U
Urologia 2015 ; 82 ( 1 ): 46-53 DOI: 10.5301/uro.5000099
ORIGINAL ARTICLE
ISSN 0391-5603
Isolation and characterization of cancer stem cells in renal cell carcinoma Giuseppe Lucarelli, Vanessa Galleggiante, Monica Rutigliano, Antonio Vavallo, Pasquale Ditonno, Michele Battaglia Department of Emergency and Organ Transplantation - Urology, Andrology and Kidney Transplantation Unit, University of Bari, Bari - Italy
Abstract Recently, several studies have investigated the presence of cancer stem cells in kidney cancer, performed characterization, and compared their profile with the normal stem cell counterparts. CD133, alone or in combination with other molecular markers, has been used to isolate normal and cancer stem cells from different sources, including renal carcinoma; however, it is still a matter of debate whether CD133+ cells really represent the main tumorigenic population within the heterogeneous pool of cancer cells that characterize this tumor. In this review, we summarize and discuss the current findings related to cancer stem cells isolation in renal cell carcinoma, focusing on controversies about their origin and the identification of a specific marker. Keywords: CD133, CTR2, Kidney injury, Renal cell carcinoma, Stem cells
Introduction The heterogeneity of tumors has long been recognized. According to the stochastic or clonal evolution model of cancer, the intrinsic differences between tumor cells are driven by ongoing genetic changes that can be positively or negatively selected. On the basis of this model, it is presumed that many cells with different phenotypes within a tumor cell population are capable of proliferating extensively and forming a neoplastic mass. An alternative view initially emerged in the 1960s and 1970s and was revived in the 1990s, thanks to work on hematologic malignancies and breast cancer (1-3). In particular, different experiments cast some doubt on the concept that all the cancer cells in a neoplastic tissue were biologically equivalent and therefore equally able to be the source of a progeny of cancer cells. According to this new model, a tumor can be understood in much the same terms as normal epithelial tissue, in which a relatively small population of self-renewing stem cells is able to generate a large number of descendant cancer cells with phenotypically different characteristics and with a limited proliferative potential in vivo. Consistently with this view, the term cancer stem cells (CSCs) identifies a subpopulation of cancer cells that possess the following characteristics: a clonogenic ability, the expression of stem cell markers, differentiation into cells of different Accepted: October 29, 2014 Published online: November 22, 2014 Corresponding Author: Giuseppe Lucarelli, MD, PhD University of Bari piazza G. Cesare, 11 70124 Bari, Italy [email protected]
lineage, growth in the form of nonadhesive spheroids, and the ability in vivo to generate serially transplantable tumors that reflect the heterogeneity of the primary cancer (Fig. 1). In this review, we summarize and discuss the current findings related to CSCs isolation in renal cell carcinoma (RCC), focusing on controversies about their origin and the identification of a specific marker.
Renal adult stem cells and their role in kidney injury repair Many studies have explored the molecular events associated with the development of tubular atrophy and of interstitial fibrosis resulting as a consequence of an acute or chronic renal injury. For example, in the case of chronic unilateral ureteral obstruction, a tubular and interstitial injury can be observed, which results from the activation of different pathways, including an increase in monocyte chemotactic protein-1 (MCP-1) expression and a decrease in epidermal growth factor (EGF) expression by tubular cells. The progression of tubulointerstitial injury leads to chronic renal damage, characterized by tubular cells apoptosis, inflammatory cell infiltration, and interstitial fibrosis (4, 5). In this scenario, the regeneration of injured portions of the tubules may occur through mechanisms that rely on the intrinsic ability of resident progenitors or dedifferentiated resident cells to proliferate and replace the damaged tissue. Approaches based on stem/progenitor cell administration or pharmacological modulation can be viewed as a promising option that may foster this intrinsic renal regeneration. The presence of resident cells with the capability to differentiate into nephron cells and to contribute to renal repair has been shown in several murine studies (6-10). Although these cells were identified with different markers and were localized in different parts of the nephron, they all shared the ability to resist apoptotic damage and to proliferate during renal injury. When injected in vivo, these adult renal stem/progenitor cells © 2014 Wichtig Publishing
Lucarelli et al
Fig. 1 - According to cancer stem cell (CSC) model, tumors can be understood in much the same terms as a normal tissue, in which a relatively small population of self-renewing stem cells are able to generate a large number of descendant cancer cells with phenotypically different characteristics. CSCs are thought to be transformed from normal stem cells, progenitors cells, and/or differentiated cells by genetic/epigenetic mutations.
