Cancer Stem Cell Research Research Articles

Editorial introductions

Editorial introductions

Three-dimensional cell culture model utilization in cancer stem cell research.

Three-dimensional (3D) cell culture models are becoming increasingly popular in contemporary cancer research and drug resistance studies. Recently, scientists have begun incorporating cancer stem cells (CSCs) into 3D models and modifying culture components in order to mimic in vivo conditions better. Currently, the global cell culture market is primarily focused on either 3D cancer cell cultures or stem cell cultures, with less focus on CSCs. This is evident in the low product availability officially indicated for 3D CSC model research. This review discusses the currently available commercial products for CSC 3D culture model research. Additionally, we discuss different culture media and components that result in higher levels of stem cell subpopulations while better recreating the tumor microenvironment. In summary, although progress has been made applying 3D technology to CSC research, this technology could be further utilized and a greater number of 3D kits dedicated specifically to CSCs should be implemented.

525 - Salinomycin affects Golgi apparatus function in cancer stem-like cells

Background: Cancer stem cells (CSC) are an immortal tumor-initiating population that can self-renew and has pluripotent capacity. Despite their small number, CSCs are believed to be critical for tumor initiation, progression and metastasis, as well as for the treatment failure and disease relapse. CSCs have been confirmed to exist in various types of tumors which include leukemia as well as various solid cancers. There are several obstacles involved with CSC research among which are their low number within tumor cell population and their relative instability in culture. Recently, a breast CSC model was established by experimental induction of epithelial to mesenchymal transition (EMT) in immortalized human mammary epithelial cells (HMLE). Using this model salinomycin was identified to be selectively toxic towards epithelial CSCs. Salinomycin is a K+/H+ exchanger that can affect cation transport across different membranes present in the cell. However the exact mechanism of the observed selectivity remains largely unknown. We have noticed that salinomycin affects posttranslational modifications of membrane proteins in the fore mentioned CSC model. This lead us to the hypothesis that salinomycin induces Golgi apparatus stress that affects secretory pathway which may represent a key vulnerability of EMT cells. Of note, monensin, another ionophore with chemical structure similar to that of salinomycin, is a well described inhibitor of Golgi function. Materials and Methods: We used HMLE cells with knocked-down E-cadherin (HMLEshEcad) that went through EMT and display characteristics of stem cells. To test our hypothesis we performed western blotting, confocal microscopy and flow cytometry after staining with fluorescently labeled Golgirelated antibodies. Results: The treatment of stem-like HMLEshEcad cells with submicromolar concentrations of salinomycin causes dysfunctional maturation of N-cadherin. In addition, we observed similar changes in processing of other plasma membrane proteins that could be accounted for aberrant Golgi apparatus function. The same effect was not observed in control cells. Immunostaining confirmed changes of Golgi apparatus morphology and function. Conclusion: Our results indeed point that salinomycin differentially affects mesenchymal and epithelial cells due to their different dependence on the Golgi apparatus function. We believe that agents that promote endoplasmic reticulum and Golgi apparatus malfunction should be further explored for their potential to selectively eradicate cancer cells that have undergone an EMT.

