Systematic validation of BACH1 antibodies enables the identification of BACH1 localisation as predictor of prognosis in early-stage lung cancer

The transcription factor BACH1 is a key regulator of cellular stress responses, metabolism, and tumour progression, and has emerged as a promising therapeutic target in several diseases, particularly metastatic cancer, inflammatory disease and cardiovascular conditions. As interest in BACH1 biology and its clinical relevance grows, the field would benefit from robust, well-characterised reagents enabling reliable detection of BACH1 across diverse experimental platforms, including analysis of human tissue. Establishing such tools is essential for advancing mechanistic studies and recognising the potential of BACH1 as a prognostic biomarker. We performed the first systematic validation of commercially available antibodies against human BACH1 across multiple experimental platforms, including western blotting, immunohistochemistry, and multiplex immunofluorescence. To ensure robustness, we validated antibodies using complementary approaches, including BACH1-knockout and knockdown cell lines, pharmacological modulators of BACH1 stability and localisation, and multiple extraction conditions. Their performance in immunohistochemistry was further tested using fixed cell pellets from wild-type and BACH1-deficient cells. Validated antibodies were subsequently applied to normal human tissues and lung cancer tissue microarrays to investigate BACH1 expression and subcellular localisation, and quantitative multiplex single-cell immunofluorescence analysis was performed in a cohort of early-stage lung adenocarcinoma patients with clinical follow-up. We demonstrate that subcellular localisation of BACH1 in early-stage lung adenocarcinoma correlates with patient prognosis, underscoring its clinical and translational significance. Our study delivers a rigorously validated toolkit for the BACH1 research community, removes a major barrier to progress in the field, and highlights the potential of BACH1 localisation as a biomarker in early-stage lung adenocarcinoma.