Background The relationship between intratumoral fungi and the progression of colorectal cancer (CRC) is largely unclear. We aim to determine the structure of the fungal community within a neoplasia (adenoma or CRC) and its association with genetic mutation in adenoma and CRC.

Methods 261 tissue biopsies from two different region cohorts of CRC (n=21) and adenoma (n=21) patients (multiple biopsies from adjacent and tumor tissues per individual) were collected. Microbial profiling was performed using 18S ribosomal RNA gene sequencing with subsequent investigation of microbiota diversities and heterogeneity. Correlations between microbiota dysbiosis and host genetic alterations (KRAS mutations and microsatellite instability) were also analysed in all tumour biopsies. In addition, an inter-kingdom network was established to elucidate the relationship of bacteria and fungi.

Results Alpha and beta diversity analyses showed significant differences between CRC and adenoma in both cohorts. We analysed the fungi composition in each cohort to generate an overview of the microbiota profile at the phylum level and the main phylum were Basidiomycota and Ascomycota. We then discovered that fungal communities within neoplasia were heterogeneous. Moreover, we found that the intra-neoplasia variation in the abundance of individual microbes changed along the adenoma-carcinoma sequence. At the fungal genus level, 11 fungi (e.g, Malassezia, Aspergillus and Candida) were enriched in CRCs and 1 fungus (Collapsidium) was depleted in CRC compared to adenoma, which was verified in cohort 2. There was a significant difference in intra-neoplasia microbiota (e.g, Hanseniaspora and Saccharomyces) between biopsies with and without KRAS mutation (P<0.05) or microsatellite instability (P<0.05), indicating the association of intratumoral microbial heterogeneity with host genetic alteration. Multi-kingdom networks showed that the link between fungi and bacteria was progressively stronger along with the adenoma-carcinoma sequence. Cooccurrence interactions between fungi and bacteria are mostly contributed by fungal Ascomycota and bacterial Proteobacteria in CRC, while Malasseziaceae and Aspergillaceae act as crucial hubs.

Conclusions This study provides an intra-neoplasia fungal profile of CRC carcinogenesis and explores fungal dysbiosis with host genetic alteration. We also revealed the fungi and bacteria interplay in contributing to CRC progression.