(ARPCs) contributed to injury repair by integrating into the tubular cells and interstitial space, and when transplanted into the metanephric kidney, they integrated into the epithelial components of the nephron (6-11). To date, two apparently distinct populations of multipotent ARPCs have been isolated from the human kidney: the first by Bussolati et al (12), from the tubule/interstitium, and the second by Sagrinati et al (13), from Bowman’s capsule. A genomic characterization of these CD133+/CD24+ ARPCs from glomeruli and tubules showed nonsignificant differences in their gene expression profile, suggesting that tubules and glomerula-derived ARPCs represent a genetically homogeneous population (14). These multipotent CD133+/CD24+ cells show characteristics typical of adult stem cells, such as clonogenicity and the expression of stem cell markers, and exhibit a multipotent differentiation ability by generating tubular epithelium-like, osteogenic-like, adipocytelike, and neuron-like cells in vitro and in vivo. They are scattered throughout the proximal tubule and show a specific gene expression (15). They also have a distinct morphology characterized by less cytoplasm, fewer mitochondria, the lack of baso-lateral invaginations, and a mature brush border, which is consistent with a less differentiated proximal tubule phenotype (16). Importantly, these cells proliferate after kidney injury in patients with acute or chronic tubular damage and account for the majority of the regenerated tubules (12, 16, 17). Moreover, Angelotti et al have recently shown that a subpopulation of CD133+/CD24+ renal progenitors lacking the expression of vascular adhesion molecule 1 (CD106) have a strong propensity to differentiate toward tubular cells in vitro (18). In vivo CD133+/CD24+/CD106- cells were specifically localized in the proximal tubule and distinct areas of the distal convoluted tubule, but were never found in the collecting duct. The exact nature of CD133+ progenitors is uncertain: they could represent a pre-existing population able to survive injury or else a dedifferentiated population that acquires progenitor characteristics after damage. Moreover, the dif© 2014 Wichtig Publishing
47
ferent populations of CD133+ reported in different nephron segments exhibit different properties, and possibly different functions. Cortical CD133+ cells in proximal tubules have been reported to proliferate in response to renal damage (16, 17). In fact, the number of CD133+ cells was increased in the tubules of transplanted patients undergoing delayed graft function as a result of acute renal injury, as well as in tubules of patients with proteinuric glomerular diseases (17). When injected into mice with glycerol-induced acute renal injury, these CD133+/ CD24+ ARPCs contributed to tubular regeneration, suggesting the feasibility of cell therapy with human isolated CD133+ renal progenitors. In damaged tissues, the release of particular factors called “damage-associated molecular pattern molecules” has been shown, which activate Toll-like receptors (TLRs) and trigger downstream activation of transcription factors that regulate the expression of survival genes or proinflammatory cytokines (19, 20). Sallustio et al have shown that TLR2 is expressed in ARPCs, functioning as a damage sensor in these cells. In fact, ARPCs increase the secretion of MCP-1, interleukin (IL)-6, and IL-8 in response to TLR2 stimulation, as well as their proliferation rate, plausibly via autocrine signaling (14). Moreover, tubular ARPCs protect RPTECs (primary renal proximal tubule epithelial cells) from cisplatin-induced toxicity by preventing apoptosis and enhancing the proliferation of surviving cells, and it has been shown that these repair processes are mediated by the secretion of inhibin-A protein, and microvesicle-shuttled decorin, inhibin-A, and cyclin D1 mRNAs (21). Taken together, these findings suggest that tubular ARPCs could work as directors of the renal regenerative process, controlling the three main kidney repair pathways: the proliferation and differentiation of resident ARPCs, the dedifferentiation and subsequent proliferation of surviving tubular cells, and the recall of bone marrow-derived stem cells.
Isolation and characterization of renal cancer stem cells Renal cell carcinoma accounts for approximately 3% of all adult malignancies. It is estimated that in 2014, 63,920 new cases will be diagnosed and 13,860 patients will die of RCC in the United States (22). RCC is increasingly diagnosed at an early stage in many countries, but despite these advances, up to 30% of patients have metastatic disease at the diagnosis, and around 20-30% of subjects undergoing surgery will suffer recurrence. In recent years, there has been a growing interest in tumor markers in different urologic tumors, not only for diagnostic purposes but also to improve the predictive power of clinical and pathological parameters (23-25). In this context, an in-depth understanding of RCC biology has led to the discovery of several circulating biomarkers associated with different features of RCC biology, including carbonic anhydrase IX (CAIX), hypoxia-inducible factor-1α (HIF1α), CA15-3, and C-reactive protein (CRP) (26-29). Moreover, drug development in RCC continues to improve as a better understanding of RCC biology emerged, providing further insight into the complex signaling networks as potential therapeutic targets. Recently, several studies have investigated the presence of CSCs in kidney cancer, performed characterization, and compared their profile with the normal stem cell counterparts.
