Cell Content Research Articles

Pseudostellaria heterophylla improves intestinal microecology through modulating gut microbiota and metabolites in mice.

Pseudostellaria heterophylla is a Chinese medicine and healthy edible that is widely used to for its immunomodulatory, antioxidant, antidiabetic and antitussive properties. However, the potential function of P. heterophylla in intestinal microecology remains unclear. In this study, we investigated the impact of P. heterophylla on immune functions and evaluated its potential to regulate the gut microbiota and metabolome. The results showed that P. heterophylla significantly increased the content of red blood cells, total antioxidant capacity and expression of immune factors, and decreased platelet counts when compared to the control under cyclophosphamide injury. In addition, P. heterophylla altered the diversity and composition of the gut bacterial community; increased the abundance of potentially beneficial Akkermansia, Roseburia, unclassified Clostridiaceae, Mucispirillum, Anaeroplasma and Parabacteroides; and decreased the relative abundance of pathogenic Cupriavidus and Staphylococcus in healthy mice. Metabolomic analyses showed that P. heterophylla significantly increased the content of functional oligosaccharides, common oligosaccharides, vitamins and functional substances. Probiotics and pathogens were regulated by metabolites across 11 pathways in the bacterial-host co-metabolism network. We demonstrated that P. heterophylla increased the abundance of probiotics and decreased pathogens, and further stimulated host microbes to produce beneficial secondary metabolites for host health. Our studies highlight the role of P. heterophylla in gut health and provide new insights for the development of traditional Chinese medicine in the diet. © 2024 Society of Chemical Industry.

Abstract 6644: aPD1 treatment of urothelial cancers is synergistically enhanced by activating anti tumor immunity using vascular targeted photodynamic therapy

Abstract Background: Recurrence and progression rate following surgical treatment of locally advanced urothelial cancer is high, underscoring the unmet need to develop an effective, well tolerated treatment strategies, particularly for high grade upper tract urothelial cancer (HG-UTUC). The treatment success of low grade UTUC by vascular targeted photodynamic therapy (VTP) with WST11 (Padeliporfin) in phase I clinical trials led the FDA to approve Phase 3 trial in a fast track, orphan drug designation for low grade UTUC (ENLIGHT). Recently, treatment with immune checkpoint inhibitor (ICIs) of the PD1/PD-L1 axis has been approved for treatment of HG-UTUC. Yet, the failure rate of aPD1 therapy in HG-UTUC is high.We hypothesized that immune checkpoint inhibition needs to be complemented by the massive priming and accrual of newly activated anti-tumor immune cells through tumor immunogenic cell death. Early evidence indicated that WST11 VTP promotes anti-tumor immunity suggesting that VTP/aPD1 combinations may significantly improve the treatment success of HG-UTUC. Methods: Mice bearing s.c. MB49-Luc, were subjected to WST11 VTP at 14d post implantation at suboptimal dose of drug or partial area of illumination. 4 or 5 doses of aPD1 were intraperitonealy administrated every 3 days starting at 1d post VTP (aPD1+1), or every 3 days till 10 days post-treatment starting at 2 days before VTP (aPD1-2). Tumor progression was followed up to 90 days post treatment using IVIS. Hematoxylin-eosin staining, and multiplex immunohistochemistry (mIHC) were performed on controls and treated animals. Results: WST11 VTP with suboptimal drug dose, aPD1+1, and aPD1-2 resulted in 17%, 23% and 43% cure at day 90 post VTP. VTP at suboptimal drug dose combined with aPD1+1 or aPD1-2, resulted in 50% and 77% cure rate at 90 days, respectively. VTP with optimal drug dose (5mg/kg) but partial illumination resulted in 20% cure rate while combining this treatment modality with aPD1-2 resulted in 90% animal cure rate at day 90.At 24h post VTP mIHC showed a 2-3 fold increase of cytotoxic T cells, dendritic and NK cells, and a ~4 folds overexpression of PD1 and PD-L1 in the treated tumor tissue. At 2 days post aPD1-2 initiation, PD1 and PD-L1 apparent expression dropped to 0 and 2 folds higher than control, respectively. Following the combined VTP and aPD1-2 treatment, both PD1 and PDL-1 contents dropped down to zero and the CD8 content reached 3 folds enhancement compared to control. Conclusions: These observations indicate that elevating the content of anti-tumor immune cells in the tumor microenvironment by VTP in combination with ICI synergistically enhances the therapeutic effect providing a much higher complete cure compared with the individual therapies. The cure rate extent strongly depends on the synchronization of the two modalities providing guidelines for the clinical management of HG-UTUC treatment. Citation Format: Lilach Agemy, Keren Sasson, Tamar Yechezkel, Rachel Hamri, Raz Robas, David Kain, Avigdor Scherz. aPD1 treatment of urothelial cancers is synergistically enhanced by activating anti tumor immunity using vascular targeted photodynamic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6644.

