Trefoil Factor Research Articles

Modulation of the Intestinal Barrier Integrity and Repair by Microbiota Extracellular Vesicles through the Differential Regulation of Trefoil Factor 3 in LS174T Goblet Cells.

Trefoil factor 3 (TFF3) plays a key role in the maintenance and repair of intestinal mucosa. TFF3 expression is upregulated by the microbiota through TLR2. At the posttranscriptional level, TFF3 is downregulated by miR-7-5p. Reduced TFF3 levels have been detected in the damaged tissue of IBD patients. Here, we investigate the regulation of TFF3 expression by microbiota extracellular vesicles (EVs) in LS174T goblet cells using RT-qPCR and inhibitors of the TLR2 or PI3K pathways. To evaluate the subsequent impact on epithelial barrier function, conditioned media from control and vesicle-stimulated LS174T cells were used to treat Caco-2 monolayers. The barrier-strengthening effects were evaluated by analysing the expression and subcellular distribution of tight junction proteins, and the repairing effects were assessed using wound-healing assays. The results showed a differential regulation of TFF3 in LS174T via EVs from the probiotic EcN and the commensal ECOR12. EcN EVs activated the TFF3 production through TLR2 and downregulated miR7-5-p through PI3K. Consistently, high levels of secreted TFF3 reinforced the tight junctions and stimulated wound healing in the Caco-2 cells. ECOR12 EVs did not cause these effects. TFF3 is a potential therapeutic target in IBD. This study contributes to understanding the molecular players (microbiota EVs) connecting gut microbes to health and may help in designing better nutritional interventions based on microbiota bioactive compounds.

Real-world implementation of non-endoscopic triage testing for Barrett's oesophagus during COVID-19.

The Coronavirus pandemic (COVID-19) curtailed endoscopy services, adding to diagnostic backlogs. Building on trial evidence for a non-endoscopic oesophageal cell collection device coupled with biomarkers (Cytosponge), an implementation pilot was launched for patients on waiting lists for reflux and Barrett's oesophagus surveillance. (i) To review reflux referral patterns and Barrett's surveillance practices. (ii) To evaluate the range of Cytosponge findings and impact on endoscopy services. Cytosponge data from centralized laboratory processing (trefoil factor 3 (TFF3) for intestinal metaplasia (IM), haematoxylin & eosin for cellular atypia and p53 for dysplasia) over a 2-year period were included. A total of 10 577 procedures were performed in 61 hospitals in England and Scotland, of which 92.5% (N = 9784/10 577) were sufficient for analysis. In the reflux cohort (N = 4074 with gastro-oesophageal junction sampling), 14.7% had one or more positive biomarkers (TFF3: 13.6% (N = 550/4056), p53: 0.5% (21/3974), atypia: 1.5% (N = 63/4071)), requiring endoscopy. Among samples from individuals undergoing Barrett's surveillance (N = 5710 with sufficient gland groups), TFF3-positivity increased with segment length (odds ratio = 1.37 per cm (95% confidence interval: 1.33-1.41, P < 0.001)). Some surveillance referrals (21.5%, N = 1175/5471) had ≤1 cm segment length, of which 65.9% (707/1073) were TFF3 negative. Of all surveillance procedures, 8.3% had dysplastic biomarkers (4.0% (N = 225/5630) for p53 and 7.6% (N = 430/5694) for atypia), increasing to 11.8% (N = 420/3552) in TFF3+ cases with confirmed IM and 19.7% (N = 58/294) in ultra-long segments. Cytosponge-biomarker tests enabled targeting of endoscopy services to higher-risk individuals, whereas those with TFF3 negative ultra-short segments could be reconsidered regarding their Barrett's oesophagus status and surveillance requirements. Long-term follow-up will be important in these cohorts.

Salivary trefoil factor family peptide 3 (TFF3) and flow rate in persons with and without obstructive sleep apnea: A preliminary study.

Obstructive sleep apnea (OSA) is one of the most common chronic diseases. Trefoil factor family 3 (TFF3) peptides are secreted by major and minor salivary glands and may be involved in the pathogenesis of OSA. This study aimed to evaluate salivary TFF3 and flow rate between those with and without OSA. This was a prospective experimental study that enrolled patients with OSA and non-OSA. Total unstimulated saliva was collected, the salivary flow rate was measured, and theTFF3 level was analyzed by using a modified sandwich enzyme-linked immunosorbent assay. Baseline characteristics, TFF3 level, and salivary flow rate were compared between both groups. Factors associated with theTFF3 level and flow rate were computed by using multivariate linear regression analysis. Twenty-eight participants were recruited in the study: 20 patients with OSA (71.42%) and 8 non-OSA as control. The TFF3 and salivary flow rates between both groups of non-OSA versusOSA were comparable (TFF3 non-OSA 61.06 vs.OSA 96.00 ng/mg; p = .276 and flow rate non-OSA0.40 vs. OSA 0.35 mL/min; p = .320). Factors associated with theTFF3 level were neck circumference with a negative coefficient of -16.419 (p = .042). For the salivary flow rate, only age was a significant factor with the coefficient of -0.013 (p = .044). TFF3 and salivary flow rate were comparable between patients with OSA and non-OSA.The factor associated with TFF3 level was neck circumference, while age was negatively associated with the salivary flow rate in patients with OSA.

