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A significant proportion of colorectal cancer (CRC) patients develop peritoneal metastases (PM) in the course of their disease. PMs are associated with a poor quality of life, significant morbidity and dismal disease outcome. To improve care for this patient group, a better understanding of the molecular characteristics of CRC-PM is required. Here we present a comprehensive molecular characterization of a cohort of 52 patients. This reveals that CRC-PM represent a distinct CRC molecular subtype, CMS4, but can be further divided in three separate categories, each presenting with unique features. We uncover that the CMS4-associated structural protein Moesin plays a key role in peritoneal dissemination. Finally, we define specific evolutionary features of CRC-PM which indicate that polyclonal metastatic seeding underlies these lesions. Together our results suggest that CRC-PM should be perceived as a distinct disease entity.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies. Effective treatment options are currently lacking, and targeted agents have so far demonstrated limited efficacy. It has been speculated that a rare population of cancer stem cells (CSCs) drives growth, therapy resistance, and rapid metastatic progression in PDAC. These CSCs are characterized by expression of genes associated with immature cells, and demonstrate high clonogenicity in vitro and tumorigenic potential in vivo. However, the unique relevance of CSCs in established PDAC tissue has not been determined. Here, we used a marker-independent stochastic clonal labeling system, in combination with a quantitative model of tumor expansion, to uncover the growth dynamics of PDAC tissue. We found that in situ all PDAC cells display clonogenic potential, which is in stark contrast to a strict hierarchical CSC model. Furthermore, we uncover that the proximity to activated cancer-associated fibroblasts determines which tumor cells actively drive tumor expansion. This means that the microenvironment is a dominant actor in defining the clonogenic activity of PDAC cells. Indeed, manipulating the PDAC associated stroma by Hedgehog pathway inhibition, altered the mode of tumor growth. These results reveal how tumor-stroma crosstalk shapes the growth dynamics and clonal architecture of these malignancies, and how perturbance of the stroma impacts on important aspects of PDAC biology.

Significance Colorectal cancer (CRC) is a heterogeneous disease, with significant variation in genotype and phenotype within each individual tumor. This intratumor heterogeneity emerges during tumor development due to clonal evolution and in part can explain therapy resistance in CRC. However, a detailed understanding of the spatiotemporal development of tumors underlying cancer evolution and intratumor heterogeneity remains absent. Here, we use lineage-tracing experiments of human CRC cells transplanted into immunocompromised mice, in combination with computational modeling, to study the growth mode of CRC. We found that the clonal position is crucial for clonal outgrowth. This demonstrates that, in addition to the genetic composition, the environment and the geometry of tumor growth play a significant role in shaping tumor evolution., 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.

Survival rates of cancer patients vary widely within and between malignancies. While genetic aberrations are at the root of all cancers, individual genomic features cannot explain these distinct disease outcomes. In contrast, intra-tumour heterogeneity (ITH) has the potential to elucidate pan-cancer survival rates and the biology that drives cancer prognosis. Unfortunately, a comprehensive and effective framework to measure ITH across cancers is missing. Here, we introduce a scalable measure of chromosomal copy number heterogeneity (CNH) that predicts patient survival across cancers. We show that the level of ITH can be derived from a single-sample copy number profile. Using gene-expression data and live cell imaging we demonstrate that ongoing chromosomal instability underlies the observed heterogeneity. Analysing 11,534 primary cancer samples from 37 different malignancies, we find that copy number heterogeneity can be accurately deduced and predicts cancer survival across tissues of origin and stages of disease. Our results provide a unifying molecular explanation for the different survival rates observed between cancer types., Intratumour heterogeneity (ITH) is associated with worse prognosis in cancer, and efficient frameworks to measure it are needed. Here the authors develop a method to estimate copy number heterogeneity, and propose that it is driven by chromosomal instability and can predict pan-cancer survival.

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.

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.