Modeling evolutionary dynamics of epigenetic mutations in hierarchically organized tumors

The cancer stem cell (CSC) concept is a highly debated topic in cancer research. While experimental evidence in favor of the cancer stem cell theory is apparently abundant, the results are often criticized as being difficult to interpret. An important reason for this is that most experimental data that support this model rely on transplantation studies. In this study we use a novel cellular Potts model to elucidate the dynamics of established malignancies that are driven by a small subset of CSCs. Our results demonstrate that epigenetic mutations that occur during mitosis display highly altered dynamics in CSC-driven malignancies compared to a classical, non-hierarchical model of growth. In particular, the heterogeneity observed in CSC-driven tumors is considerably higher. We speculate that this feature could be used in combination with epigenetic (methylation) sequencing studies of human malignancies to prove or refute the CSC hypothesis in established tumors without the need for transplantation. Moreover our tumor growth simulations indicate that CSC-driven tumors display evolutionary features that can be considered beneficial during tumor progression. Besides an increased heterogeneity they also exhibit properties that allow the escape of clones from local fitness peaks. This leads to more aggressive phenotypes in the long run and makes the neoplasm more adaptable to stringent selective forces such as cancer treatment. Indeed when therapy is applied the clone landscape of the regrown tumor is more aggressive with respect to the primary tumor, whereas the classical model demonstrated similar patterns before and after therapy. Understanding these often counter-intuitive fundamental properties of (non-)hierarchically organized malignancies is a crucial step in validating the CSC concept as well as providing insight into the therapeutical consequences of this model.     

Direct in vivo evidence for tumor propagation by glioblastoma cancer stem cells

High-grade gliomas (World Health Organization grade III anaplastic astrocytoma and grade IV glioblastoma multiforme), the most prevalent primary malignant brain tumors, display a cellular hierarchy with self-renewing, tumorigenic cancer stem cells (CSCs) at the apex. While the CSC hypothesis has been an attractive model to describe many aspects of tumor behavior, it remains controversial due to unresolved issues including the use of ex vivo analyses with differential growth conditions. A CSC population has been confirmed in malignant gliomas by preferential tumor formation from cells directly isolated from patient biopsy specimens. However, direct comparison of multiple tumor cell populations with analysis of the resulting phenotypes of each population within a representative tumor environment has not been clearly described. To directly test the relative tumorigenic potential of CSCs and non-stem tumor cells in the same microenvironment, we interrogated matched tumor populations purified from a primary human tumor transplanted into a xenograft mouse model and monitored competitive in vivo tumor growth studies using serial in vivo intravital microscopy. While CSCs were a small minority of the initial transplanted cancer cell population, the CSCs, not the non-stem tumor cells, drove tumor formation and yielded tumors displaying a cellular hierarchy. In the resulting tumors, a fraction of the initial transplanted CSCs maintained expression of stem cell and proliferation markers, which were significantly higher compared to the non-stem tumor cell population and demonstrated that CSCs generated cellular heterogeneity within the tumor. These head-to-head comparisons between matched CSCs and non-stem tumor cells provide the first functional evidence using live imaging that in the same microenvironment, CSCs more than non-stem tumor cells are responsible for tumor propagation, confirming the functional definition of a CSC.     

Emergent behaviors from a cellular automaton model for invasive tumor growth in heterogeneous microenvironments

