Tumor-related fungi and crosstalk with gut fungi in the tumor microenvironment

Fungal dysbiosis in colorectal cancer (CRC)

CRC is a common malignancy involving the colon and rectum and threatens the lives of approximately 1.8 million people every year^16,17. Inflammatory bowel disease (IBD) increases the risk of developing CRC and over the past decade a significant understanding of the pathogenesis and treatment of colorectal tumors in IBD has been achieved^3,18–20. CRC that occurs in individuals with IBD is often considered a prototype of inflammation-induced carcinogenesis¹⁸. Chronic inflammation results in oxidative stress-induced DNA damage, which may activate the oncogenes and inactivate the tumor suppressor genes. Markers of oxidative damage and DNA double-strand breaks gradually increase in the inflammation-dysplasia-carcinoma sequence¹⁸. IBD patients may develop sporadic tumors, with chronic inflammation being a key factor in risk stratification. Understanding this process helps manage inflammation in IBD patients, which in turn helps prevent cancer development.

Changes in fungal populations have been linked to CRC development^16,21,22. Fungal dysbiosis occurs in patients with colon polyps and CRC with a higher diversity in patients with polyps compared to healthy individuals²³. Intestinal fungal abundance is higher in the CRC group than in the polyp and control groups. C. albicans was reported to be the most abundant intestinal fungus in the cancer group²⁴. An analysis of mucosal-adherent fungi showed that the three major fungal phyla (Ascomycota, Basidiomycota, and Glomeromycota) were present in all samples⁸. Most fungi observed in the studies were from Ascomycetes^8,21,22,25 followed by Basidiomycota was second in both the case and control groups. The Basidiomycetes-to-Ascomycetes ratio was higher in the CRC group than the control group^8,17,22,26, indicating that fungal dysbiosis increases with disease progression²⁶. Differences in fungal composition were observed between colorectal adenomas and neighboring tissues along with a decrease in diversity in the adenomas^8,17,22. There was a significant reduction in Saccharomycetes and Pneumocystis fungi in CRC, with an enrichment of Malasseziomytes^22,26. Six fungi genera, including Acremonium, Malassezia, Moniliophthtora, Rhodotorula, Thielaviopsis, and Pisolithus, occur in individuals with CRC^22,26. Enrichment of the above six genera resulted in changes in the composition of CRC-specific fungi^22,26. In a study involving the fungal core group, one group consisted of 10 fungi enriched in CRC, while the other group was comprised of 23 fungi depleted in CRC²¹. Aspergillus rambellii is the most enriched fungus, while A. kawachii is the most depleted fungus in CRC when compared to healthy subjects^8,22. Twenty-four fungal species with varying levels of abundance were identified during screening among patients with CRC and adenomas. Among these 24 fungi, 1 species was enriched in CRC (A. rambellii), while 6 species were depleted in CRC (A. kawachii, Fusarium pseudograminearum, Lentinula edodes, Hanseniaspora guilliermondii, Saitoella complicata, and Cryptococcus neoformans). Compared to adenoma patients, the abundance of some fungi changed by > 1.4-fold compared to patients with adenomas. A total of 10 fungal species with differential abundances were identified between adenoma patients and healthy individuals. Among the 10 fungi, 5 species had an increase in abundance of at least 1.5-fold in adenoma patients²¹. Moreover, there was a significant increase in the abundance of C. albicans in CRC patients^8,24. Overall, the number of opportunistic pathogens increased and the biodiversity changed in the disease group. Candida may, in some cases, promote or accelerate the process of disease metastasis. This may be because Candida can affect immune system function, thus increasing the risk of disease metastasis. Alternatively, Candida may directly disrupt the tissue barrier, providing a pathway for disease transfer. The presence of Candida may result in decreased cell adhesion. This may be because Candida can secrete acetaldehyde and candidalysin that interfere with cell-to-cell connections, leading to a loosening of the tight bonds between cells. Reduced cell adhesion may impair tissue integrity and make it more vulnerable to disease. Thus, Candida is considered a predictor that its presence can predict certain adverse outcomes⁷.

The changes in abundance of these fungi in CRC patients may provide new clues that help us understand the pathogenesis of CRC. Depletion or enrichment of these fungi may be closely related to the occurrence and development of CRC and potentially serve as a biomarker for CRC or participate in the pathologic process underlying CRC.

Thirteen fungi, listed as key biomarkers for the diagnosis of CRC, had a positive association in all samples^25,27. This finding indicates that in addition to bacteria, fecal fungi could also serve as an important reference for the non-invasive diagnosis of CRC patients. Significant differences in the abundance of some fungal species between patients with CRC and healthy individuals can be detected based on fecal sample analysis. These fungi may be closely related to the occurrence and development of CRC with the potential to become new biomarkers for CRC diagnosis^21,22.

In a colon tumor containing C. albicans, the tumor is characterized by downregulated transcription of tumor suppressor genes, extracellular matrix receptor interactions, and genes associated with focal adhesion⁸.

