Murine fecal microbiota transfer models selectively colonize human microbes and reveal transcriptional programs associated with response to neoadjuvant checkpoint inhibitors

Abstract

Human gut microbial species found to associate with clinical responses to immune checkpoint inhibitors (ICIs) are often tested in mice using fecal microbiota transfer (FMT), wherein tumor responses in recipient mice may recapitulate human responses to ICI treatment. However, many FMT studies have reported only limited methodological description, details of murine cohorts, and statistical methods. To investigate the reproducibility and robustness of gut microbial species that impact ICI responses, we performed human to germ-free mouse FMT using fecal samples from patients with non-small cell lung cancer who had a pathological response or nonresponse after neoadjuvant ICI treatment. R-FMT mice yielded greater anti-tumor responses in combination with anti-PD-L1 treatment compared to NR-FMT, although the magnitude varied depending on mouse cell line, sex, and individual experiment. Detailed investigation of post-FMT mouse microbiota using 16S rRNA amplicon sequencing, with models to classify and correct for biological variables, revealed a shared presence of the most highly abundant taxa between the human inocula and mice, though low abundance human taxa colonized mice more variably after FMT. Multiple Clostridium species also correlated with tumor outcome in individual anti-PD-L1-treated R-FMT mice. RNAseq analysis revealed differential expression of T and NK cell-related pathways in responding tumors, irrespective of FMT source, with enrichment of these cell types confirmed by immunohistochemistry. This study identifies several human gut microbial species that may play a role in clinical responses to ICIs and suggests attention to biological variables is needed to improve reproducibility and limit variability across experimental murine cohorts.

Keywords: Biological variables; Fecal microbiota transfer; Gut microbiome; Immune checkpoint inhibitors; Lung cancer; Neoadjuvant.

Fig. 1

Fecal microbiota transfer (FMT) of human R donor stool enhances response to anti-PD-L1 treatment compared to human NR stool in syngeneic tumor-bearing germ-free (GF) mice. (a) Experimental schema for B16F10 and MC38 syngeneic GF mouse models: GF mice were given oral gavage of 4% w/v human stool on D-14, subcutaneous syngeneic tumor injections on D0, and isotype or 5 mg/kg anti-PD-L1 injections beginning on D10 and (b) tumors volumes at the end of experiment, either D19 (B16F10) or D21-22 (MC38). Graphs represent an aggregate of 3–4 individual experiments per cell line with each point representing a single mouse. (c) Data in B separated by sex of the mice. Mean and SD are shown. Statistical comparisons were performed using Mann–Whitney U Test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 2

Relative abundance of shared genus and species-level OTUs between the human stools and colonized mice are correlated post-fecal microbiota transfer (FMT). (a) Original human stool and 4% w/v inoculation slurry for each experiment prepared independently from human responder (R-FMT) and nonresponder (NR-FMT) stool. CS053, CS062, CS080 represent independent experiments. Species above 1% relative abundance are shown. (b) Taxonomic comparison at the genus level for human and mouse by Venn diagram and using Spearman correlation (graphs in lower panels). Cutoffs for inclusion: relative abundance > 0.01% and ubiquity > 34%. (c) Taxonomic comparison at the species/operational taxonomic unit (OTU) level for human and mouse by Venn diagram and using Spearman correlation (graphs in lower panels). The inclusion criteria were the same as genus level. (d) Heatmap with relative species abundances shown for donor human stool, inocula for individual experiments, and individual mice. Statistical comparisons were performed using Mann–Whitney U Test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data shown in B, C, and D include all mice used in the MC38 model across 3 independent experiments. + indicates OTUs referenced in manuscript

Fig. 3

Multiple variables, including experiment, cage, and age, can affect anti-tumor responses in murine fecal microbiota transfer (FMT) in the MC38 syngeneic GF mouse model. (a) Individual MC38 tumor growth curves are shown for each experiment and condition (n = 3). ICI group is indicated by human responder (R) FMT or nonresponder (NR) FMT. Mean and SEM are shown. Statistical comparison performed across both time and group using two-way ANOVA: *** p < 0.001, **** p < 0.0001. (b) Species/OTU overlap from individual experiments. Number of reads: R = 8,143, NR = 15,000. (c) Principal coordinate analysis (PCoA) of species/OTU from each experiment. Dots represent cages within each experiment. (d) PCoA for each cage across experiments for R- and NR-FMT mice. (e) Final tumor volume for mice < 10 weeks old vs > 10 weeks old. N = 7–22 mice/condition. F. Final tumor volume data further subdivided by sex. N = 6–13 female mice/condition; 0–10 male mice/condition. Statistical comparisons were performed using Mann–Whitney U Test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

