Journal: EMBO Reports

Article Title: Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2

doi: 10.15252/embr.202051913

Figure Lengend Snippet: A Box plot showing the quantitative protein expression results of nanoLC‐MS/MS analysis reveals the significantly higher presence of Nid1 and Fbln2 in the Ndrg4 −/− compared to the Ndrg4 +/+ ENS cell secretome ( n = 4; NSAF, normalized spectral abundance factor). Data are analyzed with R version 3.5.2 and the ibb R package. Each dot within the box plot represents the NSAF of an individual sample; the inside band reflects the median, and the bottom and top of the box the first and third quartile, respectively. The whiskers reflect the minimum and maximal values within 1.5× the interquartile range. NSAF values were compared using the Mann–Whitney U ‐test. B Data derived from the Linnarsson mouse database (http://mousebrain.org/) display the expression of Nid1 and Fbln2 within cells of the central, peripheral, and enteric nervous system. Unit color scale: Expression (log(UMI)); UMI, unique molecular identifier. Abbreviations: ENS, enteric nervous system (i.e., enteric neurons and glial cells); OL, oligodendrocytes; Exc N, excitatory neurons in CNS; Hippo, hippocampus; MB, midbrain; HB, hindbrain; Str, striatum; Hyp, hypothalamus; BG, basal ganglia; OB, olfactory bulb; CB, cerebellum; SC, spinal cord; PNS, peripheral nervous system; Glia, glial cells; and Vasc, vasculature. C–E Representative immunohistochemistry (C) and more detailed immunofluorescence (D, E) labeling indicate that Nid1 (C, D) and Fbln2 (C, E) are expressed within the myenteric plexus and by primary ENS cells ((C), n = 3, black arrowheads, scale bars, 50 µm; (D, E), n = 3, scale bars, 10 µm).

Article Snippet: To assess the influence of the two identified extracellular matrix (ECM) proteins, Nidogen‐1 (NID1) and Fibulin‐2 (FBLN2), on the proliferation and migration capacities of the human CRC cell lines HCT116 and Caco‐2, a mixture of NID1 (2.0 μg/ml in PBS, 2570‐ND‐50, R&D systems) and FBLN2 (1.0 μg/ml in PBS, 9559‐FB‐050, R&D Systems) was added to the cell culture medium.

Techniques: Expressing, Tandem Mass Spectroscopy, MANN-WHITNEY, Derivative Assay, Immunohistochemistry, Immunofluorescence, Labeling

Journal: EMBO Reports

Article Title: Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2

doi: 10.15252/embr.202051913

Figure Lengend Snippet: Representative microscopic views show that NID1 and FBLN2 are highly expressed in the human colonic ganglia (black arrowheads) and interconnecting nerve fibers, and in a limited level throughout the epithelium ( n = 3). Scale bar, 50 µm. Addition of NID1&FBLN2 to the Matrigel® dome significantly enhanced the relative growth rate of HIOs after 5 days of culture as compared to the PBS control condition. Compared to the PBS control condition (white), stimulation with NID1&FBLN2 (black) enhanced the proliferation rate of HCT116 cells after 72 h, but did not affect Caco‐2 cell proliferation. The migration rate of HCT116 cells was already significantly increased 24 h after addition of NID1&FBLN2 (black), whereas the migration of Caco‐2 cells only significantly increased after 48 h of culture with NID1&FBLN2 (black). Box plot reflecting the quantitative nanoLC‐MS/MS analysis results shows the up‐regulation of NID1 and FBLN2 in the colorectal cancer tissue secretome ( n = 17) compared to the normal colon tissue secretome ( n = 17). Data are analyzed with R version 3.5.2 and the ibb R package. Within the box plots, each dot represents the log10‐transformed raw count of an individual sample; the inside band is the median value; the bottom and top of the box the first and third quartile, respectively; and the whiskers reflect the minimum and maximal values within 1.5× the interquartile range. Representative images of human colonic cancer epithelium ( n = 3) show a high scattered expression of NID1 and FBLN2 within the cancerous epithelium. Scale bar, 50 μm. Data information: Data in (B) are derived from three independent experiments and represented as relative mean percentage ± SEM versus day 0, with the P ‐value determined using a two‐tailed, unpaired t ‐test. Data in (C and D) are derived from three independent experiments and are represented as mean ± SEM, with P ‐values determined using a two‐tailed, unpaired t ‐test. To compare the paired normal colon and CRC secretome data in (E) ( n = 17), a paired Beta‐Binominal test, taking into account the sample origin (i.e., comparing protein signatures between tissues derived from the same patient), was performed (Pham & Jimenez, ; de Wit et al, ). Source data are available online for this figure.

