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- Lundberg, Martin, et al.
(författare)
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Multiplexed Homogeneous Proximity Ligation Assays for High-throughput Protein Biomarker Research in Serological Material
- 2011
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 10:4, s. M110.004978-
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Tidskriftsartikel (refereegranskat)abstract
- A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pM sensitivity each consuming only 1 mu l of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.
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| 2. |
- Nordgaard, Cathrine, et al.
(författare)
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Metallopeptidase inhibitor 1 (TIMP-1) promotes receptor tyrosine kinase c-Kit signaling in colorectal cancer
- 2019
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Ingår i: Molecular Oncology. - : Wiley. - 1574-7891 .- 1878-0261. ; 13:12, s. 2646-2662
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Tidskriftsartikel (refereegranskat)abstract
- Colorectal cancer (CRC) is the third most prevalent cancer worldwide causing an estimated 700 000 deaths annually. Different types of treatment are available for patients with advanced metastatic colorectal cancer, including targeted biological agents, such as cetuximab, a monoclonal antibody that targets EGFR. We have previously reported a study indicating multiple levels of interaction between metallopeptidase inhibitor 1 (TIMP-1) and the epidermal growth factor (EGF) signaling axis, which could explain how TIMP-1 levels can affect the antitumor effects of EGFR inhibitors. We also reported an association between TIMP-1-mediated cell invasive behavior and KRAS status. To gain insight into the molecular mechanisms underlying the effects of TIMP-1 in CRC, we examined by transcriptomics, proteomics, and kinase activity profiling a matched pair of isogenic human CRC isogenic DLD-1 CRC cell clones, bearing either an hemizygous KRAS wild-type allele or KRAS G13D mutant allele, exposed, or not, to TIMP-1. Omics analysis of the two cell lines identified the receptor tyrosine kinase c-Kit, a proto-oncogene that can modulate cell proliferation and invasion in CRC, as a target for TIMP-1. We found that exposure of DLD-1 CRC cells to exogenously added TIMP-1 promoted phosphorylation of c-Kit, indicative of a stimulatory effect of TIMP-1 on the c-Kit signaling axis. In addition, TIMP-1 inhibited c-Kit shedding in CRC cells grown in the presence of exogenous TIMP-1. Given the regulatory roles that c-Kit plays in cell proliferation and migration, and the realization that c-Kit is an important oncogene in CRC, it is likely that some of the biological effects of TIMP-1 overexpression in CRC may be exerted through its effect on c-Kit signaling.
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