Introduction

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide¹. The majority of CRC deaths are attributed to disease progression and metastasis², therefore, understanding the intricate mechanisms underlying CRC progression is imperative for improving patient outcomes. Numerous factors have been proposed to be involved in CRC metastasis, including the gut microbiota, a diverse community of microorganisms (including bacteria, viruses, and fungi) closely linked to human health and cancer^3–8. In particular, dysbiosis of the fungal microbiota is implicated in various cancer types^9–14. For instance, the absence of receptor proteins responsible for recognizing fungi has been linked to the promotion of colorectal tumorigenesis^15,16. Among the known fungi, Candida albicans (C. albicans), an opportunistic pathogenic fungus commonly found in the human intestinal tract, can proliferate under conditions of immunosuppression or microbiome dysbiosis and leads to candidiasis¹⁷. Once adhering to host cells via adhesins, C. albicans transitions from its yeast form to a hyphal form, enabling it to penetrate host tissues and invade epithelial barriers^18–20. Moreover, C. albicans secretes virulence factors, such as candidalysin, which can directly damage host cells and activate host immune responses²¹. In CRC, C. albicans is more frequently detected in tumors compared to non-cancerous tissues, indicating a potential role in tumorigenesis and progression²². Recent studies have uncovered important interplay between C. albicans and the tumor microenvironment^23,24,25. In particular, Zhu and colleagues showed that in mouse models, C. albicans colonization induces higher tumor load and more severe colitis²⁵. However, involvement of C. albicans in CRC progression and the molecular mechanism are still largely elusive.

Previous studies have shown that Candida albicans can activate MAPK and NF-κB signaling and has been implicated in modulating hypoxia responses in gut^26,27, however, its roles in the interaction with CRC cells remain to be fully elucidated. In this study, we employed cancer cells and CRC-derived organoids to investigate the cellular responses elicited by C. albicans and to determine whether its virulence factor, candidalysin, contributes to the stabilization of HIF-1α and the ensuing pro-tumorigenic events in CRC.

Results

C. albicans is associated with CRC progression

We recruited four colorectal cancer patients (two in stage II and the other two in stage III) and collected the post-surgery tumor samples. We found that fungi were present in both stage III patients, while not in stage II patients (Figure 1a). Moreover, presence of fungi surrounding tumor cells was also observed (Figure 1b). To validate this result, we investigated its presence and abundance in colorectal adenocarcinoma samples from The Cancer Genome Atlas (TCGA) project²⁸. As shown in Figure 1c-d, the abundance of C. albicans is significantly increased in late-stage CRC patients and correlates with poorer prognosis. To elucidates the influence of C. albicans on CRC, we co-cultured the CRC cell line HCT116 with C. albicans. The results showed that C. albicans significantly enhanced cell migration (Figure 1e-f). This effect persisted even in the presence of colon bacterium Bacteroides fragilis, which is known to suppress the growth of C. albicans²⁶. We then examined epithelial-mesenchymal transition (EMT), a crucial process in cancer progression and metastasis. Analysis of EMT marker gene expression confirmed that C. albicans infection reduced E-cadherin levels while increasing Vimentin and Slug expression (Figure 1g, Suppl. Figure S1a). Immunofluorescence staining revealed a reduction in E-cadherin at infection sites, with co-localization of E-cadherin signals and the C. albicans cell wall, suggesting a direct interaction between C. albicans and the HCT116 cell membrane (Figure 1h, Suppl. Figure S1b). Collectively, these findings suggest that C. albicans contributed to late-stage CRC progression by promoting cell migration and EMT.

Figure 1.

C. albicans stabilizes HIF-1α and activates hypoxia response in CRC cells

To delineate the molecular mechanisms by which C. albicans facilitate cell migration, we conducted whole transcriptome sequencing (RNA-seq) on HCT116 cells co-cultured with C. albicans. As a result, we obtained significant up-regulation of 173 genes, which were potentially induced by C. albicans infection (Figure 2a, Suppl. Table S1). Of note, various pro-tumor cytokines, including TNF, CXCL8, CCL22, and IL6 family gene, as well as their corresponding receptors, were found to be up-regulated (Figure 2a,k, Suppl. Tables S2,S3), suggesting that C. albicans may actively contribute to the proinflammatory tumor microenvironment, thereby facilitating cancer progression²⁹. Functional annotations of the up-regulated genes revealed enrichment in multiple metabolic pathways associated with tumor development, including hypoxia response, angiogenesis, epidermal growth factor signaling, and glucose metabolism (Suppl. Figure S2a and Table S4). Gene Set Enrichment Analysis (GSEA) further highlighted the up-regulation of the HIF-1α signaling pathway (Figure 2b, Suppl. Table S5). The up-regulation of key genes involved in HIF-1α signaling pathway, including HK2, LDHA, PDK1, and PFKFB1, were confirmed by qRT-PCR (Figure 2a,c, Suppl. Figure S2b). Moreover, treatment of HCT116 cells with L-mimosine, a compound that stabilizes HIF-1α, also significantly upregulated the expression of VEGFA, HK2, LDHA, PDK1, and PFKFB4 genes (Figure 2d, Suppl. Figure S2c, S3b). To reinforce the findings, we cultured HCT116 cells under hypoxia condition and performed RNA-seq, revealing that 83.8% (145 genes) of the genes up-regulated by C. albicans were also upregulated under hypoxia (Suppl. Figure S2d).

