Gastrointestinal (GI) diseases represent a clinically significant burden, necessitating precise diagnostic approaches to optimize patient outcomes. Conventional histopathological diagnosis suffers from limited reproducibility and diagnostic variability. To overcome these limitations, we develop Digepath, a specialized foundation model for GI pathology. Our framework introduces a dual-phase iterative optimization strategy combining pretraining with fine-screening, specifically designed to address the detection of sparsely distributed lesion areas in whole-slide images. Digepath is pretrained on over 353 million multi-scale images from 210,043 H&E-stained slides of GI diseases. It attains state-of-the-art performance on 33 out of 34 tasks related to GI pathology, including pathological diagnosis, protein expression status prediction, gene mutation prediction, and prognosis evaluation. We further translate the intelligent screening module for early GI cancer and achieve near-perfect 99.70% sensitivity across nine independent medical institutions. This work not only advances AI-driven precision pathology for GI diseases but also bridge critical gaps in histopathological practice.

Background:HIV-associated lymphomas (HALs) exhibit aggressive features and poorer prognosis compared to HIV-negative lymphomas. However, their molecular and clinicopathological characteristics remain unclear in the antiretroviral therapy (ART) era. Methods:We retrospectively analyzed 208 lymphoma patients (57 HALs, 151 HIV-negative lymphomas) diagnosed between July 2019 and March 2024. Quantitative immunohistochemistry evaluated expression levels of Ki67, CD10, BCL6, MUM1, BCL2, and MYC. Independent prognostic factors were identified using multivariate Cox regression analysis, and a survival prediction model was validated by receiver operating characteristic (ROC) curve analysis. Results:HALs exhibited significantly higher proliferative activity (Ki67 AOD: 0.92 vs. 0.82, P < 0.001), more advanced disease stages (Ann Arbor stage III/IV: 77.2% vs. 60.0%, P = 0.022), and increased Epstein-Barr virus (EBV) positivity (51.1% vs. 17.9%, P < 0.001). Immunophenotyping revealed a GCB-like phenotype in HALs, characterized by elevated CD10 and BCL6 expression and decreased MUM1 and BCL2 expression. Patients with HALs had significantly shorter survival (median: 32.1 vs. 46.1 months, P < 0.001). Multivariate analysis identified Ki67 AOD (hazard ratio [HR] = 3.04, 95% confidence interval [CI]: 3.85-10.85), International Prognostic Index (IPI) (HR = 9.35, 95% CI: 4.20-20.82), and ART duration (protective, HR = 0.29/year, 95% CI: 0.19-0.45) as independent prognostic factors. The survival model demonstrated strong predictive accuracy (1-year area under the curve [AUC] = 0.831). Conclusions:HALs exhibit distinct molecular profiles-including elevated EBV infection, a GCB-like phenotype, increased Ki67 AOD, and decreased BCL2 expression-that contribute to significantly poorer survival compared to HIV-negative lymphomas. Integrating Ki67 AOD and IPI scores into prognostic models may enhance individualized prognosis and optimize treatment strategies for HAL patients.

Background:Pleomorphic giant cell adenocarcinoma (PGCA) of the prostate is a rare, aggressive variant characterized by multinucleated giant cells, sarcomatoid features, and resistance to conventional therapies. Despite its recognition in the WHO 2016 guidelines, the molecular drivers and clinicopathological correlates of PGCA remain poorly characterized. This study presents the first integrative clinicogenomic profiling of PGCA, revealing a novel prognostic gene signature with direct implications for diagnosis and treatment. Methods:We conducted comprehensive clinicopathological and genomic analyses of a treatment-refractory PGCA case using histology, immunohistochemistry (IHC), whole-exome sequencing (WES), clonal evolution modeling, and multicohort validation. IHC assessed key prostate cancer markers (AR, AMACR, KLK3, PTEN, NKX3-1, VIM), while WES compared somatic alterations in PGCA, adjacent adenocarcinoma, and stromal tissue. Public datasets (prostate_dkfz_2018, prad_tcga, prad_mcspc_mskcc_2020) were used for external validation. Results:PGCA displayed profound pleomorphism, necrosis, and complete loss of luminal markers (AR/AMACR/KLK3), along with strong vimentin (VIM) expression, consistent with epithelial-mesenchymal transition. WES revealed PGCA-specific mutations enriched in cell-cycle and inflammatory response pathways, distinct from metabolic alterations in the adjacent adenocarcinoma. Clonal evolution analysis showed divergent progression from a shared ancestral clone. Importantly, mutations in ADAMTS7, CDH1, DRD5, MGAT5, and TP53 emerged as a robust five-gene signature predictive of biochemical recurrence, metastasis, and poor survival, validated across multiple independent cohorts. Conclusion:Our study provides the first molecular roadmap of prostatic PGCA to date, establishing a novel five-gene prognostic signature and revealing fundamental insights into its pathogenesis through divergent evolution from conventional adenocarcinoma. These insights offer new opportunities for precise diagnosis, prognostic stratification, and targeted therapeutic strategies for this lethal prostate cancer variant.

The somatic cell count (SCC) is widely used to assess milk quality and diagnose intramammary infections. Several whey proteins have been shown to correlate significantly with SCC and are considered potential indicators of udder health. However, the relationship between milk whey proteins and SCC has not been fully elucidated. In this study, milk samples were grouped into five categories based on SCC levels. Subsequently, whey proteins were identified using a label-free proteomics approach, and the differential abundance of proteins was validated through a selected reaction monitoring (SRM) method. The levels of various proteins, including azurocidin 1 and kininogen-2, exhibited an increase, whereas topoisomerase I, tropomyosin-1, and desmin showed a significant decrease depending on the SCCs. Principal component analysis unveiled that these proteins contributed to the developmental alterations in milk proteins. A majority of these differentially abundant proteins were associated with response to stimulus, localization, and defense response. Our results provide fundamental information on the SCC that can be utilized for evaluating milk quality and serve as potential indicators for detecting intramammary infections.

