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.
Stromal cell-derived factor-1 (SDF-1/CXCL12), the main ligand for CXCR4, is overexpressed in human cancer. This study addressed the precise contribution of SDF-1 to gastric carcinogenesis.
SDF-1 ...transgenic mice were created and a Helicobacter-induced gastric cancer model was used in combination with H/K-ATPase-IL-1β mice. Gastric tissue was analysed by histopathology and cells isolated from the stomach were analysed by molecular biological methods.
Analysis of the H/K-ATPase/SDF-1 transgenic (SDF-Tg) mice showed that SDF-1 overexpression results in significant gastric epithelial hyperproliferation, mucous neck cell hyperplasia and spontaneous gastric dysplasia (wild-type mice 0/15 (0%) vs SDF-Tg mice 4/14 (28.6%), p=0.042, Fisher exact test) but has minimal effects on inflammation. SDF-Tg mice also showed a dramatic expansion of α-smooth muscle actin-positive myofibroblasts and CXCR4-expressing gastric epithelial cells in the progenitor zone, both of which preceded the development of significant gastritis or dysplasia. Gremlin 1-expressing mesenchymal stem cells, the putative precursors of myofibroblasts, were also increased within the dysplastic stomachs of SDF-Tg mice and showed chemotaxis in response to SDF-1 stimulation. SDF-1 overexpression alone resulted in minimal recruitment of haematopoietic cells to the gastric mucosa, although macrophages were increased late in the disease. When SDF-Tg mice were crossed with H/K-ATPase-IL-1β mice or infected with Helicobacter felis, however, there were dramatic synergistic effects on recruitment of bone marrow-derived cells and progression to preneoplasia.
Activation of the SDF-1/CXCR4 axis can contribute to early stages of carcinogenesis primarily through recruitment of stromal cells and modulation of the progenitor niche.
We have previously described a synergistic interaction between hypergastrinemia and Helicobacter felis infection on gastric corpus carcinogenesis in FVB/N mice housed under specific-pathogen-free ...conditions. However, gastrin-deficient (GAS-KO) mice on a mixed C57BL/6/129Sv genetic background maintained in conventional housing were reported to develop spontaneous gastric antral tumors. Therefore, we investigated the role of gastrin in Helicobacter -associated gastric carcinogenesis in H. felis- infected mice on a uniform C57BL/6 background housed in specific-pathogen-free conditions. Hypergastrinemic transgenic (INS-GAS) mice, GAS-KO mice, and C57BL/6 wild-type mice were infected with H. felis for either 12 or 18 months. At 12 months postinfection, INS-GAS mice had mild corpus dysplasia, while B6 wild-type mice had either severe gastritis or metaplasia, and GAS-KO mice had only mild to moderate gastritis. At 18 months postinfection, both INS-GAS and B6 wild-type mice had both severe atrophic gastritis and corpus dysplasia, while GAS-KO mice had severe gastritis with mild gastric atrophy, but no corpus dysplasia. In contrast, both GAS-KO and B6 wild-type mice had mild to moderate antral dysplasia, while INS-GAS mice did not. H. felis antral colonization remained stable over time among the three groups of mice. These results point to a distinct effect of gastrin on carcinogenesis of both the gastric corpus and antrum, suggesting that gastrin is an essential cofactor for gastric corpus carcinogenesis in C57BL/6 mice.
Oral cancer patients suffer pain at the site of the cancer. Calcitonin gene related polypeptide (CGRP), a neuropeptide expressed by a subset of primary afferent neurons, promotes oral cancer growth. ...CGRP also mediates trigeminal pain (migraine) and neurogenic inflammation. The contribution of CGRP to oral cancer pain is investigated in the present study. The findings demonstrate that CGRP-immunoreactive (-ir) neurons and neurites innervate orthotopic oral cancer xenograft tumors in mice. Cancer increases anterograde transport of CGRP in axons innervating the tumor, supporting neurogenic secretion as the source of CGRP in the oral cancer microenvironment. CGRP antagonism reverses oral cancer nociception in preclinical oral cancer pain models. Single-cell RNA-sequencing is used to identify cell types in the cancer microenvironment expressing the CGRP receptor components, receptor activity modifying protein 1
and calcitonin receptor like receptor (CLR, encoded by
).
and
transcripts are detected in cells expressing marker genes for Schwann cells, endothelial cells, fibroblasts and immune cells.
and
transcripts are more frequently detected in cells expressing fibroblast and immune cell markers. This work identifies CGRP as mediator of oral cancer pain and suggests the antagonism of CGRP to alleviate oral cancer pain.
