Detection of Cadherin-17 in human colon cancer LIM1215 cell secretome and tumour xenograft-derived interstitial fluid and plasma

Colorectal cancer (CRC), one of the most prevalent cancers in the western world, is treatable if detected early. However, 70% of CRC is detected at an advanced stage. This is largely due to the inadequacy of current faecal occult blood screening testing and costs involved in conducting population-based colonoscopy, the 'gold standard' for CRC detection. Another biomarker for CRC, carcinoembryonic antigen, while useful for monitoring CRC recurrence, is ineffective, lacking the specificity required early detection of CRC. For these reasons there is a need for more effective blood-based markers for early CRC detection. In this study we targeted glycoproteins secreted from the human colon carcinoma cell line LIM1215 as a source of potential CRC biomarkers. Secreted candidate glycoproteins were confirmed by MS and validated by Western blot analysis of tissue/tumour interstitial fluid (Tif) from LIM1215 xenograft tumours grown in immunocompromised mice. Overall, 39 glycoproteins were identified in LIM1215 culture media (CCM) and 5 glycoproteins in LIM1215 tumour xenograft Tif; of these, cadherin-17 (CDH17), galectin-3 binding protein (LGALS3BP), and tyrosine-protein kinase-like 7 (PTK7) were identified in both CM and glycosylation motifs. Swiss-Prot was used to annotate Tif. Many of the glycoproteins identified in this study (e.g., AREG, DSG2, EFNA1, EFNA3, EFNA4, EPHB4, ST14, and TIMP1) have been reported to be implicated in CRC biology. Interestingly, the cadherin-17 ectodomain, but not full length cadherin-17, was identified in CM, Tif and plasma derived from mice bearing the LIM1215 xenograft tumour. To our knowledge, this is the first report of the cadherin-17 ectodomain in plasma. In this study, we report for the first time that the presence of full-length cadherin-17 in exosomes released into the CM. This article is part of a Special Issue entitled: An Updated Secretome. © 2013 Published by Elsevier B.V

Secretome-based proteomics reveals sulindac-modulated proteins released from colon cancer cells

Although experiments in rodents and human population-based studies have demonstrated the efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) such as sulindac in colorectal cancer (CRC) prevention, a detailed knowledge of the underlying mechanism of action of this drug is limited. To better understand the chemopreventitive effects of sulindac, especially early sulindac-induced apoptotic events, we used the CRC cell line LIM1215 as an experimental model, focusing on proteins secreted into the LIM1215 culture medium-i.e., the secretome. This subproteome comprises both soluble-secreted proteins and exosomes (30-100 nm diameter membrane vesicles released by several cell types). Selected secretome proteins whose expression levels were dysregulated by 1 mM sulindac treatment over 16 h were analyzed using 2-D DIGE, cytokine array, Western blotting, and MS. Overall, 150 secreted proteins were identified, many of which are implicated in molecular and cellular functions such as cell proliferation, differentiation, adhesion, invasion, angiogenesis, metastasis, and apoptosis. Our secretome-based proteomic studies have identified several secreted modulators of sulindac-induced apoptosis action (e.g., Mac-2 binding protein, Alix, 14-3-3 isoforms, profilin-1, calumenin/Cab45 precursors, and the angiogenic/tumor growth factors interleukin 8 (IL-8) and growth related oncogene (GRO-α)) that are likely to improve our understanding of the chemopreventitive action of this NSAID in CRC. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Colon tumour secretopeptidome: Insights into endogenous proteolytic cleavage events in the colon tumour microenvironment

