Cancer stem cells (CSCs) are defined as cells with self-renewal ability, tumor-initiating capacity, and the ability to give rise to more differentiated progeny. Though the frequency of these cells may vary among various kinds of tumors, they often represent a minor subset of tumor cells endowed exclusively with tumor-initiating ability. CSCs in various cancers are mainly identified using cell-surface markers expressed on corresponding normal stem cells. CSCs are frequently enriched in advanced and aggressive tumors, and cells isolated from distant metastases often show a CSC phenotype. In pancreatic cancer, a metastasis specific subset of CSCs (mCSCs) was identified and characterized with a CD133⁺CXCR4⁺ cell phenotype. Depleting these cells from the tumor inhibited tumor metastasis, providing evidence for a CSC role in mediating cancer metastasis. In several cases, residual tumors left after conventional cancer therapy showed enriched CSCs, which were often found to arbitrate resistance response to radiation and chemotherapy. Hence, it is essential to identify and characterize CSCs to develop new cancer therapeutics, which target this population.

Introduction

Methods to Identify and Characterize TICs

Several methods have been proposed to identify, isolate and characterize CSCs. The two main strategies that are employed for the detection and characterization of CSCs include marker-dependent (or marker-based) and marker-independent strategies. Both methods have inherent advantages and disadvantages. Marker-dependent strategies still occupy the center stage in the CSC field, despite marker-independent strategies gaining prominence recently. Owing to the similarities between normal stem cells and CSCs, CSCs have been identified in several cancers using the same sets of cell surface markers that identify normal stem cells. For example, in acute myeloid leukemia (AML), the CD34⁺CD38 marker combination was used to identify AML CSC, and the same combination also identifies normal HSCs.² According to the original definition of CSC, a cell can be classified as a CSC only when it is prospectively isolated and implanted into an immunocompromised mouse to show that the cell can induce serially transplantable tumors. Owing to this limitation, though several techniques such as immunohistochemistry (IHC) can be used to detect CSCs in various tissues but cannot be used to prospectively purify CSC and test their stem cell properties.¹⁶ The two most predominant methods that are used to prospectively purify and characterize CSCs include flow cytometry (FACS) and magnetic activated cell sorting (MACS). FACS has played a critical role in identifying CSCs both by marker-dependent and by marker-independent strategies such as side population and aldefluor assay. In solid tumors, CSCs were initially identified in breast cancer (BCa) using a CD44⁺CD24 marker combination; these CD44⁺CD24 cells were purified from BCa samples using FACS and shown to possess stem cell properties.⁷

Side population (SP), a marker-independent strategy, was originally used to enrich normal HSC and leukemia SC but was later used extensively to enrich CSCs in several solid tumors.^17-19 SP technique is based on the ability of stem cells to exclude Hoechst dye 33342 and appear as a side population compared with the normal differentiated cells, which take up the dye and appear as the main population. The ATP binding cassette (ABC) transporter family proteins such as MDR1 and ABCG2 are responsible for dye exclusion and the side population phenotypic profile. These markers are highly expressed on normal as well as CSCs and usually SP population overlaps with CSCs in several cancers.²⁰ For example, LAPC9, a prostate cancer (PCa) xenograft tumor, was found to have 0.1% SP cells; when these cells were injected into NOD/SCID mice, they were found to be highly tumorigenic (100-1000 times higher) compared with non-SP cells.²⁰ However, in the same study, no detectable SP was found in several other PCa cell lines (PC3, Du145, LAPC4 etc.) analyzed. Both marker-based strategies and SP analyses have played an invaluable role in identifying CSCs and advancing the field of CSCs. However, in a few cases, no differences were observed in tumor-induction capabilities of marker-positive and SP compared with respective marker-negative and non-SP populations²¹ stressing the need for the development of new methods and techniques to identify and characterize CSCs. In contrast to SP, a novel approach based on the ability of stem cells to retain PKH26,²² a lipophilic fluorescent dye which stains quiescent cells, was used for the isolation of human normal mammary stem cells and to further characterize breast CSCs.²³ The aldefluor assay is yet another marker-independent strategy originally employed for the isolation of HSCs, but has recently been used to identify and characterize CSCs in various cancers.²⁴ Stem cells have been identified to show increased aldehyde dehydrogenase (ALDH) activity, which can be measured by the aldefluor assay. ALDH⁺ cells can be identified using the aldefluor assay by measuring the conversion of an ALDH substrate (BODIPY aminoacetaldehyde) to its fluorescent reaction product (BODIPY aminoacetate).

