Proliferative State Research Articles

Abstract TP298: Cellular Senescence and Microglial Dysfunction are Linked to Progressive Cognitive Deficits in the Chronic Phase of Ischemic Stroke

Improvements in acute stroke treatment, including endovascular thrombectomy and critical care management, have increased survival rates after stroke. However, many survivors have significant neurological deficits, as stroke remains one of the leading causes of long-term disability. Despite this, the chronic progression and long-term sequelae of ischemic stroke pathology remains woefully understudied. This study examined long-term progressive deficits in young adult male C57Bl/6 mice at 3 and 6 months after a 60-minute transient middle cerebral artery occlusion (MCAO) or sham surgery to identify secondary injury processes, and neuropathology of chronic stroke human brains. Depressive-like behavior persisted for 6 months post-stroke (PS) (p= <0.0001). Cognitive function progressively worsened from 3 to 6 months as seen by a continual reduction in the discrimination index in the novel object recognition task (p=.0063). Deficits in memory retention in mice 6-months PS were observed using the fear-conditioning test (p=.0084). Brain atrophy was evident by MicroCT imaging. Histopathological assessment revealed increased microglial activation in the hippocampus and frontal cortex in both mouse and human brains chronically after stroke. Interestingly, we also observed microglia activation proximal to apoptotic neurons in the brains of stroke mice, as shown by TUNEL assay. TUNEL co-labeling with a neuronal marker showed the same neuronal apoptosis in human samples from patients with chronic stroke, in both the hippocampus and thalamus. Disease-associated microglial (DAM) phenotypes were marked by both increased proliferative status and senescent-like phenotypes, including elevated production of cytokines and other proinflammatory molecules. Our findings demonstrate that experimental stroke accelerates inflammaging in the brain, which induces premature senescence within the chronic infarct microenvironment. Cellular senescence and chronic neurodegenerative signatures likely contribute to progressive cognitive impairment following stroke. The chronic and evolving neurological consequences of stroke may offer viable targets for delayed treatment strategies to reduce risk of vascular dementia and neurodegenerative disease.

Abstract WP316: Single-Cell RNA Sequencing Reveals a Massive Alteration in the Molecular Landscape of Microglia After Ischemic Stroke

Introduction: Ischemic stroke, a prevalent and often devastating disease, remains poorly treated. Neuroinflammation is a key element of stroke pathophysiology and represents a promising therapeutic target. Among the central components driving neuroinflammation, microglia - the primary resident immune cells within the brain parenchyma - are key players in mediating the immune response in the stroke brain. However, how microglia respond to stroke is incompletely understood. In this study, we took advantage of a mouse genetic tool (ie, Tmem119-CreERT2), sorted microglia, and performed single-cell RNA sequencing (scRNA-seq) analysis using a mouse model of permanent stroke. Moreover, as stroke exhibits sexual dimorphism, we included both male and female mice. Methods: Young male and female Ai6/TMEM119-CreERT2;Ai6 (Tmem119-Ai6) mice with microglia labeled with ZsGreen underwent permanent middle cerebral artery occlusion (pMCAO) or sham surgery. On day 3 after stroke, ZsGreen+ cells (microglia) in the brain were isolated by FACS, and used for scRNA-seq analysis. Results: First, we confirmed that in Tmem119-Ai6 mice, expression of fluorescent ZsGreen was almost exclusively in microglia in the brain, with minimal presence in blood cells. Our unbiased scRNA-seq analysis of ZsGreen+ cells revealed 13 distinct microglial clusters after stroke. Importantly, 4 major stroke-related microglial clusters were identified. One cluster shared a gene signature with the reported disease-associated microglia (DAM) subset. The other 3 clusters were characterized by an interferon (IFN) response signature, a highly proliferative state, and a macrophage-like profile, respectively. RNA in situ hybridization analysis showed that cells from these clusters mainly resided within the stroke boundaries. Notably, our scRNA-seq data suggest no major sexual dimorphism in the microglial response to permanent stroke. Conclusions: This is the first scRNA-seq study with a focus on microglia in the context of sex and stroke. We propose that 4 microglial subsets may be deemed as common stroke-associated microglia. Future work will be designed to elucidate the functional significance of these subsets and their potential implications for stroke therapy.