Abstract 7636: Quantitative analysis of colorectal adenocarcinoma images obtained by single-shot 17-plex staining followed by imaging with the Orion™ spatial biology platform

Abstract Background To understand the tumor microenvironment (TME), high resolution imaging of multiple biomarkers with whole slide context can be used as a basis for downstream biomarker quantitation and predicting patient outcomes1. Here we investigate a sample of invasive colorectal adenocarcinoma using whole slide, single-step high-plex staining and imaging at single-cell resolution followed by quantitative analysis. Methods The slide was stained in one round with a 17-plex immune-oncology panel then imaged in one round with the Orion™ spatial biology platform. Quantitative analysis was performed on the resulting TIFF file using the following pipeline:- Manually annotate regions of interest (ROI) on the tissue- Segment whole slide images into cells with UnMICST and S3segmenter - Import mask and clean data in QuPath - Generate primary feature data table with QuPath - Classify cells into populations in QuPath - Generate spatial biomarkers - Compare cell populations and their spatial interactions in the different ROIs. Results Data revealed a distinction between normal colonic epithelium, well-differentiated adenocarcinoma with immune cell collection, and an infiltrating border of the carcinoma. Differences in immune cell content and spatial organization were measured, and the infiltrating border found to contrast with other tumor regions by showing a lower proliferative fraction (Ki-67, nuclear) and differences in E-cadherin and cytokeratin expression patterns. Conclusions These data highlight the importance of sufficient plex, resolution and whole slide context to derive reliable spatial biomarkers of potential prognostic value. Further, hours vs. days speed for whole-section multiplexed biomarker quantitation, along with same-section conventional chromogenic analysis, makes this approach suited to multi-patient clinical studies. 1 JR Lin et al., High-plex immunofluorescence imaging and traditional histology of the same tissue section for discovering image-based biomarkers, Nat Cancer, 4, 1036-1052 (2023). Citation Format: Edward Lo, Tad George, Selena Larkin. Quantitative analysis of colorectal adenocarcinoma images obtained by single-shot 17-plex staining followed by imaging with the Orion™ spatial biology platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7636.

Abstract 2275: Reducing artifactual somatic variant calls from formalin-fixed paraffin-embedded specimens by using DEEPOMICS FFPE, a bioinformatic approach based on deep neural networks

Abstract Formalin-fixed, paraffin-embedded (FFPE) tissue specimens are routinely used in pathological diagnosis. However, formalin induced artificial mutations make it challenging to accurately analyze next-generation sequencing data. Existing FFPE filtering tools tend to categorize all low-frequency variants as artifacts, making it difficult to identify true variants with low allele frequencies, compromising analysis of tumors with low tumor cell content. To address this issue, we developed DEEPOMICS FFPE, an AI model designed to distinguish true variants from artifacts. We gathered paired exome genome sequencing data from both fresh frozen and FFPE samples of 24 tumors from public sources. These data were used to train and validate the model, with a split of 70% for training and 30% for validation. The deep neural network model, comprising three hidden layers, was trained using input features derived from the outputs of the MuTect2 caller. We employed the SHapley Additive exPlanations algorithm to identify relevant features, and then fine-tuned them based on the training outcomes. We assessed the performance of the final DEEPOMICS FFPE model in comparison to existing models (MuTect filter, FFPolish, and SOBDetector) using well-defined test datasets. Our findings revealed 43 distinguishing characteristics for FFPE artifacts. Refining the quantification of these characteristics resulted in improved model performance. DEEPOMICS FFPE successfully filtered out 99.6% of artifacts while retaining 85.2% of true variants, achieving an F1-score of 87.8 in the validation dataset, surpassing the performance of existing tools. Furthermore, the model demonstrated consistent performance even for variants with low allele frequencies, exhibiting a specificity of 0.998, suggesting its capability to identify subclonal variants. Future enhancement of the model will include the ability to use other variant callers and analysis of whole genome sequencing data. This newly developed tool has potential applications in designing capture panels for personalized circulating tumor DNA assays and identifying candidate neoepitopes for personalized vaccine development. DEEPOMICS FFPE is freely accessible for research purposes on the web at http://deepomics.co.kr/ffpe. Citation Format: Dong-hyuk Heo, Inyoung Kim, Heejae Seo, Seong-Gwang Kim, Minji Kim, Jiin Park, Hongsil Park, Seungmo Kang, Juhee Kim, Soonmyung Paik, Seong-Eui Hong. Reducing artifactual somatic variant calls from formalin-fixed paraffin-embedded specimens by using DEEPOMICS FFPE, a bioinformatic approach based on deep neural networks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2275.

