Protein multiplex sandwich immunoassay: A reliable and objective method for complete molecular characterization of breast cancer from core needle biopsies

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 20025-20025 ◽  
Author(s):  
G. Sauer ◽  
N. Schneiderhahn-Marra ◽  
M. Bãuerle ◽  
C. Kurzeder ◽  
K. Koretz ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Protein Microarray ◽  
Sandwich Immunoassay ◽  
Therapeutic Options ◽  
Clinical Samples ◽  
General Interest ◽  
Data Sets ◽  
Microarray Technology ◽  
Core Needle ◽  
Marker Proteins

20025 Background: Within the last years, protein microarrays have been developed to quantify a large number of parameters present in a given sample simultaneously. Such miniaturised and parallelised sandwich immunoassays are of general interest for all proteomic and diagnostic approaches in which several parameters have to be determined from small samples. We describe the development of a bead-based flow cytometry that represents a convenient approach for rapid multiplex detection of functional target molecules from breast cancer biopsies. Methods: Briefly, sonographically guided core needle biopsies (CNB) were performed from 120 breast cancer masses from different patients. All biopsies were carried out with an automated core biopsy device fitted with 14-gauge (22 mm excursion) needles. Tumor samples were frozen on at −70 Celsius and pulverized. Proteins were extracted, allowing the simultaneous quantification of more than hundred proteins by a bead-based multiplex sandwich immunoassay. Results: The total amount of extracted protein from tumor tissues (mean weight of 20 mg) ranged from 300 to 1000 μg. We demonstrate appropriate sensitivity, reproducibility, and robustness for this protein microarray technology to characterize clinical samples and generate reliable data sets. A set of multiplexed sandwich immunoassays based on Luminex beads were developed that identify marker proteins indicative of prognosis or response to therapeutic options. Up to almost hundred parameters were identified and quantified simultaneously of which estrogen, progesterone and HER2 status in the multiplex assay was highly significant correlated to results obtained by immunohistochemistry (IHC) (p < 0.01). Conclusions: Our results demonstrate that this new bead-based protein microarray technology rapidly and reliably characterizes tissue samples to generate data sets of molecular marker. Therefore, this technology may once replace traditional IHC in determining marker proteins indicative of prognosis or response to therapeutic options. No significant financial relationships to disclose.

Integrated Quantitative Analysis of the Phosphoproteome and Transcriptome in Tamoxifen-resistant Breast Cancer*

2020 ◽  
Author(s):  
Lungwani Muungo
Keyword(s):  
Breast Cancer ◽  
High Rate ◽  
System Level ◽  
Clinical Samples ◽  
Transcriptome Data ◽  
Data Set ◽  
Reporter Assays ◽  
Treated Breast ◽  
Mcf 7 ◽  
Resistant Cells

Quantitative phosphoproteome and transcriptome analysisof ligand-stimulated MCF-7 human breast cancer cells wasperformed to understand the mechanisms of tamoxifen resistanceat a system level. Phosphoproteome data revealed thatWT cells were more enriched with phospho-proteins thantamoxifen-resistant cells after stimulation with ligands.Surprisingly, decreased phosphorylation after ligand perturbationwas more common than increased phosphorylation.In particular, 17?-estradiol induced down-regulation inWT cells at a very high rate. 17?-Estradiol and the ErbBligand heregulin induced almost equal numbers of up-regulatedphospho-proteins in WT cells. Pathway and motifactivity analyses using transcriptome data additionallysuggested that deregulated activation of GSK3? (glycogensynthasekinase 3?) and MAPK1/3 signaling might be associatedwith altered activation of cAMP-responsive elementbindingprotein and AP-1 transcription factors intamoxifen-resistant cells, and this hypothesis was validatedby reporter assays. An examination of clinical samples revealedthat inhibitory phosphorylation of GSK3? at serine 9was significantly lower in tamoxifen-treated breast cancerpatients that eventually had relapses, implying that activationof GSK3? may be associated with the tamoxifen-resistantphenotype. Thus, the combined phosphoproteomeand transcriptome data set analyses revealed distinct signal

scholarly journals The Mechanical Fingerprint of Circulating Tumor Cells (CTCs) in Breast Cancer Patients