Although techniques such as IHC do not allow the functional characterization of CSCs, they are nonetheless invaluable in detecting the presence of CSCs in large-scale patient sample studies, provided the CSC markers have already been identified for the cancer of interest. Using IHC, Balic et al.,²⁵ for the first time showed that BCa cells which migrated to bone marrow show a CD44⁺CD24 BCa CSC phenotype, and the relative percentage of CSCs observed in bone marrow metastases was quite higher than primary tumors (65% vs 10%). In addition, methods such as clonal and clonogenic assays, which can be performed ex vivo or in vitro, are widely used to identify and characterize CSCs. Previously, our group showed that PC3 cells when plated at clonal density, similar to primary keratinocytes, give rise to holoclones, meroclones and paraclones.²⁶ Holoclones are tightly-packed clones and contain stem and progenitor cells whereas mero/para clones contain more mature and differentiated cells. This study provided the first direct evidence that cells isolated from holoclones not only have the ability to induce tumors but also can form serially transplantable tumors. In contrast, mero/paraclones fail to initiate tumor development. Furthermore, PC3 holoclones showed increased expression of CD44 and α2β1 integrin, two well characterized PCa CSC markers, compared with mero/para clones. Clonogenic/sphere formation assays are based on the ability of stem cells to form spheres under anchorage independent culture conditions. Several groups utilized sphere formation assays to isolate CSCs from CNS tumors,⁸ melanoma,²⁷ and pleural effusions from BCa patients²⁸ and demonstrated that sphere forming cells have the ability to initiate tumor development in immunocompromised mice. CSCs were also identified based on rapid adherence to type 1 collagen,²⁹ differential invasive capacity in transwell cultures³⁰ and their ability to retain long-term BrdU label (label retaining cells; LRC) and other DNA binding dyes such as PKH26, Vibrant Dil, CFSE etc.³¹

TICs and Cancer Metastasis

CSCs have been identified in leukemias^2,6 and in several solid tumors,^7-10 and have been shown to induce tumor development and to be capable of long-term tumor propagation. The role of CSCs in primary tumor development is widely studied and well established. However, studying their role in mediating tumor metastasis is critical, as metastasis is the primary culprit responsible for most of cancer-related patient deaths.³² To develop any meaningful CSC based cancer therapies, it is very important to address the following questions: Are CSCs responsible for cancer metastasis? Are CSCs identified in each tumor type the same as the cells that seed metastasis at a distant site, or do they comprise a subset of such cells? Or perhaps they are completely different from the CSCs that seed primary tumors? The fact that CSCs in BCa were originally identified mainly using metastatic samples provided the preliminary evidence for the existence of CSCs in metastasis.³³ However, a pioneering study by Hermann et al.,³⁴ in pancreatic cancer provided the very first direct evidence for the existence of metastatic CSCs (mCSCs). They showed that CD133 identifies pancreatic CSCs and CD133⁺ cells have the ability to initiate tumor development. However, only a minor subset of these CD133⁺ cells, which expresses both CD133 and CXCR4, is more invasive in vitro and has the ability to metastasize in vivo. It is interesting to note that both CD133⁺CXCR4 and CD133⁺CXCR4⁺ cells were similarly tumorigenic and induce tumor development but only CD133⁺CXCR4⁺ cells showed liver metastasis. The same authors also discovered CD133⁺/ CXCR4⁺ migrating CSCs in ascites punctuates from patients with colon cancer and peritoneal carcinosis.³⁵ Similarly, circulating THY1⁺ CSC-like cell population that might directly contribute to metastasis have been detected in patients with liver cancer, and this population could induce tumors in immunocompromised mice.³⁶

A recent study uncovered an unexpected cellular heterogeneity among colon cancer tumor initiating cells (TICs), which contain three different types of TICs.³⁷ These include self-renewing long-term TICs (LT-TICs), transient amplifying cells (T-TACs) with limited or no self-renewal capacity and delayed contributing TICs (DC-TICs), which were not active initially but activated in secondary or tertiary mice. Among these TICs, only LT-TICs harbored extensive self-renewing capacity, initiated tumor development and maintained serial tumor transplantation ability. Furthermore, these cells almost exclusively induced metastasis at distant sites; this provides additional evidence that metastatic CSCs may represent a minor subset of CSCs.

