Computational Pathology Reveals Extracellular-Matrix Imaging Biomarkers of Therapy Response in Preclinical Breast Cancer

Background/Objectives: Histological biomarkers of therapy response remain incompletely defined in preclinical breast cancer models. We evaluated whether quantitative immunofluorescence imaging and multimodal machine learning (ML) could identify image-derived tissue biomarkers associated with response in a 4T1 murine breast cancer therapy-response setting. Methods: We analysed 1696 immunofluorescence images across nine immunofluorescence staining panels: aPDL1, alpha-smooth muscle actin-CD31, alpha-smooth muscle actin-Ki67, CD3-CD31, CD8-Ki67, collagen–hyaluronic acid (HA), granzyme B-CD8, HMGB1, and pimonidazole/hypoxia. A Python image-analysis algorithm extracted 117 RGB-channel, intensity, morphometric, and cross-channel spatial features per image. The modelling framework included image-only deep learning (DL), tabular ML on extracted histological features, and image-plus-tabular gated fusion. Models were evaluated using stratified cross-validation, train-fold-only class balancing, bootstrap confidence intervals, permutation testing, nested feature-selection sensitivity analysis, and grouped leakage controls. Results: Histological staining panels classified treatment-response status across the nine-panel benchmark. Collagen–HA was the strongest staining (AUC 0.954), followed by alpha-smooth muscle actin-Ki67 (AUC 0.851) and hypoxia (AUC 0.837). DL achieved the best performance in eight of nine stainings. In 346 collagen–HA images, combined collagen and HA features achieved AUC 0.848, collagen-only features AUC 0.833, and hyaluronic-acid-only features AUC 0.805. In 104 animals with matched hypoxia and collagen–hyaluronic-acid images, extracellular-matrix features achieved AUC 0.985, hypoxia-only features achieved AUC 0.929, and adding hypoxia did not improve extracellular-matrix-only prediction. Conclusions: Quantitative extracellular-matrix imaging provides a strong preclinical signal for therapy-response stratification in 4T1 breast cancer. The findings are hypothesis-generating and require independent preclinical and human validation before clinical translation.