Figure 2.

Although the transcription level was unaffected, we observed the accumulation of HIF-1α in the nucleus and cytoplasm as early as 3 hours post-infection with immunofluorescence staining (Figure 2d, Suppl. Figure S3a,b). Western blots analysis further demonstrated that after C. albicans infection, HIF-1α increased in HCT116 and another two colorectal cell lines, SW48 and Caco2, while the timing of upregulation varies among these three cell lines (Figure 2e,f). PHD-mediated ubiquitination is the primary mechanism regulating HIF-1α protein stabilization, we observed that although the total level of HIF-1α increased, the ratio of its ubiquitinated form to total HIF-1α decreased. This indicates that C. albicans activates hypoxia responses by stabilizing HIF-1α protein in cancer cells (Suppl. Figure S3c,d). Angiogenesis, a critical process in cancer metastasis regulated by vascular endothelial growth factor (VEGF)³⁰, was also influenced by HIF-1α. qRT-PCR revealed an upregulation of VEGFA gene in the presence of C. albicans (Figure 2g). Western blot and immunofluorescence analyses further confirmed enhanced production of VEGFA protein in tumor cells co-cultured with C. albicans (Figure 2h,i). In addition to VEGFA, the expressions of chemokine C-X-C motif ligand 8 (CXCL8) and C-X-C motif chemokine receptor 4 (CXCR4) were both upregulated in tumor cells following exposure to C. albicans (Figure 2j,k). These findings suggested that C. albicans infection may modulate the tumor microenvironment by promoting angiogenesis. Collectively, these findings suggested that C. albicans infection induces the hypoxia response pathway in colorectal cancer cells, thereby facilitated angiogenesis and metabolism adaptation for cancer progression.

C. albicans activated c-Myc to promote HIF-1α pathway

Besides the genes shown in Figure 2c, the transcriptome data also uncovered a significant up-regulation of AP-1 (Activator protein 1) family and c-Myc genes in response to C. albicans infection, which we validated using qRT-PCR (Figure 3a). Considering the important roles of AP-1 in cell differentiation, migration, and proliferation³¹, the expressions of c-Jun and c-Myc were further substantiated by Western blots. For c-Jun, expression levels increased at 12 hours post-infection in HCT116 cells and at 24 hours in SW48 cells following C. albicans infection (Figure 3b). Similarly, C. albicans infection significantly elevated the expression of c-Myc in both HCT116 and SW48 cancer cell lines at 12 hours post-infection (Figure 3c). Notably, a high multiplicity of infection (MOI) accelerated the cellular response, observable as early as 6 hours post-infection (Suppl. Figure S4a).

Figure 3.

To determine the regulatory hierarchy in the up-regulations of c-Jun, c-Myc, and HIF-1α, we constructed HIF-1α knock down cell line and found that C. albicans still robustly upregulated c-Jun and c-Myc levels (Suppl. Figure S4b). L-mimosine upregulated c-Jun but not c-Myc, suggesting a bidirectional regulatory relationship between c-Jun and HIF-1α (Suppl. Figure S4c). Treatment of HCT116 cells with the c-Jun inhibitor SP600125 or knock down of c-Jun expression inhibited the cellular accumulation of HIF-1α after C. albicans infection (Figure 3d, Suppl. Figure S4d,e); similarly, treatment with the c-Myc inhibitor 10058-F4 or KJ-Pyr9 inhibited the cellular accumulation of HIF-1α after C. albicans infection (Figure 3e, Suppl. Figure S4f). The results suggested that c-Jun and c-Myc act upstream in the regulatory cascade leading to HIF-1α up-regulation. Moreover, only inhibition of c-Myc with 10058-F4 or KJ-Pyr9 suppressed HIF-1α downstream gene expression following C. albicans infection (Suppl. Figure S4g). While c-Myc was reported to post-transcriptionally induce HIF-1α protein and target gene expression³², our time-course experiments with cycloheximide treatment further revealed that C. albicans infection may significantly stabilizes HIF-1α protein through c-Myc (Figure 3f-g, 4e-f). These finding indicate that c-Myc is the main regulator to mediate the cell hypoxia response to C. albicans.

