Immunoscreening of the extracellular proteome of colorectal cancer cells

<p>Abstract</p> <p>Background</p> <p>The release of proteins from tumors can trigger an immune response in cancer patients involving T lymphocytes and B lymphocytes, which results in the generation of antibodies to tumor-derived proteins. Many studies aim to use humoral immune responses, namely autoantibody profiles, directly, as clinical biomarkers. Alternatively, the antibody immune response as an amplification system for tumor associated alterations may be used to indicate putative protein biomarkers with high sensitivity. Aiming at the latter approach we here have implemented an autoantibody profiling strategy which particularly focuses on proteins released by tumor cells in vitro: the so-called secretome.</p> <p>Methods</p> <p>For immunoscreening, the extracellular proteome of five colorectal cancer cell lines was resolved on 2D gels, immobilized on PVDF membranes and used for serological screening with individual sera from 21 colorectal cancer patients and 24 healthy controls. All of the signals from each blot were assigned to a master map, and autoantigen candidates were defined based of the pattern of immunoreactivities. The corresponding proteins were isolated from preparative gels, identified by MALDI-MS and/or by nano-HPLC/ESI-MS/MS and exemplarily confirmed by duplex Western blotting combining the human serum samples with antibodies directed against the protein(s) of interest.</p> <p>Results</p> <p>From 281 secretome proteins stained with autoantibodies in total we first defined the "background patterns" of frequently immunoreactive extracellular proteins in healthy and diseased people. An assignment of these proteins, among them many nominally intracellular proteins, to the subset of exosomal proteins within the secretomes revealed a large overlap. On this basis we defined and consequently confirmed novel biomarker candidates such as the extreme C-terminus of the extracellular matrix protein agrin within the set of cancer-enriched immunorectivities.</p> <p>Conclusions</p> <p>Our findings suggest, first, that autoantibody responses may be due, in large part, to cross-presentation of antigens to the immune system via exosomes, membrane vesicles released by tumor cells and constituting a significant fraction of the secretome. In addition, this immunosecretomics approach has revealed novel biomarker candidates, some of them secretome-specific, and thus serves as a promising complementary tool to the frequently reported immunoproteomic studies for biomarker discovery.</p

Significance of Liquid Biopsy for Monitoring and Therapy Decision of Colorectal Cancer

PURPOSE: Despite therapeutic improvements, all patients with nonresectable metastatic colorectal cancer (mCRC) acquire resistance to treatment probably due to the growth of mutated clones. In contrast to tissue-based studies, liquid biopsies have enabled the opportunity to reveal emerging resistance to treatment by detecting mutated clones and noninvasively monitoring clonal dynamics during therapy. METHODS: The courses of three patients with mCRC who were initially RAS wild-type were monitored longitudinally using liquid biopsy with long-term follow-up of up to 20 sequential samples. Detection of fragmented RAS mutated circulating cell-free DNA (cf)DNA in plasma was performed by BEAMing. In addition, plasma digital droplet PCR was used to detect and quantify BRAF and PIK3CA mutated cfDNA. Changes of mutational load were correlated with imaging data. RESULTS: A combination of liquid biopsy and radiological imaging enabled visualization of the occurrence of clonal redistribution after discontinuation of anti-EGFR mAb therapy, as well as emerging RAS mutations during therapy with anti-EGFR mAb indicating resistance. Furthermore, we found that growth of RAS mutated clones is independent of direct selective pressure by anti-EGFR therapy, which is a significant and new finding of this study. CONCLUSIONS: Our findings demonstrated the whole spectrum of clonal selection and redistribution of mutated cell clones leading to acquired resistance. Given our observation that the growth of RAS mutated clones can evolve even in the absence of anti-EGFR mAb therapy, there is a clear imperative to monitor RAS mutations in serial blood draws in all RAS wild-type patients in general and independent of the therapy

High-level inducible Smad4-reexpression in the cervical cancer cell line C4-II is associated with a gene expression profile that predicts a preferential role of Smad4 in extracellular matrix composition-3

<p><b>Copyright information:</b></p><p>Taken from "High-level inducible Smad4-reexpression in the cervical cancer cell line C4-II is associated with a gene expression profile that predicts a preferential role of Smad4 in extracellular matrix composition"</p><p>http://www.biomedcentral.com/1471-2407/7/209</p><p>BMC Cancer 2007;7():209-209.</p><p>Published online 12 Nov 2007</p><p>PMCID:PMC2186346.</p><p></p>robe

High-level inducible Smad4-reexpression in the cervical cancer cell line C4-II is associated with a gene expression profile that predicts a preferential role of Smad4 in extracellular matrix composition-1