Abstract A010: Longitudinal histology and spatial transcriptomics profiling in targeted treatment of melanoma patient-derived models interrogates persister and resistant cell populations

Abstract Over half of BRAF-mutant melanoma patients with initial response to targeted therapy will recur with resistant disease. It is thought that recurrence arises from the ability of resistant clonal populations to enter and exit a slow-cycling persister state, evading treatment in quiescence before returning to a proliferative state. However, clonal transcriptional profiles and their spatial relationships in the tumor environment are not well characterized. Through longitudinal profiling of tumors from pre-treatment through the growth of resistant tumors, we track clonal lineages and transcriptional state changes to identify recurrent evolutionary patterns. BRAF mutant patient-derived melanoma xenografts were treated with BRAF/MEK inhibitors through maximum tumor size reduction and re-growth. Spatial transcriptomics (ST) and H&E histological staining were performed at five timepoints across 94 and 133 days of PDX models WM4237 and WM4007 treatment, tracking clonal populations with intact tissue structure. Deep learning feature extraction on high-resolution H&E-stained images allowed the detection of histological features that co-localize with expression levels. A novel computational workflow performed integration of samples across time points, pathway enrichment analysis, and pseudotemporal ordering, allowing spatial recreation of copy number variation based clonal phylogenies and cell fate trajectory inference during the treatment course. We characterize the clonal populations by the burden of accumulated copy number variations and establish clonal phylogeny. With the added temporal resolution, ST profiling during melanoma treatment enables observation of a global shift where all lineages begin the transition into the persister transcriptional state. The drug-sensitive cells are eliminated due to treatment, and distinct transcriptional states arise due to metabolic plasticity regardless of the lineage of origin. The clones of the transient persister state constitute the minimal residual disease, where melanoma cells have an increase in invasive capacity and a slowing of the cell cycle. The ability to survive through the persister state and re-emerge into a proliferating clone where the cell cycle is upregulated varies by lineage. We observe that the transcriptional changes during emergence from dormancy show decreased invasiveness and continued reliance on oxidative respiration versus glycolysis. We compare the re-emerged resistant cell populations and pre-treatment samples to pinpoint the changes in the transcriptional state of the MAPK signaling pathway and find that sets of upregulated genes differ by the model, WM4237 and WM4007. We connect deep learning imaging features extracted from longitudinal histology profiling to ST profiles to associate histology data with the cell cycle activation, invasiveness, hypoxia state, and metabolic mode across the timepoints. We observe that the imaging features alone can be potentially used to track the evolution of the tumor cell population phenotypes during drug treatment. Citation Format: Sergii Domanskyi, Todd B. Sheridan, Brian J. Sanderson, SungHee Park, Jessica Kaster, Haiyin Li, Olga Anczukow, Meenhard Herlyn, Jeffrey H. Chuang, Jill C. Rubinstein. Longitudinal histology and spatial transcriptomics profiling in targeted treatment of melanoma patient-derived models interrogates persister and resistant cell populations [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A010.

E2F1 mediates competition, proliferation and response to cisplatin in cohabitating resistant and sensitive ovarian cancer cells.

Tumor heterogeneity is one of the key factors leading to chemo-resistance relapse. It remains unknown how resistant cancer cells influence sensitive cells during cohabitation and growth within a heterogenous tumors. The goal of our study was to identify driving factors that mediate the interactions between resistant and sensitive cancer cells and to determine the effects of cohabitation on both phenotypes. We used isogenic ovarian cancer (OC) cell lines pairs, sensitive and resistant to platinum: OVCAR5 vs. OVCAR5 CisR and PE01 vs. PE04, respectively, to perform long term direct culture and to study the phenotypical changes of the interaction of these cells. Long term direct co-culture of sensitive and resistant OC cells promoted proliferation (p < 0.001) of sensitive cells and increased the proportion of cells in the G1 and S cell cycle phase in both PE01 and OVCAR5 cells. Direct co-culture led to a decrease in the IC50 to platinum in the cisplatin-sensitive cells (5.92 µM to 2.79 µM for PE01, and from 2.05 µM to 1.51 µM for OVCAR5). RNAseq analysis of co-cultured cells showed enrichment of Cell Cycle Control, Cyclins and Cell Cycle Regulation pathways. The transcription factor E2F1 was predicted as the main effector responsible for the transcriptomic changes in sensitive cells. Western blot and qRT-PCR confirmed upregulation of E2F1 in co-cultured vs monoculture. Furthermore, an E2F1 inhibitor reverted the increase in proliferation rate induced by co-culture to baseline levels. Our data suggest that long term cohabitation of chemo-sensitive and -resistant cancer cells drive sensitive cells to a higher proliferative state, more responsive to platinum. Our results reveal an unexpected effect caused by direct interactions between cancer cells with different proliferative rates and levels of platinum resistance, modelling competition between cells in heterogeneous tumors.

