Fig. 1

Detailed workflow for AKT1^low quiescent cancer cell (QCC) identification using tyramide signal amplified immunofluorescence (TSA-IF) automated microscopy coupled with computational image analysis. a Consolidated standards of reporting trials (CONSORT) diagram for QCC analysis of tissues from a cohort of patients with triple negative breast cancer (TNBC). b Scheme demonstrating steps in TSA-IF staining of tumor sections. c Scheme demonstrating steps for automated image acquisition using the Vectra platform (Perkin Elmer) and inForm software. Individual images at × 20 undergo spectral isolation for each fluorescent marker (4',6-diamidino-2-phenylindole (DAPI), pan-AKT, H3K9me2, HES1) (i) to allow for generation of a tissue mask that segregates tumor and stroma (ii) followed by a cellular segmentation (iii) that allows for extraction of fluorescence intensities for each cellular segment e.g. nucleus, cytoplasm. d Scheme demonstrating establishment of fluorescence intensity thresholds for each marker (pan-AKT, H3K9me2, HES1) and identification of QCCs based on pre-established thresholds to allow for generation of digital tumor maps and determination of QCC percentage and QCC cluster index. NACT neoadjuvant chemotherapy, ER+ estrogen receptor positive, HRP horseradish peroxidase