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Published on: September 5, 2018
Relative adenosine production assay suitable for 2D and 3D tumor cell culture
Apple Hui Min Tay1, Andreas Lundqvist1
1Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden.
Abstract:
Adenosine (ADO), an anti-inflammatory and immunosuppressive metabolite, plays a crucial role in mediating purinergic signaling alongside adenosine triphosphate (ATP) and adenosine monophosphate (AMP) within the tumor microenvironment. Dysregulated ADO signaling has been implicated in tumor immune evasion and progression, highlighting the importance of measuring ADO production. This method chapter presents a protocol for assessing ADO levels in both two- and three- dimensional tumor cell culture conditions. The protocol employs a competitive AMP blockade strategy, where excessive AMP is introduced to inhibit CD73-mediated conversion of AMP to ADO, enabling the quantification of relative ADO production. Given ADO's potent immunosuppressive properties and its influence on various immune responses, accurate measurement of ADO production is crucial for understanding its role in tumor immune evasion and for guiding the development of targeted immunotherapeutic strategies.
Insights
This study introduces a new method to measure adenosine (ADO) production in tumor cells. Accurately quantifying ADO is vital for understanding its role in immune evasion and developing cancer immunotherapies.
Area of Science:
- Immunology
- Biochemistry
- Cancer Biology
Background:
- Adenosine (ADO) is an immunosuppressive metabolite crucial in purinergic signaling within the tumor microenvironment.
- Dysregulated ADO signaling contributes to tumor immune evasion and progression.
- Accurate measurement of ADO production is essential for therapeutic development.
Purpose of the Study:
- To present a novel protocol for quantifying adenosine production.
- To enable ADO level assessment in both 2D and 3D tumor cell cultures.
- To support research into ADO's role in tumor immunology and immunotherapy.
Main Methods:
- A competitive adenosine monophosphate (AMP) blockade strategy is employed.
- Excessive AMP is added to inhibit CD73-mediated AMP to ADO conversion.
- This allows for the relative quantification of ADO production.
Main Results:
- The protocol successfully quantifies relative ADO production in cell cultures.
- The method is applicable to both 2D and 3D tumor models.
- Provides a reliable means to assess ADO generation under specific conditions.
Conclusions:
- This method provides a crucial tool for researchers studying ADO in cancer.
- Accurate ADO measurement aids in understanding tumor immune evasion mechanisms.
- Facilitates the development of targeted immunotherapeutic strategies against cancer.
