Table 1:

Utility of various assays in evaluating ITH

Technique	Phenotypic	Genomic	Compositional	Spatial	Single-cell resolution?
Hematoxylin and Eosin	Yes, architecture and morphology	No	Yes, can identify broad microenvironment features (tumor, stroma)	Yes, but only if morphologically evident	Yes
Single or double IHC/IF	Yes, architecture, morphology, a small number of biomarkers.	No	Yes, can identify cell types based on broad cell-specific markers (cytokeratin, vimentin)	Yes, if morphological evident and/or biomarker-specific	Yes
Multiplex immunostaining	Yes, architecture, morphology, 7–10 biomarkers.	No	Yes, can identify some specific cell types based on a handful of biomarkers markers	Yes, if morphological evident and/or biomarker-specific	Yes
Mass-tagged imaging	Yes, architecture, morphology, up to 100 biomarkers.	No	Yes, can identify many novel cell phenotypes	Yes, if morphological evident and/or biomarker-specific	Yes
Karyotyping	No	Yes, chromosomal aberrations only (usually chromosome level)	No	No	Yes, but usually only a small number of cells
FISH	No	Yes, chromosomal aberrations, gene locus aberrations	No	Yes, if performed on embedded tissues	Yes, but usually only a small number of cells
NGS (bulk tumor)	No	No, but may infer using computational tools	No	No	No, but may infer using computational tools
NGS (multi-region bulk)	No	Yes, by region sampled	Yes, if sampling microenvironment	Yes, by region sampled	No
NGS (multi-region single cell)	No	Yes	Yes, if isolating single cells from both tumor and microenvironment	Yes, by spatial distribution of single cells sampled (if sampling intact tissue section)	Yes