Human Colonic Fibroblasts Research Articles

Inhibition of intestinal inflammation and fibrosis by Scutellaria Baicalensis georgi and Boswellia serrata in human epithelial cells and fibroblasts.

Inflammatory bowel disease, including Crohn's disease and ulcerative colitis, manifests with chronic intestinal inflammation and frequent sequential fibrosis. Current pharmacological therapies may show harmful side effects and are not useful for prevention or resolution of fibrosis. Thus, the use of alternative therapies is emerging as a novel useful approach. Previous results suggest that Scutellaria baicalensis Georgi (SBG) and Boswellia serrata (BS) display anti-inflammatory properties. The aim of this study was to investigate in intestinal epithelial cells and fibroblasts the anti-inflammatory and anti-fibrotic potential of SBG and BS, alone or in combination. Human colorectal adenocarcinoma cells (HT29), human intestinal epithelial cells (HIEC6) and human colon fibroblasts (CCD-18Co) were used. Cells were pretreated with SBG and BS and then exposed to pro-inflammatory and pro-fibrotic cytokines. SBG and BS extracts significantly decreased pro-inflammatory cytokine expression and improved epithelial restitution in HT29 and HIEC6 cells. Besides, fibrotic marker expression, including SNAIL, ACTA2, ZNF281, was strongly reduced. Colon myofibroblasts treated with SBG and BS showed a significant decrease of fibrotic markers as well. SBG and BS extracts significantly reduce inflammation and impair fibrosis in intestinal epithelial cells and colon myofibroblasts. No cooperative effect is observed.

Sialic acid detection and theranostic activity of phenylboronic acid-based fluorescent probe in human colorectal adenocarcinoma HT-29 cells

A new probe, 4-(((3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)imino)methyl)phenyl)boronic acid (R4B) was prepared by facile condensation of 4-formylphenylboronic acid and rhodamine B hydrazide. R4B was characterized by spectroscopic methods and single crystal X-ray diffraction. The sensor R4B solution turned pink and emitted orange fluorescence only in the presence of sialic acid but remained colorless and non-fluorescent otherwise. The sugar recognition performance was investigated via UV–vis and fluorescence spectroscopic studies. Our results revealed that R4B has good affinity and selectivity for sialic acid over common monosaccharides, with a detection limit as low as 10−7 M. Furthermore, R4B selectively inhibited growth of human colorectal adenocarcinoma HT-29 (IC50 <20 µM) without significant cytotoxicity to normal human colon fibroblasts CCD-18Co. Treatment with R4B suppressed HT-29 colony formation via mitochondrial apoptosis in a time-dependent manner. Cellular imaging studies also revealed the ability of R4B as a fluorescence dye to detect intracellular sialic acid and showed mitochondria-tracking ability in HT-29 cells. In summary, R4B is a potential theranostic for the detection of intracellular sialic acid during the early incubation period, followed by induction of cancer apoptotic cell death at a later treatment point.

Plasma-Derived Extracellular Vesicles and Non-Extracellular Vesicle Components from APCMin/+ Mice Promote Pro-Tumorigenic Activities and Activate Human Colonic Fibroblasts via the NF-κB Signaling Pathway.

Colorectal cancer (CRC) is the third most prevalent cancer worldwide. Current studies have demonstrated that tumor-derived extracellular vesicles (EVs) from different cancer cell types modulate the fibroblast microenvironment to contribute to cancer development and progression. Here, we isolated and characterized circulating large EVs (LEVs), small EVs (SEVs) and non-EV entities released in the plasma from wild-type (WT) mice and the APCMin/+ CRC mice model. Our results showed that human colon fibroblasts exposed from APC-EVs, but not from WT-EVs, exhibited the phenotypes of cancer-associated fibroblasts (CAFs) through EV-mediated NF-κB pathway activation. Cytokine array analysis on secreted proteins revealed elevated levels of inflammatory cytokine implicated in cancer growth and metastasis. Finally, non-activated cells co-cultured with supernatant from fibroblasts treated with APC-EVs showed increased mRNA expressions of CAFs markers, the ECM, inflammatory cytokines, as well as the expression of genes controlled by NF-κB. Altogether, our work suggests that EVs and non-EV components from APCMin/+ mice are endowed with pro-tumorigenic activities and promoted inflammation and a CAF-like state by triggering NF-κB signaling in fibroblasts to support CRC growth and progression. These findings provide insight into the interaction between plasma-derived EVs and human cells and can be used to design new CRC diagnosis and prognosis tools.

TL1A Promotes Fibrogenesis in Colonic Fibroblasts via the TGF-β1/Smad3 Signaling Pathway.

