A novel nano-immunoassay method for quantification of proteins from CD138-purified myeloma cells: biological and clinical utility.
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ABSTRACT: Protein analysis in bone marrow samples from patients with multiple myeloma has been limited by the low concentration of proteins obtained after CD138+ cell selection. A novel approach based on capillary nano-immunoassay could make it possible to quantify dozens of proteins from each myeloma sample in an automated manner. Here we present a method for the accurate and robust quantification of the expression of multiple proteins extracted from CD138-purified multiple myeloma samples frozen in RLT Plus buffer, which is commonly used for nucleic acid preservation and isolation. Additionally, the biological and clinical value of this analysis for a panel of 12 proteins essential to the pathogenesis of multiple myeloma was evaluated in 63 patients with newly diagnosed multiple myeloma. The analysis of the prognostic impact of CRBN/Cereblon and IKZF1/Ikaros mRNA/protein showed that only the protein levels were able to predict progression-free survival of patients; mRNA levels were not associated with prognosis. Interestingly, high levels of Cereblon and Ikaros proteins were associated with longer progression-free survival only in patients who received immunomodulatory drugs and not in those treated with other drugs. In conclusion, the capillary nano-immunoassay platform provides a novel opportunity for automated quantification of the expression of more than 20 proteins in CD138+ primary multiple myeloma samples.
Project description:Oligonucleotide therapeutics use short interfering RNA (siRNA) or antisense oligonucleotide (ASO) molecules to exploit endogenous systems-neutralizing target RNA to prevent subsequent protein translation. While the potential clinical application is vast, delivery efficiency and extrahepatic targeting is challenging. Bioanalytical assays are important in building understanding of these complex relationships. The literature currently lacks description of robust and sensitive methods to measure siRNA and ASOs in complex biological matrices. Described herein is a non-enzymatic hybridization-based immunoassay that enables quantification of individual siRNA strands (antisense or sense) in serum, urine, bile, and liver and kidney homogenates. Assay utility is also demonstrated in ASOs. The assay improves upon previous works by abolishing enzymatic steps and further incorporating Locked Nucleic Acid (LNA) nucleotide modifications to increase analyte hybridization affinity and improve sensitivity, specificity, and robustness. We report an assay with an ultrasensitive dynamic range of 0.3 to 16,700 pM for siRNA in serum. The assay was submitted to full qualification for accuracy and precision in both serum and tissue matrices and assay performance was assessed with single and mixed analytes. The reliable LNA-hybridization-based approach removes the need for matrix sample extraction, enrichment or amplification steps which may be impeded by more advanced chemical modifications.
Project description:Comparison of gene expression profiles between CD138+ and CD138- populations from human myeloma cell lines RPMI-8226 and NCI-H929. We used Affymetrix human gene 1.0ST array and analyzed with GeneSpring GX.
Project description:Although recent advances have substantially improved the management of multiple myeloma, it remains an incurable malignancy. We now demonstrate that anti-CD138 molecules genetically fused to type I interferons (IFN) synergize with the approved therapeutic bortezomib in arresting the proliferation of human multiple myeloma cell lines both in vitro and in vivo. The anti-CD138-IFNα14 fusion protein was active in inducing increased expression of signal transducer and activator of transcription 1 (STAT1) and its phosphorylation while the cell death pathway induced by bortezomib included generation of reactive oxygen species. Interferon regulatory factor 4 (IRF4), an important survival factor for myeloma cells, was down regulated following combination treatment. Induction of cell death appeared to be caspase-independent because treatment with inhibitors of caspase activation did not decrease the level of cell death. The observed caspase-independent synergistic cell death involved mitochondrial membrane depolarization, and poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, and resulted in enhanced induction of apoptosis. Importantly, using 2 different in vivo xenograft models, we found that combination therapy of anti-CD138-IFNα14 and bortezomib was able to cure animals with established tumors (7 of 8 using OCI-My5 or 8 of 8 using NCI-H929). Thus, the combination of anti-CD138-IFNα with bortezomib shows great promise as a novel therapeutic approach for the treatment of multiple myeloma, a malignancy for which there are currently no cures.