Interference of pseudorabies virus infection on functions of porcine granulosa cells via apoptosis modulated by MAPK signaling pathways

BackgroundPseudorabies virus (PRV) is one of the major viral pathogens leading to reproductive disorders in swine. However, little is known about the effects of PRV infection on porcine reproductive system. Ovarian granulosa cells are somatic cells surrounding oocytes in ovary and required for folliculogenesis. The present study aimed to investigate the interference of PRV on functions of porcine ovarian granulosa cells in vitro.MethodsPrimary granulosa cells were isolated from porcine ovaries. To investigate the PRV infectivity, transmission electron microscopy (TEM) was used to check the presence of viral particles, and the expression of viral gE gene was detected by quantitative real-time PCR (qPCR) in PRV-inoculated cells. After PRV infection, cell viability was detected by MTS assay, Ki67 for proliferative status was determined by immunofluorescence assay (IFA), cell cycle and apoptosis were detected by flow cytometry, and progesterone (P4) and estradiol (E2) were determined by radioimmunoassay. The checkpoint genes of cell cycle and apoptosis-related proteins were studied by qPCR and western blotting.ResultsVirus particles were observed in the nucleus and cytoplasm of PRV-infected granulosa cells by TEM imaging, and the expression of viral gE gene increased in a time-dependent manner post infection. PRV infection inhibited cell viability and blocked cell cycle at S phase in porcine granulosa cells, accompanied by decreases in expression of Ki67 protein and checkpoint genes related to S phase. Radioimmunoassay revealed decreased levels in P4 and E2, and the expressions of key steroidogenic enzymes were also down-regulated post PRV-infection. In addition, PRV induced apoptosis with an increase in Bax expression and activation of caspase 9, and the phosphorylation of JNK, ERK and p38 MAPKs were significantly up-regulated in porcine ovarian granulosa cells post PRV infection.ConclusionsThe data indicate that PRV causes infection on porcine ovarian granulosa cells and interferes the cell functions through apoptosis, and the MAPK signaling pathway is involved in the viral pathogenesis.

Effects of Combinatory In Vitro Treatment with Immune Checkpoint Inhibitors and Cytarabine on the Anti-Cancer Immune Microenvironment in De Novo AML Patients.

Despite substantial progress in the diagnostic and therapeutic procedures, acute myeloid leukaemia (AML) still constitutes a significant problem for patients suffering from its relapses. A comprehensive knowledge of the disease's molecular background has led to the development of targeted therapies, including immune checkpoint inhibitors, and demonstrated beneficial effects on several types of cancer. Here, we aimed to assess in vitro the potential of the immune checkpoint blockage for supporting anti-cancer responses to the AML backbone therapy with cytarabine. PBMCs of AML patients were collected at admission and, following the therapy, eight complete remission (CR) and eight non-responders (NR) subjects were selected. We assessed the effects of the in vitro treatment of the cells with cytarabine and the immune checkpoint inhibitors: anti-CTLA-4, anti-PD-1, anti-PD-L1. The study protocol allowed us to evaluate the viability of the cancer and the immune cells, proliferation status, phenotype, and cytokine release. Anti-PD-L1 antibodies were found to exert the most beneficial effect on the activation of T cells, with a concomitant regulation of the immune balance through Treg induction. There was no direct influence on the blast cells; however, the modulation of the PD-1/PD-L1 axis supported the expansion of lymphocytes. Changes in the response between CR and NR patients might result from the differential expression of PD-1 and PD-L1, with lower levels in the latter group. The tested blockers appear to support the anti-cancer immune responses rather than directly improve the effects of cytarabine. In conclusion, checkpoint proteins' modulators might improve the anti-cancer responses in the tumour environment.

Integrated proteogenomic characterization of glioblastoma evolution

The evolutionary trajectory of glioblastoma (GBM) is a multifaceted biological process that extends beyond genetic alterations alone. Here, we perform an integrative proteogenomic analysis of 123 longitudinal glioblastoma pairs and identify a highly proliferative cellular state at diagnosis and replacement by activation of neuronal transition and synaptogenic pathways in recurrent tumors. Proteomic and phosphoproteomic analyses reveal that the molecular transition to neuronal state at recurrence is marked by post-translational activation of the wingless-related integration site (WNT)/ planar cell polarity (PCP) signaling pathway and BRAF protein kinase. Consistently, multi-omic analysis of patient-derived xenograft (PDX) models mirror similar patterns of evolutionary trajectory. Inhibition of B-raf proto-oncogene (BRAF) kinase impairs both neuronal transition and migration capability of recurrent tumor cells, phenotypic hallmarks of post-therapy progression. Combinatorial treatment of temozolomide (TMZ) with BRAF inhibitor, vemurafenib, significantly extends the survival of PDX models. This study provides comprehensive insights into the biological mechanisms of glioblastoma evolution and treatment resistance, highlighting promising therapeutic strategies for clinical intervention.