Fungal dysbiosis in pancreatic cancer

Fungi may enter the pancreas from the intestinal cavity through several pathways and subsequently proliferate. This abnormal colonization of fungi may be closely related to the occurrence and development of pancreatic ductal adenocarcinoma (PDA)^26,28,29. In animal models of PDA and human studies involving PDA the fungal content in the pancreas is significantly increased by approximately 3000 times compared to the pancreas in normal physiologic states^3,28. Specifically, the fungal communities infiltrating PDA tumors are rich in Malassezia species in rodents and humans^{3,19,28,30,31}. The presence of mannose-binding lectin (MBL), which binds to fungal wall glycans to allow complement cascade activation, is responsible for neoplastic promotion, while MBL or C3 deletion in the extra-tumoral compartment or C3aR knockdown in cancer cells has tumor-protective effects, even in the presence of Malassezia, which leads to PDA promotion via the complement cascade through MBL activation^{3,19,26–28,30,32–34}. Another study showed that intra-tumoral fungi (Malassezia globosa or Alternaria alternata) enhance IL-33 secretion in PDA cancer cells through the dectin-1-mediated activation of the non-receptor tyrosine kinase (src)-spleen tyrosine kinase (Syk)-caspase recruitment domain protein 9 (CARD 9)-nuclear factor kappa B (NF-κB) pathway. Secreted IL-33 recruits and activates Th 2 and ILC 2, which stimulates tumor growth through secretion of tumor-promoting cytokines, such as IL-4, IL-5, and IL-13 in the tumor microenvironment (TME)³⁵. Other pro-inflammatory cytokines, including IL-18, IL-8, IL-6, and Th22, and chemokines, including C-C motif chemokine ligand 17 (CCL17), were also significantly increased after exposure to Malazzia, thereby exacerbating local inflammation³⁶. Malassezia colonization has been shown to induce CARD9-S12N polymorphism and to strongly enhance the release of cytokines, such as IL-10 or tumor necrosis factor-alpha (TNF-α) in wild-type (WT) or Card9−/− colitis mice compared to negative groups³⁷. In addition, Malassezia can activate the NLRP3 inflammasome through Dectin2/CARD9 signaling, accelerating the production of IL-1β and aggravating inflammation³⁶. Malassezia also promotes degradation of the extracellular matrix (ECM). Malassezia may promote tumor invasion and metastasis, in which integrin aggregation promotes focal adhesion, strengthens the ERK and PI3K pathways, and promotes cell proliferation and invasion³⁶.

More research is needed to determine the role of fungi in PDA. This discovery opens up new avenues for PDA diagnosis and treatment. For example, could detecting fungal content in the pancreas assist in PDA diagnosis? Could antifungal treatments help to intervene in PDA progression? These questions warrant further investigation in future studies.

Fungal dysbiosis in liver cancer

The gut and liver communicate bidirectionally via the biliary system and the portal vein. The gut microbiota and microbial products have a critical role in modulating liver function^32,38.

In comparison to healthy controls and cirrhotic patients, hepatocellular carcinoma (HCC) patients exhibit intestinal fungal dysregulation and significant enrichment of opportunistic fungi, such as Malassezia and C. albicans. Alpha diversity analysis revealed reduced fungal diversity in HCC and cirrhosis patients compared to healthy controls. Beta diversity analysis showed a notable separation clustering among the three groups³². Among these groups, the abundance of C. albicans was significantly higher in stage III–IV HCC patients compared to stage I–II³². C. albicans reprograms HCC metabolism and promotes NLRP6-dependent HCC progression³⁹. HCC can also be caused by M. furfur^3,32.

It is known that aflatoxins (a fungal toxin produced by the secondary metabolism of A. favus and A. parasiticus) induce HCC^32,40. Aflatoxins are usually ingested and cleared quickly from the blood. Experiments have shown that AFB1 damages intestinal epithelial cells, leading to destruction of intestinal structures⁴⁰. Metabolism of the CYP450 system forms DNA adducts that lead to mutations in the p53 tumor suppressor gene, a key mechanism in tumor development. AFB1 also induces ROS, inhibits protein, RNA, and DNA synthesis, and ultimately leads to apoptosis of microglia by activating NF-κB during oxidation^38,40,41.

Fungal dysbiosis in gastric cancer

The hallmark of GC-associated mycobiome imbalance is alteration in fungal composition and ecology. C. albicans dysbiosis is associated with gastric carcinogenesis³⁴. Candida and Alternaria are significantly increased in GC, suggesting an important role in distinguishing between GC and non-GC tissues and potentially serving as accurate biomarkers^3,8,30.

Fungal dysbiosis in lung cancer

The lungs were once thought to be sterile in a healthy state, leading to research into the lung microbiome to lag behind other aspects of human microbiomics^5,42–45. Blastomyces dermitidis/gilchristii are associated with tumor tissue in lung cancer^6–8. Researchers found evidence of Blastomyces DNA in 6 out of 50 patients with squamous cell lung cancer⁶. Intratumor mycobiome dysbiosis is correlated with lung adenocarcinoma (LUAD) pathology and is enriched with a live fungus (A. sydowii). Tumor-resident A. sydowii promotes lung tumor progression by inducing an immunosuppressive TME⁴⁶.

Fungal dysbiosis in other cancers

Narunsky-Haziza et al.⁷ characterized cancer mycobiomes in tissues and blood. All tumor types (breast cancer, lung cancer, melanoma, ovarian cancer, colon cancer, GBM, bone cancer and pancreatic cancer) tested had a higher fungal load than negative controls and the fungal load differed among tumor types. Fungi in human tumors have been observed by staining microarrays of melanoma and pancreas, breast, lung, and ovarian cancer tissues. Interestingly, images showed cancer type-specific localization patterns. Although fungal staining was mainly evident within cancer cells in pancreas, breast, and ovarian cancer, staining was mostly localized to macrophages in melanoma and lung cancer. The associations of fungi with clinical parameters, including overall survival in breast cancer, progression-free survival in ovarian cancer, the immunotherapy response in melanoma tumors, and detection of early-stage cancers, support the clinical utility as potential biomarkers and therapeutic targets⁷.

