Changes In Cellular Phenotype Research Articles

Targeted Drug Therapy for Senescent Cells Alleviates Unilateral Ureteral Obstruction-Induced Renal Injury in Rats

Hydronephrosis resulting from unilateral ureteral obstruction (UUO) is a common cause of renal injury, often progressing to late-stage renal fibrosis or even potential renal failure. Renal injury and repair processes are accompanied by changes in cellular senescence phenotypes. However, the mechanism is poorly understood. The purpose of this study is to clarify the changes in senescence phenotype at different time points in renal disease caused by UUO and to further investigate whether eliminating senescent cells using the anti-senescence drug ABT263 could attenuate UUO-induced renal disease. Specifically, renal tissues were collected from established UUO rat models on days 1, 2, 7, and 14. The extent of renal tissue injury and fibrosis in rats was assessed using histological examination, serum creatinine, and blood urea nitrogen levels. The apoptotic and proliferative capacities of renal tissues and phenotypic changes in cellular senescence were evaluated. After the intervention of the anti-senescence drug ABT263, the cellular senescence as well as tissue damage changes were re-assessed. We found that before the drug intervention, the UUO rats showed significantly declined renal function, accompanied by renal tubular injury, increased inflammatory response, and oxidative stress, alongside aggravated cellular senescence. Meanwhile, after the treatment with ABT263, the rats had a significantly lower number of senescent cells, attenuated renal tubular injury and apoptosis, enhanced proliferation, reduced oxidative stress and inflammation, improved renal function, and markedly inhibited fibrosis. This suggests that the use of the anti-senescence drug ABT263 to eliminate senescent cells can effectively attenuate UUO-induced renal injury. This highlights the critical role of cellular senescence in the transformation of acute injury into chronic fibrosis.

Autocorrelation analysis of a phenotypic screen reveals hidden drug activity

Phenotype based screening is a powerful tool to evaluate cellular drug response. Through high content fluorescence imaging of simple fluorescent labels and complex image analysis phenotypic measurements can identify subtle compound-induced cellular changes unique to compound mechanisms of action (MoA). Recently, a screen of 1008 compounds in three cell lines was reported where analysis detected changes in cellular phenotypes and accurately identified compound MoA for roughly half the compounds. However, we were surprised that DNA alkylating agents and other compounds known to induce or impact the DNA damage response produced no measured activity in cells with fluorescently labeled 53BP1—a canonical DNA damage marker. We hypothesized that phenotype analysis is not sensitive enough to detect small changes in 53BP1 distribution and analyzed the screen images with autocorrelation image analysis. We found that autocorrelation analysis, which quantifies fluorescently-labeled protein clustering, identified higher compound activity for compounds and MoAs known to impact the DNA damage response, suggesting altered 53BP1 recruitment to damaged DNA sites. We then performed experiments under more ideal imaging settings and found autocorrelation analysis to be a robust measure of changes to 53BP1 clustering in the DNA damage response. These results demonstrate the capacity of autocorrelation to detect otherwise undetectable compound activity and suggest that autocorrelation analysis of specific proteins could serve as a powerful screening tool.

Abstract PO3-05-01: Locoregional Management Based on Mode of Progression for HR+/HER2- metastatic breast cancer Treated with Combined CDK4/6 inhibitor with Aromatase inhibitor

