Adult Epithelial Cells Research Articles

Amelioration of Congenital Tufting Enteropathy in EpCAM (TROP1)-Deficient Mice via Heterotopic Expression of TROP2 in Intestinal Epithelial Cells.

TROP1 (EpCAM) and TROP2 are homologous cell surface proteins that are widely expressed, and often co-expressed, in developing and adult epithelia. Various functions have been ascribed to EpCAM and TROP2, but responsible mechanisms are incompletely characterized and functional equivalence has not been examined. Adult intestinal epithelial cells (IEC) express high levels of EpCAM, while TROP2 is not expressed. EpCAM deficiency causes congenital tufting enteropathy (CTE) in humans and a corresponding lethal condition in mice. We expressed TROP2 and EpCAM in the IEC of EpCAM-deficient mice utilizing a villin promoter to assess EpCAM and TROP2 function. Expression of EpCAM or TROP2 in the IEC of EpCAM knockout mice prevented CTE. TROP2 rescue (T2R) mice were smaller than controls, while EpCAM rescue (EpR) mice were not. Abnormalities were observed in the diameters and histology of T2R small intestine, and Paneth and stem cell markers were decreased. T2R mice also exhibited enlarged mesenteric lymph nodes, enhanced permeability to 4 kDa FITC-dextran and increased sensitivity to detergent-induced colitis, consistent with compromised barrier function. Studies of IEC organoids and spheroids revealed that stem cell function was also compromised in T2R mice. We conclude that EpCAM and TROP2 exhibit functional redundancy, but they are not equivalent.

Lipidomics reveal the protective effects of a vegetable-derived isothiocyanate against retinal degeneration

Background:Age-related macular degeneration (AMD) is a leading cause of blindness in the ageing population. Without effective treatment strategies that can prevent disease progression, there is an urgent need for novel therapeutic interventions to reduce the burden of vision loss and improve patients’ quality of life. Dysfunctional innate immune responses to oxidative stress observed in AMD can be caused by the formation of oxidised lipids, whilst polyunsaturated fatty acids have shown to increase the risk of AMD and disease progression in affected individuals. Previously, our laboratory has shown that the vegetable-derived isothiocyanate, L-sulforaphane (LSF), can protect human adult pigment epithelial cells from oxidative damage by upregulating gene expression of the oxidative stress enzyme Glutathione-S-Transferase µ1. This study aims to validate the protective effects of LSF on human retinal cells under oxidative stress conditions and to reveal the key players in fatty acid and lipid metabolism that may facilitate this protection.Methods:Thein vitrooxidative stress model of AMD was based on the exposure of an adult retinal pigment epithelium-19 cell line to 200µM hydrogen peroxide. Percentage cell proliferation following LSF treatment was measured using tetrazolium salt-based assays. Untargeted fatty acid profiling was performed by gas chromatography-mass spectrometry. Untargeted lipid profiling was performed by liquid chromatography-mass spectrometry.Results:Under hydrogen peroxide-induced oxidative stress conditions, LSF treatment induced dose-dependent cell proliferation. The key fatty acids that were increased by LSF treatment of the retinal cells include oleic acid and eicosatrienoic acid. LSF treatment also increased levels of the lipid classes phosphatidylcholine, cholesteryl ester and oxo-phytodienoic acid but decreased levels of phosphatidylethanolamine lipids.Conclusions:We propose that retinal cells at risk of oxidative damage and apoptosis can be pre-conditioned with LSF to regulate levels of selected fatty acids and lipids known to be implicated in the pathogenesis and progression of AMD.

Abstract IA25: Adult deletion of Lats1/2 leads to sarcomatoid renal cell carcinoma

