Inhibition Of TGF-β Pathway Research Articles

CCDC113 promotes colorectal cancer tumorigenesis and metastasis via TGF-β signaling pathway

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality worldwide. Although CRC patients’ survival is improved with surgical resection and immunotherapy, metastasis and recurrence remain major problems leading to poor prognosis. Therefore, exploring pathogenesis and identifying specific biomarkers are crucial for CRC early diagnosis and targeted therapy. CCDC113, a member of CCDC families, has been reported to play roles in ciliary assembly, ciliary activity, PSCI, asthma and early lung cancer diagnosis. However, the functions of CCDC113 in CRC still remain unclear. In this study, we find that CCDC113 is significantly highly expressed in CRC. High expression of CCDC113 is significantly correlated with CRC patients’ poor prognosis. CCDC113 is required for CRC tumorigenesis and metastasis. RNA-seq and TCGA database analysis indicate that CCDC113 is positively correlated with TGF-β signaling pathway. TGF-β signaling pathway inhibitor galunisertib could reverse the increased proliferation and migration ability of CRC cells caused by CCDC113 overexpression in vitro and in vivo. These results indicate that CCDC113 promotes CRC tumorigenesis and metastasis via TGF-β signaling pathway. In conclusion, it is the first time to explore the functions and mechanisms of CCDC113 in CRC tumorigenesis and metastasis. And CCDC113 may be a potential biomarker and therapeutic target for CRC intervention.

Down-regulation of CYTL1 attenuates bleomycin-induced pulmonary fibrosis in mice by inhibiting M2 macrophage polarization via the TGF-β/CCN2 axis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by chronic inflammation, lung tissue fibrotic changes and impaired lung function. Pulmonary fibrosis 's pathological process is thought to be influenced by macrophage-associated phenotypes. IPF treatment requires specific targets that target macrophage polarization. Cytokine-like 1(CYTL1) is a secreted protein with multiple biological functions first discovered in CD34+ haematopoietic cells. However, its possible effects on IPF progression remain unclear. This study investigated the role of CYTL1 in IPF progression in a bleomycin-induced lung injury and fibrosis model. In bleomycin-induced mice, CYTL1 is highly expressed. Moreover, CYTL1 ablation alleviates lung injury and fibrosis in vivo. Further, downregulating CYTL1 reduces macrophage M2 polarization. Mechanically, CYTL1 regulates transforming growth factor β (TGF-β)/connective tissue growth factor (CCN2) axis and inhibition of TGF-β pathway alleviates bleomycin-induced lung injury and fibrosis. In conclusion, highly expressed CYTL1 inhibits macrophage M2 polarization by regulating TGF-β/CCN2 expression, alleviating bleomycin-induced lung injury and fibrosis. CYTL1 could, therefore, serve as a promising IPF target.

Increased vascular smooth muscle cell senescence in aneurysmal Fibulin-4 mutant mice

Aortic aneurysms are dilatations of the aorta that can rupture when left untreated. We used the aneurysmal Fibulin-4R/R mouse model to further unravel the underlying mechanisms of aneurysm formation. RNA sequencing of 3-month-old Fibulin-4R/R aortas revealed significant upregulation of senescence-associated secretory phenotype (SASP) factors and key senescence factors, indicating the involvement of senescence. Analysis of aorta histology and of vascular smooth muscle cells (VSMCs) in vitro confirmed the senescent phenotype of Fibulin-4R/R VSMCs by revealing increased SA-β-gal, p21, and p16 staining, increased IL-6 secretion, increased presence of DNA damage foci and increased nuclei size. Additionally, we found that p21 luminescence was increased in the dilated aorta of Fibulin-4R/R|p21-luciferase mice. Our studies identify a cellular aging cascade in Fibulin-4 aneurysmal disease, by revealing that Fibulin-4R/R aortic VSMCs have a pronounced SASP and a senescent phenotype that may underlie aortic wall degeneration. Additionally, we demonstrated the therapeutic effect of JAK/STAT and TGF-β pathway inhibition, as well as senolytic treatment on Fibulin-4R/R VSMCs in vitro. These findings can contribute to improved therapeutic options for aneurysmal disease aimed at reducing senescent cells.