Secretion of fungal carcinogenic metabolites

In 2016 the first cytolysin toxin produced by the human pathogenic fungus, C. albicans, was given the name candidalysin⁸. Candidalysin is a cytolytic enzyme that damages the host cell membrane by forming a porous structure, resulting in the release of LDH and the flow of calcium into the cytoplasm, which damages the host cell membrane. These signaling pathways trigger hazard response signaling pathways that activate epithelial immunity and the secretion of IL-1β by macrophages⁵⁸. C. albicans and candidalysin activate the human epithelial EGFR receptor, whereas fungal mutants lacking candidalysin exhibit a weaker ability to induce EGFR phosphorylation during oropharyngeal candidiasis in mice⁵⁹. EGFR activation leads to the induction of the c-Fos transcription factor in the candidalysin-induced mitogen-activated protein kinase (MAPK) signaling pathway, ultimately triggering the recruitment of neutrophils via G-CSF and GM-CSF, as shown in Figure 2. This process depends on the release of EGFR ligands and an influx of calcium^40,47,59. In addition, candidalysin indirectly activates EGFR through a complex mechanism of matrix metalloproteinases and EGFR ligands, leading to downstream immune activation⁶⁰. EGFR may be activated in an atypical manner, thereby affecting intracellular processes when cells are exposed to stress stimuli, such as transport, autophagy, and energy metabolism. This atypical EGFR activity may be related to cancer cell survival and treatment resistance. Specifically, atypical EGFR activity may help cancer cells adapt to environmental changes, enhance the ability to survive, and reduce the sensitivity to anticancer drugs. One of the best-studied metabolic changes in cancer cells is the increase in glycolysis, even in the presence of oxygen, which is also known as the Warburg effect⁶¹. Lim et al.⁶² reported that EGF signaling activates the first step of glycolysis but blocks the last step, leading to the accumulation of metabolic intermediates in this pathway. In addition, Lim et al.⁶² showed that one of these intermediates, fructose 1,6 diphosphate (F1,6BP), directly binds and enhances the activity of EGFR, thereby enhancing the inhibition of local cytotoxic T cell activity caused by a lactic acid overdose, which promotes cancer cell growth and metastasis⁶². The PI3K signaling pathway regulates key metabolic activities in EGFR-mutated LUAD. The PI3K/AKT/mTOR signaling pathway is crucial for regulating aerobic glycolysis in EGFR-mutated LUAD cells. EGFR activation triggers a series of reactions that activate PI3K, further activating the PI3K/AKT/mTOR signaling pathway. This signaling pathway provides tumor cells with a significant amount of energy and biosynthetic precursors required for tumor growth and invasion⁶³. Compelling evidence has shown that Akt, a serine/threonine kinase downstream of PI3K, is involved in the Warburg effect⁶⁴. Active Akt inhibits autophagy and blocks GLUT1 endocytosis. The latter results in increased GLUT1 levels at the plasma membrane, which increases glucose uptake⁶¹. In cancer cells, Akt induces GLUT1 and GLUT3 expression and is an established mediator for the translocation of GLUT4 to the plasma membrane, which stimulates glucose transport⁶⁴.

C. albicans also produces nitrosamines and metabolizes ethanol into acetaldehyde³. Nitrosamines activate specific proto-oncogenes that trigger abnormal proliferative changes in oral epithelial cells or cancers⁶⁵. Acetaldehyde, a carcinogenic substance, can cause mutations by binding directly to DNA and proteins, induce oxidative stress, and lead to DNA damage and genetic mutations⁴⁰, as shown in Figure 2. Acetaldehyde treatment induces oxidative stress and Ca²⁺ overload in SH-SY5Y cells, leading to the phosphorylation of Drp1 at Ser616 and translocation of Drp1 to mitochondria, subsequently causing excessive fission and fragmentation of mitochondria. The resulting imbalance of mitochondrial dynamics eventually impairs mitochondrial function and causes cytotoxicity. The mitochondrial electron transport chain is the major source of intracellular ROS. When the mitochondrial respiratory chain complex is damaged, ROS can be overproduced and accumulated, causing oxidative stress and triggering more mitochondrial alterations (e.g., ΔΨm disruption and ATP depletion)⁶⁶.

Bacterial species and fungi often form multi-microbial biofilms⁴⁰. These biofilms act as a barrier that protects microbes from the host immune system and potentially promotes local inflammatory responses^3,8,28. Studies show that mucosal biofilms in the colon directly trigger CRC^3,8. Therefore, biofilms are strongly associated with CRC because biofilms evade host immune responses, increase epithelial cell permeability or promote pre-cancerous inflammation, and have a strong ability to invade and spread^3,40.

Fungi activate the immune response

The immune system detects ubiquitous fungi and requires a good balance between pro- and anti-inflammatory signals to maintain a stable host-fungal relationship. A disruption of this balance can lead to pathologic consequences⁶⁷. Fungi are usually effectively cleared by a functioning immune system. However, patients with impaired immune systems and patients receiving immunosuppressive therapy have increased susceptibility to fungal infections, which are associated with higher mortality^60,68. By masking or subverting the host detection systems, fungi may avoid inflammation, which contributes to fungal adaptation and opportunism⁶⁷. The cell wall of C. albicans is rich in various complex carbohydrate structures, including chitin, glucan, and mannan. These components can all be considered pathogen-associated molecular patterns (PAMPs). PAMPs are recognized by the host immune system during the infection process, triggering a series of immune responses^19,69,70. A PRR is a multiline-encoded receptor that recognizes a variety of pathogen-associated molecules expressed by invading microorganisms (PAMPs)⁶⁸. Four receptor families can help us identify a wide range of species, including fungi^68,69.

