Fusobacterium nucleatum infection modulates the transcriptome and epigenome of HCT116 colorectal cancer cells in an oxygen-dependent manner

Abstract

Fusobacterium nucleatum, a gram-negative oral bacterium, has been consistently validated as a strong contributor to the progression of several types of cancer, including colorectal (CRC) and pancreatic cancer. While previous in vitro studies have shown that intracellular F. nucleatum enhances malignant phenotypes such as cell migration, the dependence of this regulation on features of the tumor microenvironment (TME) such as oxygen levels are wholly uncharacterized. Here we examine the influence of hypoxia in facilitating F. nucleatum invasion and its effects on host responses focusing on changes in the global epigenome and transcriptome. Using a multiomic approach, we analyze epigenomic alterations of H3K27ac and global transcriptomic alterations sustained within a hypoxia and normoxia conditioned CRC cell line HCT116 at 24 h following initial infection with F. nucleatum. Our findings reveal that intracellular F. nucleatum activates signaling pathways and biological processes in host cells similar to those induced upon hypoxia conditioning in the absence of infection. Furthermore, we show that a hypoxic TME favors F. nucleatum invasion and persistence and therefore infection under hypoxia may amplify malignant transformation by exacerbating the effects induced by hypoxia alone. These results motivate future studies to investigate host-microbe interactions in tumor tissue relevant conditions that more accurately define parameters for targeted cancer therapies.

Fig. 1. F. nucleatum infects hypoxia conditioned cells at an increased rate and elicits increased cytokine secretion in comparison to infection of normoxia conditioned cells.

a Flow cytometry analysis of infection of normoxic and hypoxic conditioned cells infected with Fnn for 1 and 4 h, and a plot of the measured oxygen concentration during hypoxia conditioning. b Plot of median fluorescence obtained from flow cytometry using fluorescently stained Fnn (FM-1-43) indicates significantly increased infection of hypoxic cells in normoxia and hypoxia compared infection of normoxic cells in normoxia and hypoxic conditions. c Fnn cell counts in hypoxia at 1 and 4 h are greater than cell counts in normoxia indicating an increased proportion of proliferating bacteria in hypoxia. d Cytokine secretions normalized to cell counts (pM/million cells) of IL-8 and CXCL1 are significantly increased upon Fnn infection of hypoxic cells compared to normoxic cells at 4 h after infection. e Immunofluorescence confocal microscopy of Fnn infected HCT116 cells 24 h after initial infection period reveals persistence of intracellular Fnn. (Staining: Host membrane red: Membrite 568, Host nucleus blue: NucBlue, Fnn green: FM 1-43FX). Scale bar 20μm. Statistical analysis was performed using one-way ANOVA followed by Šidák’s multiple comparisons test (ns: not significant, *p < 0.05, ****p < 0.0001). Symbols (ς, ψ, ћ, †) denote the matched pairwise comparisons made using Šidák’s multiple comparisons test. Source data are provided in Supplementary Data 1.

Fig. 2. Hypoxia conditioning impacts dynamics of F. nucleatum and E. coli infection of HCT116 cells.

a Overview of the experimental protocol for hypoxia conditioning, including pretreatment, infection, continued incubation, sample extraction, and processing. Icons adapted from BioRender.com and schematic designed in Affinity Designer. b ChIP-seq tracks and RNA-seq tracks for selected markers of hypoxia: LDHA and SLC2A1 loci for normoxia and hypoxia conditioned samples.

Fig. 3. Analysis of differential enhancers reveals F. nucleatum infection specific gene regulation based on oxygen environment.

a Number of genes related to differential enhancers, FDR = 0.05 used as cutoff. b–d Selected relevant GO terms enriched from NN-HN, NN-NF, and NN-HF specific differential enhancer-related genes. e–g Volcano plots depicting genes associated with differential enhancers of NN-HN, NN-NF, and NN-HF. The differential enhancers were annotated to their nearest genes. The green points refer to genes linked to enhancers with increased signal compared to NN and purple points refer to those linked to enhancers with decreased signal. Abbreviations: NN Normoxia—No Bacteria, NF Normoxia—infection with Fnn, NE Normoxia—infection with E. coli, HN Hypoxia—No Bacteria, HF Hypoxia—infection with Fnn, and HE Hypoxia—infection with E. coli. Source data are provided in Supplementary Data 2.

Fig. 4. Transcriptomic analysis reveals differential gene expression in hypoxic and normoxic cells upon F. nucleatum infection.

a Number of differentially expressed genes, FDR = 0.05 used as cutoff. b Gene set variation analysis (GSVA) of Fnn and E.coli infection of HCT116 cells in normoxia and hypoxia. c Volcano plots showing genes associated with differential expressed genes of NN-HN, NN-NF, NN-HF, NN-NE, and NN-HE. d Volcano plots showing DEGs specifically associated with the hypoxia pathway in GSVA for NN-HN, NN-NF, and NN-HF. The green points refer to genes with upregulation compared to NN and purple points refer to those with downregulation. Abbreviations: NN Normoxia - No Bacteria, NF Normoxia—infection with Fnn, NE Normoxia—infection with E. coli, HN Hypoxia—No Bacteria, HF Hypoxia—infection with Fnn, and HE Hypoxia—infection with E. coli. Source data are provided in Supplementary Data 3.

Fig. 5. Transcription factor motif analysis in hypoxic and normoxic F. nucleatum infected cells reveals oxygen dependent variation in binding.

Heatmaps show motifs that are significantly enriched (p < 1 × 10⁻⁵) in one condition but not enriched (p > 1 × 10⁻⁴) in the other. a NN and HN. b NN and NF. c NN and HF. Abbreviations: NN: Normoxia—No Bacteria, NF Normoxia—infection with Fnn, HN Hypoxia—No Bacteria, and HF Hypoxia—infection with Fnn. Source data are provided in Supplementary Data 4.

Fig. 6. Summary schematic depicting F. nucleatum intracellular infection and subsequent host HCT116 colorectal cancer (CRC) cell response in normoxia is similar to hypoxia conditioning and amplifies malignant transformation.

Schematic and cell representation created and designed in Affinity Designer.