Immunomodulatory Peptide LW‐9 Enhances Doxorubicin Efficacy by Downregulating PD‐L1 and Promoting Intratumoral T‐Cell Responses in Murine Breast Cancer

Spider venom‐derived peptides have emerged as promising therapeutic agents due to their high specificity and ability to modulate cellular and immunological pathways. LW‐9, a peptide isolated from Phoneutria nigriventer venom, was identified as a potential immunomodulatory molecule with antitumor activity. This study evaluated the therapeutic effects of LW‐9 in a murine breast cancer model using EO771 cells. Treatment with LW‐9 significantly delayed tumour progression, with enhanced efficacy observed when combined with the chemotherapeutic agent doxorubicin. The therapeutic dose of LW‐9 (1 μg/kg) showed a favourable safety profile, with no significant alterations in hepatic or renal biochemical markers, including AST, ALT, creatinine, and urea. In addition, LW‐9 improved animal well‐being, as indicated by increased food intake and maintenance of body weight compared with doxorubicin‐treated controls. Immunological analyses demonstrated that LW‐9, particularly in combination with doxorubicin, promoted substantial remodelling of the tumour microenvironment. Flow cytometry revealed reduced PD‐L1 expression and increased frequencies of CD3 + and CD3 + /GITR + T cells, indicating enhanced antitumor immune activation. Immunohistochemical analysis confirmed increased lymphocytic infiltration within tumour tissue. Tumours maintained a stable ER‐negative, PR‐positive, HER2‐negative, and EGFR‐positive phenotype across all groups, suggesting that LW‐9‐mediated antitumor activity occurs independently of classical molecular subtype alterations. Collectively, these findings demonstrate that LW‐9 enhances chemotherapy efficacy by modulating the tumour immune microenvironment, supporting its potential as a novel immunoadjuvant strategy for breast cancer therapy.