Article Snippet: To assess the influence of the two identified extracellular matrix (ECM) proteins, Nidogen‐1 (NID1) and Fibulin‐2 (FBLN2), on the proliferation and migration capacities of the human CRC cell lines HCT116 and Caco‐2, a mixture of NID1 (2.0 μg/ml in PBS, 2570‐ND‐50, R&D systems) and FBLN2 (1.0 μg/ml in PBS, 9559‐FB‐050, R&D Systems) was added to the cell culture medium.

Techniques: Control, Migration, Tandem Mass Spectroscopy, Transformation Assay, Expressing, Derivative Assay, Two Tailed Test

Journal: Genes to Cells

Article Title: Alternative Splicing of FBLN2 Generates a Prometastatic Extracellular Matrix in Gastrointestinal Cancers by Determining N‐Glycosylation of Fibulin 2

doi: 10.1111/gtc.70027

Figure Lengend Snippet: Variant 2 is the major FBLN2 splice variant expressed in the fibroblasts of gastrointestinal cancers. (A) Schematic representation of the human FBLN2 gene. Exon (E) 9 (magenta) is included or excluded in variant 1 (v1) or variant 2 (v2) mRNAs, respectively. (B) Difference in splicing of FBLN2 exon 9 between normal (N) and primary tumor (T) tissue for 16 types of cancer in the TCGASpliceSeq database. Cancer type abbreviations are as in Table . PSI, percent spliced‐in. The values at the bottom indicate the number of tumor (T) and normal (N) tissues examined. (C, D) Box plots for PSI values of FBLN2 exon 9 determined from RNA‐seq data for normal (N), primary tumor (T), or metastatic liver tumor (M) tissue for four selected cancer types in TCGA (C) or for CRC in GSE50760 (D). (E) RT‐qPCR analysis of the expression of FBLN2 v1 and v2 in normal and primary tumor tissue isolated from CRC patients. Data are means ± SEM ( n = 7 patients). (F) Expression profiles for FBLN2 in CRC tissue determined by scRNA‐seq analysis ( GSE178341 ). The color intensity in the left plot represents the abundance of FBLN2 mRNA as shown by Log (TP10K + 1). TP10K + 1 indicates transcripts per 10 thousand plus one reads. The colors in the right plot correspond to cell identities. ILC, innate lymphoid cell; NK, natural killer. (G) Violin plots for the expression level of FBLN2 in five stromal cell types determined by scRNA‐seq analysis as in (F). (H) Representative immunohistochemical staining of FBLN2 in a tissue section containing normal epithelium isolated from a CRC patient. Boxed regions in the left image are shown at higher magnification in the middle and right images. BV, blood vessel; EL, epithelial layer; LP, lamina propria; MM, muscularis mucosae. Scale bars, 300 μm (left) and 100 μm (middle and right). (I) RT‐qPCR analysis of FBLN2 v1 and v2 expression in primary fibroblasts isolated from normal (N) or primary tumor (T) tissue of gastrointestinal cancer patients. Data are means ± SEM ( n = 6 patients). * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. (not significant) by the Wilcoxon rank sum test followed by Benjamini–Hochberg correction for multiple testing (B, C), by one‐way analysis of variance (ANOVA) followed by Tukey's post hoc test (D), or by the paired t test (E, I).

