Disseminating cells in human oral tumours possess an EMT cancer stem cell marker profile that is predictive of metastasis in image-based machine learning.

Cancer stem cells (CSCs) undergo epithelial-mesenchymal transition (EMT) to drive metastatic dissemination in experimental cancer models. However, tumour cells undergoing EMT have not been observed disseminating into the tissue surrounding human tumour specimens, leaving the relevance to human cancer uncertain. We have previously identified both EpCAM and CD24 as CSC markers that, alongside the mesenchymal marker Vimentin, identify EMT CSCs in human oral cancer cell lines. This afforded the opportunity to investigate whether the combination of these three markers can identify disseminating EMT CSCs in actual human tumours. Examining disseminating tumour cells in over 12,000 imaging fields from 74 human oral tumours, we see a significant enrichment of EpCAM, CD24 and Vimentin co-stained cells disseminating beyond the tumour body in metastatic specimens. Through training an artificial neural network, these predict metastasis with high accuracy (cross-validated accuracy of 87-89%). In this study, we have observed single disseminating EMT CSCs in human oral cancer specimens, and these are highly predictive of metastatic disease.

When oral cancers metastasise ­ that is, when tumour cells invade other parts of the body ­ they typically do so by first colonizing the lymph nodes present in the neck. As this event significantly reduces chances of survival, oral cancer patients often have their neck lymph nodes removed to prevent the spread of the disease. However, this surgery carries risks and leads to longer hospital stays, stressing the need for better ways to predict which oral tumours will metastasise. Evidence from lab-grown cells and mice studies suggest that, in oral cancer, metastasis occurs when some cells in the original tumour go through a process called the epithelial-mesenchymal transition (EMT for short). This transformation allows the cells to detach from the tumour and become invasive. However, it has so far been difficult to observe this process in actual human tumours; this is partly because cells undergoing EMT stop producing the proteins that scientists rely on to distinguish cancer and healthy cells. To address this knowledge gap, Youssef et al. focused on three proteins: two tumour markers, EpCAM and CD24; and Vimentin, which is produced in greater quantities in the invasive mesenchymal state. Previous work had shown that a specific population of oral tumour cells can continue to express all three proteins even when adopting a mesenchymal identity through EMT. Based on this knowledge, Youssef et al. hypothesised that tracking Vimentin, EpCAM and CD24 using fluorescence microscopy would allow them to identify metastasising cells in human samples. An analysis of over 12,000 images from 74 tumours obtained from surgeries revealed that, in the metastatic samples, the cells detaching from primary tumours were more likely to express these three proteins. Finally, Youssef et al. used these images to train a machine learning algorithm. When applied to data from new oral cancer patients, the programme was able to predict whether their tumours were likely to spread with 89% accuracy. If confirmed by further work, and in particular on larger samples, these findings could in the future help clinicians decide which patients with oral cancer would benefit the most from surgery to remove neck lymph nodes.

CD73 controls Myosin II-driven invasion, metastasis, and immunosuppression in amoeboid pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis because of its high propensity to metastasize and its immunosuppressive microenvironment. Using a panel of pancreatic cancer cell lines, three-dimensional (3D) invasion systems, microarray gene signatures, microfluidic devices, mouse models, and intravital imaging, we demonstrate that ROCK-Myosin II activity in PDAC cells supports a transcriptional program conferring amoeboid invasive and immunosuppressive traits and in vivo metastatic abilities. Moreover, we find that immune checkpoint CD73 is highly expressed in amoeboid PDAC cells and drives their invasive, metastatic, and immunomodulatory traits. Mechanistically, CD73 activates RhoA-ROCK-Myosin II downstream of PI3K. Tissue microarrays of human PDAC biopsies combined with bioinformatic analysis reveal that rounded-amoeboid invasive cells with high CD73-ROCK-Myosin II activity and their immunosuppressive microenvironment confer poor prognosis to patients. We propose targeting amoeboid PDAC cells as a therapeutic strategy.

