Deep-Learning-Based Hepatic Ploidy Quantification Using H&E Histopathology Images.

Polyploidy, the duplication of the entire genome within a single cell, is a significant characteristic of cells in many tissues, including the liver. The quantification of hepatic ploidy typically relies on flow cytometry and immunofluorescence (IF) imaging, which are not widely available in clinical settings due to high financial and time costs. To improve accessibility for clinical samples, we developed a computational algorithm to quantify hepatic ploidy using hematoxylin-eosin (H&E) histopathology images, which are commonly obtained during routine clinical practice. Our algorithm uses a deep learning model to first segment and classify different types of cell nuclei in H&E images. It then determines cellular ploidy based on the relative distance between identified hepatocyte nuclei and determines nuclear ploidy using a fitted Gaussian mixture model. The algorithm can establish the total number of hepatocytes and their detailed ploidy information in a region of interest (ROI) on H&E images. This is the first successful attempt to automate ploidy analysis on H&E images. Our algorithm is expected to serve as an important tool for studying the role of polyploidy in human liver disease.

A Deep Learning Approach for Histology-Based Nucleus Segmentation and Tumor Microenvironment Characterization.

Microscopic examination of pathology slides is essential to disease diagnosis and biomedical research. However, traditional manual examination of tissue slides is laborious and subjective. Tumor whole-slide image (WSI) scanning is becoming part of routine clinical procedures and produces massive data that capture tumor histologic details at high resolution. Furthermore, the rapid development of deep learning algorithms has significantly increased the efficiency and accuracy of pathology image analysis. In light of this progress, digital pathology is fast becoming a powerful tool to assist pathologists. Studying tumor tissue and its surrounding microenvironment provides critical insight into tumor initiation, progression, metastasis, and potential therapeutic targets. Nucleus segmentation and classification are critical to pathology image analysis, especially in characterizing and quantifying the tumor microenvironment (TME). Computational algorithms have been developed for nucleus segmentation and TME quantification within image patches. However, existing algorithms are computationally intensive and time consuming for WSI analysis. This study presents Histology-based Detection using Yolo (HD-Yolo), a new method that significantly accelerates nucleus segmentation and TME quantification. We demonstrate that HD-Yolo outperforms existing WSI analysis methods in nucleus detection, classification accuracy, and computation time. We validated the advantages of the system on 3 different tissue types: lung cancer, liver cancer, and breast cancer. For breast cancer, nucleus features by HD-Yolo were more prognostically significant than both the estrogen receptor status by immunohistochemistry and the progesterone receptor status by immunohistochemistry. The WSI analysis pipeline and a real-time nucleus segmentation viewer are available at https://github.com/impromptuRong/hd_wsi.

Deep learning in digital pathology for personalized treatment plans of cancer patients.

Over the past decade, many new cancer treatments have been developed and made available to patients. However, in most cases, these treatments only benefit a specific subgroup of patients, making the selection of treatment for a specific patient an essential but challenging task for oncologists. Although some biomarkers were found to associate with treatment response, manual assessment is time-consuming and subjective. With the rapid developments and expanded implementation of artificial intelligence (AI) in digital pathology, many biomarkers can be quantified automatically from histopathology images. This approach allows for a more efficient and objective assessment of biomarkers, aiding oncologists in formulating personalized treatment plans for cancer patients. This review presents an overview and summary of the recent studies on biomarker quantification and treatment response prediction using hematoxylin-eosin (H&E) stained pathology images. These studies have shown that an AI-based digital pathology approach can be practical and will become increasingly important in improving the selection of cancer treatments for patients.

Enhanced Pathology Image Quality with Restore-Generative Adversarial Network.