Abstract 5995: How FDA-approved drug, Aprepitant, induces immunogenic death of cancer cells

Abstract Aprepitant is a long-standing FDA approved anti-nausea drug, recently re-purposed as an early-stage anticancer agent; how aprepitant kills cancer cells was not well studied. We find that aprepitant kills human and mouse cancer cells by hyperactivating the stress response pathway, the anticipatory unfolded protein response (a-UPR), thereby inducing immune cell activating necrotic cell death. Aprepitant is a small molecule neurokinin-1 receptor (NK-1R) antagonist widely used to prevent nausea and vomiting in cancer patients undergoing chemotherapy. Recently aprepitant has shown promise in mouse xenograft studies of colon, lung and pancreatic cancer and melanoma. Since aprepitant was thought to trigger release of calcium stored in the lumen of the endoplasmic reticulum, the step that we showed triggers hyperactivation of the a-UPR by ErSO family anticancer agents, we explored the role of the a-UPR in aprepitant action. Aprepitatnt robustly activates the PERK arm of the UPR increasing p-PERK and peIF2α, inhibiting protein synthesis. Notably, aprepitant treatment results in ATP depletion and activation of AMPK. Aprepitant induced rapid cell death was not blocked by apoptosis inhibitors or necroptosis inhibitors. In both standard 2-dimensional cell culture and 3-dimensional organoid culture aprepitant-treated breast cancer cells displayed typical features of necrotic cell death, including cell swelling, membrane rupture, and cytoplasmic vacuolization. Cell death by necrosis and membrane rupture leads to release of immune cell activating cell contents termed damage-associated molecular patterns (DAMPs). Aprepitant treatment induces release of the classic DAMPs, HMGB1 and ATP and medium from aprepitant-treated breast cancer cells enhances migration of differentiated human THP-1 macrophage. Thus aprepitant and other necrosis inducing therapies elicit immunogenic cell death (ICD) and may have the potential to enhance adjuvant immunotherapy. Genome-wide CRISPR screens with negative selection against aprepitant and other studies are being used to explore the pathways of aprepitant induced cell death and evaluate its therapeutic efficacy. Since FDA-approved aprepitant has been widely used for many years, identifying its pathway of action will both accelerate its clinical transition as an anticancer agent and help identify patients whose genetic profiles make them most likely to benefit from aprepitant therapy. Citation Format: Xinyi Dai, Junyao Zhu, Sofia Perry, Qianjin Jiang, David J. Shapiro. How FDA-approved drug, Aprepitant, induces immunogenic death of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5995.

Abstract 2437: Distinct DNA methylation patterns in male urogenital cancers

Abstract Background: Male urogenital cancers including prostate cancer (PCa), bladder cancer (BLCA) and kidney renal cancer (KIRC), account for 38% of the total number of new male cancers. Previous studies have shown that changes in DNA methylation patterns are associated with the occurrence and progression of these cancers. Here, we performed whole-genome bisulfite methylation sequencing (WGBS) on tissue samples from these cancers to comprehensively characterize the methylation patterns to provide insights into the potential biological functions of methylation and to identify possible early detection biomarkers. Methods: Tissue samples were collected from patients with PCa, BLCA and KIRC who had not received any treatment prior to surgery from Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine. Samples with the cancer cells content ≥30% in cancer tissues and no cancer cells in the adjacent tissues was deemed qualified. A total of 43 samples were sequenced by whole-genome bisulfite methylation sequencing (WGBS) with a depth of 30X, including PCa, BLCA, KIRC cancer tissues, and their adjacent cancer tissues. Results: Distinct methylation patterns were observed in the 3 cancers. By comparing cancer and adjacent tissues, the numbers of differentially methylated sites (DMSs) were 21,195 (31% hyperDMSs and 69% hypoDMSs), 587,010 (23% hyperDMSs and 77% hypoDMSs) and 7,855,162 (5% hyperDMSs and 95% hypoDMSs) in PCa, BLCA and KIRC, respectively. The distribution of hyperDMSs and hypoDMSs across various chromosomes was relatively consistent in KIRC and BLCA. However, in PCa, the hypoDMSs were mainly located on chromosome 8 (5265/14666, 36%). Among the three cancers, only 2-3% of hypoDMSs were located in gene promoter regions, while the proportions of hyperDMSs in gene promoter regions were higher with 4%, 8% and 17% for PCa, BLCA and KIRC, respectively. Among the above DMSs, 1901 DMSs-related genes were shared by PCa, BLCA and KIRC and 251, 4252, 2475 DMSs-related genes were unique in PCa, BLCA and KIRC, respectively. Gene Ontology analysis revealed that the shared DMSs-related genes were enriched in Wnt signaling pathway and positive regulation of MAPK cascade. Conclusions: Our present study demonstrated the differential methylation patterns in male urogenital cancers, which may provide insights into the potential biological functions of methylation and to identify possible early detection biomarkers. Citation Format: Guopeng Yu, Jiangyi Wang, Long Li, Wenchuan Xie, Yushan Liu, Qing Yang, Bin Xu. Distinct DNA methylation patterns in male urogenital cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2437.