Species associated with MC38 tumor response in anti-PD-L1 treated R-FMT mice. C57BL/6 germ-free mice received human fecal microbiota transfers (FMT), followed by injection of MC38 tumor cells and treatment with the indicated isotype or anti-PD-L1 antibody. (a) R-FMT mice were categorized based on percent change in tumor volume: growth > 25% from day 10 were murine tumor progressors (MT-P, yellow, n = 15) and growth < 25% were murine tumor nonprogressors (MT-NP, blue, n = 13). (b) Bacterial species/OTUs enriched in MT-NP or MT-P. Statistical comparisons were performed using Mann–Whitney U Test: *p < 0.05, **p < 0.01. C. Correction for experimental variables (experiment, cage, and age) using a generalized linear model (GLM). OTUs above dotted line maintained significance after correction and are color coded based on direction of enrichment as shown in 4B. + indicates OTU label is truncated and full name is presented in Tables S3 and S4. Dotted line represents adjusted p < 0.05

Fig. 5

Species associated with MC38 tumor response in anti-PD-L1 treated NR-FMT mice. C57BL/6 germ-free mice received human fecal microbiota transfers (FMT), followed by injection of MC38 tumor cells and treatment with the indicated isotype or anti-PD-L1 antibody. (a) NR-FMT mice were categorized based on percent change in tumor volume: growth > 25% from day 10 were murine tumor progressors (MT-P, yellow, n = 15) and growth < 25% were murine tumor nonprogressors (MT-NP, blue, n = 13). (b) Bacterial species/OTUs enriched in MT-NP or MT-P. Statistical comparisons were performed using Mann–Whitney U Test: *p < 0.05, **p < 0.01. (c) Correction for experimental variables (experiment, cage, and age) using a generalized linear model (GLM). OTUs above dotted line maintained significance after correction and are color coded based on direction of enrichment as shown in 5B. + indicates OTU label is truncated and full name is presented in Tables S3 and S4. Dotted line represents adjusted p < 0.05

Fig. 6

Murine MC38 tumors show upregulation of multiple immune pathways and other checkpoint inhibitors in mice receiving human responder fecal microbiota transfer (R-FMT). RNA was extracted from murine tumors and sequenced using an Illumina platform with normalization across all tumors (n = 12). (a) Significant differentially expressed genes (DEG) were identified using DESeq2 from R-FMT tumors treated with anti-PD-L1 vs isotype antibody. List shown in part A is derived from R-FMT mice but is also shown for all nonresponder (NR)-FMT mice. (b) Significant DEGs from R-FMT mice treated with anti-PD-L1 vs isotype antibody were analyzed by Gene Ontology (GO). Bars indicate fold enrichment for top 10 categories. (c) Significant DEGs from murine MT-NP vs MT = -P, independent of human FMT donor (n = 6), were analyzed by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and top 10 pathways are shown with their enrichment score (ES). Tables S5–7 display complementary data. Abbreviations: MT-P, murine tumor progressor; MT-NP, murine tumor nonprogressor

Fig. 7

Murine tumors from anti-PD-L1 treated fecal microbiota transfer (human responder, R-FMT) mice showed increased CD8 + T cell and NK cell infiltration as well as increased smooth muscle actin. Representative images from each group are shown. Quantification for each stain is shown on the right of images. Mean and SEM are shown; n = 36 tumors across all groups. Statistical comparisons were performed using Mann–Whitney U Test: *p < 0.05, **p < 0.01. Antibodies: CD8α (CD8 + T cells), KLRB1 (NK cells), CD11c (dendritic cells), αSMA (smooth muscle actin). Abbreviations: MT-P, murine tumor progressor; MT-NP, murine tumor nonprogressor