Understanding tumor invasion and metastasis is of crucial importance for both fundamental cancer research and clinical practice. In vitro experiments have established that the invasive growth of malignant tumors is characterized by the dendritic invasive branches composed of chains of tumor cells emanating from the primary tumor mass. The preponderance of previous tumor simulations focused on non-invasive (or proliferative) growth. The formation of the invasive cell chains and their interactions with the primary tumor mass and host microenvironment are not well understood. Here, we present a novel cellular automaton (CA) model that enables one to efficiently simulate invasive tumor growth in a heterogeneous host microenvironment. By taking into account a variety of microscopic-scale tumor-host interactions, including the short-range mechanical interactions between tumor cells and tumor stroma, degradation of the extracellular matrix by the invasive cells and oxygen/nutrient gradient driven cell motions, our CA model predicts a rich spectrum of growth dynamics and emergent behaviors of invasive tumors. Besides robustly reproducing the salient features of dendritic invasive growth, such as least-resistance paths of cells and intrabranch homotype attraction, we also predict nontrivial coupling between the growth dynamics of the primary tumor mass and the invasive cells. In addition, we show that the properties of the host microenvironment can significantly affect tumor morphology and growth dynamics, emphasizing the importance of understanding the tumor-host interaction. The capability of our CA model suggests that sophisticated in silico tools could eventually be utilized in clinical situations to predict neoplastic progression and propose individualized optimal treatment strategies.     

Cell-to-cell signaling influences the fate of prostate cancer stem cells and their potential to generate more aggressive tumors

An increasing number of malignancies has been shown to be initiated and propelled by small subpopulations of cancer stem cells (CSC). However, whether tumor aggressiveness is driven by CSC and by what extent this property may be relevant within the tumor mass is still unsettled. To address this issue, we isolated a rare tumor cell population on the basis of its CD44(+)CD24(-) phenotype from the human androgen-independent prostate carcinoma cell line DU145 and established its CSC properties. The behavior of selected CSC was investigated with respect to the bulk DU145 cells. The injection of CSC in nude mice generated highly vascularized tumors infiltrating the adjacent tissues, showing high density of neuroendocrine cells and expressing low levels of E-cadherin and β-catenin as well as high levels of vimentin. On the contrary, when a comparable number of unsorted DU145 cells were injected the resulting tumors were less aggressive. To investigate the different features of tumors in vivo, the influence of differentiated tumor cells on CSC was examined in vitro by growing CSC in the absence or presence of conditioned medium from DU145 cells. CSC grown in permissive conditions differentiated into cell populations with features similar to those of cells held in aggressive tumors generated from CSC injection. Differently, conditioned medium induced CSC to differentiate into a cell phenotype comparable to cells of scarcely aggressive tumors originated from bulk DU145 cell injection. These findings show for the first time that CSC are able to generate differentiated cells expressing either highly or scarcely aggressive phenotype, thus influencing prostate cancer progression. The fate of CSC was determined by signals released from tumor environment. Moreover, using microarray analysis we selected some molecules which could be involved in this cell-to-cell signaling, hypothesizing their potential value for prognostic or therapeutic applications.     

Transformation of Epithelial Ovarian Cancer Stemlike Cells into Mesenchymal Lineage via EMT Results in Cellular Heterogeneity and Supports Tumor Engraftment

Ovarian cancers are heterogeneous and contain stemlike cells that are able to self-renew and are responsible for sustained tumor growth. Metastasis in the peritoneal cavity occurs more frequently in ovarian cancer than in other malignancies, but the underlying mechanism remains largely unknown. We have identified that ovarian cancer stemlike cells (CSCs), which were defined as side population (SP) cells, were present in patients’ ascitic fluid and mesenchymally transformed cell lines, ES-2 and HO-8910PM. SP cells, which were sorted from both cell lines and implanted into immunocompromised mice, were localized to the xenografted tumor boundary. In addition, SP cells exhibited an epithelial phenotype and showed a distinct gene expression profile with reduced expression of cell adhesion molecules (CAMs), indicating that SP cells exert an important role in ovarian cancer progression on the basis of their delicate interaction with the surrounding microenvironment and anatomical localization in tumors. In contrast, non-SP cells exhibited a more mesenchymal phenotype and showed more increased invasive potential than SP cells. This heterogeneity was observed as an endogenous transformation via the epithelial–mesenchymal transition (EMT) process. Inhibition of the EMT process by Snail1 silencing reduced the SP cell frequency, and affected their invasive capacity and engraftment. These findings illustrate the interplay between epithelial ovarian CSCs and the EMT, and exert a link to explain tumor heterogeneity and its necessity for ovarian cancer maintenance, metastasis and progression.     

