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The enteric neurotransmitter acetylcholine governs important intestinal epithelial secretory and immune functions through its actions on epithelial muscarinic Gq-coupled receptors such as M3R. Its role in the regulation of intestinal stem cell function and differentiation, however, has not been clarified. Here, we find that nonselective muscarinic receptor antagonism in mice as well as epithelial-specific ablation of M3R induces a selective expansion of DCLK1-positive tuft cells, suggesting a model of feedback inhibition. Cholinergic blockade reduces Lgr5-positive intestinal stem cell tracing and cell number. In contrast, Prox1-positive endocrine cells appear as primary sensors of cholinergic blockade inducing the expansion of tuft cells, which adopt an enteroendocrine phenotype and contribute to increased mucosal levels of acetylcholine. This compensatory mechanism is lost with acute irradiation injury, resulting in a paucity of tuft cells and acetylcholine production. Thus, enteroendocrine tuft cells appear essential to maintain epithelial homeostasis following modifications of the cholinergic intestinal niche., Acetylcholine regulates intestinal epithelial secretion via muscarinic Gq-coupled receptors but its role in cell differentiation is unclear. Here, the authors show that Prox1-positive endocrine cells are sensors for the cholinergic intestinal niche and can trigger increased differentiation of enteroendocrine DCLK1-positive tuft cells.

TFF2 is a small, secreted protein with anti-inflammatory properties. We previously have shown that TFF2 gene delivery via adenovirus (Ad-Tff2) suppresses colon tumor growth in colitis associated cancer. Therefore, systemic administration of TFF2 peptide could potentially provide a similar benefit. Because TFF2 shows a poor pharmacokinetic, we sought to modify the TFF2 peptide in a manner that would lower its clearance rate but retain bioactivity. Given the absence of a sequence-based prediction of TFF2 functionality, we chose to genetically fuse the C-terminus of TFF2 with the carboxyl-terminal peptide of human chorionic gonadotropin β subunit, and inserted into adenoviral vector that expresses Flag. The resulting Ad-Tff2-CTP-Flag construct translates into a TFF2 fused with two CTP and three Flag motifs. Administered Ad-Tff2-CTP-Flag decreased tumorigenesis and suppressed the expansion of myeloid cells in vivo. The fusion peptide TFF2-CTP-Flag delivered by adenovirus Ad-Tff2-CTP-Flag as well purified recombinant fusion TFF2-CTP-Flag was retained in the blood longer compared with wild-type TFF2 delivered by Ad-Tff2 or recombinant TFF2. Consistently, purified recombinant fusion TFF2-CTP-Flag suppressed expansion of myeloid cells by down-regulating cyclin D1 mRNA in vitro. Here, we demonstrate for the very first time the retained bioactivity and possible pharmacokinetic advantages of TFF2 with a modified C-terminus.

Mist1 was recently shown to identify a discrete population of stem cells within the isthmus of the oxyntic gland within the gastric corpus. Chief cells at the base of the gastric corpus also express Mist1. The relevance of Mist1 expression as a marker of specific cell populations within the antral glands of the distal stomach, however, is unknown. Using Mist1-CreERT mice, we revealed that Mist1+ antral cells, distinct from the Mist1+population in the corpus, comprise long-lived progenitors that reside within the antral isthmus above Lgr5+or CCK2R+cells. Mist1+ antral progenitors can serve as an origin of antral tumors induced by loss of Apc or MNU treatment. Mist1+ antral progenitors, as well as other antral stem/progenitor population, express Cxcr4, and are located in close proximity to Cxcl12 (the Cxcr4 ligand)-expressing endothelium. During antral carcinogenesis, there is an expansion of Cxcr4+ epithelial cells as well as the Cxcl12+perivascular niche. Deletion of Cxcl12 in endothelial cells or pharmacological blockade of Cxcr4 inhibits antral tumor growth. Cxcl12/Cxcr4 signaling may be a potential therapeutic target., National Institute of Health (U.S.) (grant U54CA126513), National Institute of Health (U.S.) (grant R01CA093405), National Institute of Health (U.S.) (grant R01CA120979), National Institute of Health (U.S.) (grant R01DK052778), Clyde Wu Family Foundation

Cancers of the gastrointestinal (GI) tract are among the most frequent and most lethal cancers worldwide. An important reason for this high mortality is that early disease is typically asymptomatic, and patients often present with advanced, incurable disease. Even in high-risk patients who routinely undergo endoscopic screening, lesions can be missed due to their small size or subtle appearance. Thus, current imaging approaches lack the sensitivity and specificity to accurately detect incipient GI tract cancers. Here we report our finding that a single dose of a high-sensitivity surface-enhanced resonance Raman scattering nanoparticle (SERRS-NP) enables reliable detection of precancerous GI lesions in animal models that closely mimic disease development in humans. Some of these animal models have not been used previously to evaluate imaging probes for early cancer detection. The studies were performed using a commercial Raman imaging system, a newly developed mouse Raman endoscope, and finally a clinically applicable Raman endoscope for larger animal studies. We show that this SERRS-NP-based approach enables robust detection of small, premalignant lesions in animal models that faithfully recapitulate human esophageal, gastric, and colorectal tumorigenesis. This method holds promise for much earlier detection of GI cancers than currently possible and could lead therefore to marked reduction of morbidity and mortality of these tumor types.