The IL-1β-p65 axis stimulates quiescent odontogenic epithelial cell rests via TGF-β signalling to promote cell proliferation of the lining epithelia in radicular cysts: A laboratory investigation.

Cyst formation of the jaws is frequently accompanied by the proliferation of odontogenic epithelial cells located in the periodontal ligament (PDL), which consists of heterozygous cells and includes the most fibroblasts. The lining epithelium of radicular cyst, an odontogenic cyst of inflammatory origin, is derived from the proliferation of the remnants of the Hertwig epithelial root sheath (odontogenic epithelial cell rests of Malassez; ERMs) in the PDL. ERMs are maintained at a lower proliferative state under physiological conditions, but the regulatory mechanisms underlying the inflammation-dependent enhanced-proliferative capabilities of ERMs are not fully understood. The aim of this study was to evaluate the effects of cytokine pathway association between TGF-β signalling and IL-1β signalling on the regulation of odontogenic epithelial cell proliferation using radicular cyst pathological specimens and odontogenic epithelial cell lines. Immunofluorescence analyses were performed to clarify the expression levels of Smad2/3 and Ki-67 in ERMs of 8-week-old mouse molar specimens. In radicular cyst (n = 52) and dentigerous cysts (n = 6) specimens from human patients, the expression of p65 (a main subunit of NF-κB), Smad2/3 and Ki-67 were investigated using immunohistochemical analyses. Odontogenic epithelial cells and PDL fibroblastic cells were co-cultured with or without an inhibitor or siRNAs. Odontogenic epithelial cells were cultured with or without TGF-β1 and IL-1β. The proliferative capabilities and Smad2 phosphorylation levels of odontogenic epithelial cells were examined. Immunohistochemically, Smad2/3-positivity was increased, and p65-positivity and Ki-67-positivity were decreased both in ERMs and in the epithelial cells in dentigerous cysts, a non-inflammatory developmental cyst. In contrast, p65-positive cells, along with the expression of Ki-67, were increased and Smad2/3-positive cells were decreased in the lining epithelia of radicular cysts. Co-culture experiments with odontogenic epithelial cells and PDL fibroblastic cells revealed that PDL cells-derived TGF-β1/2 and their downstream signalling suppressed odontogenic epithelial cell proliferation. Moreover, TGF-β1 stimulation induced Smad2 phosphorylation and suppressed odontogenic epithelial cell proliferation, while IL-1β stimulation reversed these phenotypes through p65 transactivation. These results suggest that IL-1β-p65 signalling promotes odontogenic epithelial cell proliferation through suppressing TGF-β-Smad2 signalling, which would be involved in the pathogenesis of radicular cysts.

Neutrophil extracellular traps promoting fibroblast activation and aggravating limb ischemia through Wnt5a pathway.

Although the formation of NETs contributes to cancer cell invasion and distant metastasis, its role in the pathological progression of limb ischemia remains unknown. This study investigated the functional significance of NETs in cell-cell crosstalk during limb ischemia. The changes of cell subsets in lower limb ischemia samples were detected by single-cell RNA sequencing. The expression of neutrophil extracellular traps (NETs) related markers in lower limb ischemia samples was detected by immunohistochemistry and Western blotting. The signaling pathway of NETs activation in fibroblasts was verified by immunofluorescence, PCR and Western blotting. Through single-cell RNA sequencing (scRNA-seq), we identified 9 distinct cell clusters, with significantly upregulated activation levels in fibroblasts and neutrophils and phenotypic transformation of smooth muscle cells (SMCs) into a proliferative state in ischemic tissue. At the same time, the interaction between fibroblasts and smooth muscle cells was significantly enhanced in ischemic tissue. NETs levels rise and fibroblast activation is induced in ischemic conditions. Mechanistically, activated fibroblasts promote smooth muscle cell proliferation through the Wnt5a pathway. In ischemic mice, inhibition of Wnt5a mitigated vascular remodeling and subsequent ischemia. These findings highlighting the role of cell-cell crosstalk in ischemia and vascular remodeling. We found that the NETs-initiated fibroblast-SMC interaction is a critical regulator of limb ischemia via Wnt5a pathway, a potential therapeutic target for the treatment.

Characterizing Rare Dormant and Cycling Lineages Using the Watermelon System.

Barcode-based lineage tracing approaches enable molecular characterization of clonal cell families. Barcodes that are expressed as mRNA can be used to deconvolve lineage identity from single-cell RNA sequencing transcriptional data. Here we describe the Watermelon system, which facilitates the simultaneous tracing of lineage, transcriptional, and proliferative state at a single cell level.

