Cancer Stem Cells from Solid Tumors: New Tools to Fight Cancer

Marcello Maugeri-Sacc,1 Ann Zeuner,1 and Ruggero De Maria1,2

Background The first experimental evidence supporting the cancer stem cell (CSC) theory came from the identification of a tumorigenic population of human leukemia-initiating cells with stemlike features. Relying on a similar methodological approach combining flow cytometry analysis of primary tumor cells and serial orthotropic transplantation into immunocompromised mice, CSCs have been identified in a variety of solid tumors (1). Among others, CSCs have been isolated from the most prevalent neoplastic diseases including colorectal adenocarcinoma (CRC), non-small cell lung cancer (NSCLC) and breast cancer (BC), as well as from rare but lethal tumors such as glioblastoma multiforme (GBM), neuroblastoma, sarcoma and thyroid carcinoma (24). Recently, the refinement of methodological protocols for cell isolation and expansion, together with the advent of high-throughput array technologies for gene and protein analysis are shedding new light on the functional properties of CSCs. Increasing evidence points to CSCs as responsible not only for tumor generation and auto-perpetuation, but also for tumor recurrence and for the limited efficacy of chemotherapy. While the intrinsic chemoresistance of this cellular fraction is raising questions about the effectiveness of conventional anticancer agents, the functional characterization of the CSC population is revealing innovative pharmacological strategies for cancer targeting. Discussion The therapeutic implications of the CSCs concept have allowed to refine methodological protocols in order to increase the isolation rates and to improve stem cell enrichment for functional characterization. (Fig. 1). The following are the operational criteria defining CSCs: A distinct repertoire of cell surface markers. Ability to grow as spheres in non-adherent cultures (tumor spheres). Ability to recapitulate the parental tumor in immunocompromised mice. The ability to actively exclude the dye HOECHST 33342, relying on the high expression of multidrug resistance (MDR) pumps, has been also adopted to isolate CSCs and define them as side population. Various surface markers have been exploited to sort the CSCs population. CD133 is widely used to isolate CSCs from a variety of solid tumors including CRC (5), NSCLC (6) and GBM (7), whereas the CD44+/CD24low marker panel is adopted for purifying

Authors Affiliations: 1Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanit, Rome, Italy, 2 Mediterranean Institute of Oncology, Catania, Italy 2011 American Association for Cancer Research.

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Fig. 1. Isolation and expansion of CSCs. Tumor specimens are mechanically and enzymatically dissociated and the resulting cancer cells are cultured in a serum-free medium supplemented with epidermal growth factor and broblast growth factor. In this condition, CSCs are expanded and grow as spheres in nonadherent cultures. Subsequently, the tumorigenic ability is evaluated in immunocompromised mice.

BC-initiating cells (8). However, a single marker definition may expose to a misleading interpretation of experimental results. For instance, CD133 GBM cells have been demonstrated to be tumorigenic in animal models to a similar extent as the CD133+ fraction (9). Moreover, both CD44+CD24+ and CD133+CXCR4+ pancreatic cancer cells encompass the operative criteria of CSCs (10, 11). If the identification of reliable markers for purification represents a drawback in CSCs research, combination of two or more markers could allow to overcome this intrinsic limitation. Accordingly, a highly tumorigenic cellular sub-fraction of BC-initiating cell has been isolated by combining the CD44+/CD24low phenotype with aldehyde dehydrogenase 1 positivity (12). Beyond isolation issues, efficient CSCs expansion is essential to obtain a comprehensive molecular profile. In general, tumor specimens are mechanically and enzymatically dissociated and the resulting cancer cells are cultured in a serum-free medium supplemented with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Under these culture conditions, CSCs from several tumors can grow as spheres and expand unlimitedly. More recently, a good efficiency in establishing and propagating glioma CSCs has been achieved

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Fig. 2. Venn diagrams showing the percentage of statistically similar endpoints evaluated by RPPM analysis resulted by the comparison of patient tumors with CTSC-derived xenografts (on the left) and with commercially available CRC cell line-derived (on the right). Abbreviations; RPPM: reverse phase phosphoprotein microarray, colon CTSCs: colon tumor stem cells.

