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Published as doi: 10.1096/fj.08-102590.
 (The FASEB Journal. 2008;22:3696-3705.)
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Identification of a tumor-initiating stem cell population in human renal carcinomas

Benedetta Bussolati1, Stefania Bruno1, Cristina Grange, Ugo Ferrando and Giovanni Camussi2

Department of Internal Medicine, Center for Molecular Biotechnology and Center for Research in Experimental Medicine, Turin, Italy

2Correspondence: Cattedra di Nefrologia, Dipartimento di Medicina Interna, Ospedale Maggiore S. Giovanni Battista, Corso Dogliotti 14, 10126 Turin, Italy. E-mail: giovanni.camussi{at}unito.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The purpose of the present study was to search for the presence of a tumor-initiating stem cell population in renal carcinomas. Based on the recent identification of mesenchymal stem cells in normal kidneys, we sorted cells expressing the mesenchymal stem cell marker CD105 from 5 human renal carcinomas. Because the CD105+ but not the CD105– population showed enhanced tumorigenicity when injected in severely compromised immunodeficient (SCID) mice, we cloned and characterized CD105+ cells and evaluated their stemness, differentiative ability, and serial tumor generation. Characterization of the phenotype of CD105+ clones revealed several stem cell properties: 1) clonogenic ability, 2) expression of nestin, Nanog, Oct4 stem cell markers, and lack of differentiative epithelial markers, 3) ability to grow in nonadhesive spheroids, 4) in vitro differentiation into epithelial and endothelial cell types, and 5) generation in vivo of serially transplantable carcinomas containing an undifferentiated CD105+ tumorigenic and a differentiated CD105– nontumorigenic population. In addition, some vessels present in carcinomas generated from CD105+ clones were of human origin, suggesting the capability of tumor-initiating stem cells to in vivo differentiate also in endothelial cells. In conclusion, we demonstrate that CD105+ cells and clones derived from renal carcinomas were enriched in tumor-initiating cells with stem characteristics.—Bussolati, B., Bruno, S., Grange, C., Ferrando, U., Camussi, G. Identification of a tumor-initiating stem cell population in human renal carcinomas.


Key Words: angiogenesis • CD105 • tumorigenesis • kidney tumor • progenitors • mesenchymal stem cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EMERGING EVIDENCE SHOWED THAT the capacity of a tumor to grow and propagate resides in a small population of tumor cells, termed cancer stem cells or tumor-initiating cells (1) . In particular, a tumor-initiating population able to sustain and maintain the tumor was identified in several solid tumors, such as breast, brain, colon, pancreatic, prostate, and ovary cancers as well as in melanomas (2 3 4 5 6 7 8 9 10) . Although the specific markers may differ from one tumor to another, tumor-initiating cells are characterized by their ability to form new serially transplantable tumors in immunodeficient mice and to display stem/progenitor cell properties such as ability for self-renewal and capacity to reestablish tumor heterogeneity (11) . The identification of the tumor-initiating stem cell population has a great impact in the understanding of tumor biology as well as in tumor therapy. However, no study has addressed the possible presence of tumor stem cells in renal carcinomas.

Renal carcinoma is a common form of urologic tumor, representing 3% of total human malignancies, with a high metastatic index at the diagnosis and a high rate of relapse (12) . Renal carcinoma is resistant to radiation and chemotherapies, and at the moment surgery is the only curative option (12) . Thus, new drugs that interfere with the tumor cell biology are needed (13) . The aim of the present study was to identify a tumor-initiating stem cell population in renal carcinomas.

In the search for a tumor stem cell, researchers have exploited the similarities between normal and tumor stem cells of the same tissue, because many of the molecules expressed by normal stem cells have been found in their malignant counterparts (14) .

