Spatiotemporal regulation of clonogenicity in colorectal cancer xenografts.

Cancer evolution is predominantly studied by focusing on differences in the genetic characteristics of malignant cells within tumors. However, the spatiotemporal dynamics of clonal outgrowth that underlie evolutionary trajectories remain largely unresolved. Here, we sought to unravel the clonal dynamics of colorectal cancer (CRC) expansion in space and time by using a color-based clonal tracing method. This method involves lentiviral red-green-blue (RGB) marking of cell populations, which enabled us to track individual cells and their clonal outgrowth during tumor initiation and growth in a xenograft model. We found that clonal expansion largely depends on the location of a clone, as small clones reside in the center and large clones mostly drive tumor growth at the border. These dynamics are recapitulated in a computational model, which confirms that the clone position within a tumor rather than cell-intrinsic features, is crucial for clonal outgrowth. We also found that no significant clonal loss occurs during tumor growth and clonal dispersal is limited in most models. Our results imply that, in addition to molecular features of clones such as (epi-)genetic differences between cells, clone location and the geometry of tumor growth are crucial for clonal expansion. Our findings suggest that either microenvironmental signals on the tumor border or differences in physical properties within the tumor, are major contributors to explain heterogeneous clonal expansion. Thus, this study provides further insights into the dynamics of solid tumor growth and progression, as well as the origins of tumor cell heterogeneity in a relevant model system.

A mouse model for peritoneal metastases of colorectal origin recapitulates patient heterogeneity.

The peritoneum is a common site of dissemination in patients with colorectal cancer. In order to identify high-risk patients and improve therapeutic strategies, a better understanding of the peritoneal dissemination process and the reasons behind the high heterogeneity that is observed between patients is required. We aimed to create a murine model to further elucidate the process of peritoneal dissemination and to provide an experimental platform for further studies. We developed an in vivo model to assess patterns of peritoneal dissemination of 15 colorectal cancer cell lines. Immune deficient mice were intraperitoneally injected with 10,000 human colorectal cancer cells. Ten weeks after injection, or earlier in case of severe discomfort, the mice were sacrificed followed by dissection including assessment of the outgrowth and localization of peritoneal metastases. Furthermore, using a color-based clonal tracing method, the clonal dynamics of peritoneal nodules were observed. The different cell lines showed great variation in the extent of peritoneal outgrowth, ranging from no outgrowth to localized or widespread outgrowth of cells. An association between KRAS pathway activation and the formation of peritoneal metastases was identified. Also, cell line specific tumor location preferences were observed, with similar patterns of outgrowth in anatomically related areas. Furthermore, different patterns regarding clonal dynamics were found, varying from monoclonal or polyclonal outgrowth to extensively dispersed polyclonal lesions. The established murine model recapitulates heterogeneity as observed in human peritoneal metastases, which makes it a suitable platform for future (intervention) studies.

Stem cells in homeostasis and cancer of the gut.

The intestinal epithelial lining is one of the most rapidly renewing cell populations in the body. As a result, the gut has been an attractive model to resolve key mechanisms in epithelial homeostasis. In particular the role of intestinal stem cells (ISCs) in the renewal process has been intensely studied. Interestingly, as opposed to the traditional stem cell theory, the ISC is not a static population but displays significant plasticity and in situations of tissue regeneration more differentiated cells can revert back to a stem cell state upon exposure to extracellular signals. Importantly, normal intestinal homeostasis provides important insight into mechanisms that drive colorectal cancer (CRC) development and growth. Specifically, the dynamics of cancer stem cells bear important resemblance to ISC functionality. In this review we present an overview of the current knowledge on ISCs in homeostasis and their role in malignant transformation. Also, we discuss the existence of stem cells in intestinal adenomas and CRC and how these cells contribute to (pre-)malignant growth. Furthermore, we will focus on new paradigms in the field of dynamical cellular hierarchies in CRC and the intimate relationship between tumor cells and their niche.

Inhibition of notch signaling in glioblastoma targets cancer stem cells via an endothelial cell intermediate.