ObjectiveLong-standing chronic pancreatitis is an established risk factor for pancreatic ductal adenocarcinoma (PDAC). Interleukin-1β (IL-1β) has been associated in PDAC with shorter survival. We employed murine models to investigate the mechanisms by which IL-1β and chronic pancreatitis might contribute to PDAC progression.DesignWe crossed LSL-Kras+/G12D;Pdx1-Cre (KC) mice with transgenic mice overexpressing IL-1β to generate KC-IL1β mice, and followed them longitudinally. We used pancreatic 3D in vitro culture to assess acinar-to-ductal metaplasia formation. Immune cells were analysed by flow cytometry and immunohistochemical staining. B lymphocytes were adoptively transferred or depleted in Kras-mutant mice. B-cell infiltration was analysed in human PDAC samples.ResultsKC-IL1β mice developed PDAC with liver metastases. IL-1β treatment increased Kras+/G12Dpancreatic spheroid formation. CXCL13 expression and B lymphocyte infiltration were increased in KC-IL1β pancreata. Adoptive transfer of B lymphocytes from KC-IL1β mice promoted tumour formation, while depletion of B cells prevented tumour progression in KC-IL1β mice. B cells isolated from KC-IL1β mice had much higher expression of PD-L1, more regulatory B cells, impaired CD8+T cell activity and promoted tumorigenesis. IL-35 was increased in the KC-IL1β pancreata, and depletion of IL-35 decreased the number of PD-L1+B cells. Finally, in human PDAC samples, patients with PDAC with higher B-cell infiltration within tumours showed significantly shorter survival.ConclusionWe show here that IL-1β promotes tumorigenesis in part by inducing an expansion of immune-suppressive B cells. These findings point to the growing significance of B suppressor cells in pancreatic tumorigenesis.

Background & Aims The intestinal epithelium is maintained by long-lived intestinal stem cells (ISCs) that reside near the crypt base. Above the ISC zone, there are short-lived progenitors that normally give rise to lineage-specific differentiated cell types but can dedifferentiate into ISCs in certain circumstances. However, the role of epithelial dedifferentiation in cancer development has not been fully elucidated. Methods We performed studies with Bhlha15-CreERT, Lgr5-DTR-GFP, Apcflox/flox, LSL-Notch (IC), and R26-reporter strains of mice. Some mice were given diphtheria toxin to ablate Lgr5-positive cells, were irradiated, or were given 5-fluorouracil, hydroxyurea, doxorubicin, or dextran sodium sulfate to induce intestinal or colonic tissue injury. In intestinal tissues, we analyzed the fate of progeny that expressed Bhlha15. We used microarrays and reverse-transcription PCR to analyze gene expression patterns in healthy and injured intestinal tissues and in tumors. We analyzed gene expression patterns in human colorectal tumors using The Cancer Genome Atlas data set. Results Bhlha15 identified Paneth cells and short-lived secretory precursors (including pre-Paneth label-retaining cells) located just above the ISC zone in the intestinal epithelium. Bhlha15+ cells had no plasticity after loss of Lgr5-positive cells or irradiation. However, Bhlha15+ secretory precursors started to supply the enterocyte lineage after doxorubicin-induced epithelial injury in a Notch-dependent manner. Sustained activation of Notch converts Bhlha15+ secretory precursors to long-lived enterocyte progenitors. Administration of doxorubicin and expression of an activated form of Notch resulted in a gene expression pattern associated with enterocyte progenitors, whereas only sustained activation of Notch altered gene expression patterns in Bhlha15+ precursors toward those of ISCs. Bhlha15+ enterocyte progenitors with sustained activation of Notch formed intestinal tumors with serrated features in mice with disruption of Apc. In the colon, Bhlha15 marked secretory precursors that became stem-like, cancer-initiating cells after dextran sodium sulfate–induced injury, via activation of Src and YAP signaling. In analyses of human colorectal tumors, we associated activation of Notch with chromosome instability-type tumors with serrated features in the left colon. Conclusions In mice, we found that short-lived precursors can undergo permanent reprogramming by activation of Notch and YAP signaling. These cells could mediate tumor formation in addition to traditional ISCs.

In many solid tumors, parasympathetic input is provided by the vagus nerve, which has been shown to modulate tumor growth. However, whether cholinergic signaling directly regulates progression of pancreatic ductal adenocarcinoma (PDAC) has not been defined. Here, we found that subdiaphragmatic vagotomy in LSL-Kras+/G12D;Pdx1-Cre (KC) mice accelerated PDAC development, whereas treatment with the systemic muscarinic agonist bethanechol restored the normal KC phenotype, thereby suppressing the accelerated tumorigenesis caused by vagotomy. In LSL-Kras+/G12D;LSL-Trp53+/R172H;Pdx1-Cre mice with established PDAC, bethanechol significantly extended survival. These effects were mediated in part through CHRM1, which inhibited downstream MAPK/EGFR and PI3K/AKT pathways in PDAC cells. Enhanced cholinergic signaling led to a suppression of the cancer stem cell (CSC) compartment, CD11b+ myeloid cells, TNFα levels, and metastatic growth in the liver. Therefore, these data suggest that cholinergic signaling directly and indirectly suppresses growth of PDAC cells, and therapies that stimulate muscarinic receptors may be useful in the treatment of PDAC. Significance: Subdiaphragmatic vagotomy or Chrm1 knockout accelerates pancreatic tumorigenesis, in part via expansion of the CSC compartment. Systemic administration of a muscarinic agonist suppresses tumorigenesis through MAPK and PI3K/AKT signaling, in early stages of tumor growth and in more advanced, metastatic disease. Therefore, CHRM1 may represent a potentially attractive therapeutic target. Cancer Discov; 8(11); 1458–73. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333

// Xiaowei Chen 1, 2, 3 , Michael J. Churchill 2 , Karan K. Nagar 1, 2 , Yagnesh H. Tailor 1, 2 , Timothy Chu 1, 2 , Brittany S. Rush 2 , Zhengyu Jiang 1, 2 , Edwin B.C. Wang 1, 2 , Bernhard W. Renz 1, 2 , Hongshan Wang 1, 2 , Ming Chiu Fung 3 , Daniel L. Worthley 1, 2 , Siddhartha Mukherjee 2 , Timothy C. Wang 1, 2 1 Division of Digestive and Liver Disease, Columbia University, New York, NY, USA 2 Department of Medicine and Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA 3 Division of Biology, School of Life Science, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China Correspondence to: Timothy C. Wang, e-mail: tcw21@columbia.edu Keywords: histamine deficiency, mast cell, IL-17, myeloid-derived suppressor cell, colorectal cancer Received: July 17, 2015 Accepted: September 15, 2015 Published: September 26, 2015 ABSTRACT Food allergy can influence the development of colorectal cancer, although the underlying mechanisms are unclear. While mast cells (MC) store and secrete histamine, immature myeloid cells (IMC) are the major site of histidine decarboxylase (HDC) expression, the enzyme responsible for histamine production. From our earlier work, we hypothesized that histamine is central to the association between allergy and colorectal carcinogenesis through its influence on the MC-MDSC axis. Here, we show that in wild type (WT) mice, ovalbumin (OVA) immunization elicits a typical T H 2 response. In contrast, in HDC−/− mice, the response to OVA allergy is skewed towards infiltration by IL-17 expressing MCs. This response is inhibited by histamine treatment. The HDC−/− allergic IL-17-expressing MCs promote MDSC proliferation and upregulation of Cox-2 and Arg-1. OVA allergy in HDC−/− mice increases the growth of colon tumor cells in both the MC38 tumor cell implantation model and the AOM/DSS carcinogenesis model. Taken together, our results show that histamine represses IL-17-expressing MCs and their subsequent activation of MDSCs, attenuating the risk of colorectal cancer in the setting of food allergy. Targeting the MC-MDSC axis may be useful for cancer prevention and treatment in patients, particularly in those with food allergy.