Dang-Gui-Si-Ni decoction facilitates wound healing in diabetic foot ulcers by regulating expression of AGEs/RAGE/TGF-β/Smad2/3.

Diabetic foot ulcer (DFU) is a predominant complication of diabetes mellitus with poor prognosis accompanied by high amputation and mortality rates. Dang-Gui-Si-Ni decoction (DSD), as a classic formula with a long history in China, has been found to improve DFU symptoms. However, mechanism of DSD for DFU therapy remains unclear with no systematic elaboration. In vivo, following establishment of DFU rat model, DSD intervention with low, medium and high doses was done, with Metformin (DM) as a positive control group. With wound healing detection, pathological changes by HE staining, inflammatory factor expression by ELISA and qRT-PCR, oxidative stress levels by ELISA, and AGEs/RAGE/TGF-β/Smad2/3 expression by Western blot were performed. In vitro, intervention with LY2109761 (TGF-β pathway inhibitor) based on DSD treatment in human dermal fibroblast-adult (HDF-a) cells was made. Cell viability by CCK8, migration ability by cell scratch, apoptosis by flow cytometry, and AGEs/RAGE/TGF-β/Smad2/3 expression by Western blot were measured. DFU rats exhibited elevated AGEs/RAGE expression, whereas decreased TGF-β1 and p-Smad3/Smad3 protein expression, accompanied by higher IL-1β, IL-6, TNF-α levels, and oxidative stress. DSD intervention reversed above effects. Glucose induction caused lower cell viability, migration, TGF-β1 and p-Smad3/Smad3 protein expression, with increased apoptosis and AGEs/RAGE expression in HDF-a cells. These effects were reversed after DSD intervention, and further LY2109761 intervention inhibited DSD effects in cells. DSD intervention may facilitate wound healing in DFU by regulating expression of AGEs/RAGE/TGF-β/Smad2/3, providing scientific experimental evidence for DSD clinical application for DFU therapy.

Abstract 4252: Evaluation of NXI-101 in non-small cell lung cancer (NSCLC) using patient-derived organotypic tumor spheroids (PDOTS): Targeting a novel tumorigenic pathway

Abstract ONCOKINE-1 (OK-1), a soluble protein that is released by tumor cells, is implicated in the resistance of immunotherapy. Here we evaluated the efficacy of NXI-101, a monoclonal antibody that binds and neutralizes OK-1, in a model of patient-derived organotypic tumor spheroids (PDOTS) derived from non-small cell lung carcinoma (NSCLC) patients. NSCLC PDOTS were treated with NXI-101 and the effects of the drug were evaluated on day 3 post-treatment. Cytotoxicity was measured by Hoechst/Propidium Iodide staining and quantified by measuring the area-weighted percentage of cell death. Significant cytotoxicity was observed in 2 out of 10 patient tumors (20%) as a monotherapy and 4 out of 10 (40%) when used in combination with atezolizumab. The therapeutic response for both treatments was positively correlated (p<0.05) with the level of OK-1 expression, determined by immunohistochemistry using tissue sections taken on the day of tumor specimen collection. NXI-101 alone and in combination with atezolizumab had immunomodulatory effects that were associated with cytotoxic response. For example, transcriptomic analysis, Nanostring IO 360 Panel, indicated that the drug treatments resulted in the inhibition of TGF-β pathways and stimulated a number of immune pathways in 3 of the 4 responsive PDOTS. These pathways include interferon-α, JAK-STAT, and dendritic cell activation markers. We also observed evidence of activation of these same pathways in 2 of the 6 non-responsive PDOTS. These data support the prospects of NXI-101 as a first-in-class therapeutic for the treatment of NSCLC when used alone or in combination with an immune checkpoint inhibitor. Moreover, the findings highlight the PDOTS platform's substantial value in capturing patient-specific tumor response variability and providing robust support for the mechanistic evaluation of cancer therapeutics. Citation Format: Julia Vail, Hyung Gun Maeng, Je Woong Jang, Kyungjin Boo, Je-In Youn, Alyssa Martin, Aaron Goldman, Kyoung Wan Yoon, Michael A. Perricone. Evaluation of NXI-101 in non-small cell lung cancer (NSCLC) using patient-derived organotypic tumor spheroids (PDOTS): Targeting a novel tumorigenic pathway [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 4252.

