Moritz Middelhoff

Nerve Growth Factor Promotes Gastric Tumorigenesis through Aberrant Cholinergic Signaling

Within the gastrointestinal stem cell niche, nerves help to regulate both normal and neoplastic stem cell dynamics. Here, we reveal the mechanisms underlying the cancer-nerve partnership. We find that Dclk1+ tuft cells and nerves are the main sources of acetylcholine (ACh) within the gastric mucosa. Cholinergic stimulation of the gastric epithelium induced nerve growth factor (NGF) expression, and in turn NGF overexpression within gastric epithelium expanded enteric nerves and promoted carcinogenesis. Ablation of Dclk1+ cells or blockade of NGF/Trk signaling inhibited epithelial proliferation and tumorigenesis in an ACh muscarinic receptor-3 (M3R)-dependent manner, in part through suppression of yes-associated protein (YAP) function. This feedforward ACh-NGF axis activates the gastric cancer niche and offers a compelling target for tumor treatment and prevention.

Three-Dimensional Gastrointestinal Organoid Culture in Combination with Nerves or Fibroblasts: A Method to Characterize the Gastrointestinal Stem Cell Niche

The gastrointestinal epithelium is characterized by a high turnover of cells and intestinal stem cells predominantly reside at the bottom of crypts and their progeny serve to maintain normal intestinal homeostasis. Accumulating evidence demonstrates the pivotal role of a niche surrounding intestinal stem cells in crypts, which consists of cellular and soluble components and creates an environment constantly influencing the fate of stem cells. Here we describe different 3D culture systems to culture gastrointestinal epithelium that should enable us to study the stem cell niche in vitro in the future: organoid culture and multilayered systems such as organotypic cell culture and culture of intestinal tissue fragments ex vivo. These methods mimic the in vivo situation in vitro by creating 3D culture conditions that reflect the physiological situation of intestinal crypts. Modifications of the composition of the culture media as well as coculturing epithelial organoids with previously described cellular components such as myofibroblasts, collagen, and neurons show the impact of the methods applied to investigate niche interactions in vitro. We further present a novel method to isolate labeled nerves from the enteric nervous system using Dclk1-CreGFP mice.

Dclk1-expressing tuft cells: critical modulators of the intestinal niche?

Dclk1-expressing tuft cells constitute a unique intestinal epithelial lineage that is distinct from enterocytes, Paneth cells, goblet cells, and enteroendocrine cells. Tuft cells express taste-related receptors and distinct transcription factors and interact closely with the enteric nervous system, suggesting a chemosensory cell lineage. In addition, recent work has shown that tuft cells interact closely with cells of the immune system, with a critical role in the cellular regulatory network governing responses to luminal parasites. Importantly, ablation of tuft cells severely impairs epithelial proliferation and tissue regeneration after injury, implicating tuft cells in the modulation of epithelial stem/progenitor function. Finally, tuft cells expand during chronic inflammation and in preneoplastic tissues, suggesting a possible early role in inflammation-associated tumorigenesis. Hence, we outline and discuss emerging evidence that strongly supports tuft cells as key regulatory cells in the complex network of the intestinal microenvironment.

β2 Adrenergic-Neurotrophin Feedforward Loop Promotes Pancreatic Cancer

Catecholamines stimulate epithelial proliferation, but the role of sympathetic nerve signaling in pancreatic ductal adenocarcinoma (PDAC) is poorly understood. Catecholamines promoted ADRB2-dependent PDAC development, nerve growth factor (NGF) secretion, and pancreatic nerve density. Pancreatic Ngf overexpression accelerated tumor development in LSL-Kras+/G12D;Pdx1-Cre (KC) mice. ADRB2 blockade together with gemcitabine reduced NGF expression and nerve density, and increased survival of LSL-Kras+/G12D;LSL-Trp53+/R172H;Pdx1-Cre (KPC) mice. Therapy with a Trk inhibitor together with gemcitabine also increased survival of KPC mice. Analysis of PDAC patient cohorts revealed a correlation between brain-derived neurotrophic factor (BDNF) expression, nerve density, and increased survival of patients on nonselective β-blockers. These findings suggest that catecholamines drive a feedforward loop, whereby upregulation of neurotrophins increases sympathetic innervation and local norepinephrine accumulation.

