Relative adenosine production assay suitable for 2D and 3D tumor cell culture.

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.