Expanding the Chemical Space of Transforming Growth Factor-β (TGFβ) Receptor Type II Degraders with 3,4-Disubstituted Indole Derivatives.

The TGFβ type II receptor (TβRII) is a central player in all TGFβ signaling downstream events, has been linked to cancer progression, and thus, has emerged as an auspicious anti-TGFβ strategy. Especially its targeted degradation presents an excellent goal for effective TGFβ pathway inhibition. Here, cellular structure-activity relationship (SAR) data from the TβRII degrader chemotype 1 was successfully transformed into predictive ligand-based pharmacophore models that allowed scaffold hopping. Two distinct 3,4-disubstituted indoles were identified from virtual screening: tetrahydro-4-oxo-indole 2 and indole-3-acetate 3. Design, synthesis, and screening of focused amide libraries confirmed 2r and 3n as potent TGFβ inhibitors. They were validated to fully recapitulate the ability of 1 to selectively degrade TβRII, without affecting TβRI. Consequently, 2r and 3n efficiently blocked endothelial-to-mesenchymal transition and cell migration in different cancer cell lines while not perturbing the microtubule network. Hence, 2 and 3 present novel TβRII degrader chemotypes that will (1) aid target deconvolution efforts and (2) accelerate proof-of-concept studies for small-molecule-driven TβRII degradation in vivo.

Prodigiosin Inhibits Transforming Growth Factor β Signaling by Interfering Receptor Recycling and Subcellular Translocation in Epithelial Cells.

Prodigiosin (PG) is a naturally occurring polypyrrole red pigment produced by numerous microorganisms including some Serratia and Streptomyces strains. PG has exhibited promising anticancer activity; however, the molecular mechanisms of action of PG on malignant cells remain ambiguous. Transforming growth factor-β (TGF-β) is a multifunctional cytokine that governs a wide array of cellular processes in development and tissue homeostasis. Malfunctions of TGF-β signaling are associated with numerous human cancers. Emerging evidence underscores the significance of internalized TGF-β receptors and their intracellular trafficking in initiating signaling cascades. In this study, we identified PG as a potent inhibitor of the TGF-β pathway. PG blocked TGF-β signaling by targeting multiple sites of this pathway, including facilitating the sequestering of TGF-β receptors in the cytoplasm by impeding the recycling of type II TGF-β receptors to the cell surface. Additionally, PG prompts a reduction in the abundance of receptors on the cell surface through the disruption of the receptor glycosylation. In human Caucasian lung carcinoma cells and human hepatocellular cancer cell line cells, nanomolar concentrations of PG substantially diminish TGF-β-triggered phosphorylation of Smad2 protein. This attenuation is further reflected in the suppression of downstream target gene expression, including those encoding fibronectin, plasminogen activator inhibitor-1, and N-cadherin. SIGNIFICANCE STATEMENT: Prodigiosin (PG) emerges from this study as a potent TGF-β pathway inhibitor, disrupting receptor trafficking and glycosylation and reducing TGF-β signaling and downstream gene expression. These findings not only shed light on PG's potential therapeutic role but also present a captivating avenue towards future anti-TGF-β strategies.

A83-01 and DMH1 effects in the zebrafish spermatogonial niche: Unraveling the roles of TGF-β and BMP signaling in the Fsh-mediated spermatogonial fate

Transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling has fundamental roles in the regulation of the stem cell niche for both embryonic and adult stem cells. In zebrafish, male germ stem cell niche is regulated by follicle-stimulating hormone (Fsh) through different members of the TGF-β superfamily. On the other hand, the specific roles of TGF-β and BMP signaling pathways are unknown in the zebrafish male germ stem cell niche. Considering this lack of information, the present study aimed to investigate the pharmacological inhibition of TGF-β (A83-01) and BMP (DMH1) signaling pathways in the presence of recombinant zebrafish Fsh using testicular explants. We also reanalyzed single cell-RNA sequencing (sc-RNA-seq) dataset from adult zebrafish testes to identify the testicular cellular sites of smad expression, and to understand the physiological significance of the changes in smad transcript levels after inhibition of TGF-β or BMP pathways. Our results showed that A83-01 potentiated the pro-stimulatory effects of Fsh on spermatogonial differentiation leading to an increase in the proportion area occupied by differentiated spermatogonia with concomitant reduction of type A undifferentiated (Aund) spermatogonia. In agreement, expression analysis showed lower mRNA levels for the pluripotency gene pou5f3, and increased expression of dazl (marker of type B spermatogonia and spermatocyte) and igf3 (pro-stimulatory growth factor) following the co-treatment with TGF-β inhibitor and Fsh. Contrariwise, the inhibition of BMP signaling nullified the pro-stimulatory effects of Fsh, resulting in a reduction of differentiated spermatogonia and increased proportion area occupied by type Aund spermatogonia. Supporting this evidence, BMP signaling inhibition increased the mRNA levels of pluripotency genes nanog and pou5f3, and decreased dazl levels when compared to control. The sc-RNA-seq data unveiled a distinctive pattern of smad expression among testicular cells, primarily observed in spermatogonia (smad 2, 3a, 3b, 8), spermatocytes (smad 2, 3a, 8), Sertoli cells (smad 1, 3a, 3b), and Leydig cells (smad 1, 2). This finding supports the notion that inhibition of TGF-β and BMP signaling pathways may predominantly impact cellular components within the spermatogonial niche, namely spermatogonia, Sertoli, and Leydig cells. In conclusion, our study demonstrated that TGF-β and BMP signaling pathways exert antagonistic roles in the zebrafish germ stem cell niche. The members of the TGF-β subfamily are mainly involved in maintaining the undifferentiated state of spermatogonia, while the BMP subfamily promotes spermatogonial differentiation. Therefore, in the complex regulation of the germ stem cell niche by Fsh, members of the BMP subfamily (pro-differentiation) should be more predominant in the niche than those belonging to the TGF-β (anti-differentiation). Overall, these findings are not only relevant for understanding the regulation of germ stem cell niche but may also be useful for expanding in vitro the number of undifferentiated spermatogonia more efficiently than using recombinant hormones or growth factors.

Hepsin promotes breast tumor growth signaling via the TGFβ-EGFR axis.

Hepsin, a type II transmembrane serine protease, is commonly overexpressed in prostate and breast cancer. The hepsin protein is stabilized by the Ras-MAPK pathway, and, downstream, this protease regulates the degradation of extracellular matrix components and activates growth factor pathways, such as the hepatocyte growth factor (HGF) and transforming growth factor beta (TGFβ) pathway. However, how exactly active hepsin promotes cell proliferation machinery to sustain tumor growth is not fully understood. Here, we show that genetic deletion of the gene encoding hepsin (Hpn) in a WAP-Myc model of aggressive MYC-driven breast cancer inhibits tumor growth in the primary syngrafted sites and the growth of disseminated tumors in the lungs. The suppression of tumor growth upon loss of hepsin was accompanied by downregulation of TGFβ and EGFR signaling together with a reduction in epidermal growth factor receptor (EGFR) protein levels. We further demonstrate in 3D cultures of patient-derived breast cancer explants that both basal TGFβ signaling and EGFR protein expression are inhibited by neutralizing antibodies or small-molecule inhibitors of hepsin. The study demonstrates a role for hepsin as a regulator of cell proliferation and tumor growth through TGFβ and EGFR pathways, warranting consideration of hepsin as a potential indirect upstream target for therapeutic inhibition of TGFβ and EGFR pathways in cancer.

Effects of prenatal acetaminophen exposure at different stages, doses and courses on articular cartilage of offspring mice.

