Pancreatic Ductal Adenocarcinoma (PDAC) remains one of the most lethal malignancies, projected to become the second leading cause of cancer death by 2040. The poor and heterogeneous prognosis, particularly in the Locally Advanced PDAC (LAD-PDAC) subgroup, highlights the urgent need for robust pre-treatment predictive biomarkers. Recent evidence implicates the intratumoral microbiota and metabolic reprogramming in disease progression and therapeutic response. This study aimed to characterize and integrate the microbial and metabolic signatures in pre-treatment tumor biopsies of LAD-PDAC patients, leveraging high-resolution sequencing to improve taxonomic clarity. 

A prospective cohort of LAD-PDAC patients (n=65) undergoing Endoscopic Ultrasound-Guided Fine Needle Biopsy (EUS-FNB) was enrolled. The microbial community structure was characterized using 16S rRNA gene sequencing of the extended V1–V9 regions to achieve higher species-level resolution compared to standard protocols. Untargeted metabolomics analysis was performed to identify the functional chemical landscape. Alpha and Beta diversity metrics were correlated with clinical and morphological variables, including surgical status, tumor size reduction following neoadjuvant therapy, and basal glucose levels. 

High-resolution sequencing successfully identified 267 genera and 767 species, enriched for taxa typically associated with the skin, oral cavity, and gut (e.g., C. acnes, K. pneumoniae, S. aureus, H. pylori, E. faecalis). The metabolomic profiling revealed 413 known metabolites, dominated by lipids and lipid-like molecules (~22.6%) and organic acids and derivatives (~23.6%). Crucially, the Beta-diversity of the microbial community emerged as a significant pre-treatment determinant: the microbial composition was significantly distinct in patients who later underwent surgical resection compared to those who did not (p=0.026). Furthermore, patients who showed a dimensional reduction in tumor size post-therapy presented a significantly different microbial profile compared to non-responders (p=0.031). No single microbial taxa or metabolite was found to be significantly discriminant after correction for multiple comparisons.

Our findings demonstrate that the overall structure and diversity of the intratumoral microbiota, rather than specific single markers, is a pre-existing biological feature significantly associated with critical clinical outcomes (resectability and response) in LAD-PDAC. The use of V1–V9 sequencing validated a highly complex microbial ecosystem. These results strongly advocate for the development of integrated multi-omics and Network Analysis models to uncover the functional interplay between microbes and metabolites, ultimately paving the way for personalized therapeutic stratification.