<p><b>Copyright information:</b></p><p>Taken from "High-level inducible Smad4-reexpression in the cervical cancer cell line C4-II is associated with a gene expression profile that predicts a preferential role of Smad4 in extracellular matrix composition"</p><p>http://www.biomedcentral.com/1471-2407/7/209</p><p>BMC Cancer 2007;7():209-209.</p><p>Published online 12 Nov 2007</p><p>PMCID:PMC2186346.</p><p></p>of TGF-β. c) Short-term TGF-β effects on cell cycle distribution were assessed in the high-expressor clone 28-8 as compared to HaCaT cells used as a positive control. Bars show mean values +/- standard deviation from three cultures per cell line and condition in one experiment. Similar results were obtained in repeated experiments. d) Cell growth was assessed by subcutaneous injection in nude mice. The open bars and hatched bars show the mean tumour mass +/- standard deviation of 6 tumours, each, after 2 (clones 18 und 18-2) or 4 (clones 28, 28-8 and 28-14) weeks of growth in mice that received drinking water without and with the addition of doxycycline. Statistical significance is indicated. Similar results were obtained in repeated experiments

High-level inducible Smad4-reexpression in the cervical cancer cell line C4-II is associated with a gene expression profile that predicts a preferential role of Smad4 in extracellular matrix composition-7

<p><b>Copyright information:</b></p><p>Taken from "High-level inducible Smad4-reexpression in the cervical cancer cell line C4-II is associated with a gene expression profile that predicts a preferential role of Smad4 in extracellular matrix composition"</p><p>http://www.biomedcentral.com/1471-2407/7/209</p><p>BMC Cancer 2007;7():209-209.</p><p>Published online 12 Nov 2007</p><p>PMCID:PMC2186346.</p><p></p> five days. b) Time course of Smad4-induction in the "low-expressor" clone 18-2 and in the high-expressor clone 28-14 at the mRNA level. c) Time course of Smad4-induction in clones 18-2 and 28-14 at the protein level. d) Titration of Smad4-mRNA levels in clones 18-2 and 28-14 as compared to Smad4-reexpressing SW480 cell clones. The latter clones have previously been shown to express (sub-)physiological Smad4 levels. e) Titration of Smad4 protein levels in clones 18-2, 28-14 and 28-8

High-level inducible Smad4-reexpression in the cervical cancer cell line C4-II is associated with a gene expression profile that predicts a preferential role of Smad4 in extracellular matrix composition-4

<p><b>Copyright information:</b></p><p>Taken from "High-level inducible Smad4-reexpression in the cervical cancer cell line C4-II is associated with a gene expression profile that predicts a preferential role of Smad4 in extracellular matrix composition"</p><p>http://www.biomedcentral.com/1471-2407/7/209</p><p>BMC Cancer 2007;7():209-209.</p><p>Published online 12 Nov 2007</p><p>PMCID:PMC2186346.</p><p></p>ions. Shown as examples are sections of filters containing the probes for fibronectin and BigH3. The induction of PAI-1 was detectable but less clear because a very strong neighbouring signal faded over the PAI-1 locus (data not shown). b) Corresponding filters to those shown in figure 4 were hybridised with probes for BigH3, fibronectin and PAI-1. c) Smad4-levels were titrated in clone 28-8 through dilution of doxycycline. Northern blotting with the same probes as used in (b). d) TGF-β induction of 28-8 cells was repeated in the presence of cycloheximid

High-level inducible Smad4-reexpression in the cervical cancer cell line C4-II is associated with a gene expression profile that predicts a preferential role of Smad4 in extracellular matrix composition-0

<p><b>Copyright information:</b></p><p>Taken from "High-level inducible Smad4-reexpression in the cervical cancer cell line C4-II is associated with a gene expression profile that predicts a preferential role of Smad4 in extracellular matrix composition"</p><p>http://www.biomedcentral.com/1471-2407/7/209</p><p>BMC Cancer 2007;7():209-209.</p><p>Published online 12 Nov 2007</p><p>PMCID:PMC2186346.</p><p></p> five days. b) Time course of Smad4-induction in the "low-expressor" clone 18-2 and in the high-expressor clone 28-14 at the mRNA level. c) Time course of Smad4-induction in clones 18-2 and 28-14 at the protein level. d) Titration of Smad4-mRNA levels in clones 18-2 and 28-14 as compared to Smad4-reexpressing SW480 cell clones. The latter clones have previously been shown to express (sub-)physiological Smad4 levels. e) Titration of Smad4 protein levels in clones 18-2, 28-14 and 28-8

High-level inducible Smad4-reexpression in the cervical cancer cell line C4-II is associated with a gene expression profile that predicts a preferential role of Smad4 in extracellular matrix composition-6

<p><b>Copyright information:</b></p><p>Taken from "High-level inducible Smad4-reexpression in the cervical cancer cell line C4-II is associated with a gene expression profile that predicts a preferential role of Smad4 in extracellular matrix composition"</p><p>http://www.biomedcentral.com/1471-2407/7/209</p><p>BMC Cancer 2007;7():209-209.</p><p>Published online 12 Nov 2007</p><p>PMCID:PMC2186346.</p><p></p>down clones, in which Smad4 expression was reduced to below 10% were hybridised with the BigH3 specific probe