Previous studies showed that chondrodysplasia has intrauterine origin. Although prenatal acetaminophen exposure (PAcE) can cause nervous and reproductive abnormalities in offspring, its effect on cartilage is uninvestigated. Herein, mice were treated with different doses and courses of acetaminophen at various gestational stages (100 or 400mg/kg∙d on gestational days 10-12 (GD10-12), 400mg/kg∙d on GD12 or GD15-17) based on clinical administration and conversion between humans and mice. Fetal knee joints were harvested on GD18 to analyze cartilage morphology, chondrocyte proliferation and apoptosis, and matrix content, synthesis and degradation. Results showed that 400mg/kg∙d acetaminophen exposure during GD10-12 decreased chondrocyte numbers, safranin O staining, proliferation and matrix synthesis, without elevating matrix degradation and apoptosis. Low-dose, single-course, or late-pregnancy exposure had no effect on above indexes. Moreover, Tgfβ pathway was inhibited, showing a positive correlation with the expression of Col2a1, Acan, Ki67, and Pcna. Overall, clinical doses of PAcE can inhibit chondrocyte proliferation and matrix synthesis, causing fetal mice chondrodysplasia, especially after multi-course exposure of 400mg/kg∙d acetaminophen during GD10-12, the mechanism of which might involve Tgfβ pathway inhibition. This study provides an experimental basis for assessing fetal developmental toxicity and standardizing the clinical use of acetaminophen during pregnancy.

Scalable Generation of Pre-Vascularized and Functional Human Beige Adipose Organoids.

Obesity and type 2 diabetes are becoming a global sociobiomedical burden. Beige adipocytes are emerging as key inducible actors and putative relevant therapeutic targets for improving metabolic health. However, in vitro models of human beige adipose tissue are currently lacking and hinder research into this cell type and biotherapy development. Unlike traditional bottom-up engineering approaches that aim to generate building blocks, here a scalable system is proposed to generate pre-vascularized and functional human beige adipose tissue organoids using the human stromal vascular fraction of white adipose tissue as a source of adipose and endothelial progenitors. This engineered method uses a defined biomechanical and chemical environment using tumor growth factor β (TGFβ) pathway inhibition and specific gelatin methacryloyl (GelMA) embedding parameters to promote the self-organization of spheroids in GelMA hydrogel, facilitating beige adipogenesis and vascularization. The resulting vascularized organoids display key features of native beige adipose tissue including inducible Uncoupling Protein-1 (UCP1) expression, increased uncoupled mitochondrial respiration, and batokines secretion. The controlled assembly of spheroids allows to translate organoid morphogenesis to a macroscopic scale, generating vascularized centimeter-scale beige adipose micro-tissues. This approach represents a significant advancement in developing in vitro human beige adipose tissue models and facilitates broad applications ranging from basic research to biotherapies.

Transcriptomic evidence for tumor-specific beneficial or adverse effects of TGFβ pathway inhibition on the prognosis of patients with liver cancer.

Therapeutic targeting of the Transforming Growth Factor beta (TGFβ) pathway in cancer represents a clinical challenge since TGFβ exhibits either tumor suppressive or tumor promoting properties, depending on the tumor stage. Thus, treatment with galunisertib, a small molecule inhibitor of TGFβ receptor type 1, demonstrated clinical benefits only in subsets of patients. Due to the functional duality of TGFβ in cancer, one can hypothesize that inhibiting this pathway could result in beneficial or adverse effects depending on tumor subtypes. Here, we report distinct gene expression signatures in response to galunisertib in PLC/PRF/5 and SNU-449, two cell lines that recapitulate human hepatocellular carcinoma (HCC) with good and poor prognosis, respectively. More importantly, integrative transcriptomics using independent cohorts of patients with HCC demonstrates that galunisertib-induced transcriptional reprogramming in SNU-449 is associated with human HCC with a better clinical outcome (i.e. increased overall survival), while galunisertib-induced transcriptional reprogramming in PLC/PRF/5 is associated with human HCC with a worse clinical outcome (i.e. reduced overall survival), demonstrating that galunisertib could indeed be beneficial or detrimental depending on HCC subtypes. Collectively, our study highlights the importance of patient selection to demonstrate a clinical benefit of TGFβ pathway inhibition and identifies Serpin Family F Member 2 (SERPINF2) as a putative companion biomarker for galunisertib in HCC.