After the recognition of PAMPs by PRRs, PRRs activate a series of complex intracellular signaling pathways. These signaling pathways involve key molecules, such as adaptor proteins, kinases, and transcription factors, which together convert external signals into intracellular biochemical reactions. The signals induced by PRRs activate gene expression and promote the synthesis of a range of molecules, including cytokines, cell adhesion molecules, and immune receptors, as shown in Figure 3.

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Figure 3

Fungi promote the development of cancer by affecting the immune system. ①: Pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), C-type lectin receptors (CLRs), and the galectin family proteins, are expressed on myeloid cell surfaces, including surfaces of macrophages, neutrophils, and dendritic cells and recognize various pathogen-associated molecules expressed by invading microorganisms (pathogen-associated molecular patterns, PAMPs). When macrophages recognize Candida albicans by cell surface CLR, the increase in the abundance of Candida albicans induces the upregulation of macrophage glycolysis through the HIF-1 pathway, leading to the secretion of interleukin-7 (IL-7). IL-7 then induces the production of IL-22 by RORγt innate lymphoid cells (ILC3) through the aryl hydrocarbon receptor and STAT3. This process promotes inflammation-associated tumorigenesis and tumor cell proliferation. ②: When PRRs, including CLRs, in myeloid cell recognize and bind to specific components on the fungal cell wall, the Syk-CARD9-nuclear factor-κB (NF-κB) axis promotes the assembly of the inflammasome, driving the production of pro-IL-1β and pro-IL-18. When the NLRP3 inflammasome is activated during this process, a complex is formed with caspase-1. This complex cleaves the pro-IL-1β and pro-IL-18 fragments and converts the fragments into mature forms (IL-1β and IL-18). These cytokines further drive ILC3 and Th1 cell responses, resulting in the production of IL-22 and IFN-γ, respectively. IFN-γ subsequently triggers phagocyte activation, collectively promoting inflammation-associated tumorigenesis and tumor cell proliferation. Additionally, as a myeloid cell-specific protein in the downstream of various PRRs, CARD9 mediates the stimulation TH17 by IL-6 or TNF-α, subsequently leading to the production of IL-17 that triggers neutrophil recruitment. ③: Mannose-binding lectin (MBL) binds to fungal cell wall glycans, such as Malassezia globosa, allowing complement cascade activation that acts on cancer cells to promote cancer. The C3 convertase complex splits C3 molecules into C3a and C3b. C3a binds to C3aR on the tumor cell membrane and promotes tumor cell proliferation and invasion. ④: Alternaria alternata promotes tumor growth and triggers IL-33 secretion, inducing a type 2 immune response and innate lymphoid cell 2 (ILC2), which stimulate tumor growth by secreting tumor-promoting cytokines, such as IL-4, IL-5, and IL-13, that act on tumor-associated macrophages to participate in immunosuppressive tumor-promoting activity. ⑤: In macrophages with CARD9 deficiency, Candida tropicalis promotes the production of IL-6 and GM-CSF. Subsequently, this cytokine production leads to increased accumulation of MDSCs, which suppress cytotoxic lymphocytes, thereby supporting tumor immune evasion. ⑥: Debaryomyces hansenii stimulates a type I interferon (IFN) response in macrophages, which leads to production of C-C motif chemokine 5 (CCL5) and chronic inflammation that prevents wound healing. PAMPs, pathogen-associated molecular patterns; PRRs, pattern recognition receptors; CLRs, C-type lectin receptors; HIF-1, hypoxia-inducible factor-1; AhR, aryl hydrocarbon receptor; STAT3, signal transducer and activator of transcription 3; C3, complement component 3; C3a, fragment of cleaved C3; C3aR, C3a receptor; SYK, spleen tyrosine kinase; CARD9, caspase recruitment domain-containing protein 9; NF-κB, nuclear factor-kappa B; NLRP3, NLR family pyrin domain containing 3; IL-4, interleukin 4; IL-5, interleukin 5; IL-6, interleukin 6; IL-13, interleukin 13; IL-7, interleukin 7; IL-17, interleukin 17; IL-18, interleukin 18; IL-22, interleukin 22; IL-33, interleukin 33; ILC2, group 2 innate lymphoid cell; ILC3, group 3 innate lymphoid cell; MDSC, myeloid-derived suppressor cell; CTLs, cytotoxic T lymphocytes; Th1, T helper 1 cell; Th2, T helper 2 cell; Th17, T helper 17 cell; TNF-α, tumor necrosis factor α; IFN-γ, interferon γ; IL-1β, interleukin-1β; CCL5, c-c motif chemokine 5.