Whole slide imaging is becoming a routine procedure in clinical diagnosis. Advanced image analysis techniques have been developed to assist pathologists in disease diagnosis, staging, subtype classification, and risk stratification. Recently, deep learning algorithms have achieved state-of-the-art performances in various imaging analysis tasks, including tumor region segmentation, nuclei detection, and disease classification. However, widespread clinical use of these algorithms is hampered by their performances often degrading due to image quality issues commonly seen in real-world pathology imaging data such as low resolution, blurring regions, and staining variation. Restore-Generative Adversarial Network (GAN), a deep learning model, was developed to improve the imaging qualities by restoring blurred regions, enhancing low resolution, and normalizing staining colors. The results demonstrate that Restore-GAN can significantly improve image quality, which leads to improved model robustness and performance for existing deep learning algorithms in pathology image analysis. Restore-GAN has the potential to be used to facilitate the applications of deep learning models in digital pathology analyses.

In vivo CD8+ T cell CRISPR screening reveals control by Fli1 in infection and cancer.

Improving effector activity of antigen-specific T cells is a major goal in cancer immunotherapy. Despite the identification of several effector T cell (TEFF)-driving transcription factors (TFs), the transcriptional coordination of TEFF biology remains poorly understood. We developed an in vivo T cell CRISPR screening platform and identified a key mechanism restraining TEFF biology through the ETS family TF, Fli1. Genetic deletion of Fli1 enhanced TEFF responses without compromising memory or exhaustion precursors. Fli1 restrained TEFF lineage differentiation by binding to cis-regulatory elements of effector-associated genes. Loss of Fli1 increased chromatin accessibility at ETS:RUNX motifs, allowing more efficient Runx3-driven TEFF biology. CD8+ T cells lacking Fli1 provided substantially better protection against multiple infections and tumors. These data indicate that Fli1 safeguards the developing CD8+ T cell transcriptional landscape from excessive ETS:RUNX-driven TEFF cell differentiation. Moreover, genetic deletion of Fli1 improves TEFF differentiation and protective immunity in infections and cancer.

The expression and clinical significance of murine double minute 2, lysosome-associated membrane protein 1, and P-glycoprotein in pediatric acute lymphoblastic leukemia.

BACKGROUND: To analyze the expression and clinical significance of murine double minute 2 (MDM2), lysosome-associated membrane protein (LAMP1) and P-glycoprotein (P-gp) in children with acute lymphoblastic leukemia (ALL). METHODS: Thirty-three children with ALL who were admitted to our hospital between January 2017 and January 2018 were enrolled as the ALL group. The expression of MDM2, LAMP1 and P-gp was compared between the two groups, as well as between ALL patients with different clinical characteristics. Logistic regression was used to analyze the risk factors that affect the prognosis and survival of ALL patients. Kaplan-Meier survival curves were used to analyze the correlations of MDM2, LAMP1 and P-gp on the prognosis and survival of ALL patients. RESULTS: The expression levels of MDM2, LAMP1 and P-gp in the ALL group were higher than those in the control group (P<0.05). The average survival time of the group with low expression of MDM2 was (34.92±0.56) months, the average survival time of the group with high expression of MDM2 was (31.32±0.42) months, and the difference was statistically significant (P<0.05). The average survival time of the group with low expression of LAMP1 was (36.71±0.55) months, the average survival time of the group with high expression of LAMP1 was (29.87±0.40) months, the difference was statistically significant (P<0.05). The average survival time of the group with low expression of P-gp was (36.29±0.41) months, the average survival time of the group with high expression of P-gp was (26.46±0.37) months, and the difference was statistically significant (P<0.05). CONCLUSIONS: Abnormal expression levels of MDM2, LAMP1 and P-gp protein are related to the occurrence and development of ALL, and are closely related to patient prognosis and survival. Therefore, MDM2, LAMP1and P-gp can serve as molecular markers for predicting the prognosis of children with ALL.

eIF5B drives integrated stress response-dependent translation of PD-L1 in lung cancer.

