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Background Oestrogen receptor-positive (ER+) breast cancer (BC) is traditionally considered immunologically ‘cold’ and has demonstrated limited benefit from immune checkpoint inhibitor (ICI) therapy, with recent advances seen only in select high-risk, early-stage ER +/HER2−patients treated with chemotherapy-ICI combinations.1 2 Oncolytic viruses, with their dual ability to lyse tumour cells and remodel the tumour microenvironment, offer a promising strategy to enhance immunogenicity. We aimed to evaluate the efficacy and immunological effects of an oncolytic Lister strain Vaccinia virus (VacV000; developed at QMUL and VacVBTX) in preclinical models of ER+ BC.Methods Human and murine BC cell lines were cultured in 2D or 3D for 1 or 4 days, respectively, before VacV000 or vehicle treatment. After 72 hours, viability and ATP release were assessed via CellTiter-Glo, and surface calreticulin by flow cytometry. For in vivo studies, 2×105 cells were injected into the fourth right mammary fat pad of 6–8-week-old mice. Once tumours reached 25–200 mm3, VacV000 or its vehicle were administered intratumourally every 3 days (3 doses of 108 PFU). Survival endpoint was reached when tumours measured 15 mm in any direction. RNA-seq, TCR-seq, spatial profiling and flow cytometry analyses were completed one day post-final dose.Results VacV000 exhibited broad infectivity and cytotoxicity across ER+ BC cell lines in vitro, with increased markers of immunogenic cell death, specifically ATP release and surface calreticulin. In vivo, intratumoural VacV000 significantly reduced tumour burden and extended survival in both HRM1 and TSAE1 models (HRM1: 26 vs 20 days, p=0.0042; TSAE1: 24 vs 22 days, p=0.0062) (figure 1). RNA-seq of treated tumours showed an increased inflammatory response with elevated interferon-gamma and interferon-alpha response compared to vehicle control. This was associated with higher expression of Cd274, which codes for PD-L1, overexpressed by CD45− cells as assessed by flow cytometry. We also found an enrichment in CD8+ T cells by deconvolution analysis, validated by flow cytometry and further supported by elevated numbers of unique TCR clonotypes in VacV000-treated tumours. Spatial profiling confirmed that VacV000 converted immune-excluded ER+ tumours into immune-inflamed phenotypes, marked by increased CD8+ T cells within the tumour core (figure 2).Conclusions Our work provides evidence supporting VacV000 as an immunomodulatory agent capable of reprogramming the immune landscape of ER+ BC. By converting ‘cold’ tumours into ‘hot’ ones, VacV000 holds promise for synergistic use with other immunotherapies.Acknowledgements We would like to thank all members of the Breast Cancer Now Histopathology Core Facility and the ICR Flow Cytometry Facility for the helpful services provided for this work. We are also grateful to Pier-Luigi Lollini and Jean Zhao for granting permission to use TSAE1 and HRM1 cell lines.References Loi S, Salgado R, Curigliano G, et al. Neoadjuvant nivolumab and chemotherapy in early estrogen receptor-positive breast cancer: a randomized phase 3 trial. Nat Med. 2025;31:433–441.Cardoso F, O’Shaughness J, Liu Z, et al. Pembrolizumab and chemotherapy in high-risk, early-stage, ER+/HER2− breast cancer: a randomized phase 3 trial. Nat Med. 2025;31:442–448.Ethics Approval The study was approved by The Institute of Cancer Research’s Animal Welfare and Ethical Review Body, and in accordance with Home Office Regulations under the Animals (Scientific Procedures) Act 1986, licence number PP7490522.Abstract 896 Figure 1In vivo efficacy of intratumoural VacV000 in preclinical ER+ breast cancer modelsAbstract 896 Figure 2VacV000 induces an increased infiltration of CD8+ T cells into the core of ER+ breast tumours