Article Snippet: For the transwell migration assay, the lower side of the filter membrane (diameter of 6.5 mm, pore size of 8 μm; 3422, Corning) was treated with FN1 (F0895, Sigma‐Aldrich), recombinant FBLN2 v2 (9559‐FB‐050, R&D Systems), or BSA (017‐23294, Fujifilm Wako) for 10 min and then allowed to dry for 30 min. HCT 116 cells were suspended in serum‐free McCoy's 5A medium, seeded at a density of 5 × 10 4 per membrane, and allowed to migrate for 24 or 48 h at 37°C and under 5% CO 2 .

Techniques: Variant Assay, RNA Sequencing, Quantitative RT-PCR, Expressing, Isolation, Immunohistochemical staining, Staining

Journal: Genes to Cells

Article Title: Alternative Splicing of FBLN2 Generates a Prometastatic Extracellular Matrix in Gastrointestinal Cancers by Determining N‐Glycosylation of Fibulin 2

doi: 10.1111/gtc.70027

Figure Lengend Snippet: Splicing of FBLN2 exon 9 determines N‐glycosylation of FBLN2 protein. (A, B) 24N‐T fibroblasts infected (or not) with a recombinant lentivirus encoding FBLN2‐3 × FLAG v1 or v2 were subjected to immunoblot analysis with antibodies to FLAG (A) or to RT‐qPCR analysis of FBLN2 mRNA (B). FN1 expression was analyzed as a loading control in (A). Data in (B) are means ± SEM ( n = 3 independent experiments). (C, D) 24N‐T fibroblasts engineered as in (A) were subjected to immunoprecipitation (IP) with antibodies to FLAG, and the resulting precipitates were subjected to SDS‐PAGE and staining with Oriole fluorescent dye (C) or to immunoblot analysis (IB) of HSPA5 (D). The arrowhead in (C) indicates the position of HSPA5 coprecipitated with FBLN2‐3 × FLAG v2. (E) FBLN2‐3 × FLAG v1 or v2 prepared from culture supernatants of 24N‐T fibroblasts engineered as in (A) was treated (or not) with the indicated glycosidases and then subjected to immunoblot analysis of FLAG. (F) HEK293T cells expressing FBLN2‐3 × FLAG v1 or v2 were treated with various concentrations of tunicamycin, after which medium (culture supernatant) and cell lysate (cells attached to culture dish) were prepared and subjected to immunoblot analysis of FLAG. Oriole staining of the SDS‐PAGE gel and immunoblot analysis of β‐actin were performed as loading controls for medium and cell lysate fractions, respectively. (G) Schematic representation of human FBLN2 v1 and v2 proteins. Triangles indicate the positions of putative N‐glycosylation sites. The positions of cbEGF‐like domains and anaphylatoxin (AT) modules are also shown. (H) Immunoblot analysis of FLAG for HEK293T cells expressing WT or mutant versions of FBLN2‐3 × FLAG v1 or v2 (upper panel). Cell lysates were fractionated by SDS‐PAGE in the presence (ConA gel) or absence (Standard gel) of concanavalin A. Schematic representations of N‐glycosylation sites for v1 (bottom left) and v2 (bottom right) are also shown. (I) FBLN2‐3 × FLAG v1 or v2 immunoprecipitated from 24N‐T fibroblasts engineered as in (A) was subjected to SDS‐PAGE and stained for glycoproteins (upper) or total proteins (lower). The different mobility of protein size markers between glycoprotein gel (upper) and total protein gel (lower) is likely due to the use of distinct molecular weight standards, in which the 180 kDa marker band is glycosylated. (J) Quantification of signal intensity as in (I). Data are means ± SEM ( n = 4 independent experiments). * p < 0.05, *** p < 0.001 by Student's t test (B, J).