Abstract Background - Standard management for HR+/HER2- metastatic breast cancer (MBC) comprises concurrent aromatase inhibitor (AI) and cyclin-dependent kinase 4/6 inhibitor (CDK4/6i), providing improved progression-free (PFS) and overall survival over anti-estrogen alone. Eventual resistance mechanisms potentially include phenotypic changes, clonal selection, and new mutations. We theorized that limited progressive disease (LPD) would be more common on combination therapy due to eventual new mutations or clonal selection, in comparison to generalized progressive disease (GPD) occurring more frequently on AI alone due to phenotypic cellular changes across all disease sites. Potentially loco-regional treatment (LRT) options could significantly extend disease control in selected patients on combined therapy. Methods - Patterns of progressive disease (PD) for 55 historical AI-only treated controls (group A) were compared to those for 60 patients on combined treatment with an AI and a CDK4/6 inhibitor (group B) for first-line management of HR+/HER2- MBC. Mode of progression was classified as general (GPD - PD in the majority of lesions) or limited (LPD - PD in up to three lesions in a single organ). All LPDs were assessed for suitability for LRT at time of progression. PFS was assessed for those with LPD who continued present treatment after locoregional therapy. Results - LPD occurred in 13 of 55 patients (23.6%) in group A compared to 26 of 60 (43%) in group B (p = 0.026). For LPD patients, all 13 cases in group A were considered appropriate for LRT but only 3 (23.1%) received it and all patients also switched systemic therapy. In group B, 23 of 26 LPD patients were considered suitable for LRT and 16 (57.7%) received it, of whom 11 then continued first line systemic treatment. For these 11 group B patients median PFS was 12+ months (range 3 to 36+ months). LRT consisted of RT in 14 cases (bone in 13 cases, lymph node in one case) and resection in one (isolated brain metastasis). Conclusion - Patients treated with combined CDK4/6i and AI for first line HR+/HER- MBC were significantly more likely to develop LPD than those on single agent anti-estrogen, where local therapy could often be delivered with no change to systemic therapy. This strategy, applied to 11 of 60 combination treated patients (18%), delivered a median PFS in excess of a year. In contrast more general PD was more commonly observed in patients treated with AI alone, warranting change in systemic therapy. Citation Format: Andrew Redfern, Ngie Law, Indunil Weerasena, Lisa Spalding, Hilary Martin. Locoregional Management Based on Mode of Progression for HR+/HER2- metastatic breast cancer Treated with Combined CDK4/6 inhibitor with Aromatase inhibitor [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-05-01.

Liver X receptor activation in chronic myelogenous leukemia cells yields distinct mass fingerprints by whole cell MALDI-TOF MS, which correspond to changes in cell viability, gene expression, and differentiation markers

Liver X receptor (LXR) agonists, including synthetic ligands or naturally-occurring oxysterols, exert promising lipid-modulating, anti-inflammatory, and anti-tumor effects. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) whole cell mass spectrometry (MS) shows promise in chronic disease and food science research, clinical diagnostics of cancer cells and bacterial pathogens, and drug/nutraceutical discovery due to its relatively low-cost, rapid, reliable, and high-throughput ability to identify unique cell phenotypes and differentiation states of immune cells that play essential roles in chronic metabolic and inflammatory disease pathophysiology. However, optimization of whole cell MALDI-TOF MS-based methods to assess cellular changes in response to pharmaceutical and/or nutraceutical treatment warrants further study. Thus, we investigated whether whole cell MALDI-TOF MS could detect cellular phenotype changes of human stem-like erythroleukemia K562 cells – a model for chronic myelogenous leukemia (CML) – induced by the LXR agonist TO-901317, a synthetic ligand that mimics effects of naturally-occurring oxysterols, which are oxidized cholesterol derivatives formed under conditions of high cellular cholesterol concentrations and in food products. TO-901317 dose- and time-dependently altered mRNA expression of cholesterol flux genes, including ATP-binding cassette transporter A1 (ABCA1), LDL-receptor (LDLR), HMG-CoA reductase (HMGCR), and reduced cellular cholesterol content by 22.9% at 24 hours. TO-901317 additionally dose- and time-dependently impacted K562 cell viability, number, size, and reduced mRNA expression of the anti-apoptotic gene BCL2L1 by 43.3%. These effects corresponded to induced gene expression of erythroid markers and hemoglobin content, suggesting that LXR activation may promote erythroid lineage differentiation. Whole cell MALDI-TOF MS mass fingerprinting revealed distinct proteomic phenotypes between TO-901317 vs. untreated cells, and identified histone 2A and histone H4 as uniquely expressed proteins. These findings not only elucidate a novel role for LXR agonists in a model of CML, but demonstrate that mass fingerprints obtained by whole cell MALDI-TOF mass spectrometry effectively distinguish between treatment-induced cell phenotypes.

