Pimonidazole-alkyne conjugate for sensitive detection of hypoxia by Cu-catalyzed click reaction

Abstract

Hypoxia is involved in various diseases, such as cancers. Pimonidazole has often been used as the gold-standard marker to visualize hypoxic regions. Pimonidazole labels hypoxic regions by forming a covalent bond with a neighboring protein under hypoxic conditions in the body, which is detected by immunohistochemistry performed on tissue sections. To date, some pimonidazole-fluorophore conjugates have been reported as fluorescent probes for hypoxia imaging that do not require immunostaining. They are superior to pimonidazole because immunostaining can produce high background signals. However, large fluorophores in the conjugates may alter the original biodistribution and reactivity. Here, we report a new hypoxia marker, Pimo-yne, as a pimonidazole-alkyne conjugate. Pimo-yne has a similar hypoxia detection capability as pimonidazole because the alkyne tag is small and can be detected by Cu-catalyzed click reaction with azide-tagged fluorescent dyes. We successfully visualized hypoxic regions in tumor tissue sections using Pimo-yne with reduced background signals. The detected regions overlapped well with those detected by pimonidazole immunohistochemistry. To further reduce the background, we employed a turn-on azide-tagged fluorescent dye. Graphical abstract

Click3D: Click reaction across deep tissues for whole-organ 3D fluorescence imaging

“…Following the intraperitoneal administration of 5-EU, mouse kidneys were harvested after 5 hours. The organs were then stained with Cy5-PEG 4 -azide, a hydrophilic dye we previously developed ( 23 ), using the Click3D method and cleared using BABB for 3D imaging ( Fig. 3B ).…”

“…We used Pimo-yne, a clickable probe for detecting hypoxia that we previously developed for 2D section imaging ( Fig. 4A ) ( 23 ). Pimo-yne is an alkyne-tagged derivative of pimonidazole (Pimo), a gold standard for staining hypoxic regions ( 25 ), and exhibits similar staining properties to Pimo ( 23 ).…”

“…4A ) ( 23 ). Pimo-yne is an alkyne-tagged derivative of pimonidazole (Pimo), a gold standard for staining hypoxic regions ( 25 ), and exhibits similar staining properties to Pimo ( 23 ). Under hypoxic conditions, the nitroimidazole moiety of pimonidazole is reduced to an electrophilic species, which then covalently binds to nucleophilic residues in neighboring proteins, enabling Pimo-yne to label hypoxic regions ( 23 , 26 ).…”

“…For vascular staining, an AF647-labeled anti-CD31 antibody was administered intravenously shortly before perfusion. The tissues were then subjected to Click3D staining with Cy3-PEG 4 -azide, a hydrophilic dye ( 23 ), and cleared using an aqueous RIMS, CUBIC-R+(M) ( 31 ). The aqueous RIMS was used to observe fluorescent proteins.…”