Circulating proteins and peripheral artery disease risk: observational and Mendelian randomization analyses.

We conducted observational and Mendelian randomization (MR) analyses to explore the associations between blood proteins and risk of peripheral artery disease (PAD). The observational cohort analyses included data on 257 proteins estimated in fasting blood samples from 12 136 Swedish adults aged 55-94 years who were followed up for incident PAD via the Swedish Patient Register. Mendelian randomization analyses were undertaken using cis-genetic variants strongly associated with the proteins as instrumental variables and genetic association summary statistic data for PAD from the FinnGen study (11 924 cases and 288 638 controls) and the Million Veteran Program (31 307 cases and 211 753 controls). The observational analysis, including 86 individuals diagnosed with incident PAD during a median follow-up of 6.6-year, identified 13 proteins [trefoil factor two, matrix metalloproteinase-12 (MMP-12), growth differentiation factor 15, V-set and immunoglobulin domain-containing protein two, N-terminal prohormone brain natriuretic peptide, renin, natriuretic peptides B, phosphoprotein associated with glycosphingolipid-enriched microdomains one, C-C motif chemokine 15, P-selectin, urokinase plasminogen activator surface receptor, angiopoietin-2, and C-type lectin domain family five member A] associated with the risk of PAD after multiple testing correction. Mendelian randomization analysis found associations of T-cell surface glycoprotein CD4, MMP-12, secretoglobin family 3A member 2, and ADM with PAD risk. The observational and MR associations for T-cell surface glycoprotein CD4 and MMP-12 were in opposite directions. This study identified many circulating proteins in relation to the development of incident PAD. Future studies are needed to verify our findings and assess the predictive and therapeutic values of these proteins in PAD.

Human TFF2-Fc fusion protein alleviates DSS-induced ulcerative colitis in C57BL/6 mice by promoting intestinal epithelial cells repair and inhibiting macrophage inflammation.

The clinical drugs for ulcerative colitis mainly affect the inflammatory symposiums with limited outcomes and various side effects. Repairing the damaged intestinal mucosa is a promising and alternative strategy to treat ulcerative colitis. Trefoil factor family 2 (TFF2) could repair the intestinal mucosa, however, it has a short half-life in vivo. To improve the stability of TFF2, we have prepared a new fusion protein TFF2-Fc with much stability, investigated the therapeutic effect of TFF2-Fc on ulcerative colitis, and further illustrated the related mechanisms. We found that intrarectally administered TFF2-Fc alleviated the weight loss, the colon shortening, the disease activity index, the intestinal tissue injury, and the lymphocyte infiltration in dextran sulfate sodium (DSS)-induced colitis mice. In vitro, TFF2-Fc inhibited Caco2 cells injury and apoptosis, promoted cellular migration, and increased the expression of Occludin and ZO-1 by activating P-ERK in the presence of H2O2 or inflammatory conditioned medium (LPS-RAW264.7/CM). Moreover, TFF2-Fc could reduce lipopolysaccharide (LPS)-induced production of inflammation cytokines and reactive oxygen species in RAW264.7 cells, and also inhibits the polarization of RAW264.7 cells to M1 phenotype by reducing glucose consumption and lactate production. Taken together, in this work, we have prepared a novel fusion protein TFF2-Fc, which could alleviate ulcerative colitis in vivo via promoting intestinal epithelial cells repair and inhibiting macrophage inflammation, and TFF2-Fc might serve as a promising ulcerative colitis therapeutic agent.