Intestinal fibrosis is a refractory complication of inflammatory bowel disease (IBD). Tumor necrosis factor ligand-related molecule-1A (TL1A) is important for IBD-related intestinal fibrosis in a dextran sodium sulfate (DSS)-induced experimental colitis model. This study aimed to explore the effects of TL1A on human colonic fibroblasts. A trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis model of LCK-CD2-TL1A-GFP transgenic (Tg) or wild-type (WT) mice was established to determine the effect and mechanism of TL1A on intestinal fibrosis. The human colonic fibroblast CCD-18Co cell line was treated concurrently with TL1A and human peripheral blood mononuclear cell (PBMC) supernatant. The proliferation and activation of CCD-18Co cells were detected by BrdU assays, flow cytometry, immunocytochemistry and Western blotting. Collagen metabolism was tested by Western blotting and real-time quantitative polymerase chain reaction (RT-qPCR). The level of collagen metabolism in the TNBS+ethyl alcohol (EtOH)/Tg group was greater than that in the TNBS+EtOH/WT group. Transforming growth factor-β1 (TGF-β1) and p-Smad3 in the TNBS+EtOH/Tg group were upregulated as compared with those in the TNBS+EtOH/WT group. The proliferation of CCD-18Co cells was promoted by the addition of human PBMC supernatant supplemented with 20 ng/mL TL1A, and the addition of human PBMC supernatant and TL1A increased CCD-18Co proliferation by 24.4% at 24 h. TL1A promoted cell activation and increased the levels of COL1A2, COL3A1, and TIMP-1 in CCD-18Co cells. Treatment of CCD-18Co cells with TL1A increased the expression of TGF-β1 and p-Smad3. TL1A promotes TGF-β1-mediated intestinal fibroblast activation, proliferation, and collagen deposition and is likely related to an increase in the TGF-β1/Smad3 signaling pathway.

CCR2/CCR5 antagonist cenicriviroc reduces colonic inflammation and fibrosis in experimental colitis.

Cenicriviroc (CVC) is a CCR2/CCR5 antagonist that has been shown to be effective in the treatment of inflammatory and fibrotic diseases. Our study evaluated its efficacy in colitis. Mouse models of DSS-induced acute and chronic colitis were established. The efficacy of CVC in colitis was assessed by disease activity index (DAI) scores, histological assessment of inflammation and fibrosis, and expression assays of key molecules. In in vitro experiments, HT29 cell line was exposed to TNFα to study inflammatory signaling in intestinal epithelial cells. CCD-18Co colonic myofibroblasts and human primary colonic fibroblasts were activated by TGFβ1 to mimic fibroblast activation. In HT29 cells, CVC significantly reduced mRNA expression of CCL5 (P<0.01) but had no effect on CCL2. Furthermore, CVC reduced downstream CX3CL1 (P<0.01) and TNFα (P<0.05) expression, thereby inhibiting inflammatory progression. In acute colitis mice, CVC significantly reduced DAI scores and serum TNFα levels (P<0.05) and attenuated colonic inflammation as shown by HE staining. Meanwhile, CVC had no adverse effects on the liver, heart, and kidney of mice. On the other hand, in cellular models of chronic colitis, CVC decreased the expression of fibrosis markers, including FN, CTGF, α-SMA, and MMP9, and inhibited TGFβ1-induced fibrotic activation (P<0.01). In addition, CVC attenuated colonic fibrosis in chronic colitis mice. Moreover, CVC significantly promoted autophagy, which contributed to its regulation of inflammation. CVC significantly inhibited inflammation through CCL5/CCR5 signaling without damaging vital organs and suppressed fibrotic activation in chronic colitis, suggesting its great potential to relieve colonic inflammation and fibrosis.

The TNFSF12/TWEAK Modulates Colonic Inflammatory Fibroblast Differentiation and Promotes Fibroblast-Monocyte Interactions.

Fibroblasts acquire a proinflammatory phenotype in inflammatory bowel disease, but the factors driving this process and how fibroblasts contribute to mucosal immune responses are incompletely understood. TNF superfamily member 12 (TNFSF12, or TNF-like weak inducer of apoptosis [TWEAK]) has gained interest as a mediator of chronic inflammation. In this study, we explore its role as a driver of inflammatory responses in fibroblasts and its contribution to fibroblast-monocyte interaction using human primary colonic fibroblasts, THP-1 and primary monocytes. Recombinant human TWEAK induced the expression of cytokines, chemokines, and immune receptors in primary colonic fibroblasts. The TWEAK upregulated transcriptome shared 29% homology with a previously published transcriptional profile of inflammatory fibroblasts from ulcerative colitis. TWEAK elevated surface expression of activated fibroblast markers and adhesion molecules (podoplanin [PDPN], ICAM-1, and VCAM-1) and secretion of IL-6, CCL2, and CXCL10. In coculture, fibroblasts induced monocyte adhesion and secretion of CXCL1 and IL-8, and they promoted a CD14high/ICAM-1high phenotype in THP-1 cells, which was enhanced when fibroblasts were prestimulated with TWEAK. Primary monocytes in coculture with TWEAK-treated fibroblasts had altered surface expression of CD16 and triggering receptor expressed on myeloid cells-1 (TREM-1) as well as increased CXCL1 and CXCL10 secretion. Conversely, inhibition of the noncanonical NF-κB pathway on colonic fibroblasts with a NF-κB-inducing kinase small molecule inhibitor impaired their ability to induce a CD14high phenotype on monocytes. Our results indicate that TWEAK promotes an inflammatory fibroblast-monocyte crosstalk that may be amenable for therapeutic intervention.

