Journal: Cancer Science

Article Title: Antitumor effects of novel mAbs against cationic amino acid transporter 1 (CAT1) on human CRC with amplified CAT1 gene

doi: 10.1111/cas.14741

Figure Lengend Snippet: Correlation between gene amplification and mRNA expression of SLC7A1/CAT1 and CAT1 small interfering RNA (siRNA)–mediated growth inhibition of colorectal cancer (CRC) cells. A, Relative mRNA expression (ratio from a mean value of normal tissues) in normal and cancer tissues. B, Correlation between gene amplification and mRNA expression of SLC7A1. C, CAT1 mRNA levels in HCT116 and LS1034 cells treated with CAT1 siRNA for 24 hours. D, CRC cell growth 48 hours after treatment with CAT1 siRNA

Article Snippet: Human CRC cell lines (Caco2, COLO 201, COLO 205, COLO 320DM, HCT116, HT29, LS1034, LS123, LS174T, LS180, RKO, RKO‐E6, SNU‐C1, SW1116, WiDr), PK‐1, PK‐59, PANC‐1, MIA PaCa‐2 pancreatic, BT20, BT474 breast, A549, NCI‐H2170 lung cancers, and P3U1 mouse myeloma cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Amplification, Expressing, Small Interfering RNA, Inhibition

Journal: Cancer Science

Article Title: Antitumor effects of novel mAbs against cationic amino acid transporter 1 (CAT1) on human CRC with amplified CAT1 gene

doi: 10.1111/cas.14741

Figure Lengend Snippet: Specificity of anti‐CAT1 monoclonal antibodies (mAbs) against human cancer cell lines. A, Representative flow cytometry (FCM) histograms of human colorectal cancer (CRC) cell lines stained with anti‐CAT1 mAbs. B and C, Different human CRC cell lines (B), and pancreatic, breast, and lung cancer cell lines (C) were reacted with CA2 or isotype‐matched control mAb, followed by incubation with phycoerythrin (PE)‐labeled anti‐rat IgG polyclonal antibody (pAb). The fluorescence intensity of individual cells was measured by FCM

Article Snippet: Human CRC cell lines (Caco2, COLO 201, COLO 205, COLO 320DM, HCT116, HT29, LS1034, LS123, LS174T, LS180, RKO, RKO‐E6, SNU‐C1, SW1116, WiDr), PK‐1, PK‐59, PANC‐1, MIA PaCa‐2 pancreatic, BT20, BT474 breast, A549, NCI‐H2170 lung cancers, and P3U1 mouse myeloma cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Bioprocessing, Flow Cytometry, Staining, Control, Incubation, Labeling, Fluorescence

Journal: American Journal of Cancer Research

Article Title: CRP and soluble CTLA4 are determinants of anti-PD1 resistance in gastrointestinal cancer

doi: 10.62347/NQBL9998

Figure Lengend Snippet: CRP injection increases sCTLA4 in the sera of mouse tumor models, leading to failure of anti-PD1 therapy. BALB/c mice were subcutaneously (s.c.) implanted with murine colorectal cancer Colon26 cells (5 × 105), and were intraperitoneally (i.p.) injected with anti-CTLA4 mAb, anti-PD1 mAb, or mouse IgG (mIgG) as a control at 10 mg/kg on days 5 and 9 after tumor implantation. CRP (200 µg) or PBS as a control was i.p. injected into the mice during the therapy (days 5, 7, and 9). Data on day 15. A. Increase of sCTLA4 in tumor-implanted mice after CRP injection. Isolated sera were tested for mouse sCTLA4 by ELISA (n = 3). Photos show CTLA4 expression in tumor tissues (scale = 50 µm). B. Anti-PD1 therapy fails in the CRP-injected mice (n = 5). C. Immune cells infiltrating in the tumor tissues (n = 5). The gating strategy for flow cytometric analysis is shown below. D. Splenic T-cell proliferation (n = 3). E. Cytotoxic activity of splenic cytotoxic T cells (n = 3). F. CRP stimulation upregulates CTLA4/sCTLA4 production in tumor cells. Tumor supernatants cultured with CRP (30 µg/ml) or PBS (24 hours) as a control were tested for sCTLA4 by ELISA (n = 3). Photos show membrane-bound CTLA4 (PE) and PDL1 (FITC) expressions on Colo201 cells (scale = 100 µm). G. Tumor-derived sCTLA4 suppresses CTL induction. Tumor supernatants or recombinant CTLA4 (1 µg/mL) were added to the CTL induction system, and the sorted CD8+ T cells were tested for the cytotoxic activity (n = 3). Graphs show means ± SDs. *P < 0.01, **P < 0.05 versus control without CRP by the Mann-Whitney test. Representative data of an experiment out of three independent experiments with consistent results.

Article Snippet: Human GC KATO III cells and colorectal cancer Colo201 cells were purchased from ATCC.

