Journal: Nature Microbiology

Article Title: Gut microbiota-derived hexa-acylated lipopolysaccharides enhance cancer immunotherapy responses

doi: 10.1038/s41564-025-01930-y

Figure Lengend Snippet: Mice were treated with PMB, broad-spectrum antibiotics (ABX) or control drinking water (control (Ctrl) H 2 O) for 2 weeks. a , Abundance of bacterial taxa measured in mouse faecal metagenomes as the number of classified reads at genus level normalized to faecal weight (left) (Pre versus ABX, *** P = 0.0001; Ctrl H 2 O versus ABX, **** P < 0.0001; ABX versus PMB, *** P = 0.0002) and principal component analysis of the treatment groups at the phylum level showing the differential effects on the mouse gut microbiota (right). Ellipses were drawn with a 99% confidence interval. b , c , Relative abundance of Gammaproteobacteria (Pre versus ABX, **** P < 0.0001; Pre versus PMB, **** P < 0.0001; Ctrl H 2 O versus ABX, ** P = 0.0010; Ctrl H 2 O versus PMB, ** P = 0.0029) ( b ) and LPS-encoding non-Gammaproteobacteria (Pre versus ABX, **** P < 0.0001; Ctrl H 2 O versus ABX, **** P < 0.0001; ABX versus PMB, *** P = 0.0003) ( c ) in the faecal metagenomes of treated mice shown as the mean of classified reads normalized to faecal weight. Pre, n = 20; Ctrl H 2 O, n = 10; ABX, n = 12; and PMB, n = 19 mice per group pooled from 2 experimental repeats in a – c . d , Indicated mice were pre-treated for 2 weeks with ABX or PMB in their drinking water. MC38 colorectal adenocarcinoma cells were subcutaneously implanted, and tumour growth was measured at serial time points following tumour implantation. Indicated animals were treated with anti-PD-1 i.p. starting at day 10 after tumour implantation (anti-PD-1 versus Isotype ctrl, ** P = 0.0054; anti-PD-1 versus Abx mix + anti-PD-1, * P = 0.0478; anti-PD-1 versus PMB + anti-PD-1, * P = 0.0318). Data shown were pooled from 2 experimental repeats with n = 10 mice per group. e , MC38 colorectal adenocarcinoma cells were subcutaneously implanted into PMB pre-treated or untreated mice. Measurements were taken at indicated time points post implantation. Anti-PD-1 was administered i.p. starting at day 10 after tumour implantation, and the TLR4 inhibitor TAK-242 was administered i.p. during anti-PD-1 therapy (anti-PD-1 versus Isotype ctrl, **** P < 0.0001; anti-PD-1 versus PMB + anti-PD-1, * P = 0.0162; anti-PD-1 versus anti-PD-1 + TAK242, **** P < 0.0001). Data shown were pooled from 2 experimental repeats with n = 10 mice per group. f , Representative tumour pictures. g , Representative flow cytometry measurements of indicated T-cell phenotypes from tumours in f (exact P values provided in Source Data Fig. ). Kruskal–Wallis test comparing all to all with data presented as box from the 25th to 75th percentiles, bars at median and whiskers from minimum to maximum in a – c . Two-way ANOVA comparing all to anti-PD-1 in d and e , and Brown–Forsythe with post hoc Dunnett’s test comparing all to anti-PD-1 in g , with bars and error representing mean and s.e.m.

Article Snippet: THP-1 cells (THP1-Dual Reporter Cells, TLR2 KO Dual Reporter THP-1 Cells, TLR4 KO Dual Reporter THP-1 Cells and THP1-Dual MD2-CD14-TLR4 over-expressing THP-1 cells) were purchased from InvivoGen.

Techniques: Control, Flow Cytometry

Journal: Nature Microbiology

Article Title: Gut microbiota-derived hexa-acylated lipopolysaccharides enhance cancer immunotherapy responses

doi: 10.1038/s41564-025-01930-y

Figure Lengend Snippet: Mice were treated with polymyxin B (PMB), broad-spectrum antibiotics (ABX) or control drinking water for two weeks. a , Abundance of lpxJ -encoding bacterial taxa (Pre vs ABX, **** P < 0.0001; Pre vs PMB, ** P = 0.0087; Ctrl H2O vs ABX, **** P < 0.0001; Ctrl H2O vs PMB, *** P = 0.0009) measured in mouse fecal metagenomes, shown as average number of classified reads at species level normalized to fecal weight, before and after treatment. n = 20 (Pre), 10 (Ctrl H 2 O), 12 (ABX) and 19 (PMB) mice per group pooled from 2 experimental repeats. b and c , Schematics for in vivo ABX, PMB and TAK-242 (TLR4 inhibitor) treatment regimens in the context of anti-PD-1 immunotherapy. Kruskal-Wallis test ( a ). **P < 0.01, ***P < 0.001, ****P < 0.0001 .

Article Snippet: THP-1 cells (THP1-Dual Reporter Cells, TLR2 KO Dual Reporter THP-1 Cells, TLR4 KO Dual Reporter THP-1 Cells and THP1-Dual MD2-CD14-TLR4 over-expressing THP-1 cells) were purchased from InvivoGen.