The signals induced by PRRs not only help recruit and activate more immune cells but also enhance the ability of cells to kill pathogens¹⁴. C-type lectin domain family 7 member A (Dectin-1), CARD9, IL-17, and IL-22 have been identified as key components in the host defense against fungal infections. Mutations in these molecular components have been linked to increased susceptibility to fungal infections in humans¹². CLRs, such as Dectin-1, -2, and Mincle, have a major role in anti-fungal immunity because CLRs are specific to various carbohydrate-rich moieties in the fungal cell wall⁴⁷. Dectin-1 is a well-defined CLR that recognizes beta-1,3-glucan present in the cell walls of Aspergillus, Candida, and many Saccharomyces and Trichosporon^68,71. When Dectin-1 recognizes and binds to specific components on the fungal cell wall, Dectin-1 signals through the Syk-CARD9-nuclear factor-κB (NF-κB) axis to promote the assembly of the inflammasome, driving the production of pro-IL-1β and pro-IL-18. During this process, when the NLRP3 inflammasome is activated, the NLRP3 inflammasome forms a complex with caspase-1 or in some cases, caspase-8. This complex cleaves the pro-IL-1β and pro-IL-18 fragments, converting them into mature forms, such as IL-1β and IL-18^23,40. Bergmann et al.⁷² reported that IL-1β regulates the production of IL-22 by group 3 innate lymphoid cells (ILC3s), ultimately promoting tumorigenesis by activating the epithelial signaling transducer and activator of transcription 3 (STAT3). C. albicans promotes the proliferation of intestinal epithelial cells (IECs) in a Dectin-1- and WNT-dependent manner through its interaction with Dectin-1 in IECs. The abnormal activation of the Wnt/β-catenin signaling pathway is closely related to the tumorigenesis of multiple organs, including the abnormalities triggered by mutations in genes, such as APC or CTNNB1 (encoding β-catenin)^23,24,73,74, as shown in Figure 2. C. albicans infection in mouse colorectal tissue activates the Wnt pathway. IECs may recognize C. albicans activation of the Wnt pathway through Dectin-1, thus promoting the occurrence of CRC. C. albicans induces the proliferation of IECs by activating the Wnt signaling pathway and the Wnt pathway contributes to the development of CRC²⁴. Wnt binds to Frizzled receptor and attracts LRP binding, which then undergoes phosphorylation. After Dvl binds to phosphorylated LRP, Axin is attracted to LRP, leading to disruption of the destruction complex. This disruption blocks the β-TrCP ubiquitination, resulting in the stabilization of β-catenin. Stabilized β-catenin is transported to the nucleus to activate transcription factors, which regulate target genes and ultimately lead to cell proliferation and differentiation⁷⁵. It should be noted that the specific mechanism by which fungi directly activate the Wnt pathway is not fully understood. While most studies have focused on Wnt signaling in mammalian cells, fungal activation of the Wnt pathway may involve more complex interaction and regulatory networks. In addition, different species of fungi may activate the Wnt pathway through different mechanisms, so specific cases need to be analyzed.

Genetic polymorphisms in CLEC7A, which encodes the antifungal receptor, Dectin-1, are associated with ulcerative colitis severity in humans^12,19,71. A Dectin-1 deficiency increases the severity of dextran sodium sulphate (DSS) colitis, which enhances the general expansion of fungi, such as Candida and Trichomonas, by suppressing the immune response to pathogens^{12,19,47,71,76,77}. CLEC7A−/− mice experienced weight loss and histologic changes, manifesting as mucosal erosion, crypt destruction, infiltration of inflammatory cells, and increased production of TNF-α, IFN-γ, and IL-17 in the colon⁷¹. TNF-α exhibits tumorigenic characteristics by activating representative c-JunN terminal kinase (JNK) and NF-κB signaling pathways, leading to enhanced epithelial-to-mesenchymal transformation (EMT) and accelerating tumor cell invasion³⁶. Antifungal therapy eliminates this susceptibility, further supporting the important role of intestinal fungal immunity in colitis^5,12.

Increased tumorigenesis is associated with the C. albicans burden in Dectin-3−/− mice. The increase in abundance of C. albicans induces upregulation of macrophage glycolysis through the HIF-1 pathway, leading to the secretion of IL-7. IL-7 then induces the production of IL-22 by RORγt ILC3s through the aryl hydrocarbon receptor and STAT3^3,23,40,78. CARD9 is a signaling molecule downstream of Dectin-1 and Mincle, Dectin-3, DCIR, DC-SIGN, and Lox-1, and CARD9 loss has also been linked to human susceptibility to IBD^8,12,19,71.

CARD9 deficiency results in impaired bactericidal ability and increased fungal burden, especially of C. tropicalis. Subsequently, CARD9 deficiency leads to increased accumulation of myeloid-derived suppressor cells (MDSCs), which suppress effector T cells and promote the development of CRC^{3,19,23,78,79}. C. tropicalis induces NLRP3 inflammasome activation in MDSCs via Dectin-3⁸⁰. Mannans are highly conserved components in the fungal cell wall where Candida induces Th17 and IL-23 responses by activating the PRRs, thereby exacerbating GVHD and mouse colitis⁷⁶. The mannoprotein portion of C. albicans has specifically been shown to enhance tumor adherence by stimulating endothelial cells and inflammation^14,51. Blocking the mannose receptor reduces the ability of C. albicans to influence tumor cell adhesion and inhibits IL-18 synthesis^8,14,51.

Colonization of the stomach in mice by C. albicans leads to the expansion of regulatory T cell populations, which are associated with immunosuppressive effects in the host⁴⁸. C. albicans increases the production of IL-17 and IL-23 in the stomach and oral tissues in mice⁴⁸. IL-23 promotes angiogenesis, which contributes to tumor initiation and growth. Furthermore, this cytokine antagonizes IL-12 and IFN-γ, both of which are crucial in Th1-mediated antitumor immune responses¹⁴. Thus, Candida colonization can enhance the inflammatory response by increasing the levels of these cytokines. Th17 cells are known to produce IL-17, which is necessary to fight off C. albicans. Although the pathologic mechanisms mediated by Il-17 are unknown, the role of IL-17 in inducing antimicrobial peptides, pro-inflammatory cytokines, and downstream neutrophil recruitment is considered significant⁶⁰. IL-17 promotes gastrointestinal tumorigenesis by binding to its receptor and this signal activates ERK, p38 MAPK, and NF-κB signaling pathways. IL-17 stimulates the proliferation of tumorigenic enterocytes that just lost expression of the APC tumor suppressor, as well as the proliferation of colon epithelial cells, further supporting the malignant transformation of mice⁸¹.