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Prominin-1 (CD133), alone or in combination with other molecular markers, has been used to isolate normal stem cells and CSCs from different sources, including bone marrow, brain, prostate, and kidney (30). CD133+ cells have stemness characteristics such as self-renewal, a differentiation ability, and high proliferation rate, and are able to generate tumors in murine xenograft models. However, it is still a matter of debate whether CD133+ cells really represent the main tumorigenic population within the heterogeneous pool of cancer cells that characterize a tumor. In this scenario, CD133 has been used as a marker for the identification of CSCs in RCC. In 2006, Bruno et al identified a population of CD133+/CD34cells in tissue specimens derived from patients with RCC (31). The lack of CD34 expression indicated that these cells were different from endothelial progenitor cells derived from circulating elements. These findings were also confirmed by the lack of expression of other hematopoietic markers such as CD45, CD14, and KDR/VEGFR2. Moreover, these renal CD133+/CD34- cells expressed the renal embryonic marker Pax-2 and the mesenchymal stem cell markers CD44, CD29, and CD73 but unlike mesenchymal stem cells, they were CD105-. Taken together, these findings suggested that these renal CD133+/CD34- cells did not derive from hematopoietic CD133+/CD34+ stem cells and that they were quite different from bone marrow derived mesenchymal stem cells. So, what is the role of these CD133+/CD34- progenitor cells in RCC? We now know that tumors assemble vasculature by means of different strategies. Cancer cells themselves, as well as myofibroblasts in the tumor-associated stroma, can release chemotactic factors that help to recruit circulating endothelial precursor cells into the neoplastic stroma. This recruitment is sustained by the release of Vascular endothelial growth factor (VEGF), which induces their endothelial differentiation and incorporation into tumor vessels. It has also been suggested that bone marrow derived cells may differentiate into vessel pericytes rather than endothelial cells, and contribute to tumor vascularization by releasing growth factor. Another mechanism that seems to operate in tumor angiogenesis is the engagement of tissue-resident normal stem cells. In this scenario, Bruno et al showed that CD133+/CD34resident progenitors cells were able to differentiate into endothelial cells in the presence of RCC-derived growth factors and to promote RCC development and angiogenesis. In fact, in vivo experiments demonstrated that CD133+/CD34- derived endothelial cells were able to form functional vessels connected with the mouse vascular system. Moreover, cotransplantation of these cells with K1 tumor cells enhanced tumor engraftment and growth. However, when CD133+/ CD34- cells were injected subcutaneously in immunodeficient mice, they were not able to form tumors (31). These findings indicated that the CD133+/CD34- cells were not a tumor-initiating cells population and therefore they could not be identified as RCC CSCs. It has been suggested that CD133+/CD34- cells isolated in RCC could represent a normal renal stem cell population that coexists with the more differentiated renal cancer cells and can support tumor growth and vascularization. On the basis of the recent identification of stem cells with mesenchymal characteristics within adult renal tissue, Bussolati et al investigated whether a mesenchymal stem cell
Stem cells in renal cell carcinoma
population was present in RCC and whether this population could have CSC-like features (32). In particular, by means of magnetic cell sorting, these authors identified a subpopulation of cells in RCC expressing the mesenchymal marker CD105. Their phenotypic characterization revealed that these CD105+ cells also expressed a group of markers that are characteristic of mesenchymal stem cells (CD29, CD73, CD44, CD90, CD146, and vimentin). Moreover, CD105+ cells expressed the embryonic stem cell markers (Nanog, Oct 4, Musashi, Nestin, and the embryonic renal marker Pax2) and were negative for the epithelial marker pan-CK, and for the CD133 and CD24 antigens. These cell populations had a clonogenic ability and when plated in sphere-generating medium, were able to grow in nonadhesive conditions and to generate spheres. They were able to differentiate in vitro on both endothelial and epithelial cells but had no ability to generate adipogenic or osteogenic cells. In addition, when CD 105+ cell clones were injected subcutaneously in immunocompromised SCID (Severe combined immunodeficiency) mice, they were able to generate serially transplantable tumors in vivo (32). Taken together, these data provide further evidence that RCC-derived CD105+ cells represent a tumor-initiating cell population that may originate from resident renal stem cells with mesenchymal characteristics but not from CD133+ cells. However, their origin from mutated neoplastic stromal/ mesenchymal cells or from bone marrow derived stem cells cannot be excluded. Interestingly, the treatment of these CD105+ CSCs with IL-15 was able to induce their stable differentiation into a nontumorigenic epithelial cell population that was more sensitive to cytotoxic agents than the parental CSCs (33). These findings show that it is possible to direct the epithelial differentiation of renal CSCs, increase their sensitivity to chemotherapy, and so suggest that the administration of IL15 could be a possible cytokine-based strategy to target CSCs. Another approach to identifying stem cell like populations is cytofluorimetric evaluation of Hoechst 33342 dye uptake. This assay, developed by Goodell et al in 1996, allows the identification of a cell population called a side population (SP) that has the ability to extrude the vital DNA dye, Hoechst 33342, thanks to specific membrane transporters (the ATP-binding cassette transporters family members) characteristically associated with stemness (34). Many studies have shown that CSCs of some solid tumors are present in SP cells. Using this technique, Addla et al isolated and characterized SP and non-SP populations from normal and malignant (RCC) human kidney tissue (35). Phenotypic characterization revealed that the SP cells isolated using the Hoechst 33342 dye efflux model were enriched by cells with stem-like characteristics. In fact, the RCC-SP cells showed an increased proliferative potential, clonogenicity, and a sphere-forming ability in 3D cultures. In addition, these cells expressed the mesenchymal SC markers CD44 and CD29 and the embryonal renal marker PAX-2, but were CD133 negative. On the basis of these findings, the authors suggested that the loss of CD133 could be a very early event in SC differentiation and possibly in malignant transformation (35). Most importantly, this study revealed the presence of different subpopulations of SP cells, although their role in RCC pathogenesis is still unknown, nor has any specific surface marker yet been identified. © 2014 Wichtig Publishing
Lucarelli et al
Using the same approach, Huang et al identified the SP cells from an established human RCC cell line (769P cells) and showed that these cells had CSC-like properties (self-renewal and multi-lineage differentiation), including the ability to form tumors in NOD/SCID mice after their serial transplantation (36). Moreover, in this study, it was shown that the 769P SP phenotype was determined by the expression of ABCB1 transporter, a surface protein that probably contributes to the chemo and radioresistance properties of this cell population (36). Recently, Ueda et al used two different approaches to identify CSCs in RCC cell lines: the traditional Hoechst 33342 dye efflux assay and the enzymatic approach based on the evaluation of aldehyde dehydrogenase 1 (ALDH1) activity (37). In particular, these authors identified SP cells with CSC-like properties in the ACHN renal cancer cell line. Interestingly, these ACHN SP cells were CD105 negative and these findings, unlike the results by Bussolati et al (32), suggest that CD105 may not be a universal marker for renal CSCs. Moreover, analysis of the ALDH1 activity showed that ACHN SP cells expressed a higher level of activity than non-SP cells. In addition, analysis of ALDH1 positive cells in the ACHN population revealed that these cells had a higher tumorigenic potential than ALDH1-negative cells. These findings indicate that ALDH1+ cells, rather than SP cells, exhibit renal CSC properties in the ACHN renal cancer cell line (37). The sphere culture system is a useful alternative method for isolating CSCs from many human cancers and cell lines. Many studies have shown that CSCs can be enriched in the spheres when cultured in serum-free medium supplemented with EGF and basic fibroblast growth factor (bFGF) (38-40). Using this functional assay, from SK-RC-42 RCC cell lines, Zhong et al selected a sphere-forming population of cells with stemness characteristics such as a self-renewal ability, tumorigenicity, and resistance to chemotherapy and radiotherapy (41). However, because the expression of CD44, CD24, CD105, and CD133 was not associated with these sphere-forming cells, the authors concluded that these stem cell markers could not be used to isolate SK-RC-42-derived CSCs. Finally, Gassenmaier et al demonstrated that CXC chemokine receptor 4 (CXCR4) identified a subpopulation of tumorinitiating cells in the RCC-53 renal cancer cell line, and that CXCR4+ cells formed fivefold more tumor spheres and grew significantly faster than CXCR4- cells when transplanted in NOD/SCID mice (42). Interestingly, CXCR4+ cells were more resistant to the tyrosine kinase inhibitors sunitinib, sorafenib, and pazopanib, and, unlike the findings by Bussolati et al, they were CD105 negative.