In situ distribution of markers for stemness, quiescence, proliferation and differentiation in the human cornea and limbus

Aims/Purpose: Identify the distribution of the most commonly used markers for stemness, quiescence, proliferation, and differentiation in limbal compartments such as human limbal epithelial crypts, posterior and anterior limbus, as well as the central and posterior cornea.Methods: Corneal‐limbal biopsies from cadaver human donors (n = 3) embedded in paraffin were proceeded for immunohistochemistry fluorescence and examined for stemness/progenitor markers (p63α, SOX9, ABCG2, N‐cadherin), proliferation markers (ki‐67), quiescence marker (CEBPD), differentiation markers (CX43, KRT3, KRT12, KRT13), and epithelial marker (E‐cadherin). Images were obtained by Zeiss Axio Observer. Fluorescent images were obtained using the Zeiss Axio Imager M1 fluorescence microscope (ZEISS, Oberkochen, Germany).Results: The stemness/progenitor markers p63α and SOX9 were found in basal and suprabasal cells of limbal epithelial crypts and limbal epithelium, whereas the ABCG2 and N‐cadherin were exclusive for limbal basal epithelium in all limbal structures. Proliferation marker Ki‐67 was most abundant in suprabasal layers of the limbus and rarely found in the posterior cornea, while absent in anterior cornea. Quiescence marker CEBPD was positive in the basal cells of both, limbus and cornea. Differentiation markers KRT3 and KRT12 were positive in the whole cornea and intermediate and superficial layers in anterior and posterior limbus, while rarely present in limbal epithelial crypts. However, CX43 was staining almost all suprabasal layers in the limbus as well as basal and suprabasal layers in the cornea. KRT13 was exclusive for superficial layer of the anterior and posterior limbus layers staining conjunctival cells.Conclusions: Here we identify the localization of the most common markers used to determine the stemness, quiescence, proliferation, and differentiation status to unravel the in situ distribution of these markers in the limbal epithelial crypts, posterior and anterior limbus, as well as the central and posterior cornea. The study accumulated new knowledge about the distribution of these markers and the corneal‐limbal organization.

13C Tracer Analysis and Metabolomics in Dormant Cancer Cells.

Over the last two decades, major advances in the field of tumor dormancy have been made. Yet, it is not completely understood how dormant disseminated tumor cells survive and transition to a proliferative state to generate a metastatic lesion. On the other hand, metabolic rewiring has been shown to influence metastasis development through the modulation of both intracellular signaling and the crosstalk between metastatic cells and their microenvironment. Thus, studying the metabolic features of dormant disseminated tumor cells has gained importance in understanding the dormancy process. Here, we describe a method to perform metabolomics and 13C tracer analysis in 3D cultures of dormant breast cancer cells.

Investigating the Spatial Distribution of Proliferating Cells in Primary Ovarian Cancers.

Ovarian cancer (OC) accounts for about 4% of female cancers globally. While Ki67-immunopositive (Ki67+) cell density is commonly used to assess proliferation in OC, the two-dimensional (2D) distribution pattern of these cells is poorly understood. This study explores the 2D distribution pattern of Ki67+ cells in primary OC tissues and models the proliferation process to improve our understanding of this hallmark of cancer. A total of 100 tissue cores, included in a tissue microarray (TMA) representing 5 clear cell carcinomas, 62 serous carcinomas, 10 mucinous adenocarcinomas, 3 endometrioid adenocarcinomas, 10 lymph node metastases from OC, and 10 samples of adjacent normal ovary tissue, were stained using a standardized immunohistochemical protocol. The computer-aided image analysis system assessed the 2D distribution pattern of Ki67+ proliferating cells, providing the cell number and density, patterns of randomness, and cell-to-cell closeness. Three computer models were created to simulate behavior and responses, aiming to gain insights into the variations in the proliferation process. Significant differences in Ki67+ cell density were found between low- and high-grade serous carcinoma/mucinous adenocarcinomas (p = 0.003 and p = 0.01, respectively). The Nearest Neighbor Index of Ki67+ cells differed significantly between high-grade serous carcinomas and endometrioid adenocarcinomas (p = 0.01), indicating distinct 2D Ki67+ distribution patterns. Proxemics analysis revealed significant differences in Ki67+ cell-to-cell closeness between low- and high-grade serous carcinomas (p = 0.002). Computer models showed varied effects on the overall organization of Ki67+ cells and the ability to preserve the original 2D distribution pattern when altering the location and/or density of Ki67+ cells. Cell proliferation is a hallmark of OCs. This study provides new evidence that investigating the Ki67+ cell density and 2D distribution pattern can assist in understanding the proliferation status of OCs. Moreover, our computer models suggest that changes in Ki67+ cell density and their location are critical for maintaining the 2D distribution pattern.

