Expression Of Protein Biomarkers Research Articles

NGS panels' capture of biomarkers associated with targeted therapies in clinical trials.

e13508 Background: ASCO and NCCN guidelines strongly encourage participation in clinical trials though participation rates remain low. Although up to 60% of trials require biomarker information as a prerequisite to participation, guidelines are silent on biomarker testing to assess eligibility. To help inform policy development, we analyzed commercially available NGS panels to assess the capture rate of the specific gene variants required for targeted therapies in clinical trials. Methods: Publicly available gene variant information on 12 high volume NGS tissue and plasma commercial assays representative of panel size was accessed. The JAX-CKB database was queried to identify genomic prerequisites in active phase I-III clinical trials; mRNA and protein expression biomarkers were excluded. We used unpaired t-testing to compare the ability of large versus small panels to detect variants required for targeted treatments. (Somatic gene variant annotations and related content have been powered by The Jackson Laboratory Clinical Knowledgebase (JAX-CKBTM)). Results: Overall, 185 genes were linked to eligibility for targeted therapies in clinical trials (Table). Large panel assays (>80 genes) had a higher average capture rate compared to smaller panels, (84% vs. 46%, p value < 0.000002). Similarly, capture rates in select cancers were higher in larger panels: lung (85% vs 59%), breast (92% vs 54%), prostate (86% vs 34%), pancreatic (84% vs 54%), colorectal (88% vs 52%). All results were statistically significant (p < 0.05). Smaller panels were more likely to exclude large genes (e.g. BRCA), genome-wide signatures (e.g. TMB), fusions, and copy number variants. Conclusions: NGS-based panels with more than 80 genes had higher capture rates for biomarkers required for clinical trials of targeted therapies. Clinicians searching for all relevant treatment options for patients should include large genomic sequencing panels to maximize identification of potential clinical trial eligibility. Professional societies and policy makers interested in enhancing clinical trial enrollment rates should consider the panel size-related variability in capture rates of genetic variants when developing recommendations. [Table: see text]

Tobacco Smoke Condensate Induces Morphologic Changes in Human Papillomavirus-Positive Cervical Epithelial Cells Consistent with Epithelial to Mesenchymal Transition (EMT) with Activation of Receptor Tyrosine Kinases and Regulation of TGFB.

High-risk human papillomavirus (HR-HPV; HPV-16) and cigarette smoking are associated with cervical cancer (CC); however, the underlying mechanism(s) remain unclear. Additionally, the carcinogenic components of tobacco have been found in the cervical mucus of women smokers. Here, we determined the effects of cigarette smoke condensate (CSC; 3R4F) on human ectocervical cells (HPV-16 Ect/E6E7) exposed to CSC at various concentrations (10-6-100 μg/mL). We found CSC (10-3 or 10 μg/mL)-induced proliferation, enhanced migration, and histologic and electron microscopic changes consistent with EMT in ectocervical cells with a significant reduction in E-cadherin and an increase in the vimentin expression compared to controls at 72 h. There was increased phosphorylation of receptor tyrosine kinases (RTKs), including Eph receptors, FGFR, PDGFRA/B, and DDR2, with downstream Ras/MAPK/ERK1/2 activation and upregulation of common EMT-related genes, TGFB SNAI2, PDGFRB, and SMAD2. Our study demonstrated that CSC induces EMT in ectocervical cells with the upregulation of EMT-related genes, expression of protein biomarkers, and activation of RTKs that regulate TGFB expression, and other EMT-related genes. Understanding the molecular pathways and environmental factors that initiate EMT in ectocervical cells will help delineate molecular targets for intervention and define the role of EMT in the initiation and progression of cervical intraepithelial neoplasia and CC.

Aptamer-based protein molecule detection via cyclic reverse transcription coupling with self-priming hairpin-triggered CRISPR-Cas12a system

Protein biomarkers (e.g. thrombin) are of great significance for the biological process of the organism, and its aberrant expression is closely associated with the development of diseases. With thrombin, a serine protease that plays a crucial role in maintaining homeostasis and promoting blood clotting, as detection target, this study introduces a novel approach for sensitive and accurate measurement of protein biomarker expression by utilization of cyclic reverse transcription (CRT) in combination with the self-priming hairpin-triggered CRISPR-Cas12a system. In this method, an elegantly designed sensing probe is utilized to specifically bind with the thrombin protein and convert the protein signals to nucleic acids signals, following by the CRT and CRISPR-Cas12a system-based signal amplification strategy. Taking the merit of the two-stage amplification, this assay has the capability to detect thrombin at the fM level. In addition, due to the aptamer sequence’s strong selectivity to thrombin protein and the dual-check process in the signal amplification process (first in the CRT and second in the CRISPR system), the proposed test demonstrates exceptional specificity in detecting thrombin. By re-designing the sensing probe, the established method could be extended to various protein biomarker detection. Ultimately, this assay has successfully enabled the accurate evaluation of biomarker levels in constructed clinical samples, showing significant potential for application in the realm of clinical molecular diagnosis.