Mesenteric adipose-derived exosomal TINAGL1 enhances intestinal fibrosis in Crohn's Disease via SMAD4

IntroductionCrohn's Disease (CD) is a chronic inflammatory condition characterized by intestinal fibrosis, severely impacting patient quality of life. The molecular mechanisms driving this fibrosis remain inadequately understood. Recent evidence implicates mesenteric adipose tissue (MAT) in CD pathogenesis, particularly through its exosome secretion, which may influence fibrogenic pathways. Understanding the role of MAT-derived exosomes is crucial for unraveling these molecular processes. ObjectivesThis study aims to elucidate the role of MAT-derived exosomes in CD-related intestinal fibrosis. We focus on investigating their molecular composition and the potential impact on fibrosis progression, with an emphasis on identifying novel therapeutic targets. MethodsWe induced chronic intestinal inflammation in mice using dinitrobenzene sulfonic acid (DNBS), simulating CD-like fibrosis. Exosomes were isolated from DNBS-treated mice (MG) and normal controls (NG) for characterization using electron microscopy and proteomic analysis. Additionally, human colonic fibroblasts were exposed to exosomes from CD patients and healthy individuals, with subsequent assessment of fibrogenesis through proteomic and RNA sequencing analyses. ResultsProteomic analyses revealed a significant activation of the TGF-β signaling pathway in MG-treated mice compared to controls, correlating with enhanced intestinal fibrosis. In vitro experiments demonstrated that colonic fibroblasts exposed to CD patient-derived exosomes exhibited increased fibrogenic activity. Protein docking and co-immunoprecipitation studies suggested a critical interaction between TINAGL1 and SMAD4, enhancing fibrosis. Importantly, in vivo experiments corroborated that recombinant TINAGL1 protein exacerbated DNBS-induced intestinal fibrosis. ConclusionOur findings highlight the pivotal role of MAT-derived exosomes, particularly those carrying TINAGL1, in the progression of intestinal fibrosis in CD. The involvement of the TGF-β signaling pathway, especially the SMAD4 protein, offers new insights into the molecular mechanisms of CD-related fibrosis and presents potential targets for therapeutic intervention.

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Abstract Inflammation-associated fibroblasts (IAFs) are a cell population that emerges in tissue inflammation. In inflammatory bowel diseases (IBD), IAFs correlate with disease severity and are enriched in colon tissue of patients refractory to anti-TNF therapy. IAFs can amplify tissue inflammation and mediate pro-fibrotic matrix remodeling and therefore represent a novel cellular target for treating inflammatory diseases like IBD. To explore the cellular and molecular pathways that mediate transition to an IAF-like phenotype, we developed a co-culture of human PBMCs and primary colonic fibroblasts to study stromal-immune crosstalk in promoting IAFs. Culturing primed PBMCs with fibroblasts in direct contact drives fibroblast transition to an IAF phenotype through upregulation of inflammatory mediators, evidenced by modulation of gene expression and secreted protein profiles. Single-cell RNAseq revealed IAF development is influenced by PBMC priming conditions. Exposure to LPS-primed PBMCs promoted a matrix-remodeling IAF phenotype while PHA-primed PBMCs promoted an immunomodulatory IAF. IAF activation can be dampened by inhibitors, including TNF and JAK inhibitors. This co-culture system can therefore be used to define mechanisms mediating IAF transition and identify IAF states that can be tailored to accentuate pathways for focused interrogation. We aim to leverage this system to identify novel targets and test prospective therapies to modulate IAF transition and downstream activity.

The TNF superfamily factor TWEAK promotes colonic inflammatory fibroblast differentiation and fibroblast-monocyte interaction