The microRNA target site profile is a novel biomarker in the immunotherapy response.

MicroRNAs (miRNAs) bind on the 3' untranslated region (3'UTR) of messenger RNAs (mRNAs) and regulate mRNA expression in physiological and pathological conditions, including cancer. Thus, studies have identified miRNAs as potential biomarkers by correlating the miRNA expression with the expression of important mRNAs and/or clinical outcomes in cancers. However, tumors undergo pervasive 3'UTR shortening/lengthening events through alternative polyadenylation (APA), which varies the number of miRNA target sites in mRNA, raising the number of miRNA target sites (numTS) as another important regulatory axis of the miRNA binding effects. In this study, we developed the first statistical method, BIOMATA-APA, to identify predictive miRNAs based on numTS features. Running BIOMATA-APA on The Cancer Genome Atlas (TCGA) and independent cohort data both with immunotherapy and no immunotherapy, we demonstrated for the first time that the numTS feature 1) distinguishes different cancer types, 2) predicts tumor proliferation and immune infiltration status, 3) explains more variation in the proportion of tumor-infiltrating immune cells, 4) predicts response to immune checkpoint blockade (ICB) therapy, and 5) adds prognostic power beyond clinical and miRNA expression. To the best of our knowledge, this is the first pan-cancer study to systematically demonstrate numTS as a novel type of biomarker representing the miRNA binding effects underlying tumorigenesis and pave the way to incorporate miRNA target sites for miRNA biomarker identification. Another advantage of examining the miRNA binding effect using numTS is that it requires only RNA-Seq data, not miRNAs, thus resulting in high power in the miRNA biomarker identification.

Correlation analysis of dynamic enhanced energy spectrum CT parameters with Ki-67 high expression in hepatocellular carcinoma

Objective: To investigate the correlation between dynamic enhanced energy spectral CT parameters and Ki-67 high expression in hepatocellular carcinoma (HCC). Methods: A cross-sectional study. This retrospective case-control study analyzed the clinical data of 101 patients with pathologically confirmed HCC in Xiamen Hospital of Traditional Chinese Medicine and Zhongshan Hospital of Xiamen University from December 2017 to March 2023. These 101 patients included 84 males and 17 females, and the age[M(Q1, Q3)] was 59.0(49.0,66.0)years. These patients were divided into two groups according to the immunohistochemical Ki-67 expression levels in tumor tissues: the high expression group (Ki-67%>20%, n=59) and the low expression group (Ki-67%≤20%, n=42).CT values on 70 keV and 140 keV monochromatic energy images (HU70 keV-a, HU140 keV-a, HU70 keV-p, HU140 keV-p) and water density (Dwater-a, Dwater-p) were measured in arterial phase and portal vein phase, and the difference of HU70 keV, HU140 keV, Dwater values between portal vein and arterial phase (ΔHU70 keV, ΔHU140 keV, ΔDwater), as well as ratio of HU70 keV, HU140 keV, Dwatervalues between portal vein and arterial phase (HU70 keVratio, HU140 keVratio, Dwaterratio) were calculated. Spearman correlation analysis was used to analyze the correlation between the CT spectral parameters and Ki-67%. Multivariate logistic regression model was used to determine the factors associated with high expression of Ki-67. The receiver operating characteristics (ROC) curves were used to indicate the efficacy of dynamic enhanced spectral CT in evaluating Ki-67 high expression in HCC. Results: The high Ki-67 expression group revealed higher alpha fetal protein levels, larger tumor diameter and more irregular tumor shape compared with the low Ki-67 expression group,and the differences were statistically significant (all P<0.05). Spearman correlation analysis showed that the HU140 keV-p, Dwater-p, HU70 keV ratio, HU140 keV ratio, Dwater ratio, ΔHU70 keV, ΔHU140 keV, ΔDwater were positively correlated with Ki-67 positivity rate (r:0.31-0.50, all P<0.05). The spectral CT parameters (HU70 keV-p, HU140 keV-p, Dwater-p, HU70 keV ratio, HU140 keV ratio, Dwater ratio, ΔHU70 keV, ΔHU140 keV, ΔDwater) in high Ki-67 expression group were significantly higher than those in low Ki-67 expression group (all P<0.05). Multivariate logistic regression model shows that Dwater-p(OR=1.16, 95%CI: 1.05-1.29, P=0.005), ΔHU140 keV(OR=1.39, 95%CI: 1.20-1.62, P<0.001) and irregular tumor morphology (OR=5.25, 95%CI: 1.61-17.12, P=0.006) were correlative factors for high Ki-67 expression. The HU140 keV ratio and ΔHU140 keV alone evaluated the highest AUC of high Ki-67 high expression in HCC, which were 0.82 (95%CI: 0.74-0.90), the sensitivity were 61.0%, and the specificity were 88.1% and 85.7%. The combined analysis of Dwater-p, ΔHU140 keV and irregular tumor morphology had an increased AUC of 0.88 (95%CI: 0.81-0.95) in assessment high Ki-67 expression, with the sensitivity of 84.7% and the specificity of 78.6%. Conclusions: Dynamic enhanced spectral CT parameters were positively correlated with the Ki-67 expression in HCC. Spectral CT provides a non-invasive method to evaluate the proliferation status of HCC cells, and the efficiency could be improved by multi-parameter analysis combining spectral CT parameters and morphologic features.

