Journal: iScience

Article Title: SARS-CoV-2 envelope protein regulates innate immune tolerance

doi: 10.1016/j.isci.2024.109975

Figure Lengend Snippet:

Article Snippet: On day 1, depending on the experiment, monocytes were stimulated with 1 μg/mL, 10 ng/mL, 1 ng/mL or 10 pg/mL of recombinant SARS-CoV-2 envelope protein (Novus Biologicals, Centennial, CO, USA), 1 μg/mL of SARS-CoV-2 spike recombinant protein (Sino Biological, Wayne, PA, USA) or 1 μg/mL of nucleocapsid protein (Sino Biological) for 24 h. On day 2, the media from each well was transferred to a microcentrifuge tube and centrifuged at 300 g for 10 min at room temperature.

Techniques: Recombinant, Selection, Isolation, Software

Journal: eBioMedicine

Article Title: Neural synaptic vesicle autoimmunity following aerosolized porcine neural tissue exposure: insights into autoimmune inflammatory polyradiculoneuropathy

doi: 10.1016/j.ebiom.2025.106053

Figure Lengend Snippet: Confirmation of synaptophysin- and GAP43-IgG in patients with OIPN by ELISA and CBA . a) Fifteen of 20 patients with OIPN sera tested positive for synaptophysin-IgG by ELISA compared to none of the control cohorts. b) Representative immunofluorescent images of IgG binding to GFP-tagged synaptophysin-transfected COS7 cells. Commercial synaptophysin-IgG and OIPN-IgG (magenta) co-localises (white in merge) with GFP-tagged synaptophysin protein (green). c) Twelve of 20 sera from patients with OIPN tested positive for GAP43-IgG by ELISA. d) Representative immunofluorescent images of IgG binding (magenta) to GFP-tagged GAP43 transfected COS7 cells. Colocalisation (white in merge) is observed for commercial antibody and OIPN-IgG with GFP-GAP43 protein. b and d) DNA is labelled with DAPI, blue. Healthy adult human IgG fails to bind to transfected cells. Note: No IgG bound to non-transfected cells (solitary blue nuclei). Scale bars, 20 μm. Key: OIPN, occupational inflammatory polyradiculoneuropathy; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescence protein; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Immulon 2HB plates were coated (10 ng/well) with PhIP-Seq-identified antigens: synaptophysin peptide 224-313 (Mayo Clinic Proteomics Core Facility) or GAP43 recombinant protein (Novus, #NBP2-53033) diluted in 0.01 M PBS, pH 7.4.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Binding Assay, Transfection, Fluorescence

Journal: Disease Models & Mechanisms

Article Title: Development of a physiologically relevant and easily scalable LUHMES cell-based model of G2019S LRRK2-driven Parkinson's disease

doi: 10.1242/dmm.048017

Figure Lengend Snippet: Phosphorylation of bona fide LRRK2 substrates in LUHMES clones. (A) Representative WB results showing the phosphorylation of two LRRK2 bona fide substrates, LRRK2 Ser1292 and RAB10 Thr73, in naïve and clonal LUHMES cells (L10WT and L14GS). LUHMES cells were differentiated for up to 4 days, and levels of total and phosphorylated LRRK2 and RAB10 were analyzed each day. GAPDH was used to ensure equal loading. The protein corresponding molecular mass (in kDa) is indicated on the left side of the panel. (B) Quantification of pRAB10 levels. Results from six independent experiments. Error bars show mean±s.d.

Article Snippet: Control proteins were recombinant full-length human his-tagged RAB10 (Novus Biological, catalog no. NBP2-23392) and human full-length LRRK2 (ThermoFisher Scientific, catalog no. 15383806).

Techniques: Phospho-proteomics, Clone Assay

Journal: Disease Models & Mechanisms

Article Title: Development of a physiologically relevant and easily scalable LUHMES cell-based model of G2019S LRRK2-driven Parkinson's disease

