Table 1.

Summary of CAR-T efficacy tests [17, 18]

Type	Method	Description
In vitro	qPCR	Quantitative PCR is used to measure mRNA expression of CAR. Pros Robust and low cost. Cons Indirect indicator of CAR expression on CAR-T cell surface. No assessment of cytotoxicity.
	ELISA	ELISA assay on protein expression of CAR is performed. Pros Robust and low cost. Direct indicator of CAR expression on CAR-T cell surface. Cons No assessment of cytotoxicity.
	Flow cytometry	Flow cytometry is performed after CAR labeling. Pros Robust and low cost. Direct indicator of CAR expression on CAR-T cell surface. Cons No assessment of cytotoxicity.
	Antibody microarrays	A panel of antibodies targeted against cytokines of anti-tumor effector (Granzyme B, IFN-γ, MIP-1α, TNF-α), stimulatory (GM-CSF, IL-2, IL-8), regulatory (IL-4, IL-13, IL-22), and inflammatory (IL-6, IL-17A) functions is used. Pros Indirect indicator of the immune response triggered by CAR-T cells. Cons Higher cost than other in vitro techniques. Reliance on microarray quality.
	51Cr release assay	51Cr labeling is used to measure cell viability of cancer cells in co-cultures with and without CAR-T cells. Pros Direct indicator of CAR-T cell cytotoxicity. Cons Usage of radioactive substance. Requirement for a large pool of CAR-T and cancer cells.
	BATDA assay	BATDA Reagent is used to label cancer cells for cell viability measurement Pros Direct indicator of CAR-T cell cytotoxicity Cons Requirement for a large pool of CAR-T and cancer cells
In vivo	Mouse xenograft model	Relevant genetically engineered mouse model(s) or implantation of relevant cancer cells/tissue into immunocompromised mice is used Pros Direct indicator of CAR-T cell cytotoxicity in vivo Cons High cost and time consuming CAR-T efficacy is affected by physiological and environmental factors that may be irrelevant to human patients
In vitro	Droplet microfluidics	CAR-T cells and cancer cells are co-cultured on a droplet microfluidic chip, followed by single cell counting to evaluate cancer cell viability Pros Direct indicator of CAR-T cytotoxicity Observation of CAR-T expansion Visualization of the interaction between CAR-T and cancer cells via phenotype analysis Microvolume requirement for both CAR-T and cancer cells Low cost Cons Limitation on data acquisition frequency due to microscope capability Requirement for sophisticated image processing techniques