Abstract 6562: Comprehensive liquid biopsy: Multi-analyte evaluation of cellular and plasma components of blood in prostate cancer from a single blood collection tube

Abstract Background Liquid biopsy samples offer a non-invasive method for investigating drug targets and biomarkers in solid tumors. In this study, we describe a comprehensive liquid biopsy analysis of whole blood to identify circulating tumor cells (CTCs), large extracellular vesicles and CTC fragments, and CTC-white blood cell hybrids; quantify protein biomarker expression on CTCs; perform targeted mutation profiling on CTCs and cell-free DNA (cfDNA) from plasma; and analyze protein expression on white blood cells (WBCs) from a single 7.5 mL blood sample. This array of tests was applied to blood samples from patients with Stage IV prostate cancer to demonstrate clinical feasibility. Methods A novel dual biomarker CTC assay was developed to measure protein expression of PSMA and ARv7 on CTCs from stage IV prostate cancer patients. Nucleated cells from clinical samples were processed to microscope slides using AccuCyte®, an unbiased, density-based separation method that also allows for plasma collection for downstream analysis. Slides were fixed and stained with antibodies to cytokeratin, EpCAM, and CD45 to detect CTCs and exclude WBCs, and with PSMA and ARv7 to evaluate protein biomarker expression on identified CTCs. The slides were imaged with the CyteFinder® automated immunofluorescence scanning microscope that identifies CTCs for confirmation by trained reviewers and quantitatively measures protein mean fluorescence intensity (MFI) on any cells detected on the slides (CTCs, CTC-WBC hybrids, large extracellular vesicles and CTC fragments and WBCs). Individual CTCs and control pools of WBCs from clinical samples with at least 4 CTCs were picked using CytePicker®, a needle-based system used to isolate single cells. cfDNA was isolated from plasma removed during the AccuCyte® process using a QIAamp MinElute ccfDNA kit. Picked CTCs, WBC pools, and isolated ctDNA were sequenced using the OncoZoom Cancer HotSpot panel from Paragon Genomics. Results Nine progressing Stage IV prostate cancer patient blood samples were stained with a novel PSMA/ARv7 dual biomarker assay. CTCs were identified in all clinical samples (median 4, range 1 - 384). PSMA positivity in clinical samples ranged from 0% to 75%, and ARv7 positivity ranged from 33% to 100%, and. Additional analysis using QuPath was performed to determine PSMA/ARv7 protein expression on WBCs and CTC fragments. ctDNA analysis from 5 individual samples was compared to sequencing results from individual CTCs from the same samples. Conclusions Using a single 7.5 mL blood liquid biopsy, we can measure protein expression on enumerated CTCs and quantify these same protein biomarkers on WBCs and CTC fragments/oncosomes, determine the genetic makeup of the tumor through the sequencing of cfDNA, and monitor genetic mutations in intact CTCs in circulation. Citation Format: Jon Ladd, Erin Bayer, Brock Bartels, Arista Tischner, Rachel Ponting, Eric Kaldjian, Arturo Ramirez. Comprehensive liquid biopsy: Multi-analyte evaluation of cellular and plasma components of blood in prostate cancer from a single blood collection tube [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 6562.

Abstract 1070: Overcoming deficiencies in the early detection of high grade serous ovarian cancer: Evaluating exosomal proteins as novel biomarkers of disease

