Identification of cancer stem cells provides novel tumor models for drug discovery

Abstract

Cancer stem cells (CSCs) have received considerable attention from the research community since they were first reported in human acute myeloid leukemia 15 years ago. Accumulating evidence suggests that CSCs are responsible for tumor initiation and progression, drug resistance, and metastasis in both liquid and solid tumors. These findings lead to the development of novel compounds targeting CSC populations that is becoming increasingly important for eradicating CSCs in heterogeneous tumor masses and to cure the cancer. Since 2003, we have participated in CSC studies and encountered crucial early events in the field. This article reviews the history of CSC biology, clarifies the term and its definition, and further addresses the issue of how to utilize CSCs in therapeutic target discovery and drug development based on our substantial experience.

Keywords

cancer stem cell / tumor model / drug discovery

Introduction

The discovery of many cancers as organized hierarchies sustained by CSCs at their apex has generated much interest from the cancer research community in recent years. Two models have been developed to explain tumor development, namely, the stochastic model and the hierarchy model. The stochastic model proposes that tumors are biologically homogeneous. Theoretically, regardless of their distinct phenotypes, tumor cells are tumorigenic, i.e., they are capable of regenerating tumors in xenotransplantation systems and, presumably, in humans as well. The hierarchy model proposes that tumors are organized similar to normal tissues, which are maintained by self-renewing stem cells [1]. In this model, only CSCs are able to self-renew to generate identical daughter cells, thereby sustaining tumor growth and progression. In addition to CSCs, most tumor cells are considered differentiated cell populations that become either less tumorigenic or non-tumorigenic. As with normal tissues, differentiated cancer cells eventually lose their capability to proliferate and have to be replenished by new cells derived from CSCs. The concept of CSCs further explains tumor heterogeneity, a key feature of cancer.

Modern history of CSC biology

Many malignancies, both solid and hematological, exhibit significant heterogeneity in their tumor tissues. Tumor cells vary widely in their biological phenotypes, including tumorigenic potentials in xenotransplantation systems, cell surface markers, as well as sensitivity to chemotherapy and radiation therapy, among others. Such observations led to the search for tumor-initiating cell populations, which may be solely responsible for tumor initiation and development.

The historical development of this field goes back several decades. Mouse embryonic stem cells were successfully isolated in vitro in the 1980s, but CSCs in experimental animal systems were not identified until approximately a decade later (Fig. 1). The discovery of a CD34⁺/CD38^- tumor-initiating cell population in human leukemia was the first experimental evidence in CSC research [2]. Dick et al. demonstrated that only a small fraction of leukemia cells are able to propagate xenografted leukemia in irradiated severe combined immunodeficient (SCID) mice, indicating the existence of CSCs distinguishable from other tumor cell populations. Human embryonic stem cells were successfully derived in vitro in 1998 [3]. Research advances in stem cell biology have prompted researchers to continue the search for similar cell populations in malignant tumor tissues using stem cell assays. In 2001, after conducting their research in the laboratory of stem cell biologist Irving Weissman, Al-Hajj et al. identified CD44⁺/CD24^-/low tumor cells as a tumor-initiating cell population in human breast tumors. They published a landmark review article in Nature [1], and their experimental results were later published in PNAS [4]. Within the same year, Singh et al. identified a CD133⁺ CSC in brain tumors [5]. They further confirmed the characteristics of the brain CSC by in vivo limiting dilution assay in 2004 [6]. In 2005, our research team reported a CD20-expressing tumor cell population with stem cell properties in human melanomas [7]. In 2006, a CD44⁺ prostate CSC [8], a CD44⁺ head and neck CSC [9], and a CD133⁺ liver CSC [10] were reported. The year 2007 was another fruitful period for CSC research. Li et al. identified a CD44⁺/CD24⁺ CSC in human pancreatic cancers [11]. Hermann et al. reported a metastatic CD133⁺/CXCR4⁺ CSC in human pancreatic cancers as well [12]. O’Brien et al. [13] and Ricci-Vitiani et al. [14] independently identified CD133⁺ CSCs in human colorectal cancers. Dalerba et al. reported a distinct CD44⁺/CD166⁺ CSC in human colorectal cancers [15]. Monzani et al. showed that CD133⁺/ABCG2⁺ cells represent CSCs in human melanomas [16]. Finally, Eramo et al. discovered a CD133⁺ CSC in lung cancers [17]. In the following year (2008), a CD44⁺/CD117⁺ CSC in ovarian cancers [18], an ABCB5⁺ CSC in human melanomas [19], and a CD90⁺/CD44⁺ CSC in liver cancers [20] were identified. In 2009, CD133⁺ CSCs were reported for Ewing’s sarcoma [21] and ovarian cancers [22]. Afterwards, increasing evidence for the existence of CSCs in various tumor types has been continually reported. The present article focuses on earlier publications on the subject with the support of in vivo tumorigenic data.

