Single-cell analysis of tumors: Creating new value for molecular biomarker discovery of cancer stem cells and tumor-infiltrating immune cells

Biomarker-driven individualized treatment in oncology has made tremendous progress through technological developments, new therapeutic modalities and a deeper understanding of the molecular biology for tumors, cancer stem cells and tumor-infiltrating immune cells. Recent technical developments have led to the establishment of a variety of cancer-related diagnostic, prognostic and predictive biomarkers. In this regard, different modern OMICs approaches were assessed in order to categorize and classify prognostically different forms of neoplasia. Despite those technical advancements, the extent of molecular heterogeneity at the individual cell level in human tumors remains largely uncharacterized. Each tumor consists of a mixture of heterogeneous cell types. Therefore, it is important to quantify the dynamic cellular variations in order to predict clinical parameters, such as a response to treatment and or potential for disease recurrence. Recently, single-cell based methods have been developed to characterize the heterogeneity in seemingly homogenous cancer cell populations prior to and during treatment. In this review, we highlight the recent advances for single-cell analysis and discuss the challenges and prospects for molecular characterization of cancer cells, cancer stem cells and tumor-infiltrating immune cells.     

Salivary gland stem cells: A review of development,regeneration and cancer

Salivary glands are responsible for maintaining the health of the oral cavity and are routinely damaged by therapeutic radiation for head and neck cancer as well as by autoimmune diseases such as Sjögren's syndrome. Regenerative approaches based on the reactivation of endogenous stem cells or the transplant of exogenous stem cells hold substantial promise in restoring the structure and function of these organs to improve patient quality of life. However, these approaches have been hampered by a lack of knowledge on the identity of salivary stem cell populations and their regulators. In this review we discuss our current knowledge on salivary stem cells and their regulators during organ development, homeostasis and regeneration. As increasing evidence in other systems suggests that progenitor cells may be a source of cancer, we also review whether these same salivary stem cells may also be cancer initiating cells.     

Metabolic flexibility and cell hierarchy in metastatic cancer

Cancer is characterized by disturbed homeostasis of self-renewing cell populations, and their ability to seed and grow in multiple microenvironments. This overarching cellular property of metastatic cancer emerges from the contentious cancer stem cell hypothesis that underpins the more generic hallmarks of cancer (Hanahan and Weinberg, 2000) and its subsequent add-ons. An additional characteristic, metabolic flexibility, is related to concepts developed by Warburg and to subsequent work by mid 20th century biochemists who elucidated the bioenergetic workings of mitochondria. Metabolic flexibility may circumvent limitations inherent in the increasingly popular but erroneous view that aerobic glycolysis is a universal property of cancer cells. Cancer research in the second half of the 20th century was largely the domain of geneticists and molecular biologists using reductionist approaches. Integrated approaches that address cancer cell hierarchy and complexity, and how cancer cells adapt their metabolism according to their changing environment are now beginning to emerge, and these approaches promise to address the poor mortality statistics of metastatic cancer.     

Cell sorting in cancer research—Diminishing degree of cell heterogeneity

Increasing evidence of intratumor heterogeneity and its augmentation due to selective pressure of microenvironment and recent achievements in cancer therapeutics lead to the need to investigate and track the tumor subclonal structure. Cell sorting of heterogeneous subpopulations of tumor and tumor-associated cells has been a long established strategy in cancer research. Advancement in lasers, computer technology and optics has led to a new generation of flow cytometers and cell sorters capable of high-speed processing of single cell suspensions. Over the last several years cell sorting was used in combination with molecular biological methods, imaging and proteomics to characterize primary and metastatic cancer cell populations, minimal residual disease and single tumor cells. It was the principal method for identification and characterization of cancer stem cells. Analysis of single cancer cells may improve early detection of tumors, monitoring of circulating tumor cells, evaluation of intratumor heterogeneity and chemotherapeutic treatments. The aim of this review is to provide an overview of major cell sorting applications and approaches with new prospective developments such as microfluidics and microchip technologies.     

Bladder cancer stem cells

Stem cells are undifferentiated cells that renew themselves while simultaneously producing differentiated tissue- or organspecific cells through asymmetric cell division. The appreciation of the importance of stem cells in normal tissue biology has prompted the idea that cancers may also develop from a progenitor pool (the "cancer stem cell (CSC) hypothesis"), and this idea is gaining increasing acceptance among scientists. CSCs are sub-populations of cancer cells responsible for tumor initiation, differentiation, recurrence, metastasis, and drug resistance. First identified in the hematopoietic system, CSCs have also been discovered in solid tumors of the breast, colon, pancreas, and brain. Recently, the tissue-specific stem cells of the normal urothelium have been proposed to reside in the basal layer, and investigators have isolated phenotypically similar populations of cells from urothelial cancer cell lines and primary tumors. Herein, we review the CSC hypothesis and apply it to explain the development of the two different types of bladder cancer: noninvasive ("superficial") carcinoma and invasive carcinoma. We also examine potential approaches to identify CSCs in bladder cancer as well as therapeutic applications of these findings. While exciting, the verification of the existence of CSCs in bladder cancer raises several new questions. Herein, we identify and answer some of these questions to help readers better understand bladder cancer development and identify reasonable therapeutic strategy for targeting stem cells.     

