Figure 6. In vitro treatment of tumor-derived organoids with STm^ΔaroA.

Tumor organoids derived from CAC-induced tumors and Apc^min/+ tumors were established and infected with GFP-expressing STm^ΔaroA for 2 hours. Infection was washed off, and then, organoids were cultured with gentamycin for a further 24 hours. (A) Merged bright-field and fluorescence microscope image of organoids within matrigel after 24 hours of infection shows association of STm^ΔaroA with tumor organoids. Scale bar: 50 μm. (B) CFU of STm^ΔaroA per well after 24 hours of infection. (C and D) qPCR analysis of the indicated transcripts in CAC-derived (C) and Apc^min/+ tumor (D) organoids. Representative of > 3 independent experiments with 4 independently derived organoid lines, with between 3 and 5 technical replicates per experiment. One replicate is 1 well of a 24-well plate culture with a 50 μL Matrigel dome. (E and F) Tumor organoid metabolites were assessed by GC-MS, OPLS-DA analysis, and pathway analysis of metabolites with a VIP score > 1. (G) CAC-derived tumor organoids were cultured with live or heat-killed (dead) STm^ΔaroA or with supernatant (SN) of STm^ΔaroA grown in organoid culture medium and the indicated mRNAs analyzed by qPCR. (H) Analysis of succinate levels in tumor organoids treated as described in G. Individual 2-tailed Student’s t tests (C and D) or Kruskal-Wallis tests (G and H) were performed. Data are shown as mean ± SD.