Definition of CSCs

As the evidence for CSCs continually emerged, in the winter of 2006, a group of pioneer researchers in the field gathered at a workshop sponsored by the American Association for Cancer Research in Lansdowne, Virginia, to discuss the future directions of CSC research [23]. The participants all recognized an immediate need to provide guidance for CSC research. After reviewing the existing published results through presentations and/or discussion, the majority of the participants agreed to use the term cancer stem cells (CSCs) instead of tumor-initiating cells to describe the cell population. Globally, CSCs better reflect the properties of the cell population, including tumor-initiating potentials, drug resistance, and capabilities to differentiate and self-renew. More specifically, drug resistance, differentiation, and self-renewal are the properties shared by both normal and malignant stem cells (Table 1). Thereafter, the term CSCs has received broad acceptance.

At the workshop, CSCs were defined as “those cells within a tumor that possess the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor. Cancer stem cells can thus only be defined experimentally by their ability to recapitulate the generation of a continuously growing tumor” [23]. In addition to their unique tumorigenic potentials, CSCs are capable of (1) self-renewal for generating identical daughter cells to sustain tumor growth and (2) differentiation for repopulating heterogeneous tumor cell populations in a tumor. Furthermore, resistance to conventional therapies is also well accepted as a property of CSCs [23]. Glioblastoma CSCs are resistant to chemotherapeutic agents [24]. Colon CSCs gain their resistance to apoptosis via an interleukin-4-mediated mechanism [25]. CSCs in hepatocellular carcinomas confer chemoresistance by preferential expression of the Akt/PKB survival pathway [26]. Breast CSCs [27] and glioma stem cells [28] have been shown to be less sensitive to radiation. Furthermore, analysis of patient-derived xenograft tumors of colorectal cancer revealed that CSCs are enriched after chemotherapy, indicating a resistant feature of the cells [29]. CSCs have also been correlated with epithelial-to-mesenchymal transition [30] and cancer metastasis [31,32].

Identification of CSCs: cell surface markers and in vitro prorogation under stem cell conditions

In general, CSCs can be prospectively identified and isolated by two approaches: cell sorting based on putative CSC markers (i.e., CD133, CD44, and EpCAM), side population, and expression of aldehyde dehydrogenase 1 (ALDH-1) and spheroid cultures under stem cell conditions. Representative earlier studies describing the identification of CSCs in various tumor types using both methodologies are summarized in Fig. 2.

CSCs expressing ALDH-1 or unique CSC markers may be separated from the rest of the tumor cell population (Fig. 2). Putative CSCs may be identified by their ability to effectively efflux certain dyes. The so-called side population, which shows reduced fluorescence, can be identified as CSCs by labeling tumor cells with Hoechst 33342 and measuring blue and red emissions from the dye (Fig. 2). Further in vivo tumorigenesis studies are performed to demonstrate the unique or enhanced tumorigenic potentials of the sorted subpopulation. Cells may be sorted by fluorescence-activated cell sorting (FACS) or bead-based cell sorting technologies. FACS normally results in relatively pure cell populations but requires expensive cell sorters and experienced operators, whereas bead-based cell sorting is more convenient and flexible. However, the purity of the resulting population under the latter system may not be ideal and will still require validation by flow cytometry. In our experience, FACS may yield cell populations with purity greater than 90% and bead technology often results in 50%-80% purified cells.

Theoretically, only one CSC is required to generate the secondary tumor in xenotransplantation systems. However, due to the complexity of xenotransplantation systems, consistently generating tumors by implanting only one CSC in a recipient mouse continues to be a challenge. Species differences between implanted tumor cells (human) and the host (murine) play an important role in the formation of xenograft tumors. Another important factor contributing to the successful transplantation of human cancer cells is the specific strain of mice used in the study. Use of nude mice is economical, but they only display a significantly reduced number of T lymphocytes and the rest of the immune system remains intact. SCID mice present with an impaired ability to make T or B lymphocytes or activate some components of the complement system and cannot efficiently reject tumors and transplants, resulting in a higher tumor uptake rate. NOD/SCID (or NSG) mice lack mature T cells, B cells, and natural killer cells and have thus been extensively used to identify tumorigenic CSCs in limiting dilution assay. NOG (NOD/Shi-scid/IL-2Rγnull) is a new-generation severely immunodeficient strain. NOG mice have just been used recently to study CSCs. Therefore, the minimal number of CSCs required for generating xenograft tumors in mice largely depends on the viability of the cells and the strains of mice used. Implantation routes (i.e., tumor microenvironment) may also contribute to the discrepancy in tumorigenic potentials of tumor cells. Subcutaneous implantation is convenient; however, the resulting ectopic tumor microenvironment, which differs from that of the original tumor, certainly limits the growth of non-cutaneous tumors. Renal capsule implantation better facilitates tumor growth and improves tumor uptake, but the renal capsule is still a foreign microenvironment for tumor types other than kidney cancer. In general, the surgical technologies for orthotopic implantation of various tumors need to improve.

