Table 1.

Milestone discoveries in cancer research using 3D imaging.

Cancer discovery	Impact	Microscopy	Sample preparation	Study
Macroscale
Tumour cell dissemination via lymph node blood vessels	New active process for metastasis at distant site	Light sheet Multiphoton	Fixed; CUBIC IVM	Brown et al (2018); Pereira et al (2018)
Previously unrecognized micrometastases and degree of overlap with therapeutic antibody distribution	Metastatic burden assessment and treatment evaluation	Light sheet	Fixed, whole animal; CUBIC, DISCO	Kubota et al (2017); Pan et al (2019)
Epithelial mechanical forces that lead to aggressive forms of cancer	Biophysical prediction and potential targeting of tumour aggressiveness	Confocal	Fixed; FLASH	Messal et al (2019); Fiore et al (2020)
Archival
Heterogeneity in lymphatic microvasculature and identification of vascular versus lymphatic invasion	Tumour staging and patient stratification	Light sheet	FFPE; DIPCO	Tanaka et al (2017); Tanaka et al (2018)
Her2‐enriched membrane protrusions in breast cancer biopsies	Biomarker ultrastructure that might relate to tumour aggressiveness	Super‐resolution; STORM	FFPE	Creech et al (2017)
High dimensionality
Relation between tumour cells and fibroblasts in hypoxic tumour environment	Cancer cell functional profiling in relation to the TME	Spinning disc Confocal	Fixed; RNA multiplexing ExSeq	Alon et al (2021)
Developmental trajectory and heterogeneity mapping of Wilms tumour	Identification of new tumour cell subset	Multispectral confocal	Fixed; mLSR‐3D/FUnGI	van Ineveld et al (2021)
Time resolved
Tumour plasticity and clonal evolution restriction in breast tumours	Clonal evolution of cancer	Multispectral confocal	Fixed; LSR‐3D/FUnGI	Rios et al (2019)
Stemness represents a reversible tumour cell state that can be gained or lost over time	Cancer stem cell plasticity in tumour progression	Multiphoton	IVM; mammary imaging windows	Zomer et al (2013)
Colorectal cancer metastasis linked to mutations that allow growth independence of niche signals	Driver mechanism of late‐stage cancer progression	Multiphoton	IVM; abdominal imaging windows	Fumagalli et al (2017)
Glioma cell functional interaction endows stemness and therapy resistance. Connections with neurons potentiates invasive growth	Importance of functional interconnections between tumour cells and environmental cells in tumour progression	Multiphoton; scanning electron microscopy	IVM; cranial imaging windows; optogenetics	Osswald et al (2015); Venkataramani et al (2019); Xie et al (2021)
Interplay with extracellular matrix regulates breast tumour dissemination and cooperation with macrophages supports intravasation	Role of the TME in tumour metastasis	Multiphoton	IVM; mammary imaging windows; skin‐fold windows	Harney et al (2015); Ilina et al (2020)
In vivo functional heterogeneity of CAR T cells and action radius of anti‐PD1	Cancer immunotherapy mode of action and targets to improve efficacy	Confocal; multiphoton	IVM; skin‐fold windows	Arlauckas et al (2017); Cazaux et al (2019)
Organoids
Highly heterogeneous and multi‐phasic Wilms tumour‐derived organoid cultures	Resemblance of human cancer organoid biobank to tumour tissue	Multispectral; confocal	Fixed; mLSR‐3D/FUnGI	Calandrini et al (2020)
Widespread chromosomal instability in human cancer, yet with heterogenous outcome for cell death	Functional heterogeneity shaping human tumour evolution	Confocal; spinning disc	Live	Bolhaqueiro et al (2019)
Tumour cells actively eliminate healthy cells by JNK‐dependent growth promotion	Mechanism of tumour cell competition in human cancer	Confocal; spinning disc	Live and fixed	Krotenberg Garcia et al (2021)
Widespread behavioural heterogeneity in engineered T cells targeting human cancer organoids	Opportunities to improve treatment efficacy by skewing towards T cell functional behaviour	Multispectral; confocal	Live	preprint: Dekkers et al (2021)