EGFR and TLR are involved in C. albicans-mediated cell response

Next, we aimed to identify the cell membrane receptor responsible for C. albicans-mediated response. Specifically, genes related to the regulation of EGF-activated receptor activity were enriched following C. albicans infection (Suppl. Figure S2a). Indeed, both RNA-seq and qRT-PCR confirmed that C. albicans induced the expression of amphiregulin (AREG), a member of the epidermal growth factor (EGF) family and a known ligand of EGFR (Figure 4a). To explore the role of EGFR in the response to C. albicans, we treated HCT116 cells with the EGFR kinase inhibitor AG-1478. As a result, AG-1478 significantly reduced the activation of c-Myc and half-lives of HIF-1α after C. albicans infection, indicating inhibition of the response triggered by C. albicans (Figure 4b-f); Given the complex regulatory network between HIF-1α, c-Jun, c-Myc and the mitogen-activated protein kinase (MAPK) pathway^32–38, we investigated the phosphorylation statuses of Erk and P38 (gene symbol: MAPK14), two important compartments of the EGFR-MAPK pathway. Western blots analysis revealed that C. albicans infection significantly increased the phosphorylation of Erk (Figure 4g), and treatment with the ERK inhibitor SCH772984 effectively suppressed the phosphorylation of Erk, consequently inhibiting the cellular responses triggered by C. albicans. On the other hand, phosphorylation of P38 remained unaffected after C. albicans infection, and treatment with the P38 inhibitor adezmapimod did not produce any significant inhibitory effect (Figure 4h), indicating a selective role of the ERK pathway in the cellular response to C. albicans infection.

Figure 4.

Notably, despite treatment with AG1478, the levels of HIF-1α and c-MYC in C. albicans-infected cells remained significantly elevated compared to the uninfected control group (Figure 4b). We hypothesized that this residual effect might be due to the continued activation of cell surface Toll-like receptors (TLRs) or C-type lectin receptors (CLRs). Despite the low expression of TLR and CLR in HCT116 cells (Suppl. Figure S5a, S6a), we successfully generated stable knockdown cell lines for key receptors and pathways associated with fungal recognition, including TLR2, TLR6, TLR9, MYD88, Dectin-1, and SYK (Suppl. Figure S5b). Western blot results showed that neither the suppression of genes nor treatment with the TLR2 inhibitor MMG11 or the Dectin1 inhibitor laminarin impaired the cell responses following C. albicans infection (Figure 5a, Suppl. Figure S5b-e, S6b-d). However, when cells were treated with a combination of receptor inhibitors targeting both EGFR and TLR2, the activations of HIF-1α and c-MYC induced by C. albicans were completely suppressed (Figure 5b). Furthermore, we investigated the status of NF-κB, a key pathway responsive to TLR activation, after C. albicans infection^39–41. Both immunofluorescence staining and western blot analyses revealed an increase in the phosphorylation level of p65 (gene symbol: RELA), a principal subunit of the NF-κB complex, following C. albicans infection. This led to nuclear translocation of p65, which modulated the expression of several downstream genes, including c-Jun and HIF-1α (Figure 5c-g). Additionally, combined EGFR and TLR2 inhibitors completely reversed the enhanced cell migration induced by C. albicans (Figure 5h,i). Collectively, the results suggested that C. albicans modulated the intracellular stability of HIF-1α by activating EGFR-ERK and TLR-NF-κB pathways, which further up-regulated a cohort of cellular transcription factors, including AP-1 components and c-Myc.

Figure 5.

Candidalysin is essential for eliciting colorectal cancer cell responses

To explore the virulence factor responsible for inducing cell responses, we co-cultured C. albicans with HCT116 cells and harvested C. albicans at various time points post-infection. qRT-PCR assays revealed that Als3, Hwp1, and Ece1 genes were significantly up-regulated in post-infection C. albicans (Figure 6a). Als3 and Hwp1 are known to be involved in the adhesion of C. albicans to epithelial cells, while Ece1 encodes the virulence factor candidalysin. Interestingly, the expression level of Ece1 showed a >1,000-fold increase (Figure 6a-b). In fact, during the early stages of mucosal surface infection by C. albicans, candidalysin is released into the invasion pocket created by the penetrating hyphae²¹, where it activates danger-response and damage-protection pathways in host cells, including the activation of the epidermal growth factor receptor (EGFR) in epithelial cells and the NLRP3 inflammasome in macrophages^42,43. Hence, these results suggest that candidalysin might play a critical role interacting with HCT116 cells.

Figure 6.