The culture conditions designated for propagation of normal stem cells have been successfully adopted to cultivate malignant stem cells (Fig. 2). The specific culture conditions for neural stem cells seem to be one of the first to be discovered [33,34]. The specificities of these conditions include growth factors (i.e., EGF, bFGF) and depletion of serum, which facilitate the proliferation of stem cells and prevent them from differentiation. In 2003, two independent laboratories applied the culture conditions for neural stem cells to cultivate brain CSCs [5,35]. Similar serum-free culture conditions have been used to derive CSCs from cancers of the breast [27,36], bone [37], ovaries [18,38], lung [17], prostate [39], colon [14,25], and pancreas [40].

Due to our unique experience with human embryonic stem cells, we developed a serum-free culture medium based on a recipe suitable for human embryonic stem cells to isolate malignant stem cell populations from human melanomas [7]. The culture medium is first conditioned by mouse embryonic fibroblasts and thus enriched in the growth factors and cytokines secreted by these cells. Such a conditioned medium was subsequently proven able to support the prorogation of normal human hair follicle stem cells [41] and colorectal CSCs [42].

Under serum-free conditions, interestingly, CSCs frequently form non-adherent, three-dimensional spheroids. Therefore, these cultures are often referred to as tumor spheroid cells, which are notably not CSCs yet, rendering further characterization of their CSC properties necessary (e.g., tumorigenic potentials in vivo, differentiation and self-renewal capabilities, drug resistance, relatively quiescent state, etc.). The key to prorogating CSCs in vitro is making an expandable cell population available for studies over a long period. Several unique characteristics of in vitro cultured CSCs have been observed: First, tumor spheroid cultures are propagated in three-dimensional structures, which better represent in vivo tumors in comparison with traditional monolayer cultures. Second, serum-free conditions attempt to maintain the undifferentiated state of tumor cells. For drug discovery, particularly, cultured CSCs provide a consistent cell source for the discovery of additional targets for therapeutic development. For example, in one of our studies on cultured CD133⁺ colorectal CSCs, additional cell surface markers were successfully identified for future development as therapeutic targets [42]. In addition, the availability of cultured CSCs makes it possible to extensively test therapeutic compounds targeting CSCs in vitro [43]. Furthermore, cultured CSCs can be used to generate in vivo xenograft tumor models for antitumor efficacy studies of a drug that passed in vitro studies [44].

Tumor spheroids formed by CSCs under serum-free conditions are not homogenous cell populations. Various fractions of spheroid cells retain the expression of CSC markers, whereas the rest of the cell population is negative for these markers. Therefore, tumor spheroids should be considered CSC-enriched cell populations, not pure CSC ones. Accordingly, tumor spheroid CSCs are a dynamic cell population consisting of primitive CSCs and differentiated cancer cells at various stages.

Despite the controversy as to whether bona fide CSCs may be maintained in vitro, the existing evidence on the propagation of CSC-like cells under serum-free conditions in vitro considerably improves our understanding of cancer biology. More importantly, it significantly enhances our capabilities in anticancer drug development because we do not have to be limited by traditional cancer lines. The latter types barely mimic patient tumors in clinical practice most likely because they mainly represent differentiated cancer cells. Second, CSCs cultured in stem cell conditions seem to more closely mirror the phenotype and genotype of primary tumors compared with serum-cultured cell lines [45,46]. Another advantage of cultured CSCs is that they are capable of metastasis. In our laboratory, after subcutaneous implantation in SCID mice, cultured CD133⁺ colorectal CSCs caused metastases to multiple distal tissues and organs, including lung and liver (unpublished data). These metastatic CSCs are very useful tools with which we could develop therapeutic agents targeting tumor metastasis.