Drug resistance and tumor recurrence remain clinical challenges in the treatment of urothelial carcinoma (UC). However, the underlying mechanism is not fully understood. Here, we performed single-cell RNA sequencing and identified a subset of urothelial cells with epithelial-mesenchymal transition (EMT) features (EMT-UC), which is significantly correlated with chemotherapy resistance and cancer recurrence. To validate the clinical significance of EMT-UC, we constructed EMT-UC like cells by introducing overexpression of two markers, Zinc Finger E-Box Binding Homeobox 1 (ZEB1) and Desmin (DES), and examined their histological distribution characteristics and malignant phenotypes. EMT-UC like cells were mainly enriched in UC tissues from patients with adverse prognosis and exhibited significantly elevated EMT, migration and gemcitabine tolerance in vitro. However, EMT-UC was not specifically identified from tumorous tissues, certain proportion of them were also identified in adjacent normal tissues. Tumorous EMT-UC highly expressed genes involved in malignant behaviors and exhibited adverse prognosis. Additionally, tumorous EMT-UC was associated with remodeled tumor microenvironment (TME), which exhibited high angiogenic and immunosuppressive potentials compared with the normal counterparts. Furthermore, a specific interaction of COL4A1 and ITGB1 was identified to be highly enriched in tumorous EMT-UC, and in the endothelial component. Targeting the interaction of COL4A1 and ITGB1 with specific antibodies significantly suppressed tumorous angiogenesis and alleviated gemcitabine resistance of UC. Overall, our findings demonstrated that the driven force of chemotherapy resistance and recurrence of UC was EMT-UC mediated COL4A1-ITGB1 interaction, providing a potential target for future UC treatment.

Precise diagnosis of early prostate cancer (PCa) is critical for preventing tumor progression. However, the diagnostic outcomes of currently used markers are far from satisfactory due to the low sensitivity or specificity. Here, we identified a diagnostic subpopulation in PCa tissue with the integrating analysis of single-cell and bulk RNA-seq. The representative markers of this subpopulation were extracted to perform intersection analysis with early-PCa-related gene module generated from weighted correlation network analysis (WGCNA). A total of 24 overlapping genes were obtained, the diagnostic roles of which were validated by distinguishing normal and tumorous prostate samples from the public dataset. A least absolute shrinkage and selection operator (LASSO) model was constructed based on these genes and the obtained 24-gene panel showed high sensitivity and specificity for PCa diagnosis, with better identifying capability of PCa than the commercially used gene panel of Oncotype DX. The top two risk factors, TRPM4 and PODXL2, were verified to be highly expressed in early PCa tissues by multiplex immunostaining, and PODXL2 was more sensitive and specific compared to TRPM4 and the pathologically used marker AMACR for early PCa diagnosis, suggesting a novel and promising pathology marker.

In the present study, a novel derivative, IOP-LA, was prepared by hybridizing antioxidant lipoic acid (LA) and our recently reported antioxidative marine phidianidine B-inspired indole/1,2,4-oxadiazole derivative. Our results demonstrated that IOP-LA could protect vascular endothelial cells (VECs) from oxidized low-density lipoprotein (oxLDL)-induced oxidative stress by activating the Nrf2 pathway, inhibit the production of atherosclerotic plaque, and promote the stability of atherosclerotic plaque in apoE-/- mice. Moreover, the protective effect of IOP-LA was superior to LA at the same concentration. Mechanistic studies revealed that IOP-LA significantly inhibited the increase of reactive oxygen species (ROS) levels and the translocation of nuclear factor kappa-B (NF-κB) nuclear induced by oxLDL through the nuclear factor erythroid2-related factor 2 (Nrf2) pathway. In summary, the data demonstrate that IOP-LA, as a new antioxidant, protects VECs from oxLDL-induced oxidative stress by activating the Nrf2 pathway. It is worth noting that this study provides a promising lead compound for the prevention and treatment of atherosclerosis.

Elderly rheumatoid arthritis (ERA) population faces multiple treatment dilemma. Here we aim to investigate if Gancao Nourishing-Yin decoction (GCNY) added to methotrexate (MTX) exhibit better effects in an ERA mice model. ERA mice model was established by adding D-galactose (Dgal) to collagen-induced arthritis (CIA) mice. The model was then assigned into control group (CIA + Dgal), MTX treatment group (MTX), GCNY treatment group (GCNY), and integrative treatment group (MTX + GCNY). Pathological scoring was performed to evaluate the severity between the groups. Proteomic analysis was applied to investigate the secretory phenotype of the ERA mouse model and the underlying mechanism of GCNY, MTX and their combination. Representative cytokines related to proteomic results were further validated by ELISAs. CIA + Dgal mice showed more aggressive joints damage than the CIA mice. Besides changes in the inflammatory pathway such as Pi3k-Akt signaling pathway in both model, differential expressed proteins (DEPs) indicated metabolism-related pathways were more obvious in CIA + Dgal mice. Low-dose MTX failed to show pathological improvement in CIA + Dgal mice, while GCNY improved joints damage significantly. Besides down-regulated inflammation-related targets, GCNY-regulated DEPs (such as Apoc1 3, Grk2 and Creb3l3) were broadly enriched in metabolism-related pathways. MTX + GCNY showed the best therapeutic effect, and the DEPs enriched in a variety of inflammatory,metabolism and osteoclast differentiation signaling pathway. Notably, MTX + GCNY treatment up-regulated Dhfr, Cbr1, Shmt1 involved in folic acid biosynthesis and anti-folate resistance pathways indicated a coincidence synergic action. ELISAs confirmed CPR and Akt that elevated in CIA + Dgal mice were significantly ameliorated by treatments, and adding on GCNY elevated folic acid levels and its regulator Dhfr. Aging aggravated joints damage in CIA, which probably due to metabolic changes rather than more severe inflammation. GCNY showed significant effects in the ERA mice model especially when integrated with MTX to obtain a synergic action.

PurposeEsophageal squamous cell carcinoma (ESCC) remains one of the most common causes of cancer death due to the lack of effective therapeutic options. New targets and the targeted drugs are required to be identified and developed.MethodsHighly expressed genes in ESCA were identified using the edgeR package from public datasets. Immunostaining assay verified the high expression level of EFNA1 in ESCC. CCK-8, colony formation and wound healing assays were performed to examine the role of EFNA1 and EPHA2 in ESCC progression. Cell cycle was analyzed by flow cytometry and autophagy activation was determined by autophagolysosome formation using transmission electron microscopy. The small molecule targeting to EFNA1 was identified by molecular docking and the anti-tumor effects were verified by in vitro and in vivo models with radiation treatment.ResultsEFNA1 was highly expressed in esophageal cancer and significantly associated with poor prognosis. Downregulation of EFNA1 remarkably inhibited cell proliferation and migration. Furthermore, decreased EFNA1 significantly suppressed the expression of cMYC along with its representative downstream genes involved in cell cycle, and activated autophagy. Similar effects on ESCC progression were obtained from knockdown of the corresponding receptor, EPHA2. The potential small molecule targeting to EFNA1, salvianolic acid A (SAA), could significantly suppress ESCC progression and increase the sensitivity to radiotherapy.ConclusionWe revealed that EFNA1 facilitated the ESCC progression via the possible mechanism of activating cMYC-modulated cell proliferation and suppressing autophagy, and identified SAA as a potential drug targeting EFNA1, providing new options for the future treatments for ESCC patients.