Project description:Multiple myeloma (MM), a plasma cell malignancy, is the second most prevalent hematologic malignancy in the US. Although much effort has been made trying to understand the etiology and the complexities of this disease with the hope of developing effective therapies, MM remains incurable at this time. Because of their antiproliferative and proapoptotic activities, interferons (IFNs) have been used to treat various malignancies, including MM. Although some success has been observed, the inherent toxicities of IFNs limit their efficacy. To address this problem, we produced anti-CD138 antibody fusion proteins containing either IFN?2 or a mutant IFN?2 (IFN?2(YNS)) with the goal of targeting IFN to CD138-expressing cells, thereby achieving effective IFN concentrations at the site of the tumor in the absence of toxicity. The fusion proteins inhibited the proliferation and induced apoptosis of U266, ANBL-6, NCI-H929, and MM1-144 MM cell lines. The fusion proteins decreased the expression of IFN regulatory factor 4 (IRF4) in U266. In addition, the fusion proteins were effective against primary cells from MM patients, and treatment with fusion proteins prolonged survival in the U266 murine model of MM. These studies show that IFN? antibody fusion proteins can be effective novel therapeutics for the treatment of MM.
Project description:The canonical plasma cell marker CD138 (syndecan-1) is highly expressed on the myeloma cell surface, but its functional role in vivo is unclear, as well as the ontogeny of CD138-high and CD138-negative (neg) myeloma cells. In this study we used an in vivo murine Vk*MYC myeloma model where CD138 is heterogeneously expressed depending on tumor size. We find that in comparison to CD138-neg myeloma cells, the CD138-high subset of myeloma cells is highly proliferative, less apoptotic, and enhanced IL-6R signaling, which is known to promote survival. In addition CD138-high myeloma engrafts better than its CD138-neg counterpart. In contrast, CD138-neg cells are more motile both in vitro and in vivo, and more readily disseminate and spread to other bones in vivo than CD138-high subset. Neutralizing CD138 rapidly triggers migration of myeloma cells in vivo and leads to intravasation, which results in increased dissemination to other bones. Both murine and human myeloma cells can rapidly recycle CD138 surface expression through endocytic trafficking, in response to serum levels. Blocking CD138 enhances myeloma sensitivity to bortezomib chemotherapy and significantly reduces tumor size compared to bortezomib treatment alone. Thus, our data show that CD138 surface expression dynamically regulates a switch between growth vs. dissemination for myeloma, in response to nutrient conditions.
Project description:Comparison of gene expression profiles between CD138+ and CD138- populations from human myeloma cell lines RPMI-8226 and NCI-H929. We used Affymetrix human gene 1.0ST array and analyzed with GeneSpring GX. CD138+ and CD138- subsets of cells were separated using FACS and clonogenecity of both population were compared. The RNA was extracted and hybridized with Affymetrix Human Gene 1.0ST array.
Project description:We present a highly sensitive bioanalytical microarray assay that enables the analysis of small genomic sample material. By combining an optimized cDNA purification step with single molecule cDNA detection on the microarray, the platform has improved sensitivity compared to conventional systems, allowing amplification-free determination of expression profiles with as little as 600ng total RNA. Total RNA from cells was reverse transcribed into fluorescently labeled cDNA and purified employing a precipitation method that minimizes loss of cDNA material. The microarray was scanned on a fluorescence chip-reader with single molecule sensitivity. Using the newly developed platform we were able to analyze the RNA expression profile of a subpopulation of rare multiple myeloma CD138 negative progenitor (MM CD138(neg)) cells. The high-sensitivity microarray approach led to the identification of a set of 20 genes differentially expressed in MM CD138(neg) cells. Our work demonstrates the applicability of a straight-forward single-molecule DNA array technology to current topics of molecular and cellular cancer research, which are otherwise difficult to address due to the limited amount of sample material.
Project description:Regeneration of immunosensors is a longstanding challenge. We have developed a re-usable troponin-T (TnT) immunoassay based on localised surface plasmon resonance (LSPR) at gold nanorods (GNR). Thermosensitive poly(N-isopropylacrylamide) (PNIPAAM) was functionalised with anti-TnT to control the affinity interaction with TnT. The LSPR was extremely sensitive to the dielectric constant of the surrounding medium as modulated by antigen binding after 20 min incubation at 37 °C. Computational modelling incorporating molecular docking, molecular dynamics and free energy calculations was used to elucidate the interactions between the various subsystems namely, IgG-antibody (c.f., anti-TnT), PNIPAAM and/or TnT. This study demonstrates a remarkable temperature dependent immuno-interaction due to changes in the PNIPAAM secondary structures, i.e., globular and coil, at above or below the lower critical solution temperature (LCST). A series of concentrations of TnT were measured by correlating the λLSPR shift with relative changes in extinction intensity at the distinct plasmonic maximum (i.e., 832 nm). The magnitude of the red shift in λLSPR was nearly linear with increasing concentration of TnT, over the range 7.6 × 10-15 to 9.1 × 10-4 g/mL. The LSPR based nano-immunoassay could be simply regenerated by switching the polymer conformation and creating a gradient of microenvironments between the two states with a modest change in temperature.