Abstract 7569: Characterizing pathways for metastasis of retinoblastoma in forebrain organoids

Abstract Retinoblastoma (Rb) is a childhood cancer of the eye, which usually presents in children under the age of five. When detected early, Rb has favorable prognosis in developed countries, but metastasis into the central nervous system (CNS) through the optic nerve correlate with a high mortality rate. Even if detected early, the available harsh and unspecific treatments can cause serious side effects, and enucleations are still performed frequently. There is still uncertainty regarding the mechanism for metastasis into the central nervous system. We performed bioinformatic analysis of single cell sequencing data for retinoblastoma, retinal tissues, optic nerve, and brain, to identify potential pathways and cell-cell communications fostering Rb invasion into the optic nerve and brain. Our analysis revealed several potential metastatic drivers, including immune modulation involving MIF, secreted by the Rb cells, as well as cell-adhesion pathways promoting a change in cellular phenotype. Given that animal models do not faithfully recapitulate human Rb initiation and progression, we developed a novel human iPSC-derived forebrain organoid model to investigate mechanisms of Rb CNS metastasis. Microinjections of GFP-labelled Rb cells allow to track growth and spread. In this fully human model including microglial cells, we can, for the first time, study the Rb forebrain interactions and pathways involved. RNA-seq analysis of Rb cells growing in forebrain organoids shows increased activity in neuron projection development and neuron migration, overlapping with the observed phenotype switch of invading Rb. Together, we present a novel iPSC-derived human forebrain model to monitor growth and spread of metastatic Rb, and describe new pathways involved in invading and metastasizing Rb. Citation Format: Yulia Shtanko, Sara Rodriguez, Benjamin Schachner, Emily Adis, Samantha McLaughlin, Sebastian Borges, James Dollar, Ashley Zuniga, Gabriela Quintana, Sofia Lopez, Dawn Owens, Chloe Moulin, Steffanie Braun, Gulum Yenisehirli, Daniel Pelaez, J. William Harbour, Stefan Kurtenbach. Characterizing pathways for metastasis of retinoblastoma in forebrain organoids [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 7569.

Regulatory Pathways in Growth Plate Chondrocytes that Are Impacted by Matrix Vesicle microRNA Identified by Targeted RISC Pulldown and Sequencing of the Resulting Transcriptome.

During endochondral bone formation, growth plate chondrocytes are differentially regulated by various factors and hormones. As the cellular phenotype changes, the composition of the extracellular matrix is altered, including the production and composition of matrix vesicles (MV) and their cargo of microRNA. The regulatory functions of these MV microRNA in the growth plate are still largely unknown. To address this question, we undertook a targeted bioinformatics approach. A subset of five MV microRNA was selected for analysis based on their specific enrichment in these extracellular vesicles compared to the parent cells (miR-1-3p, miR-22-3p, miR-30c-5p, miR-122-5p, and miR-133a-3p). Synthetic biotinylated versions of the microRNA were produced using locked nucleic acid (LNA) and were transfected into rat growth plate chondrocytes. The resulting LNA to mRNA complexes were pulled down and sequenced, and the transcriptomic data were used to run pathway analysis pipelines. Bone and musculoskeletal pathways were discovered to be regulated by the specific microRNA, notably those associated with transforming growth factor beta (TGFβ) and Wnt pathways, cell differentiation and proliferation, and regulation of vesicles and calcium transport. These results can help with understanding the maturation of the growth plate and the regulatory role of microRNA in MV.

Using Dimensionality Reduction to Visualize Phenotypic Changes in High-Throughput Microscopy.

High-throughput microscopy has enabled screening of cell phenotypes at unprecedented scale. Systematic identification of cell phenotype changes (such as cell morphology and protein localization changes) is a major analysis goal. Because cell phenotypes are high-dimensional, unbiased approaches to detect and visualize the changes in phenotypes are still needed. Here, we suggest that changes in cellular phenotype can be visualized in reduced dimensionality representations of the image feature space. We describe a freely available analysis pipeline to visualize changes in protein localization in feature spaces obtained from deep learning. As an example, we use the pipeline to identify changes in subcellular localization after the yeast GFP collection was treated with hydroxyurea.