Gastrin is secreted from a subset of neuroendocrine cells residing in the gastric antrum known as G cells, but low levels are also expressed in fetal pancreas and intestine and in many solid ...malignancies. Although past studies have suggested that antral gastrin is transcriptionally regulated by inflammation, gastric pH, somatostatin, and neoplastic transformation, the transcriptional regulation of gastrin has not previously been demonstrated in vivo. Here, we describe the creation of an enhanced green fluorescent protein reporter (mGAS-EGFP) mouse using a bacterial artificial chromosome that contains the entire mouse gastrin gene. Three founder lines expressed GFP signals in the gastric antrum and the transitional zone to the corpus. In addition, GFP(+) cells could be detected in the fetal pancreatic islets and small intestinal villi, but not in these organs of the adult mice. The administration of acid-suppressive reagents such as proton pump inhibitor omeprazole and gastrin/CCK-2 receptor antagonist YF476 significantly increased GFP signal intensity and GFP(+) cell numbers in the antrum, whereas these parameters were decreased by overnight fasting, octreotide (long-lasting somatostatin ortholog) infusion, and Helicobacter felis infection. GFP(+) cells were also detected in the anterior lobe of the pituitary gland and importantly in the colonic tumor cells induced by administration with azoxymethane and dextran sulfate sodium salt. This transgenic mouse provides a useful tool to study the regulation of mouse gastrin gene in vivo, thus contributing to our understanding of the mechanisms involved in transcriptional control of the gastrin gene.
Abstract
Despite remarkable responses to immune checkpoint blockade across multiple tumor types, the clinical benefit in colorectal cancer (CRC) is limited to microsatellite unstable tumors. PD-L1 ...expression is a negative prognostic marker in CRC but correlates with a better response to PD-1 blockade. Here, we investigated the role of PD-L1 in colorectal tumorigenesis and evaluated the utility of targeting myeloid-derived suppressor cells (MDSCs) in combination with PD-1 blockade in mouse models of CRC. We generated knockin mice that conditionally express the murine Pdl1 gene (R26-LSL-Pdl1-EGFP) and crossed them with LysM-Cre mice to overexpress PD-L1 specifically in the myeloid lineage. Mice received azoxymethane (AOM; 10 mg/kg i.p.) followed one week later with 2.5% dextran sodium sulfate (DSS) in the drinking water for 7 days. AOM/DSS-treated control mice formed tumors at 10 weeks and developed adenocarcinoma at 17 weeks post-AOM. LysM-Cre; R26-PD-L1 mice treated with AOM/DSS showed markedly enhanced colorectal tumorigenesis, with a significant increase in tumor number and size and enlarged spleen. In both groups, AOM/DSS treatment led to a significant expansion of myeloid cells, particularly CD11b+Gr-1+ MDSCs, in the colon and spleen, along with decreased NK cells compared with untreated mice. Notably, AOM/DSS-treated LysM-Cre; R26-PD-L1 mice showed decreased intratumoral CD8+ T cell infiltration compared to control tumors. Furthermore, there was a significant decrease in the percentage of Ki-67+ cells in intratumoral CD8+ T cells, indicating attenuated anti-tumor immunity. Trefoil factor 2 (TFF2), a secreted anti-inflammatory peptide, inhibits colon tumor growth by suppressing the expansion of CD11b+Gr-1+ MDSCs. TFF2 fused with two carboxyl-terminal peptide and three Flag motifs (TFF2-CTP-Flag) prolonged the circulation time in blood but retained bioactivity. We induced tumors in R26-PD-L1 mice with AOM/DSS, administered fusion recombinant TFF2-CTP-Flag (300 ug i.p.) and/or anti-PD-1 (RMP1-14; 200 ug i.p.) three times a week starting at 10 weeks and 14 weeks, respectively, and examined tumors at 18 weeks post-AOM. R26-PD-L1 mice treated with anti-PD-1 + TFF2-CTP showed a marked reduction in tumor growth while anti-PD-1 monotherapy failed to suppress growth. The combination of anti-PD-1 and TFF2-CTP significantly increased tumor-infiltrating CD8+ T cells and concomitantly decreased intratumoral regulatory T cells and CD11b+Gr-1+ myeloid cells. These early findings suggest that TFF2 augments the response rate of CRC to PD-1 blockade, possibly through suppressing MDSC expansion, supporting the potential of TFF2-CTP in combination I-O treatment for CRC. We are currently testing the efficacy of combined TFF2-CTP and anti-PD-1 therapy in the AOM/DSS model with PD-L1-overexpressing LysM-Cre; R26-PD-L1 mice.
