Extended Data Fig. 8: 3-IAA and FIRINOX treatment decreases autophagy in vivo.
From: Microbiota-derived 3-IAA influences chemotherapy efficacy in pancreatic cancer
a, Normalized expression of ROS-producing or degrading enzymes found in mRNA sequencing data of tumours obtained from mice treated with FIRINOX compared to tumours treated with FIRINOX + 3-IAA (n = 3 biological replicates each; p values: GPX3 (0.00012), GPX7 (1.2*10−8), NOX4 (0.023)). b, KPC tumour cells were cultured in the presence of FIRINOX (3.2 μM Oxaliplatin, 5.6 μM Irinotecan and 19.2 μM 5-FU) +/− 10 μM of 3-IAA and 400 mU/ml MPO (n = 4 biological replicates). RNA expression of GPX 3 and 7 was measured using qPCR after 24 h of treatment. Expression is depicted as relative to a housekeeping gene. c, Scramble control-transfected cells, Gpx3 or Gpx7 knockdown cells were treated with +/− FIRINOX (3.2 μM Oxaliplatin, 5.6 μM Irinotecan and 19.2 μM 5-FU) +/− 10 μM of 3-IAA and MPO and ROS was assessed using flow cytometry (n = 3 or 4 biological replicates). d, as in c, except cell numbers were calculated using flow cytometry (n = 4 biological replicates). e, SPF mice were injected with orthotopic tumours using scramble control or GPX3 KD cells and were treated with +/− FIRINOX at day eleven of the experiment (n = 5 or 6). Tumour size was measured at day eight after FIRINOX treatment. f, GSEA enrichment plot depicting positive enrichment of reactome pathway autophagy in total tumours obtained from mice treated with FIRINOX compared to tumours treated with FIRINOX and 3-IAA (n = 3 biological replicates). g, KPC tumour cells were orthotopically injected into SPF mice and mice were treated with either 3-IAA and FIRINOX or FIRINOX alone (n = 3 each). One day after FIRINOX treatment, intratumoral proteins were analysed and compared. Proteins downregulated in tumours from 3-IAA and FIRINOX-treated mice were analysed for enriched KEGG pathways. The top 10 pathways are depicted and the full list of proteins and up- or downregulated pathways is provided in SI tables 1 to 3. h, KPC tumour cells were orthotopically injected into SPF mice. Mice were treated +/− FIRINOX (d11), +/− 3-IAA (d9–13) and p62/SQSTM1 staining was analysed at day three after treatment. Statistics show the number of p62/SQSTM1high cells per field (n = 5 each). Representative pictures are shown. The scale bar represents 100 μm. i,j, KPC tumour cells were orthotopically injected into gnotobiotic mice colonized with R or NR microbiota (n = 3 or 4). Mice were treated +/− FIRINOX and LC3-l/ll (i) or p62/SQSTM1 (j) staining was analysed at day 20 of the experiment. Statistics show the number of LC3-l/llhigh or p62/SQSTM1high cells per field (n = 4). Tumours were pooled from three individual experiments with different donors each. Representative pictures are shown. The scale bar represents 100 μm. k, KPC tumour cells were orthotopically injected into SPF mice and treated and analysed as in h, except that staining for cleaved caspase-3 (CC3, apoptosis) was applied. The scale bar represents 50 μm. l, SPF mice were orthotopically injected with GFP-LC3B-RFP reporter cells and treated with FIRINOX + 3-IAA, trehalose or FIRINOX + 3-IAA + trehalose (n = 5 each). Tumours were analysed for GFP/RFP ratios via bright field imaging. The GFP/RFP ratio is shown for each respective group. m, SPF mice were injected with (mST-ATG4B) or mSt control cells. Mice received either doxycycline treatment via diet for seven days (d5 to d12) or standard food and were treated +/− FIRINOX (n = 4 or 5 each). Tumour size at day 18 of the experiment is depicted. Representative pictures show tumours at the end of the experiment. Scale bar shows one centimetre. One (a,f–k) or one out of two independent experiments (b–e,l) or three independent experiments with different lengths of doxycycline treatment (m) are shown. Each symbol represents one mouse or one in vitro replicate. Bars indicate SEM (a–e,m) or median (h–l), significant p-values are indicated and were determined as indicated in the methods section (a,f,g), by two-tailed Mann–Whitney test (b) one-way ANOVA followed by Dunnett’s (c, l) or Tukey’s (d, e, i, j, m) post-hoc test or by Kruskal–Wallis test followed by Dunn’s (h,k) post-hoc test.
