Table 1 –

Summary of the advantages/limitations of MDOTS/PDOTS microfluidic culture models relative to other in vivo and in vitro cancer models. Several typical references are provided for each type of culture.

Type culture	Cancer models		Characteristics/Advantages		Limitations
In vitro Ex vivo	3D microfluidic culture of: 26–32	Culture Conditions	Ideal to study immune-tumor interaction in 3D microenvironment Capable of modeling complex tumor microenvironment (TME) and extra-cellular matrix (ECM) Use of patient-derived and mouse specimens (PDOTS, MDOTS) or cell lines (cell line spheroids) Dynamic multicellular co-culture Reproduces paracrine and contact interactions Accounts for 3-dimensional cancer cell growth Mimics local in vivo organization Medium-term culture (1–2 weeks)	Culture Limitations	Inability to recapitulate biological in vivo interactions within the entire animal (except for body on a chip platforms) Variability in number of spheroids within the device Difficult to maintain long-term culture (months) Difficult to provide correct cell culture medium Risk of contamination during handling
	Cell line spheroids 18,29,30,32 MDOTS 16,17	Material & Methods	Requires low number of cells Ability to modulate cytokine/gradients Reduces reagents Possibility to include fluid flow stimuli with pumps	Technical issues	Low reproducibility and variability in data (PDOTS) Inability to reproduce same experiments (PDOTS) unless after “Bio-banking” of sample and create cell lines from patient
	PDOTS 16,17	Results & Potentiality	Microfluidic devices are scalable (size, number of cells) Reproducible experiments (cell line, MDOTS) Imaging in real-time Capable of evaluating drug toxicity and drug metabolism Live/dead assays Cytokine profiling High reproducibility with same mouse background (MDOTS) Can be applied for migration studies (immune cells) Ease of bulk protein RNA collections Low cost (cell line spheroids) Potential for personalized medicine		Low reproducibility and variability in data (PDOTS) Inability to reproduce same experiments (PDOTS) unless after “Bio-banking” of sample and create cell lines from patient Difficult to evaluate/extract results Requires cell sorting to collect protein lysate and RNA from each cell populations Requires experienced operator(s) and training Low throughput screening (Potential medium to high throughput screening) High cost (MDOTS, PDOTS)
In vitro Ex vivo	2D standard cell culture 31–35	Culture Conditions	Ideal to study single cancer cell autonomous processes Use of patient-derived and commercial cell line Simple technical culture Reproducible experiments Low-, Medium- to long-term culture	Culture Limitations	Inability to recapitulate biological in vivo interaction within entire human body Static 2-dimensional culture Lack of the TME Fails to account for 3-dimensional cancer cell growth
		Material & Methods	Require cells, cell culture medium and culture dishes		Lack ECM Lack immune cells Potential genetic changes of cancer cells over time No multicellular co-culture No possibility to include fluid flow stimuli with pumps Low-throughput screening
		Results & Potentiality	Potential change of the genetic background of original cancer cells Live/dead assay Cytokine profiling Imaging in real-time Easy methods to collect protein lysates and RNAs Easy evaluate/extract results Capable of evaluating drug toxicity and drug metabolism Low costs High-throughput screening (up to 96- or 384 well plates)	Technical issues
In vitro Ex vivo	Standard Transwell culture 36–40	Culture Conditions	Ideal to study paracrine signaling, chemotaxis (immune cells) and vascular permeability (drugs) Modulate cytokine/gradients bullet technical culture Dynamic multicellular co-culture 2D coating with ECM Medium- to long-term culture	Culture Limitations	Inability to recapitulate biological in vivo interaction Do not mimic contact interactions in the TME Low mimic of in vivo organization
		Material & Methods	Require cells, cell culture medium, Transwell insert (membrane) and culture wells Possible apply trans-endothelial flow with custom made/commercial platforms	Technical issues	Gravity force (g) may affect results Difficult imaging (depends on membrane transparency)
		Results & Potentiality	Capable of evaluating drug toxicity and drug metabolism Cytokine profiling Easy collect protein lysate and RNA from each cell population without sorting Easy/reproducible results Low costs High-throughput screening (up to 96- or 384 well plates)
In vitro In vivo Ex vivo	Circulating Tumor Cells (CTCs) 24,41–46	Culture Conditions	Not invasive methods of isolation from blood Multicellular co-culture Medium-term culture (1–2 weeks) Versatile and compatible with multiple platforms and type of culture (3D culture, Organoids, in vivo mouse models)	Culture Limitations	Difficult to provide protocols/medium for culture Lacks native immune and stromal cells Possible different biology between circulating tumor cells and tumor within native microenvironment
		Material & Methods	Use of patient-derived specimens	Technical issues	Often present only in patients with large disease burden Takes time to propagate sufficient material for drug screening/testing Difficult evaluate/extract results Medium- to high costs Low to medium throughput screening
		Results & Potentiality	Potential for personalized medicine Imaging in real-time Following propagation, CTCs can be used for anti-neoplastic drug testing
In vitro Ex vivo	Organoids 21,22,47–49	Culture Conditions	Ideal to Recapitulate the pathophysiology of the original tumor Model complex tumor microenvironment TME Single/Multicellular co-culture Account for 3-dimensional cancer cell growth Mimic in vivo organization Multiple methods of isolation from peripheral blood’ Amenable to repeat evaluation (‘living biobank”) Medium- to long-term culture (up to months)	Culture Limitations	Lacks native immune and stromal elements Takes time to propagate sufficient material for drug screening/testing Difficult to provide correct protocols/cell culture medium risk of contamination for high handling level
		Material & Methods	Use of patient-derived specimens or cell lines	Technical issues	Difficult to evaluate/extract results Require cell sorting to collect protein lysate and RNA from each cell populations (multi-cellular organoids) Low to medium throughput screening
		Results & Potentiality	Imaging in real-time Capable of evaluating drug toxicity and drug metabolism Easy - Bulk protein lysates and RNA extractions Low to medium costs Potential for personalized medicine
In vivo	Xenografts Mouse Models 23, 31, 33, 50–54	Culture Conditions	Ideal to study biological in vivo interaction within the entire animal body In vivo culture system using patient-derived specimens Account for 3-dimensional cancer cell growth Mimic in vivo organization and TME Multicellular co-culture Long-term culture (over months) Incompatible with high-throughput screening Fluid flow stimuli by in vivo circulation Require mouse and animal facility	Culture Limitations	Time and labor-intensive Challenging imaging in real-time Requires experienced operator(s) and training Genetic differences between species Complex infrastructure and specific technical skills required Lack of immune cells
		Material & Methods	Require mouse and animal facility	Technical issues	Require long culture to quantify results Challenging variability in data Collect protein lysate and RNA after sacrifice mouse High costs Low throughput screening
		Results & Potentiality	Patient derived Xenograft (PDXs) derived from mouse models can be cultured in 3D microfluidic device or grown as Organoids
In vivo	Immune-Competent Mouse Models 31, 33, 54–56	Culture Conditions	In vivo culture system using patient-derived specimens Biological in vivo interaction within the entire animal body Include immune interactions Account for 3-dimensional cancer cell growth Mimic in vivo organization and TME Long-term culture (over months)	Culture Limitations	Time and labor-intensive Challenging imaging in real-time Requires experienced operator(s) and training Complex infrastructure and specific technical skills required Limited number of potential drug combinations Drugs and therapeutic antibodies against mouse targets may differ from human targets Only mouse cells study
		Material & Methods	Require mouse and animal facility	Technical issues	Require long culture to quantify results Challenging variability in data Collect protein lysate and RNA after sacrifice mouse High costs Low throughput screening
		Results & Potentiality	Capable of modeling heterogeneity of in vivo response and resistance Capable of evaluating drug toxicity and drug metabolism Fluid flow stimuli by in vivo circulation Useful for evaluating drugs whose mechanism of action takes time (e.g. epigenetic modifying agents) MDOTS derived from immune-competent mouse models can be cultured in 3D microfluidic device or grown as Organoids