To investigate the function of candidalysin during C. albicans-colon cell interaction, we generated an Ece1 knockout strain (termed as “Ece1Δ/Δ” hereafter) using the CRISPR/Cas9 system in C. albicans (Suppl. Figure S7a-b). This strain, which cannot synthesize candidalysin, was assessed for its ability to penetrate HCT116 cells using a differential fluorescence assay^21,44. The Ece1Δ/Δ strain maintained the capacity to adhere to and enter HCT116 cells, similarly to the wild-type strain (Figure 6c-d). However, it failed to activate HIF-1α and c-Myc in the tumor cells (Figure 6e,f). In contrast, when HCT116 cells were treated with synthetic candidalysin, we observed a significant upregulation of c-Jun, c-Myc, and HIF-1α expression levels at 3μM for both uninfected HCT116 cells and those infected by Ece1Δ/Δ strain C. albicans (Figure 6g,h). Moreover, treated with candidalysin activated MAPK pathway and showed promoted cell migration (Figure 6i, j). Together, these results indicate that C. albicans regulates tumor cells through the secretion of candidalysin, which activates MAPK and hypoxia pathways to promote tumor cell migration.

C. albicans induce hypoxia responses in colon organoids and clinical specimens

To investigate whether C. albicans has the same effect on tumor samples, we collected three paired tumors and non-malignant colon tissues originating from CRC patients, which were isolated and cultured into organoids. After co-culturing C. albicans with the organoids for 12 hours, we also detected significant aggregation of HIF-1α in the cells, as evidenced by both Western blot and immunofluorescence staining, in both non-malignant intestinal organoids and tumor organoids (Figure 7a-c, Suppl. Figure S8a). Treatment of the organoids from colorectal cancer patients with c-Jun inhibitor SP600125 and c-Myc inhibitor 10058-F4 significantly suppressed the expression of HIF-1α (Figure 7d, Suppl. Figure S8b). Moreover, we observed that C. albicans hyphae penetrated the epithelium of colon organoids, reaching the lumen (Figure 7e). For the specimens analyzed in Figure 1a, we found higher levels of fungi near the colon mucosa in stage III samples compared to stage II sample, along with a notable aggregation of HIF-1α (Figure 7f, Suppl. Figure S9). Collectively, our findings suggest that C. albicans may contribute to the development of CRC by inducing HIF-1α aggregation in adjacent cells, thereby triggering hypoxia-related responses that create a microenvironment conducive to tumor progression.

Figure 7.

Interactions between Candida and tumor cells exhibit both species and cellular specificity

Lastly, we explored the cellular responses of CRC cells to other fungus besides C. albicans. To do this, we conducted co-culture experiments using HCT116 cells and two other fungal species, Saccharomyces cerevisiae and Candida tropicalis, to assess their adhesive capacities and effects on cancer cells. As shown in Figure 8a, C. tropicalis and S. cerevisiae exhibited minimal adhesion to cancer cells. Western blots were then performed to examine the subsequent effects of infection with these fungi, as well as a mixture of fungal cell wall polysaccharides and heat-killed Candida species. As a result, C. albicans and C. tropicalis exhibited the most potent ability to activate the hypoxia pathway, with C. albicans showing the strongest effect on the activation of c-Myc and c-Jun (Figure 8b). We hypothesized that the variations in cellular responses might be linked to the diversities of the Ece1 gene in the fungal genomes. Indeed, phylogenetic analysis revealed that evolutionarily, the Ece1 genes in various Candida species were highly conserved, whereas they differed significantly from other fungus. (Suppl. Figure S10). Of note, C. tropicalis did not express the ECE1 homolog after interaction with HCT116 cells for 1 hour (Suppl. Figure S11).

Figure 8.

Additionally, we investigated the specificity of the response to C. albicans by co-culturing it with a variety of cancer cell lines. Intriguingly, we found that C. albicans not only activated the hypoxia pathway in CRC cells but also significantly induced similar responses in breast cancer cell line HCC1937 and the gastric carcinoma cell line NUGC-3. In contrast, C. albicans failed to elicit responses in melanoma cell line A2058, the breast ductal carcinoma cell line MCF-7, and the human embryonic kidney cell line 293T (HEK293T) (Figure 8c). To explore the underlying mechanism responsible for these differences, we analyzed EGFR expression. As shown in Figure 8d, e, Suppl. Figure. S12, the results indicated a correlation between the expression and phosphorylation levels of EGFR and the phenotypic response to C. albicans infection. This was consistent with our earlier finding that C. albicans facilitates tumor cell migration through an EGFR-mediated pathway (Figure 4). Together, these findings suggest that candidalysin triggered EGFR/TLR2 - ERK/NF-κB - HIF-1α signaling pathway to induce hypoxia response, which promotes the progression of CRC (Figure 9).

Figure 9.