We have identified in normal renal tissue a population of CD133+ stem cells able to differentiate in vitro and in vivo into endothelial and epithelial cells (15) . However, CD133+ renal tumor-derived progenitor cells were not tumorigenic in vivo but rather supported angiogenesis and tumor growth in the presence of tumor cells (16) . Recently, stem cells with mesenchymal characteristics have been identified within the adult kidney (17 18 19 20) . In the present study, we investigated whether a mesenchymal stem cell population was present within renal carcinomas and whether this population exhibited tumor-initiating properties.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and in vitro expansion of CD105+ cells and clones from renal tumor specimens
Studies were performed on specimens of renal carcinomas obtained from patients undergoing radical nephrectomy. Specimens were finely minced with scissors and then digested by incubation for 1 h at 37°C in Dulbecco modified Eagle medium (DMEM) containing collagenase II (Sigma-Aldrich Corp., St Louis, MO, USA). After washings in medium plus 10% fetal calf serum (FCS) (Gibco, Grand Island, NY, USA), the cell suspension was forced through a graded series of meshes to separate the cell components from stroma and aggregates. Aliquots of the cell suspension were subjected to FACS analysis for quantification of CD105+ cells. Cells were isolated, using anti-CD105 Ab coupled to magnetic beads, by magnetic cell sorting using the magnetic-activated cell sorting (MACS) system (Miltenyi Biotec, Auburn, CA, USA) from 5 renal carcinomas (histological types: 3 clear-cell type and 2 undifferentiated carcinomas). Briefly, cells were labeled with the anti-CD105 mAb for 20 min, then washed twice and resuspended in MACS buffer (PBS without Ca2+ and Mg2+, supplemented with 1% BSA and 5 mM EDTA) at a concentration of 2 x 107 cells/80 µl. After washings, cells were separated on a magnetic stainless steel wool column (Miltenyi Biotec), according to the manufacturer’s recommendations. CD105-positive cells were plated in the presence of the expansion medium, a modification of that described for multipotent adult progenitor cells (21) , consisting of DMEM LG (Invitrogen, Paisly, UK), with insulin-transferrin-selenium, 10–9M dexametasone, 100 U penicillin, 1000 U streptomycin, 10 ng/ml epidermal growth factor (EGF) (all from Sigma-Aldrich) and 5% FCS (EuroClone, Wetherby, UK). For cell cloning, single cells were deposited in 96-well plates in presence of the expansion medium. CD105– renal tumor cells were plated and maintained in DMEM with 10% FCS. All clones were used for the in vivo experiments between the second and the fifth passage. The clones were kept in culture in adhesion in the expansion medium for more than 50 passages without phenotypic changes.

Growth kinetics
Growth curves describing culture kinetics were generated as described previously (18) . The growth area occupied by clone cultures, corresponding to 25 cm2, was assumed as 1 as a matter of simplification. When the second passage took place, the split ratio at passage 1 (1:3) was multiplied by that value, meaning that at the end of passage 1 the cumulative growth area was 3 (i.e., 3 times the growth area occupied by a primary culture). At the end of the second passage, the split ratio at passage 2 (1:3) was multiplied by the cumulative growth area at passage 1 (3x3=9). This procedure was repeated for each passage, providing a theoretical growth curve that is directly proportional to the cell number.

Sphere culture conditions
To evaluate the ability of clones to growth in nonadhesive condition as floating spheres, cells were plated at 1 x 103 cells/ml in serum-free DMEM-F12 (Cambrex BioScience, Venviers, Belgium), supplemented with 10 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml EGF, 5 µg/ml insulin, and 0.4% bovine serum albumin (all from Sigma), as described (22) . Spheres were enzymatically dissociated every 7–10 days by incubation in a nonenzimatic cell dissociation solution (Sigma) for 2 min at 37°C and passaged at 1 x 103. In addition, to evaluate the clonal sphere formation, spheres were dissociated as described above and 100 cells were plated in a 96-well culture plate to obtain a single cell per well in 200 µl of growth medium; 25 µl of medium per well were added every 5 days. The number of clonal spheres for each 96-well culture plate were evaluated after 14 days of culture and expressed as percentage of spheres.

In vitro cell differentiation
To evaluate cell differentiation, clones were incubated in differentiating media. Epithelial differentiation was obtained in the presence of RPMI plus 10% FCS, without the addition of growth factors (23) . Endothelial differentiation was obtained culturing the cells in EBM medium (Cambrex BioScience) with vascular endothelial growth factor (VEGF) (10 ng/ml) (Sigma) and 10% FCS on endothelial cell attachment factor (Sigma) (15) . Osteogenic and adipogenic differentiation were induced and assessed as described (24) .

In vivo angiogenic and tumorigenic potential of renal tumor-initiating cells
To evaluate the tumorigenic potential, CD105+ and CD105– cells (1x106 to 1x102 cells) were collected and implanted subcutaneously into SCID mice (Charles River, Jackson Laboratories, Bar Harbor, ME, USA). Cells cultured in adhesion in the presence of the expansion medium and harvested using trypsin-EDTA, or sorted cells, were washed with PBS, counted in a microcytometer chamber, and resuspended in 150 µl DMEM. Cells were chilled on ice and injected subcutaneously into the left back of SCID mice via a 26-gauge needle using a 1 ml syringe. Typically, CD105+ and CD105– cells were injected into the left and right sides of the same animal. In selected experiments, cells were added to 150 µl of Matrigel at 4°C. After 1 to 8 wk, mice were sacrificed, and tumors recovered and processed for histology. In addition, tumors were digested in collagenase II and the recovered cells processed to selection of CD105+ cells. CD105+ cells were characterized by cytofluorimetric analysis or immediately injected to generate serial tumors.