ALDH adenoid cystic carcinoma cells display cancer stem cell properties and are responsible for mediating metastasis

The cancer stem cell (CSC) theory has been proposed to explain the tumor heterogeneity and carcinogenesis process. Recent studies indicate that aldehyde dehydrogenase (ALDH) activity represents a promising CSC marker. Here, we aimed to determine whether human adenoid cystic carcinoma (AdCC) also follows CSC model by exploring the CSC properties of AdCC cells expressing high level of ALDH activity. Utilizing in-vivo series transplantation assays, we found ALDH^high AdCC cells were capable of self-renewal and of generating tumors that recapitulate the heterogeneity of the parental tumor. Utilizing in-vitro assay, we found only ALDH^high AdCC cells have tumorsphere-forming ability in anchorage-independent cultures. Finally, we showed ALDH^high AdCC cells possess highly invasive capability and are responsible for mediating metastasis. These findings suggest the existence of a developmental hierarchy within human AdCC and further elucidation of the unique survival mechanism of AdCC derived CSC population may provide novel therapeutic strategies to treat AdCC.     

Tumor initiating cells: Development and critical characterization of a model derived from the A431 carcinoma cell line forming spheres in suspension

To investigate the tumor fraction with cancer stem/tumor initiating cell (CSC/TIC) characteristics, we tested the human cervical carcinoma cell lines A431, Caski and SiHa, by growth as non-adherent spheres in specific media and aldehyde dehydrogenase (ALDH) enzymatic activity. A good correlation between the two parameters was observed and the highest levels were observed in A431 cell line that was selected for characterization of the CSC/TIC fraction. A431 parental cells already displayed characteristics common to CSC/TIC, such as sphere forming efficiency, adherent holoclone formation and high ALDH activity. Non-adherent spheres maintained or increased these properties, and, in particular, ALDH-positive fraction increased from 46 to 65% and a transient induction of stem cell markers such as Nanog, Nestin and Oct4 was observed. Furthermore, a significant increase of paraclone forming cells was observed, suggesting that differentiation took place inside sphere cell populations. As compared to parental cells, spheres were characterized by: (1) a ten-fold higher verapamil-sensitive side population fraction; (2) the appearance of a podoplanin-positive subpopulation characterized by a small cell size; (3) the ability to propagate tumors in nude mice at a lower cell dose. The global gene expression analysis demonstrated a strong and reversible modulation of 'sphere' phenotype in comparison to parental and sphere cells re-induced to adherent conditions. All together our results indicated that the growth of A431 cells as a non-adherent sphere was not sufficient by itself to define a stem-like population, but it was essential for the emergence of a small population of tumor cells with CSC properties.     

肿瘤干细胞的研究进展

肿瘤干细胞(cancer/tumor stem cell,CSC/TSC)假说认为肿瘤组织中存在极少量的瘤细胞充当着干细胞的角色,它们具有自我更新能力、无限增殖能力,且能驱动肿瘤的形成和生长。近年来,随着在血液肿瘤和实体瘤中相继发现CSC/TSC存在的相关证据,对CSC/TSC的生物学特性的认识不断深入,对肿瘤的复发、转移、耐药性等特点也有了新的观点和研究方向,就近年来该方面的进展作一综述。     

The architect who never sleeps: Tumor-induced plasticity

Tumor cell plasticity is an event that has been observed in several malignancies. In fact, most of the solid tumors are characterized by cellular heterogeneity and undergo constant changes as the tumor develops. The increased plasticity displayed by these cells allows them to acquire additional properties, enabling epithelial-mesenchymal transitions, dedifferentiation and the acquisition of stem cell-like properties. Here we discuss the particular importance of an inflammatory microenvironment for the bidirectional control of cellular plasticity and the potential for therapeutic intervention.     