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs)., National Institutes of Health (U.S.) (Grant R01 RHL115145A)

Myeloid-biased hematopoietic stem cells (MB-HSCs) play critical roles in recovery from injury, but little is known about how they are regulated within the bone marrow niche. Here we describe an auto-/paracrine physiologic circuit that controls quiescence of MB-HSCs and hematopoietic progenitors marked by histidine decarboxylase (Hdc). Committed Hdc+ myeloid cells lie in close anatomical proximity to MB-HSCs and produce histamine, which activates the H2 receptor on MB-HSCs to promote their quiescence and self-renewal. Depleting histamine-producing cells enforces cell cycle entry, induces loss of serial transplant capacity, and sensitizes animals to chemotherapeutic injury. Increasing demand for myeloid cells via lipopolysaccharide (LPS) treatment specifically recruits MB-HSCs and progenitors into the cell cycle; cycling MB-HSCs fail to revert into quiescence in the absence of histamine feedback, leading to their depletion, while an H2 agonist protects MB-HSCs from depletion after sepsis. Thus, histamine couples lineage-specific physiological demands to intrinsically primed MB-HSCs to enforce homeostasis. Chen et al. show that histidine decarboxylase (Hdc) marks quiescent myeloid-biased HSCs (MB-HSCs). Daughter myeloid cells form a spatial cluster with Hdc+ MB-HSCs and secrete histamine to enforce their quiescence and protect them from depletion, following activation by a variety of physiologic insults.

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.

Objective: To investigate the role of obesity-associated inflammation and immune modulation in gastric carcinogenesis during Helicobacter-induced chronic gastric inflammation. Design: C57BL/6 male mice were infected with H felis and placed on a high-fat diet (45% calories from fat). Study animals were analysed for gastric and adipose pathology, inflammatory markers in serum, stomach and adipose tissue, and immune responses in blood, spleen, stomach and adipose tissue. Results: H felis-induced gastric carcinogenesis was accelerated in diet-induced obese mice compared with lean controls. Obesity increased bone marrow-derived immature myeloid cells in blood and gastric tissue of H felis-infected mice. Obesity also led to elevations in CD4 T cells, IL-17A, granulocyte macrophage colony-stimulating factor, phosphorylated STAT3 and prosurvival gene expression in gastric tissue of H felis-infected mice. Conversely, in adipose tissue of obese mice, H felis infection increased macrophage accumulation and expression of IL-6, C-C motif ligand 7 (CCL7) and leptin. Finally, the combination of obesity and gastric inflammation synergistically increased serum proinflammatory cytokines, including IL-6. Conclusions: Here, we have established a model to study the molecular mechanism by which obesity predisposes individuals to gastric cancer. In H felis-infected mice, obesity increased proinflammatory immune responses and accelerated gastric carcinogenesis. Interestingly, gastric inflammation augmented obesity-induced adipose inflammation and production of adipose-derived factors in obese, but not lean, mice. Our findings suggest that obesity accelerates Helicobacter-associated gastric cancer through cytokine-mediated cross-talk between inflamed gastric and adipose tissues, augmenting immune responses at both tissue sites, and thereby contributing to a protumorigenic gastric microenvironment., National Institutes of Health (U.S.) (grant 5R01CA093405-11), Columbia University Medical Center (Naomi Berrie Diabetes Center, grant P30DK063608)

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 a muscarinic acetylcholine receptor-3 (M3R)-dependent manner, in part through suppression of Yes-Associated Protein (YAP) function. This feed-forward ACh-NGF axis activates the gastric cancer niche and offers a compelling target for tumor treatment and prevention.

// Guangchun Jin 1, 2 , Kosuke Sakitani 1 , Hongshan Wang 1, 3 , Ying Jin 1 , Alexander Dubeykovskiy 1 , Daniel L. Worthley 4 , Yagnesh Tailor 1 and Timothy C. Wang 1 1 Division of Digestive and Liver Diseases, Department of Medicine, Columbia University Medical Center, New York, NY, USA 2 The Research Institute, Yanbian University Hospital, Jilin, China 3 Department of General surgery, Zhongshan Hospital, Fudan University, Shanghai, China 4 South Australian Health and Medical Research Institute, University of Adelaide, Adelaide, South Australia, Australia Correspondence to: Timothy C. Wang, email: tcw21@columbia.edu Keywords: GPR56, progastrin, proliferation, stem cell, colorectal cancer Abbreviations: Human progastrin (hGAS), G-protein coupled receptor 56 (GPR56), 5-bromo-2’-deoxyuridine (BrdU), Bone morphogenetic protein (BMP). Received: November 02, 2016 Accepted: February 22, 2017 Published: March 23, 2017 ABSTRACT Overexpression of human progastrin increases colonic mucosal proliferation and colorectal cancer progression in mice. The G-protein coupled receptor 56 (GPR56) is known to regulate cell adhesion, migration, proliferation and stem cell biology, but its expression in the gut has not been studied. We hypothesized that the promotion of colorectal cancer by progastrin may be mediated in part through GPR56. Here, we found that GPR56 expresses in rare colonic crypt cells that lineage trace colonic glands consistent with GPR56 marking long-lived colonic stem-progenitor cells. GPR56 was upregulated in transgenic mice overexpressing human progastrin. While recombinant human progastrin promoted the growth and survival of wild-type colonic organoids in vitro , colonic organoids cultured from GPR56 −/− mice were resistant to progastrin. We found that progastrin directly bound to, and increased the proliferation of, GPR56-expressing colon cancer cells in vitro , and proliferation was increased in cells that expressed both GPR56 and the cholecystokinin-2 receptor (CCK2R). In vivo , deletion of GPR56 in the mouse germline abrogated progastrin-dependent colonic mucosal proliferation and increased apoptosis. Loss of GPR56 also inhibited progastrin-dependent colonic crypt fission and colorectal carcinogenesis in the azoxymethane (AOM) mouse model of colorectal cancer. Overall, we found that progastrin binds to GPR56 expressing colonic stem cells, which in turn promotes their expansion, and that this GPR56-dependent pathway is an important driver and potential new target in colorectal carcinogenesis.

