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3, based on ELISA data (left panel) and antibody microarray data (right panel). disease-specific sensor (clinical immunoproteomics). Uniquely, the results showed that several biologically relevant proteins reflecting histological grade could be delineated. In more detail, the tentative biomarker panels could be used to i) build a candidate model classifying grade 1 vs. grade 3 tumors, ii) demonstrate the molecular heterogeneity among grade 2 tumors, and iii) potentially re-classify several of the grade 2 tumors to more like grade 1 or grade 3 tumors. This could, in the long-term run, lead to improved prognosis, by which the patients could benefit from improved tailored care. Introduction More women are diagnosed with breast cancer than any other cancer form, affecting one in eight women during their lives [1] [[https://seer.cancer.gov/]. For patients diagnosed with breast cancer, WQ 2743 histological grade is one of the most commonly used prognostic factors [2, 3]. Histological grade describes the aggressive potential of the tumor, and is a combined score based on microscopic evaluation of the tubule formation, mitotic count, and nuclear pleamorphism [2, 3]. While grade 3 tumors are the most aggressive (highly proliferative) and poorly differentiated, grade 2 tumors are moderately differentiated, and grade 1 tumors are the least aggressive (slow growing) and well-differentiated [3]. However, concerns have been raised regarding the prognostic value of histological grade [4, 5]. These concerns reflect the shortcomings associated with conventional grading of breast cancer tumors using methods based on visual evaluation [4C8]. The patient cohort with grade 2 classified tumors, representing 30 to 60% of all patients, are in particular difficult to manage, as these tumors are very heterogeneous and less informative for clinical decision making [9]. Hence, new improved means to perform histological grading of breast cancer, and especially grade of grade 2 tumors, would thus be of significant clinical value. Several attempts have been made to subgroup breast cancer based on predominantly genetic signatures (hybridization) amplified tumors and all patients with an immunohistochemical 3+, where FISH analysis could not be evaluated, were considered HER2+ d) In cases where the sum is less than the number in the group, patient data are missing Extraction of proteins from solid tumors Protein was extracted from solid breast cancer tumor tissue and stored at -80C until use. Briefly, tissue pieces (about 50 mg/sample) were homogenized in Teflon containers with a metal ball, pre-cooled in liquid nitrogen, fixating the bomb in a shaker for two 30s periods with quick cooling in liquid nitrogen between the rounds. The homogenized samples were weighted and transferred to a collection tube and stored at -80C until use. Extraction of proteins from homogenized tissue followed previously described protocol [31, 32]. Briefly, 10 l of Extraction Buffer (100g/ml Soybean trypsin inhibitor, 350 g/ml PMSF, 0.01% (w/v) BSA and 2% (w/v) Saponin) was added per mg of sample and incubated on a rocking table at 4C o/n. Samples were centrifuged at 13,000g for TCL3 5 minutes, and supernatant was transferred to a new tube and stored at -80C until use. WQ 2743 Sample extracts were thawed on ice, and buffer was changed to PBS using Zeba desalt spin columns (Pierce Rockford, IL, USA). Protein concertation was determined using Total WQ 2743 Protein Kit, Micro Lowry (Sigma-Aldrich, St. Louis, MO, USA). Biotinylation of samples was done using EZ-link Sulfo-NHS-LC Biotin (Pierce Rockford) according to a previously optimized protocol [26, 27, 32]. Briefly, samples were diluted to approximately 2 mg/ml and biotin was added at a molar ratio of protein:biotin of 1 1:15, with a final biotin concentration of 0.6 mM. Reaction between biotin and protein was done at 4C for 2 hours, and excess biotin was removed through dialysis WQ 2743 against PBS for 72 hours at 4C. Biotinylated samples were aliquoted and transferred to new tubes and stored at -20C until use. Antibodies In total, 293 human recombinant single-chain fragment variable (scFv) antibodies (Table 2) were selected from large phage display libraries [33, 34]. Out of 293 antibodies, 262 were targeted against 98 known serum antigens, reflecting mainly immunoregulatory proteins. Each target had 1C9 clones directed against it to ensure antibody.