Discussion

Recent studies have sparked broad interest in the role of gut fungi, including C. albicans, in the development of various types of cancers^23,25,45. However, whether and how fungal components directly modulate epithelial oncogenic signaling remains largely unexplored. We identify C. albicans induce hypoxia response in colorectal cancer (CRC) cells, mediated by the activation of the EGFR/TLR2–ERK/NF-κB signaling axis and stabilization of HIF-1α. This process is critically dependent on the fungal virulence factor candidalysin. While prior research has highlighted fungal modulation of tumor-associated macrophages²⁵, our study extends this concept by identifying direct fungal–epithelial crosstalk driving oncogenic signaling in CRC. Hence, by demonstrating that C. albicans activates epithelial hypoxia signaling in a species- and cell-specific manner, our study reveals a previously unrecognized mechanism through which gut fungi may contribute to CRC progression.

Hypoxia is a hallmark of cancer, promoting angiogenesis and metastasis and resistance to therapy^46–48. Under hypoxic conditions, metabolic shift in malignant cells enhance cell proliferation and invasion ability⁴⁹, as well as induce EMT and modulate the tumor microenvironment^50,51 to promote metastasis⁵². Interestingly, previous studies have shown that tumor-associated macrophages with C. albicans infection showed an increased glycolysis level through the HIF-1α-dependent pathway²⁵. Similarly, it has been reported that C. albicans can upregulate the mRNA expression of HIF1A and CAMP in HT-29 colonocytes²⁶. In our study, however, we found that the C. albicans-induced hypoxia response in colorectal cancer (CRC) cells is predominantly regulated at the protein level, indicating post-transcriptional or translational control mechanisms. Moreover, we demonstrated that this hypoxia response relies on c-Jun and c-Myc, two key cancer-associated transcription factors that regulate cell progression, apoptosis, and cellular transformation^53,54. This finding aligns with recent reports showing that c-Myc exerts considerable control over the levels and activity of HIF-1α^34,55. Besides hypoxia, C. albicans also induced VEGFA expression (Figure 1), suggesting that the fungus may contribute to the vascularization of tumors, thereby facilitating nutrient supply and waste removal to boost tumor growth. Furthermore, activation of the NF-κB pathway by C. albicans suggested a potential role in modulating the inflammatory response worthwhile for future investigations.

In addition, we demonstrated that candidalysin is the key molecular that triggers CRC cell response after C. albicans infection. Candidalysin is a peptide toxin secreted by the hyphal form of C. albicans during invasion, which helps C. albicans compete with bacterial species in the intestinal niche⁵⁶, and it is known to drive epithelial damage, immune activation, and phagocyte attraction^21,42,43,57. Recent studies have also shown that candidalysin can modulate multiple signaling pathways in various cancer types, e.g., activate the MAPK p38 mitogen-activated protein kinase through MAPK kinases and the kinase Src in oral cancer⁵⁷. Here we showed that candidalysin acted as a critical virulence factor that induces phosphorylation of EGFR to activate MAPK/ERK and downstream HIF-1α pathway. Together, these findings suggested that candidalysin, and its downstream EGFR, could serve as therapeutic targets to suppress C. albicans-induced metastasis-promoting responses for pharmacological interventions. Moreover, the species- and cell-specificity of the interactions between Candida species and tumor cells (Figure 8) highlights the need for a nuanced understanding of the tumor microbiome. Interestingly, C. tropicalis are able to induce HIF-1α accumulation, while it does not secret candidalysin (Figure 8b, Suppl. Figure S11)⁵⁸. In fact, previous studies reported that C. tropicalis induce PKM2 phosphorylation which then upregulates HIF-1α pathway⁵⁹, suggesting high diversity of fungus-tumor interplays that requires more explorations.

In conclusion, this study reveals a novel link between fungal infection and hypoxia activation in the tumor microenvironment. Given the central role of HIF-1α in metabolic adaptation, angiogenesis, and therapeutic resistance, our study provides a promising framework for understanding how microbial factors may influence CRC progression through metabolic reprogramming.

