Sensory Neuron Rewiring in Pancreatic Ductal Adenocarcinoma: from Single-Cell RNA Profiling to Extracellular Vesicles-based strategies

Background & Aims: Neuronal innervation of the pancreas has historically been characterized using marker-based classification and physiological studies, but its transcriptomic landscape remains only partially explored. A detailed molecular profile of pancreatic sensory neurons could provide insights into their role in health and disease, particularly in pancreatic ductal adenocarcinoma (PDAC), where neural remodeling influences tumor progression and pain signaling. Methods: Wild-type and PDAC mice were injected with the retrotracer Fast Blue into pancreatic or cancerous tissue. Dorsal root ganglia were cultured, and Fast Blue-positive sensory neurons were isolated, lysed, and analyzed using single-cell RNA sequencing. Data was validated using immunohistochemistry, organoid cultures and qPCR. Results: We performed transcriptomic profiling of sensory neurons innervating the pancreatic head and tail under normal and cancer conditions. Our analysis identified neurofilament-containing neurons (NF) as the predominant sensory subtype in both contexts, while non-peptidergic neurons (NP) were underrepresented in tumor-associated innervation. Differential gene expression analysis revealed a unique subset of genes upregulated in sensory neurons innervating pancreatic tumors, many linked to mitochondrial activity. Further validation also revealed the presence of transcripts transferred via extracellular vesicles (including a non-coding portion of the PDX1 gene), suggesting a novel mechanism of tumor-neuron interaction. Conclusions: Our findings provide a detailed characterization of pancreatic and pancreatic ductal adenocarcinoma sensory innervation. We identified a circulating long non-coding RNA of potential tumor origin in the PDAC mouse model, revealing new therapeutic and biomarker opportunities in pancreatic cancer.

Background & Aims: Extracellular vesicles (EVs) hold promise as therapeutic mRNA delivery tool, yet challenges remain in achieving efficient cargo loading, targeted uptake, and functional delivery. This study aimed to develop a modular EV engineering platform integrating RNA enrichment and cell-specific tropism for improved mRNA-based delivery. Methods: We employed biotechnology strategies to design EVs with three functional modules: (1) surface display of a fusogen, vesicular stomatitis virus glycoproteins (VSV-G WT and a mutant VSV-G MUT lacking cellular tropism but retaining fusogenicity); (2) active mRNA cargo loading using the MCP system (bacteriophage MS2 Coat Protein, an RNA-binding domain with high affinity and specificity for stem-loop motifs known as MS2 loops); and (3) pseudotyping via SNAP-tag-based conjugation of glycan-binding lectins (e.g., WGA and PNA). EVs were isolated by differential ultracentrifugation and their efficiency assessed via RT-qPCR and FACS reporter assays and characterized by Western blotting. Results: VSV-G WT enabled sufficient Cre mRNA delivery but lacked specificity, whereas VSV-G MUT allowed modular retargeting. WGA-conjugated VSV-G MUT EVs restored functional delivery, confirming lectin-mediated targeting. SNAP Cd8 Display MCP scaffolds effectively enriched Cre mRNA in small EVs (s-EV), outperforming other scaffolds (i.g., Transferrin SNAP MCP). Both large EVs (L-EV) and s-EV bearing both MCP domains and surface lectins (i.g., WGA) improved reporter cell expression despite significantly lower mRNA content, suggesting enhanced uptake as the primary driver. Western blot analysis revealed that EVs bearing both MCP and surface ligands were less enriched in canonical markers (e.g., Alix, TSG101) compared to their cognate EVs without the MCP, whereas SNAP-tag expression confirmed successful scaffold incorporation across all EV subtypes. Conclusions: We established a flexible EVs engineering strategy that enables combinatorial control over cargo loading and cell targeting. These findings provide a foundation for programmable EVs tailored for therapeutic mRNA delivery and highlight key parameters governing EVs function and specificity.