Techniques: Injection, Control, Tumor Implantation, Isolation, Enzyme-linked Immunosorbent Assay, Expressing, Activity Assay, Cell Culture, Membrane, Derivative Assay, Recombinant, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T cell activation by antibody-like immunoreceptors: increase in affinity of the single-chain fragment domain above threshold does not increase T cell activation against antigen-positive target cells but decreases selectivity.

doi: 10.4049/jimmunol.173.12.7647

Figure Lengend Snippet: FIGURE 4. ErbB2 expression of target cells. Cells of ErbB2-expressing lines (SK-OV-3, Colo201, MCF-7, Colo320, and HaCat) and primary cells (PBL and keratinocytes) for the control (each 1 106 cells) were incubated with an anti-ErbB2 mAb or an isotype-matched control mAb. Bound Abs were detected by an FITC-conjugated anti-mouse IgG Ab, and cells were analyzed by flow cytometry. The MFI of bound Abs was determined and is expressed as arbitrary units. Due to their immunoreactivity, the cells were characterized as high (MFI, 100), medium (MFI, 10), and low (MFI, 10) ErbB2-positive or ErbB2-negative cells, respectively.

Article Snippet: SK-OV-3 (ATCC HTB77; American Type Culture Collection, Manassas, VA), MCF-7 (ATCC HTB22) Colo201 (ATCC CCL 224), and Colo320 (ATCC CCL 220.1) were derived from various adenocarcinomas expressing different amounts of ErbB2.

Techniques: Expressing, Control, Incubation, Cytometry

Journal: Cancer Science

Article Title: KHDRBS3 promotes multi‐drug resistance and anchorage‐independent growth in colorectal cancer

doi: 10.1111/cas.14805

Figure Lengend Snippet: Targeted knockout of KHDRBS3 in CRC cell lines. A, Western blotting analysis confirmed the knockout of KHDRBS3 . B, Dose‐response curves of HCT‐116 and LoVo transfected with empty vector (EV) or KHDRBS3 ‐specific knockout vector (KO) that were treated with 5‐FU, oxaliplatin (L‐OHP) and trifluridine (FTD). Error bars represent SD. ** P < .01 from Student t test. NS: P > .05 from Student t test. C, mRNA expression levels of CD44v, MRP1, and CD44s on HCT‐116 and LoVo transfected with EV or KO. Error bars represent SD. ** P < .01 from Student t test. NS: P > .05 from Student t test

Article Snippet: Ten cell lines derived from human CRC (DLD‐1, Colo‐201, RKO, WiDr, LoVo, HCT‐116, HT‐29, SW‐480, SW‐48 and SW‐837) were purchased from the Japanese Collection of Research Bioresources Cell Bank.

Techniques: Knock-Out, Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: Cancer Science

Article Title: KHDRBS3 promotes multi‐drug resistance and anchorage‐independent growth in colorectal cancer

doi: 10.1111/cas.14805

Figure Lengend Snippet: Overexpression of KHDRBS3 in CRC cell lines. A, Western blotting analysis confirmed the overexpression of KHDRBS3. B, Dose‐response curves of RKO and WiDr transfected with empty vector (EV) or KHDRBS3‐specific overexpression vector (OE) that were treated with 5‐FU, oxaliplatin (L‐OHP) and trifluridine (FTD). Error bars represent SD. ** P < .01 from Student t test. NS: P > .05 from Student t test. C, mRNA expression levels of CD44v, MRP1, and CD44s on RKO and WiDr transfected with EV or OE. Error bars represent SD. ** P < .01 from Student t test. NS: P > .05 from Student t test

Article Snippet: Ten cell lines derived from human CRC (DLD‐1, Colo‐201, RKO, WiDr, LoVo, HCT‐116, HT‐29, SW‐480, SW‐48 and SW‐837) were purchased from the Japanese Collection of Research Bioresources Cell Bank.

Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: Cancer Science

Article Title: KHDRBS3 promotes multi‐drug resistance and anchorage‐independent growth in colorectal cancer

doi: 10.1111/cas.14805

Figure Lengend Snippet: Immunohistochemistry and Kaplan‐Meier curves for KHDRBS3 in human CRC tissues. A, Representative images of immunohistochemical staining of KHDRBS3 in tissue sections from human colorectal carcinoma (original magnification ×40 [left] and ×400 [right]). Red box: non‐tumorous area, blue box: tumorous area. B, Representative images of upper: H&E staining and lower: immunohistochemical staining of KHDRBS3 in well (Case 1: tub1) and moderately differentiated (Case 2: tub2) CRC (original magnification ×100). C, Survival analysis of KHDRBS3 in 148 CRC cases (Cohort 1). The P ‐values (log‐rank test) are shown for the survival analysis. P < .05 indicates statistical significance

Article Snippet: Ten cell lines derived from human CRC (DLD‐1, Colo‐201, RKO, WiDr, LoVo, HCT‐116, HT‐29, SW‐480, SW‐48 and SW‐837) were purchased from the Japanese Collection of Research Bioresources Cell Bank.

