Extended Data Fig. 7: 3-IAA and MPO increase ROS and decrease cell viability.

a, Concentrations of 3-IAA measured using LC–MS/MS (left) or MOI (right) of tumours isolated from SPF mice treated with FIRINOX +/− 3-IAA five hours after treatment (n = 3 biological replicates). b, Concentrations of 3-IAA (left) or MOI (right) were measured in tumours isolated from WT or Mpo^–/– bone-marrow-reconstituted mice treated with FIRINOX + 3-IAA using LC–MS/MS five hours after treatment (n = 3 or 4 biological replicates). c, SPF mice received KPC control or Ahr KD cells orthotopically and were treated with FIRINOX (d11) or FIRINOX + 3-IAA (d9–13) (n = 4 or 5). Tumour weight was assessed at day 20 of the experiment. d, KPC tumour cells were cultured in the presence of increasing dosages of 3-IAA +/− Oxaliplatin (8 μM Oxaliplatin; n = 2-3 biological replicates). ROS expression was determined via flow cytometry using CellROX dye. Hy19636 cells were cultured with increasing dosages of 3-IAA +/− 5 × 10⁴ neutrophils (e) or MPO 200mU/ml (f) and +/− FIRINOX (1.6 μM Oxaliplatin, 2.8 μM Irinotecan and 9.6 μM 5-FU; n = 2-3 biological replicates). ROS expression was assessed via flow cytometry. Only p-values for relevant groups are shown for clarity. MIA PaCa-2 (g), T3M-4 (h) or Hy19636 (i) cells were cultured with increasing dosages of 3-IAA +/− FIRINOX (3.2 μM Oxaliplatin, 5.6 μM Irinotecan and 19.2 μM 5-FU) for 6 h (n = 3 biological replicates). Viability was assessed by MTS/MTT assay and displayed as relative to untreated cells after 48 h. KPC (j) or MIA PaCa-2 (k) cells were cultured in the presence of increasing dosages of 3-IAA +/− MPO 400 mU/ml +/− 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. Hy19636 (l) or MIA PaCa-2 (m) cells were cultured with increasing dosages of MOI (100, 200 or 400 μM) +/− FIRINOX (1.6 μM Oxaliplatin, 2.8 μM Irinotecan and 9.6 μM 5-FU; n = 5 to 12 biological replicates). Viability was assessed using the MTS assay and displayed as relative to untreated cells after 24 h. n, KPC tumour cells were orthotopically injected into SPF mice and mice were left untreated or treated with either FIRINOX (d11), 3-IAA (d9–13) or 3-IAA and FIRINOX (n = 5 each). Statistics and representative pictures show orthotopic tumours three days after indicated treatment with staining for nitrotyrosine (ROS). o, SPF mice were injected with orthotopic tumours and were treated with FIRINOX or FIRINOX + 3-IAA (n = 5 each). After five hours of treatment, ROS accumulation was analysed using flow cytometry. ROS levels of cancer cells (Epcam⁺), T cells (CD45⁺CD3⁺) or myeloid cells (CD45⁺CD11b⁺) are shown. p, Irradiated and WT (n = 5) or Mpo^–/– (n = 3) bone-marrow-reconstituted mice received KPC cells orthotopically and were treated with FIRINOX or FIRINOX + 3-IAA as in o. ROS levels were assessed by flow cytometry as in o. One (a,b,n) or one out of three (d,g–i) or two (c,e,f,j–m,o,p) independent experiments are shown. Error bars indicate SEM or median (n), significant p-values are indicated and were determined by two-tailed t-test (a–c,o,p), one-way ANOVA followed by Dunnett’s (d,g–n) or Tukey’s (e,f) post-hoc test.

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