Identification and isolation of CSCs from both clinical specimens and traditional cancer cell lines

As discussed above, existing evidence suggests that CSCs may be identified from both clinical patient samples and established cancer cell lines (Fig. 2). Fresh tumor tissues obtained from clinical samples or patient-derived xenograft models undoubtedly provide the most reliable resource for CSC isolation. Discrepancy primarily lies with whether CSCs exist in cancer cell lines or not. The presence of CSCs in the C6 glioma cell line was first documented in 2004 [47]. Specific cell surface markers differentiate CSCs from the rest of the tumor cell population in some colorectal cancer lines [48]. Growth of cancer cells of glioblastoma, mammary carcinoma, and melanoma under stem cell-like conditions preferentially propagates CSCs [49]. A distinct side population of cancer cells has also been found in various types of cancer cell lines [50,51]. ALDH-expressing cells reportedly represent CSC populations in established breast cancer cells [52]. A CSC population most likely persists in some cancer lines, but careful evaluation of the CSC characteristics of the isolated population is required prior to drawing a conclusion.

In addition to enhanced tumorigenic potentials, self-renewal and differentiation capabilities, and resistance to conventional therapies, recapitulation of the histopathology of the original patient tumor should be considered as an important criterion of CSCs, including heterogeneity of the repopulated cells and unique features of the tumor. Unfortunately, lack of information on the original tumors from which the traditional cancer cell lines are derived limits evaluation of such correlation between putative CSCs and the histopathology of their original tumors. Thus, deriving CSC lines from fresh tumor samples is essential to building novel tumor models for cancer research and drug discovery.

CSC niche and tumor microenvironment

Similar to normal stem cells, malignant stem cells are maintained in a specialized microenvironment called niche. The niche plays an important role in the regulation of CSC self-renewal and fate. It is composed of stromal cells, endothelial cells, an extracellular matrix, and secreted growth factors and cytokines (Fig. 3). CSCs have been found to recruit bone marrow-derived mesenchymal stem cells (MSCs) from the circulation into the local tumor microenvironment (i.e., niche) through cytokine-mediated signals [53,54]. Within a tumor, MSCs may further differentiate into tumor-associated fibroblasts [55-57] and endothelial cells [58], which become the key components of a CSC niche. Enhancement of tumor growth and stimulation of metastasis by MSCs have been well documented [59,60]. In addition, the presence of MSCs and/or tumor-associated fibroblasts enhances the resistance of cancer cells to therapeutic agents [61].

Modeling a niche microenvironment represents a novel avenue for the development of anti-cancer drugs. Developing in vivo xenograft mouse models with a humanized microenvironment may take long; however, it is possible to mimic a niche microenvironment in vitro by co-culturing CSCs, MSCs, tumor-associated fibroblasts, macrophages, and other cell types. A recent article highlighted the importance of tumor stroma and proposed a tumor stroma platform for the identification of novel anticancer agents [62]. With available cultured colorectal CSCs, we have tested the antitumor activity of a dual PI3K/mTOR inhibitor in a co-culture system consisting of PI3CA mutant CSCs and MSCs [44]. Incorporating a niche and a tumor microenvironment into in vitro tumor models holds promise for better mimicking clinical tumors. To this end, co-culture systems of CSCs and stromal cells can be established as a novel model for drug testing (Fig. 4). Further developing such in vivo tumor models and applying them to drug testing will surely assist in our efforts in oncology drug discovery.

More importantly, identifying the CSC niche and tumor microenvironment provides a crucial target for new therapies. For example, targeting fibroblast activation protein preferentially expressed in tumor-associated fibroblasts by immunotherapy showed antitumor activities in multiple xenograft models in vivo [63]. A selective hypoxia-inducible factor-1 inhibitor increased therapeutic efficacy through blockade of hypoxia-inducible factor-1-dependent reconstitution of tumor stroma function [64]. Depletion of tumor stromal tissue by inhibition of the Hedgehog cellular signaling pathway improved the delivery of chemotherapy in a mouse model of pancreatic cancer [65].

Origin of CSCs and controversy in cell surface markers for CSCs

Transformation of a differentiated cell may generate a CSC as the cell regains the key features of CSCs. Increasing evidence indicates that a CSC may originate from stem and precursor cells. For example, spontaneous transformation of a human adult stem cell was observed in culture. Intestinal stem cells expressing prominin-1 (CD133) are reportedly susceptible to neoplastic transformation [66]. Overexpression of the nuclear receptor tailless was found to induce long-term neural stem cell expansion and glioma initiation in mice [67]. Basal cell carcinoma has been shown to originate from the long-term resident progenitor cells of the interfollicular epidermis and the upper infundibulum in a genetically engineered mouse system [68]. Another research group demonstrated that aberrant luminal progenitors are candidate target cells for basal breast tumor development in BRCA1 mutant mice [69]. Therefore, both stem cells and progenitor cells may trigger cancer after transformation.