R-loops in normal and malignant hematopoiesis

An R-loop is a nucleic acid structure consisting of a DNA : RNA hybrid and single-stranded DNA. It is formed physiologically in normal cells and is involved in transcription, replication, and gene rearrangement; in particular, it has multiple roles including in mitochondrial DNA replication and class switch recombination of immunoglobulin genes in B cells. However, accumulating evidence indicates aberrant R-loop formation in various malignancies, including hematopoietic neoplasms. The accumulation of such inappropriate R-loops can cause conflicts between transcription and DNA replication. This exacerbates genomic instability through the generation of DNA replication stress, that, in turn, leads to cellular phenotypic changes and disease progression. When RNAs are synthesized during transcription they hybridize with template DNA in cis, giving rise to R-loops. In addition, it was recently revealed that noncoding RNAs also form R-loops when bound to genomic DNA in trans. Together with such observations, new roles for the R-loop in disease development have been proposed. The relationship between inflammation and the R-loop has also attracted much attention. In this review, we will focus on the mechanisms of R-loop formation in various hematopoietic neoplasms and introduce the important findings from recent studies. Therapeutic concepts for targeting R-loop accumulation in hematopoietic neoplasms will also be discussed.

Effect of Bioactive Glasses and Basic Fibroblast Growth Factor on Dental Pulp Cells.

Ideal regeneration of hard tissue and dental pulp has been reported with the use of a combination of bioactive glass and basic fibroblast growth factor (bFGF). However, no previous study has investigated the molecular mechanisms underlying the processes induced by this combination in dental pulp cells. This study aimed to examine the cellular phenotype and transcriptional changes induced by the combination of bioactive glass solution (BG) and bFGF in dental pulp cells using phase-contrast microscopy, a cell counting kit-8 assay, alkaline phosphatase staining, and RNA sequence analysis. bFGF induced elongation of the cell process and increased the number of cells. Whereas BG did not increase ALP activity, it induced extracellular matrix-related genes in the dental pulp. In addition, the combination of BG and bFGF induces gliogenesis-related genes in the nervous system. This is to say, bFGF increased the viability of dental pulp cells, bioactive glass induced odontogenesis, and a dual stimulation with bioactive glass and bFGF induced the wound healing of the nerve system in the dental pulp. Taken together, bioactive glass and bFGF may be useful for the regeneration of the dentin-pulp complex.

Modulation of SEMA4D-modified titanium surface on M2 macrophage polarization via activation of Rho/ROCK-mediated lactate release of endothelial cells: In vitro and in vivo

SEMA4D-modified titanium surfaces can indirectly regulate macrophages through endothelial cells to achieve an anti-inflammatory effect, which is beneficial for healing soft tissues around the gingival abutment. However, the mechanism of surface-induced cellular phenotypic changes in SEMA4D-modified titanium has not yet been elucidated. SEMA4D activates the RhoA signaling pathway in endothelial cells, which coordinates metabolism and cytoskeletal remodeling. This study hypothesized that endothelial cells inoculated on SEMA4D-modified titanium surfaces can direct M2 polarization of macrophages via metabolites. An indirect co-culture model of endothelial cells and macrophages was constructed in vitro, and specific inhibitors were employed. Subsequently, endothelial cell adhesion and migration, metabolic changes, Rho/ROCK1 expression, and inflammatory expression of macrophages were assessed via immunofluorescence microscopy, specific kits, qRT-PCR, and Western blotting. Moreover, an in vivo rat bilateral maxillary implant model was constructed to evaluate the soft tissue healing effect. The in vitro experiments showed that the SEMA4D group had stronger endothelial cell adhesion and migration, increased Rho/ROCK1 expression, and enhanced release of lactate. Additionally, decreased macrophage inflammatory expression was observed. In contrast, the inhibitor group partially suppressed lactate metabolism and motility, whereas increased inflammatory expression. The in vivo analyses indicated that the SEMA4D group had faster and better angiogenic and anti-inflammatory effects, especially in the early stage. In conclusion, via the Rho/ROCK1 signaling pathway, the SEMA4D-modified titanium surface promotes endothelial cell adhesion and migration and lactic acid release, then the paracrine lactic acid promotes the polarization of macrophages to M2, thus obtaining the dual effects of angiogenesis and anti-inflammation.

Microphysiological Models of Lung Epithelium-Alveolar Macrophage Co-Cultures to Study Chronic Lung Disease.