doi: 10.1242/dmm.048017

Figure Lengend Snippet: LRRK2 kinase inhibitor MLI-2 effectively reduces phosphorylation of two bona fide LRRK2 substrates. (A) LUHMES cells were differentiated for 2 days and then treated with increasing concentrations of LRRK2 kinase inhibitor MLI-2 for 4 h. A potent reduction in the phosphorylation of LRRK2 Ser1292 and RAB10 Thr73 was observed. (B,C) Dose-response curves of the LRRK2 kinase inhibitor MLI-2 against LRRK2 pSer1292 and RAB10 pThr73 in WT and G2019S LUHMES cells. The protein corresponding molecular mass (in kDa) is indicated on the left side of the panel. (B) Half-maximal inhibitory concentration (IC 50 ) values, calculated for WT and G2019S LRRK2 by GraphPad Prism software on LRRK2 pSer1292, were found to be 21.2 pM (L10WT clone, filled circles) and 1.45 nM (L14GS clone, filled squares). (C) IC 50 values, calculated by GraphPad Prism software for WT and G2019S LRRK2 on RAB10 pThr73, were found to be 0.78 nM (L10WT clone, filled circles) and 2.31 nM (L14GS clone, filled squares). Results from three independent experiments.

Article Snippet: Control proteins were recombinant full-length human his-tagged RAB10 (Novus Biological, catalog no. NBP2-23392) and human full-length LRRK2 (ThermoFisher Scientific, catalog no. 15383806).

Techniques: Phospho-proteomics, Concentration Assay, Software

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM expression is increased in liver metastases (Met) as compared to matched primary tumors (Pri). Immunohistochemistry (IHC) for L1CAM is shown in matched normal colon, primary CRC tumor and liver metastasis sections from a representative patient. Arrows indicate L1CAM staining at the invasion front of the primary tumor. Detail of the boxed region is shown in Extended Data Fig. 1a. b, The percentage of L1CAM-expressing tumor cells in each section. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 18 paired patient samples; two-sided Wilcoxon matched-pairs signed-rank test. c, Percentage of L1CAM-expressing cells in matched pretreatment (pre-treat.) biopsies and post-treatment (post-treat.) surgically resected residual disease in patients with locally advanced rectal cancer. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 31 patients; two-sided Wilcoxon matched-pairs signed-rank test. d, Representative sections of paired pretreatment core biopsies and matched surgical resection specimens obtained after chemoradiation, from two patients with rectal adenocarcinoma, showing L1CAM expression in peripheral areas of residual adenocarcinoma after treatment. e, L1CAM immunohistochemistry in a human CRC liver metastasis resected after neoadjuvant chemotherapy, showing dense stromal infiltration and L1CAM-expressing residual tumor cell clusters. Representative of 18 samples analyzed. f, Tumor L1CAM expression is associated with greater organoid generation capacity. Median L1CAM expression is shown for freshly resected and dissociated patient CRC liver metastases measured by flow cytometry before plating of 10,000 cells in 40 μl of Matrigel using organoid medium. Organoid generation ability was assessed 14 d after plating. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 14 paired patient tumor samples; two-sided Mann-Whitney U test. g, Number of organoids (mean ± s.e.m.) grown from 10,000 L1CAMhigh or L1CAMlow cells flow-sorted from freshly resected patient CRC primary tumors (P, left) or liver metastases (Li, right), counted 14 d after surgical resection and flow sorting. From left to right, n = 3, 3, 3, 3, 3, 3, 3, 3, 3, 4, 3, 4, 4 and 11 replicates per group from each of seven patients; two-tailed Student’s t tests. h, Subcutaneous tumor volumes measured 35 d after transplantation of mice with 50,000 organoid-derived flow-sorted L1CAMhigh or L1CAMlow cells (mean ± s.e.m.); n = 5 mice per group; two-tailed Mann-Whitney U test. i, Biaxial density plot showing relative expression of L1CAM and LGR5 in 9,974 cells from four independent patient-derived metastatic CRC organoids subjected to scRNA-seq. Five clusters identified according to relative L1CAM and LGR5 expression are overlaid as colors on the density plot. j, Dual LGR5 mRNA FISH and L1CAM immunofluorescence (IF) on a patient primary CRC tissue section (top, low magnification; bottom, high magnification), showing discrete expression levels of LGR5 and L1CAM in different cell clusters, including double-positive cells. Representative field of eight tumor sections from four patients analyzed.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Expressing, Immunohistochemistry, Staining, Flow Cytometry, MANN-WHITNEY, Two Tailed Test, Transplantation Assay, Derivative Assay, Immunofluorescence

