%0 Generic
%A Thiel, Vera
%C Heidelberg
%D 2024
%F heidok:33484
%R 10.11588/heidok.00033484
%T Analysis of neuronal infiltration in healthy pancreas and  pancreatic ductal adenocarcinoma
%U https://archiv.ub.uni-heidelberg.de/volltextserver/33484/
%X Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with a current 5-year survival rate of about 8%. In early non-metastatic stages, surgical resection with chemotherapy may be curative, but because diagnosis is usually too late, surgery is useful in only 20% of cases. Current systemic chemotherapy and/or radiotherapy, targeted- or immunotherapies achieve only short survival benefit. This is probably due to a lack of understanding about the molecular regulatory mechanisms and disease-promoting factors in PDAC. One of the histopathological hallmarks of PDAC is its extensive desmoplastic tumor stroma that includes striking infiltration by peripheral nerve fibers. The peripheral nervous system (PNS) orchestrates physiological organ function during homeostasis and stress. Many cancers in the trunk including PDAC are infiltrated by PNS neurons as part of a dynamic and complex tumor microenvironment. The extent of nerve infiltration correlates directly with tumor aggressiveness. Despite these rather classical observations the molecular and cellular processes connecting infiltrating neurons and the healthy or cancerous pancreas as well as the consequences of these remain poorly understood. This is due to current technical limitations including the fact that the cell bodies of tumor infiltrating neurons are not present in the tumor mass itself, but instead are located in the paravertebral and pre-aortic ganglia of the PNS. Thus, current expression analysis, including single cell transcriptomic analyses of tumor masses exclude these neurons, as their cell bodies are located up to 2 cm distant from the actual tumor in mice.   Here, I first describe the complex array of sensory and sympathetic neurons innervating the normal pancreas and PDAC using three dimensional light sheet fluorescence microscopy imaging (LSFM) of iDISCO cleared full tissues. Second, I report my newly developed  “Trace-n-seq” method, that combines retrograde axonal tracing from tissue back to the neuronal nuclei in the peripheral ganglia with FACS-based isolation of individual labeled PNS neurons. This allows to identify and isolate individual tissue infiltrating neurons which I further analyzed by single cell RNA sequencing. In total I characterized >2000 sympathetic and sensory neurons that infiltrate the healthy pancreas and PDAC as well as various other organs. My analysis identified novel types of infiltrating neuronal cells and provide a target-tissue specific transcriptomic landscape of the PNS. My data also suggests PDAC cell mediated reprogramming of infiltrating neuronal behavior. By using the transcriptomic data, I established a Pancreatic-Cancer-Nerve signature and dissected reciprocal neuronal interactions between PDAC cells and stromal cells generating a neuro-cancer-interactome. My data reveal that the reprogramming of neuronal behavior orchestrated by PDAC cells, persists after primary tumor resection. I show evidence that physical or pharmacological inactivation of sympathetic neurons reduces tumor burden.   Moreover, I show that Taxanes induce neuropathy of PDAC-infiltrating neurons as part of their anti-cancer activity, making them a stroma targeting drug beyond its role in inhibiting cell-cycle. In fact, inhibition of neuronal infiltration by systemic or local treatment strategies in combination with nab-Paclitaxel further increased anti-tumor efficacy.  In summary, I developed a new method that allows to identify and characterize infiltrating neurons at single cell resolution and offers new insights into the characteristics of the TME of PDAC. My data provides novel insights into the networks and functions of cancer-infiltrating neurons with direct clinical relevance. This opens new insights into how infiltrating neurons and PDAC cells interact offering novel targeting options and as mechanisms might be similar in other tumor entities, possibility to identify general tumor agnostic targeting options.