The interactions between immune cells and epithelial cells influence the progression of many respiratory diseases, such as chronic obstructive pulmonary disease (COPD). In vitro models allow for the examination of cells in controlled environments. However, these models lack the complex 3D architecture and vast multicellular interactions between the lung resident cells and infiltrating immune cells that can mediate cellular response to insults. In this study, three complementary microphysiological systems are presented to delineate the effects of cigarette smoke and respiratory disease on the lung epithelium. First, the Transwell system allows the co-culture of pulmonary immune and epithelial cells to evaluate cellular and monolayer phenotypic changes in response to cigarette smoke exposure. Next, the human and mouse precision-cut lung slices system provides a physiologically relevant model to study the effects of chronic insults like cigarette smoke with the dissection of specific interaction of immune cell subtypes within the structurally complex tissue environment. Finally, the lung-on-a-chip model provides an adaptable system for live imaging of polarized epithelial tissues that mimic the in vivo environment of the airways. Using a combination of these models, a complementary approach is provided to better address the intricate mechanisms of lung disease.

Revealing invisible cell phenotypes with conditional generative modeling

Biological sciences, drug discovery and medicine rely heavily on cell phenotype perturbation and microscope observation. However, most cellular phenotypic changes are subtle and thus hidden from us by natural cell variability: two cells in the same condition already look different. In this study, we show that conditional generative models can be used to transform an image of cells from any one condition to another, thus canceling cell variability. We visually and quantitatively validate that the principle of synthetic cell perturbation works on discernible cases. We then illustrate its effectiveness in displaying otherwise invisible cell phenotypes triggered by blood cells under parasite infection, or by the presence of a disease-causing pathological mutation in differentiated neurons derived from iPSCs, or by low concentration drug treatments. The proposed approach, easy to use and robust, opens the door to more accessible discovery of biological and disease biomarkers.

Effect of multiple nonthermal plasma treatments of filamentous fungi on cellular phenotypic changes and phytopathogenicity

The effect of multiple sublethal doses of non-thermal plasma treatments on fungal cells phenotypical changes and the reduction in phytopathogenicity of Fusarium oxysporum, Botrytis cinerea, and Alternaria alternata was examined. The intensity of these changes depended on the species of fungus and the number of exposures of the mycelia to the DBD plasma. Microscopic observations showed that the plasma damaged the surface of the hyphae, increased their thickness and decreased overall dry biomass of the organisms. A decrease in pectinolytic activity was found in F. oxysporum and A. alternata, in contrast to B. cinerea, where an increase in pectinolytic activity was observed after the fifth plasma treatment. Changes in specific xylanase activity varied and were dependent on the species of fungus. The percentage of cucumber seeds germinated artificially infected with mycelium after multiple plasma treatments increased compared to those that were mycelium infected prior to plasma exposure. Plants that developed from seeds after plasma exposure were characterized by a higher biomass and longer roots and stems. Multiple treatments of the studied fungi with plasma, followed by seed infection, increased the SWVI and SWVI indexes of cucumber seedlings, but they did not reach the characteristic value of the control seeds (not infected with fungi). The reduced phytopathogenicity of the tested fungi was confirmed by artificial infestation of tomato fruits.

Assessing the performance of the Cell Painting assay across different imaging systems.

Quantitative microscopy is a powerful method for performing phenotypic screens from which image-based profiling can extract a wealth of information, termed profiles. These profiles can be used to elucidate the changes in cellular phenotypes across cell populations from different patient samples or following genetic or chemical perturbations. One such image-based profiling method is the Cell Painting assay, which provides morphological insight through the imaging of eight cellular compartments. Here, we examine the performance of the Cell Painting assay across multiple high-throughput microscope systems and find that all are compatible with this assay. Furthermore, we determine independently for each microscope system the best performing settings, providing those who wish to adopt this assay an ideal starting point for their own assays. We also explore the impact of microscopy setting changes in the Cell Painting assay and find that few dramatically reduce the quality of a Cell Painting profile, regardless of the microscope used.

Single-cell RNA sequencing reveals the heterogeneity and intercellular communication of hepatic stellate cells and macrophages during liver fibrosis.

