Table 1.

Advantages and disadvantages of in vitro and in vivo tumor cell culture models to study interactions with immune cells.

Model Type	Advantages	Disadvantages
Conventional cultures of established tumor cell lines	Relative low cost	Limited 3D interaction
	Low care to culture	Limited cell to cell interaction
	Low expertise to culture	Limited cell to matrix interaction
	Easy genetic modification	Limited microenvironment and intercellular communication
	Fast growth	Limited or lack of cellular polarization
	Minimal culture requirement	Genotypic and phenotypic selection of clones after several splitting
	Easy drug testing	Inter and intra laboratories culture selection
	Easy/scalable experimental replicates	Needs frequent authentication
	Easy co-culture experiments with immune cells
Patient-derived tumor cell suspension	Representative of the original tumor immediately after isolation	Difficult genetic stabilization (heterogeneity)
	Ideal for TME and single cell studies	Difficult to culture
		Derived cell lines only partly representing the original tumor
		Low number of cells for functional experiments
Cell lines-derived or patient-derived spheroids	Several plasticware tools to get spheroids from single cells	Relative higher cost compared to conventional cultures
	Increment of cell to cell and cell to matrix interactions	Difficulties in getting heterotypic spheroids
	Easier growth quantification compared to organoids	Reduced architectural microenvironment
	Limited culture needs	Difficulties in getting spheroids
	Cultured in well-defined media without serum	Difficulties in setting functional assays
		Difficult experimental standardization
		Need of advanced microscopy equipment for analysis
Patient-derived organoids	Partial preservation of cellular interactions and partial polarization	Reciprocal cell interaction and gradient of factors are not always polarized as in vivo
	Genetically engineered	Medium-high care to culture
	Identification of different cell types in the same organoid	High culture cost
	Cultured in well-defined media	Reduced architectural microenvironment
	Partial maintenance of genetic features and heterogeneity	Interaction with stromal components not like in vivo and difficult to set in standard organoid medium
	Culture with self-immune cells	Difficulties in setting functional experiments
		Low-medium frequency of efficient generation from patient
		Difficulties in standardization
		Needs of advanced microscopy equipment for analysis
Animal models	Genetically determined	High cost and strong specific skill
	Patient derived xenografts improve study of drug efficacy	Not necessary mirror human cell physiology
	Humanized-mice partly resemble in vivo physiology	The stromal components derive from the animal model
		Difficulties to study immune cell interactions
Cultures in artificial scaffolds and organ on chip, associated with fluidic systems	Replaces animal models or reduces the number of animals used	High cost and specific expertise requested
	Resembles more physiological conditions	Difficult to standardize
		Needs new approaches to assess functional activity