Alteration of secretory cell lineages and immune cell migration induced by acute tuft cell ablation in mouse intestine

Background: Intestinal tuft cells have only been recently investigated thoroughly due to their roles in type 2 immunity and chemosensory function. This rare population of intestinal epithelium is implicated in extensively interacting with other cell types and involved in nutrient absorption. Doublecortin-like kinase 1 (DCLK1) is widely used for tuft cell identification in the mouse intestine. However, the effects of tuft cell deletion on mouse intestines have not been fully understood. Here, we investigated the effects of tuft cell deletion on the epithelial cell lineages and overall impact on mouse intestine. Methods: Tuft cell deletion was induced in Dclk1-IRES-GFP-CreERT2/+; Rosa-DTA (Dclk1-DTA) mice by a single dose of tamoxifen injection. Cre-negative DTA/+ mice and DTA-negative Dclk1-CreERT2 mice of littermates received tamoxifen injection at same time as controls. Mouse bodyweight was monitored daily, and intestinal tissues were sampled 2, 4, or 7 days after the tamoxifen injection. Immunostaining of intestinal tissue sections was performed for lysozyme (LYZ), trefoil factor 3 (TFF3), cluster of differentiation 3 (CD3), DCLK1, mast cell protease 1 (MCPT1), Ly6G, and F4/80. Image analysis was conducted using Qupath. Results: Dclk1-DTA mice showed a significant decrease in bodyweight to 11% of their original weight on day 4 after tamoxifen injection. They showed significantly shortened small intestinal length compared to control mice, however, colonic length showed no significant difference. Immunofluorescent staining of induced Dclk1-DTA mouse tissues revealed that DCLK1+ tuft cells were decreased on day 2, almost completely deleted on day 4, and recovered on day 7. Overall brush border structures defined by villin and SGLT1 staining were intact. Correlated with the tuft cell reduction, mislocalized Paneth-like cells, identified with LYZ+ granules, were observed in the middle or upper part of villi with increasing frequency on day 4 and decreasing on day 7. To confirm goblet/Paneth intermediate cells, multiplexed immunofluorescent (MxIF) staining was employed with LYZ and TFF3. Double-positive (LYZ+/TFF3+) cells were significantly increased in the small intestine of tuft cell-depleted mice on day 4. Intraepithelial lymphocytes, mucosal neutrophils, and F4/80+ macrophages/dendritic cells did not show significant changes after the tuft cell deletion. This indicates that there is no inflammatory reactions following tuft cell deletion. Conclusion: Acute ablation of intestinal tuft cell may cause nutrient malabsorption and changes in mucosal defense response in vivo due to the alteration of cell differentiation lineage and the decrease in absorptive area. Our findings support the importance of tuft cell function in nutrient sensing and mucosal defense mechanism. NIH R01DK128190 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Intestinal activating transcription factor 4 regulates stress-related behavioral alterations via paraventricular thalamus in male mice

Chronic stress induces depression- and anxiety-related behaviors, which are common mental disorders accompanied not only by dysfunction of the brain but also of the intestine. Activating transcription factor 4 (ATF4) is a stress-induced gene, and we previously show that it is important for gut functions; however, the contribution of the intestinal ATF4 to stress-related behaviors is not known. Here, we show that chronic stress inhibits the expression of ATF4 in gut epithelial cells. ATF4 overexpression in the colon relieves stress-related behavioral alterations in male mice, as measured by open-field test, elevated plus-maze test, and tail suspension test, whereas intestine-specific ATF4 knockout induces stress-related behavioral alterations in male mice. Furthermore, glutamatergic neurons are inhibited in the paraventricular thalamus (PVT) of two strains of intestinal ATF4-deficient mice, and selective activation of these neurons alleviates stress-related behavioral alterations in intestinal ATF4-deficient mice. The highly expressed gut-secreted peptide trefoil factor 3 (TFF3) is chosen from RNA-Seq data from ATF4 deletion mice and demonstrated decreased in gut epithelial cells, which is directly regulated by ATF4. Injection of TFF3 reverses stress-related behaviors in ATF4 knockout mice, and the beneficial effects of TFF3 are blocked by inhibiting PVT glutamatergic neurons using DREADDs. In summary, this study demonstrates the function of ATF4 in the gut-brain regulation of stress-related behavioral alterations, via TFF3 modulating PVT neural activity. This research provides evidence of gut signals regulating stress-related behavioral alterations and identifies possible drug targets for the treatment of stress-related behavioral disorders.