Colorectal carcinogenesis is a multi-step process. While ~25% of colorectal cancers (CRCs) arise in patients with a family history (genetic predisposition), ~75% of CRCs are due to age-associated accumulation of epigenetic alterations which can result in the suppression of key tumor suppressor genes leading to mutations and activation of oncogenic pathways. Sporadic colon-carcinogenesis is facilitated by many molecular pathways of genomic instability which include chromosomal instability (CIN), micro-satellite instability (MSI) and CpG island methylator phenotype (CIMP), leading towards loss of homeostasis and onset of neoplastic transformation. The unopposed activation of Wnt/β-catenin pathways, either due to loss of APC function or up-regulation of related stimulatory pathways, results in unopposed hyperproliferation of colonic crypts, considered the single most important risk factor for colon carcinogenesis. Hypermethylation of CpG islands within the promoters of specific genes can potentially inactivate DNA repair genes and/or critical tumor suppressor genes. Recently, CpG methylation of the 5’ promoter of human (h) DCLK1 gene was reported in many human epithelial cancers, including colorectal cancers (CRCs), resulting in the loss of expression of the canonical long isoform of DCLK1 (DCLK1-L) in hCRCs. Instead, a shorter isoform of DCLK1 (DCLK1-S) was discovered to be expressed in hCRCs, from an alternate β promoter of DCLKL1-gene; the clinical and biological implications of these novel findings, in relation to recent publications is discussed.

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. Renz et al. show that catecholamines promote ADRB2-dependent pancreatic ductal adenocarcinoma development and secretion of neurotrophins (NT), which in turn promote tumor innervation leading to increased NE and tumor growth. Blockade of ADRB2 or NT receptors improves gemcitabine's therapeutic effect.

The regulation and stem cell origin of normal and neoplastic gastric glands are uncertain. Here, we show that Mist1 expression marks quiescent stem cells in the gastric corpus isthmus. Mist1⁺ stem cells serve as a cell-of-origin for intestinal-type cancer with the combination of Kras and Apc mutation and for diffuse-type cancer with the loss of E-cadherin. Diffuse-type cancer development is dependent on inflammation mediated by Cxcl12⁺ endothelial cells and Cxcr4⁺ gastric innate lymphoid cells (ILCs). These cells form the perivascular gastric stem cell niche, and Wnt5a produced from ILCs activates RhoA to inhibit anoikis in the E-cadherin-depleted cells. Targeting Cxcr4, ILCs, or Wnt5a inhibits diffuse-type gastric carcinogenesis, providing targets within the neoplastic gastric stem cell niche., National Institutes of Health (U.S.) (Grants 54CA126513, R01CA093405, R01CA120979, and R01DK052778)

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

Objective Progastrin is the incompletely cleaved precursor of gastrin that is secreted by G-cells in the gastric antrum. Both gastrin and progastrin bind to the CCK2 receptor ( Cckbr or CCK2R) expressed on a subset of gastric epithelial cells. Little is known about how gastrin peptides and CCK2R regulate gastric stem cells and carcinogenesis. Interconversion among progenitors in the intestine is documented, but the mechanisms by which this occurs are poorly defined. Design We generated CCK2R-CreERT mice and performed inducible lineage tracing experiments. CCK2R+ antral cells and Lgr5+ antral stem cells were cultured in a three-dimensional in vitro system. We crossed progastrin-overexpressing mice with Lgr5-GFP-CreERT mice and examined the role of progastrin and CCK2R in Lgr5+ stem cells during MNU-induced carcinogenesis. Results Through lineage tracing experiments, we found that CCK2R defines antral stem cells at position +4, which overlapped with an Lgr5 neg or low cell population but was distinct from typical antral Lgr5 high stem cells. Treatment with progastrin interconverts Lgr5 neg or low CCK2R+ cells into Lgr5 high cells, increases CCK2R+ cell numbers and promotes gland fission and carcinogenesis in response to the chemical carcinogen MNU. Pharmacological inhibition or genetic ablation of CCK2R attenuated progastrin-dependent stem cell expansion and carcinogenesis. Conclusions CCK2R labels +4 antral stem cells that can be activated and expanded by progastrin, thus identifying one hormonal trigger for gastric stem cell interconversion and a potential target for gastric cancer chemoprevention and therapy.