Glioblastoma multiforme (GBM) is a highly heterogeneous malignant tumor. Recent data suggests the presence of a hierarchical organization within the GBM cell population that involves cancer cells with stem-like behavior, capable of repopulating the tumor and contributing to its resistance to therapy. Tumor stem cells are thought to reside within a vascular niche that provides structural and functional support. However, most GBM studies involve isolated tumor cells grown under various culture conditions. Here, we use a novel three-dimensional organotypic "explant" system of surgical GBM specimens that preserves cytoarchitecture and tumor stroma along with tumor cells. Notch inhibition in explants results in decreased proliferation and self-renewal of tumor cells but is also associated with a decrease in endothelial cells. When endothelial cells are selectively eliminated from the explants via a toxin conjugate, we also observed a decrease in self-renewal of tumor stem cells. These findings support a critical role for tumor endothelial cells in GBM stem cell maintenance, mediated at least in part by Notch signaling. The explant system further highlighted differences in the response to radiation between explants and isolated tumor neurospheres. Combination treatment with Notch blockade and radiation resulted in a substantial decrease in proliferation and in self-renewal in tumor explants while radiation alone was less effective. This data suggests that the Notch pathway plays a critical role in linking angiogenesis and cancer stem cell self-renewal and is thus a potential therapeutic target. Three-dimensional explant systems provide a novel approach for the study of tumor and microenvironment interactions.

Stem cell functionality is microenvironmentally defined during tumour expansion and therapy response in colon cancer.

Solid malignancies have been speculated to depend on cancer stem cells (CSCs) for expansion and relapse after therapy. Here we report on quantitative analyses of lineage tracing data from primary colon cancer xenograft tissue to assess CSC functionality in a human solid malignancy. The temporally obtained clone size distribution data support a model in which stem cell function in established cancers is not intrinsically, but is entirely spatiotemporally orchestrated. Functional stem cells that drive tumour expansion predominantly reside at the tumour edge, close to cancer-associated fibroblasts. Hence, stem cell properties change in time depending on the cell location. Furthermore, although chemotherapy enriches for cells with a CSC phenotype, in this context functional stem cell properties are also fully defined by the microenvironment. To conclude, we identified osteopontin as a key cancer-associated fibroblast-produced factor that drives in situ clonogenicity in colon cancer.

Stem Cells: All that Is Solid Melts into Air.

The intestinal epithelium displays great resilience, as several cell populations can replenish the stem cell pool upon damage. Two studies in Cell Stem Cell extend this capacity to enteroendocrine cells, addressing the molecular basis underlying cellular plasticity observed in the intestine and the identities of putative reserve stem cells.

Bcl-2 is a critical mediator of intestinal transformation.

Intestinal tumour formation is generally thought to occur following mutational events in the stem cell pool. However, active NF-κB signalling additionally facilitates malignant transformation of differentiated cells. We hypothesized that genes shared between NF-κB and intestinal stem cell (ISCs) signatures might identify common pathways that are required for malignant growth. Here, we find that the NF-κB target Bcl-2, an anti-apoptotic gene, is specifically expressed in ISCs in both mice and humans. Bcl-2 is dispensable in homeostasis and, although involved in protecting ISCs from radiation-induced damage, it is non-essential in tissue regeneration. Bcl-2 is upregulated in adenomas, and its loss or inhibition impairs outgrowth of oncogenic clones, because Bcl-2 alleviates apoptotic priming in epithelial cells following Apc loss. Furthermore, Bcl-2 expression in differentiated epithelial cells renders these cells amenable to clonogenic outgrowth. Collectively, our results indicate that Bcl-2 is required for efficient intestinal transformation following Apc-loss and constitutes a potential chemoprevention target.

Continuous clonal labeling reveals small numbers of functional stem cells in intestinal crypts and adenomas.

Lineage-tracing approaches, widely used to characterize stem cell populations, rely on the specificity and stability of individual markers for accurate results. We present a method in which genetic labeling in the intestinal epithelium is acquired as a mutation-induced clonal mark during DNA replication. By determining the rate of mutation in vivo and combining this data with the known neutral-drift dynamics that describe intestinal stem cell replacement, we quantify the number of functional stem cells in crypts and adenomas. Contrary to previous reports, we find that significantly lower numbers of "working" stem cells are present in the intestinal epithelium (five to seven per crypt) and in adenomas (nine per gland), and that those stem cells are also replaced at a significantly lower rate. These findings suggest that the bulk of tumor stem cell divisions serve only to replace stem cell loss, with rare clonal victors driving gland repopulation and tumor growth.