Abstract Alterations of the Hippo signaling pathway have been found in patients with sarcomatoid renal cell carcinoma, a highly aggressive kidney disease with rapid progression and poor prognosis. It is unclear whether deregulated Hippo pathway is a primary cause of sarcomatoid cancers. To investigate whether conditional (kidney-specific) Hippo knockout leads to renal cancer development and/or sarcomatoid dedifferentiation, we conditionally inactivated both Lats1 and Lats2 from the adult renal proximal tubule epithelial cells. Mosaic deletion of Lats1 and Lats2 in the adult proximal tubules results in cell-autonomous sarcomatoid renal cell carcinoma ((54%) as early as 8 weeks after gene deletion) and lung metastasis. RNA sequencing of tumors compared to control renal cortices revealed massive transcriptional changes, with 2,292 genes significantly upregulated (FC>2) and 2261 genes significantly downregulated (FCË‚0.5). To visualize the cellular onset and progression of renal defects, we conducted histologic examination at different time points after induction of Lats1/2 deletion. Mutant kidneys displayed tubular atrophy with glomerular cysts. Loss of proximal tubule epithelial markers, and de novo expression of the mesenchymal marker vimentin, indicated that kidney epithelial cells had undergone an epithelial-to-mesenchymal transition (EMT). These studies demonstrate that loss of Lats1/2 rapidly leads to aggressive, metastatic sarcomatoid renal cell carcinoma. Citation Format: Antoine Reginensi, Sanjay Jain, Benjamin Humphreys, Jess Shen, Didier Hodzic, Jeff Wrana, Helen McNeill. Adult deletion of Lats1/2 leads to sarcomatoid renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA25.

SARS-CoV-2 Receptor ACE2 Is Enriched in a Subpopulation of Mouse Tongue Epithelial Cells in Nongustatory Papillae but Not in Taste Buds or Embryonic Oral Epithelium.

As a result of the COVID-19 pandemic, evidence revealed that SARS-CoV-2 infection caused taste loss at a rate higher than that of influenza. ACE2, the entry receptor of SARS-CoV-2, has been identified in the oral epithelium; however, it is unclear at what developmental stage ACE2 expression emerges and whether ACE2 is expressed in taste buds. To identify the specific developmental stage, we analyzed RNA-Seq data from embryonic and newborn mouse oral tissue. We found that robust ACE2 expression was observed in the newborn oral epithelium. In contrast, only extremely low levels, if any, of ACE2 transcripts in the embryonic stage oral tissue were found (E12.5 and E14.5). Analyses of three public scRNA-seq data sets of adult mouse tongue epithelial cells showed that receptors for various viruses were enriched in distinct clusters of tongue epithelial cells. ACE2 was enriched in a subpopulation of epithelial cells in the basal region of nongustatory filiform papillae but not in the taste papillae or taste buds. Expression of ACE2 was detected in a small proportion of type III taste cells. Our results indicate that when applied across species, nongustatory papilla epithelial cells are the prime targets for SARS-CoV-2 infection in the tongue; thus, taste loss in COVID-19 patients is likely not caused by a direct infection of SARS-CoV-2 to taste bud cells. Additionally, fetuses at different stages of development may have distinct susceptibility to SARS-CoV-2 infection.

Analysis of Thyroid Hormone Receptor α-Knockout Tadpoles Reveals That the Activation of Cell Cycle Program Is Involved in Thyroid Hormone-Induced Larval Epithelial Cell Death and Adult Intestinal Stem Cell Development During Xenopus tropicalis Metamorphosis.

Background: There are two highly conserved thyroid hormone (triiodothyronine [T3]) receptor (TR) genes, TRα and TRβ, in all vertebrates, and the expression of TRα but not TRβ is activated earlier than T3 synthesis during development. In human, high levels of T3 are present during the several months around birth, and T3 deficiency during this period causes severe developmental abnormalities including skeletal and intestinal defects. It is, however, difficult to study this period in mammals as the embryos and neonates depend on maternal supply of nutrients for survival. However, Xenopus tropicalis undergoes a T3-dependent metamorphosis, which drastically changes essentially every organ in a tadpole. Of interest is intestinal remodeling, which involves near complete degeneration of the larval epithelium through apoptosis. Concurrently, adult intestinal stem cells are formed de novo and subsequently give rise to the self-renewing adult epithelial system, resembling intestinal maturation around birth in mammals. We have previously demonstrated that T3 signaling is essential for the formation of adult intestinal stem cells during metamorphosis. Methods: We studied the function of endogenous TRα in the tadpole intestine by using knockout animals and RNA-seq analysis. Results: We observed that removing endogenous TRα caused defects in intestinal remodeling, including drastically reduced larval epithelial cell death and adult intestinal stem cell proliferation. Using RNA-seq on intestinal RNA from premetamorphic wild-type and TRα-knockout tadpoles treated with or without T3 for one day, before any detectable T3-induced cell death and stem cell formation in the tadpole intestine, we identified more than 1500 genes, which were regulated by T3 treatment of the wild-type but not TRα-knockout tadpoles. Gene Ontology and biological pathway analyses revealed that surprisingly, these TRα-regulated genes were highly enriched with cell cycle-related genes, in addition to genes related to stem cells and apoptosis. Conclusions: Our findings suggest that TRα-mediated T3 activation of the cell cycle program is involved in larval epithelial cell death and adult epithelial stem cell development during intestinal remodeling.