Background and Aims: Colonic fibroblasts have emerged as key players in inflammatory bowel disease (IBD) for their potential to acquire a pro-inflammatory phenotype, but the factors driving this process and how these fibroblasts interact with the immune system is incompletely understood. The TNF superfamily factor 12 (TNFSF12/TWEAK) has gained interest as a potential mediator of chronic inflammation and tissue remodelling in IBD. Here we explore its role as a driver of inflammatory fibroblast differentiation and as a modulator of fibroblast-monocyte interaction. Methods: Human primary colonic fibroblasts were stimulated with TWEAK or vehicle for 24 hours. fibroblasts were co-cultured with THP1 cells or primary monocytes for an additional 24 hours to investigate their interaction. RNA sequencing, immunoblotting, ELISA and flow cytometry were used for analysis. Results: TWEAK up-regulated the expression of &gt;600 and down-regulated the expression of &gt;500 genes in primary colonic fibroblasts (pAdj&lt;0.05, fold&gt;1.5). The up-regulated transcriptome was characterised by a pro-inflammatory programme including numerous cytokines, chemokines and immune receptors, which resembled an inflammatory stroma subpopulation previously identified in in ulcerative colitis (29% transcriptional alignment). The shared transcriptome included a marked signature of non-canonical NF-κB genes. At protein level, TWEAK inflicted changes in the expression of stromal markers (PDPN, PDGFRα); stimulated the secretion of IL-6, CCL2 and CXCL10; And enhanced the expression of surface adhesion molecules ICAM-1 and VCAM-1. Co-culture of THP1 cells with fibroblasts induced their adhesion and promoted a CD14high/ICAM-1high phenotype in THP1 cells, both of which were further enhanced when fibroblasts were pre-stimulated with TWEAK, while TWEAK did not directly affect adhesion or differentiation of THP1 cultured alone. Moreover, treatment of fibroblasts with a small molecule inhibitor of non-canonical NF-κB partially abrogated the fibroblast-mediated increase in CD14 expression on THP1 cells. In primary monocytes extracted from whole blood of 7 human donors, co-culture with TWEAK-treated fibroblasts promoted differentiation to a CD16intermediate, TREM-1high phenotype. Moreover, media from TWEAK-treated fibroblast-monocyte co-cultures displayed elevated levels of CXCL1 and IL-8 compared to vehicle-treated co-cultures, or to either cell type cultured alone, pointing towards reciprocal pro-inflammatory crosstalk. Conclusions: Our results indicate that TWEAK can promote pro-inflammatory polarisation in colonic fibroblasts and positions TWEAK as a mediator in the pro-inflammatory interaction between colonic fibroblasts and monocytes. This work is supported by funding from Science Foundation Ireland and The Irish Research Council under the Pathway program (21/PATH-S/9621), Enterprise Ireland, and the Conway Institute Director’s Strategic Award. This is the full abstract presented at the American Physiology Summit 2024 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.

Investigating the interplay between LIGHT and HIF in inflammatory fibroblast responses

Introduction: Intestinal fibroblast differentiation into an inflammatory phenotype likely plays a key role in Inflammatory Bowel Disease (IBD). However, the mechanisms that control this process are poorly understood. The cytokine TNFSF14 (LIGHT) drives inflammatory fibroblast responses in other diseases such as Eosinophilic esophagitis. Additionally, hypoxia, defined as low cellular or tissue levels of oxygen, is a feature of IBD progression. The use of hydroxylase inhibiting drugs which exhibit hypoxia mimetic conditions have been shown in murine models to have protective effects in IBD. Here we hypothesize the role of LIGHT to be of central importance in intestinal fibroblast differentiation and test how this inflammatory phenotype may be affected by hypoxia mimetic drugs and conversely what effect LIGHT may have on the hypoxic condition. Methods: Primary human colonic fibroblasts (18CO), the hepatic stellate cell line (LX2) and the epithelial cell line (T84) were treated with LIGHT in time course studies. 18COs were also treated with hydroxylase inhibitors Dimethyloxalylglycine (DMOG), JNJ42041935 and Roxadustat. Responses were analysed using qPCR, flowcytometry and Immunoblot-analysis. Results: LIGHT-treated 18COs upregulated CCL2 (7-fold ± increase, p=&lt;0.001, n=5), CXCL10 (14-fold ± increase, p=&lt;0.0001, n=3) and IL34 (30-fold ± increase, p=&lt;0.05 n=5) at transcript level, and surface protein expression of ICAM1 (15% ± increase at 24 hours compared to vehicle, n=4) and VCAM1 (18% ± increase at 24 hours compared to vehicle, n=4) displaying distinct kinetics. This response was absent or to a much less significant degree in LX2s and T84s. DMOG decreased CCL2 (4-fold ± decrease compared to LIGHT treated samples, p=&lt;0.001, n=5) and CXCL10 (11-fold ± decrease compared to LIGHT treated samples, p=&lt;0.0001, n=3) gene expression in 18COs when subsequently treated with LIGHT for 8 hours but did not affect other targets tested such as CSF1, CXCL5, VCAM1 and ICAM1. Immunoblot analysis of 18COs showed a decreased HIF-1α stabilization when treated with hydroxylase inhibitors in the presence of LIGHT compared to hydroxylase inhibitors alone (3 different hydroxylase inhibiting drugs n=3-4, n=11 total). Preliminary data of HIF downstream activated genes VEGFA (14-fold ± increase, p=&lt;0.05 n=3) and GLUT1 (6-fold ± increase, p=&lt;0.05 n=3) show increased gene expression in 18COs when treated with DMOG with noticeable decreases when treated with a LIGHT DMOG combination. Conclusions: Our current studies show that LIGHT is a potential mediator of colonic inflammatory fibroblast differentiation and a possible inhibitor of HIF-1α stabilization. The effects of LIGHT are either significantly weaker or not displayed in colonic epithelial cells or hepatic stellate cells, indicating LIGHT-specific effects on intestinal stroma. Hydroxylase inhibitors show selective effects on the LIGHT driven response. Collectively our data reveals a potential LIGHT-hypoxia network that may be amenable for therapeutic interventions. SFI/IRC Pathway Fellows Award to Dr. Manresa, 21/PATH-S/9621. This is the full abstract presented at the American Physiology Summit 2024 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.