Abstract 2936: INCA33890, a novel PD-1×TGFꞵR2 bispecific antibody conditionally antagonizes TGFꞵ signaling in primary immune cells co-expressing PD-1

Abstract Transforming growth factor-β (TGFβ) signaling is common in many solid tumors and is initiated by binding of the high affinity canonical ligands TGFβ1, 2, οr 3 to TGFβR2, which forms a heteromeric receptor complex with TGFβR1 (Derynck et al, Nature Review Clinical Oncology 2020). Activation of the pathway results in potent suppression of immune cell-mediated anti-tumor immunity and has been reported to predict poor response to PD-(L)1 targeted therapy in patients (Mariathasan et al, Nature 2018; Kieffer et al, Cancer Discovery 2020). However, TGFβ drug development has been hampered by the occurrence of adverse events. INCA33890 is a dual PD-1 and TGFβR2 binding bispecific Biclonics® antibody, developed to antagonize the TGFβ signaling pathway specifically in cells co-expressing PD-1 and TGFBR2. Additionally, it potently antagonizes the PD-1 axis independently of TGFβR2 co-expression. The cell-selective action of INCA33890 was designed to mitigate risks of the known adverse effects associated with TGFβ-pathway inhibition in tissues requiring active TGFβ signaling. ΙΝCΑ33890 mediates its specificity through a PD-1 binding arm with a >10-fold higher affinity relative to the TGFβR2 binding arm. Consistent with this profile, in isogenic Jurkat cells expressing TGFβR2 ± PD-1, INCA33890 potently inhibits TGFβ1-induced pSMAD activation in a PD-1-correlated manner. Additionally, in two independent PD-1 reporter assays, INCA33890 inhibited SHP recruitment and enhanced NFAT activation with a potency within an order of magnitude to that of pembrolizumab. In mixed lymphocyte reaction assays with exhausted primary human T-cells, INCA33890 was found to induce a similar level of anti-tumor cytokine production as the combination of pembrolizumab and an anti-TGFβR2 antagonist mAb. Treatment of primary ovarian ascites with INCA33890 ex vivo induced IFNγ production in all donors tested, while pembrolizumab had no activity. Similarly, in human CD34+ cell-engrafted NSG mice, INCA33890 significantly inhibited the growth of human MDA-MB-231 and A375 subcutaneous xenograft tumors, whereas pembrolizumab or an anti-TGFβR2 antibody had little or no monotherapy activity. INCA33890 had a balanced pharmacokinetic and potency profile and was well tolerated in NHPs at exposures required for pharmacodynamic and tumor growth inhibiting activity in rodents. Encouragingly, there was no evidence of adverse effects in NHPs due to TGFβ-pathway blockade. Collectively, these results provide compelling data for an effective and specific approach to simultaneously antagonizing TGFβ and PD-1 signaling in tumors. Clinical development of INCA33890 in checkpoint inhibitor-resistant and other cancers has been initiated. Citation Format: Liang-Chuan S. Wang, Rinse Klooster, Ashwini Kulkarni, Amaya Garcia de Vinuesa, Maxim Soloviev, Linda JA Hendriks, Lu Huo, Michael Weber, Arpita Mondal, Yonghong Zhao, Shane Harvey, Xin He, Hong Chang, April Horsey, Alla Volgina, Yue Zhang, Veethika Pandey, Yan-Ou Yang, Jonathan Rios-Doria, Evgeniy Eruslanov, Daniel J. Powell, Steven M. Albelda, John de Kruif, Horacio Nastri, Cecile Geuijen, Patrick A. Mayes. INCA33890, a novel PD-1×TGFꞵR2 bispecific antibody conditionally antagonizes TGFꞵ signaling in primary immune cells co-expressing PD-1 [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 2936.

Establishment and characterisation of oviductal organoids from farm and companion animals.

Organoid technology has provided a unique opportunity to study early human development and decipher various steps involved in the pathogenesis of disease. The technology is already used in clinics to improve human patient outcomes. However, limited knowledge of the methodologies required to establish organoid culture systems in domestic animals has slowed the advancement and application of organoid technology in veterinary medicine. This is particularly true for the field of reproduction and the application of assisted reproductive technologies (ART). Here, we have developed a platform to grow oviductal organoids from five domestic species - bovine, porcine, equine, feline, and canine. The organoids were grown progressively from single cells derived from the enzymatic digestion of freshly collected infundibular/fimbrial samples. The addition of WNT, TGFβ, BMP, ROCK, and Notch signalling pathway activators or inhibitors to the organoid culture medium suggested remarkable conservation of the molecular signals involved in oviductal epithelial development and differentiation across species. The gross morphology of organoids from all the domestic species was initially similar. However, some differences in size, complexity, and growth rate were subsequently observed and described. After 21days, well-defined and synchronized motile ciliated cells were observed in organoids. Histopathologically, oviductal organoids mimicked their respective native tissue. In summary, we have carried out a detailed cross-species comparison of oviductal organoids, which would be valuable in advancing our knowledge of oviduct physiology and, potentially, help in increasing the success of assisted reproductive technologies.