Uncontrolled and excessive progression of liver fibrosis is thought to be the prevalent pathophysiological cause of liver cirrhosis and hepatocellular cancer, and there are currently no effective antifibrotic therapeutic options available. Intercellular communication and cellular heterogeneity in the liver are involved in the progression of liver fibrosis, but the exact nature of the cellular phenotypic changes and patterns of interregulatory remain unclear. Here, we performed single-cell RNA sequencing on nonparenchymal cells (NPCs) isolated from normal and fibrotic mouse livers. We identified eight main types of cells, including endothelial cells, hepatocytes, dendritic cells, B cells, natural killer/T (NK/T) cells, hepatic stellate cells (HSCs), cholangiocytes and macrophages, and revealed that macrophages and HSCs exhibit the most variance in transcriptional profile. Further analyses of HSCs and macrophage subpopulations and ligand-receptor interaction revealed a high heterogeneity characterization and tightly interregulated network of these two groups of cells in liver fibrosis. Finally, we uncovered a profibrotic Thbs1+ macrophage subcluster, which expands in mouse and human fibrotic livers, activating HSCs via PI3K/AKT/mTOR signaling pathway. Our findings decode unanticipated insights into the heterogeneity of HSCs and macrophages and their intercellular crosstalk at a single-cell level, and may provide potential therapeutic strategies in liver fibrosis.

MiR-196b-5p activates NF-κB signaling in non-small cell lung cancer by directly targeting NFKBIA

BackgroundOur recent study found that QKI-5 regulated miRNA, miR-196b-5p, promotes non-small cell lung cancer (NSCLC) progression by directly targeting GATA6, TSPAN12 and FAS. However, the biological functions of miR-196b-5p in NSCLC progression and metastasis still remain elusive. MethodsCell proliferation, migration, colony formation, cell cycle assays were used to investigate cellular phenotypic changes. Quantitative real-time PCR (qRT-PCR) and western blot analyses were used to measure expressions of relative gene and protein. Interaction between QKI-5 and miR-196b-5p was determined by RNA immunoprecipitation (RIP) assay. Luciferase reporter assay was used to determine direct binding between miR-196b-5p and NFKBIA 3′-UTR. ELISA assay was used to measure secreted IL6 proteins. Mice xenograft model was used to assess the functions of NFKBIA on in vivo tumor growth. ResultsWe demonstrated that the miR-196b-5p facilitates lung cancer cell proliferation, migration, colony formation, and cell cycle by directly targeting NFKBIA, a negative regulator of NFκB signaling. Knocking down NFKBIA increases IL6 mediated phosphorylation of signal transducer and activator of transcription 3 (STAT3) to promote lung cancer cell growth by activating NFκB signaling. The expression of NFKBIA was significantly downregulated in NSCLC tissue samples, and was negatively correlated with the expression miR-196b-5p. In addition, we found that downregulated QKI-5 expression was associated with the elevated miR-224 expression in NSCLC. ConclusionsOur findings indicated that the miR-224/QKI-5/miR-196b-5p/NFKBIA signaling pathway might play important functions in the progression of NSCLC, and suggested that targeting this pathway might be an effective therapeutic strategy in treating NSCLC.

Microfluidic-based prostate cancer model for investigating the secretion of prostate-specific antigen and microRNAs in vitro

The study of prostate cancer in vitro relies on established cell lines that lack important physiological characteristics, such as proper polarization and expression of relevant biomarkers. Microphysiological systems (MPS) can replicate cancer microenvironments and lead to cellular phenotypic changes that better represent organ physiology in vitro. In this study, we developed an MPS model comprising conventional prostate cancer cells to evaluate their activity under dynamic culture conditions. Androgen-sensitive (LNCaP) and androgen-insensitive (PC3) cells were grown in conventional and 3D cultures, both static and dynamic. Cell morphology, the secretion of prostate-specific antigen, and the expression of key prostate markers and microRNAs were analyzed. LNCaP formed spheroids in 3D and MPS cultures, with morphological changes supported by the upregulation of cytokeratins and adhesion proteins. LNCaP also maintained a constant prostate-specific antigen secretion in MPS. PC3 cells did not develop complex structures in 3D and MPS cultures. PSA expression at the gene level was downregulated in LNCaP-MPS and considerably upregulated in PC3-MPS. MicroRNA expression was altered by the 3D static and dynamic culture, both intra- and extracellularly. MicroRNAs associated with prostate cancer progression were mostly upregulated in LNCaP-MPS. Overall dynamic cell culture substantially altered the morphology and expression of LNCaP cells, arguably augmenting their prostate cancer phenotype. This novel approach demonstrates that microRNA expression in prostate cancer cells is sensitive to external stimuli and that MPS can effectively promote important physiological changes in conventional prostate cancer models.

