Andrew J. Ewald

PDGFRβ + perivascular progenitor cells in tumours regulate pericyte differentiation and vascular survival

The microvasculature consists of endothelial cells and their surrounding pericytes. Few studies on the regulatory mechanisms of tumour angiogenesis have focused on pericytes. Here we report the identification of tumour-derived PDGFRβ + (platelet-derived growth factor receptor β) progenitor perivascular cells (PPCs) that have the ability to differentiate into pericytes and regulate vessel stability and vascular survival in tumours. A subset of PDGFRβ + PPCs is recruited from bone marrow to perivascular sites in tumours. Specific inhibition of PDGFRβ signalling eliminates PDGFRβ + PPCs and mature pericytes around tumour vessels, leading to vascular hyperdilation and endothelial cell apoptosis in pancreatic islet tumours of transgenic Rip1Tag2 mice.

Collective Epithelial Migration and Cell Rearrangements Drive Mammary Branching Morphogenesis

Epithelial organs are built through the movement of groups of interconnected cells. We observed cells in elongating mammary ducts reorganize into a multilayered epithelium, migrate collectively, and rearrange dynamically, all without forming leading cellular extensions. Duct initiation required proliferation, Rac, and myosin light-chain kinase, whereas repolarization to a bilayer depended on Rho kinase. We observed that branching morphogenesis results from the active motility of both luminal and myoepithelial cells. Luminal epithelial cells advanced collectively, whereas myoepithelial cells appeared to restrain elongating ducts. Significantly, we observed that normal epithelium and neoplastic hyperplasias are organized similarly, suggesting common mechanisms of epithelial growth.

GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model

How breast cancers are able to disseminate and metastasize is poorly understood. Using a hyperplasia transplant system, we show that tumor dissemination and metastasis occur in discrete steps during tumor progression. Bioinformatic analysis revealed that loss of the transcription factor GATA-3 marked progression from adenoma to early carcinoma and onset of tumor dissemination. Restoration of GATA-3 in late carcinomas induced tumor differentiation and suppressed tumor dissemination. Targeted deletion of GATA-3 in early tumors led to apoptosis of differentiated cells, indicating that its loss is not sufficient for malignant conversion. Rather, malignant progression occurred with an expanding GATA-3-negative tumor cell population. These data indicate that GATA-3 regulates tumor differentiation and suppresses tumor dissemination in breast cancer.

Three-dimensional organotypic culture: experimental models of mammalian biology and disease

Mammalian organs are challenging to study as they are fairly inaccessible to experimental manipulation and optical observation. Recent advances in three-dimensional (3D) culture techniques, coupled with the ability to independently manipulate genetic and microenvironmental factors, have enabled the real-time study of mammalian tissues. These systems have been used to visualize the cellular basis of epithelial morphogenesis, to test the roles of specific genes in regulating cell behaviours within epithelial tissues and to elucidate the contribution of microenvironmental factors to normal and disease processes. Collectively, these novel models can be used to answer fundamental biological questions and generate replacement human tissues, and they enable testing of novel therapeutic approaches, often using patient-derived cells.

Collective invasion in breast cancer requires a conserved basal epithelial program.

Carcinomas typically invade as a cohesive multicellular unit, a process termed collective invasion. It remains unclear how different subpopulations of cancer cells contribute to this process. We developed three-dimensional (3D) organoid assays to identify the most invasive cancer cells in primary breast tumors. Collective invasion was led by specialized cancer cells that were defined by their expression of basal epithelial genes, such as cytokeratin-14 (K14) and p63. Furthermore, K14+ cells led collective invasion in the major human breast cancer subtypes. Importantly, luminal cancer cells were observed to convert phenotypically to invasive leaders following induction of basal epithelial genes. Although only a minority of cells within luminal tumors expressed basal epithelial genes, knockdown of either K14 or p63 was sufficient to block collective invasion. Our data reveal that heterotypic interactions between epithelial subpopulations are critical to collective invasion. We suggest that targeting the basal invasive program could limit metastatic progression.

Morphogenesis of epithelial tubes: Insights into tube formation, elongation, and elaboration.

Epithelial tubes are a fundamental tissue across the metazoan phyla and provide an essential functional component of many of the major organs. Recent work in flies and mammals has begun to elucidate the cellular mechanisms driving the formation, elongation, and branching morphogenesis of epithelial tubes during development. Both forward and reverse genetic techniques have begun to identify critical molecular regulators for these processes and have revealed the conserved role of key pathways in regulating the growth and elaboration of tubular networks. In this review, we discuss the developmental programs driving the formation of branched epithelial networks, with specific emphasis on the trachea and salivary gland of Drosophila melanogaster and the mammalian lung, mammary gland, kidney, and salivary gland. We both highlight similarities in the development of these organs and attempt to identify tissue and organism specific strategies. Finally, we briefly consider how our understanding of the regulation of proliferation, apicobasal polarity, and epithelial motility during branching morphogenesis can be applied to understand the pathologic dysregulation of these same processes during metastatic cancer progression.

ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium

Breast cancer progression involves genetic changes and changes in the extracellular matrix (ECM). To test the importance of the ECM in tumor cell dissemination, we cultured epithelium from primary human breast carcinomas in different ECM gels. We used basement membrane gels to model the normal microenvironment and collagen I to model the stromal ECM. In basement membrane gels, malignant epithelium either was indolent or grew collectively, without protrusions. In collagen I, epithelium from the same tumor invaded with protrusions and disseminated cells. Importantly, collagen I induced a similar initial response of protrusions and dissemination in both normal and malignant mammary epithelium. However, dissemination of normal cells into collagen I was transient and ceased as laminin 111 localized to the basal surface, whereas dissemination of carcinoma cells was sustained throughout culture, and laminin 111 was not detected. Despite the large impact of ECM on migration strategy, transcriptome analysis of our 3D cultures revealed few ECM-dependent changes in RNA expression. However, we observed many differences between normal and malignant epithelium, including reduced expression of cell-adhesion genes in tumors. Therefore, we tested whether deletion of an adhesion gene could induce sustained dissemination of nontransformed cells into collagen I. We found that deletion of P-cadherin was sufficient for sustained dissemination, but exclusively into collagen I. Our data reveal that metastatic tumors preferentially disseminate in specific ECM microenvironments. Furthermore, these data suggest that breaks in the basement membrane could induce invasion and dissemination via the resulting direct contact between cancer cells and collagen I.

Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy

SUMMARY The tumor microenvironment consists of stromal cells and extracellular factors that evolve in parallel with carcinoma cells. To gain insights into the activities of stromal cell populations, we developed and applied multicolor imaging techniques to analyze the behavior of these cells within different tumor microenvironments in the same live mouse. We found that regulatory T-lymphocytes (Tregs) migrated in proximity to blood vessels. Dendritic-like cells, myeloid cells and carcinoma-associated fibroblasts all exhibited higher motility in the microenvironment at the tumor periphery than within the tumor mass. Since oxygen levels differ between tumor microenvironments, we tested if acute hypoxia could account for the differences in cell migration. Direct visualization revealed that Tregs ceased migration under acute systemic hypoxia, whereas myeloid cells continued migrating. In the same mouse and microenvironment, we experimentally subdivided the myeloid cell population and revealed that uptake of fluorescent dextran defined a low-motility subpopulation expressing markers of tumor-promoting, alternatively activated macrophages. In contrast, fluorescent anti-Gr1 antibodies marked myeloid cells patrolling inside tumor vessels and in the stroma. Our techniques allow real-time combinatorial analysis of cell populations based on spatial location, gene expression, behavior and cell surface molecules within intact tumors. The techniques are not limited to investigations in cancer, but could give new insights into cell behavior more broadly in development and disease.

Genetic mosaic analysis reveals FGF receptor 2 function in terminal end buds during mammary gland branching morphogenesis

FGF signaling is associated with breast cancer and is required for mammary placode formation in the mouse. In this study, we employed a genetic mosaic analysis based on Cre-mediated recombination to investigate FGF receptor 2 (Fgfr2) function in the postnatal mammary gland. Mosaic inactivation of Fgfr2 by the MMTV-Cre transgene enabled us to compare the behavior of Fgfr2 null and Fgfr2 heterozygous cells in the same gland. Fgfr2 null cells were at a competitive disadvantage to their Fgfr2 heterozygous neighbors in the highly proliferative terminal end buds (TEBs) at the invasion front, owing to a negative effect of loss of Fgfr2 function on cell proliferation. However, Fgfr2 null cells were tolerated in mature ducts. In these genetic mosaic mammary glands, the epithelial network is apparently built by TEBs that over time are composed of a progressively larger proportion of Fgfr2-positive cells. However, subsequently, most cells lose Fgfr2 function, presumably due to additional rounds of Cre-mediated recombination. Using an independent strategy to create mosaic mammary glands, which employed an adenovirus-Cre that acts only once, we confirmed that Fgfr2 null cells were out-competed by neighboring Fgfr2 heterozygous cells. Together, our data demonstrate that Fgfr2 functions in the proliferating and invading TEBs, but it is not required in the mature ducts of the pubertal mammary gland.

Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters

Significance Conventional models of cancer progression propose that single cells leave the primary tumor, enter the circulation, and seed clonal metastases. However, metastases can contain multiple clones, raising the question: How do polyclonal metastases form? We demonstrate that cancer cells seed distant organs as cohesive clusters, composed of two molecularly distinct subpopulations, whose proportions vary systematically during metastasis. We establish that collective dissemination is a frequent mechanism for metastasis and identify a molecular program in the most invasive, keratin 14+ (K14+) cancer cells, regulating cell–cell adhesion, cell–matrix adhesion, and immune evasion. We demonstrate that this metastatic phenotype is dependent upon K14 expression. Understanding the molecular basis of collective dissemination may therefore enable novel prognostics and therapies to improve patient outcomes. Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14+ cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14+ epithelial tumor cell clusters disseminate collectively to colonize distant organs.