In vitro cancer models as an approach to identify targetable developmental phenotypes in cancer stem cells.

Cancer therapeutics are often highly toxic to the patient, and they often elicit rapid resistance in the tumour. Recent advances have suggested a potential new way in which we may improve on this, through two important concepts: (1) that multitudinous pathway alterations converge on a limited number of cancer cellular phenotypes, and (2) that these cancer cellular phenotypes depend on reactivation of developmental processes that are only minimally active in adult tissues. This provides a rationale for pursuing an approach of 'drugging the phenotype' focussed on targeting reactivated cellular processes from embryonic development. In this concepts paper, we cover these recent developments and their implications for the development of new cancer therapeutics that can avoid patient toxicity and acquired resistance. We then propose that in vitro tumour and developmental models can provide an experimental approach to identify and target the specific developmental processes at play, with a focus on the reactivation of developmental processes in the cancer stem cells that drive tumour progression and spread. Ultimately, the aim is to identify cellular processes that are specific to developmental phenotypes, are reactivated in cancer stem cells, and are essential to tumour progression. Therapeutically targeting these cellular processes could represent a new approach of 'drugging the phenotype' that treats the tumour whilst avoiding patient toxicity or the acquisition of therapeutic resistance.

Development of an in vitro microfluidic model to study the role of microenvironmental cells in oral cancer metastasis.

Metastasis occurs when cancer cells leave the primary tumour and travel to a secondary site to form a new lesion. The tumour microenvironment (TME) is recognised to greatly influence this process, with for instance the vascular system enabling the dissemination of the cells into other tissues. However, understanding the exact role of these microenvironmental cells during metastasis has proven challenging. Indeed, in vitro models often appear too simplistic, and the study of the interactions between different cell types in a 3D space is limited. On the other hand, even though in vivo models incorporate the TME, observing cells in real-time to understand their exact role is difficult. Horizontal compartmentalised microfluidic models are a promising new platform for metastasis studies. These devices, composed of adjacent microchannels, can incorporate multiple cell types within a 3D space. Furthermore, the transparency and thickness of these models also enables high quality real-time imaging to be performed. This paper demonstrates how these devices can be successfully used for oral squamous cell carcinoma (OSCC) metastasis studies, focusing on the role of the vascular system in this process. Conditions for co-culture of OSCC cells and endothelial cells have been determined and staining protocols optimised. Furthermore, several imaging analysis techniques for these models are described, enabling precise segmentation of the different cell types on the images as well as accurate assessment of their phenotype. These methods can be applied to any study aiming to understand the role of microenvironmental cell types in cancer metastatic dissemination, and overcome several challenges encountered with current in vitro and in vivo models. Hence, this new in vitro model capable of recapitulating important aspects of the cellular complexity of human metastatic dissemination can ultimately contribute to replacing animal studies in this field.

Cancer stem cell plasticity and its implications in the development of new clinical approaches for oral squamous cell carcinoma.

Oral squamous cell carcinoma (SCC) represents a major worldwide disease burden, with high rates of recurrence and metastatic spread following existing treatment methods. Populations of treatment resistant cancer stem cells (CSCs) are well characterised in oral SCC. These populations of CSCs engage the cellular programme known as epithelial mesenchymal transition (EMT) to enhance metastatic spread and therapeutic resistance. EMT is characterised by specific morphological changes and the expression of certain cell surface markers that represent a transition from an epithelial phenotype to a mesenchymal phenotype. This process is regulated by several cellular pathways that interact both horizontally and hierarchically. The cellular changes in EMT occur along a spectrum, with sub-populations of cells displaying both epithelial and mesenchymal features. The unique features of these CSCs in terms of their EMT state, cell surface markers and metabolism may offer new druggable targets. In addition, these features could be used to identify more aggressive disease states and the opportunity to personalise therapy depending on the presence of certain CSC sub-populations.

