Oral 25-Hydroxycholesterol Suppresses 4T1 Tumour Growth in an Immune-Context-Associated Manner

Triple-negative breast cancer (TNBC) is an aggressive subtype with limited targeted-treatment options. 25-Hydroxycholesterol (25-HC) is an oxysterol involved in metabolic and immune regulation, but its antitumour effects remain context dependent. We examined whether oral 25-HC suppresses 4T1 tumour growth in immunocompetent BALB/c mice and whether host immune status modifies the response. Mice bearing 4T1 tumours received oral vehicle or 25-HC (1 or 10 mg/kg) daily for 14 days (n = 6 per group). 25-HC reduced endpoint tumour volume from 1510.6 ± 235.4 mm3 (vehicle) to 1147.2 ± 243.1 and 866.7 ± 206.6 mm3 (adjusted p = 0.027 and 0.0004); the 10 mg/kg dose also reduced tumour weight and Ki-67 positivity, increased CD8-positive T-cell infiltration and the granzyme B (GZMB)-positive fraction, reduced M2-like macrophages and raised the M1/M2 ratio. Tumour-tissue adenosine monophosphate-activated protein kinase (AMPK) signalling was altered, with p-AMPK/AMPK increased to 1.23- and 1.57-fold of control and p-STAT6/STAT6 decreased to 0.81- and 0.46-fold. Serum 25-HC increased with dose, while intratumoral 25-HC was lowest at 10 mg/kg; no dose-associated reduction was observed in BALB/c nude mice. Oral 25-HC thus suppressed 4T1 tumour growth with immune-context-associated features; however, the treatment-by-host interaction did not reach statistical significance (p = 0.06), and immune-context association of the response remains suggestive rather than established. The reciprocal serum–tumour pattern, a descriptive observation from a single time point whose cause remains unclear, generates a testable hypothesis of immune-cell-associated 25-HC handling.