Assessment of morphological changes in the gastroduodenal area of patients with chronic pancreatitis in comorbidity with chronic viral hepatitis C

Background. Viral hepatitis C (HCV) is not only as a liver disease but also as a systemic (generalized) infection that affects ot­her organs, in particular, the pancreas and gastroduodenal zone. The state of the gastrointestinal mucosa in combined course of chronic pancreatitis (CP) and chronic HCV is little studied, but extremely relevant, as it is important for the clinical course, the quality of life of patients, as well as for assessing the safety of the performed (etiotropic, etc.) and planned restorative comprehensive treatment of such patients. The purpose is to evaluate the morphological changes of the gastroduodenal mucosa of patients with chronic pancreatitis associated with chronic viral hepatitis C who were treated etiotropically. Materials and methods. Thirty patients with CP and 65 patients with CP in comorbidity with chronic HCV after etiotropic treatment of HCV were examined. Regardless of the HCV genotype, they received a course of treatment with sofosbuvir at a dose of 400 mg and velpatasvir 100 mg in the morning after meals for 3 months (some patients took daclatasvir at a dose of 60 mg also in the morning after meals for 3 months instead of velpatasvir). The period after diagnosed and treated HCV before the examination was on average (2.35 ± 0.61) years. All patients underwent a morphological study of gastric and duodenal biopsy samples obtained by esophagogastroscopy. The prepared samples were evaluated according to the international OLGA and OLGIM systems based on the evaluation of 5 gastric biopsies (1 from the corner of the stomach, 2 from the body, and 2 from the antrum) with a description of the main pathomorphological changes evaluated on a visual analog scale. Alcian yellow and toluidine blue were used to detect H.pylori. An own system for assessing the morphological state of the gastroduodenal mucosa was also proposed. Results. The detected structural changes indicate the presence of chronic atrophic gastritis with moderate dysplasia of the glandular epithelium. Stage of chronic gastritis according to the OLGA system was III. Stage of chronic gastritis according to the OLGIM system was III. Thus, the practically asymptomatic course of quite significant gastroduodenal changes in CP combined with chronic HCV requires additional therapeutic influence, as well as an assessment of comprehensive treatment based on the effect on the gastroduodenal mucosa. In case of changes in gastroduodenal mucosa in CP on the background of chronic HCV, in contrast to those with isolated CP, a significantly higher level of inflammatory infiltrate density (moderate (2+) vs. mild (1+)) is determined, which is accompanied by moderate neutrophilic infiltration (2+) vs. mild (1+) and mild lymphohistiocytic infiltration (1+) versus no such infiltration (0), the appearance of intestinal metaplasia (2+) and moderate non-metaplastic glandular atrophy (2+) versus the absence of metaplasia (0) and mild non-metaplastic glandular atrophy (1+), as well as zones of metaplastic atrophy (1+), which was manifested by the appearance of Paneth cells and goblet cells. In CP with chronic HCV, mucosa has atrophic changes. Metaplasia is manifested by a change in cellular phenotype: complete intestinal metaplasia type I–II is manifested by the appearance of Paneth cells and goblet cells; incomplete — by the presence of columnar epithelium and goblet cells; colonic metaplasia — by enlargement of the glands and the absence of Paneth cells. Conclusions. It was proved that the state of the gastric and duodenal mucosa in the group of patients with CP and chronic HCV was significantly severe than that in isolated CP: lymphohistiocytic infiltration of the mucous membrane was detected 2.68 times more often, non-metaplastic infiltration glandular atrophy (there was practically no glandular atrophy in the group of isolated CP) — 12.65 times more often, metaplastic atrophy (it was not detected in the group of CP) — 10.64 times more often, acute leukocyte infiltration — 2.31 times more often, dysplasia of the mucosal epithelium (with isolated CP, mild dysplasia was detected) — 7.30 times more often, erosion of the mucosal epithelium — 3.07 times more often, focal hyperplasia of the glands — 3.88 times more often, intestinal metaplasia — 12.89 times more often, which was practically absent in CP. In general, the total number of pathological signs of the mucous membrane in the group of CP with comorbid chronic HCV was found to be 2.76 times higher than that in isolated CP.

ZIM1 Combined with Hydrogel Inhibits Senescence of Primary PαS Cells during In Vitro Expansion.