Background An abundance of CD8+ tumor infiltrating lymphocytes (TILs) in the center of solid tumors is a reliable predictive biomarker for patients eligible for immunotherapy. Purpose To develop a computed tomography (CT)-based radiomics signature for a preoperative prediction of an abundance of CD8+ TILs in non-small-cell lung cancer (NSCLC). Material and Methods In this retrospective study, 117 consecutive patients with pathologically confirmed NSCLC were included and randomly divided into training (n = 77) and test sets (n = 40). A total of 107 radiomics features were extracted from the three-dimensional volumes of interest of each patient. Least absolute shrinkage and selection operator (LASSO) regression was used to select the strongest features for abundance of CD8+ TILs in NSCLC, and the radiomics score was constructed through a linear combination of these selected features. Receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive performance of the radiomics score. Results The radiomics score was associated with an abundance of CD8+ TILs in NSCLC, which achieved an area under the curve (AUC) of 0.83 (95% CI=0.73–0.92) and 0.68 (95% CI=0.54–0.87) in the training and test sets, respectively. The difference was not statistically significant (P = 0.20). The tumors with high CD8+ TILs tended to have heterogeneous dependences (high value of Dependence Non-Uniformity Normalized) and complicated texture (high value of Informational Measure of Correlation 1). Conclusion CT-based radiomics features have the ability to predict CD8+ TILs expression levels of an abundance of CD8+ TILs in NSCLC, which was shown to be a potential imaging biomarker for stratifying patients who may benefit from immunotherapy.