Abstract 4250: Adherent and spheroid cell models of patient-derived xenograft for drug development and translational research

Abstract Introduction: The development of new drugs in cancer therapy comprises toxicity and efficacy tests with increasing complexity. First and foremost, in vitro experiments are performed with well-established cancer cell lines, which are subsequently validated in animal experiments as prerequisite for clinical trials. Until a few years ago, there were gaps in the complexity chain between in vitro and in vivo experiments. Ethical considerations have led to use of systems like organ on a chip or mini-organ 3D in vitro models. In cancer research, cell cultures or organoids are generated from patient tumor material. However, these cultures only reflect the tumor of one patient, which is why panels of patient tumors should be used to evaluate the effectiveness of drugs on different patients. In vivo panel screens of patient derived xenografts (PDX) mouse models need high numbers of animals and takes several months. A pre-screen with in vitro models generated from in vivo PDX tissues allows large scale and faster pre-screens complementing in vivo systems for more focused in vivo analyses. Methods: Currently we have a pool of more than 600 established PDX models of 21 tumor entities. From this pool, cancer tissues of glioblastoma, mesothelioma, gastric, head/neck, lung and breast cancer were processed in single cell suspensions and cultured under defined conditions to obtain adherent cells or spheroids. The generated PDX in vitro cultures were analyzed for cellular impurities, cancer stem cell content and perpetuation of in vivo PDX characteristics. FACS analyses for tumor specific markers, chemo sensitivity assays and growth characteristics of the PDX derived cell lines (especially for glioblastoma) were analyzed. Results: From PDX tissues used, 90% grew as adherent and/or spheroid PDX derived in vitro cultures, in which mouse cells were entirely depleted. A high percentage of these cultures showed enriched cancer stem cell features and stem cell marker expression. Tumor marker expression and standard drug sensitivity data correlate to the in vivo PDX and derived in vitro cell culture models. RNAseq data were used to predict drug sensitivities in silico for untested drugs and drug combinations on our newly established PDX derived glioblastoma cell lines. Initial screens with predicted candidates were performed. Promising conditions were successfully repeated in corresponding animal PDX models. Conclusion: The newly developed technology for establishment if in vitro cell cultures from PDX efficiently generates stably growing cell lines possessing all key features of the original PDX. These cell lines can be used for initial pre-screens to optimize and improve selection of pharmacologically active drugs or drug combinations before initiating in vivo PDX studies. Citation Format: Lars Winkler, Joshua Alcaniz, Maria Stecklum, Wolfgang Walther, Jens Hoffmann. Adherent and spheroid cell models of patient-derived xenograft for drug development and translational research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4250.

Abstract 5617: Combined inhibition of ATR and ATM with tuvusertib and lartesertib (M4076) impacts the tumor microenvironment

Abstract Ataxia telangiectasia and Rad3-related protein (ATR) and ataxia telangiectasia mutated (ATM) are two of the main kinases of the DNA damage response (DDR). Recent work has demonstrated that the addition of ATM inhibitors enhances the cytotoxicity and in vivo anti-tumor efficacy of ATR inhibitors by abrogating the ATR inhibitor mediated G1 cell cycle arrest and enhancing chromosomal damage.1 Prior to causing cell death, damaged chromosome fragments can end up in the cytosol where they are detected by cytosolic nucleic acid sensors such as cGAS, resulting in the induction of inflammatory signaling pathways and an anti-tumor immune response. This suggests that the mode of action of DDR inhibitors may involve the tumor microenvironment (TME). We therefore aimed to assess the influence of combined pharmacological ATR and ATM inhibition on the TME to better understand how this could be exploited therapeutically. In line with published results in human tumor models, the treatment of murine colon cancer MC-38 cells in vitro with a combination of ATR inhibitor tuvusertib and ATM inhibitor lartesertib led to cell death. Further analysis revealed an activation of the cGAS-STING signaling pathway, an upregulation of PD-L1 and the release of inflammatory cytokines. These results confirmed the activation of immune signaling pathways upon combined inhibition of ATR and ATM. To analyze the stimulation of an anti-tumor immune response upon tuvusertib + lartesertib treatment in vivo, MC-38 cells were transplanted into immunocompetent C57BL/6J mice. Immunoprofiling by flow cytometry and immunohistochemistry staining confirmed that treatment with tuvusertib + lartesertib indeed altered the TME in this setting. In line with the in vitro findings, a significant upregulation of PD-L1 on immune and tumor cells could be observed after 24, 48, and 72 h of treatment even though MC-38 cells expressed high levels of PD-L1 already. Immunoprofiling did not reveal any changes in the TME immune cell content at these early time points. Interestingly, PD-L1 expression as well as tumor immune population changed after prolonged (23 days) treatment. PD-L1 was still expressed on tumor cells but to a significantly lesser extent and a depletion of CD8+ T-cells was observed. However, a significant increase in NK cells could be detected. In summary, we show that inhibition of the DDR through a combination of ATR and ATM inhibitors influences the TME in the MC-38 tumor model. These findings suggest an additional mode of action beyond direct cancer cell cytotoxicity. Further studies on how this may be leveraged therapeutically, for example through a combination with immune checkpoint inhibitors, are warranted. 1. Turchick A, Zimmermann A, et al. Mol Cancer Ther 2023;22(7):859-872. Citation Format: Karin Laaber, Audrey Turchick, Julia Jabs, Brian Elenbaas, Lyubomir T. Vassilev, Astrid Zimmermann. Combined inhibition of ATR and ATM with tuvusertib and lartesertib (M4076) impacts the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5617.