Role of Taurine Transporter in the Retinal Uptake of Vigabatrin.

Vigabatrin (VGB) is a first-line drug used for treatment of infantile spasms. On therapeutic dose, VGB accumulates in the retina causing permanent peripheral visual field constriction. The mechanism involved in retinal accumulation of VGB is ambiguous. In the present study, mechanism of VGB transport into retina was evaluated. VGB uptake into retina was studied in vitro using human adult retinal pigment epithelial (ARPE-19) cells as a model for outer blood retinal barrier. The VGB cell uptake studies demonstrated saturation kinetics with Km value of 13.1mM and uptake was significantly increased at pH7.4 and hyperosmolar conditions indicating involvement of carrier-mediated Na+-Cl--dependent transporter. In the presence of taurine transporter (TauT) substrates (taurine and GABA) and inhibitor guanidinoethyl sulfonate (GES), the uptake of VGB decreased significantly demonstrating contribution of TauT. The VGB retinal levels in rats were decreased by 1.5- and 1.3-folds on chronic administration of GES and taurine, respectively. In conclusion, this study demonstrated the TauT involvement in VGB uptake and accumulation in retina.

Wnt/β-catenin signaling is critical for regenerative potential of distal lung epithelial progenitor cells in homeostasis and emphysema

Wnt/β-catenin signaling regulates progenitor cell fate decisions during lung development and in various adult tissues. Ectopic activation of Wnt/β-catenin signaling promotes tissue repair in emphysema, a devastating lung disease with progressive loss of parenchymal lung tissue. The identity of Wnt/β-catenin responsive progenitor cells and the potential impact of Wnt/β-catenin signaling on adult distal lung epithelial progenitor cell function in emphysema are poorly understood. Here, we used TCF/ Lef:H2B/GFP reporter mice to investigate the role of Wnt/β-catenin signaling in lung organoid formation. We identified an organoid-forming adult distal lung epithelial progenitor cell population characterized by a low Wnt/β-catenin activity, which was enriched in club and alveolar epithelial type (AT)II cells. Endogenous Wnt/β-catenin activity was required for the initiation of multiple subtypes of distal lung organoids derived from the Wntlow epithelial progenitors. Further ectopic Wnt/β-catenin activation specifically led to an increase in alveolar organoid number; however, the subsequent proliferation of alveolar epithelial cells in the organoids did not require constitutive Wnt/β-catenin signaling. Distal lung epithelial progenitor cells derived from the mouse model of elastase-induced emphysema exhibited reduced organoid forming capacity. This was rescued by Wnt/β-catenin signal activation, which largely increased the number of alveolar organoids. Together, our study reveals a novel mechanism of lung epithelial progenitor cell activation in homeostasis and emphysema.

Copper mediates mitochondrial biogenesis in retinal pigment epithelial cells

Age related macular degeneration (AMD) is a multifactorial disease with genetic, biochemical and environmental risk factors. We observed a significant increase in copper levels in choroid-RPE from donor eyeballs with AMD. Adult retinal pigment epithelial cells (ARPE19 cells) exposed to copper in-vitro showed a 2-fold increase in copper influx transporter CTR1 and copper uptake at 50 μM concentration. Further there was 2-fold increase in cytochrome C oxidase activity and a 2-fold increase in the mRNA expression of NRF 2 with copper treatment. There was a significant increase in mitochondrial biogenesis markers PGC1β and TFAM which was confirmed by mitochondrial mass and copy number. On the contrary, in AMD choroid-RPE, the CTR1 mRNA was found to be significantly down-regulated compared to its respective controls. SCO1 and PGC1β mRNA showed an increase in choroid–RPE. Our study proposes copper to play an important role in mitochondrial biogenesis in RPE cells.

LncRNA GAS5 suppresses ER stress‑induced apoptosis and inflammation by regulating SERCA2b in HG‑treated retinal epithelial cell.