Cancer cells express high levels of PD-L1, a ligand of the PD-1 receptor on T cells, allowing tumors to suppress T cell activity. Clinical trials utilizing antibodies that disrupt the PD-1/PD-L1 checkpoint have yielded remarkable results, with anti-PD-1 immunotherapy approved as first-line therapy for lung cancer patients. We used CRISPR-based screening to identify regulators of PD-L1 in human lung cancer cells, revealing potent induction of PD-L1 upon disruption of heme biosynthesis. Impairment of heme production activates the integrated stress response (ISR), allowing bypass of inhibitory upstream open reading frames in the PD-L1 5' UTR, resulting in enhanced PD-L1 translation and suppression of anti-tumor immunity. We demonstrated that ISR-dependent PD-L1 translation requires the translation initiation factor eIF5B. eIF5B overexpression, which is frequent in lung adenocarcinomas and associated with poor prognosis, is sufficient to induce PD-L1. These findings illuminate mechanisms of immune checkpoint activation and identify targets for therapeutic intervention.

Mice With Increased Numbers of Polyploid Hepatocytes Maintain Regenerative Capacity But Develop Fewer Hepatocellular Carcinomas Following Chronic Liver Injury.

BACKGROUND & AIMS: Thirty to 90% of hepatocytes contain whole-genome duplications, but little is known about the fates or functions of these polyploid cells or how they affect development of liver disease. We investigated the effects of continuous proliferative pressure, observed in chronically damaged liver tissues, on polyploid cells. METHODS: We studied Rosa-rtTa mice (controls) and Rosa-rtTa;TRE-short hairpin RNA mice, which have reversible knockdown of anillin, actin binding protein (ANLN). Transient administration of doxycycline increases the frequency and degree of hepatocyte polyploidy without permanently altering levels of ANLN. Mice were then given diethylnitrosamine and carbon tetrachloride (CCl4) to induce mutations, chronic liver damage, and carcinogenesis. We performed partial hepatectomies to test liver regeneration and then RNA-sequencing to identify changes in gene expression. Lineage tracing was used to rule out repopulation from non-hepatocyte sources. We imaged dividing hepatocytes to estimate the frequency of mitotic errors during regeneration. We also performed whole-exome sequencing of 54 liver nodules from patients with cirrhosis to quantify aneuploidy, a possible outcome of polyploid cell divisions. RESULTS: Liver tissues from control mice given CCl4 had significant increases in ploidy compared with livers from uninjured mice. Mice with knockdown of ANLN had hepatocyte ploidy above physiologic levels and developed significantly fewer liver tumors after administration of diethylnitrosamine and CCl4 compared with control mice. Increased hepatocyte polyploidy was not associated with altered regenerative capacity or tissue fitness, changes in gene expression, or more mitotic errors. Based on lineage-tracing experiments, non-hepatocytes did not contribute to liver regeneration in mice with increased polyploidy. Despite an equivalent rate of mitosis in hepatocytes of differing ploidies, we found no lagging chromosomes or micronuclei in mitotic polyploid cells. In nodules of human cirrhotic liver tissue, there was no evidence of chromosome-level copy number variations. CONCLUSIONS: Mice with increased polyploid hepatocytes develop fewer liver tumors following chronic liver damage. Remarkably, polyploid hepatocytes maintain the ability to regenerate liver tissues during chronic damage without generating mitotic errors, and aneuploidy is not commonly observed in cirrhotic livers. Strategies to increase numbers of polypoid hepatocytes might be effective in preventing liver cancer.

Lipocalin-2 may produce damaging effect after cerebral ischemia by inducing astrocytes classical activation.

