Quantitative Evaluation of in Vivo Target Efficacy of Anti-tumor Agents via an Immunofluorescence and EdU Labeling Strategy

Abstract

Current methods used to evaluate in vivo target efficacy of selected compound include western blot to semi-quantitatively analyze protein expression. However, problems arise as it is difficult to compare in vivo target efficacy of anti-tumor agents with the same mode of action. It is therefore desirable to develop a protocol that can quantitatively display in vivo target efficacy while also providing other useful information. In this study EdU labeling was used to mark out the proliferating area. The tumor tissue was accordingly divided into proliferating and non-proliferating areas. Fifteen tumor related proteins were stained by immunofluorescence and were found to express in either the proliferating or non-proliferating areas. This allows the quantitative analysis of protein expressions within the precise area. With simple image analysis, our method gave precise percent changes of protein expression and cell proliferation between the drugs treated group and the control group. Additional information, such as, the status of protein expression can also be obtained. This method exhibits high sensitivity, and provides a quantitative approach for in vivo evaluation of target efficacy.

Salt-Inducible Kinase 1 is a potential therapeutic target in Desmoplastic Small Round Cell Tumor

“…EdU 8 h prior to sacrifice. Tumors were harvested, weighed, fixed in formalin, embedded in paraffin, sectioned (5 μm) and stained with H&E or stained for EdU using Click-iT EdU proliferation kit (Invitrogen) [ 45 ]. Double-blind evaluation was used to count Hoechst33342+ cells and EdU+ cells in the images taken at 10× magnification (Nikon Eclipse 80i microscope; NIS-Elements software, Melville, NY) from three random areas per slide.…”

Covalent labeling of nucleic acids

“…Importantly, EdU labeling indicated proliferating areas of the tumors, as validated by immunostaining. 23 An interesting application for metabolic labeling of DNA was to mark newly synthesized DNA in different viruses (adenovirus, herpes virus, vaccinia virus). During a significant part of their replication cycles, viruses do not exist in the form of morphologically distinct virus particles, but either as subviral nucleoprotein complexes, as genetic information only, or as newly-synthesized virion components.…”

“…21,22 Some studies also point out applications of nucleic acid labeling in molecular imaging of tumors and infection. 23,24 The existing and potential medical applications are in turn a driving force for biotechnology.…”

Design and Synthesis of Indoleamine 2,3-Dioxygenase 1 Inhibitors and Evaluation of Their Use as Anti-Tumor Agents

“…The mouse melanoma cells B16F10 were cultured and harvested in saline. At day 0 of the experiment, 1 × 10 6 cells were injected subcutaneously into mice, and treatment was initiated at day 1 following the mice enrolled randomly in control and experimental groups [27]. For control group, 0.5% CMCNa was orally administered every day.…”