Defining stem cell dynamics in models of intestinal tumor initiation.

Cancer is a disease in which cells accumulate genetic aberrations that are believed to confer a clonal advantage over cells in the surrounding tissue. However, the quantitative benefit of frequently occurring mutations during tumor development remains unknown. We quantified the competitive advantage of Apc loss, Kras activation, and P53 mutations in the mouse intestine. Our findings indicate that the fate conferred by these mutations is not deterministic, and many mutated stem cells are replaced by wild-type stem cells after biased, but still stochastic events. Furthermore, P53 mutations display a condition-dependent advantage, and especially in colitis-affected intestines, clones harboring mutations in this gene are favored. Our work confirms the previously theoretical notion that the tissue architecture of the intestine suppresses the accumulation of mutated lineages.

Poor-prognosis colon cancer is defined by a molecularly distinct subtype and develops from serrated precursor lesions.

Colon cancer is a clinically diverse disease. This heterogeneity makes it difficult to determine which patients will benefit most from adjuvant therapy and impedes the development of new targeted agents. More insight into the biological diversity of colon cancers, especially in relation to clinical features, is therefore needed. We demonstrate, using an unsupervised classification strategy involving over 1,100 individuals with colon cancer, that three main molecularly distinct subtypes can be recognized. Two subtypes have been previously identified and are well characterized (chromosomal-instable and microsatellite-instable cancers). The third subtype is largely microsatellite stable and contains relatively more CpG island methylator phenotype-positive carcinomas but cannot be identified on the basis of characteristic mutations. We provide evidence that this subtype relates to sessile-serrated adenomas, which show highly similar gene expression profiles, including upregulation of genes involved in matrix remodeling and epithelial-mesenchymal transition. The identification of this subtype is crucial, as it has a very unfavorable prognosis and, moreover, is refractory to epidermal growth factor receptor-targeted therapy.

Dishevelled 2 signaling promotes self-renewal and tumorigenicity in human gliomas.

Glioblastoma multiforme is the most common glioma variant in adults and is highly malignant. Tumors are thought to harbor a subpopulation of stem-like cancer cells, with the bulk resembling neural progenitor-like cells that are unable to fully differentiate. Although multiple pathways are known to be involved in glioma tumorigenesis, the role of Wnt signaling has been poorly described. Here, we show that Dishevelled 2 (Dvl2), a key component of the Wnt signaling pathway, is overexpressed in human gliomas. RNA interference-mediated depletion of Dvl2 blocked proliferation and promoted the differentiation of cultured human glioma cell lines and primary, patient-derived glioma cells. In addition, Dvl2 depletion inhibited tumor formation after intracranial injection of glioblastoma cells in immunodeficient mice. Inhibition of canonical Wnt/ß-catenin signaling also blocked proliferation, but unlike Dvl2 depletion, did not induce differentiation. Finally, Wnt5a, a noncanonical Wnt ligand, was also required for glioma cell proliferation. The data therefore suggest that both canonical and noncanonical Wnt signaling pathways downstream of Dvl2 cooperate to maintain the proliferative capacity of human glioblastomas.

Wnt activity defines colon cancer stem cells and is regulated by the microenvironment.

Despite the presence of mutations in APC or beta-catenin, which are believed to activate the Wnt signalling cascade constitutively, most colorectal cancers show cellular heterogeneity when beta-catenin localization is analysed, indicating a more complex regulation of Wnt signalling. We explored this heterogeneity with a Wnt reporter construct and observed that high Wnt activity functionally designates the colon cancer stem cell (CSC) population. In adenocarcinomas, high activity of the Wnt pathway is observed preferentially in tumour cells located close to stromal myofibroblasts, indicating that Wnt activity and cancer stemness may be regulated by extrinsic cues. In agreement with this notion, myofibroblast-secreted factors, specifically hepatocyte growth factor, activate beta-catenin-dependent transcription and subsequently CSC clonogenicity. More significantly, myofibroblast-secreted factors also restore the CSC phenotype in more differentiated tumour cells both in vitro and in vivo. We therefore propose that stemness of colon cancer cells is in part orchestrated by the microenvironment and is a much more dynamic quality than previously expected that can be defined by high Wnt activity.