Abstract
The potential application of multiplexed quantum dot labeling (MQDL) for cancer detection and prognosis and monitoring therapeutic responses has attracted the interests of bioengineers, pathologists and cancer biologists. Many published studies claim that MQDL is effective for cancer biomarker detection and useful in cancer diagnosis and prognosis, these studies have not been standardized against quantitative biochemical and molecular determinations. In the present study, we used a molecularly characterized human prostate cancer cell model exhibiting activated c-Met signaling with epithelial to mesenchymal transition (EMT) and lethal metastatic progression to bone and soft tissues as the gold standard, and compared the c-Met cell signaling network in this model, in clinical human prostate cancer tissue specimens and in a castration-resistant human prostate cancer xenograft model. We observed c-Met signaling network activation, manifested by increased phosphorylated c-Met in all three. The downstream survival signaling network was mediated by NF-κB and Mcl-1 and EMT was driven by receptor activator of NF-κB ligand (RANKL), at the single cell level in clinical prostate cancer specimens and the xenograft model. Results were confirmed by real-time RT-PCR and western blots in a human prostate cancer cell model. MQDL is a powerful tool for assessing biomarker expression and it offers molecular insights into cancer progression at both the cell and tissue level with high degree of sensitivity.
Highlights
Semi-conductor quantum dots (QDs) fluorescent nanoparticles have been recognized as one of the great recent advances for our ability to detect relevant biomarkers expressed by cells, tissues and sera [1,2,3,4,5]
Validation of activated c-Met signaling components that lead to epithelial to mesenchymal transition (EMT) by room temperature (RT)-PCR, Western blot and Single Quantum Dot Labeling, Single QD Labeling (SQDL): Comparison of gene expression between stable LNCaP-neo control and LNCaP-receptor activator of NF-kB ligand (RANKL) cells using RT-PCR and Western blot showed evidence of activated c-Met signaling through increased expression of c-Met, p-c-Met, and p-NFkB p65 (Figure 1)
These assessments of c-Met signaling activation and EMT were subjected to SQDL analyses with a specific effort to confirm if cMet activation and EMT occurred in this cell model of prostate cancer progression
Summary
Semi-conductor quantum dots (QDs) fluorescent nanoparticles have been recognized as one of the great recent advances for our ability to detect relevant biomarkers expressed by cells, tissues and sera [1,2,3,4,5]. We employed multiplexed quantum dot labeling (MQDL) to detect the activated c-Met-mediated cell signaling pathway leading to EMT, cancer growth and bone and soft tissue metastasis in a novel prostate cancer metastasis model [11,12,13]. A series of biomarkers associated with EMT, such as decreased expression of EpCAM, and increased expression of N-cadherin, vimentin and RANKL, and c-Met signal activation, including VEGF, neuropilin 1, p-c-Met and phospho(p)-NF-kB p65 [15,22], were analyzed The results of these studies showed a remarkable parallelism of EMT and c-Met activation between the prostate cancer cell model, the CRPC xenograft model and clinical prostate cancer specimens. The methodologies established in the present study could be of significant value in the near future to characterize other activated signaling pathways in clinical specimens, interrogate molecular mechanisms underlying cancer progression, identify druggable targets, and follow up the clinical response of patients to therapeutic intervention
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