Clinical and Translational MedicineVolume 13, Issue 2 e1109 LETTER TO THE EDITOROpen Access A novel autophagy activator ginsenoside Rh2 enhances the efficacy of immunogenic chemotherapy Jing Yang, Jing Yang Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, ChinaSearch for more papers by this authorWei Zhang, Wei Zhang Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, ChinaSearch for more papers by this authorLin Jia, Lin Jia College of Pharmacy, Shenzhen Technology University, Shenzhen, ChinaSearch for more papers by this authorFei Shi, Fei Shi Emergency Department, Institute of Shenzhen Respiratory Diseases, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, ChinaSearch for more papers by this authorMin Cao, Min Cao Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, ChinaSearch for more papers by this authorJichao Sun, Jichao Sun Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, ChinaSearch for more papers by this authorChengchao Xu, Chengchao Xu Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, ChinaSearch for more papers by this authorZhijie Li, Zhijie Li Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, ChinaSearch for more papers by this authorZhiqiang Cheng, Corresponding Author Zhiqiang Cheng [email protected] Department of Pathology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, China Correspondence Chuanbin Yang, Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China. Email: [email protected] Jigang Wang, Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China; Artemisinin Research Center, Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences, Beijing, 100700 China. Email: [email protected] Shan-Chao Zhao, Department of Urology, the Third Affiliated Hospital of Southern Medical University, Guangzhou, 510515 China; Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, 510500 China. Email: [email protected] Zhiqiang Cheng, Department of Pathology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology) Email: [email protected]Search for more papers by this authorShan-Chao Zhao, Corresponding Author Shan-Chao Zhao [email protected] Department of Urology, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, China Correspondence Chuanbin Yang, Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China. Email: [email protected] Jigang Wang, Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China; Artemisinin Research Center, Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences, Beijing, 100700 China. Email: [email protected] Shan-Chao Zhao, Department of Urology, the Third Affiliated Hospital of Southern Medical University, Guangzhou, 510515 China; Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, 510500 China. Email: [email protected] Zhiqiang Cheng, Department of Pathology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology) Email: [email protected]Search for more papers by this authorJigang Wang, Corresponding Author Jigang Wang [email protected] orcid.org/0000-0002-0575-0105 Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, China Artemisinin Research Center, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China Correspondence Chuanbin Yang, Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China. Email: [email protected] Jigang Wang, Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China; Artemisinin Research Center, Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences, Beijing, 100700 China. Email: [email protected] Shan-Chao Zhao, Department of Urology, the Third Affiliated Hospital of Southern Medical University, Guangzhou, 510515 China; Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, 510500 China. Email: [email protected] Zhiqiang Cheng, Department of Pathology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology) Email: [email protected]Search for more papers by this authorChuanbin Yang, Corresponding Author Chuanbin Yang [email protected] orcid.org/0000-0001-8288-4038 Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, China Correspondence Chuanbin Yang, Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China. Email: [email protected] Jigang Wang, Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China; Artemisinin Research Center, Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences, Beijing, 100700 China. Email: [email protected] Shan-Chao Zhao, Department of Urology, the Third Affiliated Hospital of Southern Medical University, Guangzhou, 510515 China; Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, 510500 China. Email: [email protected] Zhiqiang Cheng, Department of Pathology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology) Email: [email protected]Search for more papers by this author Jing Yang, Jing Yang Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, ChinaSearch for more papers by this authorWei Zhang, Wei Zhang Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, ChinaSearch for more papers by this authorLin Jia, Lin Jia College of Pharmacy, Shenzhen Technology University, Shenzhen, ChinaSearch for more papers by this authorFei Shi, Fei Shi Emergency Department, Institute of Shenzhen Respiratory Diseases, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, ChinaSearch for more papers by this authorMin Cao, Min Cao Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, ChinaSearch for more papers by this authorJichao Sun, Jichao Sun Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, ChinaSearch for more papers by this authorChengchao Xu, Chengchao Xu Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, ChinaSearch for more papers by this authorZhijie Li, Zhijie Li Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, ChinaSearch for more papers by this authorZhiqiang Cheng, Corresponding Author Zhiqiang Cheng [email protected] Department of Pathology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, China Correspondence Chuanbin Yang, Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China. Email: [email protected] Jigang Wang, Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China; Artemisinin Research Center, Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences, Beijing, 100700 China. Email: [email protected] Shan-Chao Zhao, Department of Urology, the Third Affiliated Hospital of Southern Medical University, Guangzhou, 510515 China; Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, 510500 China. Email: [email protected] Zhiqiang Cheng, Department of Pathology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology) Email: [email protected]Search for more papers by this authorShan-Chao Zhao, Corresponding Author Shan-Chao Zhao [email protected] Department of Urology, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, China Correspondence Chuanbin Yang, Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China. Email: [email protected] Jigang Wang, Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China; Artemisinin Research Center, Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences, Beijing, 100700 China. Email: [email protected] Shan-Chao Zhao, Department of Urology, the Third Affiliated Hospital of Southern Medical University, Guangzhou, 510515 China; Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, 510500 China. Email: [email protected] Zhiqiang Cheng, Department of Pathology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology) Email: [email protected]Search for more papers by this authorJigang Wang, Corresponding Author Jigang Wang [email protected] orcid.org/0000-0002-0575-0105 Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, China Artemisinin Research Center, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China Correspondence Chuanbin Yang, Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China. Email: [email protected] Jigang Wang, Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China; Artemisinin Research Center, Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences, Beijing, 100700 China. Email: [email protected] Shan-Chao Zhao, Department of Urology, the Third Affiliated Hospital of Southern Medical University, Guangzhou, 510515 China; Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, 510500 China. Email: [email protected] Zhiqiang Cheng, Department of Pathology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology) Email: [email protected]Search for more papers by this authorChuanbin Yang, Corresponding Author Chuanbin Yang [email protected] orcid.org/0000-0001-8288-4038 Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, China Correspondence Chuanbin Yang, Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China. Email: [email protected] Jigang Wang, Department of Nephrology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China; Artemisinin Research Center, Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences, Beijing, 100700 China. Email: [email protected] Shan-Chao Zhao, Department of Urology, the Third Affiliated Hospital of Southern Medical University, Guangzhou, 510515 China; Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, 510500 China. Email: [email protected] Zhiqiang Cheng, Department of Pathology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology) Email: [email protected]Search for more papers by this author First published: 02 February 2023 https://doi.org/10.1002/ctm2.1109Citations: 1 Jing Yang, Wei Zhang, Lin Jia and Fei Shi contributed equally to this work. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Dear Editor, Immunogenic cell death (ICD) caused by certain chemotherapeutic drugs, including mitoxantrone (MTX), elicits specific protective anti-tumour immunity and is, thus, regarded as an effective strategy for cancer treatment. Pharmacological enhancement of autophagy is effective in enhancing anticancer immune responses to ICD-inducing chemotherapeutic drugs. Here, we discover that ginsenoside Rh2 (G-Rh2) enhance MTX-induced hallmarks of ICD, which include increased ATP release, relocation of calreticulin (CALR) to the cell membrane and HMGB1 (high mobility group box 1) secretion. Mechanistic studies reveal that G-Rh2induces autophagy through the activation of TFEB (transcription factor EB) and TFE3 (transcription factor E3), which contributes to the synergistic effect of G-Rh2 and MTX on promoting ATP release. In addition, G-Rh2 increased endoplasmic reticulum (ER) stress with phosphorylated eukaryotic initiation factor eIF2α, which promoted MTX-induced cell surface calcineurin exposure. Consequently, G-Rh2 enhanced the in vivo anti-tumour effect of MTX in immunocompetent mice bearing MCA205 tumour with increased cytotoxic T lymphocytes (CTLs). Thus, G-Rh2 represents a promising drug candidate for treating cancers in combination with ICD-inducing chemoimmunotherapeutic drugs such as MTX. In response to certain cellular stimuli, injured or stressed cells release DAMPs on their surface to produce immunostimulatory effects, including recruiting and activating immune cells that ultimately kill cancer cells.1 This kind of regulated cell death is referred to as ICD.1, 2 ICD can be triggered by multiple chemotherapeutics such as oxaliplatin and MTX. Key hallmarks of ICD include the secretion of ATP, cell surface relocation of CALR and extracellular release of HMGB1.2 Extracellular ATP acts as a ‘find me’ molecule that recruits antigen-presenting cells to promote anticancer immunity. Cell membrane CALR acts as an ‘eat me’ molecule for dendritic cells (DCs) to capture antigens and trigger tumour-specific cytotoxic T-cell responses. Extracellular HMGB1 binds to its receptor such as TLR4 on DCs, which promotes tumour antigen processing and presentation to T cells. Thus, the induction of ICD triggers long-lasting anti-tumour immunity, and it is regarded as an effective strategy for cancer treatment.2, 3 TFEB and TFE3 are key transcription factors that regulate autophagy.4, 5 With respect to ICD, the activation of several stress pathways, including autophagy, is indispensable for intracellular ATP release.6 Induction of autophagy by several ICD inducers enhances the anticancer effects via modulating the tumour microenvironment.7 Therefore, autophagy activation to enhance the effects of chemotherapeutics on inducing ICD holds promise for anticancer therapy.8 Driven by these considerations, we sought to identify novel autophagy enhancer(s) and evaluate their roles in stimulating anticancer immunity in combination with ICD-inducing chemotherapeutics in U2OS cells (human bone osteosarcoma epithelial cells), MCA205 cells (mouse fibrosarcoma cells) and MCA205-inoculated immunocompetent mice. Here, we found that G-Rh2 upregulated the autophagy marker LC3-II levels (Figure 1A), and lysosomal inhibitor CQ further enhanced LC3-II levels (Figure 1B,C). Immunostaining results further showed that G-Rh2 increased autophagosomes and autolysosomes (Figure 1D–G). These results indicate that G-Rh2 promotes autophagy. Furthermore, G-Rh2 enhanced the nuclear accumulation of TFEB and TFE3 as reflected by immunofluorescence (Figures 1H,I and S1A,C) and western blotting (Figure S1B,D). Knock-down of the expression of both TFEB and TFE3 (Figure S1E–H, Table S1) inhibited G-Rh2-induced autophagic flux (Figure S1I). Furthermore, G-Rh2 promoted TFEB dephosphorylation (Figure S1J), and the nuclear accumulation of TFEB/TFE3 is earlier than autophagy induction (Figure S1K,L). These results suggest that G-Rh2 enhances autophagy via