Rac1 promotes kidney collecting duct integrity by limiting actomyosin activity

A polarized collecting duct (CD), formed from the branching ureteric bud (UB), is a prerequisite for an intact kidney. The small Rho GTPase Rac1 is critical for actin cytoskeletal regulation. We investigated the role of Rac1 in the kidney collecting system by selectively deleting it in mice at the initiation of UB development. The mice exhibited only a mild developmental phenotype; however, with aging, the CD developed a disruption of epithelial integrity and function. Despite intact integrin signaling, Rac1-null CD cells had profound adhesion and polarity abnormalities that were independent of the major downstream Rac1 effector, Pak1. These cells did however have a defect in the WAVE2–Arp2/3 actin nucleation and polymerization apparatus, resulting in actomyosin hyperactivity. The epithelial defects were reversible with direct myosin II inhibition. Furthermore, Rac1 controlled lateral membrane height and overall epithelial morphology by maintaining lateral F-actin and restricting actomyosin. Thus, Rac1 promotes CD epithelial integrity and morphology by restricting actomyosin via Arp2/3-dependent cytoskeletal branching.

Establishment of a New Cell Line of Canine Mammary Tumor CMT-1026

Canine mammary tumors (CMTs) have histopathological, epidemiologic and clinical characteristics similar to those in humans and are known to be one of the best models for human breast cancer (HBC). This research aimed to describe a newly established canine cell line, CMT-1026. Tumor samples were collected from a female dog exhibiting clinical mammary neoplasm, and the adherent cells were cultured. Both the histology and immunohistochemistry (IHC) of tumor samples were estimated. Cell growth, ultrastructural, cytological and immunocytochemistry (ICC) features of CMT-1026 were examined. CMT-1026 cells were inoculated into 10 female BALB/c nude mice to evaluate oncogenicity and metastatic ability. Hematoxylin-eosin (H.E.) staining of the tumors revealed an epithelial morphology. Electron microscopy was used to detect histological and cytological of smears, and ultrathin sections showed that CMT-1026 cells were polygonal and characterized by atypia and high mitotic index in the tumor, with prominent nucleoli and multinucleated cells. IHC characterization of CMT-1026 indicated ER-, PR-, HER-2, p63+, CK5/6+, and α-SMA+ epithelial cells. ICC characterization of CMT-1026 showed high expression of Claudin-1, Delta-catenin, SOX-2, and KI-67. At 2 weeks after inoculation of the CMT-1026 cells, phyma was found in 100% of the mice. The xenograft cancers showed conservation of the original H.E. features of the female dog cancer. In conclusion, CMT-1026 may be a model of canine mammary cancer that can be used in research on the pathogenesis of both CMT and HBC.

Vacuolar-type proton ATPase is required for maintenance of apicobasal polarity of embryonic visceral endoderm

The endocytic compartments keep their interior acidic through the inward flow of protons and anions from the cytosol. Acidification is mediated by a proton pump known as vacuolar-type ATPase (V-ATPase) and transporters conferring anion conductance to the organellar membrane. In this study, we analysed the phenotype of mouse embryos lacking the V-ATPase c-subunit. The mutant embryos differentiated embryonic epithelial tissues, primitive endoderm, epiblast, and extraembryonic ectoderm; however, the organisation of these epithelia was severely affected. The apical-basal polarity in the visceral endoderm layer was not properly established in the mutant embryos, resulting in abnormal epithelial morphology. Thus, the function of V-ATPase is imperative for the establishment and/or maintenance of epithelial cell polarity, which is required for early embryogenesis.

Study of the effect of Pioglitazone and Cetuximab on Cancer Stem-like Cellsenriched HT29 cell line

One of the major causes of cancer resistance to chemotherapy has been found to be the presence of Cancer Stem Cells (CSCs) in cancerous tissues. Probably, these cells are the source of cancer and the cause of malignancy and recurrence in the affected population. Therefore, it is possible to target CSCs to treat cancer. Since the percentage of CSCs in the total tumor mass is very low, so studies about these cells depend on their isolation and enrichment methods. Some studies have suggested that EMT induction in population of normal epithelial cells and cancer cells by inhibiting of E-cadherin, protects them against chemotherapy, anticancer and apoptosis drugs, moreover they get characteristics of CSCs. So in order to study CSCs can enrich them by inhibiting of E-cadherin in tumor population.In this study, we tried to examine how the effect of Pioglitazone and Cetuximab, two drugs used in chemotherapy of Colon Cancer, on CSCs enriched HT29 cell line (was called HT29-shE) in which CSCs were enriched with induction of EMT by inhibiting of E-cadherin using shRNA.For this purpose, after cell preparation EMT and CSCs markers in Pioglitazone and Cetuximab treated cells were assessed and compared with untreated cells using flow cytometry, real‐time PCR and microscopic monitoring. The findings showed mesenchymal morphology of HT29-shE changed to epithelial morphology after Pioglitazone and Cetuximab treatment, moreover E-cadherin expression increased and Vimentin expression decreased. In addition, expression of CSC markers (CD133+ and CD44+) were reduced in HT29-shE after treatment.

