Extended Data Fig. 6: Differences between responsive and non-responsive patients hold in comparison with faecal material from healthy donors and in anaerobic conditions.
A-B) Representative fluorescence microscopy images – at lower (A) and higher (B) magnification - and mean fluorescence intensity (MFI) quantification of ZO-1 (green, nCTR= 6, nNR= 2, nR= 2, nHD= 3, p = 0.000034 for CTR vs NR, p = 0.000073 for HD vs NR, p = 0.0002 for NR vs R) and E-cadherin (ECad, white, nCTR= 7, nNR= 2 nR= 2, nHD= 3, p = 0.0304 for CTR vs NR, p = 0.0327 for HD vs NR, p = 0.0034 for NR vs R) on untreated Gut-on-Chip (CTR) or after 24h treatment with faeces (10 mg/ml) from healthy donors (HD) or patients with melanoma R or NR to immunotherapy. Scale bar 100 μm and 20 μm. Bars represent mean ± SD, ordinary one-way ANOVA. C-D) Representative fluorescence microscopy images – at lower (C) and higher (D) magnification - and mean fluorescence intensity (MFI) quantification of ZO-1 (green, nCTR= 3, nNR= 3, nR= 3, p = 0.0059 for CTR ANA vs NR ANA, p = 0.0233 for NR ANA vs R ANA), β-catenin (Bcat, magenta, nCTR= 3, nNR= 2, nR= 3) and caspase-3 (Casp, yellow, nCTR= 3, nNR= 3, nR= 3) on untreated Gut-on-Chip (CTR) or after 6h treatment with faeces (10 mg/ml) from patients with melanoma R or NR to immunotherapy in anaerobic conditions. Scale bar 100 μm and 20 μm. Bars represent mean ± SD, ordinary one-way ANOVA (D upper panel). E) Image of faecal material from responder or non-responder patients cultured on agar plates. F) Colony forming unit (CFU) count in both anaerobic (ANA) and aerobic (AERO) conditions. The bacterial load from R and NR samples was comparable, however, CFUs presented different characteristics highlighting differences in the microbiota composition of the samples. G) Total LPS synthesis. The aggregated relative abundances (in a log10 base) of UniRef90 genes involved in lipopolysaccharide (LPS) synthesis within the faecal samples of patients (nNR= 5, nR= 4, p = 0.016, two-tailed Wilcoxon rank-sum test).