The tumor microenvironment contains a diverse population of myeloid cells that are recruited from bone marrow and converted to immunosuppressive cells, thus mediating tumor cell escape from immune checkpoint. We have identified a subset of dynamic bone marrow myeloid cells, which can be identified by histidine decarboxylase (Hdc) mRNA and GFP expression in Hdc-GFP transgenic mice. Gene expression profiling showed that Hdc-GFP+ CD11b+Gr1+ myeloid cells express higher levels of cell cycle promoting genes such as Ccnd2, Ccnd3 and Cdc14a, while cell proliferation repressors including Cdc14b, Cdc16 and Cdk4 were downregulated in Hdc-GFP+ myeloid cells. To further elucidate the role of Hdc+ myeloid cells in tumor progression, we performed lineage-tracing studies using Hdc-creERT2;R26-LSL-TdTomato reporter mice. Pulsed with tamoxifen, the majority of TdTomato+ cells were localized initially to a group of CD11b+Gr1+ myeloid cells representing the highest Hdc mRNA expression in bone marrow, spleen and blood. Later on, TdTomato+CD11b+Gr1- F4/80+ macrophages can be detected, indicating a hierarchy of Hdc+ myeloid cells in which Hdc+CD11b+Gr1+ myeloid cells reside at the apex. In general, CD11b+ myeloid cells have a relative short lifespan ( In a mouse model of azoxymethane (AOM)/DSS colorectal carcinogenesis, Hdc-creERT2;R26-LSL-TdTomato;R26-LSL-DTA mice were injected with 10 mg/kg AOM and followed by 3 circles of 10 days 1.5% DSS ad libitum in drinking water. We found that Hdc-TdTomato labeled a proportion of tumor infiltrating CD11b+Gr1+ myeloid cells that expressed higher levels of Arg-1, Cox2, and Pdl1 transcripts. Continuous depletion of Hdc+ myeloid cells by administration of tamoxifen chow to induce DTA (diphtheria toxin subunit A) killing in Hdc-expressing myeloid cells abrogated half of the tumor-infiltrating MDSCs and released tumoricidal CD8+ T cells (> 15-fold), leading to decreased tumor number. This tumor suppression could be rescued by Hdc+ CD11b+Gr1+ cell adoptive transfer. Serum chemokine profiling revealed that Hdc+ DTA mediated myeloid depletion also decreased serum chemokine levels, among which Cxcl13 decreased the most (>30-fold). Cxcl13 protein and Cxcl13 mRNA also decreased in the colonic tumor tissue in the Hdc+ myeloid cell depleted AOM/DSS treatment group. Along with reduction in Cxcl13 levels, we also detected a significant reduction of Foxp3+ Treg cells in the tumor frozen sections stained with antibody against Foxp3 compared to R26-LSL-TdTomato;R26-LSL-DTA controls received the same treatments. Pre-knockdown of Cxcl13 by Dicer-substrate SiRNA (DsiRNAs) in a co-culture of splenic Hdc+ CD11b+Gr1+ myeloid cells from colon tumor-bearing mice with splenic Foxp3-GFP+ Treg cells induced apoptosis and decreased numbers of GFP+ cells compared to the scramble knockdown control group. Taken together, our data suggest that Hdc marks a distinct subset of myeloid cells with greater potency for promoting tumorigenicity, in part through supporting Tregs and suppressing CD8+ Tcells in the tumor microenvironment. Citation Format: Xiaowei Chen, Yoshihiro Takemoto, Karan K. Nagar, Timothy H. Chu, Zhengyu Jiang, Wenju Chang, Richard A. Friedman, Yagnesh H. Tailor, Daniel L. Worthley, Timothy C. Wang. Histidine decarboxylase (Hdc)-expressing myeloid cells support Foxp3+ Treg cells and promote colorectal cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-272.

All developing and adult organs are supported by connective tissues. We recently demonstrated that Gremlin 1 expressing cells in the bone (osteochondroreticular stem cells) and the bowel (intestinal reticular stem cells) are connective tissue stem cells, during development and healing. The contribution of these stem cells to the desmoplasia surrounding gastrointestinal and skeletal cancers, however, is unknown. In this study we first established that typical markers of bone marrow skeletal stem cells, also identify colony forming unit-fibroblasts (CFU-Fs) in the tumour microenvironment (identified by CD45-Ter-119-CD31-CD1040a+CD105+). Next we tested the local origins of alpha-smooth muscle actin (Acta2) expressing cancer-associated fibroblasts in orthotopic (colonoscopically delivered MC38 and carcinogenesis AOM/DSS) mouse models of colorectal cancer. Using transgenic mouse models to lineage trace and report the connective tissues in the bone and bowel, including Grem1-creERT;R26-LSL-ZsGreen; Acta2-RFP and our Acta2-CreERT line, we found that normal Grem1-expressing and Acta2-expressing cells each contribute to some, but not all, of the reactive cancer-associated fibroblasts surrounding our mouse models of cancer. Whilst, neoplastic cells appear to make a significant contribution to cancer-associated fibroblasts in some other cancers, using an epithelial specific Cre (K19-cre) there was no contribution of epithelium to Acta2-expressing cells in our AOM-DSS colorectal cancer models. We investigated Grem1 and Acta2 derived cells from the tumor microenvironment and found that these cells were clonagenic. Finally, we compared their capacity to support the in vitro growth of colorectal normal and neoplastic organoids compared to other colonic mesenchymal cell types. We are currently examining the role of these cells on expanding intestinal stem cells in normal and neoplastic gastrointestinal glands and examining the secreted factors from these cells that are relevant to tumor initiation and spread. Citation Format: Tamsin Lannagan, Susan Woods, Laura Vrbanac, Miao Yang, Jia Ng, Tongtong Wang, Yagnesh Tailor, Samuel Asfaha, Timothy Wang, Daniel L. Worthley. Desmoplasia stem and progenitor cells within the tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1721.