Hyperglycemia impairs the retinal functions in patients with diabetic retinopathy (DR). Downregulation of long non‑coding RNA growth arrest‑specific transcript 5 (lncRNA GAS5) expression in diabetes affects glucose intake and insulin signaling. Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) mediates the regulation of endoplasmic reticulum (ER) stress and apoptosis in high glucose (HG)‑treated podocytes. Therefore, the present study aimed to investigate the roles of lncRNA GAS5 and SERCA2 in retinal pigment epithelium cells exposed to HG. GAS5 expression levels were detected using reverse transcription‑quantitative PCR. In addition, the expression levels of SERCA2b, ER stress‑related proteins, pro‑inflammatory factors and apoptotic proteins were determined by western blot analysis, ELISA or flow cytometry. The results showed that HG treatment induced ER stress in ARPE‑19 human adult retinal pigment epithelial cells by upregulating the expression levels of phosphorylated (p)‑protein kinase R‑like ER kinase, p‑eukaryotic initiation factor 2α, activating transcription factor 4 and CCAAT/enhancer‑binding protein homologous protein. In addition, HG treatment induced apoptosis by increasing Bax, Bad and caspase 12, and by decreasing Bcl‑2 levels expression levels. Moreover, HG treatment induced inflammation by upregulating tumor necrosis factor‑α, interleukin (IL)‑1β and IL‑6 expression. However, GAS5 and SERCA2b overexpression significantly decreased ER stress‑related apoptosis and inflammation, whereas SERCA2b knockdown significantly reversed the inhibitory effect of GAS5 on ER stress, apoptosis and inflammation. The results of the present study indicated that GAS5 may suppress ER stress‑induced apoptosis and inflammation by regulating SERCA2b in HG‑treated cells. These data suggested that GAS5 may serve a vital role in the pathogenesis of DR, and it may be considered a potential target for DR therapy.

Direct activation of tRNA methyltransferase-like 1 (Mettl1) gene by thyroid hormone receptor implicates a role in adult intestinal stem cell development and proliferation during Xenopus tropicalis metamorphosis

BackgroundThyroid hormone (T3) plays an important role in vertebrate development. Compared to the postembryonic development of uterus-enclosed mammalian embryos, T3-dependent amphibian metamorphosis is advantageous for studying the function of T3 and T3 receptors (TRs) during vertebrate development. The effects of T3 on the metamorphosis of anurans such as Xenopus tropicalis is known to be mediated by TRs. Many putative TR target genes have been identified previously. Among them is the tRNA methyltransferase Mettl1.ResultsWe studied the regulation of Mettl1 gene by T3 during intestinal metamorphosis, a process involves near complete degeneration of the larval epithelial cells via apoptosis and de novo formation of adult epithelial stem cells and their subsequent proliferation and differentiation. We observed that Mettl1 was activated by T3 in the intestine during both natural and T3-induced metamorphosis and that its mRNA level peaks at the climax of intestinal remodeling. We further showed that Mettl1 promoter could be activated by liganded TR via a T3 response element upstream of the transcription start site in vivo. More importantly, we found that TR binding to the TRE region correlated with the increase in the level of H3K79 methylation, a transcription activation histone mark, and the recruitment of RNA polymerase II by T3 during metamorphosis.ConclusionsOur findings suggest that Mettl1 is activated by liganded TR directly at the transcriptional level via the TRE in the promoter region in the intestine during metamorphosis. Mettl1 in turn regulate target tRNAs to affect translation, thus facilitating stem cell formation and/or proliferation during intestinal remodeling.

Elevated PDK1 Expression Drives PI3K/AKT/MTOR Signaling Promotes Radiation-Resistant and Dedifferentiated Phenotype of Hepatocellular Carcinoma.