Abstract 5561: Whole slide Imaging Mass Cytometry allows the rapid profiling of the immune landscape of histopathologically aggressive prostate tumors

Abstract Patients with Gleason pattern 4 or 5 prostate tumors have a high risk of biochemical recurrence and worse survival. Affected by their local environment, dynamic immune cell phenotypes can both hinder or drive cancer progression. To understand the proteomic environments that facilitate specific immune cell responses and interactions in advanced prostate cancer, we have spatially resolved the immune cell content and metabolic features of Gleason pattern 4 and 5 and subpatterns of prostate tumors. To scale quantitative multiplexed histopathology measurements to human disease, we have applied a new high-throughput whole slide modality of high-plex Imaging Mass CytometryTM to simultaneously quantify immune cells, metabolic states, prostate specific pathways, and biomarkers in 125 whole slide sections from 42 patients over 100 cm2 of tissue. It is essential to image whole slide sections in order to fully evaluate the complete heterogeneity of these cancer samples. In parallel, multi-region macrodissection shotgun proteomics and both glycan and extracellular matrix MALDI imaging mass spectrometry have quantified the proteome associated with specific immune cell environments. Together, we identify localized proteomic microenvironments associated with specific tumor supportive or inhibitory immune cell content in human tumors that may help differentiate which high-risk patients will have a rapid biochemical recurrence following radical prostatectomy. Citation Format: Jennifer L. Gorman, Lydia Y. Liu, Jordan P. Hartig, Nikesh Parsotam, Amanda Khoo, Vladimir Ignatchenko, Sarah Asbury, Somi Afiuni, Ricardo Gonzalez, Michael J. Geuenich, Caitlin F. Harrigan, Yuju Lee, Jianan Chen, Liang Lim, Qanber Raza, Peggi M. Angel, Kieran Campbell, Stanley K. Liu, Michelle R. Downes, Richard R. Drake, Thomas Kislinger, David King, Hartland W. Jackson. Whole slide Imaging Mass Cytometry allows the rapid profiling of the immune landscape of histopathologically aggressive prostate tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5561.

The Inhibitory Effect of Early Pregnancy Factor on Red Meat Neu5Gc-Mediated Antibody Production in CMAH-/- Mice.

The meat derived from mammals such as cows, sheep, and pigs is commonly referred to as red meat. Recent studies have shown that consuming red meat can activate the immune system, produce antibodies, and subsequently develop into tumors and cancer. This is due to the presence of a potential carcinogenic compound in red meat called N-ethanol neuraminic acid (Neu5Gc). Neu5Gc is a common sialic monosaccharide in mammals, synthesized from N-acetylneuraminic acid (Neu5Ac) in the body and typically present in most mammals. However, due to the lack of the CMAH gene encoding the cytidine 5'-monophosphate Neu5Ac hydroxylase, humans are unable to synthesize Neu5Gc. Compared to primates such as mice or chimpanzees, the specific loss of Neu5Gc expression in humans is attributed to fixed genome mutations in CMAH. Although Neu5Gc cannot be produced, it can be introduced from specific dietary sources such as red meat and milk, so it is necessary to use mice or chimpanzees that knock out the CMAH gene instead of humans as experimental models. Further research has shown that early pregnancy factor (EPF) has the ability to regulate CD4+T cell-dependent immune responses. In this study, we established a simulated human animal model using C57/BL6 mice with CMAH gene knockout and analyzed the inhibitory effect of EPF on red meat Neu5Gc-induced CMAH-/- C57/BL6 mouse antibody production and chronic inflammation development. The results showed that the intervention of EPF reduced slow weight gain and shortened colon length in mice. In addition, EPF treatment significantly reduced the levels of anti Neu5Gc antibodies in the body, as well as the inflammatory factors IL-6 and IL-1β, TNF-α and the activity of MPO. In addition, it also alleviated damage to liver and intestinal tissues and reduced the content of CD4 cells and the expression of B cell activation molecules CD80 and CD86 in mice. In summary, EPF effectively inhibited Neu5Gc-induced antibody production, reduced inflammation levels in mice, and alleviated Neu5Gc-induced inflammation. This will provide a new re-search concept and potential approach for developing immunosuppressants to address safety issues related to long-term consumption of red meat.

Classifying Raman Spectra of Colon Cells Based on Machine Learning Algorithms

Colorectal cancer is very widespread in developed countries. Its diagnosis partly depends on pathologists’ experience and their laboratories’ instrumentation, producing uncertainty in diagnosis. The use of spectroscopic techniques sensitive to the cellular biochemical environment could aid in achieving a reliable diagnosis. So, we used Raman micro-spectroscopy, combined with a spectral analysis by means of machine learning methods, to build classification models, which allow colon cancer to be diagnosed in cell samples, in order to support such methods as complementary tools for achieving a reliable identification of colon cancer. The Raman spectra were analyzed in the 980–1800 cm−1 range by focusing the laser beam onto the nuclei and the cytoplasm regions of single FHC and CaCo-2 cells (modelling healthy and cancerous samples, respectively) grown onto glass coverslips. The comparison of the Raman intensity of several spectral peaks and the Principal Component Analysis highlighted small biochemical differences between healthy and cancerous cells mainly due to the larger relative lipid content in the former cells with respect to the latter ones and to the larger relative amount of nucleic acid components in cancerous cells compared with healthy ones. We considered four classification algorithms (logistic regression, support vector machine, k nearest neighbors, and a neural network) to associate unknown Raman spectra with the cell type to which they belong. The built machine learning methods achieved median values of classification accuracy ranging from 95.5% to 97.1%, sensitivity values ranging from 95.5% to 100%, and specificity values ranging from 93.9% to 97.1%. The same median values of the classification parameters, which were estimated for a testing set including unknown spectra, ranged between 93.1% and 100% for accuracy and between 92.9% and 100% for sensitivity and specificity. A comparison of the four methods pointed out that k nearest neighbors and neural networks better perform the classification of nucleus and cytoplasm spectra, respectively. These findings are a further step towards the perspective of clinical translation of the Raman technique assisted by multivariate analysis as a support method to the standard cytological and immunohistochemical methods for diagnostic purposes.