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM inhibition rescues the increase in organoid generation secondary to REST inhibition. MSK107Li organoids stably expressing the indicated shRNAs were grown in the presence or absence of doxycycline for 7 d before measuring cell viability (luminescence relative to day 0; in box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 6 organoid cultures per group; two-sided Mann-Whitney U tests). b, Relative mRNA levels of L1CAM and REST on day 0, normalized to GAPDH, in organoid-derived cells transduced with lentiviruses directing expression of the indicated shRNAs in the presence or absence of doxycycline. Data are shown as the mean ± s.e.m.; n = 4 organoid cultures per group; two-sided Student’s t tests. c, Left: schematic diagram showing how loss of epithelial integrity induces L1CAM expression during wound healing and tumor invasion, ultimately driving metastatic relapse. Right schematic diagram showing that loss of membrane E-cadherin in cells detached from their epithelial niche downregulates and displaces REST from the L1CAM enhancer, thus enabling L1CAM expression.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Inhibition, Stable Transfection, Expressing, MANN-WHITNEY, Derivative Assay, Transduction, Membrane

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, Immunohistochemistry for L1CAM (top) and Ki67 (bottom) in serial sections of a representative human CRC primary tumor invasion front showing an inverse relationship between L1CAM and Ki67 expression. Representative of 16 tumors analyzed. b, Immunohistochemistry for L1CAM (top) and Ki67 (bottom) in serial sections of a representative post-treatment human CRC liver metastasis demonstrating L1CAMhighKi67low cells in organized epithelial structures and L1CAMhighKi67high cells in disrupted epithelia. Representative of 16 tumors analyzed. c, Percentage of L1CAMhigh and L1CAMlow cells that are also Ki67high in regions of intact versus disrupted glandular epithelial architecture within CRCs. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 64, 54, 28 and 34 independent fields from 16 patient tumors; two-sided Mann-Whitney U tests. d, Immunohistochemistry for L1CAM (top) and Ki67 (bottom) in serial sections of MSK107Li matched surgically resected patient CRC liver metastasis, metastasis-derived organoids and organoid-derived subcutaneous xenograft. Dashed red lines indicate the tumor-stromal boundary. Box plots indicate the percentage of Ki67+ cells among L1CAMhigh cells in the indicated sections. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 7, 9 and 9 independent fields; two-sided Mann-Whitney U tests. e,f, L1CAM is induced during normal epithelial organoid formation. Relative L1CAM mRNA levels (mean ± s.e.m.) are shown for human (e) and mouse (f) cells freshly isolated from intact colons or collected after 14 d of growth in organoid conditions. Data were normalized to GAPDH mRNA levels. n = 4 crypts or organoid cultures from each of three patients or mice; two-sided Student’s t tests. g, Relative LGR5 mRNA levels (mean ± s.e.m.) in human cells freshly isolated from intact colons or collected after 14 d of growth in organoid conditions. Data were normalized to GAPDH mRNA levels. n = 4 crypts or organoid cultures from each of three patients; two-sided Student’s t tests. h, L1CAM is induced during epithelial regeneration after colitis. C57BL/6J mice were given 3.5% DSS in their drinking water for 5 d, inducing maximal colitis by day 7, and were then maintained on water without DSS for 12 d. Mice were killed at each of the indicated time points, and their colons were collected, sectioned and either stained with Kreyberg-Jareg stain (blue, mucin; pink, collagen) or subjected to immunohistochemistry for L1CAM. Representative images of three independent experiments are shown. i, High-magnification view showing detail of L1CAM immunohistochemical staining throughout the length of the intestinal crypt, representative of three mice each from three independent experiments.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Immunohistochemistry, Expressing, MANN-WHITNEY, Derivative Assay, Isolation, Staining, Immunohistochemical staining