Interconnected high-dimensional landscapes of epithelial-mesenchymal plasticity and stemness in cancer.

Establishing macrometastases at distant organs is a highly challenging process for cancer cells, with extremely high attrition rates. A very small percentage of disseminated cells have the ability to dynamically adapt to their changing micro-environments through reversibly switching to another phenotype, aiding metastasis. Such plasticity can be exhibited along one or more axes-epithelial-mesenchymal plasticity (EMP) and cancer stem cells (CSCs) being the two most studied, and often tacitly assumed to be synonymous. Here, we review the emerging concepts related to EMP and CSCs across multiple cancers. Both processes are multi-dimensional in nature; for instance, EMP can be defined on morphological, molecular and functional changes, which may or may not be synchronized. Similarly, self-renewal, multi-lineage potential, and resistance to anoikis and/or therapy may not all occur simultaneously in CSCs. Thus, understanding the complexity in defining EMP and CSCs is essential if we are to understand their contribution to cancer metastasis. This will require a more comprehensive understanding of the non-linearity of these processes. These processes are dynamic, reversible, and semi-independent in nature; cells traverse the inter-connected high-dimensional EMP and CSC landscapes in diverse paths, each of which may exhibit a distinct EMP-CSC coupling. Our proposed model offers a potential unifying framework for elucidating the coupled decision-making along these dimensions and highlights a key set of open questions to be answered.

Portrait of a CAF: The story of cancer-associated fibroblasts in head and neck cancer.

Complex interactions take place during cancer formation and progression. In this regard, there has been increasing focus on the non-malignant cells that make up the tumour microenvironment (TME), and how they interact with malignant tumour cells. TME is highly heterogeneous and has a major influence on tumour behaviour and therapy response. Cancer-associated fibroblasts (CAFs), one of the main components of the TME, establish dangerous liaisons with cancer cells and other components of the TME to shape a tumour-supportive environment in many types of cancer. Head and neck squamous cell carcinoma (HNSCC) encompass the malignant neoplasms arising from the mucosal lining of the oral cavity, pharynx and larynx. The TME of HNSCC contributes to tumour progression and this stromal compartment may be an interesting target for treatment. There is an emerging picture of the behaviour of CAFs in HNSCC; how they affect and are affected by the TME. We aim to summarise and discuss the current understanding of CAFs in head and neck cancer, exploring CAF activation and heterogeneity, and interaction with cancer cells and other cells within the TME.

Hybrid epithelial/mesenchymal phenotypes promote metastasis and therapy resistance across carcinomas.

Cancer metastasis and therapy resistance are the major unsolved clinical challenges, and account for nearly all cancer-related deaths. Both metastasis and therapy resistance are fueled by epithelial plasticity, the reversible phenotypic transitions between epithelial and mesenchymal phenotypes, including epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). EMT and MET have been largely considered as binary processes, where cells detach from the primary tumor as individual units with many, if not all, traits of a mesenchymal cell (EMT) and then convert back to being epithelial (MET). However, recent studies have demonstrated that cells can metastasize in ways alternative to traditional EMT paradigm; for example, they can detach as clusters, and/or occupy one or more stable hybrid epithelial/mesenchymal (E/M) phenotypes that can be the end point of a transition. Such hybrid E/M cells can integrate various epithelial and mesenchymal traits and markers, facilitating collective cell migration. Furthermore, these hybrid E/M cells may possess higher tumor-initiation and metastatic potential as compared to cells on either end of the EMT spectrum. Here, we review in silico, in vitro, in vivo and clinical evidence for the existence of one or more hybrid E/M phenotype(s) in multiple carcinomas, and discuss their implications in tumor-initiation, tumor relapse, therapy resistance, and metastasis. Together, these studies drive the emerging notion that cells in a hybrid E/M phenotype may occupy 'metastatic sweet spot' in multiple subtypes of carcinomas, and pathways linked to this (these) hybrid E/M state(s) may be relevant as prognostic biomarkers as well as a promising therapeutic targets.

