Brain metastases affect 10–30% of metastatic breast cancer patients. Limited treatment options and poor clinical outcomes highlight the unmet need for effective therapies. The lymphatic vasculature plays a critical role in cancer progression, serving as the primary route for metastasis and promoting an immunosuppressive microenvironment by inhibiting cytotoxic T cells and dendritic cell maturation. However, its role in brain metastases remains unclear. Here, we investigate whether modulating VEGFR-3, a key receptor in endothelial lymphatic cells, can normalize lymphatic vasculature, influence immune cell infiltration in the tumor microenvironment (TME), and reduce tumor growth and metastases.

VEGFR-3 expression was assessed in EO771 and 4T1 breast cancer (BC) and 2H-11 lymphatic endothelial cell lines. Cell tolerance to VEGFR-3 inhibitor (VEGFR-3i), SAR131675, was evaluated in vitro using increasing compound concentrations. VEGFR-3i was tested ex vivo in 2D and 3D cultures of BC TME-derived cells. BC and breast brain metastases (BBM) in vivo models were generated via orthotopic inoculation of murine EO771 and 4T1 cell lines. For BBM, the primary tumor was surgically removed, mimicking clinical conditions, followed by intracranial inoculation. VEGFR-3i was administered orally, intravenously, or intraperitoneally. Lymph nodes (draining and non-draining), primary tumors, and BBM were collected at various time points to assess VEGFR-3 expression and perform immune profiling via spectral flow cytometry.

In vitro studies showed minimal VEGFR-3 expression among cell lines, as expected, with a dose-dependent reduction in metabolic activity following SAR131675 treatment. Cytotoxic effects occurred only at higher concentrations, with triple-negative 4T1 being the most sensitive. In 2D primary cultures, VEGFR-3 expression was higher in BBM than primary BC, underscoring its critical role in metastases. In 3D-spheroid models, BBM displayed increased aggressiveness compared to BC, as evidenced by greater growth and sprouting. However, VEGFR-3 modulation effectively controlled BC spheroid growth and sprouting, with a moderate effect on BBM. In vivo, VEGFR3i reduced tumor volume and reprogrammed the TME.

These findings suggest that normalizing lymphatic vasculature through VEGFR3 modulation can reshape the tumor-immune-lymphatic landscape, fostering a more effective anti-tumor immune response and potentially enhancing immunotherapy efficacy.