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM immunohistochemistry in a representative colon section from an L1CAMΔIEC mouse maintained on water with DSS, showing L1CAM staining restricted to submucosal neurons and no L1CAM expression in the epithelial cells of the crypt. Representative of three independent mice. b–f, L1CAM deficiency impairs epithelial healing following DSS-induced colitis. L1CAMfl/y and L1CAMΔIEC mice were given 3.5% DSS in their drinking water for 5 d, followed by 12 d of water without DSS before being killed. b, Kaplan-Meier survival curves. n = 26 mice per group from three independent experiments; two-sided Mantel-Cox test. c, Disease activity index (composite of weight loss, diarrhea and rectal bleeding) measured at the time of maximal colitis on day 7. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 26 mice per group; two-sided Mann-Whitney U test. d, Histological score (composite of inflammation, mucosal denudation and crypt dysmorphia) on day 14. L1CAMfl/y, n = 24; L1CAMΔIEC, n = 12. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; two-sided Mann-Whitney U test. e, Representative histological sections with Kreyberg-Jareg staining (blue, mucin; pink, collagen) and immunohistochemical staining for L1CAM showing denudation of mucin-producing crypts in L1CAMΔIEC mice on day 14. Panels on the right show higher magnification of less damaged areas of the colon exhibiting loss of L1CAM immunostaining in crypts of L1CAMΔIEC mice. Representative of three independent experiments. f–l, L1CAM deficiency in the progeny of LGR5-expressing cells impairs epithelial healing after DSS-induced colitis. f, Immunohistochemistry for GFP in a representative colon section from an L1CAMΔLGR5 mouse killed after 5 d of daily tamoxifen treatment. n = 3 mice. g, WTΔLGR5 and L1CAMΔLGR5 mice were treated as in f and killed, and their colon crypts were isolated and seeded for organoid generation. Representative flow cytometry assessment is shown of L1CAM expression 24 h after seeding. n = 2 mice per genotype. h, Schematic of experimental design. Mice were treated with tamoxifen for 5 d to induce Lgr5-GFP-IRES-creERT2 expression and subsequently treated with 3% DSS for 5 d, with maximal colitis by day 7. They were then maintained on water without DSS for a further 12 d before being killed. i, Representative histological sections with Kreyberg-Jareg staining (blue, mucin; pink, collagen) and immunohistochemical staining for L1CAM showing denudation of mucin-producing crypts in the distal colon in L1CAMΔLGR5 mice on day 14, while crypts are restored in L1CAMfl/y and WTΔLGR5 mice. The panels on the right show higher magnification of less damaged areas of the colon exhibiting loss of L1CAM immunostaining in crypts in L1CAMΔLGR5 mice in comparison to L1CAMfl/y and WTΔLGR5 mice. Representative of 20 evaluable mice from two independent experiments. j, Kaplan-Meier plot showing cumulative survival of L1CAMfl/y, WTΔLGR5 and L1CAMΔLGR5 mice. n = 33 mice from two independent experiments; two-sided Mantel-Cox tests. k, Disease activity index (composite of weight loss, diarrhea and rectal bleeding) measured at the time of maximal colitis on day 7. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 33 mice from two independent experiments; two-sided Mann-Whitney U tests. l, Histological scores (composite of inflammation, mucosal denudation and crypt dysmorphia) on day 14. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 20 evaluable mice from two independent experiments; two-sided Mann-Whitney U tests.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Immunohistochemistry, Staining, Expressing, Activity Assay, MANN-WHITNEY, Immunohistochemical staining, Immunostaining, Isolation, Flow Cytometry, Comparison