Inhibition of TGF-β pathway improved the pluripotency of porcine pluripotent stem cells.

Porcine pluripotent stem cells had been derived from different culture systems. PeNK6 is a porcine pluripotent stem cell line that we established from an E5.5 embryo in a defined culture system. Signaling pathways related with pluripotency had been assessed in this cell line, and TGF-β signaling pathway-related genes were found upregulated significantly. In this study, we elucidated the role of the TGF-β signaling pathway in PeNK6 through adding small molecule inhibitors, SB431542 (KOSB) or A83-01 (KOA), into the original culture medium (KO) and analyzing the expression and activity of key factors involved in the TGF-β signaling pathway. In KOSB/KOA medium, the morphology of PeNK6 became compact and the nuclear-to-cytoplasm ratio was increased. The expression of the core transcription factor SOX2 was significantly upregulated compared with cell lines in the control KO medium, and the differentiation potential became balanced among three germ layers rather than bias to neuroectoderm/endoderm as the original PeNK6 did. The results indicated that inhibition of TGF-β has positive effects on the porcine pluripotency. Based on these results, we established a pluripotent cell line (PeWKSB) from E5.5 blastocyst by employing TGF-β inhibitors, and the cell line showed improved pluripotency.

CD4+ T Cell Regulatory Network Underlies the Decrease in Th1 and the Increase in Anergic and Th17 Subsets in Severe COVID-19.

In this model we use a dynamic and multistable Boolean regulatory network to provide a mechanistic explanation of the lymphopenia and dysregulation of CD4+ T cell subsets in COVID-19 and provide therapeutic targets. Using a previous model, the cytokine micro-environments found in mild, moderate, and severe COVID-19 with and without TGF-β and IL-10 was we simulated. It shows that as the severity of the disease increases, the number of antiviral Th1 cells decreases, while the the number of Th1-like regulatory and exhausted cells and the proportion between Th1 and Th1R cells increases. The addition of the regulatory cytokines TFG-β and IL-10 makes the Th1 attractor unstable and favors the Th17 and regulatory subsets. This is associated with the contradictory signals in the micro-environment that activate SOCS proteins that block the signaling pathways. Furthermore, it determined four possible therapeutic targets that increase the Th1 compartment in severe COVID-19: the activation of the IFN-γ pathway, or the inhibition of TGF-β or IL-10 pathways or SOCS1 protein; from these, inhibiting SOCS1 has the lowest number of predicted collateral effects. Finally, a tool is provided that allows simulations of specific cytokine environments and predictions of CD4 T cell subsets and possible interventions, as well as associated secondary effects.

Human primed and naïve PSCs are both able to differentiate into trophoblast stem cells.

The recent derivation of human trophoblast stem cells (TSCs) from placental cytotrophoblasts and blastocysts opened opportunities for studying the development and function of the human placenta. Recent reports have suggested that human naïve, but not primed, pluripotent stem cells (PSCs) retain an exclusive potential to generate TSCs. Here we report that, in the absence of WNT stimulation, transforming growth factor β (TGF-β) pathway inhibition leads to direct and robust conversion of primed human PSCs into TSCs. The resulting primed PSC-derived TSC lines exhibit self-renewal, can differentiate into the main trophoblast lineages, and present RNA and epigenetic profiles that are indistinguishable from recently established TSC lines derived from human placenta, blastocysts, or isogenic human naïve PSCs expanded under human enhanced naïve stem cell medium (HENSM) conditions. Activation of nuclear Yes-associated protein (YAP) signaling is sufficient for this conversion and necessary for human TSC maintenance. Our findings underscore a residual plasticity in primed human PSCs that allows their invitro conversion into extra-embryonic trophoblast lineages.

Theranostic mesoporous platinum nanoplatform delivers halofuginone to remodel extracellular matrix of breast cancer without systematic toxicity.