Influence of the Infant Feeding on the Taxonomy of the Gut Microbiome and the Trefoil Factors Level in Children and Adolescents

Changes in the gut microbiome are recognized as an important component of obesity in both adults and children. One factor in the gut microbiome formation is the infant feeding type, which may also have a prolonged effect on the microbial community. Breast milk contributes to the formation of mucosal tolerance to the intestinal microbiota. In turn, trefoil factors (TFF2 and TFF3) are important components of the mucosal barrier. The aim was to study the composition of the gut microbiota and the trefoil factors level in the blood of children and adolescents with obesity, depending on the infant feeding type. The study included 93 non-obese children (Group 1) and 92 obese children (Group 2). Serum TFF2 and TFF3 levels were determined by enzyme immunoassay in each study participant. The taxonomic composition of the fecal microbiome was determined by metagenomic sequencing of the 16S rRNA gene. In general, the taxonomic composition of the gut microbiota in Groups 1 and 2 was similar. However, Group 2 had less by [Prevotella], Epulopiscium and Haemophilus and more by Clostridium and Catenibacterium. Neither obesity nor the infant feeding type of influenced the serum concentration of TFF2 and TFF3. However, the infant feeding has a prolonged effect on the gut microbiota, and in Group 2 this effect was less pronounced. In Group 1, breastfeeding led to the formation of a complete mucosal tolerance to the microbiome, which did not occur with mixed and bottle feeding. In Group 2, most of the “TFFs–gut microbiome” associations were positive, indicating an unfavorable interaction between intestinal wall and microbiome in obese children and adolescents. Thus, infant feeding type seems to be a weak but significant factor in the gut microbiome formation in children and adolescents, which also affects the formation of mucosal tolerance to the intestinal microbiota.

Effect of the Infant Feeding Type on Gut Microbiome Taxonomy and Levels of Trefoil Factors in Children and Adolescents

Effect of the Infant Feeding Type on Gut Microbiome Taxonomy and Levels of Trefoil Factors in Children and Adolescents

Proteomics profiling for the global and acetylated proteins of papillary thyroid cancers

BackgroundPapillary thyroid carcinoma (PTC) is the most common endocrine malignancy cancer among the malignancies of thyroid. Despite of wide usages of proteomics in PTC, the profile of acetylated proteins in PTC remains unsettled, which is helpful for understanding the carcinogenesis mechanism and identifying useful biomarkers for PTC.MethodsThe surgically removed specimens of cancer tissues (Ca-T) and adjacent normal tissues (Ca-N) from 10 female patients pathological diagnosed as PTC (TNM stage III) were enrolled in the study. After preparing the pooled extracts of the whole proteins and the acetylated proteins from 10 cases, TMT labeling and LC/MS/MS methods were applied to the assays of global proteomics and acetylated proteomics separately. Bioinformatics analysis, including KEGG, gene ontology (GO) and hierarchical clustering were performed. Some differentially expressed proteins (DEPs) and differentially expressed acetylated proteins (DEAPs) were validated by individual Western blots.ResultsControlled with the normal tissues adjacent to the lesions, 147 out of 1923 identified proteins in tumor tissues were considered as DEPs in global proteomics, including 78 up-regulated and 69 down-regulated ones, while 57 out of 311 identified acetylated proteins in tumor tissues were DEAPs in acetylated proteomics, including 32 up-regulated and 25 down-regulated, respectively. The top 3 up- and down-regulated DEPs were fibronectin 1, KRT1B protein and chitinase-3-like protein 1, as well as keratin, type I cytoskeletal 16, A-gamma globin Osilo variant and Huntingtin interacting protein-1. The top 3 up- and down-regulated DEAPs were ribosomal protein L18a-like protein, alpha-1-acid glycoprotein 2 and eukaryotic peptide chain release factor GTP-binding subunit ERF3A, as well as trefoil factor 3, thyroglobulin and histone H2B. Functional GO annotation and KEGG pathway analysis based on the DEPs and DEAPs showed completely different changing pictures. Contrary to the top 10 up- and -down regulated DEPs, most of which were addressed in PTC and other types of carcinomas, changes of the majority DEAPs were not mentioned in the literatures.ConclusionsTaken the profiling of the global and acetylated proteomics together will provide more broad view of protein alterations on the carcinogenesis and new direction for selecting biomarker for diagnosis of PTC.

IgG Fc-binding protein positively regulates the assembly of pore-forming protein complex βγ-CAT evolved to drive cell vesicular delivery and transport

Cell membranes form barriers for molecule exchange between the cytosol and the extracellular environments. βγ-CAT, a complex of pore-forming protein BmALP1 (two βγ-crystallin domains with an aerolysin pore-forming domain) and the trefoil factor BmTFF3, has been identified in toad Bombina maxima. It plays pivotal roles, via inducing channel formation in various intracellular or extracellular vesicles, as well as in nutrient acquisition, maintaining water balance, and antigen presentation. Thus, such a protein machine should be tightly regulated. Indeed, BmALP3 (a paralog of BmALP1) oxidizes BmALP1 to form a water-soluble polymer, leading to dissociation of the βγ-CAT complex and loss of biological activity. Here, we found that the B.maxima IgG Fc-binding protein (FCGBP), a well-conserved vertebrate mucin-like protein with unknown functions, acted as a positive regulator for βγ-CAT complex assembly. The interactions among FCGBP, BmALP1, and BmTFF3 were revealed by co-immunoprecipitation assays. Interestingly, FCGBP reversed the inhibitory effect of BmALP3 on the βγ-CAT complex. Furthermore, FCGBP reduced BmALP1 polymers and facilitated the assembly of βγ-CAT with the biological pore-forming activity in the presence of BmTFF3. Our findings define the role of FCGBP in mediating the assembly of a pore-forming protein machine evolved to drive cell vesicular delivery and transport.