Techniques: Immunohistochemistry, Immunohistochemical staining, Staining

Journal: Nature communications

Article Title: Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen.

doi: 10.1038/ncomms4591

Figure Lengend Snippet: Figure 2 | Establishment of ExoScreen to detect the EVs. (a) Analysis of the size distribution in the serum of healthy donors (n ¼ 3) and colorectal cancer patients (n ¼ 3) by the NanoSight nanoparticle tracking system. (b) Detection of EVs or monomeric recombinant CD63 protein by ExoScreen using a CD63 antibody. EV protein concentration was measured via the Qubit system. The concentration of recombinant CD63 was adjusted with that of protein in EVs purified from HCT116 CM. Error bars are s.e.m. (n ¼ 3 for each condition). (c) Correlation between ExoScreen measurements for CD9 positive EVs, CD63 positive EVs or CD63/CD9 double-positive EVs and EV protein concentration in a dilution series. EV protein concentration was measured via the Qubit system. EVs were purified from HCT116 cell CM. The addition of biotinylated CD9 or CD63 antibodies without acceptor beads conjugated to antibodies is denoted ‘bCD9 only’ or ‘bCD63 only’, while ‘aCD9 only’ or ‘aCD63 only’ means addition of only acceptor beads conjugated to CD9 or CD63 antibodies without biotinylated antibodies. The addition of biotinylated antibodies and acceptor beads conjugated antibodies is denoted ‘bCD9/aCD9’ or ‘bCD63/aCD63’. Right panel shows the addition of biotinylated CD63 antibodies and acceptor beads conjugated CD9 antibodies. Error bars are s.e.m. (n ¼ 3 for each condition). (d) Evaluation of ExoScreen specificity against purified EVs from HCT116 cell treated with or without 0.05% and 0.5% Triton X-100. Two hundred fifty ng of EVs were detected by ExoScreen using CD9 antibodies. Error bars are s.e.m. (n ¼ 3 for each condition). (e) Evaluation of ExoScreen specificity against EVs from HCT116 cells treated with (Prok( þ )) or without (Prok( )) Proteinase K. Two hundred fifty ng of EVs were detected by ExoScreen using CD9 antibodies. Error bars are s.e.m. (n ¼ 3 for each condition). Data are representative of at least three independent experiments each.

Article Snippet: Human colorectal cancer cell lines (HCT116 cells, HCT15 cells, HT29 cells, COLO201 cells, COLO205 cells, WiDr cells and SW1116 cells) and normal colon fibroblast cell line CCD-18Co cells were purchased from American Type Culture Collection.

Techniques: Recombinant, Protein Concentration, Concentration Assay

Journal: Nature communications

Article Title: Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen.

doi: 10.1038/ncomms4591

Figure Lengend Snippet: Figure 6 | Analysis of circulating CD147 and CD9 double-positive EVs in healthy donors or colorectal cancer patient sera. (a) Serum levels of CD147/ CD9 double-positive EVs in colorectal cancer patients without any purification. The panel shows a scatter plot for healthy donors (n ¼ 191) and colorectal cancer patients (n ¼ 194). The P-value was calculated by using Wilcoxon rank-sum test. (b) Changes in serum levels of CD147/CD9 double-positive EVs in colorectal cancer patients (stage I or II: n ¼ 15) before (preoperation) and after (postoperation after 7–34 days) surgical removal of the tumor. Box lengths represent the interquartile range (first to third quartiles). The line in the center of the boxes represents the median value. Data represented by the asterisks are extreme values (greater than three times the interquartile range over the third quartile). The P-value was calculated by using Wilcoxon signed-rank test. (c) The results of ExoScreen detection of circulating EVs (left panel) and purified circulating EVs (right panel) in sera from healthy donors (n ¼ 10) and colorectal cancer patients (n ¼ 35) using CD147 and CD9 antibodies. The panels show the signal intensities from each samples measured for CD147/CD9 double-positive EVs using ExoScreen. (d) Receiver operating characteristic curves between healthy donors and colorectal cancer patients assesing by CD147/CD9 double-positive EVs (left panel), CEA and CA19-9 (right panel). left panel, CD147/CD9 double-positive EVs (healthy donors versus colorectal cancer patients; AUC: 0.820); right panel, CEA (AUC: 0.669); CA19-9 (AUC: 0.622) Data are representative of at least three independent experiments each. AUC, area under the curve.

Article Snippet: Human colorectal cancer cell lines (HCT116 cells, HCT15 cells, HT29 cells, COLO201 cells, COLO205 cells, WiDr cells and SW1116 cells) and normal colon fibroblast cell line CCD-18Co cells were purchased from American Type Culture Collection.

Techniques: