C. albicans contributes to late-stage CRC progression.

(a,b) Representative histological images of tumor tissues from 4 patients stained by hematoxylin and eosin (H&E), antibodies against β-glucan. Arrowheads represented positive areas for β-glucan. Scale bars indicated 50 μm. (c) Abundance of C. albicans in different stages of colorectal adenocarcinoma samples in TCGA project. P-values were calculated using Mann-Whitney U tests. (d) Survival curve of colorectal adenocarcinoma patients in TCGA project with or without C. albicans infection. P-value was calculated using Log-rank test. (e, f) Wound-healing assay for migration capability of PBS-, B. fragilis-, C. albicans-, and B. fragilis with C. albicans-treated HCT116 cells, and comparison of gap sizes (mean ± s.e.m.; n = 3), One-way ANOVA followed by Tukey’s multiple comparison test was used for comparisons. (g) Protein-level expressions of epithelial-mesenchymal transition (EMT) marker genes in HCT116 cells. (h) Immunofluorescence images showing the expression of EMT marker genes in HCT116 cells. White arrowhead represented fungal hypha. Scale bars represented 10 μm.

C. albicans induces hypoxia response pathway of colorectal cancer cells.

(a) Differentially expressed genes in HCT116 cells after C. albicans infection for 12 hours versus PBS control, and (b) Gene Set Enrichment Analysis (GSEA) analysis showing up-regulation of “response to hypoxia” signaling pathway after C. albicans infection. (c) RNA-level expression of glycolysis genes in HCT116 cells cultured with C. albicans. (d) Immunofluorescence images showing HIF-1α expression after C. albicans infection of HCT116 cells. (e, f) Protein-level expression of HIF-1α in HCT116, SW48, and Caco2 cells after C. albicans infections. One-way ANOVA followed by Tukey’s multiple comparison test was used for comparisons. (g) RNA-level, (h) Protein-level expression, and (i) immunofluorescence of VEGFA gene in HCT116 cells after C. albicans infection. (j) RNA-level expression of IL-8 and CXCR4 genes HCT116 cells after C. albicans infection. (k) Concentration of IL-8 protein in cell culture supernatant. In (c, g, h, j, k), expressions were shown as mean ± s.e.m. (n=3), and t-tests were used to compare the expression levels between C. albicans infection and PBS control. *p < 0.05, **p< 0.01, ***p< 0.001. Scale bars indicated 10 μm.

C. albicans up-regulate c-Myc and c-Jun to promote HIF-1α pathway.

(a) RNA-level expression of AP-1 and c-Myc in HCT116 cells cultured with C. albicans for 12 hours. (b) Protein-level expressions of c-Jun in HCT116 and SW48 cells cultured with C. albicans for 12 and 24 hours. (c) Protein-level expressions of c-Myc in HCT116 and SW48 cells cultured with C. albicans for 12 and 24 hours. (d) Protein-level expressions of HIF-1α, c-Myc, and c-Jun in HCT116 cells after C. albicans infection and treated with c-Jun inhibitor SP600125 (with two dosage levels) for 12 hours. (e) Protein-level expressions of HIF-1α, c-Myc, and c-Jun in HCT116 cells after C. albicans infection and treated with c-Myc inhibitor 10058-F4 for 12 hours. (f) Protein-level expressions of HIF-1α, c-Myc, and c-Jun in HCT116 cells after C. albicans infection and treated cycloheximide. (g) The fitted curve of HIF-1α relative abundance after cycloheximide treatment. In (a, b), t-tests were used to compare the expression levels after C. albicans infection versus PBS control; in (d, e), one-way ANOVA followed by Tukey’s multiple comparison tests were used to compare various groups. n.s.: p>=0.05, *p < 0.05, **p< 0.01, ***p< 0.001.

EGFR-ERK is involved in C. albicans-mediated cancer cell response.

(a) RNA-level expressions of EGFR ligands in HCT116 cells cultured with C. albicans for 12 hours. (b-d) Protein-level expressions of p-EGFR, HIF-1α, c-Myc, and c-Jun in HCT116 cells treated with EGFR inhibitor AG-1478 (with two dosage levels) for 12 hours. (e) Protein-level expressions of HIF-1α in HCT116 cells after C. albicans infection and treated with cycloheximide. (f) The fitted curve of HIF-1α relative abundance after cycloheximide treatment. (g) Protein-level expressions of p-Erk and p-P38 in HCT116 cells after C. albicans infection. (h) Protein-level expressions of HIF-1α, c-Myc, and c-Jun in HCT116 cells treated with P38 inhibitor adezmapimod or Erk inbihitor SCH772984 for 12 hours. In (a,e), expressions between C.albicans infection and PBS control were compared using t-tests; in (c,d,f), one-way ANOVA followed by Tukey’s multiple comparison tests were used to compare various groups. **p< 0.01, ***p< 0.001.

C. albicans modulated the stability of HIF-1α by activating EGFR-ERK and TLR-NF-κB pathways.

(a, b) Protein-level expression of p-EGFR, HIF-1α, and c-Myc in HCT116 cells after C. albicans infections with AG-1478/MMG11/Laminarin treatments. (c) Expression of p-P65 in HCT116 cells (whole cell or nucleus), and (d) comparison of p-P65 protein level in HCT116 nuclei cultured with C. albicans for 12h. (e) Immunofluorescence images showing p-P65 localization, (f) relative signal intensities of the respective emission fluorescence along the lines drawn in (e), and (g) comparison of nuclear fluorescence ratios in HCT116 cells after C. albicans infection. Scale bars represent 10 μm. (h) Wound-healing assay measuring migration capability of HCT116 cells after C. albicans infection with or without AG-1478/MMG11 treatment, and (i) comparison of gap sizes of different groups. In (d, g) t-tests were used to compare the two groups; in (a, b, i), one-way ANOVA followed by Tukey’s multiple comparison tests were used. *p < 0.05.

Candidalysin is critical for eliciting colorectal cancer cells response.

(a, b) Expressions of CaECE1, CaALS3 and CaHWP1 cultured with HCT116 cells at 0 to 90 minutes post infection measured by (a) qRT-PCR and (b) RT-PCR. (c) Fluorescence staining of C. albicans wild-type and ECE1Δ/Δ hyphae adherent to HCT116 cells. Scale bars were 50 μm. (d) Fluorescence staining of C. albicans wild-type and ECE1Δ/Δ hyphae invading through HCT116 cells. White arrowheads indicate hyphal invading the cells. Scale bars were 10 μm. (e, f) Protein-level expressions of HIF-1α, c-Jun, and c-Myc in HCT116 cells co-cultured with wildtype or ECE1Δ/Δ strain C. albicans for 12 hours. (g, h) Protein-level expressions of HIF-1α, c-Jun, and c-Myc in HCT116 cells co-cultured with ECE1Δ/Δ strain C. albicans and treated with or without candidalysin. (i) Activation of MAPK pathway in HCT116 cells treated with candidalysin. (j) Wound-healing assay for migration capability of HCT116 cells treated with candidalysin (3 μM). In (f, h), ANOVA followed by Tukey’s multiple comparison test was used to compare different groups. In (j), t-tests were used to compare the two groups.

Candida albicans induces hypoxia response pathway of colon organoids and CRC samples.

(a, b) Protein-level expression and immunofluorescence of HIF-1α in normal and CRC organoids after C. albicans infection. (c) Quantification of HIF-1α protein levels in (a). (d) HIF-1α expression after C. albicans infection and treated with c-Jun inhibitor SP600125 or c-Myc inhibitor 10058-F4 in CRC organoids. (e) Immunofluorescence images of CRC organoids after C. albicans infection. White and black scale bars represented 50 and 10 μm, respectively. (f) Representative histological images of tumor tissues stained by hematoxylin and eosin (H&E), antibodies against β-glucan and HIF-1α. Scale bars indicated 50 μm. In (c), t-tests were used to compare normal or tumor organoids after C. albicans infections versus PBS controls.

The interactions between Candida albicans and tumor cells exhibit both species and cellular specificity.

(a) Fluorescence staining of C. albicans, C. tropicalis, and S. cerevisiae adhesion to HCT116 cells. Black arrowheads indicated yeast or hyphal adherent to HCC116 cells. Scale bars were 50 μm.(b) Protein-level expressions of HIF-1α, c-Myc, and c-Jun in HCT116 cells after infections of various types of microbiota. (c) Protein-level expressions of EGFR, p-EGFR, HIF-1α, and c-Myc in various cancer cells after C. albicans infections. (d) Quantifications of p-EGFR and (e) HIF-1α expressions in (c). In (d), One-way ANOVA followed by Tukey’s multiple comparison test were used to compare across cells; in (e), t-tests were used to compare expressions between C. albicans infection and PBS control of the same cell type. *p < 0.05, **p< 0.01, ***p< 0.001.

Schematic diagram of the C. albicans infection during colorectal cancer progression.

This diagram depicts the proposed signaling pathway activated by C. albicans through the secretion of its peptide toxin, candidalysin. Upon interaction with colorectal cancer (CRC) cells, C. albicans engages epidermal growth factor receptor (EGFR) and toll-like receptor 2 (TLR2), triggering downstream signaling cascades. These interactions result in the activation of the mitogen-activated protein kinase (MAPK) pathway and nuclear factor kappa B (NF-κB), both of which are key mediators of cellular stress and inflammatory responses. The coordinated activation of these pathways leads to stabilization and accumulation of hypoxia-inducible factor 1-alpha (HIF-1α) through c-Myc, thereby inducing a hypoxic transcriptional program even under normoxic conditions. This hypoxia-like response contributes to the pro-tumorigenic microenvironment and may promote colorectal cancer progression.