Cancer cells stemness: A doorstep to targeted therapy

Recent advances in research on cancer have led to understand the pathogenesis of cancer and development of new anticancer drugs. Despite of these advancements, many tumors have been found to recur, undergo metastasis and develop resistance to therapy. Accumulated evidences suggest that small population of cancer cells known as cancer stem cells (CSC) are responsible for reconstitution and propagation of the disease. CSCs possess the ability to self-renew, differentiate and proliferate like normal stem cells. CSCs also appear to have resistance to anti-cancer therapies and subsequent relapse. The underlying stemness properties of the CSCs are reliant on multiple molecular targets such as signaling pathways, cell surface molecules, tumor microenvironment, apoptotic pathways, microRNA, stem cell differentiation, and drug resistance markers. Thus an effective therapeutic strategy relies on targeting CSCs to overcome the possible tumor relapse and chemoresistance. The targeted inhibition of these stem cell biomarkers is one of the promising approaches to eliminate cancer stemness. This review article summarizes possible targets of cancer cell stemness for the complete treatment of cancer.     

c-Myc Is Required for Maintenance of Glioma Cancer Stem Cells

Background

Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells.

Methodology/Principal Findings

Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G₀/G₁ phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice.

Conclusions/Significance

These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.     

An adult tissue-specific stem cell molecular phenotype is activated in epithelial cancer stem cells and correlated to patient outcome

Recent studies have shown that embryonic stem cell-like molecular phenotypes are commonly activated in human epithelial primary tumors and are linked to adverse patient prognosis. However it remains unclear whether these correlations to outcome are linked to the differentiation status of the human primary tumours1 or represent molecular reminiscences of epithelial cancer stem cells. In addition, while it has been demonstrated that leukemic cancer stem cells re-acquire an embryonic stem cell-like phenotype, the molecular basis of stem cell function in epithelial cancer stem cells has not been investigated. Here we show that a normal adult tissue-specific stem cell molecular phenotype is commonly activated in epithelial cancer stem cells and for the first time provide evidence that enrichment in cancer stem cells-specific molecular signatures are correlated to highly aggressive tumor phenotypes in human epithelial cancers.     

Old disease, new culprit: tumor stem cells in cancer

Eloquent studies from hematopoietic systems have provided proof that cancer arises from a tumor stem cell that possesses self-renewing properties. Until recently, it was believed that this tumor stem cell was unique to leukemic disorders; evidence now suggests that solid tumors also harbor cancer stem cells that are capable of initiating tumor growth in immunodeficient animals with as few as 10 cells. Consequently, the term "tumor-initiating cell" is now gaining favor within the field. Here, we conceptually discuss the current theories regarding tumor-initiating cells and their involvement in the development and progression of human malignancies. Special attention is given to laboratory techniques and strategies currently exploited to isolate tumor-initiating cells from larger populations, including their inherent strengths and weaknesses. The biological relevance of a tumor-initiating subpopulation is also pondered and arguments regarding their origin are presented. The therapeutic promise of targeting tumor-initiating cells is certainly eminent and we weigh the advantages of targeting this subpopulation. Lastly, the field of cancer stem cells appears to be well-placed to make significant strides over the next decade and we discuss potential obstacles that must be negotiated to achieve those objectives. The realization of these goals will undoubtedly further our understanding of this complex disease and should eventually lead to improved therapies in the not-so-distant future.     

The biology of human breast epithelial progenitors

Current evidence suggests that similar to other tissues in the human body mammary epithelia cells are being maintained by the unique properties of stem cells, undifferentiated as well as lineage-restricted progenitors. Because of their longevity, proliferation and differentiation potentials these primitive breast epithelial cells are likely targets of transforming mutations that can cause them to act as cancer initiating cells. In this context, understanding the molecular mechanisms that regulate the normal functions of the human breast epithelial stem cells and progenitors and how alterations to these same mechanisms can confer a cancer stem cell phenotype on these rare cell populations is crucial to the development of new and more effective therapies again breast cancer. This review article will examine the current state of knowledge about the isolation and characterization of human breast epithelial progenitors and their relevance to breast cancer research.     

Paving the road for lung stem cell biology: bronchioalveolar stem cells and other putative distal lung stem cells

New discoveries in stem cell biology are making the biology of solid tissues increasingly complex. Important seminal studies demonstrating the presence of damage-resistant cell populations together with new isolation and characterization techniques suggest that stem cells exist in the adult lung. More detailed in vivo molecular and cellular characterization of bronchioalveolar stem cells (BASCs), other putative lung stem and progenitor cells, and differentiated cells is needed to determine the lineage relationships in adult lung. Lung diseases such as cystic fibrosis or chronic obstructive pulmonary disease, as well as the most common form of lung cancer in the United States, all involve apparent bronchiolar and alveolar cell defects. It is likely that the delicate balance of stem, progenitor, and differentiated cell functions in the lung is critically affected in patients with these devastating diseases. Thus the discovery of BASCs and other putative lung stem cells will lay the foundation for new inroads to understanding lung biology related to lung disease.     