TFE3 and TFEB. FIGURE 1Open in figure viewerPowerPoint Ginsenoside Rh2 (G-Rh2) induces autophagy via transcription factor EB (TFEB) and transcription factor E3 (TFE3) activation: (A) G-Rh2 increases LC3-II levels. U2OS cells were exposed to different doses of G-Rh2 (1, 5 and 10 μM) for 16 h, and LC3-II was measured; (B and C) G-Rh2 induces autophagic flux. U2OS cells were exposed to G-Rh2 (10 μM) with or without CQ (50 μM, added at last for 3 h) for 16 h, LC3-II was measured (B) and quantified by ImageJ (C); (D and E) G-Rh2 increases LC3 puncta. After treating U2OS cells transiently expressing GFP-LC3 with G-Rh2 for 16 h, LC3 puncta was visualized (D) and quantified (E); (F and G) G-Rh2 increases autolysosomes. After treating U2OS cells transiently expressing GFP-RFP-LC3 with G-Rh2 (10 μM) for 16 h, LC3 puncta was recorded (F) and red-only puncta (autolysosome) was quantified (G). Scale bar: 15 μm; (H and I) G-Rh2 induces the relocation of TFEB and TFE3 from the cytoplasm into the nucleus. U2OS cells transiently expressing 3XFlag-TFEB or GFP-N1-TFE3 were incubated with indicated doses of G-Rh2 (1, 5 and 10 μM) for 16 h. The distribution of TFEB in cells was detected by fluorescence microscope. Scale bar: 15 μm. *p < .05; **p < .01 We further determined whether G-Rh2 induces hallmarks of ICD with or without a low concentration of MTX (MTXlow). G-Rh2 or MTX slightly but significantly reduced intracellular ATP release, and G-Rh2 combined with MTXlow substantially reduced the intracellular ATP contents (Figure 2A,B). Autophagy deficiency by knocking down ATG5 (Figure 2C–E) attenuated G-Rh2 plus MTXlow-induced decrease in intracellular ATP contents as reflected by quinacrine staining (Figure 2F)9 and the release of extracellular ATP contents (Figure 2G). Similarly, the combination of G-Rh2 and MTX-induced decrease of intracellular ATP and increase of extracellular ATP was inhibited in TFE3- and TFEB-knocked-down cells (Figure 2H,I). These findings demonstrate that the synergistic effect of G-Rh2 and MTX on ATP release depends on autophagy induction. FIGURE 2Open in figure viewerPowerPoint Ginsenoside Rh2 (G-Rh2) enhances autophagy-dependent ATP release: (A) G-Rh2 reduces intracellular ATP contents. U2OS cells were exposed to vehicle control, G-Rh2 (10 μM), a low dose of MTXlow (1 μM) or their combination for 16 h. The intracellular ATP contents were examined by quinacrine staining. MTXhigh (5 μM) was used as a positive control. Scale bar: 15 μm; (B) quantification data in (A) shows that G-Rh2 promotes mitoxantrone (MTX)-induced reduction of intracellular ATP contents; (C–E) after transfected U2OS cells with siRNA to knock down the expression of key autophagy gene ATG5, ATG5 and LC3-II levels were measured (C) and quantified (D and E); (F and G) the inhibition of autophagy through knocking down of the expression of ATG5 attenuates G-Rh2 plus MTX-induced ATP release. After ATG5 knocking down, U2OS cells were treated with vehicle control, G-Rh2 (10 μM), MTXlow (1 μM) or the combination of G-Rh2 (10 μM) and MTXlow (1 μM) for 16 h, the intracellular ATP levels were measured by quinacrine staining (F) and the extracellular ATP contents were measured by a bioluminescent assay kit (G); (H and I) inhibition of autophagy by transcription factor EB (TFEB)/transcription factor E3 (TFE3) knockdown attenuates G-Rh2 plus MTX-induced ATP release. After TFE3 and TFEB knockdown, U2OS cells were incubated with G-Rh2, MTXlow, or the combination of G-Rh2 (10 μM) and MTXlow (1 μM) for 16 h, and the intracellular ATP contents were measured by quinacrine staining (H), and the extracellular ATP contents were measured by a bioluminescent assay kit (I). Scale bar: 15 μm. *p < .05, **p < .01 Furthermore, G-Rh2 increased MTXlow-induced cell surface exposure of CALR as reflected by immunostaining and flow cytometry analysis (Figures 3A–C and S2A,B). The combination of G-Rh2 and MTXlow also increased an HMGB1 release (Figure 3D–G). To determine how G-Rh2 and MTX induce cell surface CALR exposure, we next showed that G-Rh2 increased ER stress, especially PERK/p-eIF2α/ATF4 axis (Figure S3A–H). We discovered that PERK knock-down reduced G-Rh2-induced ER stress (Figure S3I–K) and comprised G-Rh2 plus MTX-caused cell surface CALR exposure (Figure 3H,I). Interestingly, the inhibition of ER stress by 4-PBA (4-phenylbutyric acid) also attenuated cell surface relocation of CALR (Figure S3L). These results indicate that ER stress is indispensable for the role of G-Rh2 in enhancing MTX-induced cell surface relocation of CALR. Apart from ICD, the combination of G-Rh2 and MTX also induced cell apoptosis, and this effect was further enhanced by the lysosomal inhibitor CQ (Figure S4A,B), suggesting that the apoptosis may also be involved in anticancer effects. To understand the crosstalk of autophagy and ER stress during ICD, we found that the inhibition of lysosomal functions by CQ did not further enhance ER stress (Figure S4C), and ER stress inhibitor 4-PBA attenuated autophagy in response to G-Rh2 (Figure S4D). Consistently, CQ did not enhance G-Rh2 plus MTX-induced cell surface CALR exposure (Figure S4E) but attenuated G-Rh2 plus MTX-induced ATP release (Figure S4F), supporting a critical role of autophagy in promoting ATP release. Furthermore, though apoptosis inhibitor Z-VAD-FMK inhibits G-Rh2 plus MTX-induced apoptosis (Figure S5B), Z-VAD-FMK did not inhibit G-Rh2 plus reduction of intracellular ATP levels (Figure S5A), and cell surface CALR exposure (Figure S5C,D), further strengthen the hypothesis that ICD rather than apoptosis is involved in the anti-tumour effect of G-Rh2 plus MTX. FIGURE 3Open in figure viewerPowerPoint Ginsenoside Rh2 (G-Rh2) enhances the cell surface exposure of calreticulin (CALR) and the release of HMGB1 (high mobility group box 1) in the presence of mitoxantrone (MTX): (A) G-Rh2 enhances MTX-induced cell surface relocation of CALR. U2OS cells transiently expressing CALR-KDEL-RFP were treated with a low concentration of MTXlow (1 μM) with or without G-Rh2 (10 μM) for 16 h. The cell surface CALR was visualized by a confocal microscope. MTXhigh (5 μM) was used as a positive control. Scale bar: 15 μm; (B) quantification of cell surface CALR exposure in (A); (C) G-Rh2 enhances MTX-induced cell surface CALR exposure measured by flow cytometry. After drug treatment as shown in (A), U2OS cells were collected for the detection of endogenous CALR exposure via flow cytometry; (D–G) G-Rh2 enhances MTX-induced HMGB1 release. Intracellular HMGB1 was detected by immunostaining after drug treatment for 24 h (D) and quantified (E). Extracellular HMGB1 contents in cell culture medium were detected by western blotting after drug treatment (F) and quantified (G). Briefly, an equal amount of cell culture medium was precipitated using trichloroacetic acid followed by western blotting analysis. Scale bar: 15 μm; (H and I) inhibition of endoplasmic reticulum (ER) stress by knocking down of PERK, a key molecule in ER stress, attenuates G-Rh2-induced cell surface CALR exposure as determined by flow cytometry. *p < .05; **p < .01 To confirm the conserved synergistic effects of G-Rh2 and MTX in enhancing ICD, we showed that in immunosurveillance MCA205 mouse fibrosarcoma cells, G-Rh2 also enhanced autophagy (Figure S6A), induced ER stress (Figure S6B,C). Consistently, G-Rh2 enhanced MTXlow-induced cell surface CALR exposure, HMGB1 release from the nucleus, and extracellular ATP release (Figure S6D–I), suggesting that G-Rh2 also promotes MTX-induced ICD in MCA205 fibrosarcoma cells. MCA205 cells inoculated in mice are well characterized as a suitable model for the investigation of immune response, and the tumour infiltration on the skin can also be considered to be orthotopic.10 We next determined the synergistic anti-tumour role of G-Rh2 in combination with MTX by inoculating MCA205 cells into immunocompetent C57 mice followed by drug treatment as shown in the schematic model (Figure 4A). We showed that G-Rh2, MTX and a combination of G-Rh2 and MTX did not affect mice's body weight (Figure 4B), but the combination treatment significantly mitigated tumour growth (Figure 4C,D). Importantly, the combination treatment increased the abundance of CTLs while exerting minimal effect on that of regulatory T cells (Tregs) (Figures 4E,F and S7). Consequently, this combination treatment increased the CTL/Treg ratio (Figure 4G), suggesting that G-Rh2 and MTX synergistically promote anti-tumour immunity by tipping the immune balance and reprogramming the tumour microenvironment. FIGURE 4Open in figure viewerPowerPoint Ginsenoside Rh2 (G-Rh2) promotes the efficacy of anticancer chemotherapy in mice: (A) schematic diagram of drug treatment in MCA205 mouse fibrosarcomas-bearing mice. When tumours became palpable, mice received systemic intraperitoneal injection of G-Rh2, mitoxantrone (MTX) alone or their combination. At least seven mice per group; (B) treatment of mice with G-Rh2, MTX alone or their combination does not affect mice's body weight; (C and D) G-Rh2 plus MTX treatment significantly inhibits tumour growth (C) and reduces tumour size (D) of MCA205 fibrosarcomas in mice; (E) G-Rh2 plus MTX increases the ratio of CD3+CD8+ cytotoxic T lymphocytes (CTLs); (F) treatment of mice with G-Rh2, MTX alone or in combination does not affect the ratio of CD4+ FOXP3+ Treg cells; (G) treatment of mice with the combination of G-Rh2 and MTX increases the ratio of CD3+ CD8+ T lymphocytes over Treg cells; *p < .05; **p < .01. (H) a schematic model illustrating the effects of G-Rh2 in enhancing MTX-induced immunogenic cell death (ICD) and promoting its anti-tumour effects via reprogramming the tumour microenvironment. G-Rh2 enhances MTX-induced hallmarks of ICD, such as ATP release, cell surface calreticulin (CALR) exposure and HMGB1 (high mobility group box 1) release. Mechanistically, G-Rh2 promotes MTX-induced ATP release via transcription factor EB (TFEB)/transcription factor E3 (TFE3)-mediated autophagy, and G-Rh2 facilitates MTX-induced cell surface CALR exposure via activating endoplasmic reticulum (ER) stress through PERK/p-eIF2α/ATF4 axis. As such, G-Rh2 synergizes with MTX to increase the abundance of CTLs in tumours, which ultimately promotes the in vivo anti-tumour effects of chemotherapeutic ICD-inducer MTX. Overall, this study illustrates that G-Rh2 is responsible for TFE3/TFEB-mediated autophagy activation and ER-stress induction with phosphorylated eIF2α, and it synergizes with immunogenic chemotherapeutic drug MTX to enhance MTX-induced ICD, which consequently facilitates the anti-tumour effect of MTX in immunocompetent mice in vivo (Figure 4G). Our findings provide mechanistic insights into how G-Rh2 synergizes with MTX to amplify its effects on ICD induction and anti-tumour activity and provide a novel link between G-Rh2-activated TFEB/TFE3-dependent autophagy induction and ICD-involved anti-tumour effect. Our discovery indicates that G-Rh2 is a novel drug candidate for improving the anti-tumour effects of immunogenic chemotherapies. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (81902787, 82003721, 82074098, 82274182, 81841001), National Key Research and Development Program of China (2020YFA0908000), Shenzhen Science and Technology Innovation Commission (JCYJ20210324114014039, JCYJ20210324115800001), China Postdoctoral Science Foundation (2020M683182) and Guangdong Basic and Applied Basic Research Foundation (2020A1515110549). CONFLICTS OF INTEREST There are no conflicts of interest between all authors. Open Research DATA AVAILABILITY STATEMENT Data are available on reasonable request from the authors. Supporting Information Filename Description ctm21109-sup-0001-Figures.docx3.3 MB Figures ctm21109-sup-0002-SuppMat.docx50.6 KB Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. REFERENCES 1Zhou J, Wang G, Chen Y, Wang H, Hua Y, Cai Z. Immunogenic cell death in cancer therapy: present and emerging inducers. J Cell Mol Med. 2019; 23: 4854- 4865. 2Kroemer G, Galassi C, Zitvogel L, Galluzzi L. Immunogenic cell stress and death. Nat Immunol. 2022; 23: 487- 500. 3Xia H, Green DR, Zou W. Autophagy in tumour immunity and therapy. Nat Rev Cancer. 2021; 21: 281- 297. 4Zhang W, Xu C, Sun J, Shen H-M, Wang J, Yang C. Impairment of the autophagy-lysosomal pathway in Alzheimer's diseases: pathogenic mechanisms and therapeutic potential. Acta Pharm Sin B. 2022; 12: 1019- 1040. 5Yang C, Su C, Iyaswamy A, et al. Celastrol enhances transcription factor EB (TFEB)-mediated autophagy and mitigates Tau pathology: implications for Alzheimer's disease therapy. Acta Pharm Sin B. 2022; 12: 1707- 1722. 6Michaud M, Martins I, Sukkurwala AQ, et al. Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice. Science. 2011; 334: 1573- 1577. 7Pietrocola F, Pol J, Vacchelli E, et al. Caloric restriction mimetics enhance anticancer immunosurveillance. Cancer Cell. 2016; 30: 147- 160. 8Amaravadi RK, Kimmelman AC, Debnath J. Targeting autophagy in cancer: recent advances and future directions. Cancer Discov. 2019; 9: 1167- 1181. 9Liu P, Zhao L, Pol J, et al. Crizotinib-induced immunogenic cell death in non-small cell lung cancer. Nat Commun. 2019; 10: 1486. 10Zitvogel L, Pitt JM, Daillère R, Smyth MJ, Kroemer G. Mouse models in oncoimmunology. Nat Rev Cancer. 2016; 16: 759- 773. Volume13, Issue2February 2023e1109 FiguresReferencesRelatedInformation