Identification of CD133+/CD24+/CTR2+ cells as a cancer cell population with stem cell like features in RCC CD133 (Prominin-1) has been one of the most frequently used biomarkers in CSC-related research, and the first studies reported the isolation of CD133+ CSCc from brain tumors (38). Since then, the isolation of putative CSCs from a wide range of tumors by the use of anti-CD133 antibodies has been reported. However, studies of the utility of CD133 as a CSC biomarker are still ongoing because there have been several reports that CD133− cells are also able to give rise to tumors in immunodeficient mice (43-49). As stated above, © 2014 Wichtig Publishing
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in human RCC, Bussolati et al (32) isolated a tumor-initiating cell population that was CD105+ but CD133-/CD24-. Similar findings were reported by Addla et al in SP cells isolated using the Hoechst 33342 dye efflux assay (35). However, the use of CD105 as a renal CSC marker was questioned in later studies that showed how other putative subpopulations of cells with CSC-like properties were CD105- (37, 42). Moreover, Canis et al showed that stable transfection of CD133 in the human embryonic kidney 293 (HEK293) cell line induced tumor-initiating properties in these cells. In addition, HEK293 CD133high transfectants, when injected into SCID mice, generated tumors with at least a 1000-fold increased frequency as compared with CD133low cells (50). In this scenario, our group recently described and characterized a population of resident CD133+/CD24+ cancer cells (termed RDCs: RCC-derived cells) in patients with clear cell RCC (51). These RDCs showed the same clonogenic multipotency and self-renewal ability as their normal counterparts (i.e., tubular ARPCs), and differed from bone marrow derived mesenchymal cells because they did not express the typical membrane markers (CD90-, CD105-). Moreover, RDCs were able to differentiate into epithelial cells, osteocytes, or adipocytes in the presence of specific growth factors. The stemness of this population was confirmed by the expression of several stemness proteins (such as Nanog, Sox2, GATA4, and FoxA2) as compared with CD133- cancer cells and tubular ARPCs, and the tumorigenic ability of these RDCs was evaluated using the in vitro soft agar assay. In fact, only RDCs were able to generate colonies associated with malignant transformation. It has been shown that the microenvironment can affect CD133 expression in cancer cells; in this context, an enrichment of CD133+ CSCs has been described in lung, pancreatic, and brain tumors in low oxygen concentration conditions and in association with HIF-1α upregulation (52-55). Analysis of different areas of primary gliomas has revealed that CD133+ cells were mainly localized in the inner core of the neoplastic mass (56). Similarly, we found the presence of RDCs in the central region of tumor samples, wherein conditions were more hypoxic (unpublished data). These findings support the hypothesis that the presence of a hypoxic niche could affect the survival and persistence of these cell population. As angiogenesis is one of the most important hallmarks of RCC, the RDCs-associated angiogenic response was evaluated using the in vivo chick embryo chorioallantoic membrane (CAM) technique (57). This assay demonstrated that RDCs were able to trigger an angiogenic response, as shown by numerous allantoic vessels that developed radially toward the RDC implants in a “spoked-wheel” pattern. Finally, using DNA microarray analysis, we studied the gene expression profile of these RDCs, with the aim of identifying a membrane marker that could discriminate this neoplastic population from other cell types, namely tubular ARPCs, mesenchymal stem cells, and renal proximal tubule epithelial cells (RPTECs). Statistical analysis identified 72 genes that discriminated RDCs from tubular ARPCs, and among these, CTR2 (SLC31A2) was the membrane protein-coding gene showing the highest fold change (Fig. 2). To validate these findings, we evaluated CTR2 protein expression in the RDCs isolated from a different set of RCC patients by flow cytometry analysis and immunofluorescence (Fig. 3). The in situ colocalization study of
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Stem cells in renal cell carcinoma
Fig. 2 - (A) Profile plots showing 149 modulated genes that discriminate RDCs from tubular ARPCs with a false discovery rate (FDR)
Urologia 2015 ; 82 ( 1 ): 46-53 DOI: 10.5301/uro.5000099
ORIGINAL ARTICLE
ISSN 0391-5603
Isolation and characterization of cancer stem cells in renal cell carcinoma Giuseppe Lucarelli, Vanessa Galleggiante, Monica Rutigliano, Antonio Vavallo, Pasquale Ditonno, Michele Battaglia Department of Emergency and Organ Transplantation - Urology, Andrology and Kidney Transplantation Unit, University of Bari, Bari - Italy
Abstract Recently, several studies have investigated the presence of cancer stem cells in kidney cancer, performed characterization, and compared their profile with the normal stem cell counterparts. CD133, alone or in combination with other molecular markers, has been used to isolate normal and cancer stem cells from different sources, including renal carcinoma; however, it is still a matter of debate whether CD133+ cells really represent the main tumorigenic population within the heterogeneous pool of cancer cells that characterize this tumor. In this review, we summarize and discuss the current findings related to cancer stem cells isolation in renal cell carcinoma, focusing on controversies about their origin and the identification of a specific marker. Keywords: CD133, CTR2, Kidney injury, Renal cell carcinoma, Stem cells
Introduction The heterogeneity of tumors has long been recognized. According to the stochastic or clonal evolution model of cancer, the intrinsic differences between tumor cells are driven by ongoing genetic changes that can be positively or negatively selected. On the basis of this model, it is presumed that many cells with different phenotypes within a tumor cell population are capable of proliferating extensively and forming a neoplastic mass. An alternative view initially emerged in the 1960s and 1970s and was revived in the 1990s, thanks to work on hematologic malignancies and breast cancer (1-3). In particular, different experiments cast some doubt on the concept that all the cancer cells in a neoplastic tissue were biologically equivalent and therefore equally able to be the source of a progeny of cancer cells. According to this new model, a tumor can be understood in much the same terms as normal epithelial tissue, in which a relatively small population of self-renewing stem cells is able to generate a large number of descendant cancer cells with phenotypically different characteristics and with a limited proliferative potential in vivo. Consistently with this view, the term cancer stem cells (CSCs) identifies a subpopulation of cancer cells that possess the following characteristics: a clonogenic ability, the expression of stem cell markers, differentiation into cells of different Accepted: October 29, 2014 Published online: November 22, 2014 Corresponding Author: Giuseppe Lucarelli, MD, PhD University of Bari piazza G. Cesare, 11 70124 Bari, Italy [email protected]
lineage, growth in the form of nonadhesive spheroids, and the ability in vivo to generate serially transplantable tumors that reflect the heterogeneity of the primary cancer (Fig. 1). In this review, we summarize and discuss the current findings related to CSCs isolation in renal cell carcinoma (RCC), focusing on controversies about their origin and the identification of a specific marker.
Renal adult stem cells and their role in kidney injury repair Many studies have explored the molecular events associated with the development of tubular atrophy and of interstitial fibrosis resulting as a consequence of an acute or chronic renal injury. For example, in the case of chronic unilateral ureteral obstruction, a tubular and interstitial injury can be observed, which results from the activation of different pathways, including an increase in monocyte chemotactic protein-1 (MCP-1) expression and a decrease in epidermal growth factor (EGF) expression by tubular cells. The progression of tubulointerstitial injury leads to chronic renal damage, characterized by tubular cells apoptosis, inflammatory cell infiltration, and interstitial fibrosis (4, 5). In this scenario, the regeneration of injured portions of the tubules may occur through mechanisms that rely on the intrinsic ability of resident progenitors or dedifferentiated resident cells to proliferate and replace the damaged tissue. Approaches based on stem/progenitor cell administration or pharmacological modulation can be viewed as a promising option that may foster this intrinsic renal regeneration. The presence of resident cells with the capability to differentiate into nephron cells and to contribute to renal repair has been shown in several murine studies (6-10). Although these cells were identified with different markers and were localized in different parts of the nephron, they all shared the ability to resist apoptotic damage and to proliferate during renal injury. When injected in vivo, these adult renal stem/progenitor cells © 2014 Wichtig Publishing
Lucarelli et al
Fig. 1 - According to cancer stem cell (CSC) model, tumors can be understood in much the same terms as a normal tissue, in which a relatively small population of self-renewing stem cells are able to generate a large number of descendant cancer cells with phenotypically different characteristics. CSCs are thought to be transformed from normal stem cells, progenitors cells, and/or differentiated cells by genetic/epigenetic mutations.