Article Snippet: For the transwell migration assay, the lower side of the filter membrane (diameter of 6.5 mm, pore size of 8 μm; 3422, Corning) was treated with FN1 (F0895, Sigma‐Aldrich), recombinant FBLN2 v2 (9559‐FB‐050, R&D Systems), or BSA (017‐23294, Fujifilm Wako) for 10 min and then allowed to dry for 30 min. HCT 116 cells were suspended in serum‐free McCoy's 5A medium, seeded at a density of 5 × 10 4 per membrane, and allowed to migrate for 24 or 48 h at 37°C and under 5% CO 2 .

Techniques: Glycoproteomics, Infection, Recombinant, Western Blot, Quantitative RT-PCR, Expressing, Control, Immunoprecipitation, SDS Page, Staining, Mutagenesis, Molecular Weight, Marker

Journal: Genes to Cells

Article Title: Alternative Splicing of FBLN2 Generates a Prometastatic Extracellular Matrix in Gastrointestinal Cancers by Determining N‐Glycosylation of Fibulin 2

doi: 10.1111/gtc.70027

Figure Lengend Snippet: FBLN2 v2 is less stable and secreted to a lesser extent compared with v1. (A–C) 24N‐T fibroblasts infected with a recombinant retrovirus encoding FBLN2‐3 × FLAG v1 or v2 were subjected to RT‐qPCR analysis of FBLN2 mRNA (A) or to cellular fractionation followed by immunoblot analysis of FLAG (B, C). Quantitative data are means ± SEM ( n = 4 independent experiments). (D–G) 24N‐T fibroblasts engineered as in (A) were incubated with cycloheximide (100 μg/mL) for the indicated times (D) or with various concentrations of MG132 for 6 h (F), after which cell lysates were subjected to immunoblot analysis of FLAG or β‐actin (loading control). Arrowheads indicate the positions of the intracellular forms of FBLN2. Signal intensity for the intracellular forms of v1 and v2 was also determined (E, G), with the data presented as means ± SEM ( n = 4 independent experiments). ** p < 0.01, *** p < 0.001, n.s. by Student's t test (A, C), by repeated measures ANOVA (E), or by two‐way ANOVA (G).

Article Snippet: For the transwell migration assay, the lower side of the filter membrane (diameter of 6.5 mm, pore size of 8 μm; 3422, Corning) was treated with FN1 (F0895, Sigma‐Aldrich), recombinant FBLN2 v2 (9559‐FB‐050, R&D Systems), or BSA (017‐23294, Fujifilm Wako) for 10 min and then allowed to dry for 30 min. HCT 116 cells were suspended in serum‐free McCoy's 5A medium, seeded at a density of 5 × 10 4 per membrane, and allowed to migrate for 24 or 48 h at 37°C and under 5% CO 2 .

Techniques: Infection, Recombinant, Quantitative RT-PCR, Cell Fractionation, Western Blot, Incubation, Control

Journal: Genes to Cells

Article Title: Alternative Splicing of FBLN2 Generates a Prometastatic Extracellular Matrix in Gastrointestinal Cancers by Determining N‐Glycosylation of Fibulin 2

doi: 10.1111/gtc.70027

Figure Lengend Snippet: The ECM environment of CRC tissue exhibits low FBLN2 and high FN1 abundance. (A) Lysates prepared from normal (N) or primary tumor (T) tissue of CRC patients were subjected to immunoblot analysis of FBLN2 with the indicated antibodies. The gel was also stained with Oriole fluorescent dye to allow adjustment for protein loading. (B) Quantification of signal intensity as in (A). Data are means ± SEM ( n = 9 patients). (C) Representative immunohistochemical staining of extracellular FBLN2 (HPA001934) in a section containing primary tumor (T) and adjacent normal (N) tissue isolated from a CRC patient. The boxed regions in the upper image are shown at higher magnification in the lower images. FBLN2 signals in the lamina propria (LP) are indicated by arrowheads in the lower left image. BV, blood vessel; EL, epithelial layer; MM, muscularis mucosae. Scale bars, 1 mm (upper) and 100 μm (lower). (D) Representative immunofluorescence analysis of FBLN2 and FN1 in 21N‐T (upper) or 24N‐T (lower) fibroblasts isolated from rectal cancer or gastric cancer patients, respectively. DNA was stained with 4′,6‐diamidino‐2‐phenylindole (DAPI). Scale bars, 50 μm. (E) Box plots for FN1 mRNA level based on transcripts per million (TPM) in normal (N) and primary tumor (T) tissue for COADREAD in TCGA. (F) Immunoblot analysis of FN1 in lysates prepared from normal (N) and primary tumor (T) tissue of CRC patients. The gel was stained for total proteins with Oriole fluorescent dye. (G) Box plots for FN1 mRNA level based on fragments per kilobase of exon per million mapped reads (FPKM) in normal (N), primary CRC tumor (T), and metastatic liver tumor (M) tissue determined by RNA‐seq analysis ( GSE50760 ) as in Figure . * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. by the paired t test (B), Wilcoxon rank sum test (E) or by one‐way ANOVA followed by Tukey's post hoc test (G).