An alginate-based platform for cancer stem cell research

As the primary determinants of the clinical behaviors of human cancers, the discovery of cancer stem cells (CSCs) represents an ideal target for novel anti-cancer therapies (Kievit et al., 2014). Notably, CSCs are difficult to propagate in vitro, which severely restricts the study of CSC biology and the development of therapeutic agents. Emerging evidence indicates that CSCs rely on a niche that controls their differentiation and proliferation, as is the case with normal stem cells (NSCs). Replicating the in vivo CSC microenvironment in vitro using three-dimensional (3D) porous scaffolds can provide means to effectively generate CSCs, thus enabling the discovery of CSC biology. This paper presents our study on a novel alginate-based platform for mimicking the CSC niche to promote CSC proliferation and enrichment. In this study, we used a versatile mouse 4T1 breast cancer model to independently evaluate the matrix parameters of a CSC niche – including the material’s mechanical properties, cytokine immobilization, and the composition of the extracellular matrix’s (ECM’s) molecular impact – on CSC proliferation and enrichment. On this basis, the optimal stiffness and concentration of hyaluronic acid (HA), as well as epidermal growth factor and basic fibroblast growth factor immobilization, were identified to establish the platform for mimicking the 4T1 breast CSCs (4T1 CSCs) niche. The 4T1 CSCs obtained from the platform show increased expression of the genes involved in breast CSC and NSC, as compared to general 2D or 3D culture, and 4T1 CSCs were also demonstrated to have the ability to quickly form a subcutaneous tumor in homologous Balb/c mice in vivo. In addition, the platform can be adjusted according to different parameters for CSC screening. Our results indicate that our platform offers a simple and efficient means to isolate and enrich CSCs in vitro, which can help researchers better understand CSC biology and thus develop more effective therapeutic agents to treat cancer. Statement of SignificanceAs the primary determinants of the clinical behaviors of human cancers, the discovery of cancer stem cells (CSCs) represents an ideal target for novel anti-cancer therapies. However, CSCs are difficult to propagate in vitro, which severely restricts the study of CSC biology and the development of therapeutic agents. Emerging evidence indicates that CSCs rely on a niche that controls their differentiation and proliferation, as is the case with normal stem cells (NSCs). Replicating the in vivo CSC microenvironment in vitro using three-dimensional (3D) porous scaffolds can provide means to effectively generate CSCs, thus enabling the discovery of CSC biology. In our study, a novel alginate-based platform were developed for mimicking the CSC niche to promote CSC proliferation and enrichment.

At the Crossroads of Cancer Stem Cells, Radiation Biology, and Radiation Oncology.

Reports that a small subset of tumor cells initiate and sustain tumor growth, are resistant to radiation and drugs, and bear specific markers have led to an explosion of cancer stem cell research. These reports imply that the evaluation of therapeutic response by changes in tumor volume is misleading, as volume changes reflect the response of the sensitive rather than the resistant tumorigenic cell population. The reports further suggest that the marker-based selection of the tumor cell population will facilitate the development of radiation treatment schedules, sensitizers, and drugs that specifically target the resistant tumorigenic cells that give rise to treatment failure. This review presents evidence that contests the observations that cancer stem cell markers reliably identify the subset of tumor cells that sustain tumor growth and that the marker-identified population is radioresistant relative to the marker-negative cells. Experimental studies show that cells and tumors that survive large radiation doses are not more radioresistant than unirradiated cells and tumors, and also show that the intrinsic radiosensitivity of unsorted colony-forming tumor cells, in combination with the fraction of unsorted tumor cells that are tumor initiating, predicts tumor radiocurability.

Cancer Stem Cells in the Thyroid.

The cancer stem cell (CSC) model posits that CSCs are a small, biologically distinct subpopulation of cancer cells in each tumor that have self-renewal and multi-lineage potential, and are critical for cancer initiation, metastasis, recurrence, and therapy-resistance. Numerous studies have linked CSCs to thyroid biology, but the candidate markers and signal transduction pathways that drive thyroid CSC growth are controversial, the origin(s) of thyroid CSCs remain elusive, and it is unclear whether thyroid CSC biology is consistent with the original hierarchical CSC model or the more recent dynamic CSC model. Here, we critically review the thyroid CSC literature with an emphasis on research that confirmed the presence of thyroid CSCs by in vitro sphere formation or in vivo tumor formation assays with dispersed cells from thyroid cancer tissues or bona fide thyroid cancer cell lines. Future perspectives of thyroid CSC research are also discussed.

Cancer stem cells: a metastasizing menace!

Cancer is one of the leading causes of death worldwide, and is estimated to be a reason of death of more than 18 billion people in the coming 5 years. Progress has been made in diagnosis and treatment of cancer; however, a sound understanding of the underlying cell biology still remains an unsolved mystery. Current treatments include a combination of radiation, surgery, and/or chemotherapy. However, these treatments are not a complete cure, aimed simply at shrinking the tumor and in majority of cases, there is a relapse of tumor. Several evidences suggest the presence of cancer stem cells (CSCs) or tumor‐initiating stem‐like cells, a small population of cells present in the tumor, capable of self‐renewal and generation of differentiated progeny. The presence of these CSCs can be attributed to the failure of cancer treatments as these cells are believed to exhibit therapy resistance. As a result, increasing attention has been given to CSC research to resolve the therapeutic problems related to cancer. Progress in this field of research has led to the development of novel strategies to treat several malignancies and has become a hot topic of discussion. In this review, we will briefly focus on the main characteristics, therapeutic implications, and perspectives of CSCs in cancer therapy.

Breast Cancer Stem Cell Isolation.

Cells within the tumor are highly heterogeneous. Only a small portion of the cells within the tumor is capable to generate a new tumor. These cells are called cancer stem cells. Theoretically, cancer stem cells are originally from normal stem cells or early progenitor cells which accumulate the random mutations and undergo an altered version of the normal differentiation process. The cancer stem cell drives tumor progression and its recurrence. Thus, the technique to identify and purify the cancer stem cell is the key in any cancer stem cell research. In this protocol, we provide the basic technology of identification and purification of breast cancer stem cells as well as further functional assays to help the researchers achieve their research goals.

CSCdb: a cancer stem cells portal for markers, related genes and functional information.

Cancer stem cells (CSCs), which have the ability to self-renew and differentiate into various tumor cell types, are a special class of tumor cells. Characterizing the genes involved in CSCs regulation is fundamental to understand the mechanisms underlying the biological process and develop treatment methods for tumor therapy. Recently, much effort has been expended in the study of CSCs and a large amount of data has been generated. However, to the best of our knowledge, database dedicated to CSCs is not available until now. We have thus developed a CSCs database (CSCdb), which includes marker genes, CSCs-related genes/microRNAs and functional annotations. The information in the CSCdb was manual collected from about 13 000 articles. The CSCdb provides detailed information of 1769 genes that have been reported to participate in the functional regulation of CSCs and 74 marker genes that can be used for identification or isolation of CSCs. The CSCdb also provides 9475 annotations about 13 CSCs-related functions, such as oncogenesis, radio resistance, tumorigenesis, differentiation, etc. Annotations of the identified genes, which include protein function description, post-transcription modification information, related literature, Gene Ontology (GO), protein-protein interaction (PPI) information and regulatory relationships, are integrated into the CSCdb to help users get information more easily. CSCdb provides a comprehensive resource for CSCs research work, which would assist in finding new CSCs-related genes and would be a useful tool for biologists.Database URL: http://bioinformatics.ustc.edu.cn/cscdb

The Role of Biomaterials on Cancer Stem Cell Enrichment and Behavior

The theory of cancer stem cells (CSCs) and their role in cancer metastasis, tumorigenicity and resistance to therapy is slowly shifting the emphasis on the search for cancer cure: more evidence is surfacing that a successful therapy should be geared against this rare cancer cell population. Unfortunately, CSCs are difficult to culture in vitro which severely limits the progress of CSC research. This review gives a brief overview of CSCs and their microenvironment, with particular focus on studies that used in vitro biomaterial-based models and biomaterial/CSC interfaces for the enrichment of CSCs. Biomaterial properties relevant to CSC behaviors are also addressed. While the discussed research field is still in its infancy, it appears that in vitro cancer models that include a biomaterial can support CSC enrichment and this has proved indispensable to the study of their biology as well as the development of novel cancer therapies.

Characteristics of liver cancer stem cells and clinical correlations

Liver cancer is an aggressive malignant disease with a poor prognosis. Patients with liver cancer are usually diagnosed at an advanced stage and thus miss the opportunity for surgical resection. Chemotherapy and radiofrequency ablation, which target tumor bulk, have exhibited limited therapeutic efficacy to date. Liver cancer stem cells (CSCs) are a small subset of undifferentiated cells existed in liver cancer, which are considered to be responsible for liver cancer initiation, metastasis, relapse and chemoresistance. Elucidating liver CSC characteristics and disclosing their regulatory mechanism might not only deepen our understanding of the pathogenesis of liver cancer but also facilitate the development of diagnostic, prognostic and therapeutic approaches to improve the clinical management of liver cancer. In this review, we will summarize the recent advances in liver CSC research in terms of the origin, identification, regulation and clinical correlation.