The Forms of the Lectin Tff2 Differ in the Murine Stomach and Pancreas: Indications for Different Molecular Functions.

The lectin TFF2 belongs to the trefoil factor family (TFF). This polypeptide is typically co-secreted with the mucin MUC6 from gastric mucous neck cells, antral gland cells, and duodenal Brunner glands. Here, TFF2 fulfills a protective function by forming a high-molecular-mass complex with the MUC6, physically stabilizing the mucus barrier. In pigs and mice, and slightly in humans, TFF2 is also synthesized in the pancreas. Here, we investigated the murine stomach, pancreas, and duodenum by fast protein liquid chromatography (FPLC) and proteomics and identified different forms of Tff2. In both the stomach and duodenum, the predominant form is a high-molecular-mass complex with Muc6, whereas, in the pancreas, only low-molecular-mass monomeric Tff2 was detectable. We also investigated the expression of Tff2 and other selected genes in the stomach, pancreas, and the proximal, medial, and distal duodenum (RT-PCR analysis). The absence of the Tff2/Muc6 complex in the pancreas is due to a lack of Muc6. Based on its known motogenic, anti-apoptotic, and anti-inflammatory effects, we propose a protective receptor-mediated function of monomeric Tff2 for the pancreatic ductal epithelium. This view is supported by a report that a loss of Tff2 promotes the formation of pancreatic intraductal mucinous neoplasms.

Amphibian pore-forming protein βγ-CAT drives extracellular nutrient scavenging under cell nutrient deficiency

Nutrient acquisition is essential for animal cells. βγ-CAT is a pore-forming protein (PFP) and trefoil factor complex assembled under tight regulation identified in toad Bombina maxima. Here, we reported that B.maxima cells secreted βγ-CAT under glucose, glutamine, and pyruvate deficiency to scavenge extracellular proteins for their nutrient supply and survival. AMPK signaling positively regulated the expression and secretion of βγ-CAT. The PFP complex selectively bound extracellular proteins and promoted proteins uptake through endolysosomal pathways. Elevated intracellular amino acids, enhanced ATP production, and eventually prolonged cell survival were observed in the presence of βγ-CAT and extracellular proteins. Liposome assays indicated that high concentration of ATP negatively regulated the opening of βγ-CAT channels. Collectively, these results uncovered that βγ-CAT is an essential element in cell nutrient scavenging under cell nutrient deficiency by driving vesicular uptake of extracellular proteins, providing a new paradigm for PFPs in cell nutrient acquisition and metabolic flexibility.

Abstract 3023: Identifying colorectal cancer biomarkers using a targeted proteomics platform with extensive coverage

Abstract Despite advances in treatment, colorectal cancer remains one of the leading causes of cancer-specific death. To decrease colorectal cancer mortality, better and more cost-efficient tools for risk prediction and early detection are needed. One area that has shown promise is using protein biomarkers, measured in blood, to detect individuals with early-stage disease or at increased risk. The primary aim of this study was therefore to identify novel protein biomarkers associated with colorectal cancer risk and the secondary aim was to validate protein biomarkers that had been described in previous studies. We included data and samples from 185 incident colorectal cancer cases and 185 controls (matched on age, sex, sampling date, and the number of freeze-thaw cycles) from the Northern Sweden Health and Disease Study (NSHDS). Samples were collected between six years and three months prior to the diagnosis date of each case and concentrations of 1536 individual proteins were measured in plasma samples using the Olink Explore 1536 platform (Olink Proteomics). Prior to statistical analyses, 249 proteins were excluded, either due to not passing quality control or having more than 50% of the concentrations below the limit of detection. In addition, we also excluded extreme outliers. Conditional logistic regression was used to estimate Odds Ratios (ORs) and 95% confidence intervals (95% CI) for the association between protein concentrations and colorectal cancer risk. Multivariable models were adjusted for predetermined colorectal cancer risk factors, including body mass index, alcohol consumption, physical activity, smoking status, and level of education. When considering p&amp;lt;0.005 as a cut-off, we identified 20 proteins that were associated with colorectal cancer risk. Our top hit was TFF3 (Trefoil Factor 3) which was positively associated with colorectal cancer at a high significance level (multivariable OR per SD: 1.84 95% CI: 1.38 - 2.45). TFF3 was significant in men (FDR&amp;lt;0.1), but not in women (pheterogeneity &amp;lt;0.05). In addition, several of the top 20 proteins such as AREG, MMP7, and CXCL16 have been linked to the diagnosis or prognosis of colorectal cancer in earlier studies. To conclude, we identified one novel protein biomarker, TFF3, strongly associated with colorectal cancer risk and validated several proteins previously linked to the diagnosis or prognosis of colorectal cancer. Citation Format: Justin Harbs, Matilda Rentoft, Xijia Liu, Beatrice S. Melin, Sophia Harlid, Bethany Van Guelpen. Identifying colorectal cancer biomarkers using a targeted proteomics platform with extensive coverage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3023.

