SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load. Consequently, there was a need to introduce standards to support estimation of SARS-CoV-2 viral load in diagnostic specimens. In a collaborative study, INSTAND established two reference materials (RMs) containing heat-inactivated SARS-CoV-2 with SARS-CoV-2 RNA loads of ~107 copies/mL (RM 1) and ~106 copies/mL (RM 2), respectively. Quantification was performed by RT-qPCR using synthetic SARS-CoV-2 RNA standards and digital PCR. Between November 2020 and February 2021, German laboratories were invited to use the two RMs to anchor their Ct values measured in routine diagnostic specimens, with the Ct values of the two RMs. A total of 305 laboratories in Germany were supplied with RM 1 and RM 2. The laboratories were requested to report their measured Ct values together with details on the PCR method they used to INSTAND. This resultant 1,109 data sets were differentiated by test system and targeted gene region. Our findings demonstrate that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that they are treated as being unique to an individual laboratory. For this reason, clinical guidance based on viral loads should not cite Ct values. The RMs described were a suitable tool to determine the specific laboratory Ct for a given viral load. Furthermore, as Ct values can also vary between runs when using the same instrument, such RMs could be used as run controls to ensure reproducibility of the quantitative measurements.
BackgroundPancreatic cancer development is associated with characteristic alterations like desmoplastic reaction and immune escape which are mediated by the cell-cell communication mechanism and by the microenvironment of the cells. The whole of released components are important determinants in these processes. Especially the extracellular vesicles released by pancreatic cancer cells play a role in cell communication and modulate cell growth and immune responses.ResultsHere, we present the proteomic description of affinity purified extracellular vesicles from pancreatic tumour cells, compared to the secretome, defined as the whole of the proteins released by pancreatic cancer cells. The proteomic data provide comprehensive catalogues of hundreds of proteins, and the comparison reveals a special proteomic composition of pancreatic cancer cell derived extracellular vesicles. The functional analysis of the protein composition displayed that membrane proteins, glycoproteins, small GTP binding proteins and a further, heterogeneous group of proteins are enriched in vesicles, whereas proteins derived from proteasomes and ribosomes, as well as metabolic enzymes, are not components of the vesicles. Furthermore proteins playing a role in carcinogenesis and modulators of the extracellular matrix (ECM) or cell-cell interactions are components of affinity purified extracellular vesicles.ConclusionThe data deepen the knowledge of extracellular vesicle composition by hundreds of proteins that have not been previously described as vesicle components released by pancreatic cancer cells. Extracellular vesicles derived from pancreatic cancer cells show common proteins shared with other vesicles as well as cell type specific proteins indicating biomarker candidates and suggesting functional roles in cancer cell stroma interactions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-014-0050-5) contains supplementary material, which is available to authorized users.
In pancreatic cancer, there is a clear unmet need to identify new serum markers for either early diagnosis, therapeutic stratification or patient monitoring. Proteomic analysis of tumor cell secretomes is a promising approach to indicate proteins released from tumor cells in vitro. Ectodomain shedding of transmembrane proteins has previously been shown to contribute significant fractions the tumor cell secretomes and to generate valuable serum biomarkers. Here we introduce a soluble form of the giant cadherin Fat1 as a novel biomarker candidate. Fat1 expression and proteolytic processing was analyzed by mass spectrometry and Western blotting using pancreatic cancer cell lines as compared to human pancreatic ductal epithelial cells. RNA expression in cancer tissues was assessed by in silico analysis of publically available microarray data. Involvement of ADAM10 (A Disintegrin and metalloproteinase domain-containing protein 10) in Fat1 ectodomain shedding was analyzed by chemical inhibition and knockdown experiments. A sandwich ELISA was developed to determine levels of soluble Fat1 in serum samples. In the present report we describe the release of high levels of the ectodomain of Fat1 cadherin into the secretomes of human pancreatic cancer cells in vitro, a process that is mediated by ADAM10. We confirm the full-length and processed heterodimeric form of Fat1 expressed on the plasma membrane and also show the p60 C-terminal transmembrane remnant fragment corresponding to the shed ectodomain. Fat1 and its sheddase ADAM10 are overexpressed in pancreatic adenocarcinomas and ectodomain shedding is also recapitulated in vivo leading to increased Fat1 serum levels in some pancreatic cancer patients. We suggest that soluble Fat1 may find an application as a marker for patient monitoring complementing carbohydrate antigen 19-9 (CA19-9). In addition, detailed analysis of the diverse processed protein isoforms of the candidate tumor suppressor Fat1 can also contribute to our understanding of cell biology and tumor behavior.
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