Effects of Cetuximab and Erlotinib on the behaviour of cancer stem cells in head and neck squamous cell carcinoma.

The therapeutic responses of many solid tumours to chemo- and radio-therapies are far from fully effective but therapies targeting malignancy-related cellular changes show promise for further control. In head and neck squamous cell carcinoma, the epidermal growth factor receptor (EGFR) is commonly overexpressed and investigation of agents that block this receptor indicate a limited response when used alone but an ability to enhance the actions of other drugs. The hierarchical stem cell patterns present in tumours generate cellular heterogeneity and this is further complicated by cancer stem cells (CSC) shifting between epithelial (Epi-CSC) and mesenchymal (EMT-CSC) states. To clarify how such heterogeneity influences responses to EGFR blocking, we examined the effects of Cetuximab and Erlotinib on the cell sub-populations in HNSCC cell lines. These agents reduced cell proliferation for all subpopulations but induced little cell death. They did however induce large shifts of cells between the EMT-CSC, Epi-CSC and differentiating cell compartments. Loss of EMT-CSCs reduced cell motility and is expected to reduce invasion and metastasis. EGFR blocking also induced shifts of Epi-CSCs into the differentiating cell compartment which typically has greater sensitivity to chemo/radiation, an effect expected to enhance the overall response of tumour cell populations to adjunctive therapies.

Reprogramming to developmental plasticity in cancer stem cells.

During development and throughout adult life, sub-populations of cells exist that exhibit phenotypic plasticity - the ability to differentiate into multiple lineages. This behaviour is important in embryogenesis, is exhibited in a more limited context by adult stem cells, and can be re-activated in cancer cells to drive important processes underlying tumour progression. A well-studied mechanism of phenotypic plasticity is the epithelial-to-mesenchymal transition (EMT), a process which has been observed in both normal and cancerous cells. The epigenetic and metabolic modifications necessary to facilitate phenotypic plasticity are first seen in development and can be re-activated both in normal regeneration and in cancer. In cancer, the re-activation of these mechanisms enables tumour cells to acquire a cancer stem cell (CSC) phenotype with enhanced ability to survive in hostile environments, resist therapeutic interventions, and undergo metastasis. However, recent research has suggested that plasticity may also expose weaknesses in cancer cells that could be exploited for future therapeutic development. More research is needed to identify developmental mechanisms that are active in cancer, so that these may be targeted to reduce tumour growth and metastasis and overcome therapeutic resistance.

Phenotypic Plasticity Determines Cancer Stem Cell Therapeutic Resistance in Oral Squamous Cell Carcinoma.

Cancer stem cells (CSCs) drive tumour spread and therapeutic resistance, and can undergo epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) to switch between epithelial and post-EMT sub-populations. Examining oral squamous cell carcinoma (OSCC), we now show that increased phenotypic plasticity, the ability to undergo EMT/MET, underlies increased CSC therapeutic resistance within both the epithelial and post-EMT sub-populations. The post-EMT CSCs that possess plasticity exhibit particularly enhanced therapeutic resistance and are defined by a CD44(high)EpCAM(low/-) CD24(+) cell surface marker profile. Treatment with TGFß and retinoic acid (RA) enabled enrichment of this sub-population for therapeutic testing, through which the endoplasmic reticulum (ER) stressor and autophagy inhibitor Thapsigargin was shown to selectively target these cells. Demonstration of the link between phenotypic plasticity and therapeutic resistance, and development of an in vitro method for enrichment of a highly resistant CSC sub-population, provides an opportunity for the development of improved chemotherapeutic agents that can eliminate CSCs.

Invasive oral cancer stem cells display resistance to ionising radiation.