Methods

Ethics approval and cell culture

This study had been approved by the Ethics Committee of Shenzhen Bay Laboratory, Ethics Committee of Southern University of Science and Technology, and Ethics Committee of The Seventh Affiliated Hospital, Sun Yat-Sen University. Clinical colon tumor samples were collected from Southern University of Science and Technology Hospital and The Seventh Affiliated Hospital, Sun Yat-Sen University. The human CRC cell line HCT116 (RRID:CVCL_0291), gastric adenocarcinoma cell line NUGC-3 (RRID:CVCL_1612), breast cancer cell line HCC1937 (RRID:CVCL_0290) and non-small cell lung cancer A-427 (RRID:CVCL_1055) cultured in RPMI 1640 Medium (Thermo, #C11875500BT), supplemented with 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich, #F0193), at 37°C in a humidified incubator with an atmosphere of 5% CO₂. The human CRC cell lines SW48 (RRID:CVCL_1724) and Caco2 (RRID:CVCL_0025), breast cancer cell line MCF-7 (RRID:CVCL_0031), melanoma cell line A2058 (RRID:CVCL_1059), and embryonic kidney cells HEK293T (RRID:CVCL_0063) are grown in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo, #C11995500BT) supplemented with 10% FBS, under the same conditions. For detected gene expression under hypoxia, HCT116 cells were cultured at 37[in an anaerobic chamber with an atmosphere of 0% O₂, 5% CO₂ and 95% N₂. Cells were routinely tested for mycoplasma contamination and were found to be negative. C. albicans (RRID: NCBITaxon_5476), C. tropicalis (RRID: NCBITaxon_5482), and S. cerevisiae (RRID: NCBITaxon_4932) were grown on YPD (Yeast Peptone Dextrose) agar plate (HKM, #21100). Prior to infection, a single colony was inoculated into 4 ml YPD liquid medium and incubated overnight at 30°C in a high-humidity environment. Bacteroides fragilis was cultured on columbia blood agar plate (HKM, #CP0160) and inoculated with a single colony into 5 ml BHI broth, supplemented with 25μg/ml hemin chloride and 10μg/ml vitamin K1(HKM, #SR0300). Cultures were maintained in an anaerobic chamber at 37[.

Co-culture of cells and strains

For co-culture of cell line and strains, 1×10⁶ cells were grown to confluence on 6-well plate. Before co-culture, the medium was replaced with 2 ml RPMI 1640 without FBS and supplemented with the strains, then incubated for 6 or 12 hours at 37 °C/5% CO2. For co-culture of organoids and C. albicans, approximately 200 colon organoids (∼5×10⁵ cells) were harvest from a 12-well plate. Organoids were gently washed with 1 mL of DMEM medium without FBS, then the DMEM was aspirated and replaced with 1 mL of the organoid culture medium. The medium was then replaced with 1 ml serum-free medium supplemented with C. albicans and incubated for 12 hours at 37 °C/5% CO₂. Unless otherwise specified, the MOI (multiplicity of infection) used in the infection experiments was set at a ratio of strain:cell = 1:25.

Cell migration assay

Cell migration was assessed using a wound-healing assay. Briefly, HCT116 cells were seeded in 6-well culture plates to reach approximately 90% confluence. A wound was introduced by manually scraping. Following the initial wound creation, cells were washed with phosphate-buffered saline (PBS) to remove debris and 2 ml serum-free medium supplemented with the strains was added. Cells were then incubated at 37°C in a CO2 incubator for 24 hours. Images of the wounded area were captured at 0 and 24 hours, and the gap sizes were quantified by measuring the wound area using ImageJ software. The relative gap size was calculated by comparing the wound area at 24 hours to the initial wound size.

RNA extraction and real-time PCR

Total RNA was isolated from cultured tumor cells using a commercial kit (Zymo, #R2052), followed by quantification and purity assessment with nanodrop. 1μg of total RNA were treated with DNase to eliminate DNA contamination and then converted to cDNA using a high-capacity cDNA synthesis kit (Vazyme, #R333-01). For quantitative Real-Time PCR (qPCR), gene-specific primers were used in a SYBR Green-based qPCR assay to quantify the expression levels of target genes (Vazyme, #Q712-02). The qPCR cycling conditions included an initial denaturation step, followed by 40 cycles of amplification. Relative gene expression was determined using the 2^-ΔΔCt method with β-actin served as endogenous control for normalization. The DNA primers were listed in Suppl. Table S6.

Protein extraction and western blots

Tumor cells were lysed in a radioimmunoprecipitation assay (RIPA) buffer (Beyotime, #P0013) containing protease and phosphatase inhibitors (Selleck, #B14001, #B15001). The lysate was centrifuged, and the supernatant containing the total protein was collected. Protein concentration was determined using the detergent compatible bradford protein assay kit (Beyotime, #P0006C) with bovine serum albumin (BSA) used for standard curve construction. For western blot analysis, 10μg protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% non-fat dry milk for one hour, the membrane was incubated with a primary antibody at 4[overnight, followed by a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000). Protein bands were visualized using an SuperFemto ECL Chemiluminescence Kit (Vazyme, # E423-01). Images were captured by ChemiDoc MP (Bio-rad, RRID:SCR_019037) and analyzed using ImageJ software. Human α-actin and β-tubulin were used as a loading control. The antibodies and dilutions were listed in Suppl. Table S7.

