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  • br Western blotting br Tissue

    2020-08-12


    Western blotting
    Tissue lysates of patient-derived and cell lineederived xeno-graft colon tumor samples were established and samples were placed on ice. The membrane was then incubated with Claudin-1 antibody conjugated with IRDye800CW at 4 C overnight. Removal of excess primary antibody was achieved by washing the membranes in tris-buffered saline and poly-sorbate 20 three times. The membrane was scanned with the LI-COR Odyssey Infrared Imaging System (LI-COR, model 9120) and detection and quantification of band intensities was conducted using Image Studio Lite Ver 5.2 software (LI-COR). Bands were normalized to total protein by dividing the in-tensity of the band by the intensity of the total protein from the same sample on the same blot. Beta-actin was utilized as a control.
    Establishment of colon cancer cell line tumors
    The human colon cancer cell line LS174T, obtained from the American Type Culture Collection (Manassas, VA) was utilized for establishment of subcutaneous tumors. Nude male mice were anesthetized using 0.02 mL of ketamine solution. The mice were prepped with ethanol solution and 363-24-6 were injected into the bilateral flanks. The tumors were allowed to grow until they reached 50 mm3.
    Establishment of subcutaneous patient-derived xenografts with tumors from the operating room
    Human patient colon cancers were excised from patients under standard sterile conditions in the operating room at UCSD Thornton Hospital under UCSD IRB protocol 140046. After dividing the tumor in the pathology laboratory, a portion of the tumor was placed in PBS on ice and properly transferred to the laboratory. Under standard protocols with IRB approval and informed consent from the patient, these samples were used for establishment of xenograft models. Initially, tumor fragments were implanted subcutaneously within 30 min of patient surgery over the right and left upper and lower flanks in male nude mice between 4 and 6 wk of age. Subcutaneous tumors were allowed to grow for 2-4 wk until they reached at least 50 mm3.
    Colon cancer PDOX models
    Mice were anesthetized by intramuscular injection of 0.02 mL of ketamine solution. The abdomen was prepped with 70% ethanol solution. An incision was then made vertically in the midline of the abdomen through the skin and peritoneum. The cecum was carefully exposed and a 1 mm3 tumor frag-ment was surgically implanted onto the serosal layer of the cecum using 8-0 surgical sutures (Ethicon Inc, NJ). The bowel was then returned into the peritoneal cavity, and the abdominal wall and skin was closed with 6-0 nylon surgical sutures (Ethicon Inc, NJ).11
    Mouse imaging
    For noninvasive and intravital imaging, the Pearl Trilogy Small Animal Fluorescence Imaging system was used (LI-COR). The Pearl Trilogy is equipped for sensitive imaging of NIR fluorophores at 700 nm and 800 nm. Claudin-1 antibody con-jugated to LI-COR IRdye800CW (Claudin-1-IRDye800CW) was used to image subcutaneous cell line tumors and PDOX colon tumors. Images were assessed for dose response, signal to background comparison, signal duration and phototoxicity.
    A time and dose-response study was completed on sub-cutaneous cell line models, established as described above. Three groups of three mice were administered increasing doses of Claudin-1-IRDye800CW: 12.5, 25, and 50 mg recon-stituted in 100 mL PBS, via tail vein injection. In vivo imaging of the mice was performed at 24, 48, and 72 h after intravenous
     injection. Two mice were used as controls (antibody alone, and dye alone). After imaging, all mice were euthanized and laparotomy was performed to assess internal organs.
    Once the optimal dose and timing for imaging was deter-mined, orthotopic models of cell line tumors and PDOX tumor models were established using LS174T and two patient-derived tumors. 5 mice were used in each group for a total of 15 mice. After tumors grew to at least 50 mm3, the mice were administered 25 mg Claudin-1-IRDye800CW via tail vein injection. Imaging was performed 48 h after administration of Claudin-1-IRDye800CW.
    Establishment of peritoneal carcinomatosis models
    Intraperitoneal injection of one million LS174T cells recon-stituted in 100 mL of PBS was performed with three Nu/Nu male mice. After 3 wk, mice received 25 mg of Claudin-1-IRDye800CW reconstituted in 100 mg of PBS via tail vein in-jection. Images were obtained 48 h after intravenous injection on the Pearl Trilogy Small Animal Imaging System.
    Results
    Claudin-1 expression is expressed in patient-derived tumors
    Immunoblotting using nine patient-derived tumors and LS174T colon cancer cell line revealed that eight of nine patient-derived tumors expressed varying amounts of Claudin-1 and LS174T cell lysate overexpressed Claudin-1 (Fig. 1). The cell lysates with the highest signal intensity were all patient-derived colon cancer metastases, including lung, liver, and distant colon metastases (Fig. 1).