BACKGROUND: Functions of astrocytes in the rehabilitation after ischemic stroke, especially their impacts on inflammatory processes, remain controversial. This study uncovered two phenotypes of astrocytes, of which one was helpful, and the other harmful to anoxic neurons after brain ischemia. METHODS: We tested the levels of inflammatory factors including TNF-a, IL-6, IL-10, iNOS, IL-1beta, and CXCL10 in primary astrocytes at 0 h, 6 h, 12 h, 24 h, and 48 h after OGD, grouped the hypoxia astrocytes into iNOS-positive (iNOS(+)) and iNOS-negative (iNOS(-)) by magnetic bead sorting, and then co-cultured the two groups of cells with OGD-treated neurons for 24 h. We further verified the polarization of astrocytes in vivo by detecting the co-localization of iNOS, GFAP, and Iba-1 on MCAO brain sections. Lentivirus overexpressing LCN2 and LCN2 knockout mice (#024630. JAX, USA) were used to explore the role of LCN2 in the functional polarization of astrocytes. 7.0-T MRI scanning and the modified Neurological Severity Score (mNSS) were used to evaluate the neurological outcomes of the mice. RESULTS: After oxygen-glucose deprivation (OGD), iNOS mRNA expression increased to the peak at 6 h in primary astrocytes, but keep baseline expression in LCN2-knockout astrocytes. In mice with transient middle cerebral artery occlusion (tMCAO), LCN2 was proved necessary for astrocyte classical activation. In LCN2 knockout mice with MCAO, no classically activated astrocytes were detected, and smaller infarct volumes and better neurological functions were observed. CONCLUSIONS: The results indicated a novel pattern of astrocyte activation after ischemic stroke and lipocalin-2 (LCN2) plays a key role in polarizing and activating astrocytes.

Optimization of behavioural tests for the prediction of outcomes in mouse models of focal middle cerebral artery occlusion.

Intraluminal middle cerebral artery occlusion (MCAO) is the most widely used model of stroke. We aimed to predict the outcome of MCAO using a combination of fine behavioural tests for the prediction of unsuccessful surgery in mice leading to no infarction, haemorrhage and unexpected death. MCAO was performed on adult mice under the guidance of laser-Doppler flowmetry (LDF) to warrant a decrease in regional cerebral blood flow (rCBF) in the MCA territory. Four outcomes of MCAO were defined according to histological analysis: infarction, no infarction, haemorrhage and unexpected death (death within 24h post-surgery). Fine behavioural tests including the rotarod, modified neurological severity score (mNSS), Clark general and Clark focal tests were performed separately at 6h, 12h and 24h post-stroke. A total of 94 mice were included in the analysis. The infarction rate associated with MCAO was 58.5% (55/94). After optimization of the timing and behavioural tests, we found that higher Clark focal (>17.5) or higher mNSS scores (>10) were markedly related to early death, whereas a lower mNSS score (<3.5) was indicative of a tendency to show no infarction at 6h post-stroke. After 24h post-stroke, there was a positive correlation between the infarct volume and Clark focal results. Behavioural tests could help to predict the outcomes in the MCAO mouse model.

Surfactant administration via a thin endotracheal catheter during spontaneous breathing in preterm infants.

To systematically review the clinical outcomes of surfactant administration via a thin endotracheal catheter during spontaneous breathing compared with conventional administration involving tracheal intubation, mechanical ventilation (MV), and tracheal extubation, in preterm infants. PubMed, EMBASE, and the Cochrane Library were searched to identify relevant clinical trials. Data were analyzed using the Cochrane Collaboration methods. Primary outcome measures included the incidence of MV and bronchopulmonary dysplasia (BPD). Finally, four RCTs, two prospective cohort trials, and six historical controlled trials involving 5,261 preterm infants were analyzed. In RCTs, surfactant administration though a thin catheter reduced the incidence of MV (risk ratio [RR]: 0.74; 95% confidence interval [CI]: 0.66, 0.81) in 72 hr and BPD (RR: 0.69, 95%CI: 0.50, 0.97) compared with conventional administration and in non-RCTs, there was also significant reduction in the incidence of MV (RR: 0.55, 95%CI: 0.45, 0.68) and BPD (RR: 0.70, 95%CI: 0.60, 0.82) in favor of the thin catheter group. There were no significant differences between the two procedures in terms of short-term pulmonary complications, intracranial pathology, necrotizing enterocolitis, retinopathy of prematurity, and mortality. Thus, surfactant administration via a thin endotracheal catheter to preterm infants has promising benefits, including reducing the incidences of MV and BPD, while providing comparable breathing support to conventional administration in MV. Pediatr Pulmonol. 2017; 52:844-854. © 2017 Wiley Periodicals, Inc.