Introduction and objectives: There is increasing evidence that chronic activation of sympathetic neurons can lead to increased noradrenaline levels in the tumor microenvironment (TME) in PDAC. However, the mechanisms by which adrenergic neurotransmitters are delivered to the TME are not well understood. In this study we aimed to investigate the effect of locally and systemically delivered catecholamines into the TME in two well established genetically engineered mouse models of PDAC (Pdx1-Cre/KRasG12D (KC) and Pdx1-Cre/KRasG12D/Trp53R172H (KPC)). Methods: Adrenergic signaling was induced or inhibited in KC or KPC mice and in pancreatic organoids using various pharmacological compounds. Adrenergic receptor expression was assessed by RT-PCR, WB and IHC. Downstream pathways were identified by phosphorylation assays and WB. Adrenergic signaling was modeled in vivo applying restraint stress. To elucidate the crosstalk between nerves and cancer cells, pancreatic spheres and PDAC cells were co-cultured with DRGs and neuronal plasticity was quantified. NGF secretion was measured by RT-PCR and ELISA. Sympathetic denervation of the pancreas and blockage of the 2-receptor was employed as a treatment strategy in KPC mice. Overexpression of NGF was targeted to the mouse pancreas using a novel NGF knockin mouse, which was crossed to KC and KPC mice. Results: ADRB2 was upregulated during pancreatic cancer development. Furthermore, in vitro stimulation of cells harboring an oncogenic KRas mutation with catecholamines resulted in significantly increased sphere forming efficiency. The non-specific -blocker propranolol and the selective 2-blocker ICI 118,551 inhibited this effect. Selective blockade of 2-adrenergic signaling suppressed pancreatic sphere formation caused by co-cultures with DRGs. NGF secretion was stimulated through beta2-adrenergic signaling. Furthermore, restraint stress accelerated PDAC development in KC mice. The effects of stress were prevented by treatment with the selective ADRB2 antagonist ICI 118,551. Specific β2-blocker treatment as well as sympathetic denervation in addition to GEM significantly increased the survival of mice with 3-5 mm tumors in comparison to controls treated by GEM alone. Targeted overexpression of NGF in the mouse pancreas using a novel NGF knockin mouse resulted in marked acceleration of PanIN lesions in the KC mouse and shortened overall survival in KPC mice significantly. Finally, retrospective analysis of a PDAC patient cohort revealed a extension of overall survival in PDAC patients on non-selective -blockers. Conclusions: Taken together, these studies suggest that increased catecholamines can engender a feed forward loop, whereby upregulation of NGF can lead to increased innervation and local NE accumulation, thereby enhancing tumor growth. Therapies targeting these adrenergic and NGF pathways may prove useful in the treatment and prevention of pancreatic cancer. Citation Format: Bernhard W. Renz, Marina Macchini, Yoku Hayakawa, Xiaowei Chen, Zhengyu Jiang, Takayuki Tanaka, C. Benedikt Westphalen, Yagnesh Tailor, Jens Werner, Axel Kleespies, Alina C. Iuga, Daniel L. Worthley, Kenneth P. Olive, Timothy C. Wang. A novel β 2 adrenergic-nerve growth factor feed forward loop promotes pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5111.

Intestinal crypts are maintained by long-lived intestinal stem cells (ISCs) which reside near the base of glands. Above the ISC pool, there are short-lived progenitors that can supply lineage-specific differentiated cell types into the villus. Notch and Wnt pathways play a key role for determining the stem/progenitor cell function and their cell fate, and regulating cancer development in the gastrointestine. Although it has been well established that ISCs are a major origin of intestinal cancer, it remains unknown whether intestinal progenitor population can give rise to cancer. We use Mist1-CreERT mice and induce Notch and Wnt activation by mating with LSL-Notch1-IC mice and/or Apc flox mice. We found that a bHLH transcriptional factor Mist1 is expressed in Paneth cells (Lysozyme+CD24high) as well as short-lived secretory progenitors (Lysozyme-CD24low) in the intestine. Mist1+ secretory progenitors are radio- and chemo-resistant, but do not show stem cell interconversion even after intestinal injury or Lgr5 ablation. However, aberrant Notch activation changes Mist1+ cells to Lgr5+ long-lived enterocyte progenitors, with loss of secretory lineage differentiation. Mist1+CD24low progenitor population can form intestinal organoids with Notch activation in vitro. Mist1+ secretory progenitors can give rise to intestinal cancer by simultaneous Notch and Wnt activation by loss of Apc gene, while loss of Apc in Mist1 lineage alone does not develop cancer. In the colon, Mist1 marks colonic secterory progenitors that become to lineage trace and can be a cancer-initiating cell after Notch activation or DSS-induced colonic injury. These results provide the clear evidence of cellular plasticity dependent on Notch signaling in the gut, and suggest short-lived progenitors as another cellular origin of cancer besides ISC pool. Citation Format: Yoku Hayakawa, Kosuke Sakitani, Woosook Kim, Yagnesh Tailor, Karan Nagar, Kazuhiko Koike, Samuel Asfaha, Timothy C. Wang. Mist1+ secretory progenitor cells can give rise to cancer in the intestine and colon. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 912.

Background/Aims: The parasympathetic nervous system plays an important role in the regulation of epithelial homeostasis and has also been postulated to play a role in tumorigenesis. However, the exact role of the vagus nerve in pancreatic carcinogenesis is not well understood. Here we study the effects of muscarinic signaling on pancreatic tumorigenesis in a genetically engineered mouse model of pancreatic cancer (PDx1-Cre/KRasG12D (KC). Methods: Mice were either vagotomized at 8 weeks or treated with a cholinergic agonist. Pancreatic tissue was collected and analyzed by immunohistochemistry and RT-PCR at 20 weeks of age; cells were isolated and assayed for colony and sphere forming assays. Different human (AsPC-1, BxPC-3, Mia PaCa-2, Panc-1) and murine (K-2548 and K-8282) pancreatic cancer cell lines were subjected to cholinergic and anti-cholinergic drugs and assayed by RT-PCR, Western blot and flow cytometry. Results: In pancreatic organoid cultures derived from pancreata harboring an oncogenic KRas mutation, cholinergic agonists suppressed sphere formation significantly. Furthermore, pharmacological inhibition or genetic knockout of the muscarinic M3 receptor abolished this effect in vitro. In human and murine pancreatic cancer cells, anchorage independent growth and tumor sphere forming capacity were reduced by pretreatment with cholinergic agonists. Further evaluation revealed that parasympathetic agonists decrease the CD44+CD24+EpCAM+ proved CSC's population. Vagotomy, when performed in KC mice at 8 weeks resulted in pancreatic cancer development in 20% of the animals at 20 week of age. Treatment with the direct muscarinic agonist bethanechol caused a significant delay of PanIN progression in KC mice. Conclusions: Taken together, our findings suggest that vagal innervation has a regulatory role in pancreatic tumorigenesis via M3 receptor-mediated suppression of CSCs. Since current surgical approaches to resection of PDAC from the head of the pancreas are necessarily associated with a parasympathetic denervation of the pancreas, and thereby a loss of its suppressive effect, this fact may might partly explain the high local recurrence rate of this dismal disease. Additional treatment with cholinergic agonists should be considered in future regimen. Citation Format: Bernhard W. Renz, Marina Macchini, Yoku Hayakawa, C. Benedikt Westphalen, Michael Churchill, Suchetak Kar, Xiaowei Chen, Karan Nagar, Yagnesh Tailor, Daniel L. Worthley, Alina C. Iuga, Ken P. Olive, Timothy C. Wang. Parasympathetic signaling suppresses pancreatic cancer development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5079. doi:10.1158/1538-7445.AM2015-5079