One effective strategy for treating atherosclerosis is to inhibit the injury of vascular endothelial cells (VECs) induced by oxidized low-density lipoprotein (oxLDL) and high glucose (HG). This study synthesized and evaluated a series of novel Nrf2 activators derived from the marine natural product phidianidine for their ability to protect human umbilical VECs against oxLDL- and HG-induced injury. The results of in vitro bioassays demonstrated that compound D-36 was the most promising Nrf2 activator, effectively inhibiting the apoptosis of HUVECs induced by oxLDL and HG. Furthermore, Nrf2 knockdown experiments confirmed that compound D-36 protected against oxLDL- and HG-induced apoptosis in HUVECs by activating the Nrf2 pathway. These findings provide important insights into a new chemotype of marine-derived Nrf2 activators that could potentially be optimized to develop effective anti-atherosclerosis agents.

目的 探讨老年类风湿关节炎(ERA)小鼠的分泌表型特点及甘草养阴汤联合甲氨蝶呤对ERA小鼠的干预作用和整体效应机制.方法 DBA/1 雄性小鼠共72 只,随机取10 只作为正常组;另取12 只建立胶原诱导关节炎(CIA)模型,作为RA组;剩余50 只小鼠在CIA模型基础上加D-半乳糖刺激建立ERA模型,将造模成功小鼠随机分为ERA组、甲氨蝶呤组、甘草养阴汤组、甲氨蝶呤+甘草养阴汤组.正常组、RA组、ERA组小鼠给予生理盐水灌胃,每日1 次;甲氨蝶呤组给予甲氨蝶呤1 mg/kg灌胃,每周2 次;甘草养阴汤组给予甘草养阴汤3.0 g/kg灌胃,每日1 次;甲氨蝶呤+甘草养阴汤组给予甲氨蝶呤和甘草养阴汤灌胃,药物用量及方法同甲氨蝶呤组和甘草养阴汤组.各组均灌胃4 周.对各组小鼠后肢关节炎病变进行评分,HE染色观察各组踝关节组织水肿、滑膜增生、软骨/骨侵蚀、血管翳形成、脂肪沉积情况并进行评分,采用非依赖采集(DIA)蛋白组学技术分析各组小鼠的分泌表型特点.结果 ERA组后肢肿胀更明显,关节炎评分及肢关节水肿、滑膜增生、软骨/骨侵蚀评分均明显高于RA组(P均<0.05),血管翳形成及脂肪沉积评分与RA组比较差异均无统计学意义(P均>0.05);甘草养阴汤组、甲氨蝶呤+甘草养阴汤组关节炎评分及关节水肿、软骨/骨侵蚀、脂肪沉积评分均明显低于ERA组(P均<0.05),甲氨蝶呤组关节炎评分及关节水肿、滑膜增生、软骨/骨侵蚀、血管翳形成、脂肪沉积评分与ERA组比较差异均无统计学意义(P均>0.05).ERA组与RA组比较,上调71 个蛋白,下调70 个蛋白,主要富集于中性粒细胞胞外陷阱形成、脂质和动脉粥样硬化、鞘脂信号通路等.甲氨蝶呤+甘草养阴汤除了对多种炎症因子的抑制作用,还倾向于对脂质代谢等通路靶点的调节,尤其是上调叶酸合成通路相关靶点.结论 ERA小鼠的代谢改变可能导致更为严重的关节损伤,甘草养阴汤对甲氨蝶呤治疗ERA具有减毒增效作用.