The secretopeptidome comprises endogenous peptides derived from proteins secreted into the tumour microenvironment through classical and non-classical secretion. This study characterised the low-Mr (< 3 kDa) component of the human colon tumour (LIM1215, LIM1863) secretopeptidome, as a first step towards gaining insights into extracellular proteolytic cleavage events in the tumour microenvironment. Based on two biological replicates, this secretopeptidome isolation strategy utilised differential centrifugal ultrafiltration in combination with analytical RP-HPLC and nanoLC-MS/MS. Secreted peptides were identified using a combination of Mascot and post-processing analyses including MSPro re-scoring, extended feature sets and Percolator, resulting in 474 protein identifications from 1228 peptides (≤ 1% q-value, ≤ 5% PEP) - a 36% increase in peptide identifications when compared with conventional Mascot (homology ionscore thresholding). In both colon tumour models, 122 identified peptides were derived from 41 cell surface protein ectodomains, 23 peptides (12 proteins) from regulated intramembrane proteolysis (RIP), and 12 peptides (9 proteins) generated from intracellular domain proteolysis. Further analyses using the protease/substrate database MEROPS, (http://merops.sanger.ac.uk/), revealed 335 (71%) proteins classified as originating from classical/non-classical secretion, or the cell membrane. Of these, peptides were identified from 42 substrates in MEROPS with defined protease cleavage sites, while peptides generated from a further 205 substrates were fragmented by hitherto unknown proteases. A salient finding was the identification of peptides from 88 classical/non-classical secreted substrates in MEROPS, implicated in tumour progression and angiogenesis (FGFBP1, PLXDC2), cell-cell recognition and signalling (DDR1, GPA33), and tumour invasiveness and metastasis (MACC1, SMAGP); the nature of the proteases responsible for these proteolytic events is unknown. To confirm reproducibility of peptide fragment abundance in this study, we report the identification of a specific cleaved peptide fragment in the secretopeptidome from the colon-specific GPA33 antigen in 4/14 human CRC models. This improved secretopeptidome isolation and characterisation strategy has extended our understanding of endogenous peptides generated through proteolysis of classical/non-classical secreted proteins, extracellular proteolytic processing of cell surface membrane proteins, and peptides generated through RIP. The novel peptide cleavage site information in this study provides a useful first step in detailing proteolytic cleavage associated with tumourigenesis and the extracellular environment. This article is part of a Special Issue entitled: An Updated Secretome. © 2013 Elsevier B.V

Highly-purified exosomes and shed microvesicles isolated from the human colon cancer cell line LIM1863 by sequential centrifugal ultrafiltration are biochemically and functionally distinct

Secretion and exchange of extracellular vesicles (EVs) by most cell types is emerging as a fundamental biological process. Although much is known about EVs, there is still a lack of definition as to how many naturally occurring EV subtypes there are and how their properties and functionalities might differ. This vexing issue is critical if EVs are to be fully harnessed for therapeutic applications. To address this question we have developed and describe here a sequential centrifugal ultrafiltration (SCUF) method to examine, in an unbiased manner, what EV subtypes are released in vitro into cell culture medium using the human colon carcinoma cell line LIM1863 as a model system. Using the culture medium from ~7.2×109 LIM1863 cells, SCUF was performed using hydrophilic PVDF membranes with low protein binding properties (Millipore Durapore™ Ultrafree-CL filters with 0.1, 0.22, 0.45 and 0.65μm pore size). EV particle sizing was measured using both dynamic light scattering and cryo-electron microscopy. Comparative proteome profiling was performed by GeLC-MS/MS and qualitative protein differences between EV subtypes determined by label-free spectral counting. The results showed essentially two EV subtypes; one subtype (fraction Fn1) comprised heterogeneous EVs with particle diameters of 30-1300nm, the other (fraction Fn5) being homogeneous EVs of 30-100nm diameter; based on cryo-EM both EV subtypes were round shaped. Western blot analysis showed Fn5 (SCUF-Exos) contained traditional exosome marker proteins (Alix+, TSG101+, CD81+, CD63+), while Fn1 (SCUF-sMVs) lacked these protein markers. These findings were consistent with sMVs isolated by differential centrifugation (10,000g, DC-sMVs) and exosomes (100,000g EVs depleted of 10,000g material). The buoyant density of sMVs determined by OptiPrep™ density gradient centrifugation was 1.18-1.19g/mL and exosomes 1.10-1.11g/mL. Comparative protein profiling of SCUF-Exos/-sMVs revealed 354 and 606 unambiguous protein identifications, respectively, with 256 proteins in common. A salient finding was the first report of 350 proteins uniquely identified in sMVs may of which have the potential to enable discrimination of this EV subtype from exosomes (notably, members of the septin family, kinesin-like protein (KIF23), exportin-2/chromosome segregation like-1 protein (CSE1L), and Rac GTPase-activating protein 1 (RACGAP1)). We report for the first time that both SCUF-Exos and SCUF-sMVs isolated from LIM1863 colon cancer cells induce invasion of recipient NIH3T3 cells. Interestingly, the SCUF-sMVs promote invasion to a significantly greater extent (3-fold) than SCUF-Exos. This analytical SCUF method for fractionating EVs is potentially scalable using tangential flow filtration, thereby providing a solid foundation for future in-depth functional studies of EV subtypes using diverse cell types and functional assays.</p

