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A luminescent sensor for investigating serotonin metabolism in neuroendocrine cancer
Dane H Tow1, Carlos Hf Chan2, James R Howe2
1Department of Surgery, University of Iowa Carver College of Medicine, Iowa City, IA.
Background:
Serotonin is emerging as a promising therapeutic target in tryptophan hydroxylase 1-positive tumors, but further mechanistic studies are needed to effectively target dysregulated serotonin metabolism. One challenge is a lack of methods for studying the dynamic nature of serotonin metabolism. Here, we report the development of a genetically encoded luminescent biosensor, termed iSero-Rluc, for the real-time detection of serotonin in live cells.
Methods:
The engineered serotonin binding domain (iSero) and Renilla luciferase (Rluc) reporter were cloned into yeast and mammalian expression vectors to create a fusion protein that could act as a biosensor to detect endogenous serotonin levels in live cells. The iSero-Rluc biosensor was stably expressed in the BON cell line and luciferase assays, mass spectroscopy, immunofluorescence, and Western blotting were used to study serotonin metabolism under different cell culture conditions.
Results:
The iSero-Rluc sensor detected exogenous serotonin in a yeast model. When stably expressed in the BON cell line, iSero-Rluc revealed that serotonin biosynthesis is increased in an anchorage-independent growth state and is induced upon serum starvation.
Conclusion:
The iSero-Rluc biosensor is a powerful tool in the study of tumor serotonin metabolism. It enabled real-time detection of alterations in serotonin synthesis in living cells under various growth conditions and has the potential to provide greater insight into serotonin metabolism in different stages of tumor progression and to identify therapeutic strategies to target cancer metastases and carcinoid crises.
Insights
Researchers developed a novel luminescent biosensor, iSero-Rluc, for real-time serotonin detection. This tool aids in studying serotonin metabolism in cancer, offering insights into tumor progression and therapeutic strategies.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Serotonin metabolism is a potential therapeutic target in tryptophan hydroxylase 1-positive tumors.
- Dysregulated serotonin metabolism requires further mechanistic study for effective therapeutic targeting.
- A lack of dynamic serotonin metabolism study methods hinders research.
Purpose of the Study:
- To develop a genetically encoded luminescent biosensor for real-time serotonin detection in live cells.
- To enable the study of dynamic serotonin metabolism in various cellular conditions.
- To investigate serotonin metabolism in cancer progression.
Main Methods:
- Engineered serotonin binding domain (iSero) fused with Renilla luciferase (Rluc) reporter.
- Cloning into yeast and mammalian expression vectors for biosensor creation.
- Stable expression in BON cell line, utilizing luciferase assays, mass spectroscopy, immunofluorescence, and Western blotting.
Main Results:
- The iSero-Rluc sensor successfully detected exogenous serotonin in a yeast model.
- In BON cells, iSero-Rluc revealed increased serotonin biosynthesis during anchorage-independent growth.
- Serotonin biosynthesis was observed to be induced upon serum starvation.
Conclusions:
- The iSero-Rluc biosensor is a valuable tool for studying tumor serotonin metabolism.
- It allows real-time detection of serotonin synthesis alterations in living cells.
- Potential applications include insights into tumor progression and identifying therapeutic strategies for metastasis and carcinoid crises.
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