Abstract Introduction: High-grade serous ovarian cancer (HGSOC) accounts for over 75% of all epithelial ovarian cancer and has a high mortality rate due to a lack of early detection methods. Many biomarkers have been proposed; however, none have been shown to improve detection at an early stage in this patient population. The purpose of this study is to assess whether there are unique extracellular vesicle (EV) protein signatures of early-stage HGSOC that could serve as early detection biomarkers. Methods: 250 serum samples were obtained from a multi-institutional retrospective cohort of patients with all stages of HGSOC and healthy controls. LC-MS/MS and PEA of serum samples from patients with early-stage HGSOC identified differentially expressed EVs proteins in HGSOC versus controls. Tob candidate proteins are validated by ELISA. Results: We have identified the top 10 EVs candidate proteins based on their fold change and statistical significance. When comparing early-stage HGSOC and controls using ELISA, CFH, PCP, and CCNE1 exhibited the highest Area Under the Curve (AUC) values of 0.94, 0.91, and 0.83, respectively. In contrast, MUC16 (also known as CA-125) exhibited an AUC of only 0.42 in whole serum. Furthermore, MUC16 distinguished between controls and both early and advanced-stage HGSOC in EVs, while in whole serum, it was only differentially expressed between controls and advanced-stage disease. Our results demonstrate that the predictive performance of the combined biomarkers surpasses that of any individual biomarker. Further, we integrated the ten aforementioned biomarkers, deriving the optimal combination biomarker by maximizing the corresponding Sum of Harmonic Means (SHUM) function by utilizing data from all 70 patients, results showed the optimal coefficients of the biomarkers, which maximize the diagnostic accuracy for early-stage HGSOC. The overall true positive rate (TPR) of 0.943, false positive rate (FPR) of 0.000, and Mathew's correlation coefficient (MCC) were presented for both the combined biomarker approach and individual biomarkers. The results demonstrated that the predictive performance of the combined biomarkers surpassed that of any individual biomarker. Conclusion: Expression of EV-based protein biomarkers is significantly different in early-stage HGSOC compared to both control and late-stage HGSOC. This work can be readily translated to clinical use and potentially improve the early detection of HGSOC substantially. Citation Format: Michelle Lightfoot, Kalpana Deepa Priya Dorayappan, Lianbo Yu, Colin Hisey, Takahiko Sakaue, Muralidharan Anbalagan, Casey M. Cosgrove, Larry G. Maxwell, Premal Thaker, Beth Y. Karlan, David O’Malley, Raphael E. Pollock, David E. Cohn, Rajan Gogna, Selvendiran Karuppaiyah. Overcoming deficiencies in the early detection of high grade serous ovarian cancer: Evaluating exosomal proteins as novel biomarkers of disease [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 1070.

Abstract C043: Discovery of new drug combinations for metastatic colorectal cancer

Abstract Colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide. Despite a range of therapeutic options, drug resistance remains a significant challenge in CRC treatment. Drug combination therapy offers an opportunity to enhance therapeutic responses. There are several combination drug options that have been used for CRC patients such as FOLFOX (5-FU + Oxaliplatin) and FOLFIRI (5-FU + Irinotecan). This study aimed to discover novel drug combinations for CRC by conducting high-throughput screening of 800 pharmacologically active compounds in combination with the three main chemotherapy backbones used in CRC treatment: 5-FU, SN-38, and Oxaliplatin. Initially, we performed a primary drug screen using 11 human CRC cell lines, resulting in the identification of 72 candidate drug combinations (24 candidates x 3 backbones) exhibiting synergistic effects. Subsequently, we validated these candidates in a subset of 5 cell lines. Among them, 24 drug combinations (8 candidates x 3 backbones) were selected and further evaluated in an expanded panel of 44 CRC cell lines to determine the spectrum of drug responses and identify potential mutation, gene and protein expression biomarkers, that could predict the efficacy of the drug combinations. We investigated the molecular mechanisms underlying the synergistic effects of final 4 candidates combined with chemotherapeutic agents. Chk1 inhibitor MK-8776 showed significantly higher synergy scores in combination with SN-38 in TP53 mutated cell lines, which was further validated by in vitro apoptosis analysis and in vivo xenograft models using TP53 wildtype and knockout isogenic CRC cell lines. MK-8776 abrogated SN-38-mediated G2 cell cycle arrest, resulting in an increased cytotoxicity. These findings highlight the potential of the combination treatment involving SN-38 and MK-8776 as a promising strategy for CRC patients with TP53 mutations. Citation Format: Eun-Jung In, Tao Tan, Dmitri Mouradov, Anuratha Sakthianandeswaren, Amanda Au, Kym Lowes, Oliver Sieber. Discovery of new drug combinations for metastatic colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C043.

Development of a human peripheral blood ex vivo model for rapid protein biomarker detection and applications to radiation biodosimetry.

In the event of a widespread radiological incident, thousands of people may be exposed to a wide range of ionizing radiation. In this unfortunate scenario, there will be a need to quickly screen a large number of people to assess the amount of radiation exposure and triage for medical treatment. In our earlier work, we previously identified and validated a panel of radiosensitive protein biomarkers in blood leukocytes, using the humanized-mouse and non-human primate (NHP) models. The objective of this work was to develop a high-throughput imaging flow-cytometry (IFC) based assay for the rapid measurement of protein biomarker expression in human peripheral blood samples irradiated ex vivo. In this assay design, peripheral human blood samples from healthy adult donors were exposed to 0-5 Gy X-irradiation ex vivo and cultured for up to 2 days. Samples were stained with a cocktail of surface antigens (CD66b, CD20, and CD3), fixed and permeabilized, and intracellularly stained for BAX (Bcl-2-associated X) protein, used here as a representative biomarker. Samples were interrogated by IFC, and a uniform analysis template was created to measure biomarker expression in heterogeneous and specific leukocyte subtype populations at each time point. In this human blood ex vivo model, we show that within gated populations of leukocyte subtypes, B-cells are highly radiosensitive with the smallest surviving fraction, followed by T-cells and granulocytes. Dose-dependent biomarker responses were measured in the lymphocytes, B-, and T-cell populations, but not in the granulocytes, with dose-response curves showing increasing fold changes in BAX protein expression up to Day 2 in lymphocyte populations. We present here the successful use of this ex vivo model for the development of radiation dose-response curves of a candidate protein biomarker towards future applications of dose reconstruction and biodosimetry.

