between cancer cells and T cells in LNMT promoted immune escape via the SPP1 pathway. Additionally, a C3 + fibroblast subtype enriched in the PTs was correlated with unfavorable prognosis in cervical cancer patients. These C3 + fibroblasts enhanced tumor invasiveness through activation of the GAS6/AXL pathway. This syngeneic orthotopic mouse model effectively replicates the metastatic cascade of cervical cancer. Its ability to closely mimic the tumor microenvironment makes this model a valuable immunocompetent orthotopic platform for investigating the metastatic mechanism and evaluating novel therapeutic strategies for cervical cancer.