There is a significant amount of evidence to suggest that human tumors are driven and maintained by a sub-population of cells, known as cancer stem cells (CSC). In the case of head and neck cancer, such cells have been characterised by high expression levels of CD44 cell surface glycoprotein, while we have previously shown the presence of two diverse oral CSC populations in vitro, with different capacities for cell migration and proliferation. Here, we examined the response of oral CSC populations to ionising radiation (IR), a front-line measure for the treatment of head and neck tumors. We show that oral CSC initially display resistance to IR-induced growth arrest as well as relative apoptotic resistance. We propose that this is a result of preferential activation of the DNA damagerepair pathway in oral CSC with increased activation of ATM and BRCA1, elevated levels of DNA repair proteins RAD52, XLF, and a significantly faster rate of DNA double-strand-breaks clearance 24 hours following IR. By visually identifying CSC sub-populations undergoing EMT, we show that EMT-CSC represent the majority of invasive cells, and are more radio-resistant than any other population in re-constructed 3D tissues. We provide evidence that IR is not sufficient to eliminate CSC in vitro, and that sensitization of CD44hi/ESAlow cells to IR, followed by secondary EMT blockade, could be critical in order to reduce primary tumor recurrence, but more importantly to be able to eradicate cells capable of invasion and distant metastasis.

Phenotypic plasticity and epithelial-to-mesenchymal transition in the behaviour and therapeutic response of oral squamous cell carcinoma.

It is increasingly recognised that phenotypic plasticity, apparently driven by epigenetic mechanisms, plays a key role in tumour behaviour and markedly influences the important processes of therapeutic survival and metastasis. An important source of plasticity in malignancy is epithelial-to-mesenchymal transition (EMT), a common epigenetically controlled event that results in transition of malignant cells between different phenotypic states that confer motility and enhance survival. In this review, we discuss the importance of phenotypic plasticity and its contribution to cellular heterogeneity in oral squamous cell carcinoma with emphasis on aspects of drug resistance and EMT.

Elevation in 5-FU-induced apoptosis in head and neck cancer stem cells by a combination of CDHP and GSK3ß inhibitors.

BACKGROUND: Cancer stem cells (CSCs) are involved in both tumourigenesis and in tumour recurrence after therapy. In head and neck squamous cell carcinoma (HNSCC), there are two biologically different CSC phenotypes both of which express high levels of CD44 but differ in their expression levels of epithelial-specific antigen (ESA). One phenotype is CD44(high)/ESA(high) and has epithelial features (Epi-CSCs), while the other is CD44(high) /ESA(low), has undergone epithelial to mesenchymal transition (EMT-CSCs), has mesenchymal features and is migratory (Biddle et al., 2011). CSCs are resistant to therapeutically induced apoptosis but the molecular mechanisms by which they develop apoptotic resistance remains unclear. However, glycogen synthase kinase 3ß (GSK3ß) contributes to regulation of both the self-renewal and switching of these two CSC phenotypes (Shigeishi et al., 2013). METHODS: CD44(high) /ESA(low), CD44(high) /ESA(high) and CD44(low) cells were FACS sorted from the HNSCC cell line LUC4, and 5-FU-induced apoptosis was analysed by Annexin V staining followed by flow cytometry analysis. RESULTS: CD44(high) /ESA(low) cells exhibited marked resistance to 5-FU-induced apoptosis and had high expression of dihydropyrimidine dehydrogenase (DPD). The DPD inhibitor, 5-chloro-2, 4-dihydroxypyridine (CDHP) significantly enhanced 5-FU-induced apoptosis of CD44(high)/ESA(low) cells. Inhibition of GSK3ß induced CD44(high) /ESA(low) cells to undergo mesenchymal-to-epithelial transition (MET) to CD44(high)/ESA(high) cells and pre-existing CD44(high) /ESA(high) cells to differentiate. Apoptosis induced by 5-FU was thus facilitated. Combination of both CDHP and GSK3ß inhibitors markedly enhanced 5-FU-induced apoptosis of CD44(high) /ESA(low) cells. CONCLUSIONS: Our results suggest potentially new approaches for the elimination of the therapy resistant HNSCC CSC population.