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM is required for organoid regeneration. CRC107Li organoid-derived cells were transduced with lentivirus directing the expression of either Cas9 alone or Cas9 with sgRNAs targeting L1CAM and allowed to grow under antibiotic selection for 14 d, when they were flow-sorted and seeded at a concentration of 2,000 cells per 40 μl of Matrigel in independent wells of a 96-well plate. The number of organoids (mean ± s.e.m.) established from each population 14 d after sorting and seeding is shown. From left to right, n = 10, 13 and 11 organoid cultures per group; two-tailed Mann-Whitney U test. b,c, L1CAM knockdown inhibits regrowth of multiple patient-derived organoids. Organoids derived from four patients with metastatic CRC were transduced with lentiviruses directing the expression of doxycycline (Dox)-inducible shRNA targeting L1CAM, expanded and, where indicated, treated with doxycycline for 48 h before dissociation and seeding at a concentration of 2,000 cells per 40 μl of Matrigel. Knockdown efficiencies of two independent L1CAM-targeting shRNAs in four patient-derived organoids (b) and relative cell viability on day 14 as compared to day 0 (mean ± s.e.m.) after plating of organoid-derived single cells (c) are shown. n = 6 organoid cultures per group; two-sided Student’s t tests. d, L1CAM is required for subcutaneous tumor growth in vivo. MSK107Li organoid-derived cells (50,000) expressing a doxycycline-inducible shRNA targeting L1CAM were injected subcutaneously into each flank of immunodeficient NSG mice. Where indicated, organoids were treated with doxycycline 2 d before transplantation and mice were maintained on a doxycycline diet for the duration of the experiment. Tumor volume (mean ± s.e.m.) was measured with calipers at the indicated time points after subcutaneous inoculation. n = 10 tumors from five mice per group; two-tailed Mann-Whitney U test. e, Representative image and quantification of tumor bioluminescence measured 35 d after inoculation. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 10 tumors from five mice per group; two-sided Mann-Whitney U test. f, Day 21 steady-state MSK107Li and MSK121Li organoids were incubated in medium containing 50 μM irinotecan, and L1CAM expression was measured in residual DAPI− cells 7 d later. Top- flow cytometry plots showing distribution of the data. Bottom: bars showing median fluorescence intensity of L1CAM expression in each population. From left to right, n = 6,512, 130, 8,542 and 49 cells per group, representative of three independent experiments. g, Single cells derived from CRC107Li organoids transduced with lentivirus directing expression of the indicated shRNAs were seeded at a concentration of 2,000 cells per 40 μl grown as organoids for 21 d and then treated with doxycycline and/or irinotecan (irino) as indicated. The viability assay shows the luminescence (mean ± s.e.m.) of each population relative to the luminescence at the time that drug treatment was started (day 0); n = 5 organoid cultures per group; two-sided Mann-Whitney U test. h, Solid-phase binding assay showing dose-response curves of recombinant human L1CAM-Fc binding to plates coated with equimolar concentrations of the indicated proteins. After washing, bound L1CAM-Fc was detected with horseradish peroxidase (HRP)-conjugated anti-human IgG, HRP substrate was added and OD450 was measured. Data are shown as the mean ± s.e.m; n = 5 wells per time point, representative of three independent experiments; two-tailed Mann-Whitney U test. i, L1CAM mediates the interaction of dissociated CRC cells with laminin isoforms. Single cells derived from MSK121Li organoids (3,000) cultured in the presence or absence of doxycycline to knock down L1CAM were seeded in wells coated with 30 nM of the indicated proteins. After 1 h of adhesion and extensive washing, the percentage of adherent cells (mean ± s.e.m.) was measured as the relative luminescence of each well immediately after plating. n = 10 organoid cultures per condition; two-tailed Mann-Whitney U tests.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Derivative Assay, Transduction, Expressing, Selection, Concentration Assay, Two Tailed Test, MANN-WHITNEY, shRNA, In Vivo, Injection, Transplantation Assay, Incubation, Flow Cytometry, Fluorescence, Viability Assay, Binding Assay, Recombinant, Cell Culture

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, L1CAM expression is dynamically regulated during organoid growth. L1CAM immunohistochemistry of CRC organoids of varying size shows progressive restriction of L1CAM expression to cells at the periphery and an overall decrease in L1CAM expression with increasing organoid size. Representative of six organoid lines analyzed. b, L1CAM is induced by dissociation of normal, primary tumor and metastatic human organoids. Relative L1CAM mRNA levels, normalized to GAPDH mRNA levels (mean ± s.e.m.), were measured in intact organoids versus organoid-derived single-cell suspensions plated in Matrigel and assayed 24 h after dissociation. n = 4 replicates per group; two-sided Student’s t tests. c, Time course of regenerating organoids derived from L1CAMhigh (left) and L1CAMlow (right) cells flow-sorted from day 21 MSK107Li (top) and MSK121Li (bottom) organoids. Histograms show the distribution of L1CAM expression (measured by APC fluorescence) in each population at the indicated time points after flow sorting. Representative of three independent experiments. d,e, Dynamic induction of the L1CAMhigh phenotype by a subset of pre-existing L1CAMlow cells. CRC107Li organoids were labeled with lentivirally expressed tdTomato or GFP, and flow-sorted tdTomato+GFP−L1CAMhigh and tdTomato−GFP+L1CAMlow cells were mixed in equal proportions and allowed to regrow as organoids in the presence or absence of irinotecan. Flow plots of the distribution of tdTomato- and GFP-expressing cells (d) and the relative proportions of these cells in the population (e) are shown from monitoring by flow cytometry at the indicated time points after mixing; from left to right, n = 17, 107, 68, 251, 484 and 348 DAPI− single cells representative of three independent experiments with two organoid lines; two-sided chi-squared tests. f,g, Flow cytometry contour plots showing the distribution of L1CAM expression at the indicated time points in the presence or absence of chemotherapy (f) and median fluorescence intensity (MFI) of L1CAM expression (measured by APC) in cells derived from tdTomato+GFP−L1CAMhigh and tdTomato−GFP+L1CAMlow precursors on each day (g), representative of three independent experiments with two organoid lines.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: Expressing, Immunohistochemistry, Derivative Assay, Fluorescence, Labeling, Flow Cytometry