Gamma globulin binding of hinokiflavone with anti-osteoporosis effects: A mechanistic study

Oxidative stress can trigger apoptosis and associated reduction of osteoblast activity. Hinokiflavone (HNK) with a biflavonoid-based structure might show potent antioxidant activity. However, before the application of this compound as a promising therapeutic bioactive material, its interaction with blood proteins should be investigated to further reveal its pharmacokinetic and pharmacodynamic properties. Therefore, in this paper, the interaction of HNK with γ-globulin, one of the main blood proteins, was assessed. Then, the antioxidant properties of HNK against H2O2-induced osteoblast cytotoxicity in MC3T3-E1 cells were assessed. It was shown that HNK can potentially bind to γ-globulin mostly with the aid of hydrophilic forces, with a slight effect on the protein structure. It was then determined that HNK significantly recovered cell survival and alkaline phosphatase (ALP) activity and collagen-I content in MC3T3-E1 cells exposed to H2O2. In addition, HNK decreased the production of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) in MC3T3-E1 cells triggered by H2O2. Finally, HNK was shown to control the apoptosis induction in MC3T3-E1 cells triggered by H2O2 mediated by regulation of Bax, Bcl-2 and caspase-3. Taken together, these data demonstrated that HNK with promising blood protein binding properties can show significant protective effects in MC3T3-E1 cells mediated by inhibition of osteoblast dysfunction, oxidative stress, and apoptosis.

Deferiprone-resveratrol hybrid attenuates iron accumulation, oxidative stress, and antioxidant defenses in iron-loaded human Huh7 hepatic cells.

Chronic liver diseases are complications of thalassemia with iron overload. Iron chelators are required to remove excessive iron, and antioxidants are supplemented to diminish harmful reactive oxygen species (ROS), purposing to ameliorate oxidative liver damage and dysfunctions. The deferiprone-resveratrol hybrid (DFP-RVT) is a synthetic iron chelator possessing anti-β-amyloid peptide aggregation, anti-malarial activity, and hepatoprotection in plasmodium-infected mice. The study focuses on investigating the antioxidant, cytotoxicity, iron-chelating, anti-lipid peroxidation, and antioxidant defense properties of DFP-RVT in iron-loaded human hepatocellular carcinoma (Huh7) cells. In the findings, DFP-RVT dose dependently bound Fe(II) and Fe(III) and exerted stronger ABTS•- and DPPH•-scavenging (IC50 = 8.0 and 164μM, respectively) and anti-RBC hemolytic activities (IC50 = 640μM) than DFP but weaker than RVT (p < 0.01). DFP-RVT was neither toxic to Huh7 cells nor PBMCs. In addition, DFP-RVT diminished the level of redox-active iron (p < 0.01) and decreased the non-heme iron content (p < 0.01) in iron-loaded Huh7 cells effectively when compared without treatment in the order of DFP-RVT > RVT ∼ DFP treatments (50µM each). Moreover, the compound decreased levels of hepatic ROS in a dose-dependent manner and the level of malondialdehyde, which was stronger than DFP but weaker than RVT. Furthermore, DFP-RVT restored the decrease in the GSH content and GPX and SOD activities (p < 0.01) in iron-loaded Huh7 cells in the dose-dependent manner, consistently in the order of RVT > DFP-RVT > DFP. Thus, the DFP-RVT hybrid possesses potent iron chelation, antioxidation, anti-lipid peroxidation, and antioxidant defense against oxidative liver damage under iron overload.

Heparanase promotes the onset and progression of atherosclerosis in apolipoprotein E gene knockout mice