Resistance to radiotherapy (IR), with consequent disease recurrence, continues to limit the efficacy of contemporary anticancer treatment for patients with hepatocellular carcinoma (HCC), especially in late stage. Despite accruing evidence implicating the PI3K/AKT signaling pathway in cancer-promoting hypoxia, cancerous cell proliferation and radiotherapy-resistance, it remains unclear which molecular constituent of the pathway facilitates adaptation of aggressive HCC cells to tumoral stress signals and drives their evasion of repeated IR-toxicity. This present study investigated the role of PDK1 signaling in IR-resistance, enhanced DNA damage repair and post-IR relapse, characteristic of aggressive HCC cells, while exploring potential PDK1-targetability to improve radiosensitivity. The study employed bioinformatics analyses of gene expression profile and functional protein–protein interaction, generation of IR-resistant clones, flow cytometry-based ALDH activity and side-population (SP) characterization, siRNA-mediated loss-of-PDK1function, western-blotting, immunohistochemistry and functional assays including cell viability, migration, invasion, clonogenicity and tumorsphere formation assays. We showed that the aberrantly expressed PDK1 characterizes poorly differentiated HCC CVCL_7955, Mahlavu, SK-HEP1 and Hep3B cells, compared to the well-differentiated Huh7 or normal adult liver epithelial THLE-2 cells, and independently activates the PI3K/AKT/mTOR signaling. Molecular ablation of PDK1 function enhanced susceptibility of HCC cells to IR and was associated with deactivated PI3K/AKT/mTOR signaling. Additionally, PDK1-driven IR-resistance positively correlated with activated PI3K signaling, enhanced HCC cell motility and invasiveness, augmented EMT, upregulated stemness markers ALDH1A1, PROM1, SOX2, KLF4 and POU5F1, increased tumorsphere-formation efficiency and suppressed biomarkers of DNA damage—RAD50, MSH3, MLH3 and ERCC2. Furthermore, the acquired IR-resistant phenotype of Huh7 cells was strongly associated with significantly increased ALDH activity, SP-enrichment, and direct ALDH1-PDK1 interaction. Moreover, BX795-mediated pharmacological inhibition of PDK1 synergistically enhances the radiosensitivity of erstwhile resistant cells, increased Bax/Bcl-2 apoptotic ratio, while suppressing oncogenicity and clonogenicity. We provide preclinical evidence implicating PDK1 as an active driver of IR-resistance by activation of the PI3K/AKT/mTOR signaling, up-modulation of cancer stemness signaling and suppression of DNA damage, thus, projecting PDK1-targeting as a putative enhancer of radiosensitivity and a potential new therapeutic approach for patients with IR-resistant HCC.

Dexamethasone Loaded Liposomes by Thin-Film Hydration and Microfluidic Procedures: Formulation Challenges.

Liposomes have been one of the most exploited drug delivery systems in recent decades. However, their large-scale production with low batch-to-batch differences is a challenge for industry, which ultimately delays the clinical translation of new products. We have investigated the effects of formulation parameters on the colloidal and biopharmaceutical properties of liposomes generated with a thin-film hydration approach and microfluidic procedure. Dexamethasone hemisuccinate was remotely loaded into liposomes using a calcium acetate gradient. The liposomes produced by microfluidic techniques showed a unilamellar structure, while the liposomes produced by thin-film hydration were multilamellar. Under the same remote loading conditions, a higher loading capacity and efficiency were observed for the liposomes obtained by microfluidics, with low batch-to-batch differences. Both formulations released the drug for almost one month with the liposomes prepared by microfluidics showing a slightly higher drug release in the first two days. This behavior was ascribed to the different structure of the two liposome formulations. In vitro studies showed that both formulations are non-toxic, associate to human Adult Retinal Pigment Epithelial cell line-19 (ARPE-19) cells, and efficiently reduce inflammation, with the liposomes obtained by the microfluidic technique slightly outperforming. The results demonstrated that the microfluidic technique offers advantages to generate liposomal formulations for drug-controlled release with an enhanced biopharmaceutical profile and with scalability.

Lipidomics reveal the protective effects of a vegetable-derived isothiocyanate against retinal degeneration

Background:Age-related macular degeneration (AMD) is a leading cause of blindness in the ageing population. Without effective treatment strategies that can prevent disease progression, there is an urgent need for novel therapeutic interventions to reduce the burden of vision loss and improve patients’ quality of life. Dysfunctional innate immune responses to oxidative stress observed in AMD can be caused by the formation of oxidised lipids, whilst polyunsaturated fatty acids have shown to increase the risk of AMD and disease progression in affected individuals. Previously, our laboratory has shown that the vegetable-derived isothiocyanate, L-sulforaphane (LSF), can protect human adult pigment epithelial cells from oxidative damage by upregulating gene expression of the oxidative stress enzyme Glutathione-S-Transferase µ1. This study aims to validate the protective effects of LSF on human retinal cells under oxidative stress conditions and to reveal the key players in fatty acid and lipid metabolism that may facilitate this protection.Methods:Thein vitrooxidative stress model of AMD was based on the exposure of an adult retinal pigment epithelium-19 cell line to 200µM hydrogen peroxide. Percentage cell proliferation following LSF treatment was measured using tetrazolium salt-based assays. Untargeted fatty acid profiling was performed by gas chromatography-mass spectrometry. Untargeted lipid profiling was performed by liquid chromatography-mass spectrometry.Results:Under hydrogen peroxide-induced oxidative stress conditions, LSF treatment induced dose-dependent cell proliferation. The key fatty acids that were increased by LSF treatment of the retinal cells include oleic acid and eicosatrienoic acid. LSF treatment also increased levels of the lipid classes phosphatidylcholine, cholesteryl ester and oxo-phytodienoic acid but decreased levels of phosphatidylethanolamine lipids.Conclusions:We propose that retinal cells at risk of oxidative damage and apoptosis can be pre-conditioned with LSF to regulate levels of selected fatty acids and lipids known to be implicated in the pathogenesis and progression of AMD.

Promotion of wound healing by acetate in murine colonic epithelial cell via c-Jun N-terminal kinase activation.

Mucosal healing is an important clinical goal in patients with inflammatory bowel disease. Recently, short-chain fatty acids (SCFAs) have been reported to have multifaceted effects to host. However, the effects of SCFAs on wound healing in intestinal epithelial cells are unclear. In the present study, we investigated the effects of acetate, one of the major SCFAs, on the wound healing of murine colonic epithelial cells. Young adult mouse colonic epithelial cells were used to determine the effect of acetate using wound healing assay. Mitogen-activated protein kinase and Rho kinase inhibitor were used to elucidate intracellular signal of wound healing treated with acetate. Meanwhile, Rho activation assays were utilized to measure Rho activation levels. To assess in vivo effects, C57B6 mice with dextran sodium sulfate for 7days were treated with enema administration of acetate for 7days. Body weight, disease activity index, colon length, and mucosal break ratio in histology were examined. Acetate enhanced wound healing and fluorescence intensity of actin stress fiber compared with control. These effects were canceled with pretreatment of c-Jun N-terminal kinase (JNK) inhibitor or Rho kinase inhibitor. Furthermore, JNK inhibitor reduced the activation of Rho induced by acetate. In the dextran sodium sulfate-induced colitis model, the mice with enema treatment of acetate significantly exhibited recovery. In this study, we demonstrated that acetate promoted murine colonic epithelial cell wound healing via activation of JNK and Rho signaling pathways. These findings suggested that acetate could have applications as a therapeutic agent for patients with intestinal mucosal damage, such as inflammatory bowel disease.

AQP5 enriches for stem cells and cancer origins in the distal stomach.

LGR5 marks resident adult epithelial stem cells at the gland base in the mouse pyloric stomach1, but the identity of the equivalent human stem cell population remains unknown owing to a lack of surface markers that facilitate its prospective isolation and validation. In mouse models of intestinal cancer, LGR5+ intestinal stem cells are major sources of cancer following hyperactivation of the WNT pathway2. However, the contribution of pyloric LGR5+ stem cells to gastric cancer following dysregulation of the WNT pathway-a frequent event in gastric cancer in humans3-is unknown. Here we use comparative profiling of LGR5+ stem cell populations along the mouse gastrointestinal tract to identify, and then functionally validate, the membrane protein AQP5 as a marker that enriches for mouse and human adult pyloric stem cells. We show that stem cells within the AQP5+ compartment are a source of WNT-driven, invasive gastric cancer in vivo, using newly generated Aqp5-creERT2 mouse models. Additionally, tumour-resident AQP5+ cells can selectively initiate organoid growth in vitro, which indicates that this population contains potential cancer stem cells. In humans, AQP5 is frequently expressed in primary intestinal and diffuse subtypes of gastric cancer (and in metastases of these subtypes), and often displays altered cellular localization compared with healthy tissue. These newly identified markers and mouse models will be an invaluable resource for deciphering the early formation of gastric cancer, and for isolating and characterizing human-stomach stem cells as a prerequisite for harnessing the regenerative-medicine potential of these cells in the clinic.

