Comprehensive Lipidome Profiling of Isogenic Primary and Metastatic Colon Adenocarcinoma Cell Lines

A “shotgun” lipidomics strategy consisting of sequential functional group selective chemical modification reactions coupled with high-resolution/accurate mass spectrometry and “targeted” tandem mass spectrometry (MS/MS) analysis has been developed and applied toward the comprehensive identification, characterization and quantitative analysis of changes in relative abundances of >600 individual glycerophospholipid, glycerolipid, sphingolipid and sterol lipids between a primary colorectal cancer (CRC) cell line, SW480, and its isogenic lymph node metastasized derivative, SW620. Selective chemical derivatization of glycerophosphoethanolamine and glycerophosphoserine lipids using a “fixed charge” sulfonium ion containing, d₆-<i>S</i>,<i>S</i>′-dimethylthiobutanoylhydroxysuccinimide ester (d₆-DMBNHS) reagent was used to eliminate the possibility of isobaric mass overlap of these species with the precursor ions of all other lipids in the crude extracts, thereby enabling their unambiguous assignment, while subsequent selective mild acid hydrolysis of plasmenyl (vinyl-ether) containing lipids using formic acid enabled these species to be readily differentiated from isobaric mass plasmanyl (alkyl-ether) containing lipids. Using this approach, statistically significant differences in the abundances of numerous lipid species previously identified as being associated with cancer progression or that play known roles as mediators in a range of physiological and pathological processes were observed between the SW480 and SW620 cells. Most notably, these included increased plasmanylcholine and triglyceride lipid levels, decreased plasmenylethanolamine lipids, decreased C-16 containing sphingomyelin and ceramide lipid levels, and a dramatic increase in the abundances of total cholesterol ester and triglyceride lipids in the SW620 cells compared to those in the SW480 cells

Comprehensive Lipidome Profiling of Isogenic Primary and Metastatic Colon Adenocarcinoma Cell Lines

A “shotgun” lipidomics strategy consisting of sequential functional group selective chemical modification reactions coupled with high-resolution/accurate mass spectrometry and “targeted” tandem mass spectrometry (MS/MS) analysis has been developed and applied toward the comprehensive identification, characterization and quantitative analysis of changes in relative abundances of >600 individual glycerophospholipid, glycerolipid, sphingolipid and sterol lipids between a primary colorectal cancer (CRC) cell line, SW480, and its isogenic lymph node metastasized derivative, SW620. Selective chemical derivatization of glycerophosphoethanolamine and glycerophosphoserine lipids using a “fixed charge” sulfonium ion containing, d₆-<i>S</i>,<i>S</i>′-dimethylthiobutanoylhydroxysuccinimide ester (d₆-DMBNHS) reagent was used to eliminate the possibility of isobaric mass overlap of these species with the precursor ions of all other lipids in the crude extracts, thereby enabling their unambiguous assignment, while subsequent selective mild acid hydrolysis of plasmenyl (vinyl-ether) containing lipids using formic acid enabled these species to be readily differentiated from isobaric mass plasmanyl (alkyl-ether) containing lipids. Using this approach, statistically significant differences in the abundances of numerous lipid species previously identified as being associated with cancer progression or that play known roles as mediators in a range of physiological and pathological processes were observed between the SW480 and SW620 cells. Most notably, these included increased plasmanylcholine and triglyceride lipid levels, decreased plasmenylethanolamine lipids, decreased C-16 containing sphingomyelin and ceramide lipid levels, and a dramatic increase in the abundances of total cholesterol ester and triglyceride lipids in the SW620 cells compared to those in the SW480 cells

Deep Sequencing of RNA from Three Different Extracellular Vesicle (EV) Subtypes Released from the Human LIM1863 Colon Cancer Cell Line Uncovers Distinct Mirna-Enrichment Signatures

<div><p>Secreted microRNAs (miRNAs) enclosed within extracellular vesicles (EVs) play a pivotal role in intercellular communication by regulating recipient cell gene expression and affecting target cell function. Here, we report the isolation of three distinct EV subtypes from the human colon carcinoma cell line LIM1863 – shed microvesicles (sMVs) and two exosome populations (immunoaffinity isolated A33-exosomes and EpCAM-exosomes). Deep sequencing of miRNA libraries prepared from parental LIM1863 cells/derived EV subtype RNA yielded 254 miRNA identifications, of which 63 are selectively enriched in the EVs - miR-19a/b-3p, miR-378a/c/d, and miR-577 and members of the let-7 and miR-8 families being the most prominent. Let-7a-3p*, let-7f-1-3p*, miR-451a, miR-574-5p*, miR-4454 and miR-7641 are common to all EV subtypes, and 6 miRNAs (miR-320a/b/c/d, miR-221-3p, and miR-200c-3p) discern LIM1863 exosomes from sMVs; miR-98-5p was selectively represented only in sMVs. Notably, A33-Exos contained the largest number (32) of exclusively-enriched miRNAs; 14 of these miRNAs have not been reported in the context of CRC tissue/biofluid analyses and warrant further examination as potential diagnostic markers of CRC. Surprisingly, miRNA passenger strands (star miRNAs) for miR-3613-3p*, -362-3p*, -625-3p*, -6842-3p* were the dominant strand in A33-Exos, the converse to that observed in parental cells. This finding suggests miRNA biogenesis may be interlinked with endosomal/exosomal processing.</p></div

Summary of small RNA sequencing of LIM1863 cell and extracellular vesicles.

a<p>calculated as a percentage of raw reads.</p>b<p>calculated as a percentage of clean reads.</p><p>Summary of small RNA sequencing of LIM1863 cell and extracellular vesicles.</p

miRNAs enriched in different EV subtypes.

a<p>miRNA data for human CRC tissue/biofluid <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Luo1" target="_blank">[65]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Rossi1" target="_blank">[91]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Mazeh1" target="_blank">[92]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Xie1" target="_blank">[94]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Dong2" target="_blank">[97]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Plaisier1" target="_blank">[98]</a>.</p>*<p>miRNA star sequences identified in this study, according to miRBase.</p>+<p>Enriched miRNA star sequence in EV in comparison to mature miRNA.</p>$<p>miRNA star sequence expressed greater than the mature miRNA in all four libraries.</p><p>miRNAs enriched in different EV subtypes.</p

EV-enriched miRNAs from qRT-PCR and Deep Sequencing Data.

<p>The normalized expression values were log₂ (deep sequencing) and −ΔΔCt (qRT-PCR) transformed. Comparisons between A33-Exos/CL, EpCAM-Exos/CL, and sMVs/CL are shown.</p

Human colon carcinoma cell line LIM1863 release two populations of exosomes (A33-Exos and EpCAM-Exos) and shed microvesicles (sMVs).

<p><b>(A)</b> LIM1863 cells were grown in RPMI-1640 medium supplemented with 5% FCS (exosome-depleted), 100 U/ml penicillin and 100 µg/ml streptomycin in CELLine Bioreactor classic flasks and the culture medium (CM) collected. sMVs were first isolated from the CM (yield: ∼20 mg protein, 72 µg RNA). Next, A33-Exos were isolated from the sMV-free CM via anti-A33 antibody capture (yield: ∼3 mg protein, 8.8 µg RNA) and EpCAM-Exos were isolated from the A33-Exos-depleted CM using EpCAM-coupled magnetic beads (yield: ∼3 mg protein, 9.2 µg RNA). <b>(B-D)</b> Electron microscopy images of sMVs <b>(B)</b>, A33-Exos <b>(C)</b> and EpCAM-Exos <b>(D)</b> showing a size of 150–300 nm diameter and 40–100 nm diameter for sMVs and A33−/EpCAM-Exos, respectively. Scale bar: 100 nm (n = 3). <b>(E)</b> Western blot of EVs (10 µg protein) for A33, Alix (PDCD6IP), TSG101, and CD9. <b>(F)</b> Total RNA electropherogram analysis (Agilent Bioanalyzer) from LIM1863 cells and derived EVs. Y axis of electropherogram is arbitrary fluorescence unit intensity (FU) and x axis is migration time in seconds (s) and nucleotides (nt).</p

Candidate circulating miRNA biomarkers associated with extracellular vesicles and colorectal cancer.

<p>(<b>A</b>) Three-way Venn diagram depicting miRNAs identified in LIM1863-derived EVs that are associated with published miRNA data for human CRC tissue/blood and faeces <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Luo1" target="_blank">[65]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Rossi1" target="_blank">[91]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Mazeh1" target="_blank">[92]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Xie1" target="_blank">[94]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Dong2" target="_blank">[97]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Plaisier1" target="_blank">[98]</a>. 6 miRNAs are common to all three EVs, while 32, 2, and 4 are selectively represented in A33-Exos, EpCAM-Exos, and sMVs, respectively. miRNAs indicated in <i>black</i> bold represent association with CRC, <i>red</i> represents miRNAs not identified in previous CRC reviews/studies. *denotes miRNA star (miRNA*sequence). (<b>B</b>) Three-way Venn diagram of identified miRNAs in EVs associated with published reports for miRNAs from human CRC plasma/serum/faecal samples human <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Luo1" target="_blank">[65]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Rossi1" target="_blank">[91]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Mazeh1" target="_blank">[92]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Xie1" target="_blank">[94]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Dong2" target="_blank">[97]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone.0110314-Plaisier1" target="_blank">[98]</a>. Notably, miR-21 found in our study has been reported in published in human CRC plasma, serum and faeces samples. 47, 5, 16 miRNAs found in our studies have been previously reported in CRC plasma, serum, and faecal samples, respectively. <i>Red</i> represents miRNAs in our study that are highly-enriched in EVs (63 miRNA dataset); <i>green</i> represents other miRNAs identified in current study (from our 254 miRNA dataset).</p

miRNA star sequences (miRNA*) are enriched in LIM1863-derived EVs.

<p>(<b>A</b>) 13 miR star sequences (miRNA*) selectively enriched in LIM1863-derived EVs, compared to CL (based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110314#pone-0110314-t002" target="_blank">Table 2</a>). A33-Exos (<i>blue</i>), EpCAM-Exos (<i>red</i>), sMVs (<i>green</i>), CL (<i>purple</i>). $, miR star sequences highly enriched in all four miRNA libraries. (<b>B</b>) Log₂ values for passenger strand miRNA (miRNA*)/mature strand miRNA in all four miRNA libraries (based on 254 highly-represented miRNAs). A33-Exos (<i>blue</i>), EpCAM-Exos (<i>red</i>), sMVs (<i>green</i>), CL (<i>purple</i>).$ denotes miRNA* more highly expressed than corresponding mature miRNA in all 4 libraries; *⁺ denotes miRNA* strands selectively enriched in A33-Exos only, compared to mature miRNA strand expression.</p

Characterisation of 254 highly-represented miRNAs in all four miRNA libraries (A33-Exos, EpCAM-Exos, sMVs and CL).

<p>(<b>A</b>) Distribution of known miRNA sequences in CL, sMVs, A33- and EpCAM-Exos based on normalised expression values (transcripts per million reads, TPM). (<b>B</b>) Top 10 miRNA clusters based on analysis of the 254 miRNAs identified with respect to their location on the human reference genome (hg19). (<b>C</b>) Distribution of clustered miRNAs on different human chromosomes. Clustered miRNAs (including miRNA number) were identified according to their chromosomal locations, which should be within 10 k bp on the chromosome; miRNAs from the same precursor (-5p, -3p) were only considered once. (<b>D</b>) Three-way Venn diagram depicting the 63 miRNAs enriched in sMVs, A33- and EpCAM-Exos, relative to CL. 6 miRNAs are common to all EVs, while 22 miRNAs are common to both exosomal datasets. miRNAS selectively enriched in each EV miRNA dataset: A33-Exos 32/56, EpCAM-Exos 2/25, and sMVs 4/13.</p