Multidimensional Imaging of Breast Cancer

Abstract

Breast cancer is a pathological condition characterized by high morphological and molecular heterogeneity. Not only the breast cancer cells, but also their tumor micro-environment consists of a multitude of cell types and states, which continuously evolve throughout progression of the disease. To understand breast cancer evolution within this complex environment, in situ analysis of breast cancer and their co-evolving cells and structures in space and time are essential. In this review, recent technical advances in three-dimensional (3D) and intravital imaging of breast cancer are discussed. Moreover, we highlight the resulting new knowledge on breast cancer biology obtained through these innovative imaging technologies. Finally, we discuss how multidimensional imaging technologies can be integrated with molecular profiling to understand the full complexity of breast cancer and the tumor micro-environment during tumor progression and treatment response.

Figure 1.

3D imaging technologies to uncover the spatial traits of breast cancer progression. (A) 3D diagram demonstrating the features of diverse commonly used imaging modalities to study intact breast cancer samples. The horizontal axis represents the image dimensionality (number of labels typically used to visualize different structures, cells, or molecules), the vertical axis represents the imaging scale ranging from nanometers (nm) to centimeters (cm), and the size of the dots represents image resolution ranging from subcellular (small dots) to macrostructural (large dots). Note that none of the currently available imaging technologies reach a combination of large-scale high dimensionality and high resolution. (IF) Immunofluorescence. (B) An example of panoramic imaging of the full intact mouse body using whole-body clearing and light-sheet imaging, which can be used to detect small cell numbers, such as micrometastases, in large volumes, such as the entire mouse. Scale bar, 2 cm. (Photo in B is reprinted from Pan et al. 2019 with permission from Elsevier © 2019.) (C) 3D light-sheet imaging of macrostructural components in primary breast tumors highlighting high spatial heterogeneity in vascular architecture including poorly perfused regions (region i) and well-vascularized regions (region ii) within the same tumor sample. Scale bars, 250 µm (overview panel) and 100 µm (zoom regions). (Photo C is reprinted from Dobosz et al. 2014 under the terms of a Creative Commons CC-BY-NC-ND license.) (D) Whole-mount 3D confocal imaging of an Elf5-rtTA/TetO-cre/Pten^fl/fl/p53^fl/fl/R26R-Confetti mammary gland with F-actin (blue) in a precancerous stage. Mutant cells are labeled with CFP, GFP, YFP, or RFP. Scale bar, 500 µm. (Photo in D is reprinted from Rios et al. 2019 with permission from Elsevier © 2019.) (E) Representative 3D image of breast tumor tissue derived from xenotransplanted human breast tumor organoids, immunolabeled with seven different markers and imaged using multispectral confocal imaging. Scale bar, 300 µm. (Photo E is reprinted from van Ineveld et al. 2021 with permission from the authors © 2021, who hold full copyright under exclusive license to Springer Nature America.)

Figure 2.

Dynamic intravital microscopy to uncover breast cancer dynamics at a cellular level. (A) Intravital imaging of mutant mammary cells revealed the first steps of tumorigenesis in the mammary gland where β-catenin activation drives the formation of hyperplastic regions with squamous differentiation within a few days. Scale bar, 50 µm. (Photos in A are reprinted from Lloyd-Lewis et al. 2022 under the terms of a Creative Commons Attribution 4.0 International license (CC BY 4.0).) (B) In vivo imaging of mammary tumor progression revealed several modes of invasion, including collective strand invasion (panel i) and single-cell invasion (panel ii). Scale bars, 200 µm (overviews) and 25 µm (panels i and ii). (Photos in B are reprinted from Ilina et al. 2018 under the terms of a Creative Commons CC-BY license.) (C) In vivo detection of metastatic breast cancer cells in the vasculature of the lymph node elucidating a role for the lymph node as a cancer cell hub prior to further distant metastatic spreading. Scale bar, 25 µm. (Photo in C is reprinted from Pereira et al. 2018 with permission from American Association for the Advancement of Science © 2018.) (D) Time-lapse intravital imaging of the lung vasculature allows behavioral tracking of disseminated tumors cells upon arrival in the lungs, including recirculation or apoptosis (top panels) or extravasation (bottom panels). Scale bar, 15 µm. (Photos in D are reprinted from Borriello et al. 2022 under the terms of a Creative Commons Attribution 4.0 International License (CC BY 4.0).) (E) Multiday intravital microscopy of metastatic breast cancer cells in the brain revealed that the rate-limiting step for metastatic progression is to overcome the dormancy-inducing microenvironment. Scale bars, 40 µm. (Photos in E are reprinted from Dai et al. 2022 with permission from the authors who hold the exclusive copyright license for this work published by Springer Nature.)