Immunofluorescence
Cytofluorimetric analysis was performed using the following antibodies, all fluorescein isothiocyanate (FITC) or phycoerythrin (PE) conjugated: anti-CD24, anti-CD44, anti-CD31, anti-CD146/Muc-18, anti-CD90, anti-CD73, anti-CD29, and anti-CD105 mAbs and anti-epithelial membrane antigen (EMA) mAb (all from Dako, Copenhagen, Denmark), anti-KDR mAb and anti-VEGFR3 (R&D System, Minneapolis, MN, USA) and anti-CD133 (Miltenyi Biotec). Isotype-matched FITC- or PE-conjugated control mouse G (IgG) were from Dako. Cells were incubated for 30 min at 4°C with the appropriate antibody or with the irrelevant control in PBS containing 2% heat-inactivated human serum. Cells were analyzed on a FACScan (Becton Dickinson, Franklin Lakes, NJ, USA). Ten thousand cells were analyzed at each experimental point.

Indirect immunofluorescence was performed on cells cultured on chamber slides or on cryostatic or paraffin-embedded samples of tumors recovered from SCID mice. Cells were fixed in 3.5% paraformaldehyde containing 2% sucrose and, when needed, permeabilized with Hepes-Triton X-100 buffer. The following antibodies were used: anti-von Willebrand Factor (vWF) (Dako) and anti-pan-cytokeratin (CK) Abs (Biomeda, Foster City, CA, USA), anti-CK7 mAb (Ventana Medical Systems, Illkirch, France), anti-vimentin (Sigma), Oct4, anti-Musashi, anti-Nanog Abs (Abcam, Cambridge, UK), anti-Pax2 Ab (Covance, Princeton, NJ, USA), and nestin (Chemicon, Temecula, CA, USA). Sections were stained for HLA class I polyclonal Ab (BioLegend, San Diego, CA, USA), polyclonal anti-mouse β2-microglobulin Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-human CD31 (Becton Dickinson) or anti-mouse CD31 (Abcam) mAbs. Antigen retrieval was obtained by heating at 100°C for 30 min in 1mM citrate buffer, pH 6. Alexa Fluor 488 or Texas Red goat anti-rabbit IgG and Alexa Fluor 488 or Texas Red anti-mouse IgG (all from Molecular Probes, Eugene, OR, USA) were used as secondary antibody. Confocal microscopy analysis was performed using a Zeiss LSM 5 Pascal model confocal microscope (Carl Zeiss, Oberkochen, Germany). Hoechst 33258 dye (Sigma) was added for nuclear staining.

Immunohistochemistry
Sections from paraffin-embedded blocks of human tumors obtained in SCID mice were collected onto poly-L-lysine-coated slides and stained using the following antibodies: rabbit anti-CK, anti-mouse β2-microglobulin, or rabbit anti-HLA class I Ab, anti-EMA mAb (clone E29), and anti-vimentin mAb (clone R9) (all from Dako). Endogenous peroxidase activity was blocked with 6% H2O2 for 8 min at room temperature. Primary antibodies were applied to slides overnight or for 1 h at 4°C. Horseradish peroxidase-labeled anti-rabbit or anti-mouse Envision polymers (Dako) were incubated for 1 h 30 min. The reaction product was developed using 3,3-diaminobenzidine. Omission of the primary antibody or substitution with an unrelated rabbit serum or mouse IgG served as negative control.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation and characterization of CD105+ cells and clones from renal carcinomas
Based on the recent identification of mesenchymal stem cells in normal kidneys (17 18 19) , we sorted cells expressing the mesenchymal stem cell marker CD105 from 5 human renal carcinomas. The frequency of CD105+ cells in renal carcinomas was 8.06 ± 3.3% (n=10). To test the hypothesis whether a small population in renal carcinomas is responsible for tumor generation, we evaluated the tumor-initiating ability of CD105+ and CD105– cells after xenograft in SCID mice. Before injection, isolated CD105+ and CD105– cell populations were cultured in adhesion overnight and analyzed by FACS. The CD105+ population contained 50.07 ± 4.3% CD31+ cells and 5.5 ± 1.4% CD45+ cells. This population did not express EMA. At variance, the CD105– cells expressed EMA at 98.5 ± 0.5%. As shown in Table 1 , the CD105+ population induced tumors with 100% incidence, whereas the CD105– population induced tumors with only 10% incidence.