A chemical approach to stem cell biology

Small molecule libraries have been used successfully to probe several biological systems. Recent work has translated these successes across to the field of stem cell biology. Stem cells hold promise for both modeling of early development as well as having therapeutic potential. Enhanced understanding of the molecular mechanisms that control stem cell fates as well as an improved ability to manipulate cell populations are required. Known mechanistic chemical compounds have been used with stem cells to accomplish these two goals. More recently, through the utilization of high fitness libraries in phenotype-based screens, several small molecules that control self-renewal and differentiation in stem cells have been identified. These small molecules provide useful chemical tools for both basic research and practical applications.     

Identification of a Population of Epidermal Squamous Cell Carcinoma Cells with Enhanced Potential for Tumor Formation

Epidermal squamous cell carcinoma is among the most common cancers in humans. These tumors are comprised of phenotypically diverse populations of cells that display varying potential for proliferation and differentiation. An important goal is identifying cells from this population that drive tumor formation. To enrich for tumor-forming cells, cancer cells were grown as spheroids in non-attached conditions. We show that spheroid-selected cells form faster growing and larger tumors in immune-compromised mice as compared to non-selected cells. Moreover, spheroid-selected cells gave rise to tumors following injection of as few as one hundred cells, suggesting these cells have enhanced tumor-forming potential. Cells isolated from spheroid-selected tumors retain an enhanced ability to grow as spheroids when grown in non-attached culture conditions. Thus, these tumor-forming cells retain their phenotype following in vivo passage as tumors. Detailed analysis reveals that spheroid-selected cultures are highly enriched for expression of epidermal stem cell and embryonic stem cell markers, including aldehyde dehydrogenase 1, keratin 15, CD200, keratin 19, Oct4, Bmi-1, Ezh2 and trimethylated histone H3. These studies indicate that a subpopulation of cells that possess stem cell-like properties and express stem cell markers can be derived from human epidermal cancer cells and that these cells display enhanced ability to drive tumor formation.     

In situ stem cell therapy: novel targets, familiar challenges

Tissue engineering approaches for expanding, differentiating and engrafting embryonic or adult stem cells have significant potential for tissue repair but harnessing endogenous stem cell populations offers numerous advantages over these approaches. There has been rapid basic biological progress in the identification of stem cell niches throughout the body and the molecular factors that regulate their function. These niches represent novel therapeutic targets and efforts to use them involve the familiar challenges of delivering molecular medicines in vivo. Here we review recent progress in the use of genes, proteins and small molecules for in situ stem cell control and manipulation, with a focus on using stem cells of the central nervous system for neuroregeneration.     

A Possible Explanation for the Variable Frequencies of Cancer Stem Cells in Tumors

A controversy surrounds the frequency of cancer stem cells (CSCs) in solid tumors. Initial studies indicated that these cells had a frequency ranging from to of the total cells. Recent studies have shown that this does not always seem to be the case. Some of these studies have indicated a frequency of . In this paper we propose a stochastic model that is able to capture this potential variability in the frequency of CSCs among the various type of tumors. Considerations regarding the heterogeneity of the tumor cells and its consequences are included. Possible effects on conventional treatments in clinical practice are also described. The model results suggest that traditional attempts to combat cancer cells with rapid cycling can be very stimulating for the cancer stem cell populations.     

Mechanisms that mediate stem cell self-renewal and differentiation

Stem cells have two common properties: the capacity for self-renewal and the potential to differentiate into one or more specialized cell types. In general, stem cells can be divided into two broad categories: adult (somatic) stem cells and embryonic stem cells. Recent evidence suggested that tumors may contain "cancer stem cells" with indefinite potential for self-renewal. In this review, we will focus on the molecular mechanisms regulating embryonic stem cell self-renewal and differentiation, and discuss how these mechanisms may be relevant in cancer cells.     

Platelet cancer cell interplay as a new therapeutic target

The interaction between circulating tumor cells and platelets is a key factor in cancer metastasis. These interactions, driven by a variety of receptors, support circulating tumor cells by protecting them from immune detection, cushioning them from shear stress, and promoting their arrest at the endothelium. Additionally, platelets have been shown to accumulate in the primary tumors, promoting tumor growth and angiogenesis by releasing growth factors. Furthermore, tumor cells can interact with platelets by inducing aggregation, which further protects cancer cells. However, the platelet cancer cell interplay also offers new approaches to develop targeted therapies. The accumulation of platelets in tumors has successfully been leveraged to deliver chemotherapeutics and imaging agents. Likewise, these platelet-based interactions have been utilized to target cancer cells in circulation. Although these current systems have limitations including drug loading and storage, leveraging platelet-cancer cell interactions to effectively target circulating tumor cells and tumors shows great promise for future cancer treatments.