Bone marrow stem cells (BMSCs) are a promising source of seed cells in bone tissue engineering, which needs a great quantity of cells. Cell senescence occurs as they are passaged, which could affect the therapeutic effects of cells. Therefore, this study aims to explore the transcriptomic differences among the uncultured and passaged cells, finding a practical target gene for anti-aging. We sorted PαS (PDGFR-α+SCA-1+CD45-TER119-) cells as BMSCs by flow cytometry analysis. The changes in cellular senescence phenotype (Counting Kit-8 (CCK-8) assay, reactive oxygen species (ROS) test, senescence-associated β-galactosidase (SA-β-Gal) activity staining, expression of aging-related genes, telomere-related changes and in vivo differentiation potential) and associated transcriptional alterations during three important cell culture processes (in vivo, first adherence in vitro, first passage, and serial passage in vitro) were studied. Overexpression plasmids of potential target genes were made and examed. Gelatin methacryloyl (GelMA) was applied to explore the anti-aging effects combined with the target gene. Aging-related genes and ROS levels increased, telomerase activity and average telomere length decreased, and SA-β-Gal activities increased as cells were passaged. RNA-seq offered that imprinted zinc-finger gene 1 (Zim1) played a critical role in anti-aging during cell culture. Further, Zim1 combined with GelMA reduced the expression of P16/P53 and ROS levels with doubled telomerase activities. Few SA-β-Gal positive cells were found in the above state. These effects are achieved at least by the activation of Wnt/β-catenin signaling through the regulation of Wnt2. The combined application of Zim1 and hydrogel could inhibit the senescence of BMSCs during in vitro expansion, which may benefit clinical application.

Identification and characterization of karyopherin a-2, a nuclear export protein in lung adenocarcinoma.

e21025 Background: It is emerging that the Karyopherin a-2 (KPNA2), a nucleocytoplasmic transport protein, regulates the nuclear import of macromolecules and contributes to carcinogenesis and changes in cellular phenotype. In particular, it can be a poor prognostic marker by causing aberrant subcellular localization of DNA damage response proteins or OCT4-c MYC pathway molecules depending on the type of carcinoma and inducing anticancer resistance. In some carcinomas, an association with the p53 oncogenic pathway has been reported, showing good prognostic values. We aimed to investigate the clinical significance and metabolic pathway of stable cancer associated fibroblast (CAF) and cancer cells for KPNA2. Methods: We retrospectively analyzed the data extracted from EMR for NSCLC patients receiving curative surgical resection in Uijeongbu and Bucheon St. Mary’s Hospitals (n = 165). Immunohistochemistry staining was performed on the tumor microarray samples using antibodies against KPNA2. The functional assay and gene silencing was conducted. Results: Based on the TMA staining, mean KPNA2 score was 36.5 [0-240] and median value was 10. The clinical and pathological information of the experimental group is as follows. Table 1. Scatter plots and correlation between KPNA2 expression levels and tumor invasiveness markers reveals that high KPNA2 expressor was related to lymphatic invasion (p = 0.00782), advanced stage (p = 0.0202) and current smoker (p = 0.0074). KPNA2 expression was higher in squamous cell carcinoma(SqCC) histotype than in adenocarcinoma (SqCC ratio (%), 13.5 vs 43.4 in Low exp vs High exp; p = 0.007). Patients with high expression of KPNA2 showed shorter survival than other patients (median OS (months), 72.8 vs 42.2, P < 0.0001; median RFS (months), 72.8 vs 32.1, P < 0.0011) and relevant to high recurrence (recurrence rate (%), 24.7 vs 44.7, p = 0.007). Further, high KPNA2 expression was associated with poor survival outcomes. To test the effect of KPNA2 on cancer cell invasion, we knocked down Kpna2 via shRNA. Spheroids composed of H1299, Calu-6, A549, HCC827 lung cancer cells with either Kpna2-overexpression or Kpna2-knockdown, and spheroid invasion was then monitored for two days. Eminently, the 3-D invasion of spheroids was suppressed via Kpna2 knockdown. In addition, Kpna2-overexpressing cells promoted cancer invasion to a greater extent than control. Conclusions: In conclusion, our results suggest that KPNA2 protein expression has a poor prognostic association, revealed by its association with survival outcome or invasiveness markers. Its clinical significance as an oncogenic target molecule is emerging, and further evaluation using an organoid model is in progress.