In BCa patients, CD44⁺CD24 cells can be detected readily in metastatic pleural effusions⁷ and the majority of early disseminated cancer cells detected in the bone marrow of BCa patients showed a CD44⁺CD24 phenotype; their relative percentage went up to 65% in metastasis compared with less than 10% in primary tumor.²⁵ CD44⁺ PCa CSCs also showed a high metastatic propensity compared with CD44 cells.³⁸ In addition, a 186 gene invasiveness gene signature (IGS) generated from CD44⁺CD24 BCa CSCs predicted the tendency of tumors to metastasize, and also predicted metastasis-free and overall survival in breast, medulloblastoma, lung and prostate cancers.³⁹ Furthermore, a 11-gene signature obtained from highly metastatic PCa included stemness genes such as BMI-1, which is implicated in the regulation of CSCs in leukemia as well as solid tumors.⁴⁰ These observations further reinforce the link between cells with CSC phenotype and cancer metastasis.

Since the CSC hypothesis states that only a CSC has the endowed capacity to initiate tumor development, it is fair to hypothesize that only CSCs will have the ability to reinitiate tumors at distant sites in a permissive niche.¹³ Cancer cells either need to recruit components to form a permissive niche or they themselves need to occupy the niches of normal stem cells.¹³ Cancer cells were shown to direct bone marrow derived cells (BMDCs) to future sites of metastasis even before their arrival to form a pre-metastatic niche.⁴¹ The recruitment of BMDCs to distant sites may involve cancer cell-secreted factors such as osteopontin.^13,42 Recently, it has been shown that the incoming cancer cells can directly compete with hematopoietic stem cells (HSC) for HSC niche occupancy.⁴³ PCa cells used in this study, after occupying the HSC niche, could reduce HSC numbers by driving their terminal differentiation. More interestingly, when putative PCa CSCs (CD133⁺CD44⁺) were isolated and used in competitive binding assays, they were better able to block HSC binding to osteoblasts compared with marker negative CD133CD44 cells.⁴³ Moreover, CD133⁺CD44⁺ cells, which represented only a minor fraction of the cultured cells, were significantly enriched in bone marrow 24 hours after intracardiac injection. Similar observations were made in case of the colon cancer LT-TICs discussed earlier,³⁷ which also seem to home to bone marrow and later form metastases in liver. Bone marrow served as a long-term reservoir to these cells, suggesting that metastatic cells may persist in HSCs niches and drive distant metastases.

CSCs may be intrinsically endowed with tumor-initiating and metastasis-forming ability but clearly the surrounding environment plays a critical role in providing a suitable niche to maintain their self-renewal properties, dormancy, and perhaps their tumorigenicity.⁴⁴ CSCs may also take cues from the environment to attain a migratory phenotype. Cancer cells, especially epithelial cells in solid tumors, are observed to undergo epithelial-mesenchymal transition (EMT), a process wherein epithelial cells acquire a mesenchymal phenotype in response to contextual signals they receive from the tumor microenvironment, especially from reactive stroma.¹⁵ Cancer cells undergoing EMT have also been shown to attain a CSC phenotype and acquire invasive and migratory properties.⁴⁵

Tumor Microenvironment, EMT and TICs

The relationship between the tumor microenvironment, cancer progression, and induction of metastasis has been extensively studied. CSCmicroenviromental interactions may also play a critical role in maintaining CSCs and their tumorigenicity.^44,46 Differential induction of metastasis between orthotopic and ectopic tumor implantation models exemplifies the importance of tumor microenvironment in metastasis. In 1990s, Dr. Fidlers group showed that PC-3M PCa cells injected into dorsal prostates (DP; orthotopic site) of nude mice induced robust metastasis but the same cells injected subcutaneously (s.c; ectopic) failed to metastasize despite the primary tumor induction.^47,48 This observation emphasized the importance of tumor microenvironment in inducing cancer metastasis. Similar observations were made when pancreatic cancer cells were injected orthotopically vs ectopically: only the cells injected orthotopically into the pancreas expressed metastatic potential.⁴⁹ Interestingly, CSCs also showed a similar preference for an orthotopic tumor microenvironment.⁵⁰ Higher tumor incidence was observed when stem-like glioblastoma cells were injected intracranially compared with CSCs injected subcutaneously.⁵⁰ In addition, Calabrese et al.,⁵¹ reported that Nestin⁺CD133⁺ brain CSCs and endothelial cells interact closely within a perivascular niche. They further demonstrated that increasing the number of endothelial cells in an orthotopic environment not only increased the initiation and growth of tumors but also lead to the expansion of self-renewing Nestin⁺CD133⁺ stem cells.

Our recent studies also indicate the influence of the tumor microenvironment on CSCs. PCa cells injected into orthotopic dorsal prostate exhibited enrichment of several CSC related genes such as ABCG2, CD44, CD133, and genes associated with metastatic stem cells such as CXCR4 and CD24 compared with ectopic, s.c tumors (ref. 52 and unpublished data). Functional validation using FACS, qRT-PCR, and ShRNA knockdown experiments further confirmed the enrichment of CSCs and their role in cancer metastasis. Conversely, recent studies demonstrated that stem-like cells in glioma and prostate tumors could also modify the microenvironment, thereby promoting angiogenesis and tumor progression.^53-55 Stem-like glioma cells secreted factors such as vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF-1) to promote tumor angiogenesis, and when signaling by either one of the factors was blocked, all CSC-driven angiogenesis was blocked.^53,54 Thus, similar to the symbiosis between bulk cancer cells and the tumor microenvironment, a reciprocal relationship exists between CSCs and tumor microenvironment, one which plays a critical role in tumor initiation, progression, and metastasis. Additionally, a recently uncovered relationship between EMT and CSCs in solid tumors sheds new light on the influence of tumor microenvironment in acquisition of a CSC phenotype.¹²

Using human mammary epithelial cells (HMLEs), Mani et al.,⁴⁵ demonstrated that when EMT is induced in HMLEs by ectopic expression of Snail or Twist, HMLEs acquire CD44⁺CD24 BCa CSC phenotype. In addition, TGFβ (secreted often by reactive stroma) induced EMT in HMLE cells and also resulted in the acquisition of a CSC phenotype.⁴⁵ These cells also showed increased sphere-forming ability in vitro and increased tumorigenicity when implanted in vivo, the functional readouts of CSCs. In response to EMT induction, a similar increase in CD44⁺ CD117⁺ CSCs was observed in ovarian cancers and the acquisition of stemness was found to be critical for the progression of metastasis.⁵⁶ Importantly, the fact that un-manipulated CSCs isolated directly from BCa patient tumors showed simultaneous expression of CSC and EMT markers reinforces the EMT and CSC connection.⁴⁵ In addition, cancer cells at the invasive front of solid tumors have shown features of stem cells as well as EMT⁵⁷ and poorly differentiated and aggressive tumors were enriched for embryonic stem cell gene signatures.⁵⁸ These studies further suggest that EMT and acquisition of a CSC phenotype may go hand-in-hand in cancers of epithelial origin and play an important role in mediating metastasis.

