A human GRPr-transfected Ace-1 canine prostate cancer model in mice

Abstract

BACKGROUND A versatile drug screening system was developed to simplify early targeted drug discovery in mice and then translate readily from mice to a dog prostate cancer model that more fully replicates the features of human prostate cancer. METHODS We stably transfected human cDNA of the GRPr bombesin (BBN) receptor subtype to canine Ace-1 prostate cancer cells (Ace-1huGRPr). Expression was examined by 125I-Tyr4-BBN competition, calcium stimulation assay, and fluorescent microscopy. A dual tumor nude mouse xenograft model was developed from Ace-1CMV (vector transfected Ace-1) and Ace-1huGRPr cells. The model was used to explore the in vivo behavior of two new IRDye800-labeled GRPr binding optical imaging agents: 800-G-Abz4-t-BBN, from a GRPr agonist peptide, and 800-G-Abz4-STAT, from a GRPr antagonist peptide, by imaging the tumor mice and dissected organs. RESULTS Both agents bound Ace-1huGRPr and PC-3, a known GRPr-expressing human prostate cancer cell line, with 4–13nM IC50 against 125I-Tyr4-BBN, but did not bind Ace-1CMV cells (vector transfected). Binding was blocked by bombesin. Ca2+ activation assays demonstrated that Ace-1huGPRr expressed biologically active GRPr. Both Ace-1 cell lines grew in the flanks of 100% of the nude mice and formed tumors of ~0.5 cm diameter in 1 week. In vivo imaging of the mice at 800nm emission showed GRPr+: GRPr− tumor signal brighter by a factor of two at 24 h post IV administration of 10 nmol of the imaging agents. Blood retention (4–8% ID at 1 h) was greater by a factor >10 and cumulative urine accumulation (28–30% at 4 h) was less by a factor 2 compared to a radioactive analog of the t-BBN containing agent, 177LuAMBA, probably due to binding to blood albumin, which we confirmed in a mouse serum assay. CONCLUSIONS The dual tumor Ace-1CMV/Ace-1huGRPr model system provides a rapid test of specific to nonspecific binding of new GRPr avid agents in a model that will extend logically to the known Ace-1 orthotopic canine prostate cancer model.

Development of an orthotopic canine prostate cancer model expressing human GRPr

“…Starting with a known isolated Ace‐1 canine prostate cancer cell line that expresses very little mRNA for GRPr and does not bind GRPr targeting peptides, our prior study demonstrated that the Ace‐1 cell line could be transfected with human GRPr. The resulting Ace‐1 huGRPr cells expressed the GRPr receptor on their surfaces and the receptor functioned biologically as a growth factor, grew reliably in nude mice, and could be optically imaged in vivo and microscopically with IR800‐G‐Abz4‐t‐BBN . The histopathology results reported herein definitively demonstrate the growth of these cells in the prostate glands and other tissues of immunosuppressed dogs, as well as the cells’ ability to seed to regional lymph nodes.…”

“…To place the catheter at the canine prostatic artery, we used X‐ray fluoroscopy guided with an iodinated X‐ray contrast agent, iodixanol (Visipaque 320, General Electric Healthcare, Princeton, NJ). To ensure that the highly concentrated (∼0.5 M) X‐ray agent did not block the binding of IR800‐G‐Abz4‐t‐BBN to GRPr, we duplicated our previously published radioligand binding assay, but in the presence of 0‐10% iodixanol . Ace‐1 huGRPr cells were seeded at 30 000 cells per well in a 96‐well plate in triplicate.…”

“…To ensure that the highly concentrated (∼0.5 M) X-ray agent did not block the binding of IR800-G-Abz4-t-BBN to GRPr, we duplicated our previously published radioligand binding assay, but in the presence of 0-10% iodixanol. 12 Ace-1 huGRPr cells were seeded at 30 000 cells per well in a 96-well plate in triplicate. Cell culture medium was replaced 24 h later with 100 μL of binding buffer (RPMI 1640, 20 mM HEPES, 0.1% BSA w/v, 0.5 mM PMSF, and 0.1 mg/mL bacitracin) at room temperature and the plate was then kept at 4°C for 30 min to allow the medium to cool slowly.…”

“…A canine model has been developed that uses cultured Ace‐1 canine prostate cancer cells implanted into prostate glands of immune‐suppressed beagles . We previously reported development of a novel Ace‐1 cell line that was transfected with the human (hu) gastrin‐releasing peptide receptor (GRPr) gene, producing a highly GRPr expressing and biologically functioning cell line, Ace‐1 huGRPr , that grows stably in culture and in nude mice . Nude mice xenografts were imaged with a 10 nmol intravenous dose of a GRPr‐targeted optical imaging agent, IR800‐G‐Abz4‐AMBA .…”

“…11 We previously reported development of a novel Ace-1 cell line that was transfected with the human (hu) gastrin-releasing peptide receptor (GRPr) gene, producing a highly GRPr expressing and biologically functioning cell line, Ace-1 huGRPr , that grows stably in culture and in nude mice. 12,13 Nude mice xenografts were imaged with a 10 nmol intravenous dose of a GRPr-targeted optical imaging agent, IR800-G-Abz4-AMBA. 14 Herein we report the successful translation of these developments to dogs.…”