Histidine decarboxylase (HDC)-expressing granulocytic myeloid cells induce and recruit Foxp3+ regulatory T cells in murine colon cancer

ABSTRACT The colorectal tumor microenvironment contains a diverse population of myeloid cells that are recruited and converted to immunosuppressive cells, thus facilitating tumor escape from immunoediting. We have identified a genetically and functionally distinct subset of dynamic bone marrow myeloid cells that are characterized by histidine decarboxylase (HDC) expression. Lineage tracing in Hdc-CreERT2;R26-LSL-tdTomato mice revealed that in homeostasis, there is a strong bias by HDC+ myeloid cells toward the CD11b+Ly6Ghi granulocytic lineage, which was accelerated during azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colonic carcinogenesis. More importantly, HDC+ myeloid cells strongly promoted colonic tumorigenesis, and colon tumor progression was profoundly suppressed by diphtheria toxin A (DTA)-mediated depletion of HDC+ granulocytic myeloid cells. In addition, tumor infiltration by Foxp3+ regulatory T cells (Tregs) was markedly impaired following HDC+ myeloid cell depletion. We identified an HDC+ myeloid-derived Cxcl13/Cxcr5 axis that mediated Foxp3 expression and Treg proliferation. Ablation of HDC+ myeloid cells or disruption of the Cxcl13/Cxcr5 axis by gene knockdown impaired the production and recruitment of Tregs. Cxcl13 induction of Foxp3 expression in Tregs during tumorigenesis was associated with Stat3 phosphorylation. Overall, HDC+ granulocytic myeloid cells affect CD8+ T cells directly and indirectly through the modulation of Tregs and thus appear to play key roles in suppressing tumoricidal immunity.

Abstract LB-144: Tff2 labels pancreatic progenitors that lack proliferative potential during tissue regeneration but can serve as the origin of pancreatic cancer

While controversy over the existence of adult pancreatic stem cells persists, it is now appreciated that the acinar compartment of the pancreas harbors heterogeneous progenitors. Recent single-cell analysis also demonstrated the presence of molecularly distinct, albeit morphologically identical, acinar cell sub-lineages. Previously, using lineage-tracing approach, we reported the Dclk1+ facultative progenitors that are critical for pancreatic regeneration. Here, we identified a different pancreatic progenitor-like subpopulation which is labelled by trefoil factor 2 (Tff2), a known progenitor marker and capable of tracing multiple cell lineages in the stomach. In addition, Tff2 molecules have been shown to play a suppressive role in PDAC progression. We utilized constitutive Tff2Cre and inducible Tff2CreERT2-DTR mice which were generated through modification of a BAC allele. We crossed Tff2CreERT2-DTR with reporter mice (R26R-mTmG, -tdTomato) to trace Tff2 labeled cells, and found that Tff2 labels ~2 % of the overall population in the adult acinar compartment, which showed slow proliferation (1 year, descendants Citation Format: Zhengyu Jiang, Bernhard W. Renz, Marina Macchini, Tanaka Takayuki, Ryota Takahashi, Giovanni Valenti, Woosook Kim, Wenju Chang, Yoku Hayakawa, Kosuke Sakitani, Moritz Middelhoff, Zinaida Dubeykovskaya, Timothy Chu, Karan Nagar, Yagnesh Tailor, Chythra R. Chandregowda, Akanksha Anand, Samuel Asfaha, Alina C. Iuga, Timothy C. Wang. Tff2 labels pancreatic progenitors that lack proliferative potential during tissue regeneration but can serve as the origin of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-144. doi:10.1158/1538-7445.AM2017-LB-144