Cancer stem cells and human malignant melanoma

Cancer stem cells (CSC) have been identified in hematological malignancies and several solid cancers. Similar to physiological stem cells, CSC are capable of self-renewal and differentiation and have the potential for indefinite proliferation, a function through which they may cause tumor growth. Although conventional anti-cancer treatments might eradicate most malignant cells in a tumor, they are potentially ineffective against chemoresistant CSC, which may ultimately be responsible for recurrence and progression. Human malignant melanoma is a highly aggressive and drug-resistant cancer. Detection of tumor heterogeneity, undifferentiated molecular signatures, and increased tumorigenicity of melanoma subsets with embryonic-like differentiation plasticity strongly suggest the presence and involvement of malignant melanoma stem cells (MMSC) in the initiation and propagation of this malignancy. Here, we review these findings in the context of functional properties ascribed to melanocyte stem cells and CSC in other cancers. We discuss the association of deregulated signaling pathways, genomic instability, and vasculogenic mimicry phenomena observed in melanoma subpopulations in light of the CSC concept. We propose that a subset of MMSC may be responsible for melanoma therapy-resistance, tumor invasiveness, and neoplastic progression and that targeted abrogation of a MMSC compartment could therefore ultimately lead to stable remissions and perhaps cures of metastatic melanoma.     

Stem cells in gastrointestinal cancers: The road less travelled

Cancer stem cells(CSC) are thought to be malignant cells that have the capacity to initiate and maintain tumor growth and survival. Studies have described CSC in various gastrointestinal neoplasms such as colon, pancreas and liver and gastroesophageal tumors. The mechanism by which CSC develop remains unclear. Several studies have explored the role of dysregulation of the Wnt/β-catenin, transformation growth factor-beta and hedhog pathways in generation of CSC. In this review, we discuss the various molecular abnormalities that may be related to formation of CSC in gastrointestinal malignancies, strategies to identify CSC and therapeutic strategies that are based on these concepts. Identification and targeting CSC is an intriguing area and may provide a new therapeutic option for patients with cancer including gastrointestinal malignancies. Although great progress has been made, many issues need to be addressed. Precise targeting of CSC will require precise isolation and characterization of those cells. This field is also evolving but further research is needed to identify markers that are specific for CSC.Although the application of this field has not entered the clinic yet, there continues to be significant optimism about its potential utility in overcoming cancer resistance and curing patients with cancer.     

Cancer stem cell, niche and EGFR decide tumor development and treatment response: A bio-computational simulation study

Recent research in cancer biology has suggested the hypothesis that tumors are initiated and driven by a small group of cancer stem cells (CSCs). Furthermore, cancer stem cell niches have been found to be essential in determining fates of CSCs, and several signaling pathways have been proven to play a crucial role in cellular behavior, which could be two important factors in cancer development. To better understand the progression, heterogeneity and treatment response of breast cancer, especially in the context of CSCs, we propose a mathematical model based on the cell compartment method. In this model, three compartments of cellular subpopulations are constructed: CSCs, progenitor cells (PCs), and terminal differentiated cells (TCs). Moreover, (1) the cancer stem cell niche is, considered by modeling its effect on division patterns (symmetric or asymmetric) of CSCs, and (2) the EGFR signaling pathway is integrated by modeling its role in cell proliferation, apoptosis. Our simulation results indicate that (1) a higher probability for symmetric division of CSC may result in a faster expansion of tumor population, and for a larger number of niches, the tumor grows at a slower rate, but the final tumor volume is larger; (2) higher EGFR expression correlates to tumors with larger volumes while a saturation function is observed, and (3) treatments that inhibit tyrosine kinase activity of EGFR may not only repress the tumor volume, but also decrease the CSCs percentages by shifting CSCs from symmetric divisions to asymmetric divisions. These findings suggest that therapies should be designed to effectively control or eliminate the symmetric division of CSCs and to reduce or destroy the CSC niches.     