Diosmetin-7-O-β-D-glucopyranoside suppresses endothelial-mesenchymal transformation through endoplasmic reticulum stress in cardiac fibrosis.

Diosmetin-7-O-β-D-glucopyranoside (Diosmetin-7-O-glucoside) is a natural flavonoid glycoside known to have a therapeutic application for cardiovascular diseases. Cardiac fibrosis is the main pathological change in the end stage of cardiovascular diseases. Endothelial-mesenchymal transformation (EndMT) induced by endoplasmic reticulum stress (ER stress) via Src pathways is involved in the process of cardiac fibrosis. However, it is unclear whether and how diosmetin-7-O-glucoside regulates EndMT and ER stress to treat cardiac fibrosis. In this study, molecular docking results showed that diosmetin-7-O-glucoside bound well to ER stress and Src pathway markers. Diosmetin-7-O-glucoside suppressed cardiac fibrosis induced by isoprenaline (ISO) and reduced the levels of EndMT, ER stress in mice heart. Primary cardiac microvascular endothelial cells (CMECs) were induced by transforming growth factor-β1 (TGF-β1) to perform EndMT. Diosmetin-7-O-glucoside could effectively regulate EndMT and diminish the accumulation of collagen I and collagen III. We also showed that the tube formation in CMECs was restored, and the capacity of migration was partially inhibited. Diosmetin-7-O-glucoside also ameliorated ER stress through the three unfolded protein response branches, as evidenced by organelle structure in transmission electron microscopy images and the expression of protein biomarkers like the glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP). Further analysis showed that diosmetin-7-O-glucoside could suppress the expression level of Src phosphorylation, then block EndMT with the maintenance of endothelial appearance and endothelial marker expression. These results suggested that the diosmetin-7-O-glucoside can regulate EndMT through ER stress, at least in part via Src-dependent pathways.

Deciphering Phenotypes from Protein Biomarkers for Translational Research with PIPER.

Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) has widespread clinical use for detection of inborn errors of metabolism, therapeutic drug monitoring, and numerous other applications. This technique detects proteolytic peptides as surrogates for protein biomarker expression, mutation, and post-translational modification in individual clinical assays and in cancer research with highly multiplexed quantitation across biological pathways. LC-MRM for protein biomarkers must be translated from multiplexed research-grade panels to clinical use. LC-MRM panels provide the capability to quantify clinical biomarkers and emerging protein markers to establish the context of tumor phenotypes that provide highly relevant supporting information. An application to visualize and communicate targeted proteomics data will empower translational researchers to move protein biomarker panels from discovery to clinical use. Therefore, we have developed a web-based tool for targeted proteomics that provides pathway-level evaluations of key biological drivers (e.g., EGFR signaling), signature scores (representing phenotypes) (e.g., EMT), and the ability to quantify specific drug targets across a sample cohort. This tool represents a framework for integrating summary information, decision algorithms, and risk scores to support Physician-Interpretable Phenotypic Evaluation in R (PIPER) that can be reused or repurposed by other labs to communicate and interpret their own biomarker panels.

Vinyl chloride enhances high-fat diet-induced proteome alterations in the mouse pancreas related to metabolic dysfunction.

Alterations in physiological processes in pancreas have been associated with various metabolic dysfunctions and can result from environmental exposures, such as chemicals and diet. It was reported that environmental vinyl chloride (VC) exposure, a common industrial organochlorine and environmental pollutant, significantly exacerbated metabolic-related phenotypes in mice fed concurrently with high-fat diet (HFD) but not low-fat diet (LFD). However, little is known about the role of the pancreas in this interplay, especially at a proteomic level. The present study was undertaken to examine the protein responses to VC exposure in pancreas tissues of C57BL/6J mice fed LFD or HFD, with focus on the investigation of protein expression and/or phosphorylation levels of key protein biomarkers of carbohydrate, lipid, and energy metabolism, oxidative stress and detoxification, insulin secretion and regulation, cell growth, development, and communication, immunological responses and inflammation, and biomarkers of pancreatic diseases and cancers. We found that the protein alterations may indicate diet-mediated susceptibility in mouse pancreas induced by HFD to concurrent exposure of low levels of inhaled VC. These proteome biomarkers may lead to a better understanding of pancreas-mediated adaptive or adverse response and susceptibility to metabolic disease.

