Unraveling Molecular Complexity through Single Cell approaches: From Olfactory Bulb Architecture to Dorsal Root Ganglia Remodeling in Pancreatic Cancer

The accessory olfactory system and the role of the nervous system in pancreatic cancer represent two critical yet underexplored areas in neuroscience and cancer biology. This thesis employs transcriptomic and spatial profiling techniques to investigate the cellular and molecular diversity of the olfactory bulb (OB), and examine the molecular changes in sensory neurons innervating cancer pancreas. In the first part of this work, I analyzed single-nucleus RNA sequencing (snRNA-seq) and spatial transcriptomics data to profile the cellular composition of the olfactory bulb (OB), with a particular emphasis on the distinct transcriptomic signatures of cell types in the accessory (AOB) and main olfactory bulb (MOB). By integrating datasets enriched for cells from both regions, I identified unique excitatory neuronal subpopulations in the AOB marked by genes such as Nrp2, Tbx21, and Fst, while Nrp1 specifically marked excitatory neurons in the MOB. Additionally, Trp73 marked cells within the glomerular layer of both regions. These findings reveal previously unrecognized molecular diversity within the AOB, adding to our understanding of the accessory olfactory system and provide insight into the conserved and region-specific features of the AOB and MOB. Spatial transcriptomic analysis not only validated these observations but also identified Cntn6 and Ndst4 as molecular markers for the AOB excitatory neurons. In the second part, I characterized sensory neurons innervating the pancreas in both healthy and KPC (pancreatic cancer) mouse models by analyzing single-cell RNA sequencing data generated after retrograde tracing. Neurofilament (NF) neurons emerged as the predominant DRG type, while non-peptidergic (NP) neurons were selectively reduced in KPC mice. Notably, Pdx1-CreERT2 transcripts were detected specifically in DRGs innervating the KPC pancreas, suggesting potential transfer of tumor model–derived RNA via extracellular vesicles. Gene expression analysis also revealed mitochondrial alterations, particularly in NP and peptidergic (PEP) neurons, indicating cancer-associated metabolic reprogramming. These transcriptomic findings were supported by immunofluorescence, which confirmed the relative abundance of DRG types innervating the pancreas, and by RT-qPCR, which validated the presence of transgene-derived transcripts in DRGs. Additionally, MitoRed staining combined with IF further validated mitochondrial changes at the protein level, reinforcing the transcriptional evidence of altered metabolic activity in DRGs during pancreatic cancer. Together, these findings underscore the dynamic interplay between cancer cells and the nervous system and suggest new avenues for early detection and therapeutic intervention in pancreatic cancer. Overall, this thesis advances our understanding of the molecular and cellular mechanisms underlying olfactory processing and cancer-neuron interactions.