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  		Table 1. Tumor generation from CD105+ cells and clones from renal carcinoma

As CD105 marker is also expressed by endothelial cells, fibroblasts, and tumor-infiltrating macrophages, to avoid the presence of non-neoplastic contaminating cells, we cloned the CD105+ cells after immunomagnetic separation in an expansion medium, a modification of that described for multipotent adult progenitor cells (21) . CD105+ cells showed a clonal ability around 1/15,000. Twelve clones originating from different renal cell carcinomas expressing CD105 in 100% of cells were expanded and characterized (Fig. 1 ). The CD105+ clones were negative for the endothelial or hematopoietic markers CD31, VEGF receptor 2, and CD45. Moreover, CD105 clones expressed several markers characteristic of mesenchymal stem cells, such as CD44, CD90, CD146, CD73, and CD29 and vimentin (Figs. 1 and 2 ). Moreover, they expressed the stem cell markers nestin, Nanog, Musashi, and Oct4 as well as the renal embryonic marker Pax2 and were negative for the epithelial marker pan-CK (Fig. 2A ), for the CD133 and CD24 antigens (Fig. 1) , known to be expressed on normal renal progenitors (15 , 25) , and for the endothelial marker vWF (Fig. 2A ). All 12 clones (4 derived from clear-cell carcinomas and 8 from undifferentiated carcinomas) showed the same phenotype. Typically, the clones were cultured in adhesion in the expansion medium for more than 50 passages without phenotypic changes. Figure 2B shows the growth rate of 4 different clones in the first 100 days of culture.


Figure 1
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  		Figure 1. Characterization of CD105+ clones from renal carcinomas by cytofluorimetric analysis. Representative FACS analysis of CD105+ cell clones (at the second passage) showing the expression of the mesenchymal stem cell markers CD105, CD146, CD90, CD73, CD29, and CD44 but not of CD24 and CD133. The dark lines indicate the specific antibody; the dotted lines indicate the isotypic control. Similar marker expression was detected for all 12 clones.


Figure 2
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  		Figure 2. Immunofluorescence analysis, growth curves, and sphere formation of CD105+ clones. A) Representative immunofluorescence expression by CD105+ cell clones of vimentin, of the renal embryonic marker Pax2, of the stem cell markers Oct4, nestin, Nanog, and Musashi, but not of the differentiation markers vWF and CK. Original view x650. Nuclei were counterstained with Hoechst dye. Similar marker expression was detected for all clones. B) Growth curves of representative CD105+ clones are shown for 100 days. Growth area is represented as a multiple of the area occupied by a confluent primary culture, arbitrarily set to 1 (see Materials and Methods). C) Micrograph representative of spheres generated by cell culture of a CD105+ clone (C2) in sphere medium containing EGF and FGF. Original view x200. Similar sphere-generating ability was tested for clones D2, F9, and B5.

When plated in sphere-generating medium, the CD105+ clones were able to grow in nonadhesive condition and to generate spheres (Fig. 2C ). Primary spheres, enzymatically digested after 7–10 days and replated as single-cell suspension, generated second passage spheres. To evaluate whether the ability to form spheres was maintained, cells were serially passaged using this procedure and propagated as spheres for 10 passages (approximate growth rate of the clones in spheres: 1 cell doubling every 30 h). To exclude that spheres were derived from cell aggregates, the spheres were cloned by plating 1 single cell per well into 96-well culture plates. The clonal ability was 42 ± 7%, indicating that ~1 in 2.4 cells is a sphere-generating cell.

In vitro epithelial and endothelial differentiation
CD105+ clones were plated in epithelial- and endothelial-differentiating medium to evaluate their possible differentiative ability. Two weeks after culture in epithelial differentiating medium, almost all cells acquired the expression of CK—in particular, they were positive for CK7—and maintained vimentin expression but lost the stem cell marker nestin, indicating an epithelial differentiation (Fig. 3A ). When cultured in endothelial differentiating medium, cells from the same clones acquired an endothelial phenotype after 2 wk of culture. Cells acquired expression of the endothelial markers vWF, KDR, VEGFR3, and CD31, which were negative in undifferentiated cells, maintained CD105 and CD146 expression, but were negative for CK (Fig. 3B, C ). In addition, cells lost the mesenchymal markers CD73 and vimentin. No ability to differentiate into adipogenic or osteogenic cells was found when cells were cultured in the appropriate differentiating media. Bone-marrow-derived cells were used as positive control for these differentiative conditions (not shown).