Highly-purified exosomes and shed microvesicles isolated from the human colon cancer cell line LIM1863 by sequential centrifugal ultrafiltration are biochemically and functionally distinct

Secretion and exchange of extracellular vesicles (EVs) by most cell types is emerging as a fundamental biological process. Although much is known about EVs, there is still a lack of definition as to how many naturally occurring EV subtypes there are and how their properties and functionalities might differ. This vexing issue is critical if EVs are to be fully harnessed for therapeutic applications. To address this question we have developed and describe here a sequential centrifugal ultrafiltration (SCUF) method to examine, in an unbiased manner, what EV subtypes are released in vitro into cell culture medium using the human colon carcinoma cell line LIM1863 as a model system. Using the culture medium from ~7.2×109 LIM1863 cells, SCUF was performed using hydrophilic PVDF membranes with low protein binding properties (Millipore Durapore™ Ultrafree-CL filters with 0.1, 0.22, 0.45 and 0.65μm pore size). EV particle sizing was measured using both dynamic light scattering and cryo-electron microscopy. Comparative proteome profiling was performed by GeLC-MS/MS and qualitative protein differences between EV subtypes determined by label-free spectral counting. The results showed essentially two EV subtypes; one subtype (fraction Fn1) comprised heterogeneous EVs with particle diameters of 30-1300nm, the other (fraction Fn5) being homogeneous EVs of 30-100nm diameter; based on cryo-EM both EV subtypes were round shaped. Western blot analysis showed Fn5 (SCUF-Exos) contained traditional exosome marker proteins (Alix+, TSG101+, CD81+, CD63+), while Fn1 (SCUF-sMVs) lacked these protein markers. These findings were consistent with sMVs isolated by differential centrifugation (10,000g, DC-sMVs) and exosomes (100,000g EVs depleted of 10,000g material). The buoyant density of sMVs determined by OptiPrep™ density gradient centrifugation was 1.18-1.19g/mL and exosomes 1.10-1.11g/mL. Comparative protein profiling of SCUF-Exos/-sMVs revealed 354 and 606 unambiguous protein identifications, respectively, with 256 proteins in common. A salient finding was the first report of 350 proteins uniquely identified in sMVs may of which have the potential to enable discrimination of this EV subtype from exosomes (notably, members of the septin family, kinesin-like protein (KIF23), exportin-2/chromosome segregation like-1 protein (CSE1L), and Rac GTPase-activating protein 1 (RACGAP1)). We report for the first time that both SCUF-Exos and SCUF-sMVs isolated from LIM1863 colon cancer cells induce invasion of recipient NIH3T3 cells. Interestingly, the SCUF-sMVs promote invasion to a significantly greater extent (3-fold) than SCUF-Exos. This analytical SCUF method for fractionating EVs is potentially scalable using tangential flow filtration, thereby providing a solid foundation for future in-depth functional studies of EV subtypes using diverse cell types and functional assays.</p

Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids

Exosomes are naturally occurring biological nanomembranous vesicles (̃40 to 100 nm) of endocytic origin that are released from diverse cell types into the extracellular space. They have pleiotropic functions such as antigen presentation and intercellular transfer of protein cargo, mRNA, microRNA, lipids, and oncogenic potential. Here we describe the isolation, via sequential immunocapture using anti-A33- and anti-EpCAM-coupled magnetic beads, of two distinct populations of exosomes released from organoids derived from human colon carcinoma cell line LIM1863. The exosome populations (A33-Exos and EpCAM-Exos) could not be distinguished via electron microscopy and contained stereotypical exosome markers such as TSG101, Alix, and HSP70. The salient finding of this study, revealed via gel-based LC-MS/MS, was the exclusive identification in EpCAM-Exos of the classical apical trafficking molecules CD63 (LAMP3), mucin 13 and the apical intestinal enzyme sucrase isomaltase and increased expression of dipeptidyl peptidase IV and the apically restricted pentaspan membrane glycoprotein prominin 1. In contrast, the A33-Exos preparation was enriched with basolateral trafficking molecules such as early endosome antigen 1, the Golgi membrane protein ADP-ribosylation factor, and clathrin. Our observations are consistent with EpCAM- and A33-Exos being released from the apical and basolateral surfaces, respectively, and the EpCAM-Exos proteome profile with widely published stereotypical exosomes. A proteome analysis of LIM1863-derived shed microvesicles (sMVs) was also performed in order to clearly distinguish A33- and EpCAMExos from sMVs. Intriguingly, several members of the MHC class I family of antigen presentation molecules were exclusively observed in A33-Exos, whereas neither MHC class I nor MHC class II molecules were observed via MS in EpCAM-Exos. Additionally, we report for the first time in any extracellular vesicle study the colocalization of EpCAM, claudin-7, and CD44 in EpCAM-Exos. Given that these molecules are known to complex together to promote tumor progression, further characterization of exosome subpopulations will enable a deeper understanding of their possible role in regulation of the tumor microenvironment. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc

Minor increases in Phyllostachys edulis (Moso bamboo) biomass despite evident alterations of soil bacterial community structure after phosphorus fertilization alone: Based on field studies at different altitudes

Understanding the relationships among bamboo growth, soil microbial community and phosphorus (P) fertilization may shed new light on the optimization of P application rate and ecological function of P nutrient in forest ecosystem. This study aimed to evaluate the impacts of P fertilizations (25 and 50 kg P ha−1) on Phyllostachys edulis (Moso bamboo) growths, soil nutrient contents and microbial properties at different altitudes (300 and 800 m) and to link Moso bamboo growths with abiotic and biotic factors. Compared with the blank control, P fertilizations alone generated negligible impacts on the increases of Moso bamboo biomass and soil available P contents. Bacterial and fungal community diversities kept relatively stable after P fertilizations. Contrastingly, relative to the control, 25 kg P ha−1 application significantly enhanced the relative abundance of Proteobacteria and changed bacterial community structure at low altitude. At low and high altitudes, 50 kg P ha−1 applications significantly increased the relative abundances of Ascomycota. The Moso bamboo growth might be stimulated by the increases of soil total nitrogen, available P, Proteobacteria and Ascomycota. Our results demonstrated that P fertilization alone significantly changed soil bacterial community structure but generated negligibly stimulating role in the increase of Moso bamboo biomass. © 2019 Elsevier B.V

YBX1/YB-1 induces partial EMT and tumourigenicity through secretion of angiogenic factors into the extracellular microenvironment

