Measurement of CD73 enzymatic activity using luminescence-based and colorimetric assays

CD73 is a membrane-anchored ectoenzyme that degrades extracellular AMP into adenosine, a potent immunosuppressive factor. In physiological conditions, induction of the CD73-adenosine pathway acts as natural feedback mechanism to prevent excessive immune reactions and subsequent tissue damage. In the past few years, the CD73-adenosine pathway has emerged as a major immunosuppressive mechanism by which multiple types of cancer evade anti-tumor immunity. Research from our group and others have established that blocking the CD73-adenosine pathway represents a promising approach to improve cancer immunotherapy. In this context, an increasing number of research laboratories are becoming interested in CD73 biology and in the development/characterization of CD73 inhibitors. Implementation of simple, rapid and HTS-compatible assays to evaluate CD73 enzymatic active is a critical step for any laboratory willing to study the CD73-adenosine pathway. Over the years, we developed, optimized or adapted various methodologies to assess CD73 enzymatic activity using in vitro assays. In this chapter, we describe two different in vitro assays adapted to the measurement of CD73 enzymatic activity. Both assays are simple, robust, HTS-compatible and can be used in a cell-based fashion.