Figure 3
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  		Figure 3. Epithelial and endothelial differentiation of CD105+ clones cultured in differentiating media. Cells from CD105+ clones cultured in epithelial or endothelial differentiating medium for 2 wk acquired the expression of differentiative markers and lost stem cell markers. A) Representative micrographs showing the expression of the epithelial markers CK and CK7. Cells maintained vimentin expression but lost the stem cell marker nestin. B) Representative micrographs showing the expression by immunofluorescence analysis of the endothelial marker vWF and the loss of CK and vimentin. Nuclei were counterstained with Hoechst dye. Independent experiments using 8 different cell clones were performed with similar results. C) Representative FACS analysis of the acquirement by the B5 cell clone of the endothelial markers CD31, KDR, and VEGFR3 after 14 days of culture (t=14) in respect undifferentiated CD105+ cells (t=0). CD105 was maintained, whereas the mesenchymal marker CD73 was lost. The dark lines indicate the specific antibody; the dotted lines indicate the isotypic control. Original view x650 (A, B).

Tumor-initiating ability of the CD105+ population
Four CD105+ cell clones (2 clones derived from renal clear-cell carcinomas and 2 from undifferentiated carcinomas) were injected subcutaneously in SCID mice. All clones were tumorigenic. Moreover, we found that as few as 102 CD105+ cells from clones were able to generate tumors (Table 1) . Then clones were used to generate serially transplantable tumors (Table 2 ). From primary tumors formed in SCID mice by CD105+ clones, we found the presence of CD105+ and CD105– cells, indicating that CD105+ cells were able to generate tumor heterogeneity (Fig. 4 ). We therefore sorted the CD105+ cells and compared the tumorigenic activity of the CD105+ cells vs. CD105– cells (Table 2) . CD105+ cells gave raise to secondary and tertiary tumors, whereas CD105– cells did not. Moreover, the CD105+ cells originated from serially transplanted tumors maintained the same phenotype of the primary clones (not shown).


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  		Table 2. Tumor generation in serial passages from CD105+ cell clones


Figure 4
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  		Figure 4. Serial tumor generation from CD105+ cell clones. Tumors generated in SCID mice by CD105+ clones (primary tumor) were digested, and cells recovered and processed by cytofluorimetric analysis. CD105– and CD105+ cells were obtained. CD105+ cells were sorted, recovered, and injected to obtain serial tumors.

Analysis of tumors generated by CD105+ tumor-initiating cells
After 1 wk from the injection of CD105+ clones within Matrigel, a vascular network of human vessels, as detected by the expression of human HLA class I antigen (Fig. 5A , and inset) and CD31 (not shown), was observed. The vessels were connected with the mouse vasculature, as containing blood erythrocytes. Only a few clusters of epithelial cells were seen. After 3 wk, tumors grew in multiple epithelial nodules containing several vessels (Fig. 5B ). By immunohistochemistry, the tumors generated by CD105+ cell clones expressed CK, vimentin, and EMA (Fig. 5C-F ), as described for renal carcinomas (26 , 27) . No difference was observed among tumors originated from different CD105+ clones, nor in different tumor passages. The tumor growth was also observed when tumor clones were injected subcutaneously in the absence of Matrigel. The human nature of the tumors was shown by the expression of HLA class I antigen, and not mouse β2 microglobulin (Fig. 6 ). We also analyzed whether vessels developed within the tumor may derive from the transplanted tumor-initiating cells. We found that the majority of vessels detected around and within the tumor were of murine origin because they coexpressed CD31 and the mouse β2 microglobulin, whereas some of the intratumor vessels were of human origin, as detected by HLA class I and human CD31 expression (Fig. 6) . These results suggest that renal tumor-initiating cells are able to differentiate into different cell types of the tumor, including epithelial and endothelial cells. To evaluate whether tumors derived from CD105+ clones of different types of carcinomas could recapitulate the tumor subtype of origin, 3 animals per group were injected with 2 clones from undifferentiated renal carcinomas and 2 clones from clear-cell carcinomas, respectively. The animals were sacrificed after 6–8 wk to compare the morphology and immunohistochemistry with that of the patients’ tumors. As shown in Fig. 7 , the CD105+ clones from undifferentiated tumors showed morphological aspects comparable to that of the tumor of origin. The CD105+ clones derived from clear-cell carcinomas showed the typical clear-cell aspects only in focal areas, maintaining an undifferentiated aspect in most of the tumor possibly because of the high proliferation of the tumor. By immunohistochemistry, tumors derived from different CD105+ clones expressed EMA, low molecular weight CK, and vimentin as the tumor of origin. Tumors derived from clones of undifferentiated tumors were PAS negative, whereas focal areas of PAS-positive cells were detectable in tumors derived from clones of clear-cell carcinomas (Table 3 ).


