Blank control
Frozen tissue samples were first washed to remove residual Tissue-Tek OCT with 1 mL of 1x PBS followed by centrifugation at 3000 rpm for 15 minutes at room temperature. DNA extraction was subsequently carried out using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers instructions. FFPE samples were first deparaffinized with Deparaffinization Solution (Qiagen, Hilden, Germany) and then processed using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany), following the manufacturers protocol. In both cases, DNA concentration was assessed using the Qubit 3 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) with the dsDNA High Sensitivity Assay Kit. Microbiota profiling was conducted through amplification and sequencing of the bacterial 16S rRNA gene using the Ion Torrent platform (Thermo Fisher Scientific, Waltham, MA, USA). Library preparation and sequencing were performed according to previously described protocols [28]. Briefly, 5 ng of DNA from each sample were subjected to two parallel PCR reactions using the Ion 16S Metagenomics Kit. Each reaction underwent 25 cycles under the manufacturers recommended conditions. Two primer pools targeting seven hypervariable regions of the 16S rRNA gene were used: Pool 1 (V2, V4, V8) and Pool 2 (V3, V67, V9). Amplicons from both reactions were quantified with the Qubit Fluorometer, pooled in equimolar concentrations per sample, and purified using CleanPCR beads (CleanNA, Waddinxveen, The Netherlands). Barcoded libraries were then constructed using the Ion Plus Fragment Library Kit in combination with the Ion Xpress Barcode Adapters 196 Kit. Final libraries were quantified, diluted to a concentration of 30 pM, and pooled together. Template was prepared with the Ion 510 & Ion 520 & Ion 530 Kit  Chef and loaded onto an Ion 530 Chip through the Ion Chef instrument. The loaded chip was sequenced through an Ion S5 System (Thermo Fisher Scientific, Waltham, MA, USA).