Low dose administration of mature silkworm powder induces gastric mucosal defense factors in ethanol-induced gastric injury rat model.

Gastric mucosa is important to protect the gastric damage against external factors. We previously reported the gastro-protective effects of steamed and freeze-dried mature silkworm larval powder (SMSP) in ethanol-treated rats. However, the factors that promote mucosal formation still remain unclarified. In this study, we evaluated the effect of SMSP on the restoration and maintenance of gastric mucosal layer as well as anti-inflammatory response in ethanol-induced stomach injury in rats. A significant decrease of ulcer indexes, histopathological scores and pro-inflammatory cytokine levels was observed in SMSP-treated group. In addition, SMSP protected the mucosal layer from ethanol-induced gastric damage by increasing the expression of nitric oxide synthases and heat shock proteins, along with promoting genes related gastric mucosal protection and biosynthesis including mucin 5AC and trefoil factors. These results demonstrate that SMSP attenuates the pro-inflammatory responses and strengthens the gastric mucosal layer, thus exhibiting gastro-protective effect against ethanol-induced gastric injury in rats.

Abstract 704: MDSC-targeted TFF2-MSA suppresses tumor growth and increases survival in anti-PD-1 treated MC38 and CT26.wt murine colorectal cancer models

Abstract Aims: Myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are a potential therapeutic target in immune checkpoint cancer therapy, but MDSC-targeted therapies have yet been shown to improve survival. Trefoil factor family 2 (TFF2), a secreted anti-inflammatory peptide, can suppress MDSC expansion and activate tumor immunity in part through agonism of the CXCR4 receptor. The aim of this study is to investigate whether a novel TFF2 - albumin fusion peptide (TFF2-MSA) can improve survival in anti-PD-1 treated syngeneic colorectal cancer (CRC) mouse models. Methods: Two syngeneic colon carcinoma mouse models were developed using cell lines grafted subcutaneously into mice. MC38 CRC cells were engrafted into C57BL/6 mice while CT26.wt CRC cells were implanted into BALB/C mice We generated a recombinant fusion protein, designated TFF2-MSA, which contains murine TFF2 fused to murine serum albumin (MSA), for the purpose of increasing half-life and reducing dose frequency. Mice subsequently received either TFF2-MSA or anti-PD-1 antibody (clone 29F.1A12) or both, and tumor volume, and survival were measured. At the endpoint, flow cytometry was performed to examine treatment-induced effects on immune profiles. Results: In the MC38 model, administration of TFF2-MSA suppressed tumor growth (TGI 38%), the combination of TFF2-MSA and anti-PD-1 had an additive effect and suppressed tumor growth dramatically (TGI 74%). The combination also exhibited a survival rate of 90% after 50 days, while vehicle and single TFF2-MSA therapy were 30% and 65%, respectively. The percentage of exhausted CD8+ T cells was markedly reduced in the draining lymph node by the combination treatment, as measured by flow cytometry using antibodies against LAG3, TIM3 and PD-1. In the CT26.wt model, administration of TFF2-MSA alone exhibited little effect, but the combination of anti-PD-1 and TFF2-MSA showed a profound effect. In the CT26.wt model, administration of TFF2-MSA suppressed tumor growth (TGI 17%), anti-PD-1 alone (TGI 43%) and the combination of TFF2-MSA and anti-PD-1 (TGI 54%). Conclusion: Targeting MDSCs using TFF2-MSA fusion protein synergizes well with PD-1 blockade therapy in advanced and metastatic syngeneic mouse models of colorectal cancer. In a separate abstract, additive effects between TFF2-MSA and anti-PD-1 antibody were also demonstrated in a separate ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) gastric cancer model, suggesting combination therapy may also be applicable to gastric cancer. Citation Format: Bruce L. Daugherty. MDSC-targeted TFF2-MSA suppresses tumor growth and increases survival in anti-PD-1 treated MC38 and CT26.wt murine colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 704.