Caveolin-1 is a checkpoint regulator in hypoxia-induced astrocyte apoptosis via Ras/Raf/ERK pathway.

Astrocytes, the most numerous cells in the human brain, play a central role in the metabolic homeostasis following hypoxic injury. Caveolin-1 (Cav-1), a transmembrane scaffolding protein, has been shown to converge prosurvival signaling in the central nerve system. The present study aimed to investigate the role of Cav-1 in the hypoxia-induced astrocyte injury. We also examined how Cav-1 alleviates apoptotic astrocyte death. To this end, primary astrocytes were exposed to oxygen-glucose deprivation (OGD) for 6 h and a subsequent 24-h reoxygenation to mimic hypoxic injury. OGD significantly reduced Cav-1 expression. Downregulation of Cav-1 using Cav-1 small interfering RNA dramatically worsened astrocyte cell damage and impaired cellular glutamate uptake after OGD, whereas overexpression of Cav-1 with Cav-1 scaffolding domain peptide attenuated OGD-induced cell apoptosis. Mechanistically, the expressions of Ras-GTP, phospho-Raf, and phospho-ERK were sequestered in Cav-1 small interfering RNA-treated astrocytes, yet were stimulated after supplementation with caveolin peptide. MEK/ERK inhibitor U0126 remarkably blocked the Cav-1-induced counteraction against apoptosis following hypoxia, indicating Ras/Raf/ERK pathway is required for the Cav-1's prosurvival role. Together, these findings support Cav-1 as a checkpoint for the in hypoxia-induced astrocyte apoptosis and warrant further studies targeting Cav-1 to treat hypoxic-ischemic brain injury.

Orosomucoid1: Involved in vascular endothelial growth factor-induced blood-brain barrier leakage after ischemic stroke in mouse.

Vascular endothelial growth factor (VEGF) is a promising candidate for the treatment of ischemic stroke. However, accumulating evidence demonstrated that VEGF could exacerbate blood-brain barrier (BBB) disruption after ischemic stroke. This study was designed to investigate the underlying mechanisms. In the present study, a transient (90 min) middle cerebral artery occlusion (MCAO) model was performed to induce ischemic stroke in mice. VEGF was administered intracerebroventricularly 3h after reperfusion. A gene expression microarray was utilized to investigate the differentially expressed genes among the sham, MCAO, and VEGF groups. A total of 3381 mRNAs were significantly altered by cerebral ischemia when compared with the sham group, and 15 of them were changed in the VEGF group when compared with the MCAO group. Among the 15 genes, orosomucoid (Orm) 1 was most sharply changed, and this gene has previously been reported to maintain the permeability of microvessels and integrity of the BBB. Results of the microarray showed that the expression of Orm1 increased after cerebral ischemia, whereas it decreased in response to VEGF, which was confirmed by real-time quantitative PCR, western blotting, immunohistochemistry, and immunofluorescence. The bioinformatics analysis indicated two NF-κB binding sites on the Orm1 promoter, and a super-shift assay verified that NF-κB could bind the Orm1 promoter. Results of the electrophoretic mobility shift assay (EMSA) revealed that VEGF inhibited the DNA-binding activity of NF-κB/p65. Furthermore, the elevated expression and activation of key members in the canonical NF-κB pathway induced by cerebral ischemia were also inhibited by VEGF treatment. In conclusion, this study demonstrated that decreasing the Orm1 expression via inhibition of the NF-κB pathway could be a possible mechanism involved in the aggravation of BBB disruption after stroke by VEGF.