TICs and Therapeutic Implications

CSC-targeted therapy has immense potential to alleviate tumor burden, to inhibit tumor progression, and to overcome therapeutic resistance commonly observed in response to conventional cancer therapeutics. In fact, conventional cancer therapies may enrich for CSCs in residual tumors in several cancers such as pancreatic, colorectal, HCC and lung cancer,⁵⁹ and CD133⁺ glioblastoma CSCs showed resistance to radiation therapy by preferential activation of a DNA damage response.⁶⁰ First, however, a few concerns related to CSC therapy need to be addressed. The conventional cancer therapy uses reduction of the primary tumor burden as the end point to measure the effectiveness of therapy. However, the CSC therapy, which potentially targets a rare population of cancer cells, might not show a significant reduction in the primary tumor burden, at least in the short-term. In addition, CSCs seem to use the same canonical pathways that operate in normal stem cells, and this poses a concern as CSC therapy might target the normal stem cell component which may in turn disrupt cell homeostasis and processes such as repair of damaged tissues. The first obstacle can be overcome by employing end points more relevant to CSCs such as CSC content in particular tumors, disease recurrence, or metastasis formation etc. In-depth understanding of molecular mechanisms that are functional specifically in CSCs will help overcome the harmful effects posed by drugs that target both normal stem cells as well as CSCs. A recent finding that leukemia-initiating cells depend on PTEN function where as normal HSCs do not need PTEN for their normal function raises the hope that CSC-specific drugs may be developed.⁶¹ In fact, a PCa study revealed preferential activation of the PI3K/AKT pathway in sphere-forming CD133⁺/CD44⁺ TICs, and a dual PI3K/mTOR inhibitor NVP-BEZ235 was able to decrease the viability and the sphere-forming ability of CD133⁺/CD44⁺ cells.⁶²

In theory, CSC therapeutics can be developed to target CSC self-renewal, to induce differentiation of CSCs, and to inhibit the ABC family of drug transporters (which are highly expressed in CSCs) that pump drugs out of stem cells.¹³ Several studies have proposed strategies for CSC therapy and a few have even tested them using in vivo models (see ref. 63 for a comprehensive review); these tests have mainly been performed in primary tumor systems; fewer studies have tested their efficacy on metastatic CSCs. Of particular interest is a study performed by Hermann et al.,³⁴ wherein, they showed that depleting CD133⁺CD44⁺ cells by using anti-CXCR4 antibodies or AMD3100, a specific pharmacological inhibitor of CXCR4, completely inhibited pancreatic cancer metastasis. In addition, a recent study demonstrated that CXCR4⁺/CD133⁺ cells, enriched after dacarbazine treatment, exhibited higher metastatic activity compared with CXCR4/CD133⁺ cells in malignant melanoma.⁶⁴ More importantly, blocking CXCR4 along with dacarbazine treatment inhibited both tumor growth and metastasis. As CXCR4 plays a critical role in homing HSCs to the bone marrow microenvironment,⁶⁵ AMD3100, a specific inhibitor of CXCR4, may be used to thwart the attempts of CSCs to mobilize to bone marrow. According to another recent study, PCa cells were even mobilized out of the bone marrow in response to AMD3100 treatment, and when these cells were segregated into putative PCa CSCs (CD133⁺CD44⁺) and non-CSCs (CD133^-CD44^-), the former CSC population showed higher levels of CXCR4 and homed better compared with the latter non-CSC population.⁴³ These studies clearly demonstrate that CSCs can be potentially targeted and may serve as ideal targets to control cancer metastasis.

Conclusion

Metastasis is the leading cause of cancer-related patient deaths, stressing the need to develop therapeutics to control and abrogate cancer metastasis. Ideal targets are needed to make this endeavor successful and CSCs may be those targets against which new-age cancer therapeutics can be developed. In fact, several conventional cancer therapeutics leave in their wake a smaller tumor enriched for CSCs, which eventually mediate disease recurrence and lead to metastasis. However, therapies targeted specifically against CSCs may leave more differentiated cells, which form the bulk of the tumor, untouched, but may destroy the tumors "engine." According to the emerging concept of cancer cell plasticity, however, differentiated tumor cells may be able to dedifferentiate and replenish CSCs, making it difficult to control cancer in this scenario also. Therefore, caution should be maintained while employing CSC-based therapies and conventional therapies in isolation. Instead, combinatorial therapies, which include regimens of both CSC-specific drugs and conventional cancer drugs that target CSCs as well as the bulk of the tumor, may provide a better chance for both remission and cure.

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