Human placental-derived stem cell therapy ameliorates experimental necrotizing enterocolitis

These studies demonstrate a human placental-derived stem cell (hPSC) therapeutic strategy for necrotizing enterocolitis (NEC). In an experimental model of NEC, hPSC administration improved macroscopic intestinal health, ameliorated epithelial morphology, and supported the intestinal stem cell niche. Our data suggest that hPSC are a potential therapeutic approach to attenuate established intestinal NEC damage. Further, we show hPSC are a novel research tool that can be utilized to elucidate critical neonatal repair mechanisms to overcome NEC.

Mutational inactivation of Apc in the intestinal epithelia compromises cellular organisation

ABSTRACTThe adenomatous polyposis coli (Apc) protein regulates diverse effector pathways essential for tissue homeostasis. Truncating oncogenic mutations in Apc removing its Wnt pathway and microtubule regulatory domains drives intestinal epithelia tumorigenesis. Exuberant cell proliferation is one well-established consequence of oncogenic Wnt pathway activity; however, the contribution of other deregulated molecular circuits to tumorigenesis has not been fully examined. Using in vivo and organoid models of intestinal epithelial tumorigenesis we found that Wnt pathway activity controls intestinal epithelial villi and crypt structure, morphological features lost upon Apc inactivation. Although the Wnt pathway target gene c-Myc (also known as Myc) has critical roles in regulating cell proliferation and tumorigenesis, Apc specification of intestinal epithelial morphology is independent of the Wnt-responsive Myc-335 (also known as Rr21) regulatory element. We further demonstrate that Apc inactivation disrupts the microtubule cytoskeleton and consequently localisation of organelles without affecting the distribution of the actin cytoskeleton and associated components. Our data indicates the direct control over microtubule dynamics by Apc through an independent molecular circuit. Our study stratifies three independent Apc effector pathways in the intestinal epithelial controlling: (1) proliferation, (2) microtubule dynamics and (3) epithelial morphology.This article has an associated First Person interview with the first author of the paper.

P03.29 Characterization of treatment-induced adaptive immune responses in pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy marked by poor prognosis and profound drug resistance characterized in more than 90% of cases by KRAS mutations. To recapitulate central aspects of PDAC, we employed genetically engineered mouse models presenting KrasG12D pancreas specific expression. Through a high-throughput combination drug screen with trametinib as backbone we identified a high synergism with the multikinase inhibitor nintedanib, preferentially in mesenchymal PDAC, a subtype of this disease characterized by poor prognosis and therapeutic resistance. This combinatorial treatment, that led to the induction of apoptosis in vitro and disease regression in vivo, was accompanied by a strong tumor infiltration of CD8 positive T cells.Materials and MethodsTo characterize the treatment-induced adaptive immune cell infiltration in vivo, we performed orthotopic transplantations of KRAS-driven murine PDAC cell lines presenting mesenchymal and epithelial morphology. The derived control and nintedanib + trametinib treated PDAC tumors were analyzed by multi-color immunofluorescence stainings. We compared the findings to high parameter flow cytometry results.ResultsConfocal microscopy of the immunofluorescence stainings revealed an overall increase of tumor-infiltrating lymphocytes (TIL) in the tumors upon combinatorial treatment with substantial differences in quantity and spatial distribution. Tumors derived from a PDAC cell line of epithelial morphology were characterized by few TIL mainly located at the invasive margins of the tumors, while tumors derived from a mesenchymal PDAC cell line showed a strong increase of TIL even in the center of the tumor mass. Furthermore, an increased ratio of CD8 positive cytotoxic T cells to CD4 positive helper T cells as well as a decrease of Foxp3 and CD4 positive regulatory T cells could be observed for tumors derived from the mesenchymal PDAC cell line under combinatorial treatment. To investigate if the observed recruitment of T cells was indispensable for treatment efficacy of the combinatorial therapy, we orthotopically transplanted the mesenchymal PDAC cell line in immunodeficient CD3-Knockout (CD3ko) mice and applied an analogous combinatorial treatment scheme. In the CD3ko mice, the combinatorial treatment did not lead to an increased survival or tumor regression as observed in immunocompetent mice. However, flow cytometry and immunofluorescence stainings revealed an increase of B cells upon nintedanib + trametinib treatment.ConclusionsOur findings indicate a reduced efficacy of the combinatorial treatment in T cell deficient mice, underlining the importance of T cells in treatment-induced anti-tumor responses and enlarging the understanding of the role of TIL in PDAC.Disclosure InformationJ. Heetmeyer: None. C. Falcomatà: None. S. Bärthel: None. C. Schneeweis: None. A. Coluccio: None. C. Veltkamp: None. G. Schneider: None. D. Saur: None.