ELISA to detect the IL-8 concentration

After completing the co-culture experiments, culture supernatants were collected and stored at −80 °C for future analysis. The assays were conducted according to the manufacturers’ protocols (ELabscience, #E-OSEL-H0014). In brief, IL-8 was captured by antibodies pre-coated on 96-well plates. Subsequently, biotinylated detection antibodies were applied to the wells, followed by the addition of a streptavidin-horseradish peroxidase (HRP) complex. The presence of the IL-8 was then visualized by adding a substrate solution, and the absorbance was measured at approximately 450 nm using a spectrophotometer (Promega, RRID:SCR_018614).

Whole transcriptome sequencing and data analysis

For RNA sequencing, total RNA was extracted from tumor cells using column-based purification method (Zymo, #R2052) and analyzed for quality on an Agilent 2100 Bioanalyzer. Ribosomal RNA (rRNA) was depleted using a commercially available kit (Vazyme, #N406-01), which selectively removes rRNA and enriches the mRNA fraction. The rRNA-depleted RNA was fragmented and used to construct a cDNA library following a protocol provided by VAHTS Universal V8 RNA-seq Library Prep Kit for MGI (Vazyme, #NRM605-01). The cDNA library was amplified using PCR and analyzed for size distribution on an Agilent 4200 TapeStation System (RRID:SCR_018435), then subjected to a circularization process to form a concatemers of the cDNA fragments (Vazyme, #NM201-01). After quantified using a Qubit fluorometer, the circularized library was loaded onto a sequencing flow cell and sequenced on a MGISEQ-2000 sequencer (MGI) in paired-end 150 bp mode.

RNA-seq data analysis was analyzed as in our previous study⁶¹. Briefly, raw sequencing reads were first subjected to adapter trimming and low-quality base removal with Ktrim software (version 1.5.0)⁶², and then aligned to the human reference genome (NCBI GRCh38) with STAR software (version 2.7.9a)⁶³. Gene expression quantifications were conducted using FeatureCounts software (version 2.0.3)⁶⁴. Subsequently, differential gene expression analysis was performed with DESeq2 (version 1.26.0)⁶⁵. Gene Ontology (GO) enrichment analysis was conducted with ClusterProfiler (version 4.12.6)⁶⁶. Gene Set Enrichment Analysis (GSEA) was applied to identify coordinated expression changes within biological pathways⁶⁷.

Immunofluorescence staining

For cell immunofluorescence (IF) staining, HCT116 cells were evenly distributed onto 8-well chamber slides at a density of 10⁵ cells per well. After reaching ∼90% confluence, cells were washed with phosphate-buffered saline (PBS) and 200ul serum-free medium supplemented with the 10⁵ C. albicans (MOI 1:1) was added. After 3 hours of culture in a humidified incubator (37°C/5% CO2), slides were fixed with 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilized with 0.1% Triton X-100 in PBS for 10 minutes. Non-specific binding was blocked with 5% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. Cells were incubated with the primary antibody overnight at 4°C in a humidified chamber. After washing with PBS, the slides were incubated with a fluorochrome-conjugated secondary antibody (1:1000 dilution) and for 1 hour at room temperature in the dark. C. albicans were marked with Concanavalin A-Alexa Fluor 647 or Calcofluor White. Finally, the slides were mounted using an antifade mounting medium with DAPI (Beyotime, #P0131).

For organoids staining, uninfected and infected organoids were fixed in 4% paraformaldehyde in 100 mM PBS for 1 hour, washed with PBS with 100 mM glycine, permeabilized in 0.5% Triton X-100 in PBS for 1 hour, then incubated in staining buffer (4% BSA, 0.05% Tween-20 in PBS pH 7.4, 10% goat/donkey serum) for 4 hours, followed by incubation with primary antibody for 24 hours at 4°C. After washing with PBST (PBS with 0.05% Tween-20), incubated with fluorescent secondary antibodies, phalloidin and DAPI, for 4 h at room temperature. Following additional washes, whole mounts were submerged in antifade mounting medium (Beyotime, #P0126). Images were captured using Zeiss LSM900 confocal microscope (Carl Zeiss, RRID:SCR_022263) fitted with a 63× oil immersion objective or 10× objective.

Construction of stable cell lines

Lentiviral particles were produced by co-transfecting 293T cells with the shRNA expression vector (pLKO-shRNA-EGFP vector) along with packaging (psPAX2) and envelope (pMD2.G) plasmids, using Lipofectamine 3000 Transfection Reagent (Thermos, #L3000015). The culture medium was replaced 24 hours post-transfection, and the lentiviral supernatant was collected 48 and 72 hours, filtered through a 0.45 µm filter. After HCT116 cells reach approximately 70% confluence, lentiviral particles were added to the cells in the presence of polybrene (5 µg/mL) to enhance infection efficiency. The cells were then incubated at 37°C for 24 hours. Stably transfected cells were selected using a Beckman CytoFLEX SRT Benchtop Cell Sorter (RRID:SCR_025068). The efficiency of shRNA-mediated knockdown was validated at the mRNA levels. The DNA primers for plasmid construction were listed in Table S1.