Modulation of WNT signalling by inflammatory factor TWEAK, and its consequences on fibroblast-epithelial communication

BACKGROUND AND AIMS: A single layer of epithelial cells that are supported by resident mucosal fibroblasts, provides an intestinal barrier against pathogens. In Inflammatory Bowel Disease (IBD), this barrier is disrupted by mechanisms which are poorly understood. Intestinal fibroblasts produce WNT, BMP and Notch mediators that contribute to epithelial homeostasis and an impairment on these signals results in the loss of barrier integrity which has been observed in IBD. In fact, fibroblasts producing these signals may be lost during inflammation. Yet the mediators and mechanism that drive this loss of homeostatic stromal-derived signals are unknown. TWEAK is a member of the tumour necrosis factor superfamily (TNFSF) that was found to be upregulated in IBD biopsies and dysregulated tissue repair and promote chronic inflammation in in vivo murine models. However, whether TWEAK exerts its effects on fibroblasts and how these may contribute to barrier breakdown remains unknown. My central hypothesis is that TWEAK dysregulates the crosstalk between fibroblasts and epithelial cells in the intestinal tract by impairing a homeostatic signalling network. METHODS: To assess the effect of TWEAK on WNT signalling and wound healing in fibroblasts and epithelial cells, the expression of WNT ligands and receptors were assessed. Primary human colonic fibroblasts and human colonic epithelial cells were stimulated with TWEAK at various time points and analysed by qPCR, Western blot and Flow cytometry and scratch wound assay. RESULTS: Both Fibroblasts and epithelial cells displayed abundant Fn14 surface receptor, although epithelial cells at a level lower than that found on colonic fibroblasts. Transcriptional signatures at gene RSPO2 (0.5-fold ± decrease, *p&lt;0.05, n=4), WNT5A (1 fold ± decrease, **p&lt;0.01, n=4) and BMP6 (1 fold ± decrease, ***p&lt;0.001, n=4) and protein level RSPO2 (50% ± decrease respectively, n=5) showed a TWEAK-mediated suppression after prolonged exposure (24-48h), but not in epithelial cells. Moreover, TWEAK delayed fibroblast healing capacity after 24h (50% ± decrease in wound closure, n=4) in a wound-scratch assay. CONCLUSION: TWEAK represses the expression of homeostatic factors in colonic fibroblasts but not on epithelial cells, suggesting this mechanism could be specific to the intestinal stroma. The effect that this impairment of WNT signals in stromal cells may have on their differentiation and communication with the epithelium will be subject for further study. UCD Ad Astra PhD Scholarship. This is the full abstract presented at the American Physiology Summit 2024 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.

Disruption of epithelium integrity by inflammation-associated fibroblasts through prostaglandin signaling.

Inflammation-associated fibroblasts (IAFs) are associated with progression and drug resistance of chronic inflammatory diseases such as inflammatory bowel disease (IBD), but their direct impact on epithelial cells is unknown. Here, we developed an in vitro model whereby human colon fibroblasts are induced by specific cytokines and recapitulate key features of IAFs in vivo. When cocultured with patient-derived colon organoids (colonoids), IAFs induced rapid colonoid expansion and barrier disruption due to swelling and rupture of individual epithelial cells. Colonoids cocultured with IAFs also show increased DNA damage, mitotic errors, and proliferation arrest. These IAF-induced epithelial defects are mediated by a paracrine pathway involving prostaglandin E2 and its receptor EP4, leading to protein kinase A -dependent activation of the cystic fibrosis transmembrane conductance regulator. EP4-specific chemical inhibitors effectively prevented IAF-induced colonoid swelling and restored normal proliferation and genome stability. These findings reveal a mechanism by which IAFs could promote and perpetuate IBD and suggest a therapeutic avenue to mitigate inflammation-associated epithelial injury.

Abstract 285: Targeting TXNDC5 in stromal fibroblasts resolves desmoplasia and resistance to immune checkpoint blockade in mesenchymal-type colorectal cancer