目的 探讨发生于脾脏的表皮样囊肿(SEC)临床病理特征、诊断、鉴别诊断、治疗及预后.方法 收集2017—2020年深圳市人民医院4例SEC患者的临床病理资料及随访资料,对其病理形态特点及免疫组织化学进行观察分析,并复习相关文献.结果 4例患者均为女性,发病年龄19～34岁,平均(22±3)岁.2例为体检时偶然发现,无明显症状,2例表现为左上腹胀痛不适.3例血清肿瘤标志物糖类抗原(CA)19-9水平升高,1例正常.4例均行脾全切术.大体检查肿瘤最大径6～13 cm,平均(8.8±1.5)cm.2例表现为单房囊性,2例表现为多房囊性,囊壁尚光滑,部分囊壁粗糙,灰黄,质稍硬.4例镜下形态相似,囊壁被覆分化成熟的鳞状上皮,可见角化物.其中2例部分囊壁衬覆上皮脱落,可见组织细胞反应,胆固醇结晶形成,钙盐沉积.免疫组织化学:4例CEA、P63均阳性.3例CA19-9阳性,1例为阴性.结论 SEC是脾脏的一种罕见的良性病变,儿童和青壮年多发,女性多见.患者常无症状,当囊肿较大时,可出现临床症状或破裂、出血及感染等并发症,需及时手术治疗.

BACKGROUND:Clinically, accurate pathological diagnosis is often challenged by insufficient tissue amounts and the unaffordability of additional immunohistochemical or genetic tests; thus, there is an urgent need for a universal approach to improve the subtyping of lung cancer without the above limitations. Here we aimed to develop a deep learning system to predict the immunohistochemistry (IHC) phenotype directly from whole-slide images (WSIs) to improve the subtyping of lung cancer from surgical resection and biopsy specimens.METHODS:A total of 1914 patients with lung cancer from three independent hospitals in China were enrolled for WSI-based immunohistochemical feature prediction system (WIFPS) development and validation.RESULTS:The WIFPS could directly predict the IHC status of nine subtype-specific biomarkers, including CK7, TTF-1, Napsin A, CK5/6, P63, P40, CD56, Synaptophysin, and Chromogranin A, achieving average areas under the curve (AUCs) of 0.912, 0.906, and 0.888 and overall diagnostic accuracies of 0.925, 0.941, and 0.887 in the validation datasets of total, external surgical resection specimens and biopsy specimens, respectively. The histological subtyping performance of the WIFPS remained comparable with that of general pathologists (GPs), with Cohen's kappa values ranging from 0.7646 to 0.8282. Furthermore, the WIFPS could be trained to not only predict the IHC status of anaplastic lymphoma kinase (ALK), programmed death-1 (PD-1), and programmed death ligand 1 (PD-L1), but also predict EGFR and KRAS mutation status, with AUCs from 0.525 to 0.917, as detected in separate populations.CONCLUSIONS:In this study, the WIFPS showed its proficiency as a useful complement to traditional histologic subtyping for integrated immunohistochemical spectrum prediction as well as potential in the detection of gene mutations.

Abstract Background: Esophageal squamous intraepithelial neoplasia (ESIN) is considered a precursor lesion of esophageal squamous cell carcinoma (ESCC), High grade ESIN (HGESIN)has much higher risk of progression to ESCC comparing with low grade ESIN(LGESIN). the distinction between squamous cell dysplasia and reactive changes, and HGESIN and LGESIN can sometimes be subjective and challenging. RSPO2 is a member of the R-spondin family of proteins, and was identified as either oncogenes or tumor suppressors depending on the cancer type. Methods: In the current study, we compared the immunohistochemistry(IHC) expression of newly identified marker RSPO2 with p53 and Ki-67 in different esophageal squamous lesions, and to evaluate correlations between their expression levels in these lesions. Results: IHC stains of RSPO2, p53 and Ki-6 were performed on endoscopic submucosal dissection specimens from 71 patients including 96 HGESIN, 50 LGESIN, and 160 fragments of adjacent normal squamous esophageal mucosa. RSPO2 and Ki-67 showed high sensitivity and specificity for diagnosing ESIN from normal squamous epithelium. The sensitivity of high level expression of RSPO2 in distinguishing HGESIN from LGESIN was as high as 97.92%, but specificity was only 72%. The specificity of strong staining of RSPO2 in distinguishing HGESIN from LGESIN was as high as 100%, but the sensitivity was as low as 29.17%. The specificity of high level expression of Ki-67 and p53 in distinguishing HGESIN from LGESIN was 96% and 96% respectively, but sensitivity was reduced to 56.25% and 62.5% respectively. Conclusions: This study demonstrates that both RSPO2 and Ki-67 can be useful in differential diagnosing ESIN.

Introduction To develop and validate a simple-to-use nomogram based on preoperative CT to predict spread through air space (STAS) status of stage IA lung adenocarcinoma (ADC). Methods In this retrospective study, 434 patients with pathological proven periphery stage IA lung adenocarcinoma were included, which consisted of 349 patients from center I for training group and 85 patients from Center II for test group. STAS was identified in 53 patients (40 patient in the training group and 13 patients in the test group). On the basis of preoperative CT images, 19 morphological characteristics were analyzed. Univariable analysis was used to explore the association between clinical and CT characteristics and STAS status in the training group (P < 0.002). Independent risk factors for STAS were identified using multivariable logistic regression analysis and then used to build a nomogram for preoperative predicting STAS status. Results Type of nodules, diameter of solid component, lobulation and percentage of the solid component (PSC) were associated with STAS status of peripheral stage IA lung ADCs statistical significantly. Multivariate logistics regression analysis revealed that PSC and lobulation were independent risk factors for STAS. The nomogram based on these factors achieved good predictive performance for STAS with a C-index of 0.803 in the training group and a well-fitted calibration curve. Using a cut-off value which was obtained from Youden index of the receiver operating characteristic (ROC) curve, a diagnosis accuracy of 70.6% was obtained in the test group with sensitivity, specificity, positive prediction value (PPV) and negative prediction value (NPV) of 92.3%, 66.7%, 33.3% and 98.0%, respectively. Conclusion The nomogram based on preoperative CT images could achieve good predictive performance for STAS status of lung adenocarcinomas. This simple-to-used model can facilitate surgeons for a rational operation pattern choice at bedside.

To investigate the diagnostic value of the Prostate Imaging Reporting and Data System version 2.1 (PI-RADS v2.1) for clinically significant prostate cancer (CsPCa). We also aimed to combine PI-RADS v2.1 with prostate-specific antigen (PSA) derivatives to improve the predictive value of CsPCa. We retrospectively collected relevant data who underwent standard MRI examinations of the prostate and subjected to a prostate biopsy at Shenzhen People’s hospital from November 2014 to November 2019. Included 125 cases of CsPCa and 383 cases of non-CsPCa. All cases were scored using the PI-RADS v2.1. The clinical data collected included age, PSA, free PSA/total PSA, prostate volume and PSA density (PSAD). A univariate analysis was performed to identify statistically significant indicators. Logistic regression was used to analyze the predictive value of the multi-parameter combination on CsPCa. Except age, the difference in all of indicators between the CsPCa group and non-CsPCa group was statistically significant. The PI-RADS score and PSAD value had the highest diagnostic value. Logistic regression analysis revealed that the PI-RADS score and PSAD value were independent predictors of CsPCa, with a regression model AUC of 0.935. CsPCa detection rates were low when the PI-RADS score ≤ 2 or the PI-RADS score = 3 and the PSAD value ≤ 0.33 ng/ml/ml. Combining the PI-RADS score and PSAD value improved the predictive performance of CsPCa. Patients with a PI-RADS score ≤ 2 or a PI-RADS score = 3 and a PSAD value ≤ 0.33 ng/ml/ml can avoid an unnecessary biopsy.

Inhibition of oxidized low-density lipoprotein (oxLDL)-induced vascular endothelial cell (VEC) injury is one of the effective strategies for treating atherosclerosis. In the present study, a series of novel marine phidianidine-inspired indole-1,2,4-oxadiazoles was designed, synthesized, and evaluated for their effects against oxLDL-induced injury in VECs. Among them, compound D-6, displaying the most effective protective activity, was found to inhibit oxLDL-induced apoptosis and the expression of ICAM-1 and VCAM-1 in VECs. Mechanistic studies showed that D-6 could trigger Nrf2 nuclear translocation, subsequently resulting in increased expression of Nrf2 target gene HO-1. Meanwhile, D-6 suppressed the increase of ROS level and nuclear translocation of NF-κB induced by oxLDL. Importantly, Nrf2 knockdown attenuated the inhibition effects of D-6 on oxLDL-induced apoptosis, ROS production and NF-κB nuclear translocation. Collectively, our studies demonstrated that compound D-6 protected against oxLDL-induced endothelial injury by activating Nrf2/HO-1 anti-oxidation pathway.

淋巴结活检的主要作用是对恶性肿瘤淋巴转移进行分级,明确肿瘤转移的具体范围,减少不必要的手术及术后并发症[1].术中冷冻切片能在较短时间内获得淋巴结的病理诊断结果,对手术治疗方式和手术范围具有较高的指导作用,因此越来越受到临床医师的青睐.淋巴结组织由于其细胞丰富,质地较脆,外围常有脂肪包绕,在日常冷冻切片工作中较难获取高质量切片.固定是影响冷冻切片质量的主要因素之一[2],组织固定欠佳直接导致染色质量差,严重影响诊断的准确性.本科室采用不同固定液对淋巴结冷冻切片进行HE染色,取得良好的效果,现报道如下.