Tumor Heterogeneity and Plasticity
Both genetic and phenotypic tumor heterogeneity (see Glossary) are important contributing factors to therapy resistance and tumor regrowth. During tumor progression, acquisition of mutations and the infiltration of stromal cells results in a heterogeneous population of tumor cells that harbor a variety of genetic changes and experience diverse microenvironments . Consequently, a small fraction of cells may evolve that have the intrinsic properties and a supporting environment to migrate, invade, and colonize a distant metastatic site. In patients, chemotherapy is used to reduce primary tumor burden and to target metastases that cannot be surgically removed. However, owing to the high grade of heterogeneity, and perhaps therapy-induced changes in the tumor microenvironments, single or multiple tumor cells may exist or evolve that possess qualities that give insensitivity to chemotherapy. This may later result in tumor regrowth and ultimately cause relapse.
Commonly used techniques in the clinic, such as immunohistochemistry, have provided important insights into tumor heterogeneity and metastasis in experimental mouse tumor models. Although these techniques provide spatial information, they can only provide static insights at discrete time-intervals in different mice. To reveal the dynamics of cancer heterogeneity and metastasis over time in the same mouse, intravital microscopy (IVM) techniques have been developed (Box 1). These techniques have recently been used to reveal both spatial and temporal information at the single-cell level concerning the development of tumor heterogeneity and its effect on tumor cell behavior in mice. In this review we discuss the recent insights provided by IVM mouse studies into the cause and dynamics of cancer heterogeneity, its consequences for the metastatic cascade and the development of therapy resistance.
Imaging Intrinsic Tissue Heterogeneity
All tissues, including tumors, are intrinsically heterogeneous; each comprises various cell lineages that differ in their expression profile and microenvironment, and therefore in their phenotypic behavior. Lineage-tracing experiments in healthy tissues have shown that different cell types are generated by self-renewing pools of adult stem cells. However, static pictures cannot provide information on the fate of individual stem cells [2, 3]. By imaging hair follicles and intestinal crypts in living mice, the dynamic fate of stem cells was determined. IVM showed that the location of various stem cells within the stem cell niche determines their behavior and their fate [4, 5]. For example, by combining lineage tracing and IVM in the intestine, it was shown that stem cells located at the border of the stem cell niche are likely to be repelled out of the niche. Moreover, it was demonstrated that, once stem cells are removed from their niche, they lose stemness and ultimately acquire the expression profile of one of the specialized lineages. Interestingly, when these stem cells were ablated either with a laser or genetically, the more specialized cells were able to re-enter the stem cell niche, regain stemness potential, and replenish the stem cell pool [4, 5]. Together, these IVM studies show that the expression profile of cells in tissue is heterogeneous and plastic, and that cells can switch state and fate when they experience a different niche or are located differently within the niche.
To study similar principles in cancer, tumor cells can be genetically labeled with fluorescent markers such as the multicolor brainbow [6, 7] or confetti system [2,8] (Box 2). Using these constructs it is possible to trace tumor cells and their progeny over time in their native environment. Lineage-tracing experiments in intestinal adenomas, glioblastomas, and squamous skin papillomas showed that tumors maintain a degree of tissue hierarchy in which a small pool of tumor cells with stem cell potential (referred to as cancer stem cells, CSCs) drives tumor growth [8, 9, 10]. By combining multi-day IVM through imaging windows with confetti lineage-tracing techniques, it was shown in mammary carcinoma that this small pool of CSCs is highly plastic; over time tumor cells can lose or (re)gain CSC capacity  (Figure 1 and Box 3). This IVM study, in addition to IVM studies in healthy tissue, illustrate that the intrinsic heterogeneity of tissues is dynamic, whereby states such as stemness can be gained or lost by cells as a response to a changing microenvironment or position within this environment. However, the cues that drive this plasticity are yet to be discovered………