Fig. 4
AMPK activation combined with BCL-2/XL inhibition induces MYC-associated apoptosis in breast cancer patient-derived explant cultures. a Workflow for breast cancer patient tissue-derived explant cultures (PDEc). Tumor pieces were brought to the lab directly after surgery and cut into three pieces; one for DNA/protein extraction, one for immunohistochemical (IHC) analyses and one for 3D culture. b IHC analysis of MYC in breast tumors. Paraffin-embedded samples were immunostained for MYC and ≥5 fields of view (FOV) from 1 to 3 sections scored. MYC-high tumors: >50% positive cells/FOV, MYC-low tumors: <20%-positive cells/FOV. N = 10 tumors. c Representative images of MYC-low and MYC-high PDEc cultures. The PDEc samples were cultured for 7 days, stained and imaged by confocal microscopy (IF). d MYC status before and after 3D culture. MYC positivity scored as in b. e ABT-737+A-769662-induced apoptosis in MYC-high, MYC-low, and non-cancerous (tumor adjacent tissue and reduction mammoplasty) PDEc cultures. Samples were adjusted to culture for 6 days and treated for 24 h with vehicle (DMSO), 1 μM ABT-737, 10 μM A-769662 or ABT-737+A-769662 combination. The level of apoptosis was scored from confocal immunofluorescence images. The left panel shows representative images. The graphs at right plot the quantification of apoptosis. Apoptosis was scored as 1 = <10% apoptotic cells/explant, 2 = >10% apoptotic cells/explant in a cohesive structure, 3 = >10% apoptotic cells/explant in a deteriorated structure. Each dot indicates one fragment. Horizontal lines: Average, Student’s t-test (unpaired). f Summary of the apoptotic response in PDEc. Student’s t-test (unpaired). The different shades of green represent the different MYC-low tumor samples, and the different shades of red represent the different MYC-high tumor samples. g AMPK activation upregulates BIM in PDEc culture. pACC was used as an AMPK activity marker. The sample was treated with DMSO or 10 μM A-769662 for 24 h