Targeting Cyclin-Dependent Kinase 1 Induces Apoptosis and Cell Cycle Arrest of Activated Hepatic Stellate Cells.

Liver fibrosis is the integral process of chronic liver diseases caused by multiple etiologies and characterized by excessive deposition of extracellular matrix (ECM). During liver fibrosis, hepatic stellate cells (HSCs) transform into a highly proliferative, activated state, producing various cytokines, chemokines, and ECM. However, the precise mechanisms that license HSCs into the highly proliferative state remain unclear. Cyclin-dependent kinase 1 (CDK1) is a requisite event for the transition of the G1/S and G2/M phases in eukaryotic cells. In this study, it is demonstrated that CDK1 and its activating partners, Cyclin A2 and Cyclin B1, are upregulated in both liver fibrosis/cirrhosis patient specimens and the murine hepatic fibrosis models, especially in activated HSCs. In vitro, CDK1 is upregulated in spontaneously activated HSCs, and inhibiting CDK1 with specific small-molecule inhibitors (CGP74514A, RO-3306, or Purvalanol A) orshorthairpinRNAs(shRNAs) resulted in HSC apoptosis and cell cycle arrest by regulating Survivin expression. Above all, it is illustrated that increased CDK1 expression licenses the HSCs into a highly proliferative state and can serve as a potential therapeutic target in liver fibrosis.

OIP5-AS1/CD147/TRPM7 axis promotes gastric cancer metastasis by regulating apoptosis related PI3K-Akt signaling.

To explore the mechanism of OIP5-AS1/CD147/TRPM7 axis to gastric cancer (GC) metastasis. Bioinformatic analysis was performed to pick up the candidate genes associated with regulation GC metastasis. Using GC cell lines, AGS and MKN-45 as research objects, identify the effect of candidate genes on GC metastasis, judge cell proliferation status by MTT assay and cell clone number, and detect cell migration by Transwell and Wound-healing assay. The molecular mechanism of CD147/OIP5/TRPM7 axis regulating GC metastasis was further explored by RNA sequencing. The key signaling pathways were subsequently verified by flow cytometry and WB. Bioinformatic analysis suggested OIP5-AS1/CD147/TRPM7 axis may be involving in GC metastasis. The RNA interference experiment proved that after gene interference, the proliferation ability of GC cells decreased significantly (P<0.05), which was manifested in the reduction of the number of cell clones. In addition, the migration ability of GC cells was also affected, which was based on the results of Wound Healing (P<0.05). CD147, OIP5-AS1 and TRPM7 all have harmful effects on GC cells. The relationship between OIP5-AS1 and CD147/TRPM7 was detected by RNA immunoprecipitation. Moreover, the RNA sequencing data indicated that CD147/OIP5-AS1/TRPM7 may coordinately regulate the PI3K-AKT pathway related to GC cell apoptosis, thereby affecting the proliferation and migration of GC cells. After RNA interference, the level of apoptosis increased both in AGS and MKN-45 cells. Meanwhile, the expression of pro-apoptotic proteins Caspase9 and BAX were up-regulated (P<0.05). In addition, the expression of PI3K and AKT proteins was reduced (P<0.05). The mouse tumorigenesis experiment corroborated the results of the in vitro study. OIP5-AS1/CD147/TRPM7 axis reduces GC cell proliferation by regulating apoptosis associated with PI3K-AKT signaling, further affecting cancer metastasis.