Expression of betapapillomavirus oncogenes increases the number of keratinocytes with stem cell-like properties.

Human papillomaviruses (HPV) of genus Betapapillomavirus (betaPV) are associated with nonmelanoma skin cancer development in epidermodysplasia verruciformis (EV) and immunosuppressed patients. Epidemiological and molecular studies suggest a carcinogenic activity of betaPV during early stages of cancer development. Since viral oncoproteins delay and perturb keratinocyte differentiation, they may have the capacity to either retain or confer a "stem cell-like" state on oncogene-expressing cells. The aim of this study was to determine (i) whether betaPV alters the expression of cell surface markers, such as CD44 and epithelial cell adhesion molecule (EpCAM), that have been associated with epithelial stemness, and (ii) whether this confers functional stem cell-like properties to human cutaneous keratinocytes. Fluorescence-activated cell sorter (FACS) analysis revealed an increase in the number of cells with high CD44 and EpCAM expression in keratinocyte cultures expressing HPV type 8 (HPV8) oncogenes E2, E6, and E7. Particularly through E7 expression, a distinct increase in clonogenicity and in the formation and size of tumor spheres was observed, accompanied by reduction of the epithelial differentiation marker Calgranulin B. These stem cell-like properties could be attributed to the pool of CD44(high) EpCAM(high) cells, which was increased within the E7 cultures of HPV5, -8, and -20. Enhanced EpCAM levels were present in organotypic skin cultures of primary keratinocytes expressing E7 of the oncogenic HPV types HPV5, -8, and -16 and in clinical samples from EV patients. In conclusion, our data show that betaPV may increase the number of stem cell-like cells present during early carcinogenesis and thus enable the persistence and accumulation of DNA damage necessary to generate malignant stem cells.

Sub-sets of cancer stem cells differ intrinsically in their patterns of oxygen metabolism.

The glycolytic response of hypoxic cells is primarily mediated by the hypoxia inducible factor alpha (HIF-1α) but even in the presence of abundant oxygen tumours typically show high rates of glycolysis. Higher levels of HIF-1α in tumours are associated with a poorer prognosis and up-regulation of markers of epithelial mesenchymal transition (EMT) due to HIF-1α actions. We have recently shown that EMT occurs within the CD44(high) cancer stem cell (CSC) fraction and that epithelial and EMT CSCs are distinguished by high and low ESA expression, respectively. We here show that hypoxia induces a marked shift of the CSC fraction towards EMT leading to altered cell morphology, an increased proportion of CD44(high)/ESA(low) cells, patterns of gene expression typical of EMT, and enhanced sphere-forming ability. The size of EMT fractions returned to control levels in normoxia indicating a reversible process. Surprisingly, however, even under normoxic conditions a fraction of EMT CSCs was present and maintained high levels of HIF-1α, apparently due to actions of cytokines such as TNFα. Functionally, this EMT CSC fraction showed decreased mitochondrial mass and membrane potential, consumed far less oxygen per cell, and produced markedly reduced levels of reactive oxygen species (ROS). These differences in the patterns of oxygen metabolism of sub-fractions of tumour cells provide an explanation for the general therapeutic resistance of CSCs and for the even greater resistance of EMT CSCs. They also identify potential mechanisms for manipulation of CSCs.

Maintenance of stem cell self-renewal in head and neck cancers requires actions of GSK3ß influenced by CD44 and RHAMM.