Figure 5
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  		Figure 5. CD105+ tumor-initiating cells formed tumors in SCID mice. A, B) Representative ematoxilin and eosin micrographs showing the organization of CD105+ cell clone D2 (1x104 cells) injected subcutaneously in Matrigel. Matrigel plugs were recovered after 1 or 3 wk. Several vessels connected with the mouse vasculature and containing erythrocytes and small clusters of tumor cells were observed after 1 wk (A). By immunohistochemistry vessels were positive for the human HLA class I antigen (A, inset). After 3 wk, nodules of tumor cells were present (B). C–F) Representative micrographs of tumor sections showing positivity for CK (C), vimentin (D), and EMA (E), and a negative control section (F). CD105+ cells and cell clones at different passages showed the same morphology and marker expression. Original view x100; inset, x400.


Figure 6
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  		Figure 6. Vessels and tumor formation by CD105+ cell clones subcutaneously injected in SCID mice. A) Representative micrograph showing the expression of human HLA class I antigen by tumor cells. B) Representative immunofluorescence micrographs showing colocalization of human HLA class I and human CD31 in vessels present within the CD105+ clone-generated tumors, as seen by confocal microscopy. C) Human CD31 expression by tumor vessels confirmed by immunoistochemistry using an mAb that did not cross-react with the mouse CD31. D, E) Representative immunohistochemistry for mouse β2 microglobulin showing the presence of murine vessels and isolated cells at the periphery (D) and inside (E) the tumor. F) Micrograph showing the colocalization of mouse β2 microglobulin and mouse CD31 in a peripheral vessel. Original view x100 (A, D, E); x200 (C); x650 (B, F). Data are representative of 6 experiments with similar results.


Figure 7
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  		Figure 7. Morphological appearance of tumors derived from CD105+ clones compared with the original renal tumors. A, B) Representative micrographs showing the morphological appearance of an undifferentiated renal carcinoma with glandular aspects (A) and of the tumor developed in SCID mice after 6 wk from the subcutaneous injection of 1 x 102 cells deriving from a CD105+ clone of the same tumor (B). Similar glandular aspects are visible. C, D) Representative micrographs showing the morphological appearance of an undifferentiated renal carcinoma with sarcomatoid aspects (C) and similar aspects seen in the tumor developed in SCID mice after 6 wk from the subcutaneous injection of 1 x 102 cells deriving from a CD105+ clone of the same tumor (D). E, F) Representative micrographs showing the morphological appearance of a clear-cell renal carcinoma (E) and of the tumor developed in SCID mice after 8 wk from the subcutaneous injection of 1 x 102 cells deriving from a CD105+ clone of the same tumor (E). Clear-cell changes (arrow) and glandular features (arrowheads) are present in focal areas (E). Original view x200.


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  		Table 3. Immunohistochemical comparison between patient tumor and tumor developed in SCID mice by CD105+ clones


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we found a tumor-initiating cell CD105+ population present in human renal carcinomas.

The identified CD105+ cells presented the characteristic of tumor stem cells previously described for cancer stem cells present in other tumor types (11) . In particular, the CD105+ cells 1) were clonogenic, 2) expressed stem cell markers and lacked differentiative markers, 3) could differentiate in vitro into epithelial and endothelial cell types, and 4) could generate in vivo serially transplantable tumors. These tumors, despite being derived from clones expressing mesenchymal markers, were epithelial carcinomas as the tumor of origin and were characterized by the maintenance of a CD105+ tumorigenic population and by the presence of a nontumorigenic differentiated CD105– population.

