Extended Data Fig. 6: 3-IAA and chemotherapy induce cell death in neutrophils.
From: Microbiota-derived 3-IAA influences chemotherapy efficacy in pancreatic cancer
a, FACS-sorted T cells or neutrophils were cultured for 24 h +/− 1,000 μM 3-IAA and +/− FIRINOX (3.2 μM Oxaliplatin, 5.6 μM Irinotecan and 19.2 μM 5-FU). Viability was assessed via flow cytometry (n = 3, biological replicates). b, MPO activity was determined in 50,000 FACS-sorted bone-marrow-derived neutrophils, neutrophil precursors (lineage-negative, CD115−, Ly6B+, Ly6Gint-low) from the bone marrow or PDAC-infiltrating neutrophils using a fluorometric MPO activity assay kit (n = 4, biological replicates). c, FACS-sorted bone-marrow-derived neutrophils were cultured in the presence of increasing dosages of 3-IAA +/− MPO +/− FIRINOX (3.2 μM Oxaliplatin, 5.6 μM Irinotecan and 19.2 μM 5-FU; n = 3, biological replicates). Cell numbers were quantified using flow cytometry after 48 h of culture. d, FACS-sorted neutrophil precursors were cultured in the presence of increasing dosages of 3-IAA +/− FIRINOX (3.2 μM Oxaliplatin, 5.6 μM Irinotecan and 19.2 μM 5-FU; n = 3, biological replicates). Cell numbers were quantified using flow cytometry after 48 h of culture. e, As in a, except only neutrophils were cultured in the presence of +/− Oxaliplatin (8 μM Oxaliplatin) and 1,000 μM 3-IAA, 1,000 μM 3-IPA or DMSO at similar concentrations (n = 3 to 6, biological replicates). f, As in c, except cell death of neutrophils was defined by flow cytometry using Annexin V and PI staining. Frequencies are shown relative to total neutrophils (n = 3, biological replicates). g, As in c, except NET formation was defined by SYTOX DNA staining after three hours of treatment (n = 3, biological replicates). h, Degranulation of 1 × 106 FACS-sorted bone-marrow-derived neutrophils was measured after 30 min of stimulation with FIRINOX (3.2 μM Oxaliplatin, 5.6 μM Irinotecan and 19.2 μM 5-FU), 1 mM 3-IAA + FIRINOX or FIRINOX + 1 μM fMLP using an MPO activity assay kit (n = 3, biological replicates). i, KPC tumour cells were orthotopically injected into SPF mice and mice were treated +/− 500 mg/kg 3-IAA for 5 consecutive days and FIRINOX at day 11 (n = 5 each). Immune cells from the spleen and tumour were analysed by flow cytometry at day 3 after FIRINOX treatment. Neutrophil counts in the spleen or tumour are shown. j, As in i, except only 3-IAA, but no FIRINOX was applied (n = 5 each). Each symbol represents one mouse or one in vitro replicate. One experiment (b) or one out of three (c,d) or two (a,e–j) independent experiments are shown. Error bars indicate SEM, significant p-values are indicated and were determined by one-way ANOVA followed by Dunnett’s (a–h) post-hoc test or two-tailed t-test (i,j).