Epithelial-mesenchymal transition (EMT) describes a morphogenetic program which confers mesenchymal cell properties, such as reduced cell-cell contact and increased cell migration and invasion, to epithelial cells. Here we investigate the role of the pleiotropic transcription/splicing factor and RNA-binding protein nucleasesensitive element-binding protein 1 (YBX1/YB-1) in increasing the oncogenic potential of epithelial MDCK cells. Characterization of MDCK cells expressing YBX1 (MDCKYBX1 cells) revealed a partial EMT phenotype, including cytosolic relocalization of E-cadherin, increased cell scattering, and anchorage-independent growth. Subcutaneous injection of parental MDCK cells into NOD/SCID mice did not form tumours. Critically, MDCKYBX1 cells established viable tumour xenografts, and immunohistochemical staining indicated murine vascularization by CD31+ endothelial cells. We analysed the total secretome (containing soluble and extracellular vesicles) of MDCKYBX1 cells to investigate regulation of the tumour microenvironment. YBX1 expression elevated release of secreted factors known to enhance angiogenesis (TGF-β, CSF-1, NGF, VGF, ADAM9 and ADAM17), compared to MDCK cells. Importantly, treatment with MDCKYBX1 cell-derived secretome increased recipient 2F-2B endothelial cell motility. This defines YBX1 as an oncogenic enhancer that can regulate tumour angiogenesis via release of secreted modulators into the extracellular microenvironment

Transcriptome and long noncoding RNA sequencing of three extracellular vesicle subtypes released from the human colon cancer LIM1863 cell line

Previously we reported that LIM1863 colorectal cancer (CRC) cells secrete three distinct extracellular vesicle subtypes-two subpopulations of exosomes (apical EpCAM-Exos and basolateral A33-Exos) and shed microvesicles (sMVs)-with distinct protein and miRNA signatures. Here, we extend our omics approach to understand the fundamental role of LIM1863-derived EVs by performing a comprehensive analysis of their mRNAs and long non-coding RNAs (lncRNAs) using RNA-Seq. We show that 2,389 mRNAs, 317 pseudogene transcripts, 1,028 lncRNAs and 206 short non-coding RNAs selectively distributed to (i.e., are enriched in) LIM1863 EVs, relative to the parent cell. An Ensembl/UniProtKB analysis revealed 1,937 mRNAs encode canonical proteins, 348 isoforms (including splice-variant proteins), and 119 'missing proteins' (i.e., annotated in Ensembl but not UniProtKB). Further dissection of our protein/RNA data revealed that 6/151 observed RNA binding proteins have the potential to interact with ∼75% of EV-enriched RNAs. Intriguingly, the co-existence of U1 and U2 ribonucleoproteins and their cognate snRNAs in LIM1863 EVs suggests a possible association of CRC EVs with recipient cell splicing events. Our data reveal several potential lncRNA CRC biomarkers and novel splicing/fusion genes that, collectively, will advance our understanding of EV biology in CRC and accelerate the development of EV-based diagnostics and therapeutics

Proteome profiling of exosomes derived from human primary and metastatic colorectal cancer cells reveal differential expression of key metastatic factors and signal transduction components

Exosomes are small extracellular 40-100 nm diameter membrane vesicles of late endosomal origin that can mediate intercellular transfer of RNAs and proteins to assist premetastatic niche formation. Using primary (SW480) and metastatic (SW620) human isogenic colorectal cancer cell lines we compared exosome protein profiles to yield valuable insights into metastatic factors and signaling molecules fundamental to tumor progression. Exosomes purified using OptiPrep™ density gradient fractionation were 40-100 nm in diameter, were of a buoyant density ∼1.09 g/mL, and displayed stereotypic exosomal markers TSG101, Alix, and CD63. A major finding was the selective enrichment of metastatic factors (MET, S100A8, S100A9, TNC), signal transduction molecules (EFNB2, JAG1, SRC, TNIK), and lipid raft and lipid raft-associated components (CAV1, FLOT1, FLOT2, PROM1) in exosomes derived from metastatic SW620 cells. Additionally, using cryo-electron microscopy, ultrastructural components in exosomes were identified. A key finding of this study was the detection and colocalization of protein complexes EPCAM-CLDN7 and TNIK-RAP2A in colorectal cancer cell exosomes. The selective enrichment of metastatic factors and signaling pathway components in metastatic colon cancer cell-derived exosomes contributes to our understanding of the cross-talk between tumor and stromal cells in the tumor microenvironment. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.</p