Fungal-bacterial interactions in cancer

Symbiotic fungi and bacteria co-exist in the gut in a complex and delicate ecosystem. The interaction patterns between these microorganisms in the colon are crucial for maintaining intestinal health. Therefore, studying the interaction patterns of symbiotic fungi and bacteria in the gut, as well as the potential roles in CRC, is of great significance for understanding the pathogenesis of CRC and developing new treatment methods. A complex network of interactions among microbes across kingdoms can influence cancer behavior and treatment responsiveness⁸.

In lower gastrointestinal tract tumors, there is a positive correlation between intratumoral Candida and Dialister, while a negative correlation exists with Ruminococcus, Akkermansia, and Barnesiella intestine-hominis⁸. There were fewer and mostly positive correlations between fungi and bacteria in the control group compared to the CRC group, and a stronger co-exclusion relationship was observed in CRC²². Compared to individuals with adenomas and healthy individuals, the co-occurrence interactions among fungi enriched in CRC are stronger in CRC. Similarly, the strength of fungus-bacterial interactions increases as CRC progresses²¹. As a control group, fungi and bacteria have a synergistic association.

Some novel inter-kingdom interactions that involve various fungal classes, such as Pucciniomycetes, Exobasidiomycetes, Malasseziomycetes, and the Mucoromycota phylum, have been discovered in CRC. These fungi form an important co-exclusive network with the primary bacterial species, suggesting an ecologic imbalance in the gut microbiota in CRC. Specifically, cross-kingdom co-occurrence interactions involving fungi, such as Leotiomycetes, sodium-absorbing fungi, and Eurotiomycetes, were reversed to co-exclusive interactions in CRC. This shift may indicate a disruption in gut microbiota homeostasis, probably resulting from a balanced and synergistic relationship between different kingdoms. The disruption of this relationship may further contribute to colorectal tumor development²². Conversely, co-occurrence interactions were observed between fungi (Chaetomiaceae) and bacteria (Ruminicoccaceae), as well as between fungi (Pseudeurotiaceae) and bacteria (Alteromonadaceae and Geobacteraceae). These interactions between symbiotic kingdoms contribute significantly to the homeostasis of colonic microbiota. The microbial community in the colon represents a complex ecosystem, where diverse microorganisms work synergistically to maintain the balance of the intestinal environment. This balance is crucial for human health. In the context of CRC, the interaction between bacteria and fungi becomes particularly complex. With the enrichment of bacteria, the previously positive correlation between CRC-enriched fungi (Chaetomiaceae) and CRC-lacking bacteria (Ruminococcaceae), as well as between CRC-enriched fungi (Pseudourotiaceae) and CRC-lacking bacteria (Alteromonadaceae), is no longer present. This finding reveals the unique changes in the gut microbiota in CRC. Such changes may indicate an imbalance or reconfiguration of the gut microbiota during the occurrence and development of CRC. Instead, the positive correlation between the fungus, Pseudourotiaceae, and the bacterium, Geobacteraceae, reverses to a negative correlation, despite both being CRC-enriched²².

A hallmark of gastric tumors is that Lactobacilli are only associated with Candida clusters, which also tend to be isolated from H. pylori. The significant positive correlation between Streptococci and Clostridia with Candida indicates the potential occurrence of a symbiotic or collaborative relationship between these organisms. This relationship may promote the growth and reproduction in the gastric tumor environment. However, the significant negative correlation between Streptococci and Clostridia with Saccharomyces cerevisiae suggests a competitive or inhibitory relationship between these two types of microorganisms in the gastric tumor environment. The decrease in yeast populations may be related to the occurrence and development of gastric tumors, but specific mechanisms require further investigation⁸. Fusobacterium nucleatum is a bacterium often found in the intestines of patients with CRC. F. nucleatum is closely related to the occurrence and development of CRC. The close association between A. rambellii and F. nucleatum further suggests that A. rambellii and F. nucleatum may collaborate or influence each other during the pathogenesis of CRC. Additionally, A. rambellii has been shown to promote the growth of CRC cells in vitro and facilitate tumor growth in xenograft mice, providing a new perspective on the pathogenesis of CRC. This finding indicates that modulating the intestinal microbial community, especially targeting key microorganisms, such as A. rambellii and F. nucleatum, may aid in the prevention and treatment of CRC²¹. The diagnostic panel utilizing a combination of fungal and bacterial markers has demonstrated higher accuracy for the diagnosis of intestinal diseases, emphasizing the potential value and importance of intestinal fungi in clinical applications^21,82.

The presence and changes in fungi and bacteria in the TME have been widely studied. These microorganisms have been detected in the intestine, other mucosal surfaces, and within tumors, with community composition potentially evolving alongside cancer development. These changes may be related to the occurrence, development, and metastasis of cancer. However, current research on cross-kingdom interactions between fungi and bacteria in cancer is still in its infancy, and many mechanisms are not yet fully understood. Therefore, more research is needed to explore this field in depth to understand and address cancer as a global health issue more effectively.