Citation Format: Woosook Kim, Na Fu, Phaneendra Duddempudi, Zinaida Dubeykovskaya, Steven Almo, Chandan Guha, Seth Lederman, Timothy Wang. Stabilized recombinant trefoil factor 2 (TFF2-CTP) enhances anti-tumor activity of PD-1 blockade in mouse models of colorectal cancer abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6640.
Abstract
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 <5%) with a proliferative peak at around 6 months. Analysis of Cre recombination of both Tff2CreERT2-DTR and Cre mice revealed proliferative heterogeneity among Tff2+ acinar cells. Interestingly, following caerulein-induced injury, pancreatic ductal ligation and partial pancreatectomy, Tff2 labeled cells, distinct from Dclk1+ acinar cells, did not show increased proliferation but remained unchanged or decreased in the number of clones, suggesting that Tff2+ progenitors are not the major drivers of acinar regeneration in response to injurious stimuli. Quantitative RT-PCR analysis revealed higher expression of Sox9 and c-Met in Tff2+ acinar progenitor-like cells vs. Tff2- acinar cells. Targeted expression of KRasG12D in Tff2+ cells in the adult pancreas through crosses to LSL-KRasG12D mice led to a spectrum of mPanIN formation from 6 to 12 months, but no progression to PDAC within 12 months. Upon caerulein treatment, the progression of PanIN lesions was dramatically accelerated, with approximately 30% of the mice developing aggressive PDAC. Embryonic activation of KRasG12D in Tff2+ cells in Tff2cre mice spontaneously initiated a robust PDAC in the absence of cerulein. Clonal expansion labeled by a multicolor reporter (R26R-Brainbow2.1) showed that PDACs were polyclonal, derived from multiple Tff2+ progenitor cells. Acute pancreatitis induced by caeruelein accelerated tumor development, and significantly shortened the survival of Tff2cre;LSL-Kras mice. Altogether, Tff2 labels a subpopulation of the larger acinar progenitor pool. Tff2+ acinar cells are not facultative progenitors but can serve as a cell of origin for PDAC.
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
The trefoil factor TFF2 is a member of a tripartite family of small proteins that is produced by the stomach and the colon. Recombinant TFF2, when applied intrarectally in a rodent model of hapten ...colitis, hastens mucosal healing and reduces inflammatory indexes. Additionally, TFF2 is expressed in immune organs, supporting a potential immunomodulatory and reparative role in the bowel. In this study we confirm that TFF2 is expressed in the colon and is specifically enriched in epithelial cells relative to colonic leukocytes. TFF2-deficient, but not TFF1-deficient, mice exhibit a more severe response to acute or chronic dextran sulfate (DSS)-induced colitis that correlates with a 50% loss of expression of TFF3, the principal colonic trefoil. In addition, the response to acute colitis is associated with altered expression of IL-6 and IL-33, but not other inflammatory cytokines. While TFF2 can reduce macrophage responsiveness and block inflammatory cell recruitment to the colon, the major role in limiting the susceptibility to acute colitis appears to be maintenance of barrier function. Bone marrow transfer experiments demonstrate that leukocyte expression of TFF2 is not sufficient for prevention of colitis induction but, rather, that the gastrointestinal epithelium is the primary source of TFF2. Together, these findings illustrate that epithelial TFF2 is an important endogenous regulator of gut mucosal homeostasis that can modulate immune and epithelial compartments. Because of its extreme stability, even in the corrosive gut lumen, TFF2 is an attractive candidate as an oral therapeutic scaffold for future drug development in the treatment of inflammatory bowel disease.
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