(ARPCs) contributed to injury repair by integrating into the tubular cells and interstitial space, and when transplanted into the metanephric kidney, they integrated into the epithelial components of the nephron (6-11). To date, two apparently distinct populations of multipotent ARPCs have been isolated from the human kidney: the first by Bussolati et al (12), from the tubule/interstitium, and the second by Sagrinati et al (13), from Bowman’s capsule. A genomic characterization of these CD133+/CD24+ ARPCs from glomeruli and tubules showed nonsignificant differences in their gene expression profile, suggesting that tubules and glomerula-derived ARPCs represent a genetically homogeneous population (14). These multipotent CD133+/CD24+ cells show characteristics typical of adult stem cells, such as clonogenicity and the expression of stem cell markers, and exhibit a multipotent differentiation ability by generating tubular epithelium-like, osteogenic-like, adipocytelike, and neuron-like cells in vitro and in vivo. They are scattered throughout the proximal tubule and show a specific gene expression (15). They also have a distinct morphology characterized by less cytoplasm, fewer mitochondria, the lack of baso-lateral invaginations, and a mature brush border, which is consistent with a less differentiated proximal tubule phenotype (16). Importantly, these cells proliferate after kidney injury in patients with acute or chronic tubular damage and account for the majority of the regenerated tubules (12, 16, 17). Moreover, Angelotti et al have recently shown that a subpopulation of CD133+/CD24+ renal progenitors lacking the expression of vascular adhesion molecule 1 (CD106) have a strong propensity to differentiate toward tubular cells in vitro (18). In vivo CD133+/CD24+/CD106- cells were specifically localized in the proximal tubule and distinct areas of the distal convoluted tubule, but were never found in the collecting duct. The exact nature of CD133+ progenitors is uncertain: they could represent a pre-existing population able to survive injury or else a dedifferentiated population that acquires progenitor characteristics after damage. Moreover, the dif© 2014 Wichtig Publishing
47
ferent populations of CD133+ reported in different nephron segments exhibit different properties, and possibly different functions. Cortical CD133+ cells in proximal tubules have been reported to proliferate in response to renal damage (16, 17). In fact, the number of CD133+ cells was increased in the tubules of transplanted patients undergoing delayed graft function as a result of acute renal injury, as well as in tubules of patients with proteinuric glomerular diseases (17). When injected into mice with glycerol-induced acute renal injury, these CD133+/ CD24+ ARPCs contributed to tubular regeneration, suggesting the feasibility of cell therapy with human isolated CD133+ renal progenitors. In damaged tissues, the release of particular factors called “damage-associated molecular pattern molecules” has been shown, which activate Toll-like receptors (TLRs) and trigger downstream activation of transcription factors that regulate the expression of survival genes or proinflammatory cytokines (19, 20). Sallustio et al have shown that TLR2 is expressed in ARPCs, functioning as a damage sensor in these cells. In fact, ARPCs increase the secretion of MCP-1, interleukin (IL)-6, and IL-8 in response to TLR2 stimulation, as well as their proliferation rate, plausibly via autocrine signaling (14). Moreover, tubular ARPCs protect RPTECs (primary renal proximal tubule epithelial cells) from cisplatin-induced toxicity by preventing apoptosis and enhancing the proliferation of surviving cells, and it has been shown that these repair processes are mediated by the secretion of inhibin-A protein, and microvesicle-shuttled decorin, inhibin-A, and cyclin D1 mRNAs (21). Taken together, these findings suggest that tubular ARPCs could work as directors of the renal regenerative process, controlling the three main kidney repair pathways: the proliferation and differentiation of resident ARPCs, the dedifferentiation and subsequent proliferation of surviving tubular cells, and the recall of bone marrow-derived stem cells.
Isolation and characterization of renal cancer stem cells Renal cell carcinoma accounts for approximately 3% of all adult malignancies. It is estimated that in 2014, 63,920 new cases will be diagnosed and 13,860 patients will die of RCC in the United States (22). RCC is increasingly diagnosed at an early stage in many countries, but despite these advances, up to 30% of patients have metastatic disease at the diagnosis, and around 20-30% of subjects undergoing surgery will suffer recurrence. In recent years, there has been a growing interest in tumor markers in different urologic tumors, not only for diagnostic purposes but also to improve the predictive power of clinical and pathological parameters (23-25). In this context, an in-depth understanding of RCC biology has led to the discovery of several circulating biomarkers associated with different features of RCC biology, including carbonic anhydrase IX (CAIX), hypoxia-inducible factor-1α (HIF1α), CA15-3, and C-reactive protein (CRP) (26-29). Moreover, drug development in RCC continues to improve as a better understanding of RCC biology emerged, providing further insight into the complex signaling networks as potential therapeutic targets. Recently, several studies have investigated the presence of CSCs in kidney cancer, performed characterization, and compared their profile with the normal stem cell counterparts.