Background and aimsAtherosclerosis is the primary underlying cause of myocardial infarction and stroke, which are the major causes of death globally. Heparanase (Hpse) is a pro-inflammatory extracellular matrix degrading enzyme that has been implicated in atherogenesis. However, to date the precise roles of Hpse in atherosclerosis and its mechanisms of action are not well defined. This study aims to provide new insights into the contribution of Hpse in different stages of atherosclerosis in vivo. MethodsWe generated Hpse gene-deficient mice on the atherosclerosis-prone apolipoprotein E gene knockout (ApoE−/−) background to investigate the impact of Hpse gene deficiency on the initiation and progression of atherosclerosis after 6 and 14 weeks high-fat diet feeding, respectively. Atherosclerotic lesion development, blood serum profiles, lesion composition and aortic immune cell populations were evaluated. ResultsHpse-deficient mice exhibited significantly reduced atherosclerotic lesion burden in the aortic sinus and aorta at both time-points, independent of changes in plasma cholesterol levels. A significant reduction in the necrotic core size and an increase in smooth muscle cell content were also observed in advanced atherosclerotic plaques of Hpse-deficient mice. Additionally, Hpse deficiency reduced circulating and aortic levels of VCAM-1 at the initiation and progression stages of disease and circulating MCP-1 levels in the initiation but not progression stage. Moreover, the aortic levels of total leukocytes and dendritic cells in Hpse-deficient ApoE−/− mice were significantly decreased compared to control ApoE−/−mice at both disease stages. ConclusionsThis study identifies Hpse as a key pro-inflammatory enzyme driving the initiation and progression of atherosclerosis and highlighting the potential of Hpse inhibitors as novel anti-inflammatory treatments for cardiovascular disease.

The GPR40 novel agonist SZZ15-11 improves non-alcoholic fatty liver disease by activating the AMPK pathway and restores metabolic homeostasis in diet-induced obese mice.

Non-alcoholic fatty liver is the most common cause of chronic liver disease. GPR40 is a potential therapeutic target for energy metabolic disorders. GPR40 is a potential therapeutic target for energy metabolic disorders. SZZ15-11 is a newly synthesized GPR40 agonist. In this study, we estimate the potency of SZZ15-11 in fatty liver treatment. In vivo, diet-induced obese (DIO) mice received SZZ15-11 (50 mg/kg) and TAK875 (50 mg/kg) for 6 weeks. Blood glucose and lipid, hepatocyte lipid and liver morphology were analysed. In vitro, HepG2 cells and GPR40-knockdown HepG2 cells induced with 0.3 mM oleic acid were treated with SZZ15-11. Triglyceride and total cholesterol of cells were measured. At the same time, the AMPK pathway regulating triglycerides and cholesterol esters synthesis was investigated via western blot and quantitative polymerase chain reaction in both liver tissue and HepG2 cells. SZZ15-11 was found to not only attenuate hyperglycaemia and hyperlipidaemia but also ameliorate fatty liver disease in DIO mice. At the same time, SZZ15-11 decreased triglyceride and total cholesterol content in HepG2 cells. Whether examined in the liver of DIO mice or in HepG2 cells, SZZ15-11 upregulated AMPKα phosphorylation and then downregulated the expression of the cholesterogenic key enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase and inhibited acetyl-CoA carboxylase activity. Furthermore, SZZ15-11 promotes AMPK activity via [cAMP]i accumulation. This study confirmed that SZZ15-11, a novel GPR40 agonist, improves hyperlipidaemia and fatty liver, partially via Gs signalling and the AMPK pathway in hepatocytes.

New insights into Gasdermin D pore formation.

Gasdermin D (GSDMD) is a pore-forming protein that perforates the plasma membrane (PM) during pyroptosis, a pro-inflammatory form of cell death, to induce the unconventional secretion of inflammatory cytokines and, ultimately, cell lysis. GSDMD is activated by protease-mediated cleavage of its active N-terminal domain from the autoinhibitory C-terminal domain. Inflammatory caspase-1, -4/5 are the main activators of GSDMD via either the canonical or non-canonical pathways of inflammasome activation, but under certain stimuli, caspase-8 and other proteases can also activate GSDMD. Activated GSDMD can oligomerize and assemble into various nanostructures of different sizes and shapes that perforate cellular membranes, suggesting plasticity in pore formation. Although the exact mechanism of pore formation has not yet been deciphered, cysteine residues are emerging as crucial modulators of the oligomerization process. GSDMD pores and thus the outcome of pyroptosis can be modulated by various regulatory mechanisms. These include availability of activated GSDMD at the PM, control of the number of GSDMD pores by PM repair mechanisms, modulation of the lipid environment and post-translational modifications. Here, we review the latest findings on the mechanisms that induce GSDMD to form membrane pores and how they can be tightly regulated for cell content release and cell fate modulation.

Association between Left Atrial Appendage Morphology and Clot Histology in Patients with Embolic Ischemic Stroke: An Exploratory Study.

Acute embolic ischemic stroke poses a significant healthcare challenge. Histological clot features' variability among patients with acute ischemic stroke treated by mechanical thrombectomy has potential implications for determining treatment and etiology. This study investigated the clot histological feature differences among patients who experienced cardioembolic stroke and embolic stroke of undetermined source with different left atrial appendage (LAA) morphologies. We conducted a prospective observational study involving 79 patients with acute embolic ischemic stroke undergoing mechanical thrombectomy. Computed tomography angiography images were used to classify LAA morphologies. An artificial intelligence algorithm assessed the clot fibrin and red blood cell contents. Patients with chicken-wing LAA morphology exhibited lower mean clot fibrin proportions than did those with non-chicken-wing morphology (p < 0.001). Linear regression analysis showed that chicken-wing LAA was significantly associated with a lower clot fibrin proportion (estimate, -0.177; 95% CI [-0.259, -0.096]; p < 0.001). The successful recanalization rate and first-pass effect between the groups did not differ significantly. The chicken-wing LAA morphological type is associated with lower clot fibrin contents, suggesting potentially different embolism mechanisms or diverse embolic sources, compared with the non-chicken-wing LAA types. Further studies are required to investigate this association.

