rabbit anti human fap  (Novus Biologicals)

Journal: Molecular Cancer Therapeutics

Article Title: The Recycling Collagen Receptor uPARAP Is a Unique Mediator of Stromal Drug Delivery to Carcinoma Cells

doi: 10.1158/1535-7163.MCT-25-0051

Figure Lengend Snippet: Subcutaneous xenograft models with human lung and colon carcinoma cells recapitulate the uPARAP expression patterns found in human tumors. A and B, Human EBC-1 lung carcinoma cells and HT29 colon carcinoma cells are negative for uPARAP expression in vitro . EBC-1 and HT29 cells, as well as human SaOS-2 osteosarcoma cells (positive control), were cultured in vitro . A, (Top) Analysis of uPARAP expression by Western blotting. The uPARAP band (apparent relative molecular mass ∼180,000) is absent in the two carcinoma cell lines but present in the SaOS-2 cells. (Bottom) A Coomassie-stained SDS-PAGE gel with the same loading shows a uniform content of total protein in the three samples. B, Flow cytometry analysis of the same cell types following incubation with fluorescently labeled anti-uPARAP antibody, 2h9-AF647, or isotype control antibody, IgG-AF647. A prominent uptake of 2h9-AF647 was seen in the SaOS-2 cells, whereas no signal was observed in HT29 or EBC-1 cells. C, EBC-1 and HT29 tumors have infiltrating CAFs with strong expression of uPARAP. EBC-1 cells or HT29 cells were injected subcutaneously into CB17 SCID mice as in Supplementary Fig. S5. Tumor specimens from the mice were excised at predefined endpoints (see “Materials and Methods”) and immunostained for uPARAP. Tumor cells are uPARAP-negative, whereas infiltrating fibroblasts are uPARAP-positive (arrows in right figures at high magnification). Scale bars, 500 µm (low mag); 50 µm (high mag). D and E, CAF-directed uptake of uPARAP-directed mAb after administration in vivo . Tumor-bearing mice were injected intravenously with AF647-conjugated anti-uPARAP mAb, followed by flow cytometric analysis after 24 hours. D, Analysis of tumor material from mice with the indicated subcutaneous tumors. Tumors were excised, disaggregated into single-cell suspensions, and analyzed by flow cytometry, using markers for human cells (HLA and hCD29) and for activated fibroblasts (FAP), in addition to recording the fluorescence of the AF647-conjugated anti-uPARAP. Tumor cells are uPARAP-negative, whereas a pronounced uptake of the anti-uPARAP mAb is observed on infiltrating CAFs. EBC-1, n = 6. HT29, n = 3. See Supplementary Fig. S9 for fluorescence minus one panels. E, Uptake of uPARAP-directed mAb in normal tissues. (Left) Various organs from the EBC-1 tumor–bearing mice shown in D were excised and analyzed as in D , along with the tumor material. For the normal tissues, gating was performed on the specific cell population with the highest uPARAP signal, identified using additional markers (see Supplementary Fig. S10). For each tissue, the mean fluorescence intensity (MFI) of the cell population with the most prominent anti-uPARAP mAb uptake was compared with that of the CAFs (tumor). (Right) Fluorescence signal (MFI of AF647 fluorophore) of anti-uPARAP antibody 2h9 (2h9-AF647) or isotype control antibody (IgG-AF647) in the liver, after i.v. injection of mice in the same manner. n = 3 for all tissues except for the liver with IgG-AF647, where n = 4.

Article Snippet: The following reagents for IHC, flow cytometry, and Western blotting were purchased from the indicated commercial sources: Mayer’s HTX (cat. no. 01820, Histolab), Eosin Y 0.2% (cat. no. 01650, Histolab), Tissue-Tek Tissue Clear (cat. no. 1466, Sakura Finetek), Glas Tissue-Mount (cat. no. 1467N, Sakura Finetek), Proteinase K (CAS no. 39450-01-6, cat. no. 3115879001, Roche, Sigma-Aldrich), BOND Epitope Retrieval Solution 2 (cat. no. AR9640, Triolab), Antibody Diluent Background Reducing (cat. no. S3022, Dako/Agilent), hydrogen peroxide 30% (cat. no. 107209, Merck), BOND Dewax Solution (cat. no. AR9222, Triolab), BOND Wash Solution 10× Concentrate (cat. no. AR9590, Triolab), BOND Polymer Refine Detection Kit (cat. no. DS9800, Leica Biosystems), DAB+ 2 component kit (cat. no. C09, Origene, GBI Labs), Dako EnVision+ HRP anti-rabbit secondary antibody (RRID:AB_2630375, cat. no.K4003, Agilent), Rat IgG VisUCyte HRP polymer antibody (RRID:AB_3659616, cat. no. VC005, RnD Systems), MRC2 Mouse mAb (clone OTI9G4, cat. no. TA811858, OriGene), lamin B1 antibody human specific (RRID:AB_2737035, clone 12G6, HS-404017, Synaptic Systems GmbH), Anti-mouse CD45 Brilliant Violet 605 (RRID:AB_2562342, cat. no. 103140, BioLegend), streptavidin-APC-Cy7 (cat. no. 405208, BioLegend), PE-streptavidin (cat. no. 405203, BioLegend), APC-Cy7 anti-mouse CD326 (EpCam; RRID:AB_1501158, clone G8.8, cat. no. 118217, BioLegend), PerCP/Cy5.5 anti-mouse CD31 (RRID:AB_10612742, clone 390, cat. no. 102419, BioLegend), Anti-human HLA PE (RRID:AB_314874, cat. no. 311405, BioLegend), Anti-human CD29 PerCP-Cy5.5 (RRID:AB_2715816, cat. no. 303023, BioLegend), Red Blood Cell Lysis Buffer (cat. no. 420301, BioLegend), Zombie Violet live/dead stain (cat. no. 423114, BioLegend), Rabbit anti-human FAP mAb (F1A4G; cat. no. CST-52818T, Cell Signaling Technology), Anti-human/mouse FAP biotin (RRID:AB_2057508, cat. no. BAF3715, R&D Systems), Alexa Fluor 647 Protein Labeling Kit (cat. no. A20173, Thermo Fisher Scientific), mouse IgG affinity purified (cat. no. MS-GF, Innovative Research), IRDye 800CW goat anti-mouse secondary antibody (RRID:AB_621842, cat. no. 926-32210, LI-COR Biosciences), BD Pharmingen Purified Rat Anti-Mouse CD16/CD32 Mouse BD Fc Block (RRID:AB_394656, clone 2.4G2, cat. no. 553141, BD Biosciences), and human normal immunoglobulin (Human Fc block, Privigen).

Techniques: Expressing, In Vitro, Positive Control, Cell Culture, Western Blot, Staining, SDS Page, Flow Cytometry, Incubation, Labeling, Control, Injection, In Vivo, Fluorescence