Figure 7. STm^ΔaroA treatment affects tumor organoid stem–forming capacity.

(A) Organoids were infected with STm^ΔaroA (or control) as in Figure 6 for 24 hours. They were then dissociated into a single cell suspension. An equal number was then reseeded into Matrigel and passaged weekly at an equal density for 3 weeks. MTT assay was performed at the indicated day. Representative images are shown below for the indicated days. Scale bars: 500 μm. Each point indicates an independent well. Two-way Students T-test performed. Representative of 2 experiments, data shown from Apc^min/+,SI tumor line. (B) Measurement of LDH in the cell culture supernatant after 24 hours of infection. Data shown as percentage of cell death compared with wells treated with cell lysis solution. Each data point indicates an independent well. Data are representative of 3 experiments. (C) Active caspase 3 assessed by a plate-based colorimetric assay on organoids infected as in B, with the addition of a pan-caspase inhibitor or staurosporine (STS) alone. Each point is an individual well. One-way ANOVA with Dunnett’s multiple-comparison test. Representative images to the right. Scale bar: 500 μm. Data are representative of 2 independent experiments and shown from Apc^min/+,SI tumor line. (D and E) Tumor organoids derived from Lgr5-GFP reporter mice induced with CAC were infected with mCherry-expressing STm^ΔaroA for 24 hours, as outlined. Organoids were dissociated into single cells, stained with a live/dead marker, and analyzed by flow cytometry. The percentage of cells that are infected (mCherry⁺) in the live or dead cell gate (D) and the percentage of cells from the mCherry⁺ gate that are EpCAM⁺Lgr5^– or EpCAM⁺Lgr5⁺ (E) are shown. Data are pooled from 2 independent experiments, and each point is an average from 2 wells. Data are shown as mean ± SD.