Further information and requests for resources and reagents should also be directed to the corresponding authors
Further information and requests for resources and reagents should also be directed to the corresponding authors. Ethics approval and consent to participate Mice were maintained, and experiments were performed under protocols (IACUC00001239-RN00 and IACUC00000731-RN01) approved by the Institutional Animal Care and Use Committee (IACUC). and that the JM1-24-3 mAb may serve as the basis for a potential therapeutic agent. Keywords: MUC18, CD146, Metastatic melanoma, Therapeutic antibody, Targeted therapy Background Melanoma is the most lethal form of common skin cancers. While targeted and immune-based therapies are increasingly promising, not all patients benefit, thus establishing the need to identify novel targets that can contribute to improved therapeutic strategies. Using live melanoma cell immunization and high-throughput screening (HTS) [1], we generated a novel neutralizing monoclonal antibody (mAb) directed against a melanoma cell-surface antigen, which we subsequently identified as the functional, glycosylated protein MUC18. MUC18, also known as MCAM (melanoma cell adhesion molecule), CD146 (cluster of differentiation 146), or METCAM/MelCAM (metastatic melanoma CAM), is expressed on the surface of metastatic melanoma and other cancer cells [2]. Expression of MUC18 has been demonstrated to promote tumorigenesis and tumor progression, and therapeutic targeting of AZD9496 maleate MUC18 can reduce bone metastasis in a prostate cancer model [3]. Investigations outlined here demonstrated that the mAb developed was capable of specifically interacting with MUC18 on melanoma cells, initiating downstream signaling events that were associated with inhibition of melanoma cell proliferation, migration, and invasion in vitroand reduction in tumor growth and metastasis in AZD9496 maleate vivo. Furthermore, we found that these signaling events depended on binding of the mAb to a conformational epitope on the extracellular domain of MUC18. Materials and methods Animal study A/J mice (6C8?weeks old, male) (Harlan Sprague Dawley, Inc., Indianapolis, IN) and nu/nu mice (4C8?weeks old, male) (JAX, Pub Harbor, Maine) were used AZD9496 maleate for this study. Mice were managed and experiments were performed under protocols (IACUC00001239-RN00 and IACUC00000731-RN01) authorized by the Institutional Animal Care and Use Committee (IACUC). Cell lines & cells & other resources A375 cells (main cell collection with low metastatic capacity) [4]; and A2058 and WM266-4 cells (highly metastatic melanoma cells), SP2/0 (mouse myeloma cells), EC-RF24 (immortalized human being endothelial cells) were purchased from American Type Tradition Collection (ATCC, Manassas, VA). Human being peripheral blood mononuclear cells (PBMCs) were separated by using Ficoll-Plaque Plus (Ficoll, GE Healthcare Biosciences) from healthy donor blood (Gulf Coast Regional Blood Center). Additional cell lines were gifts from collaborators. Cell lines were cultivated in serum free medium MD6 derived from Rabbit Polyclonal to DRD4 Dulbeccos altered Eagles medium (DMEM, Gibco) supplemented with 5% FBS and 1% penicillin-streptomycin. Formalin-fixed paraffin inlayed (FFPE) cells slides and cells microarrays (TMAs) were prepared from a variety of tumors and normal cells from our institutional cells standard bank under institutional review table protocols (LAB09C0197 and PA11C0957). Tunicamycin (Cat. #T7765, Sigma-Aldrich, MO) and Dynabeads conjugated with Protein A (Cat. #14311D, Invitrogen, Inc., Carlsbad, CA) were also purchased. Antigen & Antibodies Human being recombinant protein CD146 (MCAM) was purchased from OriGene Systems (Cat. #TP308937, Rockville, MD). Commercial antibodies used in this study include anti-human MUC18 (mouse mAb, Cat. #MAB932; goat polyclonal Ab, Cat. #AF932, R&D Systems, Inc., Minneapolis, MN; mouse mAb, Cat # ab233923; AbCam, Cambridge, MA) and an irrelevant mAb (served as isotype control antibody) and goat serum (IgG). FITC, HRP and Alexa fluor 647 conjugated goat anti-mouse IgG (Cat. #115C095-071, 115C035-071, 115C606-062, Jackson ImmunoResearch Lab, Western Grove, PA) served as secondary antibodies. Live-cell immunization Five million each of A375, A2058 and WM266-4 metastatic melanoma cells were injected subcutaneously (s.c.) into three A/J mice every 2 weeks ?3, followed by an intraperitoneal (i.p.) boost. Three days after boost, spleen cells from your.