Abstract 4225: Analysis of the landscape of cancer-associated neurons (CANs) in diverse cancers through retrograde tracing and single cell molecular profiling

Abstract

Abstract Hyperinnervation emerges as a common hallmark in the tumor microenvironment (TME) of most cancers, with clinical studies establishing a direct link between increased innervation and poor prognosis. Despite the acknowledged pivotal role of the TME, previous molecular profiling excluded tumor-infiltrating neurons due to the unique anatomical characteristics of peripheral neurons. While their axons extend into tumor masses, their DNA- and mRNA-containing cell bodies reside in adjacent ganglia, rendering them absent from virtually all large data sets analyzing tumor material. To fill this critical gap of the role of neurons for cancer growth, therapy resistance and metastasis, we developed a multi-layered approach to unravel the molecular basis of the neural influence in a.o. pancreatic (PDAC), colon cancer and melanoma including their metastasis. First, we unveiled a complex neural network within tumors and metastases through tissue clearing and 3D imaging of patient-derived xenografts, GEMMs, and human samples. This approach was complemented by the introduction of 'Trace-n-Seq,' a novel methodology integrating retrograde axonal tracing, FACS analysis, and single-cell RNA sequencing of neurons innervating both healthy organs and tumors. Expanding this technique unveiled the molecular profiles of over 3000 individual sympathetic, parasympathetic, and sensory neurons infiltrating various tumors, metastases, and pancreatitis, which were compared to neurons innervating healthy organs. Thereby we identified novel neuronal subtypes and observed tumor-induced neuronal reprogramming. Our single-cell data, when integrated with scRNA sequencing of PDAC, unveiled comprehensive neuronal interactions. An interactome analysis identified key genes in tumor/stroma-nerve signaling. To address the functional consequence of hyperinnervation of PDAC tumors, we show data demonstrating that surgical or pharmacological denervation of tumors caused a reduction in tumor size, concomitant with diminished stromal and increased immune compartments. Our data also suggest that the anti-cancer activity of paclitaxel -not oxaliplatin- was partially dependent on its ability to directly target CANs. In fact, in a neoadjuvant setting, PDAC patients treated with Gemcitabine/nab-paclitaxel, but not FOLFIRINOX, displayed a reduction in neuronal structures, underscoring the clinical significance of these observations. In summary, our comprehensive approach has elucidated the role of neurons in PDAC, melanoma, pancreatitis, and metastasis, leveraging innovative tools for studying the molecular programs of CANs at the single-cell level. These insights not only unveil novel mechanisms orchestrated by neuronal activity but also pave the way for imminent clinical trials, aiming to combat PDAC by targeting cancer-associated neurons. Citation Format: Vera Thiel, Simon Renders, Jasper Panten, Daniel Azorin, Nicolas Dross, Albrecht Stenzinger, Sebastian Schölch, Frank Winkler, Martin Sprick, Andreas Trumpp. Analysis of the landscape of cancer-associated neurons (CANs) in diverse cancers through retrograde tracing and single cell molecular profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4225.