Abstract 1397: Identification of a hepatocellular carcinoma cell line capable of monitoring the differentiation of CD13(+)CD166(-) cancer stem cells and effects of sorafenib

Abstract Background: Recent cancer stem cell (CSC) research has highlighted the necessity to develop drugs targeting CSCs. However, only few in vitro models are available which show CSC hierarchy and allow monitoring of CSC differentiation and screening of drugs targeting CSC. Aim: In the present study, we aimed to identify such hepatocellular carcinoma (HCC) cell line(s). Methods: Based on a hypothesis that the proportion of CSCs would decrease during culture because of their differentiation into progenitors (population switch), we tested the positivity of several markers (CD13, EpCAM, CD133, CD44, CD90, CD24, CD166) in HCC cell lines (HuH-7, Li-7, PLC/PRF/5, HLF, HLE) before and after 2 months culture. Results: Only Li-7 cells showed relevant population switch: the proportion of CD13 decreased, while that of CD166 increased during the culture. High tumorigenicity of Li-7 was lost after the population switch. CD13(+)/CD166(-) cells showed slow growth and reconstructed the bulk Li-7 populations composed of CD13(+)/CD166(-), CD13(-)/CD166(-) and CD13(-)/CD166(+) fractions, while CD13(-)/CD166(+) cells showed rapid growth but could not reproduce any other cell populations. CD13(+)/CD166(-) cells showed high ALDH activity, spheroid forming ability and resistance to 5FU. Microarray analysis demonstrated higher expression of stemness-related genes in CD166(-) than CD166(+) fraction. These results indicated the hierarchy of Li-7 cells, in which CD13(+)/CD166(-) and CD13(-)/CD166(+) cells serve as slow growing CSCs and rapid growing progenitors, respectively. Interestingly, sorafenib selectively targeted CD166(-) fraction including CD13(+) CSCs, which exhibited higher mRNA expression of FGF3 and FGF4, candidate biomarkers for sorafenib. 5-FU followed by sorafenib inhibited the growth of bulk Li-7 cells more effectively than reverse sequence or either alone. Conclusions: We identified an useful cell line Li-7 capable of monitoring the CD13 (+) CSC differentiation and screening the drugs targeting the CSCs of HCC and found that sorafenib preferentially targeted CD13(+) CSCs in vitro. Screening cell lines for “population switch” may be useful to identify novel candidate model systems for screening drugs targeting CSCs. Citation Format: Takeshi Yamada, Masato Abei, Inaho Danjoh, Ryoko Shirota, Taro Yamashita, Shinji Endo, Ichinosuke Hyodo, Yukio Nakamura. Identification of a hepatocellular carcinoma cell line capable of monitoring the differentiation of CD13(+)CD166(-) cancer stem cells and effects of sorafenib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1397. doi:10.1158/1538-7445.AM2015-1397

Cancer stem cells in hepatocellular carcinoma: Therapeutic implications based on stem cell biology.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most frequent cause of cancer-related death worldwide. Despite advances in its diagnosis and treatment, the prognosis of patients with advanced HCC remains unfavorable. Recent advances in stem cell biology and associated technologies have enabled the identification of minor components of tumorigenic cells, termed cancer stem cells (CSC) or tumor-initiating cells, in cancers such as HCC. Furthermore, because CSC play a central role in tumor development, metastasis and recurrence, they are considered to be a therapeutic target in cancer treatment. Hepatic CSC have been successfully identified using functional and cell surface markers. The analysis of purified hepatic CSC has revealed the molecular machinery and signaling pathways involved in their maintenance. In addition, epigenetic transcriptional regulation has been shown to be important in the development and maintenance of CSC. Although inhibitors of CSC show promise as CSC-targeting drugs, novel therapeutic approaches for the eradication of CSC are yet to be established. In this review, we describe recent progress in hepatic CSC research and provide a perspective on the available therapeutic approaches based on stem cell biology.

Research progress of Oct-4 in cancer stem cells

Stem cell (SC) is a class of initial cells with potential self renewal and differentiation ability. Under certain conditions, it can proliferate and differentiate into various functional cells. Nucleoprotein Oct-4, Nanog and Sox2 are three major transcription factors, which play central roles in maintaining the pluripotent characteristics and inhibiting SC differentiation. Cancer stem cell(CSC) theory supposed that there is also existing SC-CSC possessing potential self renewal and differentiation abilities in tumor tissues. It is the SC properties of CSC that result in tumor's invasive growth, metastasis and resistance to treatment. Therefore, looking for CSC and determining their markers become an important direction for cancer research. And the introduction of Oct-4, Nanog and Sox2 into CSC research field is undoubtedly the most exciting forefront. Key words: Oct-4; Cancer stem cell

Identification of a unique hepatocellular carcinoma line, Li-7, with CD13(+) cancer stem cells hierarchy and population change upon its differentiation during culture and effects of sorafenib.

BackgroundsCancer stem cell (CSC) research has highlighted the necessity of developing drugs targeting CSCs. We investigated a hepatocellular carcinoma (HCC) cell line that not only has CSC hierarchy but also shows phenotypic changes (population changes) upon differentiation of CSC during culture and can be used for screening drugs targeting CSC.MethodsBased on a hypothesis that the CSC proportion should decrease upon its differentiation into progenitors (population change), we tested HCC cell lines (HuH-7, Li-7, PLC/PRF/5, HLF, HLE) before and after 2 months culture for several markers (CD13, EpCAM, CD133, CD44, CD90, CD24, CD166). Tumorigenicity was tested using nude mice. To evaluate the CSC hierarchy, we investigated reconstructivity, proliferation, ALDH activity, spheroid formation, chemosensitivity and microarray analysis of the cell populations sorted by FACS.ResultsOnly Li-7 cells showed a population change during culture: the proportion of CD13 positive cells decreased, while that of CD166 positive cells increased. The high tumorigenicity of the Li-7 was lost after the population change. CD13(+)/CD166(−) cells showed slow growth and reconstructed the bulk Li-7 populations composed of CD13(+)/CD166(−), CD13(−)/CD166(−) and CD13(−)/CD166(+) fractions, whereas CD13(−)/CD166(+) cells showed rapid growth but could not reproduce any other population. CD13(+)/CD166(−) cells showed high ALDH activity, spheroid forming ability and resistance to 5-fluorouracil. Microarray analysis demonstrated higher expression of stemness-related genes in CD166(−) than CD166(+) fraction. These results indicated a hierarchy in Li-7 cells, in which CD13(+)/CD166(−) and CD13(−)/CD166(+) cells serve as slow growing CSCs and rapid growing progenitors, respectively. Sorafenib selectively targeted the CD166(−) fraction, including CD13(+) CSCs, which exhibited higher mRNA expression for FGF3 and FGF4, candidate biomarkers for sorafenib. 5-fluorouracil followed by sorafenib inhibited the growth of bulk Li-7 cells more effectively than the reverse sequence or either alone.ConclusionsWe identified a unique HCC line, Li-7, which not only shows heterogeneity for a CD13(+) CSC hierarchy, but also undergoes a “population change” upon CSC differentiation. Sorafenib targeted the CSC in vitro, supporting the use of this model for screening drugs targeting the CSC. This type of “heterogeneous, unstable” cell line may prove more useful in the CSC era than conventional “homogeneous, stable” cell lines.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1297-7) contains supplementary material, which is available to authorized users.

Cancer stem cells in basic science and in translational oncology: can we translate into clinical application?

Since their description and identification in leukemias and solid tumors, cancer stem cells (CSC) have been the subject of intensive research in translational oncology. Indeed, recent advances have led to the identification of CSC markers, CSC targets, and the preclinical and clinical evaluation of the CSC-eradicating (curative) potential of various drugs. However, although diverse CSC markers and targets have been identified, several questions remain, such as the origin and evolution of CSC, mechanisms underlying resistance of CSC against various targeted drugs, and the biochemical basis and function of stroma cell-CSC interactions in the so-called ‘stem cell niche.’ Additional aspects that have to be taken into account when considering CSC elimination as primary treatment-goal are the genomic plasticity and extensive subclone formation of CSC. Notably, various cell fractions with different combinations of molecular aberrations and varying proliferative potential may display CSC function in a given neoplasm, and the related molecular complexity of the genome in CSC subsets is considered to contribute essentially to disease evolution and acquired drug resistance. In the current article, we discuss new developments in the field of CSC research and whether these new concepts can be exploited in clinical practice in the future.