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Prominin-1 (CD133), alone or in combination with other molecular markers, has been used to isolate normal stem cells and CSCs from different sources, including bone marrow, brain, prostate, and kidney (30). CD133+ cells have stemness characteristics such as self-renewal, a differentiation ability, and high proliferation rate, and are able to generate tumors in murine xenograft models. However, it is still a matter of debate whether CD133+ cells really represent the main tumorigenic population within the heterogeneous pool of cancer cells that characterize a tumor. In this scenario, CD133 has been used as a marker for the identification of CSCs in RCC. In 2006, Bruno et al identified a population of CD133+/CD34cells in tissue specimens derived from patients with RCC (31). The lack of CD34 expression indicated that these cells were different from endothelial progenitor cells derived from circulating elements. These findings were also confirmed by the lack of expression of other hematopoietic markers such as CD45, CD14, and KDR/VEGFR2. Moreover, these renal CD133+/CD34- cells expressed the renal embryonic marker Pax-2 and the mesenchymal stem cell markers CD44, CD29, and CD73 but unlike mesenchymal stem cells, they were CD105-. Taken together, these findings suggested that these renal CD133+/CD34- cells did not derive from hematopoietic CD133+/CD34+ stem cells and that they were quite different from bone marrow derived mesenchymal stem cells. So, what is the role of these CD133+/CD34- progenitor cells in RCC? We now know that tumors assemble vasculature by means of different strategies. Cancer cells themselves, as well as myofibroblasts in the tumor-associated stroma, can release chemotactic factors that help to recruit circulating endothelial precursor cells into the neoplastic stroma. This recruitment is sustained by the release of Vascular endothelial growth factor (VEGF), which induces their endothelial differentiation and incorporation into tumor vessels. It has also been suggested that bone marrow derived cells may differentiate into vessel pericytes rather than endothelial cells, and contribute to tumor vascularization by releasing growth factor. Another mechanism that seems to operate in tumor angiogenesis is the engagement of tissue-resident normal stem cells. In this scenario, Bruno et al showed that CD133+/CD34resident progenitors cells were able to differentiate into endothelial cells in the presence of RCC-derived growth factors and to promote RCC development and angiogenesis. In fact, in vivo experiments demonstrated that CD133+/CD34- derived endothelial cells were able to form functional vessels connected with the mouse vascular system. Moreover, cotransplantation of these cells with K1 tumor cells enhanced tumor engraftment and growth. However, when CD133+/ CD34- cells were injected subcutaneously in immunodeficient mice, they were not able to form tumors (31). These findings indicated that the CD133+/CD34- cells were not a tumor-initiating cells population and therefore they could not be identified as RCC CSCs. It has been suggested that CD133+/CD34- cells isolated in RCC could represent a normal renal stem cell population that coexists with the more differentiated renal cancer cells and can support tumor growth and vascularization. On the basis of the recent identification of stem cells with mesenchymal characteristics within adult renal tissue, Bussolati et al investigated whether a mesenchymal stem cell
Stem cells in renal cell carcinoma
population was present in RCC and whether this population could have CSC-like features (32). In particular, by means of magnetic cell sorting, these authors identified a subpopulation of cells in RCC expressing the mesenchymal marker CD105. Their phenotypic characterization revealed that these CD105+ cells also expressed a group of markers that are characteristic of mesenchymal stem cells (CD29, CD73, CD44, CD90, CD146, and vimentin). Moreover, CD105+ cells expressed the embryonic stem cell markers (Nanog, Oct 4, Musashi, Nestin, and the embryonic renal marker Pax2) and were negative for the epithelial marker pan-CK, and for the CD133 and CD24 antigens. These cell populations had a clonogenic ability and when plated in sphere-generating medium, were able to grow in nonadhesive conditions and to generate spheres. They were able to differentiate in vitro on both endothelial and epithelial cells but had no ability to generate adipogenic or osteogenic cells. In addition, when CD 105+ cell clones were injected subcutaneously in immunocompromised SCID (Severe combined immunodeficiency) mice, they were able to generate serially transplantable tumors in vivo (32). Taken together, these data provide further evidence that RCC-derived CD105+ cells represent a tumor-initiating cell population that may originate from resident renal stem cells with mesenchymal