Article Snippet: For the transwell migration assay, the lower side of the filter membrane (diameter of 6.5 mm, pore size of 8 μm; 3422, Corning) was treated with FN1 (F0895, Sigma‐Aldrich), recombinant FBLN2 v2 (9559‐FB‐050, R&D Systems), or BSA (017‐23294, Fujifilm Wako) for 10 min and then allowed to dry for 30 min. HCT 116 cells were suspended in serum‐free McCoy's 5A medium, seeded at a density of 5 × 10 4 per membrane, and allowed to migrate for 24 or 48 h at 37°C and under 5% CO 2 .

Techniques: Western Blot, Staining, Immunohistochemical staining, Isolation, Immunofluorescence, RNA Sequencing

Journal: Genes to Cells

Article Title: Alternative Splicing of FBLN2 Generates a Prometastatic Extracellular Matrix in Gastrointestinal Cancers by Determining N‐Glycosylation of Fibulin 2

doi: 10.1111/gtc.70027

Figure Lengend Snippet: FBLN2 suppresses the adhesion and migration of CRC cells. (A) Representative results for a transwell migration assay in which HCT 116 cells were seeded on a transwell insert coated (or not) with FN1 (20 μg/mL) or FBLN2 v2 (20 μg/mL) and were then allowed to migrate for 48 h. (B) Quantification of the area of migrated cells as in (A). Data are means ± SEM ( n = 3 independent experiments). (C) Representative results for a transwell migration assay in which HCT 116 cells were seeded on a transwell insert coated (or not) with FN1 (20 μg/mL) and various concentrations of FBLN2 v2 or BSA and were then allowed to migrate for 24 h. (D) Quantification of the area of migrated cells as in (C). Data are means ± SEM ( n = 4 independent experiments). (E) Representative results for a cell adhesion assay in which HCT 116 cells were seeded in low‐attachment dishes coated (or not) with FN1 (2 μg/mL) and various concentrations of FBLN2 v2 or BSA. (F) Quantification of attached cells as in (E). (G) Model for the role of alternative splicing of FBLN2 exon 9 in the remodeling of ECM. Data are means ± SEM ( n = 3 independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. by one‐way ANOVA followed by Tukey's post hoc test (B, D, F). Scale bars, 200 μm (A, C) and 100 μm (E).

Article Snippet: For the transwell migration assay, the lower side of the filter membrane (diameter of 6.5 mm, pore size of 8 μm; 3422, Corning) was treated with FN1 (F0895, Sigma‐Aldrich), recombinant FBLN2 v2 (9559‐FB‐050, R&D Systems), or BSA (017‐23294, Fujifilm Wako) for 10 min and then allowed to dry for 30 min. HCT 116 cells were suspended in serum‐free McCoy's 5A medium, seeded at a density of 5 × 10 4 per membrane, and allowed to migrate for 24 or 48 h at 37°C and under 5% CO 2 .

Techniques: Migration, Transwell Migration Assay, Cell Adhesion Assay, Alternative Splicing