Distinct cell surface markers have been applied to isolate CSCs in certain types of cancer. For example, CD133 and CD44 have been successfully used to identify CSCs in colorectal cancers [13-15]. The reasons accounting for such distinction remain unclear. However, one possibility is that the cellular origin of an individual patient may differ from that of another, resulting in distinct cell surface markers inherited from their seeding cells. Another possibility is that CSCs are a dynamic population whose phenotypes may shift. Further investigation is necessary to better understand CSCs.

CSCs for oncology drug discovery

Targeting CSCs represents a promising new approach to develop anticancer drugs. CSCs are solely responsible for generating tumors in experimental animals. The resistance of CSCs to chemotherapy and radiation therapy further highlights the importance of eradicating them. Therefore, treatments designed to kill CSCs may be highly effective.

Cultured CSCs under serum-free conditions provide novel in vitro tumor models for anticancer drug testing. Compared with monolayer cultures of traditional cancer cell lines, their advantages have been extensively discussed in the literature. Practically, tumor spheroid cells enriched with CSCs can be dissociated into single cells and plated into 96-well plates for drug testing (Fig. 5). After treatment with candidate compounds, cell viabilities and apoptosis are further assessed. Among several cell proliferation detection systems tested in our laboratory, we found the CellTiter-Glo® Luminescent Cell Viability Assay kit from Invitrogen to be superior to MTT and alamar blue assays. As CSCs are cultivated under serum-free conditions, which facilitates their quiescent state, an extended treatment is often required to show inhibition of cell proliferation. Theoretically, reformation of tumor spheroids from single CSCs enables testing of the self-renewal capability of CSCs. However, we found that cell-cell aggregation, which is observed using a real-time imaging system, frequently occurs and thus contributes to the reformation of tumor spheroids. Further development of self-renewal assays using spheroid CSCs is clearly required.

When using CSCs as the cell source to initiate xenograft tumor models, it is important to recognize that they will start to differentiate immediately after implantation. The resulting tumors contain a fraction of CSCs at levels consistent with those of the original tumors. For example, if a CSC fraction constitutes 5% of the tumor cell population in a patient sample, the xenograft tumor derived from the CSC population would contain approximately 5% of CSCs as well. Both CSC-enriched tumor spheroid cells and FACS-sorted CSCs may be used to initiate xenograft tumors in mice for in vivo efficacy studies. However, the tumors generated are not enriched with CSCs due to spontaneous differentiation. In addition to traditional tumor growth inhibition assay through the measurement of changes in tumor sizes, novel and relevant CSC assays may be conducted to evaluate the specific effects of a compound on CSC populations after treatment. First, xenograft tumors can be resected for the detection of CSC populations by flow cytometry (Fig. 6). Changes in CSC frequency reflect the responses of CSCs to treatment with the compound. Conventional chemotherapeutic compounds have been well demonstrated to often result in increased CSC frequencies post-treatment, which is due to preferential killing of the differentiated cancer cell populations [29]. Similar results have been reported in clinical practice. For example, ALDH-expressing breast CSCs increased in patient specimens after sequential treatment with paclitaxel and epirubicin [70]. Second, the capability of the remaining CSCs after treatment can be assessed by reimplantation of tumor fragments in secondary animals. Briefly, tumor fragments of similar sizes obtained from each individual tumor are implanted into naive mice. Tumor incidence and tumor volumes are compared with those in vehicle and standard-of-care compound-treated groups. If the novel compound possesses inhibitory effects on the proliferation and/or viability of CSCs, lower tumor incidence rates and smaller tumor volumes may be observed in the group. This assay actually aims to test the self-renewal capability of CSCs that survive treatment with the tested compound.

Future directions in CSC research

The identification of CSCs in various tumor types provides targeted cellular populations for the development of more effective anticancer therapies. Specific markers for CSCs remain controversial, and one of the explanations may be that cell origins vary among patients: a patient’s primitive normal stem cells that transformed into cancer cells may differ from another patient’s progenitor cells. Therefore, functional assays, including in vivo limiting dilution, drug resistance, and self-renewal assays, play a more important role in the identification and validation of CSC populations. Along with better understanding of CSC populations, drugs that target CSCs more specifically may be discovered. It is very encouraging that many candidate compounds targeting the Wnt, Notch, and HH pathways are currently being analyzed. These pathways, including others, are often highly activated in CSCs and facilitate the self-renewal of CSCs. Theoretically, combined treatment with a compound targeting CSC-related pathways and a standard-of-care drug may achieve synergistic effects.

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