Intratumoral fungus N. crassa is associated with worsened prognosis in ovarian cancer via modulation of extracellular matrix.

Objective: Landmark studies on intratumoral fungi (ITF) have raised concerns due to irreproducible results and data-analysis errors. We aimed to determine whether ITF exist in ovarian cancer (OvCa) and, if so, whether they play a role in disease biology.

Methods: Formalin-fixed, paraffin-embedded (FFPE) OvCa samples and multiple controls underwent operational decontamination, qPCR, ITS sequencing, and post hoc data decontamination. We also leveraged updated fungal reads from TCGA generated by the TCMbio group, which addressed human-read contamination and artificial inflation, to validate findings and assess prognostic associations. A murine syngeneic OVHM model with intratumoral N. crassa injection was established. Transcriptomic and metabolomic analyses were performed to explore mechanisms.

Results: Tumor-containing blocks harbored significantly higher fungal loads than environmental controls but had loads comparable to paraffin controls. Applying a two-pass decontamination filter reduced raw sequence features from 9,289 ASVs to 659 ASVs. We focused on high-abundance features present in human tissues but absent from xenografts and paraffin controls and identified one candidate, N. crassa, associated with unfavorable prognosis in OvCa. Integrating human and murine data, we found Neurospora correlated with eosinophils, whereas N. crassa itself was not immune-related. N. crassa promoted OvCa progression with downregulation of ILK and decreased ECM-receptor interaction.

Conclusions: Most ITF signals are likely contaminants. We identified N. crassa as associated with unfavorable prognosis in OvCa, potentially via modulation of the extracellular matrix.