Understanding the colon cancer stem cells and perspectives on treatment.

An area of research that has been recently gaining attention is the relationship between cancer stem cell (CSC) biology and chemo-resistance in colon cancer patients. It is well recognized that tumor initiation, growth, invasion and metastasis are promoted by CSCs. An important reason for the widespread interest in the CSC model is that it can comprehensibly explain essential and poorly understood clinical events, such as therapy resistance, minimal residual disease, and tumor recurrence. This review discusses the recent advances in colon cancer stem cell research, the genes responsible for CSC chemoresistance, and new therapies against CSCs.

New insights into pancreatic cancer stem cells.

Pancreatic cancer (PC) has been one of the deadliest of all cancers, with almost uniform lethality despite aggressive treatment. Recently, there have been important advances in the molecular, pathological and biological understanding of pancreatic cancer. Even after the emergence of recent new targeted agents and the use of multiple therapeutic combinations, no treatment option is viable in patients with advanced cancer. Developing novel strategies to target progression of PC is of intense interest. A small population of pancreatic cancer stem cells (CSCs) has been found to be resistant to chemotherapy and radiation therapy. CSCs are believed to be responsible for tumor initiation, progression and metastasis. The CSC research has recently achieved much progress in a variety of solid tumors, including pancreatic cancer to some extent. This leads to focus on understanding the role of pancreatic CSCs. The focus on CSCs may offer new targets for prevention and treatment of this deadly cancer. We review the most salient developments in important areas of pancreatic CSCs. Here, we provide a review of current updates and new insights on the role of CSCs in pancreatic tumor progression with special emphasis on DclK1 and Lgr5, signaling pathways altered by CSCs, and the role of CSCs in prevention and treatment of PC.

Breast cancer stem cells: current advances and clinical implications.

There is substantial evidence that many cancers, including breast cancer, are driven by a population of cells that display stem cell properties. These cells, termed cancer stem cells (CSCs) or tumor initiating cells, not only drive tumor initiation and growth but also mediate tumor metastasis and therapeutic resistance. In this chapter, we summarize current advances in CSC research with a major focus on breast CSCs (BCSCs). We review the prevailing methods to isolate and characterize BCSCs and recent evidence documenting their cellular origins and phenotypic plasticity that enables them to transition between mesenchymal and epithelial-like states. We describe in vitro and clinical evidence that these cells mediate metastasis and treatment resistance in breast cancer, the development of novel strategies to isolate circulating tumor cells (CTCs) that contain CSCs and the use of patient-derived xenograft (PDX) models in preclinical breast cancer research. Lastly, we highlight several signaling pathways that regulate BCSC self-renewal and describe clinical implications of targeting these cells for breast cancer treatment. The development of strategies to effectively target BCSCs has the potential to significantly improve the outcomes for patients with breast cancer.

Pancreatic cancer stem cells: New insight into a stubborn disease

Resistance to conventional therapy and early distant metastasis contribute to the unsatisfactory prognosis of patients with pancreatic cancer. The concept of cancer stem cells (CSCs) brings new insights into cancer biology and therapy. Many studies have confirmed the important role of these stem cells in carcinogenesis and the development of hematopoietic and solid cancers. Recent studies have shown that CSCs regulate aggressive behavior, recurrence, and drug resistance in pancreatic cancer. Here, we review recent advances in pancreatic cancer stem cells (PCSCs) research. Particular attention is paid to the regulation mechanisms of pancreatic cancer stem cell functions, such as stemness-related signaling pathways, microRNAs, the epithelial-mesenchymal transition (EMT), and the tumor microenvironment, and the development of novel PCSCs targeted therapy. We seek to further understand PCSCs and explore potential therapeutic targets for pancreatic cancer.