Tumor-infiltrating immune cells and survival in head and neck squamous cell carcinoma: a retrospective computational study

The immune infiltration profiles of the tumor microenvironment have effects on the prognosis of head and neck squamous cell carcinoma (HNSCC). Whereas, HNSCC is a heterogeneous group of tumors, but past work has not taken this into consideration. Herein, we investigate the associations between survival and the function of immune cells in different tumorigenic sites of HNSCC. 1149 samples of HNSCC were collected from publicly accessible databases. Based on gene expression data, CIBERSORTx was applied to determine the proportion of 22 immune cell subpopulations. In the Cox regression model, the associations between overall survival, disease-free survival, and immune cells were examined, modeling gene expression and immune cell proportion as quartiles. Consensus cluster analysis was utilized to uncover immune infiltration profiles. Regardless of tumor sites, CD8+ T cells and activated CD4 memory T cells were associated with favorable survival, while eosinophils were the opposite. The survival of the hypopharynx, oral cavity, and larynx subsites was somewhat affected by immune cells, while the survival of the oropharynx subsite potentially was the most impacted. High expression of TIGIT, CIITA, and CXCR6 was linked to better survival, mainly in the oropharynx subsite. Immune cell clusters with four distinct survival profiles were discovered, of which the cluster with a high CD8+ T cell content had a better prognosis. The immune-infiltration pattern is related to the survival of HNSCC to varying degrees depending on the tumor sites; forthcoming studies into immune-mediated infiltration profiles will lay the groundwork for treating HNSCC with precision therapy.

A histological examination of the effects of Ferula elaeochytris extract on kidney and liver tissues in myoglobinuric acute renal failure

ABSTRACT Myoglobinuric acute renal failure (MARF) is a structural and functional disorder that occurs in the kidney following the release of muscle cell contents into the circulation. In this present study, possible protective and curative effects of Ferula elaeochytris extract against kidney and liver damage in experimentally induced MARF in a rat model were investigated. 3–4 Month-old, 200–250 g Sprague Dawley rats were divided into 8 equal groups with 7 rats per group. Group I was a no-intervention Control group. All groups except for the Group I were dehydrated for 16 hours. Following this dehydration, 50% v/v aqueous glycerol solution was injected into both hind leg muscles of the animals, at a dose of 8 ml/kg. The rats were given physiological saline (SF) once orally before the model was administered (Group II) and after the model was administered (Group V). Similarly, two different doses of Ferula elaeochytris root extract (40 mg/kg and 80 mg/kg) were dissolved in 2 ml of SF and administered orally before (Groups III and IV) and after (Group VI, VII) the model was created. Following the experimental period, kidney and liver tissues were removed from all groups, and fixed in 10% neutral formaldehyde solution for light microscopic examinations. Intracellular vacuolization, enlargement in the Bowman’s space, widespread atrophy in the tubular structures, luminal enlargement, and desquamation were detected in the kidney tissue sections of all the experimental model groups. In the liver tissue sections, was detected hepatocyte degeneration, intracellular vacuolization, irregularity in cell membrane borders, and apoptotic bodies. These histopathological consequences of MARF were evaluated for all groups, and whereas a curative effect of Ferula elaeochytris could be seen, its protective effect was higher than its curative effect.

Comparison of batch, fed-batch and continuous operation modes for scalable polyhydroxyalkanoate (PHA) production and carbon sequestration from phenol

Polyhydroxyalkanoates (PHA) are biodegradable intracellular polymers, and a renewable alternative to conventional plastics. To reduce PHA's cost, this study combined an unconventional carbon source, i.e. a toxic aromatics phenol, with an acclimated consortium, to develop a scalable PHA production process. The resultant process performance was systematically compared across three operational modes: batch, fed-batch, and continuous. Phenol toxicity caused inhibition in the batch mode, reducing the PHA synthesis rate but resulting in a high PHA content in cells (PHA% > 50 %). Fed-batch feeding alleviated such inhibition, facilitating a high PHA titer and yield from the substrate. Interestingly, a higher feeding rate in the fed-batch reactor improved its performance, probably by providing a suitable food-to-microbe ratio for the biomass. Using a continuous mode also reduced phenol toxicity, but resulted in more synthesis of non-PHA cellular materials (NPCM). The difference might have been caused by different metabolic states and the presence of intermediates in the reactors. Comparing all 14 conditions under the three modes, feeding phenol more gradually tended to channel substrate away from PHA to NPCM synthesis. The volumetric productivity generally ranged from 3 to 12 mg L−1 min−1, while the biomass productivity was between 2 and 11 mg g initial CDW−1 min−1. A 3-stage, production-harvest and starvation-production process was then designed to simulate real industrialized manufacturing, where the operational modes had a long-term effect on biomass' productivity. Fed-batch mode performed the best, as it fixed 1/3 of phenol's organic carbon in PHA product, suggesting high potential for future application.