Kamkaew, Anyanee (2015-08). TrkC Targeted Probes for Cancer Diagnosis and Therapeutics. Doctoral Dissertation.
This dissertation features a small molecule, non-peptidic, ligand designed to bind a cell surface receptor called tropomyosin receptor kinase C (TrkC). TrkC is overexpressed on various types of tumors including breast cancer and melanoma. It would be advantageous in clinical applications to conjugate the novel ligand with imaging or therapeutic agents then treat TrkC-positive cancers. For proof of concept, first, we used our ligand conjugated to a commercial cytotoxic drug, 6-mercaptopurine, then tested the agent in NIH-3T3 cells stably transfected with TrkC, and compared with wild type NIH-3T3 (TrkC-). A targeting effect was observed but we could not obtain an IC50 value for the conjugate due to lack of solubility at the concentrations that would have been required. Therefore, we chose a cytotoxic rosamine dye to conjugate with the targeting ligand. Selective cytotoxicity of the conjugate was observed, but high levels of non-specific binding also occurred. Conjugation of the TrkC-targeting ligand with a fluorescent dye may be useful for optical imaging in vivo, and they are also cytotoxic then these theranostics can be used to both image and treat the tumor. We hypothesized that BODIPY dye derivatives could be suitable candidates for this approach since they have excellent fluorescent characteristics and can be modified for photodynamic therapy (PDT). Results collected from cellular assays proved the selectivity of this probe and its fluorescence led us to where the conjugate functioned inside the living cells. Some cancer cell lines that naturally overexpress TrkC include metastatic breast cancer and metastatic melanoma. Consequently, this study was expanded from stable TrkC transfectants to include metastatic-breast and -melanoma. The results show the beneficial effects of TrkC targeting in histochemistry and cellular assays, including internalization and selective cell killing. In a mouse model, collaborators discovered our PDT probe at 10 mg/kg (one injection, intravenous tail vein) caused complete ablation of a tumor after 6 days post injection with no sign of metastasis to other organs. PDT can be effective if near-IR absorbing photosensitizers (?max >700 nm) are used to excite organs located in the deeper tissue. For this reason, the cargo was modified to aza-BODIPY dyes, which can absorb light at a longer wavelength than BODIPYs. Simultaneously, the conjugate structure was changed because we hypothesized this might lead to decreased neurotoxicity. Histological studies showed the new probe selectively stained breast tissues leaving normal tissues unstained. In vivo optical imaging also proved that the new TrkC targeting probe was effective in a murine breast cancer model. Incidentally, the spinal cord of the mouse was nicely imaged by this same reagent, presumably because TrkC+ cells are highly concentrated in the peripheral nervous system. Finally, we hypothesized that the novel near-IR targeting probe could be applied to imaging and treatment of metastatic melanoma. A rare Sinclair Swine model, available at TAMU, was part of the plan for these studies. Histology results showed selective staining by the targeting probe on melanoma tissue, as compared with normal tissue obtained from the same pig. Similarly, our probe stained tissue from human patients with metastatic melanoma tissue, but it did not stain healthy skin.