Anne C. Rios1,2, Jacco van Rheenen2,3 and Colinda L.G.J. Scheele4,5 1Princess Máxima Center for Pediatric Oncology, 3584 CS Utrecht, The Netherlands 2Oncode Institute, 3521 AL Utrecht, The Netherlands 3Department of Molecular Pathology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands 4Laboratory for Intravital Imaging and Dynamics of Tumor Progression, VIB Center for Cancer Biology, KU Leuven, 3000 Leuven, Belgium 5Department of Oncology, KU Leuven, 3000 Leuven, Belgium Correspondence: Colinda.Scheelekuleuven.be

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.