characteristics but not from CD133+ cells. However, their origin from mutated neoplastic stromal/ mesenchymal cells or from bone marrow derived stem cells cannot be excluded. Interestingly, the treatment of these CD105+ CSCs with IL-15 was able to induce their stable differentiation into a nontumorigenic epithelial cell population that was more sensitive to cytotoxic agents than the parental CSCs (33). These findings show that it is possible to direct the epithelial differentiation of renal CSCs, increase their sensitivity to chemotherapy, and so suggest that the administration of IL15 could be a possible cytokine-based strategy to target CSCs. Another approach to identifying stem cell like populations is cytofluorimetric evaluation of Hoechst 33342 dye uptake. This assay, developed by Goodell et al in 1996, allows the identification of a cell population called a side population (SP) that has the ability to extrude the vital DNA dye, Hoechst 33342, thanks to specific membrane transporters (the ATP-binding cassette transporters family members) characteristically associated with stemness (34). Many studies have shown that CSCs of some solid tumors are present in SP cells. Using this technique, Addla et al isolated and characterized SP and non-SP populations from normal and malignant (RCC) human kidney tissue (35). Phenotypic characterization revealed that the SP cells isolated using the Hoechst 33342 dye efflux model were enriched by cells with stem-like characteristics. In fact, the RCC-SP cells showed an increased proliferative potential, clonogenicity, and a sphere-forming ability in 3D cultures. In addition, these cells expressed the mesenchymal SC markers CD44 and CD29 and the embryonal renal marker PAX-2, but were CD133 negative. On the basis of these findings, the authors suggested that the loss of CD133 could be a very early event in SC differentiation and possibly in malignant transformation (35). Most importantly, this study revealed the presence of different subpopulations of SP cells, although their role in RCC pathogenesis is still unknown, nor has any specific surface marker yet been identified. © 2014 Wichtig Publishing
Lucarelli et al
Using the same approach, Huang et al identified the SP cells from an established human RCC cell line (769P cells) and showed that these cells had CSC-like properties (self-renewal and multi-lineage differentiation), including the ability to form tumors in NOD/SCID mice after their serial transplantation (36). Moreover, in this study, it was shown that the 769P SP phenotype was determined by the expression of ABCB1 transporter, a surface protein that probably contributes to the chemo and radioresistance properties of this cell population (36). Recently, Ueda et al used two different approaches to identify CSCs in RCC cell lines: the traditional Hoechst 33342 dye efflux assay and the enzymatic approach based on the evaluation of aldehyde dehydrogenase 1 (ALDH1) activity (37). In particular, these authors identified SP cells with CSC-like properties in the ACHN renal cancer cell line. Interestingly, these ACHN SP cells were CD105 negative and these findings, unlike the results by Bussolati et al (32), suggest that CD105 may not be a universal marker for renal CSCs. Moreover, analysis of the ALDH1 activity showed that ACHN SP cells expressed a higher level of activity than non-SP cells. In addition, analysis of ALDH1 positive cells in the ACHN population revealed that these cells had a higher tumorigenic potential than ALDH1-negative cells. These findings indicate that ALDH1+ cells, rather than SP cells, exhibit renal CSC properties in the ACHN renal cancer cell line (37). The sphere culture system is a useful alternative method for isolating CSCs from many human cancers and cell lines. Many studies have shown that CSCs can be enriched in the spheres when cultured in serum-free medium supplemented with EGF and basic fibroblast growth factor (bFGF) (38-40). Using this functional assay, from SK-RC-42 RCC cell lines, Zhong et al selected a sphere-forming population of cells with stemness characteristics such as a self-renewal ability, tumorigenicity, and resistance to chemotherapy and radiotherapy (41). However, because the expression of CD44, CD24, CD105, and CD133 was not associated with these sphere-forming cells, the authors concluded that these stem cell markers could not be used to isolate SK-RC-42-derived CSCs. Finally, Gassenmaier et al demonstrated that CXC chemokine receptor 4 (CXCR4) identified a subpopulation of tumorinitiating cells in the RCC-53 renal cancer cell line, and that CXCR4+ cells formed fivefold more tumor spheres and grew significantly faster than CXCR4- cells when transplanted in NOD/SCID mice (42). Interestingly, CXCR4+ cells were more resistant to the tyrosine kinase inhibitors sunitinib, sorafenib, and pazopanib, and, unlike the findings by Bussolati et al, they were CD105 negative.