of villus formation at embryonic day (E)14.5. This analysis revealed crack-like extensions of the lumen from the apical surface into the epithelium, which lengthen over time. The tips of these cracks are associated with dividing cells. Because of this association and a precedent for lumen-forming cell divisions in the zebrafish neural keel, we speculated that these cells might generate cracks by trafficking apical proteins to the cytokinetic plane. Using intestinal explant cultures in concert with nocodazole-mediated synchronization of the cell cycle, we examined the trafficking of the apical protein Ezrin during cell division. We found that Ezrin is deposited along the cytokinetic plane of divisions at crack tips, defining new villus domains and segregating daughter cells onto adjacent villi. We call these specialized cell divisions e-divisions (lumen extending). We also noted a second type of cell division, which functions in intestinal growth, but is not associated with apical cracks and does not define new villus domains. We have named these divisions g-divisions (growth building). Eand g-divisions differ cell biologically in several ways: localization of apical proteins, segregation of daughter cells, midbody morphology, and angle of the mitotic spindle relative to the nearest overlying apical surface. Ezrin null mice have fused villi with underlying isolated secondary lumens. Such a phenotype has been thought to support the secondary lumen model of normal villus morphogenesis, because it was believed that the loss of Ezrin perturbs their fusion with the main lumen. However, we have now determined that dividing cells in Ezrin null mice have mis-oriented mitotic spindles; we speculate that mis-orientation of e-divisions may in fact cause ectopic lumens and fused villi in this model. Indeed, the characteristic phenotype of Ezrin null mice can be recapitulated in wild-type intestines after treatment with blebbistatin, a myosin II inhibitor that results in mis-oriented spindles both in vitro and in intestinal explant cultures. This indicates that correct spindle orientation is indispensible for proper villus morphogenesis. Further understanding of this process will open up new avenues of in vitro bioengineering of intestinal surface, potentially providing life-saving therapies for those with intestinal failure.

Gastrin is a hormone that binds to the CCK2 receptor and promotes proximal gastric cancer, but inhibits distal gastric cancer development. However, the precise roles of the CCK2 receptor, and its alternative ligand, progastrin, in gastric carcinogenesis have not been clarified. In this study, we found that progastrin accelerated antral proliferation and carcinogenesis through CCK2R+ antral stem cell expansion, using mouse gastric cancer models and transgenic mice lines including human progastrin-overexpressing (hGAS) mice, CCK2R knockout mice, Lgr5-CreERT-IRES-GFP knockin mice, and newly generated CCK2R-BAC-CreERT mice. Progastrin promoted gastric antral cancer development induced by MNU and/or H. felis. During carcinogenesis, progastrin increased the expression of Lgr5 and gland fission in response to the chemical carcinogen MNU. Genetic ablation of CCK2R diminished these progastrin-mediated effects. In vitro 3D culture experiments revealed that progastrin, but not amidated gastrin, significantly increased gastric organoid formation and growth in Noggin-free condition, effects that were ablated by a CCK2R inhibitor YF-476 or CCK2R gene deletion. In the antrum, CCK2R was expressed in an Lgr5low cell population that displayed stemness, which could be enhanced by progastrin. In the presence of progastrin, Lgr5lowCCK2R+ cells interconverted to Lgr5hi cells. Finally, we generated a new BAC-transgenic CCK2R-CreERT murine line, and lineage tracing experiments showed that CCK2R+ cells, which reside slightly above the base of the antrum, contained long-lived stem cells in vivo and in vitro. Chemical inhibition of CCK2R attenuated progastrin-dependent cancer development in mice. In conclusion, CCK2R labels Lgr5low antral stem cells that can be activated and expanded by progastrin. These findings may help the understanding of the underlying mechanism in gastric stem cell regulation by a CCK2R signal. Citation Format: Hayakawa Yoku, Guanchun Jin, Hongshan Wang, Xiaowei Chen, Christoph B. Westphalen, Samuel Asfaha, Daniel L. Worthley, Bernhard Renz, Hiroshi Ariyama, Zinaida A. Dubeykovskaya, Yoshihiro Takemoto, Ashlesha Mulay, Yagnesh Tailor, Duan Chen, Sureshkumar Muthupalani, James G. Fox, Shigeo Takaishi, Timothy C. Wang. CCK2R marks gastric antral stem cell and mediates antral carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4795. doi:10.1158/1538-7445.AM2014-4795

Introduction: Gastrointestinal (GI) tract dysbiosis and resulting inflammation are implicated in breast carcinogenesis. CD11b+GR1+ myeloid- derived suppressor cells (MDSCs) are important drivers of breast cancer. Here we report that in APCMin/+ mice treated with enteric Helicobacter hepaticus (H. hepaticus) MDSCs cells are dramatically expanded and, along with the pathogen H. hepaticus, are directly recruited to mammary gland lymph node, where they suppress anti-carcinogenic cytotoxic T-cells and promote procarcinogenic inflammation, respectively. Methods: In our H. hepaticus and specific pathogen free colony we inoculated female APCMin/+ mice with H. hepaticus and compared neoplastic and inflammatory outcomes to the non-infected controls. Results: Successful infection was confirmed by H. hepaticus specific qPCR and fluorescence in situ hybridization (FISH). H. hepaticus was identified not only within the small intestine but also within the mammary gland lymph node and mammary tumor tissue of the treated group. In APCMin/+ mice treated with chow diet, H. hepaticus-induced procarcinogenic CD11b+GR1+ cells within the lamina propria of the small intestine, the surrounding mesenteric lymph nodes, the spleen, peripheral blood and the mammary gland lymph nodes. In H. hepaticus infected mice, this was accompanied by increased splenic expression of IL-1β, IL-6 and GM-CSF and the increased expression of IL-1β, IL-6 and GM-CSF, IL-23a and IL-17a in the small intestine. Interestingly, adding high fat diet to H. hepaticus infected mice appeared to further promote the trafficking of MDSCs from the spleen into the peripheral blood and regional lymph nodes with increased serum inflammatory cytokines production cytokines, including IL-6. This appears to have been mediated, at least in part, by adipocyte signaling to myeloid precursors. Conclusions: We show the presence of H. hepaticus in mammary gland and intestine, which correlated with mammary gland tumor incidence and increased intestinal tumor multiplicity. The co-existence of enteric H. hepaticus infection appears to promote mammary tumorigenesis through upregulation of IL-1β, IL-6 and GM-CSF and the resulting trafficking of tumor promoting CD11b+GR1+ MDSCs. We are currently examining the route and broader relevance of H. hepaticus extra-intestinal translocation and whether this has direct effects on tumorigenesis, consistent with findings in other Helicobacter spp. Citation Format: Xiaowei Chen, Daniel L. Worthley, Zhongming Ge, Yagnesh Tailor, Christian Kaufman, Lenzie Cheaney, Lesley Kline, Gladys Y. Valeriano, Christoph B. Westphalen, Susan E. Erdman, James G. Fox, Timothy C. Wang. Helicobacter hepaticus contributes to mammary gland carcinogenesis through bacterial translocation and subsequent expansion of cancer-promoting myeloid-derived suppressor cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A100.