Cells sorted from head and neck cancers on the basis of their high expression of CD44 have high potency for tumor initiation. These cells are also involved in epithelial to mesenchymal transition (EMT) and we have previously reported that cancer stem cells (CSCs) exist as two biologically distinct phenotypes. Both phenotypes are CD44(high) but one is also ESA(high) and maintains epithelial characteristics, the other is ESA(low) , has mesenchymal characteristics and is migratory. Examining CD44-regulated signal pathways in these cells we show that CD44, and also RHAMM, act to inhibit phosphorylation of glycogen synthase kinase 3ß (GSK3ß). We show that inhibitory phosphorylation reduces the formation of both "tumor spheres" and "holoclone" colonies, functional indicators of stemness. GSK3ß inhibition also reduces the expression of stem cell markers such as Oct4, Sox2, and Nanog and upregulates expression of the differentiation markers Calgranulin B and Involucrin in the CD44(high) /ESA(high) cell fraction. Transition of CSCs out of EMT and back to the epithelial CSC phenotype is induced by GSK3ß knockdown. These results indicate that GSK3ß plays a central role in determining and maintaining the phenotypes and behavior of CSCs in vitro and are likely to be involved in controlling the growth and spread of tumors in vivo.

CD44 staining of cancer stem-like cells is influenced by down-regulation of CD44 variant isoforms and up-regulation of the standard CD44 isoform in the population of cells that have undergone epithelial-to-mesenchymal transition.

CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44(high) cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44(high) population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44(high) population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44(high) population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that consideration be given to use of multiple cancer stem-like cell markers and suitable procedures for cell isolation in order that the correct populations are assayed.

Cancer stem cells and EMT in carcinoma.

The majority of deaths from carcinoma are caused by secondary growths that result from tumour invasion and metastasis. The importance of epithelial-to-mesenchymal transition (EMT) as a driver of invasion and metastasis is increasingly recognised, and recent evidence has highlighted a link between EMT and the cancer stem cells that initiate and maintain tumours and have also been implicated in invasion and metastasis. Here, we review cancer stem cells and their link with EMT, and explore the importance of this link in metastasis and therapeutic resistance of tumours. We also discuss new evidence from our laboratory demonstrating that cancer stem cells display a remarkable phenotypic plasticity that enables them to switch between an epithelial phenotype that drives tumour growth and an EMT phenotype that drives metastasis. As successful therapies must eradicate cancer stem cells in all their guises, the identification of sub-types of cancer stem cells that display therapeutic resistance and phenotypic plasticity has important implications for the future design of therapeutic strategies. The ability to assay the responses of different cancer stem cell phenotypes in vitro holds promise for the rapid development of a new generation of targeted therapies that fulfil this objective.

Cancer stem cells in squamous cell carcinoma switch between two distinct phenotypes that are preferentially migratory or proliferative.

Epithelial-to-mesenchymal transition (EMT) is an important driver of tumor invasion and metastasis, which causes many cancer deaths. Cancer stem cells (CSC) that maintain and initiate tumors have also been implicated in invasion and metastasis, but whether EMT is an important contributor to CSC function is unclear. In this study, we investigated whether a population of CSCs that have undergone EMT (EMT CSCs) exists in squamous cell carcinoma (SCC). We also determined whether a separate population of CSCs that retain epithelial characteristics (non-EMT CSCs) is also present. Our studies revealed that self-renewing CSCs in SCC include two biologically-distinct phenotypes. One phenotype, termed CD44(high)ESA(high), was proliferative and retained epithelial characteristics (non-EMT CSCs), whereas the other phenotype, termed CD44(high)ESA(low), was migratory and had mesenchymal traits characteristic of EMT CSCs. We found that non-EMT and EMT CSCs could switch their epithelial or mesenchymal traits to reconstitute the cellular heterogeneity which was characteristic of CSCs. However, the ability of EMT CSCs to switch to non-EMT character was restricted to cells that were also ALDH1(+), implying that only ALDH1(+) EMT cells had the ability to seed a new epithelial tumor. Taken together, our findings highlight the identification of two distinct CSC phenotypes and suggest a need to define therapeutic targets that can eradicate both of these variants to achieve effective SCC treatment.