Introduction

Esophageal squamous cell carcinoma (ESCC) is associated with poor prognosis despite advances in neoadjuvant immunochemotherapy (NICT). With highly variable treatment responses, identifying reliable predictive biomarkers remains crucial. While gut microbiome influences on immunotherapy efficacy are increasingly recognized, the specific role of the fungal microbiome (mycobiome) in NICT outcomes for ESCC remains unexplored, representing a significant gap in developing personalized treatment strategies.

Aims & Methods

This study aimed to characterize gut mycobiome signatures in ESCC patients and develop fungal biomarker-based predictive models for NICT response. We conducted ITS2 sequencing on 136 fecal samples collected from 68 ESCC patients (pre- and post-NICT) and 19 healthy controls (HC). Patients were stratified as responders (R, n=26) or non-responders (NR, n=42) based on tumor regression grade (TRG) scores. We performed comprehensive analyses of mycobiome diversity, composition, co-occurrence networks, and functional pathways across groups. The Boruta algorithm was employed for feature selection to identify fungal biomarkers, followed by multilayer perceptron (MLP) machine learning with five-fold cross-validation to develop predictive models. Additionally, ESCC xenograft models were established to investigate gut mycobiome-immune interactions through fecal microbiota transplantation (FMT) and targeted fungal manipulation experiments.

Results

ESCC patients demonstrated significant mycobiome dysbiosis compared to healthy controls, characterized by reduced fungal alpha diversity and distinct beta diversity patterns. At baseline, ESCC patients showed enrichment of Rhodotorula minuta, Actinomucor elegans, and several Candida species. Responders exhibited significantly higher baseline fungal diversity than non-responders, with enrichment of Meyerozyma, Candida boidinii, Trichosporon dermatis, and Cryptococcus species, whereas non-responders showed predominance of Saccharomyces, Nakaseomyces, and pathogenic Candida species (C. glabrata, C. parapsilosis, C. albicans). Responders also displayed more complex, stable fungal co-occurrence networks with higher density and clustering coefficients. Post-NICT, both groups showed partial recovery of mycobiome diversity, with significant increases in beneficial fungal taxa. Functional pathway analysis revealed distinct metabolic profiles between responders and non-responders. Using the Boruta algorithm, we identified 23 genus-level and 22 species-level fungal biomarkers that demonstrated high predictive value. The MLP model based on genus-level markers achieved AUCs of 90.2% (training) and 91.9% (test), while the species-level model showed comparable performance. In animal experiments, mice receiving R-FMT exhibited reduced tumor growth and enhanced CD8+ T cell infiltration compared to NR-FMT recipients. Notably, Candida boidinii supplementation synergized with anti-PD-1 therapy, while Saccharomyces depletion enhanced treatment sensitivity.

Conclusion

Our findings establish the gut mycobiome as a powerful predictor of NICT response in ESCC, with machine learning models achieving >90% accuracy using fungal biomarkers. Experimental validation not only confirms mechanistic links between specific fungi and antitumor immunity but also demonstrates that targeted mycobiome modulation can enhance immunotherapy efficacy. This dual approach of prediction and intervention offers a novel framework for precision oncology in ESCC, potentially transforming clinical decision-making and treatment outcomes in this challenging malignancy.

References

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Introduction

The interplay of diet and the intestinal microbiome plays a key role in developing colorectal cancer (CRC), with westernization of diets, e.g., an increase in fat and sugar and a decrease in fiber content, being the main contributor. We recently demonstrated that the endoplasmic reticulum unfolded protein response (ER-UPR) effector activating transcription factor 6 (ATF6) is associated with early and late onset of CRC in patients. Furthermore, the overexpression of ATF6 observed in a subset of CRC patients is associated with the presence of certain gut bacteria. The specific expression of activated ATF6 (nATF6) in mouse intestinal epithelial cells (nATF6^IEC) induces spontaneous development of colonic tumors in a microbiota-dependent manner, highlighting the functional relevance of ATF6 as a novel oncogenic driver.

Aims & Methods

To elucidate the adverse role of dietary fat, sugar, and fiber in tumorigenesis, we exposed nATF6^IEC mice under SPF conditions to control (chow) and westernized diets for 7 weeks after weaning. Microbial and metabolite profiles were assessed through deep metagenomic sequencing and targeted/untargeted mass spectrometry (LC-MS) analysis. Microbiota transfer of diet-exposed mice into germ-free recipients was conducted to verify the microbiota-dependent transmissibility of tumor formation. Bismuth(II)subsalicylate (BSS) was used as a dietary additive to validate the impact of bacteria-produced H₂S on tumorigenesis.

Results

Westernized high lard (HL) diet, enriched in fat and sugar and depleted in fermentable fiber, increased tumor burden in biallelic nATF6^IEC mice. Unexpectedly, HL induced de novo tumorigenesis in otherwise tumor-free monoallelic nATF6^IEC mice. Surprisingly, modifying the HL diet by supplementing dietary fiber or lowering fat content failed to protect the HL-induced tumorigenic phenotype. Metabolite profiling clearly differentiated westernized from control diets and tumor bearing from control mice. Transferring microbiota from diet-exposed mice into germ-free nATF6^IEC mice was sufficient to trigger tumorigenesis. Metagenomic profiles of westernized diet-exposed mice identified an increased relative abundance of H₂S-producing Desulfovibrio species and genes related to dissimilatory sulfate reduction. Supplementing westernized diets with the H₂S-scavenger BSS resulted in a drastic change in microbial communities as well as the prevention of tumor formation in monoallelic nATF6^IEC mice.

Conclusion

These results highlight the ability of westernized diets to enhance nATF6-related tumorigenesis while altering metabolite and microbial profiles. Westernized diets favor the growth of sulfate-reducing Desulfovibrionaceae, possibly resulting in increased H₂S production. H₂S scavenging significantly reduces diet-induced colonic tumor susceptibility, clearly demonstrating the impact of diet in shaping cancer-related microbiome alterations.