Role of tenascin C in lesion formation in early peritoneal endometriosis

To identify cytokines or extracellular matrix components that contribute to adhesion to, and invasion of, the peritoneum, proximal to lesions in the early phase of endometriosis. Laboratory-based study. University Hospital and Laboratory of Animal Science. Five women with ovarian endometrioma, 138 wild-type (WT) C57BL/6N mice, and 48 Tenascin C (Tnc) knockout (TncKO) mice. To establish a murine endometriosis model, 20 pieces of minced uterine tissue fragments from each horn were administered intraperitoneally to syngeneic mice. Three days later, endometriotic lesions and peritoneal tissues were collected. Separately, we transfected human peritoneal mesothelial cells (HMrSV5) or human endometrial stromal cells (hESCs) with Tnc small interfering ribonucleic acid. We employed a polymerase chain reaction array to profile gene expression in the murine peritoneum, in both peritoneum distal to lesions and peritoneum surrounding lesions (PSL). The expression of upregulated genes in the PSL was verified in the peritoneal samples by real-time reverse transcription-polymerase chain reaction. TncKO mice were used to investigate the role of Tnc in the development of endometriosis. We evaluated the proliferative activity or inflammatory state of lesions by Ki67 or CD3 immunostaining. Intraperitoneal distribution of macrophages was assessed by fluorescence-activated cell sorting. Using Tnc small interfering ribonucleic acid, we examined the invasive capacity of hESCs in a coculture system with HMrSV5. Tnc gene expression was significantly higher in PSL than in peritoneum distal to lesions. The weight and number of TncKO lesions in TncKO hosts were lower than those of WT lesions in WT hosts. In contrast, the weight and number of nonattached TncKO lesions in TncKO hosts were higher than those of nonattached WT lesions in WT hosts. We observed decreased Ki67-positive cells or H-scores for CD3, a lower proportion of M1 macrophages, and a higher proportion of M2 macrophages in TncKO lesions in TncKO recipients. Silencing of Tnc expression in hESCs and HMrSV5 diminished the invasivity of hESCs. Tnc may be a crucial factor in the development of early peritoneal endometriosis.

Dynamic compartmentalization of the pro-invasive transcription factor NHR-67 reveals a role for Groucho in regulating a proliferative-invasive cellular switch in C. elegans.

A growing body of evidence suggests that cell division and basement membrane invasion are mutually exclusive cellular behaviors. How cells switch between proliferative and invasive states is not well understood. Here, we investigated this dichotomy in vivo by examining two cell types in the developing Caenorhabditis elegans somatic gonad that derive from equipotent progenitors, but exhibit distinct cell behaviors: the post-mitotic, invasive anchor cell and the neighboring proliferative, non-invasive ventral uterine (VU) cells. We show that the fates of these cells post-specification are more plastic than previously appreciated and that levels of NHR-67 are important for discriminating between invasive and proliferative behavior. Transcription of NHR-67 is downregulated following post-translational degradation of its direct upstream regulator, HLH-2 (E/Daughterless) in VU cells. In the nuclei of VU cells, residual NHR-67 protein is compartmentalized into discrete punctae that are dynamic over the cell cycle and exhibit liquid-like properties. By screening for proteins that colocalize with NHR-67 punctae, we identified new regulators of uterine cell fate maintenance: homologs of the transcriptional co-repressor Groucho (UNC-37 and LSY-22), as well as the TCF/LEF homolog POP-1. We propose a model in which the association of NHR-67 with the Groucho/TCF complex suppresses the default invasive state in non-invasive cells, which complements transcriptional regulation to add robustness to the proliferative-invasive cellular switch in vivo.

Dynamic compartmentalization of the pro-invasive transcription factor NHR-67 reveals a role for Groucho in regulating a proliferative-invasive cellular switch in C. elegans

A growing body of evidence suggests that cell division and basement membrane invasion are mutually exclusive cellular behaviors. How cells switch between proliferative and invasive states is not well understood. Here, we investigated this dichotomy in vivo by examining two cell types in the developing Caenorhabditis elegans somatic gonad that derive from equipotent progenitors, but exhibit distinct cell behaviors: the post-mitotic, invasive anchor cell and the neighboring proliferative, non-invasive ventral uterine (VU) cells. We show that the fates of these cells post-specification are more plastic than previously appreciated and that levels of NHR-67 are important for discriminating between invasive and proliferative behavior. Transcription of NHR-67 is downregulated following post-translational degradation of its direct upstream regulator, HLH-2 (E/Daughterless) in VU cells. In the nuclei of VU cells, residual NHR-67 protein is compartmentalized into discrete punctae that are dynamic over the cell cycle and exhibit liquid-like properties. By screening for proteins that colocalize with NHR-67 punctae, we identified new regulators of uterine cell fate maintenance: homologs of the transcriptional co-repressor Groucho (UNC-37 and LSY-22), as well as the TCF/LEF homolog POP-1. We propose a model in which the association of NHR-67 with the Groucho/TCF complex suppresses the default invasive state in non-invasive cells, which complements transcriptional regulation to add robustness to the proliferative-invasive cellular switch in vivo.