Abstract 5088: MDSC-targeted TFF2-MSA synergizes with PD-1 blockade therapy in diffuse-type gastric cancer

Abstract Aims: Recent studies revealed myeloid-derived suppressor cell (MDSC) in the tumor microenvironment as an appealing therapeutic target to sensitize intestinal-type gastric cancer (GC) to PD-1 blockade therapy. However, whether MDSC inhibition can improve survival in the more aggressive diffuse-type GC is unknown. Trefoil factor family 2 (TFF2), a secreted anti-inflammatory peptide, suppresses MDSC expansion partly via CXCR4 receptor. Here, we developed a novel MDSC-targeted peptide TFF2-MSA and investigated whether it can synergize with anti-PD1 to prolong survival in syngeneic diffuse GC mouse models. Methods: Murine serum albumin (MSA) was appended to the murine TFF2 to generate TFF2-MSA peptide with an extended serum half-life. ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) GC cells developed from a highly malignant diffuse GC mouse model were grafted subcutaneously into HDC-GFP transgenic mice in which the more immunosuppressive histidine decarboxylase (HDC)-expressing myeloid cells were traced with GFP. These recipient mice subsequently received either TFF2-MSA or anti-PD-1 antibody or both. Further, to assess treatment efficacy in inhibiting spontaneous lung metastasis, subcutaneous flank tumors were resected in ACKP cell-grafted mice once tumors reached 500 mm3, and mice then received treatment with TFF2-MSA or anti-PD-1 or the combination. At the endpoint, flow cytometry and histopathological analyses were performed to examine immune profiles and tumor metastasis. In another model, ACKP-luc cells were implanted orthotopically to stomach submucosa, and recipient mice received the same treatments started from 1 week later to assess their efficacy. Results: While either TFF2-MSA or PD-1 antibody showed little benefit as a single agent (TGI 15% and 25% respectively, p&amp;gt;0.05), their combination dramatically suppressed ACKP s.c. tumor growth (TGI 78%, p&amp;lt;0.0001) and markedly prolonged median survival (64 days vs. 32.5 days in control) of host mice in a synergistic manner. The combination therapy efficiently reduced HDC-GFP+ MDSCs (by 67% vs. control, p&amp;lt;0.01), and profoundly increased tumor-infiltrating cytotoxic CD8+ T cells (by 18 fold, p&amp;lt;0.001). Further, while standard chemotherapy (5-fluorouracil and oxaliplatin) showed limited efficacy in this model, addition of chemotherapy to the TFF2-MSA/PD-1 antibody combination further improved treatment efficacy (TGI 91%) and extended median survival to 73 days. In addition, combination of TFF2-MSA/PD-1 antibody significantly reduced 80% lung metastasis (vs. control, p&amp;lt;0.0001), in contrast to minimal inhibition observed with either monotherapy (p&amp;gt;0.05). Finally, in the orthotopic model, the combo regimen eradicated GC in 50% mice compared to 0% in either monotherapy treatment. Conclusion: Targeting MDSCs using TFF2-MSA synergizes with PD-1 blockade therapy in advanced and metastatic syngeneic mouse models of diffuse-type GC. Citation Format: Jin Qian, Sandra Ryeom, Bruce Daugherty, Seth Lederman, Timothy C. Wang. MDSC-targeted TFF2-MSA synergizes with PD-1 blockade therapy in diffuse-type gastric cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5088.

Abstract 4346: TFF1 and TFF3 predict response to CDK4/6 inhibitors in breast cancer patients