Fungal-virus interaction in cancer

To date, the interaction between viruses and fungi remains poorly studied. The intestinal fungal diversity of patients with hepatitis B cirrhosis was higher than that of patients with chronic hepatitis B, which was higher than that of HBV carriers and healthy volunteers. There was no significant difference in intestinal fungal diversity between HBV carriers and healthy volunteers. The diversity of intestinal fungi was positively correlated with disease progression in patients with different degrees of chronic HBV infection⁸³. The abundance of fungal species was significantly reduced in the HIV/HCV co-infected group compared to the healthy control group, while no significant difference was found in the single-infected group. The opportunistic pathogenic fungal spectrum and fungal inter-relationships in the co-infected group became less characteristic but more complex than in the single-infected group. Intestinal fungal disorders occur in HIV- and HCV-infected patients and this disorder is further complicated in HIV/HCV co-infected patients. With the existence of some new fungal genera, some unique fungal interrelationships have emerged, which can be either positive or negative⁸⁴. In this era of efficacious antiretroviral therapy and consequent immune reconstitution, oropharyngeal and esophageal candidiasis remain two clinically relevant presentations in the global HIV setting. Both diseases are predominantly caused by C. albicans. HIV infection generates an environment selected for overexpression of the virulence potential by the fungus, particularly concerning the secreted aspartyl proteinases (Saps). For these reasons, new therapeutics targeting virulence factors and specific immune interventions are being addressed⁸⁵.

Symbiotic fungi regulate radiotherapy response through interaction with immune cells in the TME

It is well-established that tumor-associated immune cells rely on an immune microenvironment that promotes or hinders tumor progression. This microenvironment can be regulated in part by interactions between the immune system and the microbiome⁸⁶. Other components of the gut microbiome, particularly fungi, have recently been found to modulate inflammatory responses in various diseases, including colitis, asthma, and inflammatory colon cancer, because of the effects on dendritic and myeloid cells⁸⁶.

The effect of radiotherapy is adversely affected by the presence of symbiotic bacteria and fungi in the gut^8,86. The elimination of specific bacteria by antibiotics can significantly diminish the effectiveness of chemotherapy or immunotherapy, while the elimination of fungi may improve the efficacy of radiation therapy⁸⁶. Symbiotic fungi can bind to the receptor, Dectin-1, upregulating tumor-promoting macrophages and downregulating anti-tumor T cells, thus inhibiting the anti-tumor immune response after radiotherapy^8,86.

Active metabolites secreted by fungi indirectly influence the efficacy of immunotherapy by regulating the host immune system

Immune checkpoint inhibitors (ICIs) represent a breakthrough in cancer treatment⁸⁷. ICIs significantly improve patient outcomes in various tumors, but the efficacy has been inconsistent⁸⁸. In recent years increasing evidence has pointed out that the composition of intestinal bacteria can affect the effectiveness of ICIs, but the predictive accuracy is unsatisfactory^87,89. In contrast, the role of intestinal fungi in predicting the efficacy of tumor immunotherapy is more accurate than intestinal bacteria⁸⁷.

Recent studies have shown that the imbalance in intestinal flora caused by antibiotics may affect the efficacy of ICIs^88,90,91, the development of tolerance to PD-1 inhibitors, and significantly reduce progression-free survival and overall survival rates^90,91. Using fecal DNA analysis techniques, such as 16S and shotgun sequencing, can help uncover significant differences in microbiota composition between responsive and non-responsive patients. This disparity not only provides clues about the potential role of microbiota in tumor immunity but also lays the foundation for further investigating the mechanistic links between microbiota and anti-tumor immunity⁸⁹. The development of tumors is intricately connected to the immune system. The significant systemic impact of gut microbiota on tumor progression and treatment is likely mediated through immune system modulation. The gut microbiota is indeed an extremely complex system that profoundly affects the immune system not only through direct interactions with host cells but also via the release of various metabolites into the circulation. Specifically, short-chain fatty acids (SCFAs), important metabolites in the gut microbiota, have particularly significant effects. SCFAs have a significant role in the differentiation and activation of anti-inflammatory Treg cells or pro-inflammatory TH1 and TH17 cells. These cells have crucial roles in the immune responses and SCFAs regulate the balance, thus affecting the overall state of the immune system. For example, SCFAs may reduce inflammatory reactions by inhibiting the activity of pro-inflammatory cells or enhance immune tolerance by promoting the generation of anti-inflammatory cells. Additionally, SCFAs regulate the differentiation of plasma cells and the secretion of IgA. By modulating the differentiation of plasma cells and IgA secretion, SCFAs enhance the function of the intestinal mucosal barrier, thereby protecting the host from infection. Finally, SCFAs also affects the polarization of pro-inflammatory M1 and anti-inflammatory M2 macrophages. Macrophages are an important class of immune cells that have a vital role in inflammatory responses and immune regulation. By regulating macrophage polarization, SCFAs impact the magnitude and duration of inflammatory reactions, thus protecting the host from the damage caused by excessive inflammatory responses^{5,26,89,91,92}. A recent study focusing on patients with PD-1-responsive and non-responsive solid tumors revealed that patients with a responsive status had higher levels of SCFAs, including butyrate⁹³. It is well-known that fungi produce SCFAs, hence the impact of fungi on immunotherapy needs to be studied further.

Bacteria and bacteria secretions have an indirect anti-tumor role by regulating intestinal fungi

SCFAs produced by symbiotic bacteria can act on neighboring fungi, affecting replication and morphogenesis. Acetate, butyrate, and propionate have all been shown to inhibit the growth dynamics and mycelium formation of C. albicans and Pichia pastoris in vitro, thereby impacting stable colonization of the intestine. In addition, some strains of E. coli release soluble factors that directly kill C. albicans, adding to the well-known role of this bacterium in inhibiting C. albicans replication, biofilm formation, and intestinal colonization⁴⁷. Enterococcus faecalis is a common resident of the intestine and an important opportunistic pathogen. E. faecalis secretes the peptide, EntV, which can effectively inhibit the formation of mycelia¹⁹. Pichigia inhibits the growth of Candida, Aspergillus, Fusarium, and Cryptococcus by regulating virulence factors, such as nutrient restriction, biofilm formation, development, and adhesion. This inhibitory activity is related to secreted protein(s)^58,94,95. In the presence of Lactobacillus rhamnosus GG, cytokines prevent intestinal epithelial cell-induced apoptosis, activate the anti-apoptotic Akt/protein kinase B pathway, and inhibit pro-apoptotic P38/mitogen-activated protein kinase activation via TNF-α, IFN-γ, or IL-1⁹⁶. L. rhamnosus limits the processes required for C. albicans invasions by secreting metabolites and inhibits C. albicans by starving C. albicans of nutrients that sustain the invasion phenotype¹⁹. Therefore, studying these antibacterial substances may provide new ideas for the development of novel antifungal drugs.