The enriched collagens in the extracellular matrix (ECM) of breast cancer substantially impede drug delivery. Halofuginone (HF), a potent antifibrotic agent, was effective to deplete the collagens and remodel the ECM by inhibiting the TGFβ pathway. However, the application of HF was hindered by its strong liver toxicity. Herein, mesoporous platinum (mPt) nanoparticles were constructed to load HF as theranostic nanoplatforms. mPt had a uniform spherical structure with a diameter of 79.83 ± 6.97 nm and an average pore diameter of 20 nm and exhibited good photothermal conversion efficiency of 62.4%. The obtained HF-loaded nanoplatform (PEG@mPt-HF) showed enhanced cytotoxicity through the combination of photothermal therapy and the anti-TGFβ effect induced by HF. The animal imaging and histochemical assays confirmed the PEG@mPt-HF could efficiently deliver HF to tumors (monitored by CT) and remodel the ECM by TGFβ pathway inhibition, which resulted in increased anti-cancer efficacy. Importantly, the liver toxicity observed in HF-treated mice was negligible in those treated by PEG@mPt-HF. Overall, this study designed a theranostic nanoplatform to remodel the ECM with remarkably reduced systematic toxicity and enhance the therapeutic efficacy through combination treatment.

Efficient generation of a CYP3A4-T2A-luciferase knock-in HepaRG subclone and its optimized differentiation.

CRISPR/Cas9 has allowed development of better and easier-to-use ADME models than traditional methods by complete knockout or knock-in of genes. However, gene editing in HepaRG cells remains challenging because long-term monoclonal cultivation may alter their differentiation capacity to a large extent. Here, CRISPR/Cas9 was used to generate a CYP3A4-T2A-luciferase knock-in HepaRG subclone by Cas9-mediated homologous recombination and monoclonal cultivation. The knock-in HepaRG-#9 subclone retained a similar differentiation potential to wildtype HepaRG cells (HepaRG-WT). To further improve differentiation and expand the applications of knock-in HepaRG cells, two optimized differentiation procedures were evaluated by comparison with the standard differentiation procedure using the knock-in HepaRG-#9 subclone and HepaRG-WT. The results indicated that addition of forskolin (an adenylate cyclase activator) and SB431542 (a TGF-β pathway inhibitor) to the first optimized differentiation procedure led to better differentiation consequence in terms of not only the initiation time for differentiation and morphological characterization, but also the mRNA levels of hepatocyte-specific genes. These data may contribute to more extensive applications of genetically modified HepaRG cells in ADME studies.

Glutathione prevents high glucose-induced pancreatic fibrosis by suppressing pancreatic stellate cell activation via the ROS/TGFβ/SMAD pathway.

The activation of pancreatic stellate cells (PSCs) is the key mechanism of pancreatic fibrosis, which can lead to β-cell failure. Oxidative stress is an important risk factor for PSC activation. There is no direct evidence proving if administration of glutathione can inhibit fibrosis and β-cell failure. To explore the role of glutathione in pancreatic fibrosis and β-cell failure induced by hyperglycaemia, we established a rat model of pancreatic fibrosis and β-cell failure. The model was founded through long-term oscillating glucose (LOsG) intake and the setup of a sham group and a glutathione intervention group. In vitro, rat PSCs were treated with low glucose, high glucose, or high glucose plus glutathione to explore the mechanism of high glucose-induced PSC activation and the downstream effects of glutathione. Compared with sham rats, LOsG-treated rats had higher reactive oxygen species (ROS) levels in peripheral leukocytes and pancreatic tissue while TGFβ signalling was upregulated. In addition, as the number of PSCs and pancreatic fibrosis increased, β-cell function was significantly impaired. Glutathione evidently inhibited the upregulation of TGFβ signalling and several unfavourable outcomes caused by LOsG. In vitro treatment of high glucose for 72 h resulted in higher ROS accumulation and potentiated TGFβ pathway activation in PSCs. PSCs showed myofibroblast phenotype transformation with upregulation of α-SMA expression and increased cell proliferation and migration. Treatment with either glutathione or TGFβ pathway inhibitors alleviated these changes. Together, our findings suggest that glutathione can inhibit PSC activation-induced pancreatic fibrosis via blocking ROS/TGFβ/SMAD signalling in vivo and in vitro.