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a,b, L1CAM is not required for intestinal adenoma formation. Male APCΔIEC and L1CAM/APCΔIEC mice were killed at 3 months of age, and their colons were collected, sectioned and examined for adenoma formation. a, Number of adenomas per mouse intestine. In box plots, boxes show the 25th–75th percentile with the median and whiskers show the minimum-maximum; n = 5 mice per group; two-sided Mann-Whitney U test. b, Mean adenoma diameter per mouse. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; n = 5 mice per group; two-sided Mann-Whitney U test. c–e, L1CAM inhibition impairs orthotopic rectal tumor engraftment. NSG mice were given 3% DSS in their water for 5 d and then maintained on water without DSS for 2 d before intraluminal transplantation with 2 × 105 cells from dissociated MSK107Li organoids expressing doxycycline-inducible shRNA targeting L1CAM or control shRNA. Where indicated, organoids were treated in vitro with doxycycline starting 2 d before transplantation, and mice were maintained on a doxycycline diet. Mice were killed 90 d after transplantation, and their colons were collected and examined for tumor engraftment. c, Percentage of mice with an engrafted orthotopic tumor. From left to right, the stacked bar graphs show n = 7, 7, 10, 13, 9 and 7 mice per group from three independent experiments; two-sided chi-squared tests. d,e, Tumor diameter per engrafted mouse (in box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 6, 5, 6, 4, 5 and 1 mice from three independent experiments; two-sided Mann-Whitney U tests) (d) and representative H&E-stained sections (e). Arrows indicate tumour diameter. f,g, L1CAM inhibition impairs metastatic colonization of the liver. Cells (5 × 104) were derived from dissociated MSK107Li organoids with doxycycline-inducible expression of shRNA targeting L1CAM. Where indicated, organoids were treated with doxycycline starting 2 d before transplantation and mice were maintained on a doxycycline diet. Representative H&E-stained sections of liver metastases at the experimental endpoint (arrows indicate tumour diameter) (f) and quantification of ex vivo liver bioluminescence signal measured 60 d after transplantation (g) are shown. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 9, 10, 8, 9, 5 and 5 mice per group; two-sided Mann-Whitney U tests. h–l, L1CAM inhibition impairs local tumor expansion and metastasis from orthotopic cecal xenografts. h, Schematic of the experiment: cells (4 × 105) derived from MSK121Li organoids transduced with lentivirus directing the expression of tdTomato-luciferase and shRNA targeting L1CAM or control shRNA were injected into the cecal submucosa. Mice were monitored until cecal tumors were evident by ex vivo bioluminescence imaging 3 weeks after injection, randomized on the basis of bioluminescence signal and maintained on or off a doxycycline diet for 7 weeks before being killed. i–l, Quantification of whole-mouse bioluminescence signal (i) and ex vivo bioluminescence signal in the cecum (j), liver (k) and lung (l), normalized to bioluminescence at the time of randomization. In box plots, boxes show the 25th–75th percentile with the median, and whiskers show the minimum-maximum; from left to right, n = 5, 6, 12 and 11 mice per group; two-sided Mann-Whitney U tests. m–o, Combination of L1CAM inhibition with chemotherapy impairs tumor growth to a greater extent than chemotherapy alone. m, Schematic of the experiment. Cells (2 × 105) derived from MSK107Li organoids transduced with lentivirus directing the expression of tdTomato-luciferase and shRNA targeting L1CAM or control shRNA were injected subcutaneously; mice were randomized on the basis of bioluminescence intensity 5 weeks after injection and maintained on a doxycycline diet and/or treated weekly with irinotecan as indicated for 4 weeks before being killed. n,o, Ex vivo tumor volume (n) and mass (o), normalized to tumor bioluminescence at the time of randomization. From left to right, n = 10, 10, 8, 7, 10, 10, 10 and 9 tumors per group; mean ± s.e.m.; two-sided Mann-Whitney U tests.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: MANN-WHITNEY, Inhibition, Transplantation Assay, Expressing, shRNA, In Vitro, Staining, Derivative Assay, Ex Vivo, Transduction, Luciferase, Injection, Imaging