Establishment of human ovarian serous carcinomas cell lines in serum free media

Ovarian cancers are the fifth leading cause of cancer death among US woman. The majority of ovarian cancers belong to a category of serous adenocarcinomas. This type of cancer is often diagnosed at a late stage of the disease. Surgical debulking, followed by chemotherapy is the current treatment. Half of all patients will die within 5 years of diagnosis of the disease. Poor survival may be due to disease progression as a consequence of development of drug resistance, cancer cell heterogeneity within the tumor, or the persistence of cancer stem cells. Cancer stem cells (CSC) are defined as a minority cell type in the tumor, which retains the capacity, through asymmetric division, for self-renewal as well as differentiation into multiple cell types. Through this process, CSC can regenerate the entire tumor phenotype and subsequent metastases. Initial in vitro work in the area of solid tumor CSC biology has focused on the isolation and propagation of cells with CSC-like properties from breast and colon tumors. Breast and colon cell lines with CSC-like properties have been isolated and maintained in vitro for extended periods of time. The in vitro maintenance of these CSC requires growth in hormone-supplemented serum-free media and the use of matrix or growth as tumor spheres (Roberts, Ricci-Vitiani et al., Cammareri et al.). Based on the pioneering work generating breast and colon CSC, our lab has begun to develop methods for the establishment cell lines with CSC-like properties from additional solid tumors. In this article, we describe methods, using defined medium, which allow for the successful establishment of continuous cell cultures from a minority cell type within serous ovarian cancers. The cell lines established using these methods grow in serum-free hormone-supplemented medium either as a monolayer on a matrix, or as tumor spheres in suspension. These cells express markers previously reported for tumor stem cells, including CD44 and CD133, and form tumors that recreate the morphology of the original patient tumor when implanted in immune deficient mice. The introduction of this method will facilitate the expansion of ovarian cancer cells for investigating cancer stem cell biology as well as providing tools to aid in the development of new treatments for this deadly disease.     

Human colon cancer stem cells are enriched by insulin-like growth factor-1 and are sensitive to figitumumab

Cancer stem cells (CSCs) are recognized as contributors to cancer progression and therapeutic resistance in liquid and solid malignancies. We analyzed a panel of human colon cancer cell lines for CSC populations by side population and aldehyde dehydrogenase activity. IGF-1 enriches these putative colon CSC populations in a β-catenin-dependent manner. Chemical inhibition of Akt depletes SP cells, and conversely, the overexpression of a constitutively active mutant version of Akt is sufficient to enrich CSC populations. CP-751,871, a fully human antibody with specificity to the IGF-1 receptor, is currently being tested in clinical trials for a variety of solid tumors. CP-751,871 reduces CSC populations in colon cancer cell lines in vitro and reduces tumor growth in vivo. We have identified a novel role for IGF-1 in the enrichment of chemo-resistant CSC populations. Our results suggest that CP-751,871 has preferential activity against putative CSC populations and, therefore, may complement current standard chemotherapeutic regimens that target cycling cells.     

Evolution and Phenotypic Selection of Cancer Stem Cells

Cells of different organs at different ages have an intrinsic set of kinetics that dictates their behavior. Transformation into cancer cells will inherit these kinetics that determine initial cell and tumor population progression dynamics. Subject to genetic mutation and epigenetic alterations, cancer cell kinetics can change, and favorable alterations that increase cellular fitness will manifest themselves and accelerate tumor progression. We set out to investigate the emerging intratumoral heterogeneity and to determine the evolutionary trajectories of the combination of cell-intrinsic kinetics that yield aggressive tumor growth. We develop a cellular automaton model that tracks the temporal evolution of the malignant subpopulation of so-called cancer stem cells(CSC), as these cells are exclusively able to initiate and sustain tumors. We explore orthogonal cell traits, including cell migration to facilitate invasion, spontaneous cell death due to genetic drift after accumulation of irreversible deleterious mutations, symmetric cancer stem cell division that increases the cancer stem cell pool, and telomere length and erosion as a mitotic counter for inherited non-stem cancer cell proliferation potential. Our study suggests that cell proliferation potential is the strongest modulator of tumor growth. Early increase in proliferation potential yields larger populations of non-stem cancer cells(CC) that compete with CSC and thus inhibit CSC division while a reduction in proliferation potential loosens such inhibition and facilitates frequent CSC division. The sub-population of cancer stem cells in itself becomes highly heterogeneous dictating population level dynamics that vary from long-term dormancy to aggressive progression. Our study suggests that the clonal diversity that is captured in single tumor biopsy samples represents only a small proportion of the total number of phenotypes.     

