Targeting tumoral CXCL3 in triple-negative breast cancer: Association with tumor vasculature and immune surveillance.

32 Background: Triple Negative Breast Cancer (TNBC) accounts for 10-20% of breast cancers and is characterized by poor prognosis, early recurrence, high metastatic potential, and lack of effective targeted therapies. Hypoxia-driven immunosuppression and aberrant tumor vasculature are key hallmarks contributing to TNBC progression and resistance to therapy. The pro-angiogenic chemokine CXCL3, frequently overexpressed in aggressive breast tumors, plays a significant role in abnormal angiogenesis, tumor growth, metastasis, and immune evasion. However, the specific role of tumoral CXCL3 in regulating vascular normalization and immune surveillance remains inadequately understood. This study investigates whether targeting the CXCL3-CXCR2 axis can remodel the tumor microenvironment and enhance immunotherapeutic efficacy in TNBC. Methods: An orthotopic 4T1 murine TNBC model was used to evaluate the impact of CXCL3-CXCR2 axis blockade. Genetic knockdown of CXCL3 and pharmacological inhibition of CXCR2 using SB225002 (5 mg/kg/day, i.p.) were employed. Tumor growth kinetics were monitored. Vascular morphology (CD31, α-SMA), perfusion (lectin), and hypoxia (EF5) were assessed by immunofluorescence. Immune cell profiling was performed using flow cytometry. Proliferation (Ki-67), apoptosis (cleaved caspase-3), phospho-ERK, phospho-p38 signaling, and PD-L1 expression were evaluated. Combination therapy studies were conducted using SB225002 and anti-PD-L1 antibody (200 µg/kg/q3d/i.p.), followed by assessment of tumor progression, lung metastasis, Granzyme B expression, and survival (Kaplan-Meier analysis). Results: CXCR2 inhibition using SB225002 significantly reduced tumor growth and volume (***P < 0.001). Treatment decreased vessel diameter and density while enhancing pericyte coverage, indicating vascular normalization. Improved perfusion and reduced intratumoral hypoxia were observed (*P < 0.05). Importantly, SB225002 enhanced infiltration of anti-tumoral immune cells, including CD8⁺ T cells and M1 macrophages (***P < 0.001), while reducing regulatory T cells (Tregs) and N2 neutrophils. Reduced Ki-67 expression, downregulation of phospho-ERK and phospho-p38 signaling, increased apoptosis, and modulation of PD-L1 expression further supported its anti-tumor efficacy. Combination therapy with SB225002 and anti-PD-L1 demonstrated superior tumor control, reduced lung metastasis, increased Granzyme B expression, and significantly improved survival (*P < 0.05). Conclusions: Targeting the CXCL3-CXCR2 axis in TNBC promotes vascular normalization, reduces hypoxia, enhances anti-tumor immune surveillance, and improves the efficacy of immune checkpoint blockade, offering a promising combinatorial therapeutic strategy.