Abstract Objectives: Mesenchymal-type colorectal cancer (CRC), characterized by strong stromal infiltration and immune tolerance, resists immune checkpoint blockade and has poor outcomes. Cancer-associated fibroblasts (CAFs), abundant in tumor stroma, actively remodel the extracellular matrix (ECM), modulate immune evasion, and drive tumor progression. We have recently identified thioredoxin domain-containing protein 5 (TXNDC5), a protein disulfide isomerase (PDI), as a critical mediator of fibroblast activation and ECM remodeling in organ fibrosis. We hypothesized that TXNDC5 could contribute to fibroblast activation, stroma formation and disease progression in cancer, especially in the stroma-enriched fibrogenic mesenchymal-type CRC. Methods: Transcriptome databases of CRC were re-analyzed to determine the clinical relevance of TXNDC5. Experimentally, CRC was induced in mouse lines by azoxymethane (AOM) and dextran sulfate sodium (DSS) stimuli, a model sharing multiple characteristics with human mesenchymal-type CRC. Human colonic fibroblast line CCD-18co was used to investigate the molecular mechanisms by which TXNDC5 regulates colonic fibroblast activities. Fibroblast-specific TXNDC5 knockout (Col1a2-Cre/ERT2*TXNDC5fl/fl) mice were generated, combining with single-cell RNA sequencing analyses on AOM/DSS-induced CRC tumors in these animals, to clarify how fibroblast TXNDC5 impact tumor microenvironment, CRC progression and response to immune checkpoint blockade. Findings: TXNDC5 was predominantly expressed in stromal fibroblasts of human and mouse CRC. Fibroblast-specific deletion of Txndc5 lessened CAF activation, attenuated tumor fibrosis and reduced tumor burden in AOM/DSS-induced CRC. Mechanistically, increased TXNDC5 levels augments TGFβ signaling in CAF by post-translational stabilization of TGFBR1 through its PDI activity. In addition, deletion of Txndc5 in CAFs led to less tumor desmoplasia, decompressed tumor vessels and attenuated intra-tumoral hypoxia, thereby easing immune tolerance and increasing cytotoxic T cell infiltration in CRC. Single-cell transcriptome analysis revealed a marked change of intra-tumoral immune cell populations upon fibroblast-specific deletion of TXNDC5, shifting from myeloid-derived suppressive cells to cytotoxic tumor-infiltrating lymphocytes. Importantly, depletion of TXNDC5 in CAFs potentiated the anti-tumor effects of immune checkpoint blockade with anti-PD1 therapy in CRC. Conclusions: Our data suggest an important yet previously unrecognized role of fibroblast TXNDC5 in CRC progression, through enhancing CAF activation, stroma formation and immune escape. Combining immune checkpoint blockade with TXNDC5 deletion synergistically improved anti-tumor effects in CRC. Targeting TXNDC5, therefore, can be a novel therapeutic approach for CRC patients. Citation Format: Kai-Lin Cheng, Chih-I Chen, Shu-Han Yu, Huai-Wen Liang, Yi-Wei Tsai, Chen-Ting Hung, Yu-Shan Lin, Yi-Shiuan Tzeng, Sung-Jan Lin, Yueh-Feng Wu, Jen-Kuang Lee, Chia-Hui Yu, Shuei-Liong Lin, Shih-Yu Chen, Tzu-Tang Wei, Yun-Ju Huang, Ruey-Hwa Chen, Ching-Chow Chen, Kai-Chien Yang. Targeting TXNDC5 in stromal fibroblasts resolves desmoplasia and resistance to immune checkpoint blockade in mesenchymal-type colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 285.

Mapping the core senescence phenotype of primary human colon fibroblasts.

Advanced age is the largest risk factor for many diseases and several types of cancer, including colorectal cancer (CRC). Senescent cells are known to accumulate with age in various tissues, where they can modulate the surrounding tissue microenvironment through their senescence associated secretory phenotype (SASP). Recently, we showed that there is an increased number of senescent cells in the colons of CRC patients and demonstrated that senescent fibroblasts and their SASP create microniches in the colon that are conducive to CRC onset and progression. However, the composition of the SASP is heterogenous and cell-specific, and the precise senescence profile of colon fibroblasts has not been well-defined. To generate a SASP atlas of human colon fibroblasts, we induced senescence in primary human colon fibroblasts using various in vitro methods and assessed the resulting transcriptome. Using RNASequencing and further validation by quantitative RT-PCR and Luminex assays, we define and validate a 'core senescent profile' that might play a significant role in shaping the colon microenvironment. We also performed KEGG analysis and GO analyses to identify key pathways and biological processes that are differentially regulated in colon fibroblast senescence. These studies provide insights into potential driver proteins involved in senescence-associated diseases, like CRC, which may lead to therapies to improve overall health in the elderly and to prevent CRC.

Computational and In vitro Elucidation of Indolenine-barbituric Acid Zwitterions as Potential Chemotherapeutical Agents

Introduction: The continuous pursuit of novel chemotherapeutical agents with improved efficacy and reduced adverse effects remains a critical area of research despite advancements in chemotherapy. We have previously synthesized indolenine and barbituric acid zwitterion scaffolds 1–10 sustainably; however, their precise chemotherapeutical properties are still lacking. Methods: In this present work, we conducted in silico ADMET analyses, molecular docking calculations, DNA binding studies, and cytotoxicity assays on these zwitterions. Results and Discussion: Among the 10 zwitterions, zwitterion 3 bearing a methoxy group demonstrated the highest drug-likeness score, low toxicity, as well as no violation of Lipinski’s rule of five and Veber’s rule. Both molecular docking calculations and DNA binding studies suggested that the minor groove of DNA is the most probable molecular target of 3 among the others (i.e., topoisomerase and tubulin). In addition, zwitterion 3 exhibited selective cytotoxicity against a wide array of human cancer cell lines without noticeable effect against the normal human colon fibroblast CCD- 18Co. Conclusion: Overall, these preliminary findings from our combined computational and experimental strategy suggested that 3 remains promising for further elaboration as a chemotherapeutic agent.