Different stem cell populations have been identified in the normal and neoplastic kidney. In rodents, the presence of stem cells was identified on the base of bromodeoxyuridine retention, of stem cell marker expression (Sca-1), on the ability to extrude Hoechst dye (so-called side population), or on stringent culture conditions (for review, see ref. 28 ). In particular, multipotent mesenchymal stem cells were identified in both the tubular and glomerular components of the nephron (17 18 19) . In humans, we found a rare population of CD133+ cells that lacked the expression of hematopoietic markers (CD34 and CD45) and expressed some MSC markers, such as CD29, CD90, CD44, and CD73. Moreover, they expressed Pax-2, an embryonic renal marker (29) , suggesting their renal origin. These cells may undergo epithelial and endothelial differentiation both in vitro and in vivo (15) . Sagrinati et al. (25) isolated and characterized a population of CD133+ CD24+ cells from the Bowman’s capsule of adult human kidneys that exhibited a multipotent differentiation ability.

Within renal tumors, we previously detected the presence of CD133+ renal resident stem cells that were shown to contribute to tumor vasculogenesis and to retain the differentiative capacity of CD133+ cells from normal renal tissue. However, these cells were not tumorigenic (16) . Moreover, in the Wilms’ tumor, the identification of multiple imprinted and stemness genes suggested the origin of this tumor from tumor progenitor cells of the developing kidney (30) .

In the present study, we found that the CD105+ population present in renal cell carcinomas was enriched in tumor-initiating cells. These cells, selected for their clonogenic ability, displayed stem cell markers and differentiative ability. In addition, CD105+ clones were able to grow in suspension, as floating spheres, as described for normal and tumor stem cells in an undifferentiated state (22 , 23) . Renal CD105+ tumor-initiating cells expressed several mesenchymal stem cell markers, such as CD44, CD146, CD73, CD29, CD90, and vimentin (24) . Moreover, CD105+ clones expressed the stem cell markers nestin, Nanog, Musashi, and Oct4. At variance with mesenchymal stem cells of the bone marrow (14) , CD105+ clones expressed the renal marker Pax2 (29) and were unable to differentiate into adipocytes or osteocytes.

These data may suggest that the CD105+ cells representing a tumor-initiating cell population originated from resident renal stem cells with mesenchymal characteristics. The lack of CD133 and CD24 expression, which are present in adult and embryonic renal progenitors (15 , 25 , 31) , by tumor-initiating CD105+ clones suggests that they are not derived from the CD133+ population. Moreover, CD133+ cells previously isolated from renal tumors did not express CD105 and were not tumorigenic (16) . However, it cannot be excluded that the CD105+ stem cell population that we identified in renal carcinomas may derive from mutated stromal/mesenchymal cells of the tumor or from bone-marrow-derived stem cells. Indeed, mesenchymal stem cells derived from the bone marrow have been suggested to contribute to cancer both in human and in mouse models (32 33 34) . Moreover, mesenchymal cells present in tumor stroma were shown to be able to follow an autonomous fate, developing into rapidly growing, highly vascularized, and invasive mesenchymal tumors (35) . Reports suggest the possibility that these tumor mesenchymal cells may undergo tumorigenic phenotypical changes induced by the neoplastic microenvironment (36 37 38) . Indeed, various studies showed chromosomal alterations in tumor stroma (39) . The CD105+ tumor-initiating cells that we identified in renal carcinomas, at variance with those derived from the stroma that forms mesenchymal tumors, were able to generate carcinomas expressing an epithelial phenotype. In vivo, CD105+ cells were able to maintain the tumor-initiating CD105+ population expressing stem cell properties and lacking differentiative markers. Moreover, CD105+ clones were able to generate a progeny of differentiated CD105– cells unable to generate the tumor and expressing cytokeratin.

Tumor-initiating cells or "cancer stem cells" are characterized by their ability to display stem/progenitor cell properties: competence for self-renewal and capacity to differentiate in a heterogeneous tumor cell population (see refs. 1 , 11 ). In addition, if the idea that tumor stem cells originate from mutated stem cells of the tissue is true, it is conceivable that tumor stem cells may differentiate in different lineages. This has been shown for melanoma-derived stem/progenitor cells that are able to differentiate in multiple mesenchymal lineages, such as adypocitic, osteocytic, and chondrocytic lineages (10) . In breast tumors, the ability of stem/progenitor cells to differentiate in both ductal/luminal cells and myoepithelial cells has been extensively shown (22) .

In the present study, we demonstrate that clones of CD105+ tumor stem/progenitor cells are bipotent, being able to differentiate into tumor epithelial and endothelial cells in vitro and in vivo. These results are consistent with recent publications describing the ability of mutated mesenchymal stem cells to generate tumors, tumor adipose tissue, and tumor vasculature (40) and the differentiation of breast tumor stem/progenitor cells not only into cells of the glandular epithelium but also into the endothelial lineage (41) .