Cancers ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1119
Author(s):  
Ivonne Nel ◽  
Erik W. Morawetz ◽  
Dimitrij Tschodu ◽  
Josef A. Käs ◽  
Bahriye Aktas
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Hematopoietic Cells ◽  
Surrogate Marker ◽  
Clinical Samples ◽  
Breast Cancer Patients ◽  
Shape Restoration

Circulating tumor cells (CTCs) are a potential predictive surrogate marker for disease monitoring. Due to the sparse knowledge about their phenotype and its changes during cancer progression and treatment response, CTC isolation remains challenging. Here we focused on the mechanical characterization of circulating non-hematopoietic cells from breast cancer patients to evaluate its utility for CTC detection. For proof of premise, we used healthy peripheral blood mononuclear cells (PBMCs), human MDA-MB 231 breast cancer cells and human HL-60 leukemia cells to create a CTC model system. For translational experiments CD45 negative cells—possible CTCs—were isolated from blood samples of patients with mamma carcinoma. Cells were mechanically characterized in the optical stretcher (OS). Active and passive cell mechanical data were related with physiological descriptors by a random forest (RF) classifier to identify cell type specific properties. Cancer cells were well distinguishable from PBMC in cell line tests. Analysis of clinical samples revealed that in PBMC the elliptic deformation was significantly increased compared to non-hematopoietic cells. Interestingly, non-hematopoietic cells showed significantly higher shape restoration. Based on Kelvin–Voigt modeling, the RF algorithm revealed that elliptic deformation and shape restoration were crucial parameters and that the OS discriminated non-hematopoietic cells from PBMC with an accuracy of 0.69, a sensitivity of 0.74, and specificity of 0.63. The CD45 negative cell population in the blood of breast cancer patients is mechanically distinguishable from healthy PBMC. Together with cell morphology, the mechanical fingerprint might be an appropriate tool for marker-free CTC detection.

scholarly journals Splicing factor SRSF1 promotes breast cancer progression via oncogenic splice switching of PTPMT1

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Jun-Xian Du ◽  
Yi-Hong Luo ◽  
Si-Jia Zhang ◽  
Biao Wang ◽  
Cong Chen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Progression ◽  
High Risk Group ◽  
Clinical Samples ◽  
Binding Motif ◽  
Ki 67 ◽  
Tissue Samples

Abstract Background Intensive evidence has highlighted the effect of aberrant alternative splicing (AS) events on cancer progression when triggered by dysregulation of the SR protein family. Nonetheless, the underlying mechanism in breast cancer (BRCA) remains elusive. Here we sought to explore the molecular function of SRSF1 and identify the key AS events regulated by SRSF1 in BRCA. Methods We conducted a comprehensive analysis of the expression and clinical correlation of SRSF1 in BRCA based on the TCGA dataset, Metabric database and clinical tissue samples. Functional analysis of SRSF1 in BRCA was conducted in vitro and in vivo. SRSF1-mediated AS events and their binding motifs were identified by RNA-seq, RNA immunoprecipitation-PCR (RIP-PCR) and in vivo crosslinking followed by immunoprecipitation (CLIP), which was further validated by the minigene reporter assay. PTPMT1 exon 3 (E3) AS was identified to partially mediate the oncogenic role of SRSF1 by the P-AKT/C-MYC axis. Finally, the expression and clinical significance of these AS events were validated in clinical samples and using the TCGA database. Results SRSF1 expression was consistently upregulated in BRCA samples, positively associated with tumor grade and the Ki-67 index, and correlated with poor prognosis in a hormone receptor-positive (HR+) cohort, which facilitated proliferation, cell migration and inhibited apoptosis in vitro and in vivo. We identified SRSF1-mediated AS events and discovered the SRSF1 binding motif in the regulation of splice switching of PTPMT1. Furthermore, PTPMT1 splice switching was regulated by SRSF1 by binding directly to its motif in E3 which partially mediated the oncogenic role of SRSF1 by the AKT/C-MYC axis. Additionally, PTPMT1 splice switching was validated in tissue samples of BRCA patients and using the TCGA database. The high-risk group, identified by AS of PTPMT1 and expression of SRSF1, possessed poorer prognosis in the stage I/II TCGA BRCA cohort. Conclusions SRSF1 exerts oncogenic roles in BRCA partially by regulating the AS of PTPMT1, which could be a therapeutic target candidate in BRCA and a prognostic factor in HR+ BRCA patient.

Sign in / Sign up

Export Citation Format

Share Document