in adherent cultures with a laminin substrate (13). As a result of improved expansion protocols, the amount of CSCs that can be obtained from a single patient is nowadays suitable for functional studies and extensive characterization, which can provide comprehensive information on deregulated genes and altered intracellular pathway nodes. One of the main limitations to optimal preclinical testing of molecular targeted agents is represented by the inadequacy of cell lines in mirroring both the biological behavior and drug sensitivity of human tumors. Conversely, our recent experimental data showed that CSC-derived xenografts reproduce at molecular level the original patient tumor. In fact, reverse phase phosphoprotein microarray (RPPM) analysis of colon CSCs (CCSCs) revealed a greater concordance of considered molecular endpoints between CSCs-derived xenografts and patients tumors than NCI-60-derived tumors (Fig. 2). Thus, the possibility to expand CSCs in vitro and the generation of CSC-derived xenografts could reasonably represent a stable system to investigate the signaling network of a given patient tumor, coupled with the identification of predictive biomarkers of response. Consistent with this, the screening of libraries of pathway-targeted inhibitors on CSCs may facilitate the identification of treatments that can increase the therapeutic efficacy in preclinical models of human cancer and are ready to be tested in clinical trials (14, and our unpublished results). CSCs have been found to be resistant to conventional chemotherapy in vitro and in vivo (15, 16). The use of CSC-based xenografts in immunocompromised mice reproduces the difficult of curing advanced cancer with conventional chemotherapy in patients (17). This is in line with what observed in breast cancer patients treated with neoadjuvant chemotherapy, whose tumors seem to show a relative increased number of CSCs (18). A proficient machinery for DNA damage response and repair is

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one of the main mechanisms involved in the intrinsic chemoresistance of CSCs (19). As shown in glioblastoma tumors exposed to ionizing radiations (20), we find that CSCs exploit the activation of Chk1 and Chk2 to repair DNA and avoid the occurrence of mitotic catastrophe after exposure of chemotherapy. Thus, the use of Chk1 inhibitors may prevent DNA repair and sensitize CSCs to chemotherapeutic drugs. Another possible therapeutic strategy involving CSC targeting could be the use of differentiation-inducing agents. Differentiation therapy achieved a dramatic efficacy in the treatment of acute promyelocytic leukemia, which currently includes the combination of all-trans retinoic acid (ATRA) and anthracyclines (21). The use of ATRA in combination with chemotherapy has been recently proposed for the therapy of NSCLC (22). Recent studies in colon cancer and glioblastoma showed that bone morphogenetic protein 4 (BMP4) can promote CSCs differentiation and considerably increase the anti-tumor efficacy of chemotherapeutic drugs (17, 23). Moreover, embryonic networks have been demonstrated to be reactivated in CSCs and susceptible to efficient pharmacological inhibition. In particular, the Hedgehog and Notch pathways are among the most characterized and influent players which supervise the dynamics within the CSCs pool. For instance, it has been found that Hedgehog inhibition by cyclopamine significantly hampered sphere-forming proficiency of GBM stemlike cells (24). Finally, CSCs have been demonstrated to serve as vascular precursor through the trans-differentiation into endothelial-like cells, thus providing a direct contribution to tumor vasculature (25, 26). Such process is sustained by the Notch pathway and can be constrained by gamma-secretase inhibitors. Future Directions Since different cellular sub-populations seem to share the hallmarks of CSCs in many tumors, a major challenge is represented by the identification of more reliable markers for CSCs purification. To this end, high-throughput genomic and proteomic analysis using microarray technology could allow a better molecular definition of CSCs through, for instance, the evaluation of distribution of embryonic pathway components in the CSCs compartment compared with the remaining tumor cells. Moreover, the exhaustive characterization of deregulated genes and protein networks in CSCs may allow the identification of crucial signal transduction pathways to be targeted by specific drugs or biomolecules. The growing availability of preclinical models based on CSCs may considerably foster the development of more effective therapies, while allowing the identification of biomarker-guided evaluation of targeted agents, thus paving the way for a new generation of clinical trials based on the presence of specific molecular alterations. New insights in CSCs biology have opened new perspectives to test different compounds by taking into account their potential anti-CSC activity. Schematically, the pharmacological strategies that target CSCs could be grouped as follows: Small molecules or monoclonal antibodies inhibiting stemness-associated pathways (i.e. Hedgehog and Notch inhibitors). Chemosensitivity-restoring or enhancing agents such as DNA-repair pathway inhibitors, MDR inhibitors and differentiation-inducing agents. Agents depriving CSCs of the necessary microenvironmental support, or compounds able to avoid the contribution of CSCs in generating a tumor-supportive microenvironment.

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However, it is possible to speculate that many of the compounds mentioned above could be endowed of cytostatic, rather than cytotoxic, effects, and thus active against microscopic residual disease rather than massive metastatic tumors. Thus, it is extremely important not to underestimate long-term outcomes at the expense of rapid parameters of tumor response in proof-of-concept studies. Likewise, the subordination of adjuvant studies to results from the metastatic setting in current clinical trials methodology could obscure potential activity of anti-CSCs treatments. Finally, the adoption of CSC-related endpoints in proof-of principle clinical trials, such as pre- and post-treatment expression of cell surface markers and/or sphere-forming capability, should be considered for optimal clinical development of putative anti-CSCs agents.

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