Dihydromyricetin alleviates inflammatory bowel disease associated intestinal fibrosis by inducing autophagy through the PI3K/AKT/mTOR signaling pathway.

Intestinal fibrosis is a common complication of inflammatory bowel disease and is characterized by tissue stiffening and luminal narrowing. Dihydromyricetin (DHM) can alleviate liver fibrosis and renal interstitial fibrosis by inducing autophagy. However, whether DHM can alleviate intestinal fibrosis remains unclear. This study is aimed at evaluating the role and mechanism of action of DHM in inflammatory bowel disease-associated intestinal fibrosis. Mice were administered dextran sulfate sodium (DSS) in drinking water to induce inflammatory bowel disease-associated intestinal fibrosis. HE staining, qPCR, and Western blotting were used to analyze colon inflammation. Masson's trichrome staining, qPCR, Western blotting, and immunofluorescence staining were used to evaluate the severity of fibrosis. Transmission electron microscopy and Western blotting were used to assess the activation of autophagosomes. The human colonic fibroblast line CCD-18Co was cultured in the presence of TGF-β1 to develop a fibrotic phenotype. Immunofluorescence staining, Western blotting, and qPCR were used to assess the alteration of fibrosis markers and used to investigate whether DHM-induced autophagy was involved in the inactivation of CCD-18Co cells. Additionally, the role of the PI3K/AKT/mTOR pathway was investigated. DHM alleviated intestinal inflammation and inhibited the progression of intestinal fibrosis. Additionally, DHM induced the activation of autophagy, thereby alleviating intestinal fibrosis, and downregulated the PI3K/AKT/mTOR signaling pathway in vitro. Overall, this study demonstrated that DHM can inhibit the progression of intestinal fibrosis and activation of colonic fibroblasts by inducing autophagy through the PI3K/AKT/mTOR signaling pathway, thereby playing a preventive and therapeutic role in intestinal fibrosis.

Blastocystis sp. reduces the efficacy of 5-fluorouracil as a colorectal cancer chemotherapeutic treatment

Blastocystis is an enteric protozoan parasite with extensive genetic variation and unclear pathogenicity. It is commonly associated with gastrointestinal symptoms such as nausea, diarrhea, vomiting and abdominal pain in immunocompromised individuals. In this study, we explored the in vitro and in vivo effects of Blastocystis on the activity of a commonly used CRC chemotherapeutic agent, 5-FU. The cellular and molecular effects of solubilized antigen of Blastocystis in the presence of 5-FU were investigated using HCT116, human CRC cell line and CCD 18-Co, normal human colon fibroblast cells. For the in vivo study, 30 male Wistar rats were divided into six groups, as follows; Control Group: oral administration of 0.3 ml Jones' medium, Group A: rats injected with azoxymethane (AOM), Group A-30FU: Rats injected with AOM and administered 30 mg/kg 5-FU, Group B-A-30FU: rats inoculated with Blastocystis cysts, injected with AOM and administered 30 mg/kg 5-FU, Group A-60FU: rats injected with AOM and administered 60 mg/kg 5-FU and Group B-A-60FU: rats inoculated with Blastocystis cysts, injected with AOM and administered 60 mg/kg 5-FU. The in vitro study revealed that the inhibitory potency of 5-FU at 8 μM and 10 μM was reduced from 57.7% to 31.6% (p < 0.001) and 69.0% to 36.7% (p < 0.001) respectively when co-incubated with Blastocystis antigen for 24 h. However, the inhibitory potency of 5-FU in CCD-18Co cells was not significantly affected in the presence of Blastocystis antigen. The reduced inhibitory potency of 5-FU against cancer cell proliferation due to the presence of Blastocystis is consistent with the upregulation of expression of type 2 cytokines, transforming growth factor (TGF-β) and nuclear factor E2-related factor 2 (Nrf2) gene expression. Increased inflammation and abnormal histopathological findings along with a significant cancer multiplicity and adenoma incidence were evident in the intestine of the B-A-30FU and B-A-60FU groups when compared with the A-30FU and A-60FU groups respectively. Our in vitro and in vivo findings indicate that Blastocystis infection could potentially interfere with chemotherapy regimens such as 5-FU in CRC patients undergoing chemotherapy.

Structural insights and cytotoxicity evaluation of benz[e]indole pyrazolyl-substituted amides.

Five new compounds of benz[e]indole pyrazolyl-substituted amides (2a-e) were synthesised in low to good yields via the direct amide-coupling reaction between a pyrazolyl derivative containing a carboxylic acid and several amine substrates. The molecular structures were determined by various spectroscopic methods, such as NMR (1H, 13C and 19F), FT-IR and high-resolution mass spectrometry (HRMS). X-ray crystallographic analysis on the 4-fluorobenzyl derivative (2d) reveals the amide-O atom to reside to the opposite side of the molecule to the pyrazolyl-N and pyrrolyl-N atoms; in the molecular packing, helical chains feature amide-N‒H⋯N(pyrrolyl) hydrogen bonds. Density-functional theory (DFT) at the geometry-optimisation B3LYP/6-31G(d) level on the full series shows general agreement with the experimental structures. While the LUMO in each case is spread over the benz[e]indole pyrazolyl moiety, the HOMO spreads over the halogenated benzo-substituted amide moieties or is localised near the benz[e]indole pyrazolyl moieties. The MTT assay showed that 2e, exhibited the highest toxicity against a human colorectal carcinoma (HCT 116 cell line) without appreciable toxicity towards the normal human colon fibroblast (CCD-18Co cell line). Based on molecular docking calculations, the probable cytotoxic mechanism of 2e is through the DNA minor groove binding.

The effect of up-regulation of NLE1 gene expression on the invasion and migration of colon cancer cells and its mechanism

ObjectivesTo explore the regulatory effects of up-regulation of NLE1 gene expression in vitro and the mechanism of colon cancer cell invasion and migration. MethodsThe logarithmic growth phase human colonic fibroblasts CCD-18Co, colon cancer cells HT-29, and NLE1 interference group were transfected with NLE1 siRNA to interfere with expressing viral vectors and liposomes. The NLE1 negative control group was added with liposomes, and NLE1 was overexpressed. The expression changes of RNA interference on the target gene were analyzed. ResultsThe expression of NLE1 mRNA in colon cancer cells was significantly higher (P < 0.05). Likewise, the expression of NLE1 protein in colon cancer cells was significantly increased than in control colon cancer cell lines (p < 0.05). Compared with the NLE1 interference group and the NLE1 negative control group, the migration and invasion ability of colon cancer cells in the NLE1 overexpression group were significantly varied (P < 0.05)‘. ConclusionsUp-regulation of the NLE1 gene in colon cancer cells promotes cell migration and invasion of colon cancer cells and inhibits the apoptosis ability of colon cancer cells.

Abstract B013: Activin’s influence on the tumor microenvironment in colon cancer

Abstract Background and aims: Activin A (activin) is a key molecule that regulates cell specific and context dependent signaling in tumor growth and metastasis as well as local and systemic inflammatory responses. Understanding the link between tumor-promotive and inflammatory effects may provide a novel opportunity for targeted therapy in colorectal cancer (CRC). We have previously shown that activin signaling promotes epithelial to mesenchymal transition, tumor migration, and that serum activin correlates with advanced stage in CRC patients. Recently, activin has been implicated in enhancing CD4+ to CD8+ communications to promote cytotoxic elimination of tumor cells. We now hypothesize that activin exerts cell-specific effects in the tumor microenvironment (TME) to both promote anti-tumoral activity of immune cells and pro-metastatic behavior of tumor cells in a context-dependent manner. Methods: Western blot and transwell migration assays with and without activin were performed in human epithelial colon cancer cells with varying expression levels of ACVR2 and in human colonic fibroblasts. The influence of the canonical Smad4 pathway was elucidated in a Ts4-Cre;Apcflox;Smad4flox mouse model for CRC. We analyzed a TMA of 1055 stage II and III CRC patients from the QUASAR2 cohort to correlate activin and CD4+ expression with outcome. The TMAs were also analyzed via Digital Spatial Profiling (DSP, NanoString) to determine the immune cell heterogeneity and cell signaling patterns within the tumor microenvironment relative to activin. Results: The non-canonical pAkt pathway is activated in ACVR2-restored but not ACVR2-mutated HCT116+chr2 colon cancer cells, which leads to tumor cell migration in a PI3K-dependent manner. In vivo, ablation of downstream canonical SMAD signaling is associated with elevated activin, alpha-SMA and pAkt and increases in tissue dysplasia, intestinal stromal disorganization, and animal mortality. While there are direct pro-metastatic effects of activin on tumor cells, activin expression in the TME of stage II or III CRC patients is associated with elevated CD4 and survival benefit (High activin/high CD4: 1.665 hazard ratio, 95% Cl of ratio 1.379 to 2.010). DSP analysis confirmed co-localization of activin with immune cells in the TME which influences cell signaling patterns found in these regions. Conclusion: In colon cancer cells, activin leads to preferential prometastatic PI3K/Akt pathway activation. Within the TME, high levels of activin in the presence of CD4+ T-cells is associated with better outcomes in CRC patients. This work lays the foundation to further study context-dependent activin signaling as an adjunct or target in CRC. Citation Format: Mark B. Wiley, Jessica Bauer, Kunaal Mehrotra, David N. Church, Rachel S. Kerr, David J. Kerr, Paul Grippo, Barbara Jung. Activin’s influence on the tumor microenvironment in colon cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B013.