Spheroid Culture and Acidic Region Labeling

Spheroids were prepared and processed as previously described^{68 (link)}. Briefly, HT-29 cells (1500 cells/well) were seeded in Ultra-Low Attachment 96-well plates (Corning) in DMEM supplemented with 10% heat-inactivated FBS, 10 mM D-glucose and 2 mM L-glutamine. For immunohistochemical studies, spheroids were incubated for 24 h with 2 µM Alexa Fluor 568-conjugated pH-low insertion peptide variant 3 (pHLIP V3; NH₂-ACDDQNPWRAYLDLLFPTDTLLLDLLW-COOH^{69 (link)}) to label acidic regions. Spheroids were then washed twice in PBS, fixed in 4% PFA, harvested and embedded in OCT. Frozen sections (5 µm) were stained with either BODIPY^TM 493/503 (#D3922; Thermo Fisher Scientific) or anti-Ki67 antibody (#556003, BD Biosciences). Sections were incubated with Alexa Fluor 568-conjugated anti-mouse secondary antibodies (#A11031; Thermo Fisher Scientific), and nuclei were counterstained with DAPI. Slides were prepared with fluorescence mounting medium (Dako), and staining was visualized with a Zeiss Imager 1.0 Apotome microscope. All spheroid samples from a same experiment were imaged by using the same gain and exposure settings.