Journal: Nature cancer

Article Title: L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

doi: 10.1038/s43018-019-0006-x

Figure Lengend Snippet: a, REST ChIP-seq analysis, showing diminution of the REST peak at the L1CAM intronic enhancer in dissociated organoid-derived cells, collected 16 h after dissociation, in comparison to intact organoids. Input control is also shown. LCA10 (gray) is not expressed in CRC organoids. Two independent organoid cultures from two patient-derived organoid lines were analyzed per condition. b, Relative mRNA levels (mean ± s.e.m.) of REST and L1CAM in intact MSK107Li organoids (day 0), cells collected 24 h after dissociation and plating as single cells (day 1), and cells collected at the indicated time points during organoid regeneration. Organoids were transduced with lentivirus constitutively expressing shRNA targeting REST or control shRNA. Gene expression was normalized to GAPDH mRNA levels. Day 1 shControl versus shREST.1: P < 0.0001 (REST), P < 0.0001 (L1CAM); day 1 shControl versus shREST.2: P = 0.007 (REST), P < 0.0001 (L1CAM); n = 4 organoid cultures per sample per time point; two-sided Student’s t tests. c, Relative expression of CDH1, REST and L1CAM (mean ± s.e.m.) in intact MSK107Li organoids transduced with lentivirus constitutively expressing shRNA targeting CDH1 or control shRNA. n = 4 organoid cultures per group; two-sided Student’s t tests. d, ChIP-PCR using antibodies against REST or isotype-control immunoglobulin in intact MSK107Li organoids transduced with lentivirus constitutively expressing shRNA targeting CDH1 or control shRNA. Fold enrichment (mean ± s.e.m.) is shown relative to the corresponding 2% input. PCR primers were selected to amplify immunoprecipitated DNA at the indicated positions relative to the L1CAM transcriptional start site. P values correspond to the comparison between shControl anti-REST and shCDH1 anti-REST; n = 3 organoid cultures per condition; two-sided Student’s t tests. e, Induction of L1CAM expression by E-cadherin knockdown can be rescued by REST but not by dominant-negative REST (dnREST). Relative mRNA levels of L1CAM, CDH1 and REST are shown in MSK107Li organoids stably expressing shRNA targeting CDH1 or control shRNA as well as cDNA expressing REST or dnREST. Gene expression was normalized to the mRNA levels of GAPDH. Data are shown as the mean ± s.e.m.; n = 4 organoid cultures per group; two-sided Student’s t tests.

Article Snippet: Solid-phase L1CAM ligand binding assays used recombinant human L1CAM (human Fc tag, R&D Systems; His tag, Thermo Fisher Scientific), UltraPure BSA (Thermo Fisher Scientific), purified mouse laminin-111 (Sigma-Aldrich), purified mouse collagen IV (Cultrex, R&D Systems), purified human collagen V (Sigma-Aldrich), recombinant human tenascin C (R&D Systems) and recombinant human laminin-411, laminin-421, laminin-511 and laminin-521 (Biolamina).

Techniques: ChIP-sequencing, Derivative Assay, Comparison, Transduction, Expressing, shRNA, Immunoprecipitation, Dominant Negative Mutation, Stable Transfection

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: Computationally designed IL-21 mimic recapitulates receptor interactions with superior stability and human-mouse cross-reactivity. (A) The optimized IL-21 mimic, 21h10, is designed by recapitulating the helical bundle structure of the native hIL-21. Unstructured regions in the hIL-21 are removed, and short helices are extended to accommodate improved intramolecular packing. Some of the interface residues are conserved from the native hIL-21 for the design of initial hits. Other residues from the de novo scaffolds are further optimized, as shown. (B) Association and dissociation of 21h10 to human and murine IL-21R and 𝛾 c show concentration-dependent binding curves of 21h10. 21h10’s K D against hIL-21R is 482 pM, hIL-21R/h𝛾 c is 86.7 nM, mIL-21R is 7.87 nM, and mIL-21R/m𝛾 c is 72.8 nM. For measuring K D against hIL-21R and mIL-21R, IL-21R was immobilized on the biolayer interferometry (BLI) tip. For measuring K D against hIL-21R/h𝛾 c and mIL-21R/m𝛾 c , 𝛾 c was immobilized on the BLI tip and IL-21R was provided in solution with 1.5-fold molar excess to 21h10. Dotted lines show curve fits of raw data using a 1:1 Langmuir binding model. (C) The optimized IL-21 mimic, 21h10, exhibits monodispersed distribution when sized through size-exclusion chromatography using Superdex 75 10/300 GL column. (D) Wavelength scan from 200 nm to 250 nm confirmed 𝛼-helical secondary structure present in 21h10. Thermal melt from 25°C to 95°C with 222 nm scan revealed outstanding thermal stability of 21h10, with a T m around 75°C. (E) Crystal structure of 21h10 in complex with hIL-21R and h𝛾 c (PDB: XXXX). (F) 21h10 conserves key molecular interactions in the site I interface (left) and site IIa interface (right) observed in the structure of the human IL-21 receptor complex. (Human IL-21 receptor complex, PDB ID: 8ENT).

