Fig. 2

Localization and survival of S. typhimurium YB1 in MCTS. HT-29 MCTS were incubated with S. typhimurium YB1 (YB1, green). After 48 h, MCTS cryosections were stained for S. typhimurium (a, c Alexa Fluor^® 488-conjugated secondary antibody, green; b Alexa Fluor^® 555-conjugated secondary antibody, red) and hypoxia (a, c Hypoxyprobe, red) or apoptosis (b TUNEL, green). Nuclei were stained with Hoechst (blue). Images were acquired with a ×10 (a, b scale bars 100 µm; all three fluorescence channels are shown plus the merged image in the lower right panel) or ×100 (in c scale bar 10 µm) objective. The image in c is acquired at a location inside the hypoxic area of an MCTS. The inset in the upper right corner of c is a digital zoom on the image part marked with a white box. d Viable bacteria of S. typhimurium YB1 in HT-29 MCTS at different time points after inoculation. As controls (medium), bacteria were incubated under identical conditions in the absence of MCTS. For each strain, time-point and condition, six tumours were infected and combined for preparation of tumour lysates. Values are colony-forming units (CFU) per mL lysate and are mean ± SEM of three tumour lysates infected with independent bacterial cultures (in some cases, error bars are smaller that graph symbols). Statistical analysis was performed using Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001)