A high-throughput, 28-day, microfluidic model of gingival tissue inflammation and recovery

Nearly half of American adults suffer from gum disease, including mild inflammation of gingival tissue, known as gingivitis. Currently, advances in therapeutic treatments are hampered by a lack of mechanistic understanding of disease progression in physiologically relevant vascularized tissues. To address this, we present a high-throughput microfluidic organ-on-chip model of human gingival tissue containing keratinocytes, fibroblast and endothelial cells. We show the triculture model exhibits physiological tissue structure, mucosal barrier formation, and protein biomarker expression and secretion over several weeks. Through inflammatory cytokine administration, we demonstrate the induction of inflammation measured by changes in barrier function and cytokine secretion. These states of inflammation are induced at various time points within a stable culture window, providing a robust platform for evaluation of therapeutic agents. These data reveal that the administration of specific small molecule inhibitors mitigates the inflammatory response and enables tissue recovery, providing an opportunity for identification of new therapeutic targets for gum disease with the potential to facilitate relevant preclinical drug efficacy and toxicity testing.

Differential expression of host protein biomarkers among symptomatic clinic attendees finally diagnosed with tuberculosis and other respiratory diseases with or without latent Mycobacterium tuberculosis infection

BackgroundThere is a need for new tools for the diagnosis of tuberculosis (TB) amongst patients who present at primary health care centers with symptoms suggestive of TB. ObjectivesTo assess the abilities of selected blood-based host biomarkers to discriminate between patients who self-presented with symptoms suggestive of TB and were subsequently diagnosed with pulmonary tuberculosis (PTB), other respiratory diseases (ORD) with latent Mycobacterium tuberculosis infection (ORD_LTBI) or ORD without latent infection (ORD_NoLTBI). MethodsPresumptive TB patients (n = 161) were enrolled at a TB Clinic in Kampala, Uganda, and blood was collected. Participants were later classified as having PTB or ORD using standard microbiological confirmatory tests. Patients with ORD were subsequently classified as having LTBI or no LTBI using the QuantiFERON Gold-plus test. The concentrations of 27 host biomarkers were evaluated in patient sera using the Luminex platform, followed by an evaluation of their abilities to discriminate between PTB, ORD_LTBI, and ORD_NoLTBI. ResultsMultiple host biomarkers including IP10, IL6, IL2, IL1β, TNFα, IFNγ, and IL12p70, were significantly different between patients with PTB (n = 55), ORDs (n = 106), and between PTB and the two ORD sub-groups. A bio-signature comprising IP10, IL6, TNFα IL1β, IL1ra, and IL12p70 best diagnosed PTB disease, with an area under the ROC curve of 90. ConclusionWe identified host biomarkers that discriminated between different M.tb infection states amongst patients who presented with symptoms requiring investigation for TB. The biomarkers that showed diagnostic potential in our study may be considered as additional candidate markers for future active PTB rapid screening tests.

Prevention of Testicular Damage by Indole Derivative MMINA via Upregulated StAR and CatSper Channels with Coincident Suppression of Oxidative Stress and Inflammation: In Silico and In Vivo Validation.

Cis-diamminedichloroplatinum (II) (CDDP) is a widely used antineoplastic agent with numerous associated side effects. We investigated the mechanisms of action of the indole derivative N’-(4-dimethylaminobenzylidene)-2-1-(4-(methylsulfinyl) benzylidene)-5-fluoro-2-methyl-1H-inden-3-yl) acetohydrazide (MMINA) to protect against CDDP-induced testicular damage. Five groups of rats (n = 7) were treated with saline, DMSO, CDDP, CDDP + MMINA, or MMINA. Reproductive hormones, antioxidant enzyme activity, histopathology, daily sperm production, and oxidative stress markers were examined. Western blot analysis was performed to access the expression of steroidogenic acute regulatory protein (StAR) and inflammatory biomarker expression in testis, while expression of calcium-dependent cation channel of sperm (CatSper) in epididymis was examined. The structural and dynamic molecular docking behavior of MMINA was analyzed using bioinformatics tools. The construction of molecular interactions was performed through KEGG, DAVID, and STRING databases. MMINA treatment reversed CDDP-induced nitric oxide (NO) and malondialdehyde (MDA) augmentation, while boosting the activity of glutathione peroxidase (GPx) and superoxide dismutase (SOD) in the epididymis and testicular tissues. CDDP treatment significantly lowered sperm count, sperm motility, and epididymis sperm count. Furthermore, CDDP reduced epithelial height and tubular diameter and increased luminal diameter with impaired spermatogenesis. MMINA rescued testicular damage caused by CDDP. MMINA rescued CDDP-induced reproductive dysfunctions by upregulating the expression of the CatSper protein, which plays an essential role in sperm motility, MMINA increased testosterone secretion and StAR protein expression. MMINA downregulated the expression of NF-κB, STAT-3, COX-2, and TNF-α. Hydrogen bonding and hydrophobic interactions were predicted between MMINA and 3β-HSD, CatSper, NF-κβ, and TNFα. Molecular interactome outcomes depicted the formation of one hydrogen bond and one hydrophobic interaction between 3β-HSD that contributed to its strong binding with MMINA. CatSper also made one hydrophobic interaction and one hydrogen bond with MMINA but with a lower binding affinity of -7.7 relative to 3β-HSD, whereas MMINA made one hydrogen bond with NF-κβ residue Lys37 and TNF-α reside His91 and two hydrogen bonds with Lys244 and Thr456 of STAT3. Our experimental and in silico results revealed that MMINA boosted the antioxidant defense mechanism, restored the levels of fertility hormones, and suppressed histomorphological alterations.

Impact of automated methods for quantitative evaluation of immunostaining: Towards digital pathology.

IntroductionWe sought to develop a novel method for a fully automated, robust quantification of protein biomarker expression within the epithelial component of high-grade serous ovarian tumors (HGSOC). Rather than defining thresholds for a given biomarker, the objective of this study in a small cohort of patients was to develop a method applicable to the many clinical situations in which immunomarkers need to be quantified. We aimed to quantify biomarker expression by correlating it with the heterogeneity of staining, using a non-subjective choice of scoring thresholds based on classical mathematical approaches. This could lead to a universal method for quantifying other immunohistochemical markers to guide pathologists in therapeutic decision-making.MethodsWe studied a cohort of 25 cases of HGSOC for which three biomarkers predictive of the response observed ex vivo to the BH3 mimetic molecule ABT-737 had been previously validated by a pathologist. We calibrated our algorithms using Stereology analyses performed by two experts to detect immunohistochemical staining and epithelial/stromal compartments. Immunostaining quantification within Stereology grids of hexagons was then performed for each histological slice. To define thresholds from the staining distribution histograms and to classify staining within each hexagon as low, medium, or high, we used the Gaussian Mixture Model (GMM).ResultsStereology analysis of this calibration process produced a good correlation between the experts for both epithelium and immunostaining detection. There was also a good correlation between the experts and image processing. Image processing clearly revealed the respective proportions of low, medium, and high areas in a single tumor and showed that this parameter of heterogeneity could be included in a composite score, thus decreasing the level of discrepancy. Therefore, agreement with the pathologist was increased by taking heterogeneity into account.Conclusion and discussionThis simple, robust, calibrated method using basic tools and known parameters can be used to quantify and characterize the expression of protein biomarkers within the different tumor compartments. It is based on known mathematical thresholds and takes the intratumoral heterogeneity of staining into account. Although some discrepancies need to be diminished, correlation with the pathologist’s classification was satisfactory. The method is replicable and can be used to analyze other biological and medical issues. This non-subjective technique for assessing protein biomarker expression uses a fully automated choice of thresholds (GMM) and defined composite scores that take the intra-tumor heterogeneity of immunostaining into account. It could help to avoid the misclassification of patients and its subsequent negative impact on therapeutic care.

Anti-inflammatory potential of berberine-rich extract via modulation of inflammation biomarkers.

Berberine-rich extract (BRE) prepared from Berberis lycium root bark using green extraction approach and its marker compound berberine has a broad spectrum of clinical applications. Berberine's potential pharmacological effects include anticancer, antidiarrheal, antidiabetic, antimicrobial and anti-inflammatory activities. In current work, BRE and berberine were evaluated for their therapeutic prospects in inflammation models. The comparative effect of BRE and berberine against inflammation was determined through in vitro chemiluminescence technique. The in vivo anti-inflammatory evaluation of BRE and berberine (25, 75, and 125 mg/kg) compared to diclofenac (10mg/kg) was performed in carrageenan and formaldehyde-induced inflammation in Wistar rats. Histopathological and biochemical studies were conducted to find the comparative anti-inflammatory potential of BRE and berberine on pathological hallmarks induced by formaldehyde. Moreover, the modulatory effects on inflammatory biomarkers were also investigated through qPCR. ELISA (enzyme-linked immunoassay test assay) was performed to investigate the expression of pathological protein biomarkers like TNF-α and IL-6 and levels of antioxidant enzymes were estimated in liver homogenates. Both BRE and berberine markedly (p < .001) reduced paw diameter and inflammation in carrageenan and formaldehyde-induced inflammation. The levels of antioxidant enzymes were recovered (p < .001) by BRE and berberine treatments, and compared to the formaldehyde-treated inflammation model. Both BRE and berberine remarkably downregulated the mRNA and protein expression of inflammatory biomarkers. BRE similar to berberine mitigated the level of antioxidant enzymes in liver homogenate. The undertaken study suggests that BRE, a natural, green, and therapeutically bioequivalent to berberine could be used as an economical phytomedicine in the treatment of inflammatory disorders. PRACTICAL APPLICATIONS: Anti-inflammatory drugs like NSAIDS are associated with serious adverse effects like gastrointestinal ulcer, worsening of preexisting cardiovascular disorders, and renal failure. Therefore, there is a constant demand to develop novel, inexpensive therapeutic strategies to treat the inflammatory disorder with the least harmful effects. Pure phytochemicals with anti-inflammatory potential are costly and hard to isolate, therefore green microwave-assisted extraction technique is developed to get the rich bioequivalent extract. Berberis lycium a medicinal plant with berberine as a major bioactive constituent, has wide acceptance in traditionally used medicine and as food. Pharmacological studies revealed its hepatoprotective, anticancer, antidiabetic, and antihypertensive activities. BRE was prepared by green microwave-assisted extraction and enrichment by resin column to get a higher yield of berberine. The comparative anti-inflammatory effect of BRE and berberine was determined by in vitro and in vivo studies. Results obtained from this experimental work contribute beneficial guidance that reinforces the use of the BREto treat inflammatory disorders.