Generation of C. albicans mutant strains

ECE deletion was performed according to the method descripted by Nguyen. Briefly, Guide RNAs (gRNAs) were designed using the CRISPOR online website (http://crispor.tefor.net/). The gRNA sequences were integrated with gRNA expression cassettes by cloning-free stitching PCR. The Cas9 expression cassette was amplified from vector pADH137 (RRID:Addgene_90986). The 500bp upstream and downstream sequences of ECE1 were fused with mCherry by fusion PCR. Cas9 cassette, gRNA cassette and donor DNA were transformed into the competent cells of C. albicans using the lithium acetate transformation method. Transformants were selected on YPD agar plates containing nourseothricin to isolate stably integrated cells. Single colonies from the selection plates were picked and grown in liquid YPD medium. Genomic DNA was extracted and the ECE1 locus modification was PCR amplified using primers flanking and inner of the target site.

Tissue processing and organoid culture

Adjacent normal and tumor tissues were isolated from the resected colon segments. Healthy crypts and tumor epithelium was isolated essentially as described by Sato et al⁶⁸. Organoids from adjacent colonic tissues were cultured in optimized human intestinal organoid medium⁶⁹. The composition of medium is: advanced DMEM/F12 basal culture medium with 1% HEPES, 1% GlutaMAX, 1% Penicillin-Streptomycin, 10 ng/ml Wnt, 100 ng/ml R-Spondin, 100 ng/ml Noggin, 1× B27, 1,25 mM n-Acetyl Cysteine, 50 ng/ml human EGF, 5 μM A83-01, 3 μM SB202190, and 100 μg/ml Primocin. Tumor organoids were cultured with organoid medium minus Wnt and R-Spondin for the selection of organoids with intrinsic Wnt signaling activation. Organoids were split once a week and medium were refreshed every three days.

H&E and immunohistochemistry staining

Tissue specimens collected from colorectal cancer patients were fixed in freshly prepared 4% paraformaldehyde at 4 °C for 24 hours. After fixation, the tissues were dehydrated, embedded, and sectioned. Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) were performed using standard procedures. H&E staining involved the use of hematoxylin and eosin dyes. For immunohistochemistry (IHC), specific primary antibodies targeting β-1,3-glucan (1:100) and HIF-1α (1:200) were applied to detect the presence and localization of these markers within the tissue sections. The stained slides were scanned using the SLIDEVIEW VS200 system (Olympus, RRID:SCR_024783), and images were processed using OlyVIA software (Olympus, RRID:SCR_016167).

Survival analysis

The abundance of C. albicans in colon adenocarcinoma samples in The Cancer Genome Atlas (TCGA) was collected from Narunsky-Haziza et al. study⁶⁰, which characterized fungal populations across multiple cancer types⁶⁰. Survival analysis was performed in the “survival” package in R.

Statistical analysis

All experiments were performed in triplicate, and the data is presented as the mean ± standard error of the mean (SEM). Statistical analysis was conducted using unpaired t-test and a one-way ANOVA followed by a post-hoc Tukey test to determine the significance of differences between two groups and more than two groups respectively.

Acknowledgements

This work was supported by Guangdong Basic and Applied Basic Research Foundation (2023B1515120073), National Key R&D Program of China (2022YFA0912700), China Postdoctoral Science Foundation (2023M742421), Shenzhen Bay Scholar Fellowship (to C.D. and K.S.), and Major Program of Shenzhen Bay Laboratory (S241101004). We’d like to thank Ms. Qi Wang for technical assistance, SZBL Bioimaging Core, and SZBL High Performance Computing and Informatics Core for technical supports.

Additional information

Data availability

Raw RNA-seq data has been deposited to Gene Expression Omnibus (GEO) under accession number GSE279572 (the reviewers could access the data using the token: kvsliomkdzsxtiv; the data would be made publicly available upon acceptance of this manuscript).

Author contributions

Conception and design: W.W., K.S.; Study supervision: W.W., G.H., K.S.; Development of methodology: W.W., H.L.; Patient recruitment and clinical data analysis: C.D., N.L.; Cell culture and experiments: W.W., H.L., M.Y.; Acquisition of data: all authors; Analysis and interpretation of data: all authors; Writing the manuscript: W.W., K.S.; Review and edit of the manuscript: W.W., Y.Z., N.L., G.H., K.S.

Funding

Guangdong Basic and Applied Basic Research Foundation (2023B1515120073)

Additional files

Supplementary figures

Supplementary Tables