Table 3 Main advantages and limitations of techniques used to evaluate cell signaling pathways in 2D and 3D cultures

Gene expression analysis

qRT-PCR

• Standard technique to quantify the expression of specific genes;

• High sensitivity (detection of less than five copies of a target sequence) and high specificity;

• High ability to provide real-time monitoring of the amplification process;

• By employing suitable internal standards and calculations, mean variation coefficients range from 1 to 2%, enabling consistent analysis of minor gene expression alterations.

• In 3D-models, isolating intact RNA from cell lysates can be challenging due to the presence of extracellular matrix and tight cell–cell interactions. This difficulty can result in RNA degradation and fragmentation, ultimately reducing RNA quality;

• Designing primers that are specific, efficient, and non-dimerizing can be challenging, especially for genes with complex sequences or repetitive regions;

• The conversion of RNA to cDNA in the reverse transcription reaction is inclined to variability due to multiple reverse transcriptase enzymes with diverse characteristics, and different classes of oligonucleotides.

Valasek and Repa (2005); Maitra Roy et al. (2023)

DNA microarrays

• Enables simultaneous analysis of thousands of genes, making it ideal for large-scale gene expression studies;

• Detects the expression of both coding and non-coding genes, providing a view of the transcriptional profile of the sample

• In 3D-models, cell lysate step presented same limitation as qRT-PCR;

• Requires technical expertise and knowledge in bioinformatics;

• It may not detect genes with low expression or those that are not present on the specific array used;

• Microarrays provide information about relative abundance of molecules but do not directly measure absolute concentrations. Hybridization signals exhibit non-linearity, limiting accurate quantification across a wide range of concentrations;

• Results often need to be validated by techniques such as qRT-PCR, to confirm differential expression of identified genes.

Stoughton (2005); Bumgarner (2013); Maitra Roy et al. (2023)

RNA-seq

• Provides a comprehensive view of the transcriptome, allowing detection and quantification of all mRNA transcripts present in the sample;

• Offers high resolution, allowing the identification of subtle differences in gene expression between different samples or experimental conditions.

• In 3D-models, cell lysate step presented same limitation as qRT-PCR;

• Requires technical expertise and knowledge in bioinformatics;

• Results may require validation by techniques such as qRT-PCR, to confirm differential expression of identified genes.

Stark et al. (2019); Maitra Roy et al. (2023)

Protein expression analysis

Western blot

Standard technique to quantify the expression of specific proteins;

• High sensitivity and specificity.

• In 3D models, preparing cell lysates involves protein extraction methods, often mechanical dissociation techniques, which can lead to protein degradation or structural modifications, affecting analysis;

• The structure of 3D models may also hinder complete protein extraction, resulting in underestimated protein abundance.

• Costa et al. (2016); Gandhi et al. (2022); Maitra Roy et al. (2023)

ICC/IHC

• Allows localization of proteins within their cellular context;

• Provides both qualitative information on protein distribution and localization and quantitative data on protein expression levels through image analysis techniques;

• Can be applied to a wide range of tissue types and sample preparations, including formalin-fixed paraffin-embedded tissues, frozen sections, and cell cultures.

• Non-specific binding of antibodies to other proteins or tissue components can introduce background staining, potentially obscuring the signal of interest;

• Variations in tissue fixation and processing methods can affect antibody accessibility and staining intensity.

Taylor et al. (2013); Renshaw (2017)

Mass Spectrometry

• Simultaneous analysis of thousands of proteins in a single sample, making it ideal for large-scale proteomic studies;

• High accuracy;

• Allows relative quantification of protein expression, enabling comparison between different samples or experimental conditions.

• Cell lysate step for 3D-models presented same limitation as western blot;

• Requires technical expertise and knowledge in bioinformatics;

• Results may require validation by techniques such as western blot, to confirm proteomic analysis.

Mann et al. (2001); Aebersold and Mann (2016); Costa et al. (2016); Maitra Roy et al. (2023)

Mass cytometry

• Enables simultaneous analysis of multiple surface and intracellular markers at the single-cell level;

• Uses metal-labeled antibodies instead of fluorophores, eliminating spectral overlap issues, which are common in fluorescence-based flow cytometry;

• Allows high-dimensional profiling of heterogeneous cell populations.

• Requires highly specialized instrumentation and expertise in data acquisition and computational analysis;

• Data interpretation is complex and requires advanced bioinformatics tools.

Tanner et al. (2013); Spitzer and Nolan (2016); Bouzekri et al. (2019)