Determination of CAR T cell metabolism in an optimized protocol

Abstract

Adoptive transfer of T cells modified to express chimeric antigenic receptors (CAR) has emerged as a solution to cure refractory malignancies. However, although CAR T cell treatment of haematological cancers has now shown impressive improvement in outcome, solid tumours have been more challenging to control. The latter type is protected by a strong tumour microenvironment (TME) which might impact cellular therapeutic treatments. Indeed, the milieu around the tumour can become particularly inhibitory to T cells by directly affecting their metabolism. Consequently, the therapeutic cells become physically impeded before being able to attack the tumour. It is therefore extremely important to understand the mechanism behind this metabolic break in order to develop TME-resistant CAR T cells. Historically, the measurement of cellular metabolism has been performed at a low throughput which only permitted a limited number of measurements. However, this has been changed by the introduction of real-time technologies which have lately become more popular to study CAR T cell quality. Unfortunately, the published protocols lack uniformity and their interpretation become confusing. We herein tested the essential parameters to perform a metabolic study on CAR T cells and propose a check list of factors that should be set in order to draw sound conclusion.

Expanding CAR-T cell immunotherapy horizons through microfluidics

“…Subsequently, conventional measurements are taken to assess T cell cytotoxicity, proliferation kinetics, 176 and metabolic profile. 177 For the evaluation of CAR expression level, various techniques, such as qPCR, have been utilized to assess the mRNA expression level of CAR, ELISA to measure CAR protein expression, and flow cytometry to quantify CAR expression levels in the bulk population. Although these efficacy tests are robust, cost-effective, and easily implemented in biological laboratory settings, they depend on surrogate indicators of T cell cytotoxicity, which may not accurately reflect therapeutic outcomes.…”