Quercetin attenuates high glucose-induced injury in human retinal pigment epithelial cell line ARPE-19 by up-regulation of miR-29b.

Quercetin is a kind of distinctive bioactive flavonoid that has potent anti-oxidant, anti-inflammatory and anti-diabetic properties. The present article was designed to check the effect of quercetin on diabetic retinopathy. Adult retinal pigment epithelial cell line (ARPE)-19 cells were pre-treated with quercetin and then stimulated by high glucose. Cell damage was evaluated by CCK-8 assay, flow cytometer, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay, 2,7-dichlorofluorescein diacetate probe and western blot. The association between quercetin and miR-29b expression as well as the downstream pathways was studied by qRT-PCR and western blot. Pre-treating ARPE-19 cells with quercetin clearly attenuated high glucose-induced viability loss, apoptosis, MCP-1 and IL-6 overproduction and reactive oxygen species (ROS) generation. Quercetin down-regulated p53, Bax and cleaved-caspase-3 expression, while up-regulated CyclinD1, CDK4 and Bcl-2. miR-29b was low expressed in high glucose-treated cell, but quercetin elevated its expression. Moreover, the protective action of quercetin towards ARPE-19 cells was attenuated when miR-29b was suppressed. Also, quercetin promoted PTEN/AKT pathway, while inhibited NF-κB pathway via a miR-29b-dependent way. These data illustrated quercetin possibly possess the anti-diabetic retinopathy potential, as quercetin clearly attenuated high glucose-evoked damage in ARPE-19 cells. The protective action of quercetin may due to its regulation on miR-29b expression as well as PTEN/AKT and NF-κB pathways.

BMP Antagonists Secreted by Mesenchymal Stromal Cells Improve Colonic Organoid Formation: Application for the Treatment of Radiation-induced Injury

Radiation therapy is crucial in the therapeutic arsenal to cure cancers; however, non-neoplastic tissues around an abdominopelvic tumor can be damaged by ionizing radiation. In particular, the radio-induced death of highly proliferative stem/progenitor cells of the colonic mucosa could induce severe ulcers. The importance of sequelae for patients with gastrointestinal complications after radiotherapy and the absence of satisfactory management has opened the field to the testing of innovative treatments. The aim of this study was to use adult epithelial cells from the colon, to reduce colonic injuries in an animal model reproducing radiation damage observed in patients. We demonstrated that transplanted in vitro-amplified epithelial cells from colonic organoids (ECO) of C57/Bl6 mice expressing green fluorescent protein implant, proliferate, and differentiate in irradiated mucosa and reduce ulcer size. To improve the therapeutic benefit of ECO-based treatment with clinical translatability, we performed co-injection of ECO with mesenchymal stromal cells (MSCs), cells involved in niche function and widely used in clinical trials. We observed in vivo an improvement of the therapeutic benefit and in vitro analysis highlighted that co-culture of MSCs with ECO increases the number, proliferation, and size of colonic organoids. We also demonstrated, using gene expression analysis and siRNA inhibition, the involvement of bone morphogenetic protein antagonists in MSC-induced organoid formation. This study provides evidence of the potential of ECO to limit late radiation effects on the colon and opens perspectives on combined strategies to improve their amplification abilities and therapeutic effects.

Intravitreal injection of peptides PnPa11 and PnPa13, derivatives of Phoneutria nigriventer spider venom, prevents retinal damage.

Background: PnPa11 and PnPa13 are synthetic peptides derived from Phoneutria nigriventer spider venom, which display antinociceptive and neuroprotective properties. In this work, we evaluated the safety of intravitreal use and the neuroprotective effect of these peptides.Methods: The cytotoxicity and the antiangiogenic activity of these peptides were evaluated by the sulforhodamine-B method and chicken chorioallantoic membrane (CAM) assay, respectively. The in vivo safety was analyzed in Wistar rats that were intravitreally injected with different doses (0.50; 1.25; 2.50; 3.75 and 5.00 µg/mL) of these peptides (right eye, n = 6). The retinal function was assessed by electroretinography exams (ERG), intraocular pressure (IOP), and histological analyzes. In order to investigate the neuroprotective effect, Wistar rats received intravitreal injections (right eye, n = 6) of peptides at 1.25 µg/mL and then were exposed to blue LED light. In addition, the visual function and the retinal microstructure were verified.Results: Cytotoxicity analyses demonstrated that the peptides did not present any toxicity over ARPE-19 (adult retinal pigmented epithelial) cell line and the antiangiogenic study highlighted that the peptides promoted the reduction of blood vessels. The intravitreal injection did not cause major changes, neither induced any irreversible damage. In the retinal degeneration assay, the ERG records demonstrated that the prior treatment with PnPa11 and PnPa13 protected the retina from damage. Morphological analyses confirmed the ERG findings. Immunoblotting analyses revealed that PnPa11 increased Erk1/2, NR2A, and NR2B retinal expression after the light stress model, but did not cause Akt1 activation, while PnPa13 prevented Erk1/2 and Akt1 dephosphorylation.Conclusions: The intraocular administration of these peptides was well tolerated and presented protective activity against retinal degeneration, suggesting the potential use of these peptides as neuroprotectors in the ophthalmological field.

Renewed proliferation in adult mouse cochlea and regeneration of hair cells

The adult mammalian inner ear lacks the capacity to divide or regenerate. Damage to inner ear generally leads to permanent hearing loss in humans. Here, we present that reprogramming of the adult inner ear induces renewed proliferation and regeneration of inner ear cell types. Co-activation of cell cycle activator Myc and inner ear progenitor gene Notch1 induces robust proliferation of diverse adult cochlear sensory epithelial cell types. Transient MYC and NOTCH activities enable adult supporting cells to respond to transcription factor Atoh1 and efficiently transdifferentiate into hair cell-like cells. Furthermore, we uncover that mTOR pathway participates in MYC/NOTCH-mediated proliferation and regeneration. These regenerated hair cell-like cells take up the styryl dye FM1-43 and are likely to form connections with adult spiral ganglion neurons, supporting that Myc and Notch1 co-activation is sufficient to reprogram fully mature supporting cells to proliferate and regenerate hair cell-like cells in adult mammalian auditory organs.

Abstract C053: NP-G2-044, a novel fascin inhibitor, blocks tumor metastasis and increases antitumor immune response

Abstract Filopodia are finger-like plasma membrane protrusions that are formed upon remodeling of the actin cytoskeleton beneath the plasma membrane. Fascin protein is the main actin-bundling protein in filopodia and invadopodia which are fundamental to cell shape and motility events. Human fascin expression is low or absent in normal adult epithelial cells but highly expressed in metastatic tumors and in antigen-presenting dendritic cells within the tumor microenvironment. Elevated levels of fascin are correlated with poor prognosis and short survival due to aggressive phenotypes with increased lymph node and distant metastases. Mouse genetic studies demonstrate that deletion of fascin gene delays tumor development, slows tumor growth, reduces metastatic colonization, and increases overall survival. These mouse genetic studies provide strong evidence for fascin’s role in tumor initiation, tumor progression and tumor metastasis, making it an attractive target for drug development. Chemical libraries were screened to identify small-molecule compounds that specifically inhibit the biochemical function of fascin to bundle actin filaments. The inhibitors were optimized to produce the lead compound, NP-G2-044. Preclinical studies showed that fascin inhibitors increase the overall survival of tumor-bearing mice and reinvigorate anti-tumor immune response in syngeneic mouse models of breast, lung, pancreatic and prostate tumors. Fascin protein levels are increased in conventional dendritic cells (cDCs) in the immunosuppressive tumor microenvironment. The number of intra-tumoral activated cDCs is increased following fascin inhibitor treatment. Moreover, together with anti-PD-1 antibody, fascin inhibition markedly increases the number of Ki67+ proliferating and Granzyme-B+ activated CD8+ killer T-cells in tumor tissue. These data demonstrate that fascin inhibitors simultaneously act on tumor cells to block tumor metastasis and on intratumoral DCs to reinvigorate anti-tumor immune response. We are currently conducting a first-in-human phase 1 trial of NP-G2-044 in patients with advanced or metastatic solid tumor malignancies. Citation Format: Vincent Chung, Frank Tsai, Komal Jhaveri, Yufeng Wang, Daniel D Von Hoff, Edward G Garmey, J. Jillian Zhang, Xin-Yun Huang. NP-G2-044, a novel fascin inhibitor, blocks tumor metastasis and increases antitumor immune response [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C053. doi:10.1158/1535-7163.TARG-19-C053