In conclusion, the results of the present study indicate the presence in renal carcinomas of a tumor-initiating cell population expressing the characteristic of stem cells as defined by their in vitro and in vivo self-maintenance and differentiative abilities and by the expression of embryonic stem cell markers.


   ACKNOWLEDGMENTS
 
This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), Italian Ministry of University and Research (MIUR) COFIN and MIUR ex60% grants, the Italian Ministry of Health (Ricerca Finalizzata 02), the Progetto S. Paolo Oncologia, and the Progetti Finalizzati Regione Piemonte, Oncoprot.


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication February 14, 2008. Accepted for publication June 5, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Reya, T., Morrison, S. J., Clarcke, M. F., Weissman, I. L. (2001) Stem cells, cancer, and cancer stem cells. Nature 414,105-111[CrossRef][Medline]
  2. Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., Clarke, M. F. (2003) Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. U. S. A. 100,3983-3988[Abstract/Free Full Text]
  3. Singh, S. K., Clarke, I. D., Terasaki, M., Bonn, V. E., Hawkins, C., Squire, J., Dirks, P. B. (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res. 63,5821-5828[Abstract/Free Full Text]
  4. Singh, S. K., Hawkins, C., Clarke, I. D., Squire, J. A., Bayani, J., Hide, T., Henkelman, R. M., Cusimano, M. D., Dirks, P. B. (2004) Identification of human brain tumour initiating cells. Nature 432,396-401[CrossRef][Medline]
  5. O'Brien, C. A., Pollett, A., Gallinger, S., Dick, J. E. (2007) A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445,106-110[CrossRef][Medline]
  6. Ricci-Vitiani, L., Lombardi, D. G., Pilozzi, E., Biffoni, M., Todaro, M., Peschle, C., De Maria, R. (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445,111-115[CrossRef][Medline]
  7. Li, C., Heidt, D. G., Dalerba, P., Burant, C. F., Zhang, L., Adsay, V., Wicha, M., Clarke, M. F., Simeone, D. M. (2007) Identification of pancreatic cancer stem cells. Cancer Res. 67,1030-1037[Abstract/Free Full Text]
  8. Collins, A. T., Berry, P. A., Hyde, C., Stower, M. J., Maitland, N. J. (2005) Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res. 65,10946-10951[Abstract/Free Full Text]
  9. Bapat, S. A., Mali, A. M., Koppikar, C. B., Kurrey, N. K. (2005) Stem and progenitor-like cells contribute to the aggressive behaviour of human epithelial ovarian cancer. Cancer Res. 65,3025-3029[Abstract/Free Full Text]
  10. Fang, D., Nguyen, T. K., Leishear, K., Finko, R., Kulp, A. N., Hotz, S., Van Belle, P. A., Xu, X., Elder, D. E., Herlyn, M. (2005) A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Res. 65,9328-9337[Abstract/Free Full Text]
  11. Clarke, M. F., Dick, J. E., Dirks, P. B., Eaves, C. J., Jamieson, C. H., Jones, D. L., Visvader, J., Weissman, I. L., Wahl, G. M. (2006) Cancer stem cells–perspectives on current status and future directions: AACR workshop on cancer stem cells. Cancer Res. 66,9339-9344[Free Full Text]
  12. Lane, B. R., Kattan, M. W. (2005) Predicting outcomes in renal cell carcinoma. Curr. Opin. Urol. 15,289-297[Medline]
  13. Larkin, J. M., Chowdhury, S., Gore, M. E. (2007) Drug insight: advances in renal cell carcinoma and the role of targeted therapies. Nat. Clin. Pract. Oncol. 4,470-479[CrossRef][Medline]
  14. Liu, B. B., Qin, L. X., Liu, Y. K. (2005) Adult stem cells and cancer stem cells: tie in or tear apart?. J. Cancer Res. Clin. Oncol. 131,631-638[CrossRef][Medline]
  15. Bussolati, B., Bruno, S., Grange, C., Buttiglieri, S., Deregibus, M. C., Cantino, D., Camussi, G. (2005) Isolation of renal progenitor cells from adult human kidney. Am. J. Pathol. 166,545-555[Abstract/Free Full Text]
  16. Bruno, S., Bussolati, B., Grange, C., Collino, F., Graziano, M. E., Ferrando, U., Camussi, G. (2006) CD133+ renal progenitor cells contribute to tumor angiogenesis. Am. J. Pathol. 169,2223-2235[Abstract/Free Full Text]
  17. Gupta, S., Verfaillie, C., Chmielewski, D., Kren, S., Eidman, K., Connaire, J., Heremans, