Breast Cancer Cell Lysates Research Articles

Boosting immunotherapy efficacy: Empowering the Potency of Dendritic cells loaded with breast cancer lysates through CTLA-4 suppression

Anticancer immunotherapies with a dendritic Cell (DC) basis are becoming more popular. However, it has been suggested that the tumor's immunosuppressive mechanisms, such as inhibitory immunological checkpoint molecules, reduce the effectiveness of anticancer immunogenicity mediated by DC. Thus, overcoming immune checkpoints and inducing effective antigen-specific T-cell responses uniquely produced with malignant cells represent the key challenges. Among the inhibitory immune checkpoints, DCs' ability to mature and present antigens is decreased by CTLA-4 expression. Consequently, we hypothesized that by expressing CTLA-4 cells on DCs, the T cells' activation against tumor antigens would be suppressed when confronted with these antigens presented by DCs. In this research, by loading cell lysate of breast cancer (BC) on DCs and the other hand by inhibiting the induction of CTLA-4 using small interfering RNA (siRNA), we assessed the functional activities and phenotypes of DCs, and also the responses associated with T-cells following co-culture DC/T cell. Our research has shown that the suppression of CTLA-4 enhanced the stimulating capabilities of DCs. Additionally, CTLA-4-suppressed BC cell lysate-loaded DCs produced more IL-4 and IFN-ϒ and increased T cell induction in contrast to DCs without CTLA-4 suppression. Together, our data point to CTLA-4-suppressed DCs loaded with BC cell lysate as a potentially effective treatment method. However, further research is required before employing this method in therapeutic contexts.

Reducing Cathodic Drift during Isoelectric Focusing Using Microscale Immobilized pH Gradient Gels.

Microfluidic analytical tools play an important role in miniaturizing targeted proteomic assays for improved detection sensitivity, throughput, and automation. Microfluidic isoelectric focusing (IEF) can resolve proteoforms in lysate from low-to-single cell numbers. However, IEF assays often use carrier ampholytes (CAs) to establish a pH gradient for protein separation, presenting limitations like pH instability in the form of cathodic drift (migration of focused proteins toward the cathode). Immobilized pH gradient (IPG) gels reduce cathodic drift by covalently immobilizing the pH buffering components to a matrix. To our knowledge, efforts to implement IPG gels at the microscale have been limited to glass microdevices. To adapt IEF using IPGs to widely used microfluidic device materials, we introduce a polydimethylsiloxane (PDMS)-based microfluidic device and compare the microscale pH gradient stability of IEF established with IPGs, CAs, and a hybrid formulation of IPG gels and CAs (mixed-bed IEF). The PDMS-based IPG microfluidic device (μIPG) resolved analytes differing by 0.1 isoelectric point within a 3.5 mm separation lane over a 20 min focusing duration. During the 20 min duration, we observed markedly different cathodic drift velocities among the three formulations: 60.1 μm/min in CA-IEF, 2.5 μm/min in IPG-IEF (∼24-fold reduction versus CA-IEF), and 1.4 μm/min in mixed-bed IEF (∼43-fold reduction versus CA-IEF). Lastly, mixed-bed IEF in a PDMS device resolved green fluorescent protein (GFP) proteoforms from GFP-expressing human breast cancer cell lysate, thus establishing stability in lysate from complex biospecimens. μIPG is a promising and stable technique for studying proteoforms from small volumes.

A review on polymeric nanocomposites for the electrochemical sensing of breast cancer biomarkers

Breast cancer is a rapidly spreading disease and early detection of associated biomarkers in body fluids, particularly blood, is crucial. Electrochemical biosensors, known for their high sensitivity, selectivity, cost-effectiveness, and miniaturization potential, have emerged as a promising tool for breast cancer screening. This review article focuses on the integration of polymers and nanosized objects to enhance the performance of electrochemical biosensors in analyzing breast cancer biomarkers in serum samples, breast cancer cell lysates, and tumor tissues. Recent developments in this field have demonstrated the significant benefits of these sensors, including improved sensitivity and selectivity. By incorporating nanocomposites into the electrochemical sensing interface, signal amplification is achieved, resulting in enhanced detection limits and accuracy. The use of different dimensionalities of nanomaterials (0D, 1D, 2D, and 3D nanostructured interfaces) in polymer-based nanocomposites has further contributed to the development of sensors with unique properties for sensitive and rapid detection. The potential of electrochemical biosensors based on polymer nanocomposites to revolutionize disease management lies in their ability to enable user-friendly breast cancer screening and facilitate early detection. However, there are still limitations that need to be addressed, and further research is required to optimize the performance of these sensors. The article also discusses the future perspectives and real-world applications of electrochemical biosensors based on polymer nanocomposites while considering the challenges that lie ahead.

Direct Competitive Assay for ERBB2 Detection in Breast Cancer Cell Lysates Using 1-D Photonic Crystals-Based Biochips

Direct Competitive Assay for ERBB2 Detection in Breast Cancer Cell Lysates Using 1-D Photonic Crystals-Based Biochips

An Activity-Based Oxaziridine Platform for Identifying and Developing Covalent Ligands for Functional Allosteric Methionine Sites: Redox-Dependent Inhibition of Cyclin-Dependent Kinase 4.

Activity-based protein profiling (ABPP) is a versatile strategy for identifying and characterizing functional protein sites and compounds for therapeutic development. However, the vast majority of ABPP methods for covalent drug discovery target highly nucleophilic amino acids such as cysteine or lysine. Here, we report a methionine-directed ABPP platform using Redox-Activated Chemical Tagging (ReACT), which leverages a biomimetic oxidative ligation strategy for selective methionine modification. Application of ReACT to oncoprotein cyclin-dependent kinase 4 (CDK4) as a representative high-value drug target identified three new ligandable methionine sites. We then synthesized a methionine-targeting covalent ligand library bearing a diverse array of heterocyclic, heteroatom, and stereochemically rich substituents. ABPP screening of this focused library identified 1oxF11 as a covalent modifier of CDK4 at an allosteric M169 site. This compound inhibited kinase activity in a dose-dependent manner on purified protein and in breast cancer cells. Further investigation of 1oxF11 found prominent cation-π and H-bonding interactions stabilizing the binding of this fragment at the M169 site. Quantitative mass-spectrometry studies validated 1oxF11 ligation of CDK4 in breast cancer cell lysates. Further biochemical analyses revealed cross-talk between M169 oxidation and T172 phosphorylation, where M169 oxidation prevented phosphorylation of the activating T172 site on CDK4 and blocked cell cycle progression. By identifying a new mechanism for allosteric methionine redox regulation on CDK4 and developing a unique modality for its therapeutic intervention, this work showcases a generalizable platform that provides a starting point for engaging in broader chemoproteomics and protein ligand discovery efforts to find and target previously undruggable methionine sites.

Two-dimensional magnetic bimetallic organic framework nanosheets for highly efficient enrichment of phosphopeptides.

Highly selective enrichment and sensitive detection of phosphopeptides is pivotal for comprehensive phosphoproteomics analysis; however, it also poses a long-standing challenge. Here, a novel two-dimensional (2D) magnetic bimetallic organic framework (MOF) nanosheet with Zr-O clusters and Ti-O clusters (denoted as the Fe3O4@Zr-Ti BPDC nanosheet) is prepared via a solvothermal method and in situ deposition of Fe3O4 nanoparticles for the first time. Taking advantage of the abundant dual affinities of Zr-O and Ti-O clusters for phosphopeptides, large surface area and high chemical stability, the Fe3O4@Zr-Ti BPDC nanosheets exhibit excellent enrichment performance for phosphopeptides. Within the framework of density functional theory, the interaction between Zr-O clusters, Ti-O clusters and phosphorylated molecules was studied to find the possible reason behind the superior adsorption performance of the bimetallic MOF nanosheets. We found that electrons would migrate from Ti to Zr spontaneously after doping Ti element and enhance the electrostatic traction between Zr species and phosphorylated molecules, demonstrating that the synergistic effect of Zr-Ti was helpful to improve the enrichment efficiency for phosphopeptides. Furthermore, the Fe3O4@Zr-Ti BPDC nanosheets showed good enrichment performance in complex bio-samples, including nonfat milk, human saliva, and a breast cancer cell lysate, indicating their tremendous potential in the analysis of trace phosphorylated biomolecules in complex bio-samples.

Abstract 733: Significance of PELP1/SETDB1 axis in endocrine therapy resistance

Abstract BACKGROUND: PELP1 is commonly overexpressed in breast cancer (BCa) and is implicated in endocrine therapy resistance. However, the mechanism by which PELP1 contributes to therapy resistance remains elusive. Our ongoing studies using yeast two hybrid screen identified histone methyltransferase SETDB1 as a novel interactor of PELP1. The objective of this study is to determine the significance of PELP1 interaction with SETDB1 in BCa endocrine therapy resistance. METHODS: We used two ER+ BCa cell lines (MCF7 and ZR75) and three endocrine therapy resistant cell lines (Tamoxifen resistant MCF7-TamR, Fulvestrant resistant MCF7-FR, Tamoxifen resistant MCF7-PELP1-cyto). Additional BCa models with stable overexpression and under expression of SETDB1 and PELP1 (MCF7-SETDB1-PELP1shRNA, ZR75-SETDB1-PELP1shRNA, ZR75-SETDB1, MCF7-SETDB1, MCF7-TamR-SETDB1shRNA, MCF7-FR-SETDB1shRNA) were generated using lentiviral transduction. Functional significance of cross-talk was tested using MTT, soft agar, and colony formation assays. Mechanistic studies were conducted using immunoprecipitation, Western blotting, and RT-qPCR. RESULTS: Analyses of TCGA databases showed that SETDB1 expression is positively correlated with PELP1 expression in ER+ BCa (r=0.30, p<0.0001). Immunoprecipitation assays using multiple ER+ BCa cell lysates confirmed the interaction of SETDB1 with PELP1. Using ER+ BCa models with overexpression of SETDB1 or knockdown, we provided evidence that SETDB1 plays an important role in the proliferation of BCa cells. PELP1 knockdown attenuated SETDB1 mediated BCa cells proliferation. SETDB1 upregulation contributed to tamoxifen resistance, while PELP1 knockdown re-sensitized cells to tamoxifen therapy. Further, endocrine therapy resistant model cells (MCF7-TamR, MCF7-FR, MCF7-PELP1-cyto) showed increased expression of SETDB1, and its knockdown sensitized them to endocrine therapy. Mechanistic studies revealed that PELP1 is needed for SETDB1 mediated Akt phosphorylation and activation of its downstream targets such as S6, and Cyclin D1. CONCLUSIONS: The results of study suggest that the PELP1/SETDB1 interactions contribute to endocrine therapy resistance via aberrant activation of Akt signaling. Targeting PELP1/SETDB1 axis could represent a new therapeutic avenue to mitigate endocrine resistance. Supported by VA grant I01BX004545 (R.K. Vadlamudi) Citation Format: Zexuan Liu, Junhao Liu, Weiwei Tang, Uday P. Pratap, Kristin A. Altwegg, Behnam Ebrahimi, Xiaonan Li, Gangadhara R. Sareddy, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi. Significance of PELP1/SETDB1 axis in endocrine therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 733.

Abstract PS17-34: Proto-oncogene PELP1 interactions with SETDB1 contribute to aberrant activation of AKT1 in breast cancer

Abstract BACKGROUND: Hyperactivation of PI3K/Akt signaling is implicated in breast cancer (BCa) progression. SETDB1, a methyltransferase, is also implicated in BCa, however, the mechanism remains elusive. Recent studies have shown SETDB1 can methylate non-histone substrates such as Akt1 and contribute to its aberrant activation, thus leading to tumor progression. PELP1 is a proto-oncogene which is overexpressed in BCa and participates in both the nuclear and extra nuclear functions of various nuclear receptors. In a subset of BCa, PELP1 uniquely localizes in the cytoplasm and contributes to endocrine therapy resistance. The objective of this study is to characterize the significance of PELP1 in SETDB1 mediated Akt signaling in BCa progression. METHODS: Using yeast two-hybrid screening, we identified PELP1 as a novel interacting protein of SETDB1. Functional significance of cross-talk was tested using MTT, proliferation, stemness and colony formation assays. Mechanistic studies were conducted using immunoprecipitation, RNA-seq, shRNA, overexpression, Western blotting, and RT-qPCR. Biological significance of PELP1 and SETDB1 in endocrine therapy resistance was also examined. RESULTS: Analyses of TCGA databases showed that SETDB1 is highly expressed in BCa and associated with poor clinical outcome. Further, SETDB1 expression is positively correlated with PELP1 expression in BCa (r=0.30, p<0.0001). Immunoprecipitation assays using multiple BCa cell lysates confirmed the interaction of SETDB1 with PELP1. Using two different shRNAs targeting SETDB1 and multiple BCa model cells, we provided evidence that SETDB1 plays an important role in the proliferation of BCa cells. SETDB1 upregulation is sufficient to accelerate proliferation and PELP1 knockdown attenuated SETDB1 oncogenic functions. SETDB1 overexpression contributed to resistance to tamoxifen treatment, while PELP1 knockdown re-sensitized cells to therapy. RNA-seq identified Akt signaling pathways were activated by SETDB1 in BCa. Mechanistic studies showed that SETDB1 overexpression increased Akt phosphorylation and its downstream signaling, while PELP1 knock down attenuated SETDB1 mediated Akt activation in both MCF7 and ZR75 models. Furthermore, in BCa model cells that uniquely express PELP1 in cytoplasm, knockdown of SETDB1 reduced activation of Akt and its downstream pathways. CONCLUSIONS: Our study results suggest that the PELP1/SETDB1 interactome plays an important role in aberrant Akt activation. Drugs that target PELP1/SETDB1 axis may be useful in modulating aberrant Akt signaling. Supported by VA grant I01BX004545. Citation Format: Zexuan Liu, Junhao Liu, Weiwei Tang, Uday P. Pratap, Kristin A. Altwegg, Xiaonan Li, Suryavathi Viswanadhapalli, Ratna Vadlamudi. Proto-oncogene PELP1 interactions with SETDB1 contribute to aberrant activation of AKT1 in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-34.

Exosomes exist in nipple discharge of breast cancer.

Exosomes exist in nipple discharge of breast cancer.

A near-infrared-II fluorescence anisotropy strategy for separation-free detection of adenosine triphosphate in complex media

Fluorescence anisotropy (FA) has been widely applied for detecting and monitoring special targets in life sciences. However, matrix autofluorescence restricted its further application in complex biological samples. Herein, we report a near-infrared-II (NIR-II) FA strategy for detecting adenosine triphosphate (ATP) in human serum samples and breast cancer cell lysate, which employed NIR-II fluorescent Ag2Se quantum dots (QDs) as tags to reduce matrix autofluorescence effect and applied graphene oxide (GO) to enhance fluorescence anisotropy signals. In the presence of ATP, the recognition between NIR-II Ag2Se QDs labeled aptamer (QD-pDNA) and ATP led to the release of QD-pDNA from GO, resulting in the obvious decrease of FA values. ATP could be quantitatively detected in concentrations ranged from 3 nM to 2500 nM, with a detection limit down to 1.01 nM. This study showed that the developed NIR-II FA strategy could be applied for detecting targets in complex biological samples and had great potential for monitoring interactions between biomolecules in biomedical research.

Abstract 4371: PELP1-TFAP2C crosstalk promotes endocrine resistance in breast cancer cells

Abstract Background: A significant proportion of estrogen receptor positive (ER+) breast cancer (BC) initially respond to antiestrogens or aromatase inhibitors but become therapy resistant-BC. Development of effective therapies for endocrine therapy resistant BC represent the highest unmet need. PELP1 is a nuclear receptor coregulator, commonly overexpressed in BC, contributes to therapy resistance and correlate with poor survival. TFAP2C (transcription factor AP-2 gamma) is a known regulator of ER activity and high expression of TFAP2C is associated with a decreased response to Fulvestrant. The objective of this study is to examine the significance of PELP1-TFAP2C crosstalk in the development of breast cancer therapy resistance. Methods: We used yeast two-hybrid screen to identify PELP1 interacting transcription factors. Interaction of PELP1 and TFAP2C was confirmed by immunoprecipitation and GST pull down assays. Functional significance of the cross talk was tested using CellTiter-Glo, MTT and colony formation assays in the presence or absence of endocrine therapy. Mechanistic studies were conducted using shRNA, overexpression, Western blotting, reporter gene assays, RT-qPCR, ChIP-seq and RNA-seq analysis. Biological significance of PELP1 and TFAP2C in endocrine therapy resistance was examined using overexpression and under expression models of PELP1 and TFAP2C in multiple BC models including MCF7 and ZR75. Results: Yeast based screening of a mammary gland cDNA expression library using PELP1 as the bait identified TFAP2C as a novel interacting protein of PELP1. Immunoprecipitation assays using multiple BC cell lysates confirmed the interaction of PELP1 with TFAP2C. Using various deletions of PELP1, and by using GST pull down assays, we identified N-terminal 400-600aa region of PELP1 as the major interaction site for TFAP2C. Using RNA-seq of PELP1 knockdown BC model cells, we predicted TFAP2C as an enriched transcription factor in PELP1 regulated genes. The GSEA results from RNA-seq showed TFAP2C and PELP1 induce a subset of common genes. Reporter gene assays confirmed that PELP1 functions as a coactivator of TFAP2C. Mechanistic studies showed that TFAP2C activates both AKT and ERK pathways in ER+ cell lines, while knock down of PELP1 attenuated these effects. Overexpression of TFAP2C contributed to increased cell proliferation and endocrine therapy resistance in MCF7 and ZR75 models, while knock down of PELP1 attenuated these effects. Utilizing ZR75-TFAP2C xenograft models with WT PELP1 or PELP1 knock down, we provided genetic evidence that endogenous PELP1 is essential for TFAP2C drive n breast cancer progression in vivo. Conclusions: Collectively, our studies demonstrated that PELP1 functions as a coactivator of TFAP2C in modulating a set of ER target genes, TFAP2C functions as a transcription factor of PELP1 regulated genes and blocking the PELP1-TFAP2C axis could have therapeutic utility for treating therapy resistance. Supported by VA grant I01BX004545 (R.K. Vadlamudi) Citation Format: Junhao Liu, Suryavathi Viswanadhapalli, Mengxing Li, Uday P. Pratap, Weiwei Tang, Zexuan Liu, Yiliao Luo, Kristin A. Altwegg, Xiaonan Li, Gangadhara R. Sareddy, Rajeshwar R. Tekmal, Ratna K. Vadlamudi. PELP1-TFAP2C crosstalk promotes endocrine resistance in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4371.

Plasmonic Modulation of the Upconversion Luminescence Based on Gold Nanorods for Designing a New Strategy of Sensing MicroRNAs.

Upconversion nanoparticles (UCNPs) have potential applications in biosensing and bioimaging. However, the UCNPs-based sensors constructed by luminescence resonance energy transfer (LRET) always suffer from low quenching efficiency, hindering their application. Therefore, exploring a new strategy to resolve this issue is highly desirable. Herein, a strategy based on the surface plasmon resonance (SPR) effect of gold nanorods (AuNRs) is presented. The luminescence of UCNPs was modulated by adjusting the SiO2 thickness of AuNRs@SiO2 and the structure of UCNPs; an enhancement factor of ≈50 times was obtained. Based on the results of the SPR effect of AuNRs, we designed two kinds of potential upconversion microRNA sensors using microRNA-21 as a model to resolve the problem of the lower quenching efficiency resulting from a dye as a quencher. Studies revealed that the proposed strategy could be successfully used to construct upconversion microRNA sensors for avoiding the limitation of the low quenching efficiency. The sensitivity was ≈10 000 times higher than that of the upconversion sensor using dyes as quenchers. Importantly, the assay of microRNA-21 was successfully achieved using this sensor in human serum samples and human breast cancer cell (MCF-7) lysates. It provides a new method for designing upconversion microRNA sensors and may have potential for use in biosensing and bioimaging.

Membrane-Based Affinity Purification to Identify Target Proteins of a Small-Molecule Drug

Identifying the target proteins of small-molecule drug candidates is important for determining their molecular mechanisms of action. Porous membranes derivatized with such small molecules may provide an attractive target-identification platform due to a high protein-capture efficiency during flow through membrane pores. This work employs carbonic anhydrase II (CAII) binding to immobilized 4-(2-aminoethyl)benzenesulfonamide (AEBSA) to examine the efficiency and selectivity of affinity capture in modified membranes. Selective elution of captured protein, tryptic digestion, tandem mass spectrometry analysis, and label-free quantification (LFQ) identify CAII as the dominant AEBSA target in diluted serum or cell lysate. CAII identification relies on determining the ratio of protein LFQ intensities in sample and control experiments, where free AEBSA added to the control loading solution limits CAII capture. Global proteomics shows that the spiked CAII is the only protein with a log2 ratio consistently >2, and the detection limit for CAII identification is 0.004 wt % of the total protein in 1:4 diluted human serum or 0.024 wt % of the total protein from breast cancer cell lysates. The same approach also identifies native CAII in human kidney cell lysate as an AEBSA target. Comparison of affinity capture using membranes, Affi-Gel 10 resin or M-270 Dynabeads derivatized with AEBSA suggests that only membranes allow identification of low-abundance CAII as a target.

One-step 18F-fluorination of smart positron emission tomography tracer for sensing furin activity in tumors

IntroductionPeptide analogues have attracted considerable attention in the field of developing novel positron emission tomography (PET) imaging agents due to their unique properties. Nevertheless, the complicated radiolabeling process and fast metabolism usually pose challenges to the clinical applications of peptide-based molecular probes. Herein a novel PET tracer containing a specific peptide sequence Arg-Val-Arg-Arg (RVRR), Acetyl-Arg-Val-Arg-Arg-Cys(StBu)-Gly(AMB[18F]F3)-CBT ([18F]1), was designed and radiosynthesized using a simple and convenient one-step 18F-fluorination procedure. The smart tracer can be activated by the protease furin and then undergoes an intermolecular cyclization reaction in tumor cells, leading to improved PET imaging efficiency of tumor. MethodsThe radiosynthesis of the target tracer [18F]1 and the control tracer [18F]1-ctrl was performed under facile conditions in pyridazine-HCl buffer (pH~2.5) at 80 °C within 30 min. The enzyme-controlled condensation was studied for non-radioactive compound 1 in the human breast cancer cell lysates (MDA-MB-468). The cellular uptake of [18F]1 and [18F]1-ctrl was studied and compared by measuring the activity in MDA-MB-468 cells using a γ-counter after incubation with 37 kBq of [18F]1 or [18F]1-ctrl, respectively. In vivo behavior of [18F]1 was examined through PET imaging of MDA-MB-468 tumor-bearing mice and compared with that of [18F]1-ctrl as well as that of [18F]1 co-injected with non-radioactive compound 1. ResultsThe tracer [18F]1 was obtained with a high radiochemical yield (RCY) of 42.5 ± 1.47% and an excellent radiochemical purity (RCP > 99%). Under the activation of furin and GSH, the tracer suffered a condensation reaction to form dimers and then self-assembled into nanoparticles to produce enduring signal. The cellular uptake of [18F]1 and [18F]1-ctrl was determined to be 10.2 ± 0.37 and 1.19 ± 0.25%ID at 120 min, respectively. For in vivo PET imaging, [18F]1 exhibited the optimum tumor uptake of 2.39 ± 0.31%ID/g and the tumor-to-muscle uptake ratio of 2.93 ± 0.92 at 10 min post injection. Co-injection of [18F]1 and non-radioactive compound 1 produced a high tumor uptake ranging from 2.83 ± 0.23%ID/g to 3.40 ± 0.18%ID/g at 10 min and 60 min post injection, respectively. ConclusionsThe one-step labeling method of tracer [18F]1 showed advantage in simplifying the radiolabeling process with high RCY, which could enable a real kit process for the synthesis of 18F-radiopharmaceuticals and was significant for the large-scale production of tracers for clinical applications. PET imaging results suggested that the tracer [18F]1 had good tumor uptake and the co-injection strategy of [18F]1 with 1 could enhance the imaging signal in tumor.

A carbon nanoparticle and DNase I-Assisted amplified fluorescent biosensor for miRNA analysis

Nucleic acid-based biosensors have become powerful tools in biomedical applications. But the stability issue seriously limits their wide applications. Fortunately, the emergence of carbon nanoparticles (CNPs), which can effectively protect DNA probes from enzymatic digestion and unspecific protein binding, provides a good solution. In this work, a DNase I-aided cyclic enzymatic amplification method (CEAM) for microRNA analysis has been developed based on the coupling use of nucleic acid probes with specific molecular recognition ability as well as CNPs with excellent biostability. The method is simple and sensitive, with a detection limit down to 3.2 pM. Furthermore, satisfactory results are achieved for miRNA analysis in breast cancer cell lysate, demonstrating the applicability in disease diagnosis. The ingenious combination of CNPs and nucleic acid probes can open a new chapter in the development of versatile analytical strategies that holds great potentials for clinical diagnosis, food safety, and environmental monitoring.

Abstract P6-04-14: The role of PELP1-TFAP2C crosstalk in mediating endocrine-therapy-resistance in breast cancer

Abstract Background: A significant proportion of estrogen receptor (ER) positive (ER+) breast cancer (BC) initially respond to endocrine therapies, such as antiestrogens or aromatase inhibitors. However, ER+ BC can build up resistance to treatment progressing into therapy resistant-BC (TR-BC). Development of effective therapeutics for endocrine-therapy-resistant BC represents a significant unmet need in BC treatment options. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is oncogenic nuclear receptor coregulator, commonly overexpressed in BC. PELP1 overexpression is correlated with poorer patient survival and is associated with development of TR-BC. TFAP2C (AP2Gamma) is a known regulator of ER activity. In addition, high expression of TFAP2C is associated with a decreased response to the steroidal antiestrogen, Fulvestrant. However, it remains unknown whether PELP1 and TFAP2C crosstalk and if the PELP1/TFAP2C axis behaves synergistically to contribute to the development of therapy resistance in TR-BC. Methods: To gain insight into PELP1 signaling mechanisms, we used yeast two-hybrid screen to identify proteins that interact with PELP1. The interaction between PELP1 and TFAP2C was confirmed by immunoprecipitation using both endogenous and GFP tagged proteins. GST fusions of various domains of PELP1 were used to identify the TFAP2C interacting domain. Functional significance of the crosstalk was tested using Celltiter Glo, MTT, apoptosis, and invasion assays. Mechanistic studies were conducted using shRNA, overexpression, Western Blot, reporter gene assays, RT-qPCR, ChIP and RNA-Seq analysis. Biological significance of PELP1 and TFAP2C in endocrine-therapy-resistance was examined using overexpression and under-expression models of PELP1 and TFAP2C in multiple ER+ BC and TR-BC model cells (MCF-7, ZR-75, T-47D, MCF-7-TamR, MCF7-LTLT and ZR75-ERMT537S. Results: Screening of a mammary gland cDNA expression library using PELP1 as the bait identified TFAP2C as a novel interacting protein of PELP1. Immunoprecipitation assays utilizing multiple BC cell lysates confirmed interaction of PELP1 with TFAP2C. We also confirmed PELP1 and TFAP2C interactions using GFP and GST epitope tagged proteins. Using GST fusion of various domains of PELP1, we identified the PELP1 N-terminal domain (aa 400-600) as the major interaction site for TFAP2C. Using PELP1 knockdown BC model cell lines with RNA-Seq analysis, we identified a set of genes regulated by PELP1. The GSEA results from the RNA-Seq data predicted TFAP2C as an enriched transcription factor in a subset of PELP1 regulated genes. RT-qPCR analysis confirmed that PELP1 is needed for optimal regulation of the ER and associated ER target genes by TFAP2C. Reporter gene assays confirmed that PELP1 functions as a coregulator of TFAP2C. Overexpression of TFAP2C contributed to endocrine-therapy-resistance in BC model cells, while knockdown of PELP1 abolished TFAP2C mediated therapy resistance. Conclusions: Collectively, our studies identified PELP1 functions as a coregulator of TFAP2C in modulating ER target genes. TFAP2C functions as a transcription factor of PELP1 regulated genes and blocking the PELP1-TFAP2C axis will have therapeutic utility for treating TR-BC Citation Format: Junhao Liu, Suryavathi Viswanadhapalli, Mengxing Li, Uday P Pratap, Yiliao Luo, Kristin A Altwegg, Xiaonan Li, Gangadhara R Sareddy, Rajeshwar R Tekmal, Ratna K Vadlamudi. The role of PELP1-TFAP2C crosstalk in mediating endocrine-therapy-resistance in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-14.

Ultrasensitive immunoassay of tumor protein CA 15.3 in MCF-7 breast cancer cell lysates and unprocessed human plasma using gold nanoparticles doped on the structure of mesoporous silica

Breast cancer is the most common threat in women worldwide. Increasing death rate of diagnosed cases is the main leading cause of designing specific immunoassay for tumor marker in breast cancer. Cancer antigen 15-3 (CA 15-3) is a tumor protein for many types of cancer, most notably breast cancer. Herein, we report a sandwich-type electrochemical immunosensor based on signal amplification strategy of multiple nanocomposites to detection of CA 15-3 biomarker. The proposed immunosensor fabricated by reduced graphene oxide (rGO), poly-dopamine (PDA) and amino functionalized mesoporous silica (MCM-41-NH2) doped by gold nanoparticles composite on the glassy carbon electrode with a large surface area which was prepared a new platform to immobilization of primary antibodies and provide excellent conductivity. To further amplify the electrochemical signal, the trace tag on the foundation of gold nanoparticles (AuNPs) is coated with MCM-41-NH2 nanocomposite through thionine linking, which provides more amino groups to capture more horseradish peroxidase-labeled antibodies (HPR-Ab2) and enhances the conductivity. Under optimal conditions, the developed immunosensor exhibits excellent analytical performance for the determination of CA 15-3 with a wide linear range from 0.002 to 125 U/mL and a low limit of quantification of 0.002 U/mL. Furthermore, satisfactory results are obtained for the determination of CA 15-3 in human plasma samples, indicating the potential of the immunoassay to be applied in clinical analysis.

An innovative immunosensor for ultrasensitive detection of breast cancer specific carbohydrate (CA 15-3) in unprocessed human plasma and MCF-7 breast cancer cell lysates using gold nanospear electrochemically assembled onto thiolated graphene quantum dots

The accurate quantification of the level of breast cancer specific protein CA 15-3 in serum is crucial for cancer prognosis. This work, a novel and sensitive label-free immunoassay based on gold nanospear (Au NSs) electrochemically assembled onto thiolated graphene quantum dots (CysA/GQDs) for the detection of CA 15-3 antibodies. The CysA/Au NSs/GQDs hybrid interface provides a large surface area for the effective immobilization of CA 15-3 antigens, as well as it ascertains the bioactivity and stability of immobilized CA 15-3 antigens. Field emission scanning electron microscope (FE-SEM), and EDS photoelectron spectroscopies were used to monitor the sensor fabrication. Also, cyclic voltammetry was used to quantify the extent of Au NSs' surface coverage by CA 15-3 antigens. Square wave voltammetry (SWV) was employed to investigate the immunosensor fabrication and to monitor the binding events between CA 15–3 antigens-antibodies. Under optimized experimental conditions, the immunosensor displayed good sensitivity and specificity. The CA 15-3 were detected in a concentration as low as 0.11U/mL with a linear range from 0.16–125U/mL. The high sensitivity of the immunosensor may derive from the high loading of CA 15-3 antibodies on CysA/Au NSs/GQDs hybrid interface which increases the number of binding events. The method was successfully applied assay of the CA 15-3 in unprocessed human plasma samples. Also, proposed immunosensor was applied to the assay of CA 15-3 malignant cell line lysates (human breast adenocarcinoma cell line-MCF-7).

Cancer Diagnosis: A Universal Upconversion Sensing Platform for the Sensitive Detection of Tumour‐Related ncRNA through an Exo III‐Assisted Cycling Amplification Strategy (Small 10/2018)

In article number 1703858, Xingcai Wu, Jun-Jie Zhu, and co-workers report a universal and sensitive upconversion sensing nanoplatform for detecting tumor-related non-coding RNA. The sensitivity of the probe is improved by tuning the number of DNA units on the gold nanoparticles, and an exonuclease III-assisted target cycling amplification strategy improves the sensitivity and achieves the universality of this sensing nanoplatform. Through this method, the expression level of microRNA-21 in human breast cancer cell lysate is successfully measured, suggesting its potential in cancer diagnosis.

A Universal Upconversion Sensing Platform for the Sensitive Detection of Tumour‐Related ncRNA through an Exo III‐Assisted Cycling Amplification Strategy

Here, a sensitive and universal noncoding RNA (ncRNA) upconversion sensing nanoplatform is developed. Gold nanoparticles bearing one hairpin DNA (Hp) molecule are conjugated to the linker DNA modified NaYF4 :Yb, Er@NaYF4 upconversion nanoparticles by DNA hybridization, leading to quenching of the upconversion emission through fluorescence resonance energy transfer. A signal DNA (SDNA) sequence is designed to open Hp, recovering the upconversion emission. To achieve universality and high sensitivity of the nanoprobe, an exonuclease III (Exo III)-assisted cycling amplification strategy is introduced. A multifunctional hairpin DNA (mHp) containing ncRNA recognition sequence and SDNA sequence is designed to recognize ncRNA and trigger Exo III as a biocatalyst to stepwise disintegrate itself, releasing both ncRNA and SDNA. The released ncRNA can be reused to release more SDNA, which greatly improves the sensing sensitivity. By changing the recognition portion of mHp, various ncRNA can be detected. The sensitive detection of both homeobox (HOX) transcript antisense RNA segment and miR-21 is achieved with this novel strategy, even in human serum, indicating the universality and sensitivity of the proposed strategy. Additionally, the expression level of miR-21 in human breast cancer cell (MCF-7) lysate is successfully measured, suggesting its potential in clinical diagnosis.