Abstract Background: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6is) in combination with endocrine therapy (ET) have become the mainstay treatment for patients with hormone receptor (ER)-positive, HER2-negative metastatic breast cancer (BC). However, one-third of patients do not respond to the treatment (intrinsic resistance), leading to early tumor progression and treatment failure, and most patients eventually acquire resistance to therapy. The exact mechanisms of resistance to CDK4/6i are yet to be elucidated, and due to subsequent early (intrinsic) or late progression on CDK4/6i, long-term cures are not achieved. Methods: Transcriptomic profiles of BC cell lines that were sensitive or resistant to approved CDK4/6i, independent of subtype, were downloaded from the Cancer Cell Line Encyclopedia database. Bioinformatics analysis was performed by integrated Differential Expression and Pathway (iDEP) workflow. In parallel, transcriptomic profiles of metastatic biopsies from pre-CDK4/6i-treated BC patients (n = 54) were generated using RNA-Access technology. Key common biomarkers identified from the BC cell lines and patient tumor samples were validated by immunohistochemistry in formalin-fixed paraffin-embedded (FFPE) metastatic specimens from 73 patients prior to CDK4/6i therapy. Results: Transcriptomic profile comparisons of BC cell lines (n = 35) that were sensitive versus resistant to CDK4/6is identified Trefoil Factor 1 and 3 (TFF1 and TFF3) secretory proteins as top predictors of response. Correlation analysis showed a strong negative correlation between TFF1 and TFF3 levels to IC50 values of palbociclib and abemaciclib. Knockdown of TFF3 in CDK4/6i-sensitive BT20 cells with shRNA induced drug resistance in the cells. Transcriptomic data from 54 BC patients pre-treated with CDK4/6i showed the expression levels of TFF1 and TFF1/3 receptors (CCR4 and CCR7) were significantly increased in patients who developed late progression (&amp;gt; 6 months) compared to early progressors (&amp;lt; 3 months). In addition, IHC analysis of TFF1 and TFF3 expressions in 73 pre-treated BC patient samples showed significantly higher protein expression levels in late progressors than in early progressors. Conclusions: Our results identified and validated the common biomarkers, TFF1 and TFF3, as predictors of CDK4/6i response efficacy in ER-positive, HER2-negative BC patients. These findings provide a predictive tool capable of selecting de novo metastatic BC patients who will have the greatest benefit from the combination of CDK4/6i with endocrine treatment. Furthermore, our discovery of biomarkers in early versus late progression on CDK4/6i therapy will enable new therapeutic avenues and provide the rationale for future large-scale clinical trials. Citation Format: Linjie Luo, Yan Wang, Sophia Mastoraki, Akshara Singareeka Raghavendra, Juliana Navarro-Yepes, Nicole M. Kettner, Serena Kim, Debasish Tripathy, Kelly Hunt, Khandan Keyomarsi. TFF1 and TFF3 predict response to CDK4/6 inhibitors in breast cancer patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4346.

Oyster polysaccharides relieve DSS-induced colitis via anti-inflammatory and maintaining the physiological hypoxia

Oyster polysaccharides (OPS) possess potent anti-inflammatory properties and mediate gut microbiome. The research aimed to investigate the beneficial effect of OPS on attenuating colitis. OPS administration decreased the disease activity index and suppressed the increase in colon length. Hematoxylin and eosin staining results displayed that OPS restored the DSS-induced histopathological damage. After oral administration of OPS, myeloperoxidase activity and pro-inflammatory cytokines (TNF-α) in colitis mice were inhibited, while IL-10 was elevated. Western blotting results revealed that OPS improved the expression of tight junction proteins (ZO-1, Claudin-4, and Occludin). Additionally, OPS stabilized the expression of hypoxia-inducible factor-1α (HIF-1α) and prevented the levels of bacterial endotoxin (lipopolysaccharides). OPS activated barrier-protective genes (intestinal trefoil factor) via mediating HIF-1α. These results indicated that OPS alleviated DSS-induced colitis by inhibiting inflammation and regulating HIF-1α. OPS would be a potential candidate to alleviate DSS-induced colitis.

Identification and validation of eight estrogen-related genes for predicting prognosis of papillary thyroid cancer

Papillary thyroid cancer (PTC) is one of the most common malignant tumors in female, and estrogen can affect its progression. However, the targets and mechanisms of estrogen action in PTC remain unclear. Therefore, this study focuses on the relationship between estrogen-related genes (ERGs) expression and prognosis in PTC, particularly neuropeptide U (NMU), and its important role in tumor progression. Based on The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, differentially expressed genes (DEGs) predominantly enriched in ERGs were identified between PTC and normal tissue. Then, we identified ERGs that contributed most to PTC prognosis, including Transducer of ERBB2 1 (TOB1), trefoil factor 1 (TFF1), phospholipase A and acyltransferase 3 (PLAAT3), NMU, kinesin family member 20A (KIF20A), DNA topoisomerase II alpha (TOP2A), tetraspanin 13 (TSPAN13), and carboxypeptidase E (CPE). In addition, we confirmed that NMU was highly expressed in PTC and explored the effect of NMU on PTC cells proliferation in vitro and in vivo. The results showed that the proliferative capacity of PTC cells was significantly reduced with NMU knockdown. Moreover, the phosphorylation levels of the Kirsten rat sarcoma virus (KRAS) signaling pathway were significantly lower with NMU knockdown. These results suggest that ERGs, especially NMU, may be novel prognostic indicators in PTC.