Article Snippet: To measure the binding affinity to hIL-21R or mIL-21R, biotinylated hIL-21R (R&D Systems Cat# AVI9249) or biotinylated mIL-21R (Acro Biosystems Cat# ILR-M82E3), respectively, were immobilized on streptavidin-coated biosensors (SAForteBio) at 5 μg/mL in the octet binding buffer, with 2-fold serial dilutions of the ligand starting at 100 nM, with 300 seconds for association and another 300 seconds for dissociation.

Techniques: Concentration Assay, Binding Assay, Size-exclusion Chromatography

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: ( A ) Pre-activated human and murine CD8 + T cells were treated with increasing doses of native IL-21 and 21h10 for 20 minutes and stained with AF488-phospho-STAT1 and AF647-phospho-STAT3 for flow cytometry analysis. ( B ) Genes related to cellular signaling and phenotype are compared for expression levels between murine CD8 + T cells treated with PBS, mIL-21, or 21h10 at 100 pM or 1 nM. * indicates memory-related genes, and ** indicates effector-related genes. Murine CD8 + T cells were pre-activated with TCR signals and treated with the cytokines at different concentrations for 24 hours, and cells were harvested for RNA-sequencing. ( C ) The percentage of IFN-𝛾-secreting cells within isolated CD8 + T cells upon treating PBS, mIL-21, or 21h10 at 1 nM, was quantified using flow cytometry after 5 hours of stimulation with PMA/Ionomycin in the presence of a protein transport inhibitor. ( D ) LCMV was inoculated into healthy mice along with daily injections of PBS, mIL-21 (2.5 µg per mouse), or 21h10 (2.5 µg per mouse). LCMV-specific CD8 + T cells from PBMC and spleen were analyzed for granzyme B expression. ( E ) Computational model of 21AT36, which is redesigned from 21h10 using ProteinMPNN, to bind IL-21R but not bind 𝛾 c . The mutated residues are highlighted in red, which abolish interactions with 𝛾 c . The 𝛾 c is included in the figure to show where the interface is positioned, not to imply that 21AT36 binds to 𝛾 c . ( F ) Western blot for STAT and pSTAT with vehicle (PBS), mIL-21, 21h10, and 21AT36 in TRP1 high CD8 + T cells. ( G ) MC38-bearing mice were treated with PBS, 21AT36, 21h10, or anti-PD-1 for 14 days. ( H ) Mice were inoculated with MC38 and treated with PBS, mIL-21, 10-fold dose mIL-21, and 21h10. ( I ) Western blot for STAT and pSTAT in the spleens of mice treated with vehicle (PBS), mIL-21, and 21h10 ( J ) Normalized pSTAT3/STAT3 in spleens of treated mice over 24 hours from ( I ). ( K ) MC38 rechallenges with MC38-survivor mice from previous 21h10 or anti-PD-1 treatment.

Article Snippet: To measure the binding affinity to hIL-21R or mIL-21R, biotinylated hIL-21R (R&D Systems Cat# AVI9249) or biotinylated mIL-21R (Acro Biosystems Cat# ILR-M82E3), respectively, were immobilized on streptavidin-coated biosensors (SAForteBio) at 5 μg/mL in the octet binding buffer, with 2-fold serial dilutions of the ligand starting at 100 nM, with 300 seconds for association and another 300 seconds for dissociation.

Techniques: Staining, Flow Cytometry, Expressing, RNA Sequencing Assay, Isolation, Western Blot