Polarization of protease-activated receptor 2 (PAR-2) signaling is altered during airway epithelial remodeling and deciliation

Protease-activated receptor 2 (PAR-2) is activated by secreted proteases from immune cells or fungi. PAR-2 is normally expressed basolaterally in differentiated nasal ciliated cells. We hypothesized that epithelial remodeling during diseases characterized by cilial loss and squamous metaplasia may alter PAR-2 polarization. Here, using a fluorescent arrestin assay, we confirmed that the common fungal airway pathogen Aspergillus fumigatus activates heterologously-expressed PAR-2. Endogenous PAR-2 activation in submerged airway RPMI 2650 or NCI–H520 squamous cells increased intracellular calcium levels and granulocyte macrophage–colony-stimulating factor, tumor necrosis factor α, and interleukin (IL)-6 secretion. RPMI 2650 cells cultured at an air–liquid interface (ALI) responded to apically or basolaterally applied PAR-2 agonists. However, well-differentiated primary nasal epithelial ALIs responded only to basolateral PAR-2 stimulation, indicated by calcium elevation, increased cilia beat frequency, and increased fluid and cytokine secretion. We exposed primary cells to disease-related modifiers that alter epithelial morphology, including IL-13, cigarette smoke condensate, and retinoic acid deficiency, at concentrations and times that altered epithelial morphology without causing breakdown of the epithelial barrier to model early disease states. These altered primary cultures responded to both apical and basolateral PAR-2 stimulation. Imaging nasal polyps and control middle turbinate explants, we found that nasal polyps, but not turbinates, exhibit apical calcium responses to PAR-2 stimulation. However, isolated ciliated cells from both polyps and turbinates maintained basolateral PAR-2 polarization, suggesting that the calcium responses originated from nonciliated cells. Altered PAR-2 polarization in disease-remodeled epithelia may enhance apical responses and increase sensitivity to inhaled proteases.

ANALYSIS OF SULFUR VAPOR EXPOSURE TO THE NUMBER OF MICRONUCLEUS AND ORAL BUCCAL EPITHELIAL MORPHOLOGY

Background: The sulfur vapor consists of SO2 and CO2 which are genotoxins that may cause the damage of DNA to the micronucleus in buccal epithelial cells. Micronucleus is a mass like a nucleus, measuring one-third of the nucleus. DNA damage can also be seen from changes in the morphology of epithelial cells. Objective: This study aimed to identify the effect of sulfur vapor exposure on the number of micronucleus and morphology epithelial cells in the oral cavity on the sulfur miner. Methods: The method of this study was analytic observational with a cross-sectional approach. The total sample of this study was 24 respondents divided into 2 groups, each group contained 12 respondents. Exfoliated buccal cells were collected by scrapping the buccal mucosa. The specimens stained using Hematoxylin and Eosin. Nucleus and cytoplasmic area were examined using image J 1.40 Results: The result showed the average number of buccal mucosa micronucleus on coal miners higher (35,50) than controls (11,58). The result of Independent-measures T-test obtained significant different on the number of micronucleus between sulfur miner and controls (p=0,000). The result of Independent-measures T-test on the nuclear area and cytoplasmic area between sulfur miner and controls obtained insignificant different (p=0,379 dan p=0,616). Conclusion: Based on this study can be concluded that sulfur vapor exposure affected on the number of micronucleus on sulfur miners, but did not influence morphology of epithelial cells.

Altered polarization of PAR-2 signaling during airway epithelial remodeling

ABSTRACTBackgroundProtease-activated receptor 2 (PAR-2) is activated by proteases involved in allergy and triggers airway epithelial secretion and inflammation. PAR-2 is normally expressed basolaterally in differentiated nasal ciliated cells.ObjectiveWe tested if epithelial remodeling during diseases characterized by loss of cilia and squamous metaplasia may alter PAR-2 polarization.MethodsEndogenous PAR-2 responses were measured by live cell calcium and cilia imaging, measurement of fluid secretion, and quantification of cytokines. We utilized airway squamous cell lines, primary differentiated air-liquid interface cultures, and tissue explants. Cells were exposed to disease-related modifiers that alter epithelial morphology, including IL-13, cigarette smoke condensate, and retinoic acid deficiency. We used concentrations and exposure times that altered epithelial morphology without causing breakdown of the epithelial barrier, likely reflecting early disease states.ResultsPAR-2 signaling in airway squamous cells activated calcium and inflammatory responses. Squamous cells cultured at air liquid interface (ALI) responded to PAR-2 agonists applied both apically and basolaterally. Primary well-differentiated nasal epithelial ALI cultures responded only to basolateral PAR-2 stimulation. Primary cultures exposed to IL-13, cigarette smoke condensate, or reduced retinoic acid responded to both apical and basolateral PAR-2 stimulation. Nasal polyp tissue, but not control middle turbinate, exhibited apical calcium responses to PAR-2 stimulation. However, isolated ciliated cells from both polyp and turbinate maintained basolateral PAR-2 polarization.ConclusionsSquamous metaplasia and/or loss of cilia enhances apical PAR-2 responses. Altered PAR-2 polarization in dedifferentiated or remodeled epithelia may contribute to increased sensitivity to inhaled protease allergens in inflammatory airway diseases.