Cancer types with lower mutational load and a non-permissive tumor microenvironment are intrinsically resistant to immune checkpoint blockade. While the combination of cytostatic drugs and immunostimulatory antibodies constitutes an attractive concept for overcoming this refractoriness, suppression of immune cell function by cytostatic drugs may limit therapeutic efficacy. Here we show that targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) does not impair dendritic cell-mediated T cell priming and activation. Accordingly, combining MEK inhibitors (MEKi) with agonist antibodies (Abs) targeting the immunostimulatory CD40 receptor results in potent synergistic antitumor efficacy. Detailed analysis of the mechanism of action of MEKi shows that this drug exerts multiple proimmunogenic effects, including the suppression of M2-type macrophages, myeloid derived suppressor cells and T-regulatory cells. The combination of MEK inhibition with agonist anti-CD40 Ab is therefore a promising therapeutic concept, especially for the treatment of mutant Kras-driven tumors such as pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor entity with dismal prognosis. PDAC is characterized by an extensive desmoplastic stroma and the tumor microenvironment (TME) has been recognized to play crucial roles in tumor development, therapy resistance, progression and metastasis. Although different cell types of the TME have been molecularly analyzed, tumor-infiltrating neurons could not be considered so far, because their cell bodies are located in adjacent ganglia of the peripheral nervous system and thus they are not part of the tumor mass (Demir, Thiel et al, Nature Cancer 2020; Demir, Thiel et al. Cancer Cell 2020). One of the unique features of PDAC is the strong infiltration by nerve fibers which correlates directly with tumor malignancy. In recent years it has been shown that either neurotransmitters secreted by neurons, or modulators of neuronal infiltration secreted by cancer cells can influence tumor growth. Here we use 3D light sheet fluorescence microscopy imaging (LSFM) of full tissue cleared PDAC specimen of tumors growing in genetically engineered mouse models or patient-derived xenograft models. Our data showthat these PDACs are innervated by a complex array of sensory and sympathetic neurons. To molecularly analyse neurons innervating the tumor mass, we have developed a novel approach we termed Trace’n’seq. This combines retrograde axonal tracing and FACS analysis with single cell RNA sequencing with the aim to identify, isolate and analyse hundreds of traced neurons infiltrating tumors or their respective healthy organs. Thereby, we have identified previously undescribed neuronal cell types and provide a landscape of sympathetic and sensory nerves innervating the healthy pancreas as well as PDAC. Our single cell transcriptomic analysis revealed signs of reprogramming of neuronal behaviour orchestrated by the tumor cells. These datasets combined with single cell profiling of PDAC cells and its associated stroma provide a first complete, unbiased insight into neurons infiltrating both healthy pancreas and PDAC. Based on a comprehensive interactome analysis of the interplay between tumor cells, stroma and the neurons, we have identified various candidate genes involved in tumor-nerve signalling. We are currently performing gain-and loss- of function approaches to dissect the role of individual factors. In summary, with Trace'n'seq, we have developed a novel technology pipeline to isolate tissue innervating neurons to investigate the role of neuronal infiltration in the development and progression of PDAC. Citation Format: Vera Thiel, Simon Renders, Jasper Panten, Julian Mochayedi, Rienk Offringa, Martin Sprick, Andreas Trumpp. Trace'n'Seq: Assessing neuronal infiltration and its impact in pancreatic ductal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3650.