Techniques: Control, In Vivo

Journal: Nature Microbiology

Article Title: Gut microbiota-derived hexa-acylated lipopolysaccharides enhance cancer immunotherapy responses

doi: 10.1038/s41564-025-01930-y

Figure Lengend Snippet: MC38 colorectal adenocarcinoma cells were subcutaneously implanted into PMB pre-treated or untreated mice. Measurements were taken at indicated time points post implantation. Anti-PD-1 was administered i.p. starting at day 10 after tumor implantation, and the TLR4 inhibitor TAK-242 was administered i.p. during anti-PD-1 therapy. Flow cytometry measurements of indicated immune cells isolated from tumors of treated mice are shown. Data shown were pooled from 2 experimental repeats with n = 10 mice per group (exact p-values provided in Source Data Extended Data Figures). No differences were statistically significant by Kruskal-Wallis test.

Article Snippet: THP-1 cells (THP1-Dual Reporter Cells, TLR2 KO Dual Reporter THP-1 Cells, TLR4 KO Dual Reporter THP-1 Cells and THP1-Dual MD2-CD14-TLR4 over-expressing THP-1 cells) were purchased from InvivoGen.

Techniques: Tumor Implantation, Flow Cytometry, Isolation

Journal: Nature Microbiology

Article Title: Gut microbiota-derived hexa-acylated lipopolysaccharides enhance cancer immunotherapy responses

doi: 10.1038/s41564-025-01930-y

Figure Lengend Snippet: a – d , Wild-type, TLR2 −/− or TLR4 −/− NF-kB reporter THP-1 (human monocyte/macrophage) cells were incubated with the indicated doses of hexa- or penta-acylated LPS for 24 h. Data are from two biological replicates per concentration of LPS and are representative of at least three experimental replicates. a , NF-kB activation in response to 1 ng ml −1 hexa-acylated LPS or 1 µg ml −1 of penta-acylated LPS (Hexa LPS WT versus TLR4 −/− , * P = 0.0274). b , NF-kB activation dose–response with the indicated doses of hexa- or penta-acylated LPS. c , IL-1β (left) and IL-6 (right) secretion by THP-1 cells after 24 h treatment with the indicated doses of LPS, shown as fold increase over media controls. d , NF-kB activation with the indicated combined doses of hexa- and penta-acylated LPS (1 ng Hexa versus +10, +100 and +1,000 ng Penta, **** P < 0.0001). e , MC38 colorectal adenocarcinoma cells were subcutaneously implanted into wild-type mice, and measurements were taken at indicated time points post implantation. Indicated animals were treated with anti-PD-1 i.p. and/or oral hexa- or penta-acylated LPS or control drinking water starting at day 10 after tumour implantation (anti-PD-1 versus Isotype ctrl, *** P = 0.0002; anti-PD-1 versus anti-PD-1 + Hexa, * P = 0.0318). Data shown were pooled from 2 experimental repeats with n = 10 mice per group. f , Flow cytometry measurements of IFNγ (anti-PD-1 versus Isotype ctrl, * P = 0.0325; anti-PD-1 versus anti-PD-1 + Hexa, * P = 0.0423) and TNF (anti-PD-1 versus anti-PD-1 + Hexa, * P = 0.0352) producing CD8 + T cells infiltrating tumours of mice in e with n = 5 mice per group. Brown–Forsythe and Welch ANOVA comparing all to WT in a . One-way ANOVA with a Dunnett’s multiple comparisons test in d . Two-way ANOVA comparing all to anti-PD-1 in e and Brown–Forsythe with post hoc Dunnett’s test comparing all to anti-PD-1 in f , with bars and error representing mean and s.e.m.

Article Snippet: THP-1 cells (THP1-Dual Reporter Cells, TLR2 KO Dual Reporter THP-1 Cells, TLR4 KO Dual Reporter THP-1 Cells and THP1-Dual MD2-CD14-TLR4 over-expressing THP-1 cells) were purchased from InvivoGen.

Techniques: Incubation, Concentration Assay, Activation Assay, Control, Flow Cytometry

Journal: Nature Microbiology

Article Title: Gut microbiota-derived hexa-acylated lipopolysaccharides enhance cancer immunotherapy responses

doi: 10.1038/s41564-025-01930-y

Figure Lengend Snippet: a , NF-kB reporter human THP-1 cells that do not (WT) or do over-express CD14 and TLR4 (CD14/TLR4-OE) were incubated with the indicated doses of hexa- or penta-acylated LPS for 24 h. Representative data from n = 3 biological replicates are shown from one of two experimental repeats. No significant differences between WT and CD14/TLR4-OE were calculated by two-tailed Mann-Whitney tests comparing equivalent doses and types of LPS. b , WT NF-kB reporter THP-1 cells were incubated with the indicated doses of hexa-acylated LPS and polymyxin B (PMB) for 24 h. NF-kB activation is expressed as a percentage of maximum. The means of n = 2 biological replicates are plotted in representative plots from one of three experimental repeats. Mouse RAW 264.7 macrophages were incubated with the indicated doses of hexa- or penta-acylated LPS without ( c ) or with ( d ) indicated concentrations of polymyxin B (PMB) for 24 h. Cytokine secretion was measured by cytometric bead arrays from culture supernatants. c , IL-6 (top) and TNF-α (bottom) secretion after 24 h of the indicated doses of hexa- or penta-acylated LPS. d , IL-6 (top) and TNF- α (bottom) secretion after 24 h of the indicated doses of hexa-acylated LPS and PMB. The means of n = 2 biological replicates are plotted in representative plots from one of three experimental repeats. For all plots, data are presented as mean ± SD.

Article Snippet: THP-1 cells (THP1-Dual Reporter Cells, TLR2 KO Dual Reporter THP-1 Cells, TLR4 KO Dual Reporter THP-1 Cells and THP1-Dual MD2-CD14-TLR4 over-expressing THP-1 cells) were purchased from InvivoGen.

Techniques: Incubation, Two Tailed Test, MANN-WHITNEY, Activation Assay