Background & Aims Progastrin stimulates colonic mucosal proliferation and carcinogenesis through the cholecystokinin 2 receptor (CCK2R) - partly by increasing the number of colonic progenitor cells. However, little is known about the mechanisms by which progastrin stimulates colonic cell proliferation. We investigated the role of bone morphogenetic proteins (BMPs) in progastrin induction of colonic cell proliferation via CCK2R. Methods We performed microarray analysis to compare changes in gene expression in the colonic mucosa of mice that express a human progastrin transgene, gastrin knockout mice, and C57BL/6 mice (controls); the effects of progastrin were also determined on in vitro colonic crypt cultures from cholecystokinin 2 receptor knockout and wild-type mice. Human colorectal and gastric cancer cells that expressed CCK2R were incubated with progastrin or Bmp2; levels of β-arrestin 1 and 2 were knocked down using small interfering RNAs. Cells were analyzed for progastrin binding, proliferation, changes in gene expression, and symmetric cell division. Results The BMP pathway was down-regulated in the colons of human progastrin mice compared with controls. Progastrin suppressed transcription of Bmp2 through a pathway that required CCK2R and was mediated by β-arrestin 1 and 2. In mouse colonic epithelial cells, down-regulation of Bmp2 led to decreased phosphorylation of Smads1/5/8 and suppression of inhibitor of DNA binding 4. In human gastric and colorectal cancer cell lines, CCK2R was necessary and sufficient for progastrin binding and induction of proliferation; these effects were blocked when cells were incubated with recombinant Bmp2. Incubation with progastrin increased the number of CD44+, bromodeoxyuridine+, and NUMB+ cells, indicating an increase in symmetric divisions of putative cancer stem cells. Conclusions Progastrin stimulates proliferation in colons of mice and cultured human cells via CCK2R- and β-arrestin 1 and 2-dependent suppression of Bmp2 signaling. This process promotes symmetric cell division. © 2013 by the AGA Institute.

Introduction: The cellular origin and contribution of mesenchymal stem cells (MSCs) in the gastrointestinal tract, bone marrow and tumor microenvironment are unknown. More specific markers of MSCs are needed. We hypothesized that Grem1 expression would be a specific marker of MSCs. Methods: We developed Grem1-BAC-CreERT, αSMA-BAC-CreERT, Grem1-BAC-EGFP-peptide 2A-DTR-peptide 2A-CreERT and Vimentin-BAC-CreERT transgenic lines to characterize the origin, function and kinetics of gastrointestinal, bone marrow and tumor-associated mesenchyme. These mice were crossed to reporters, Grem1fl/fl and diphtheria toxin associated lines. The cellular fate and function of the Grem1, αSMA and Vimentin expressing cells, was analyzed by direct fluorescence microscopy and immunostaining at several time points (up to 12 months) and in several cancer models. Results: In the bone marrow, Grem1 recombination was identified in a very rare population of CD45/Ter119/CD31 triple negative cells (0.004% of all cells from compact bone extraction). In the bone marrow, Grem1-EGFP identified a discrete population of perisinusoidal cells. Grem1-recombined cells dramatically expand within MSC cultures forming large colony forming units, with a CFU-F efficiency of 3%. Grem1-recombined cells could be differentiated in vitro into adipocytes, osteoblasts, chondrocytes and αSMA(+) myofibroblasts. Furthermore, these cells are long-lived, recoverable from the bone at least 9 months after tamoxifen induction and can be transplanted. In the small intestine, 24 hours after tamoxifen induction, there were single Grem1 recombined cells found immediately subjacent to the basement membrane. These initially rare αSMA(-) recombined cells expanded over the next 12 months to completely trace the periepithelial fibroblast sheath. In both the gut and bone marrow, perinatal tamoxifen induction led to dramatically accelerated lineage tracing. Syngeneic tumorigenicity and carcinogenesis studies (including hepatic metastatic model (splenic injection of MC38, C57Bl/6 colorectal cancer cell line), MNU/Hfelis and AOM/DSS gastrointestinal cancer models) in our Grem1-BAC-specific lines revealed that Grem1 marked a cellular origin of cancer-associated mesenchyme, although many cancer-associated fibroblasts also arise from a specific αSMA+ stromal population. The exact lineage relationship between these Grem1 and αSMA+ cells is being tested. Conclusions: Grem1-expression labels a specific mesenchymal progenitor in the bone and gut that can give rise to multiple mesenchymal lineages during development and in the peritumoral stroma. We are currently examining the functional relevance of these cells and Grem1 production in supporting the normal and pathological stem cell niche in the bone marrow, gut and tumor microenvironment. Citation Format: Daniel L. Worthley, Yiling Si, Samuel Asfaha, Benedikt Westphalen, Yagnesh Tailor, Michael Churchill, Jean-Philippe Pradere, Robert Schwabe, Siddhartha Mukherjee, Timothy Wang. Gremlin 1 labels a mesenchymal progenitor in the gastrointestinal tract, bone and tumor microenvironment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2299. doi:10.1158/1538-7445.AM2013-2299