Probiotics prevent cancer by inhibiting carcinogenic production, enhancing the immune system, regulating the balance of intestinal flora, and acting directly against cancer

Probiotics are defined as a class of living organisms that result in numerous health benefits to the host when consumed in sufficient quantities^92,97,98. In particular, the role of probiotics in promoting gut health and preventing gastrointestinal diseases, including cancer, has been studied and reported extensively³⁰. Probiotics not only help maintain the balance of gut microbiota but also potentially reduce the risk of CRC. Furthermore, probiotics can be potentially used in cancer treatment to manage the side effects of traditional cancer therapies, such as surgery, radiotherapy, and chemotherapy, thereby improving the safety of these treatments^98–100. Probiotics include bacterial species, such as Lactobacillus, Bifidobacterium, and a strain of yeast (Saccharomyces boulardii)^100,101. Currently, most probiotic products contain lactic acid bacteria belonging to the genera, Lactobacillus and Bifidobacterium⁹⁹. The genus, Lactobacillus, is generally considered safe and is a source of biologically active secondary metabolites. Extracellular polysaccharides from the genus, Lactobacillus, act as antioxidants, anti-biofilms, immunomodulators, antibacterial agents, and anticancer properties^8,98,102. Most of the probiotics in the human microbiota belong to the genus, Lactobacillus, but there are also some yeast strains present in dairy and fermented products that are classified as probiotics¹⁰³.

Intestinal ecologic disorders may cause changes in mucosal barriers and immune functions, resulting in increased intestinal permeability and bacterial translocation. Therefore, bacterial products originating from the intestine, such as endotoxins, enter the bloodstream through the portal vein system and general circulation¹⁰⁴. S. boulardii significantly reduced the levels of costimulatory molecules (CD40 and CD80), as well as the lipopolysaccharide (LPS)-induced DC mobilization marker CC-chemokine receptor CCR7 (also known as CD197). This finding may contribute to the regulation of immune and inflammatory responses, thus protecting the host from potential tissue damage and inflammation. Protein(s) secreted by S. boulardii have demonstrated the anti-inflammatory potential through several mechanisms. Protein(s) secreted by S. boulardii inhibit the production of pro-inflammatory cytokines mediated by the inflammatory NF-κB. These microorganisms also simultaneously regulate the activity of ERK1/2 and p38 from the MAPK family, which further affects inflammatory response. Specifically, the microorganisms stimulate the production of IL-8, an important chemokine involved in leukocyte migration in inflammatory responses, but also lead to cell necrosis^97,105. In addition, S. boulardii inhibits the proliferation of naive T cells, which helps to control overactivation of the immune response^97,105,106. More importantly, these microorganisms activate the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ). PPAR-γ is a nuclear receptor that participates in the regulation of various biological processes, including inflammation, metabolism, and cell proliferation¹⁰⁵.

Antigen penetration caused by intestinal mucosal destruction is one of the main factors contributing to the pathogenesis of many inflammatory diseases. Treatment with S. boulardii reduced the intestinal permeability and plasma endotoxin in CTC-treated rats¹⁰⁴. Probiotics enhance intestinal barrier function by regulating the cytoskeleton and phosphorylation of tight junction proteins, such as ZO-1^96,105. S. boulardii-induced intestinal nutritional effects include enhanced brush boundary enzyme activity, increased sIgA secretion in intestinal fluids, and enhanced production of polymeric Ig receptor in villi and crypt cells^101,106,107. In addition, the release of spermidine, which is necessary for cell division and differentiation, also has specific effects on the regeneration of mucosal epithelium^107,108. Many digestive enzymes and nutrient transporters may be induced by polyamines secreted by S. boulardii, which promote RNA binding and stabilization, thereby promoting growth and protein synthesis⁹². S. boulardii regulates the gut microbiota, stimulates the production of SCFAs, inhibits the release of and various enzymes, such as lactase, alkaline phosphatase, glucose amylase, and A-glucosidase maltase, from the brush boundary and also induces the maturation of IECs^60,92,100. SCFAs, especially butyrate, inhibit histone deacetylation. This inhibitory causes the group egg white to be in a more acetylated state, thus affecting chromatin organization and activating genes involved in cell differentiation, apoptosis, and cell cycle arrest in malignant cells and downregulating the expression of inflammatory factors in intestinal mucosa cells¹⁰⁹. S. boulardii helps to increase the levels of the anti-inflammatory cytokine, IL-10, and decreases the levels of the proinflammatory cytokines, such as IL-6 and TNF-α^{92,104,106,110}. Apparently, beneficial fungi are capable of creating a favorable environment for the antitumor effect.

At present, three clinical trials of S. boulardii have been completed, and the project numbers are NCT01895530, NCT01609660, and NCT03358511. The first two studies determined the impact of probiotics on the intestinal microbiota and the association with postoperative outcome after colorectal surgery. The third study looked at modifying the gut microbiome to target breast cancer.