Identification of CD133+/CD24+/CTR2+ cells as a cancer cell population with stem cell like features in RCC CD133 (Prominin-1) has been one of the most frequently used biomarkers in CSC-related research, and the first studies reported the isolation of CD133+ CSCc from brain tumors (38). Since then, the isolation of putative CSCs from a wide range of tumors by the use of anti-CD133 antibodies has been reported. However, studies of the utility of CD133 as a CSC biomarker are still ongoing because there have been several reports that CD133− cells are also able to give rise to tumors in immunodeficient mice (43-49). As stated above, © 2014 Wichtig Publishing
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in human RCC, Bussolati et al (32) isolated a tumor-initiating cell population that was CD105+ but CD133-/CD24-. Similar findings were reported by Addla et al in SP cells isolated using the Hoechst 33342 dye efflux assay (35). However, the use of CD105 as a renal CSC marker was questioned in later studies that showed how other putative subpopulations of cells with CSC-like properties were CD105- (37, 42). Moreover, Canis et al showed that stable transfection of CD133 in the human embryonic kidney 293 (HEK293) cell line induced tumor-initiating properties in these cells. In addition, HEK293 CD133high transfectants, when injected into SCID mice, generated tumors with at least a 1000-fold increased frequency as compared with CD133low cells (50). In this scenario, our group recently described and characterized a population of resident CD133+/CD24+ cancer cells (termed RDCs: RCC-derived cells) in patients with clear cell RCC (51). These RDCs showed the same clonogenic multipotency and self-renewal ability as their normal counterparts (i.e., tubular ARPCs), and differed from bone marrow derived mesenchymal cells because they did not express the typical membrane markers (CD90-, CD105-). Moreover, RDCs were able to differentiate into epithelial cells, osteocytes, or adipocytes in the presence of specific growth factors. The stemness of this population was confirmed by the expression of several stemness proteins (such as Nanog, Sox2, GATA4, and FoxA2) as compared with CD133- cancer cells and tubular ARPCs, and the tumorigenic ability of these RDCs was evaluated using the in vitro soft agar assay. In fact, only RDCs were able to generate colonies associated with malignant transformation. It has been shown that the microenvironment can affect CD133 expression in cancer cells; in this context, an enrichment of CD133+ CSCs has been described in lung, pancreatic, and brain tumors in low oxygen concentration conditions and in association with HIF-1α upregulation (52-55). Analysis of different areas of primary gliomas has revealed that CD133+ cells were mainly localized in the inner core of the neoplastic mass (56). Similarly, we found the presence of RDCs in the central region of tumor samples, wherein conditions were more hypoxic (unpublished data). These findings support the hypothesis that the presence of a hypoxic niche could affect the survival and persistence of these cell population. As angiogenesis is one of the most important hallmarks of RCC, the RDCs-associated angiogenic response was evaluated using the in vivo chick embryo chorioallantoic membrane (CAM) technique (57). This assay demonstrated that RDCs were able to trigger an angiogenic response, as shown by numerous allantoic vessels that developed radially toward the RDC implants in a “spoked-wheel” pattern. Finally, using DNA microarray analysis, we studied the gene expression profile of these RDCs, with the aim of identifying a membrane marker that could discriminate this neoplastic population from other cell types, namely tubular ARPCs, mesenchymal stem cells, and renal proximal tubule epithelial cells (RPTECs). Statistical analysis identified 72 genes that discriminated RDCs from tubular ARPCs, and among these, CTR2 (SLC31A2) was the membrane protein-coding gene showing the highest fold change (Fig. 2). To validate these findings, we evaluated CTR2 protein expression in the RDCs isolated from a different set of RCC patients by flow cytometry analysis and immunofluorescence (Fig. 3). The in situ colocalization study of
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Stem cells in renal cell carcinoma
Fig. 2 - (A) Profile plots showing 149 modulated genes that discriminate RDCs from tubular ARPCs with a false discovery rate (FDR)
