Advantages and disadvantages of in vitro methods for preclinical drug screening for CRC

MethodsAdvantagesDisadvantagesKey typesReferences
2D cell culture systemSimple and low consumption for maintenance; rapid screening; high reproducibilityRequire considerable large panel; marked discrepancy with in vivo and clinical studies; lacks in vivo characteristicsNCI60 panel[51, 52]
Bodmer group (> 120 CRC cell lines)[53]
Co-culture of CRC cell lines with CAFs[54, 55]
Microfluidic based-cell culture systemLow reagent consumption; allow high throughput screening; stable biochemical/concentration gradient; shear stress generation; microvasculatureLack of representative functional vascular network and heterogeneous phenotype of tumor; present transient effects of cell-drug behavior; not able to withstand long-term experiment; difficult to determine individual cell effectMicrofluidic CRC culture[56–59]
Microfluidic co-culture of CRC with fibroblasts[60, 61]
3D spheroid

  • Recapitulate tumor oxygen gradients and hypoxia with multiple layers of formation

  • Retain chemoresistance behaviour with multiple layers and decreased sphericity

  • Recapitulate the multifaceted and heterogeneous TME with multiple cells culture

  • Avascular characteristic and passive chemical diffusion disrupt cells processes or functions and affect drug readout

  • Lack of size uniformity leads to data variation

  • Complex and non-standardized drug readout assays

  • Difficult to determine individual cell effect to drug

Scaffold-free spheroid[23]
Co-culture CRC spheroids with fibroblasts[60, 62–64]
MCTS[65]
3D organoids

  • Withstand long-term culture; recapitulate tumor heterogeneity with heterogeneous drug responses; reproduce molecular and cellular composition of tumor

  • Recapitulate tumor ECM stiffness; support the organoids growth and sensitivity to drug

  • Retain genomic characteristic of tumor

  • Retain inter-tumor heterogeneity; support personalized therapy

  • Specific cancer subtype modelling for precision medicine

  • Time-consuming; laborious; inefficient data analysis; intra- and inter-batch heterogeneity; low batch-to-batch reproducibility

  • Lack of cells intrinsic processes such as cell adhesion and migration can lead to inefficient drug readout

  • Limited predictive ability to certain drug response (i.e., combination based involving oxaliplatin response)

Organoids in Matrigel[66]
Organoids in chemically defined synthetic hydrogels[67]
CTCDO[68]
PDO[69–72]
Serrated CRC organoids[73–77]

NCI: National Cancer Institute; CTCDO: circulating tumor cells (CTCs)-derived organoids; MCTS: multicellular tumor spheroids; PDO: patient-derived organoid