肿瘤干细胞及其模型的研究现状及临床治疗最新进展

癌症发生最有名的模型之一肿瘤CSCcancer stem cell(CSC)模型已经被确立为一种细胞机制,有助于体现不同类型的肿瘤表型和功能的异质性。然而,最新研究突出显示了肿瘤CSC模型的复杂性和挑战性:CSC表型在不同的病人间有本质上的不同,肿瘤可能含有多种表型或遗传上不同的CSC,转移的肿瘤CSC可能由原始肿瘤CSC进展而来,肿瘤细胞可进行可逆的表型的改变。虽然在特定情况下,肿瘤CSC的概念具有临床相关性,但越来越多的证据表明,定位、鉴定出肿瘤灶中所有亚型的肿瘤CSC将对临床上预防肿瘤复发具有特殊意义。有以下几个因素可以促使异质性的发生:基因突变,后生的变化,与微环境间的相互作用以及存在或缺失细胞分级等。肿瘤异质性可以用不同的细胞机制来解释。虽然CSC具有CSC自我更新和分化的特性,但是它们不一定是正常组织CSC转化而形成的。这种CSC模型引起了科研工作者广泛的关注,我们结合新近的相关文献综述如下。     

Understanding the role of stromal fibroblasts in cancer progression

The major cellular components of tumor microenvironment, referred to as the cancer stroma, are composed of cancer-associated fibroblasts that support tumor epithelial growth, invasion and therapeutic resistance. Thus when we speak of developing therapies that address tumor heterogeneity it is not only a matter of different mutations within the tumor epithelia. While individual mutations in the stromal compartment are controversial, the heterogeneity in fibroblastic population in a single tumor is not up for debate. Cooperative interaction among heterotypic fibroblasts and tumor cells contribute to cancer progression. Therefore to tackle solid tumors, we need to understand its complex microenvironment. Here we review some seminal developments in the field of tumor microenvironment, mainly focusing on cancer-associated fibroblast.     

The cancer stem cell theory: is it correct?

The cancer stem cell hypothesis posits that tumor growth is driven by a rare subpopulation of cells, designated cancer stem cells (CSC). Studies supporting this theory are based in large part on xenotransplantation experiments wherein human cancer cells are grown in immunocompromised mice and only CSC, often constituting less than 1% of the malignancy, generate tumors. Herein, we show that all colonies derived from randomly chosen single cells in mouse lung and breast cancer cell lines form tumors following allografting histocompatible mice. Our study suggests that the majority of malignant cells rather than CSC can sustain tumors and that the cancer stem cell theory must be reevaluated.     

Understanding the role of stromal fibroblasts in cancer progression

The major cellular components of tumor microenvironment, referred to as the cancer stroma, are composed of cancer-associated fibroblasts that support tumor epithelial growth, invasion and therapeutic resistance. Thus when we speak of developing therapies that address tumor heterogeneity it is not only a matter of different mutations within the tumor epithelia. While individual mutations in the stromal compartment are controversial, the heterogeneity in fibroblastic population in a single tumor is not up for debate. Cooperative interaction among heterotypic fibroblasts and tumor cells contribute to cancer progression. Therefore to tackle solid tumors, we need to understand its complex microenvironment. Here we review some seminal developments in the field of tumor microenvironment, mainly focusing on cancer-associated fibroblast.