In Vitro Screening Method for Characterization of Macrophage Activation Responses.

Macrophage activation refers to the enhanced functionality of macrophages in response to endogenous or exogenous stimuli. Due to the existence of limitless stimuli and a multitude of receptors on macrophage surfaces, the nature of activation (or acquired functioning) can be specific to the encountering stimulus. This article describes a macrophage-activation screening platform in a 96-well format. The methodology involves the generation of bone marrow-derived macrophages, their activation into two extreme activation states, and screening of activated macrophages for expression of bonafide protein biomarkers. A high-throughput and stringent assay to determine macrophage activation markers developed in this article can be adapted for biomarker determination in pathological conditions and toxicant/drug safety screening.

Abstract 426: Development of oridonin-based proteolysis-targeting chimera (PROTAC) degraders as effective breast cancer therapeutics

Abstract Natural products provide a wealth of resources as the molecular starting point for the development of therapeutic agents, including a plethora of anticancer drugs currently in clinical use. Oridonin is a natural diterpene compound enriched in the medicinal herb Rabdosia rubescens, and its anticancer property has been extensively studied. However, the potency of oridonin against triple-negative breast cancer (TNBC) limits its potential clinical translation. Previously, we developed a class of oridonin analogues including CYD0618 and others as effective anticancer agents to inhibit tumor growth of TNBC in vitro and in vivo. Intriguingly, CYD0618 has been shown to directly bind to and inhibit STAT3 transcription factor, which is constitutively activated in the majority of human cancers. Currently there are no FDA-approved direct STAT3 inhibitors in clinical use. Based on the proteolysis-targeting chimera (PROTAC) technology, our team designed novel oridonin- and CYD0618-based PROTACs in an attempt to achieve specific STAT3 binding and protein degradation. We determined anticancer effects of oridonin- and CYD0618-PROTACs on proliferation, colony formation, and apoptosis of the TNBC cell line MDA-MB-231. We found that oridonin- and CYD0618-based PROTACs inhibited the proliferation of MDA-MB-231 cells significantly, similarly effective as the lead compound CYD0618 and much more potent than oridonin. We then examined the expression of STAT3, pSTAT3 and protein biomarkers for tumor metastasis using Western blot. STAT3 and pSTAT3 protein levels were downregulated dose- and time-dependently in MDA-MB-231 cells treated with CYD0618 and CYD0618-PROTACs, respectively. Our results demonstrate impressive effects of CYD0618-based PROTACs against TNBC cells, primarily through direct targeting of STAT3. We are in the process of further characterizing the molecular profile of CYD0618-based PROTACs by studying its interaction with STAT3 and protein degradation as well as their efficacy against TNBC tumor xenografts in vivo. Citation Format: Ruixia Ma, Pingyuan Wang, Junhai Xu, Jimin Xu, Doerte R. Fricke, Yu Xue, Hyejin Kim, Haiying Chen, Xi Liu, Jia Zhou, Qiang Shen. Development of oridonin-based proteolysis-targeting chimera (PROTAC) degraders as effective breast cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 426.

Effects of Soy-Based Infant Formula on Obesity and Neurodevelopment in an Autism Mouse Model

ObjectivesMice fed soy-based diets exhibit increased weight gain compared to mice fed casein-based diets and the effects are more pronounced in a model of fragile X syndrome (FXS; Fmr1KO). FXS is a neurodevelopmental disability characterized by intellectual impairment, seizures, autistic behavior, anxiety, and obesity. We hypothesize that high consumption of soy protein during postnatal development is a dietary exposure that increases the risk of developing obesity, particularly in vulnerable populations such as FXS. Here, we analyzed body weight as a function of mouse age, diet, and genotype to determine the effect of diet on weight gain. We also assessed plasma protein biomarker expression and behavior in mice in response to diet. Medical record and survey data were employed to identify associations between infant formula, weight gain and neurological development in children.MethodsMethods include prospective and retrospective evaluation of weight gain and neurological outcomes in mice and humans, respectively, as a function of postnatal diet.ResultsJuvenile Fmr1KO mice fed a soy protein-based rodent chow throughout gestation and postnatal development exhibit increased weight gain compared to mice fed casein-based purified ingredient diet or grain-based, low phytoestrogen chow. Adolescent and adult Fmr1KO mice fed a soy-based infant formula diet exhibited increased weight gain compared to reference diets. Increased body mass was due to increased lean mass. Wild type male mice fed soy-based infant formula exhibited increased learning in a passive avoidance paradigm and Fmr1KO male mice had a deficit in nest building. Mice consuming a soy-based infant formula diet exhibited altered expression of numerous plasma proteins including the adipose hormone leptin and the β-amyloid degrading enzyme neprilysin. Consumption of soy-based infant formula was not associated with obesity at 6 years of age in children but was associated with an increased need for support in school.ConclusionsIn conclusion, single-source, soy-based diets may contribute to the development of obesity and the exacerbation of neurological phenotypes in developmental disabilities through a β-amyloid-mediated pathway.Funding SourcesThis work was supported by the National Institutes of Health, FRAXA Research Foundation, RayBiotech and the United States Department of Agriculture.

Interpatient Heterogeneity in Drug Response and Protein Biomarker Expression of Recurrent Ovarian Cancer.

Simple SummaryRecurrent ovarian-cancer patients face low 5-year survival rates despite chemotherapy. Oncologists may choose from a variety of guideline-recommended second-line therapeutic options without knowing which one works best. Thus, therapy alterations and adjustments are often required. We analyzed the response that 30 tumor lesions had to certain treatments in our patient-derived ovarian-cancer spheroid model. In addition, we characterized samples by immunohistochemical staining for new druggable molecular targets. Our results might help in tailoring future therapies for individual patients with recurrent ovarian cancer.Recurrent ovarian-cancer patients face low 5-year survival rates despite chemotherapy. A variety of guideline-recommended second-line therapies are available, but they frequently result in trial-and-error treatment. Alterations and adjustments are common in the treatment of recurrent ovarian cancer. The drug response of 30 lesions obtained from 22 relapsed ovarian cancer patients to different chemotherapeutic and molecular agents was analyzed with the patient-derived ovarian-cancer spheroid model. The profile of druggable biomarkers was immunohistochemically assessed. The second-line combination therapy of carboplatin with gemcitabine was significantly superior to the combination of carboplatin with PEGylated liposomal doxorubicin (p < 0.0001) or paclitaxel (p = 0.0007). Except for treosulfan, all nonplatinum treatments tested showed a lesser effect on tumor spheroids compared to that of platinum-based therapies. Treosulfan showed the highest efficacy of all nonplatinum agents, with significant advantage over vinorelbine (p < 0.0001) and topotecan (p < 0.0001), the next best agents. The comparative testing of a variety of treatment options in the ovarian-cancer spheroid model resulted in the identification of more effective regimens for 30% of patients compared to guideline-recommended therapies. Recurrent cancers obtained from different patients revealed profound interpatient heterogeneity in the expression pattern of druggable protein biomarkers. In contrast, different lesions obtained from the same patient revealed a similar drug response and biomarker expression profile. Biological heterogeneity observed in recurrent ovarian cancers might explain the strong differences in the clinical drug response of these patients. Preclinical drug testing and biomarker profiling in the ovarian-cancer spheroid model might help in optimizing treatment management for individual patients.

Discovery, validation, and diagnostic ability of multiple protein-based biomarkers in saliva and gingival crevicular fluid to distinguish between health and periodontal diseases.

AimTo discover and validate differential protein biomarker expression in saliva and gingival crevicular fluid (GCF) to discriminate objectively between periodontal health and plaque‐induced periodontal disease states.Materials and MethodsOne‐hundred and ninety participants were recruited from two centres (Birmingham and Newcastle upon Tyne, UK) comprising healthy, gingivitis, periodontitis, and edentulous donors. Samples from the Birmingham cohort were analysed by quantitative mass spectrometry proteomics for biomarker discovery. Shortlisted candidate proteins were then verified by enzyme‐linked immunosorbent assay in both cohorts. Leave‐one‐out cross validation logistic regression analysis was used to identify the best performing biomarker panels.ResultsNinety‐five proteins were identified in both GCF and saliva samples, and 15 candidate proteins were selected based upon differences discovered between the donor groups. The best performing panels to distinguish between: health or gingivitis and periodontitis contained matrix metalloproteinase‐9 (MMP9), S100A8, alpha‐1‐acid glycoprotein (A1AGP), pyruvate kinase, and age (area under the curve [AUC] 0.970); health and gingivitis contained MMP9, S100A8, A1AGP, and pyruvate kinase, but not age (AUC 0.768); and mild to moderate and advanced periodontitis contained MMP9, S100A8, A1AGP, pyruvate kinase, and age (AUC 0.789).ConclusionsBiomarker panels containing four proteins with and without age as a further parameter can distinguish between periodontal health and disease states.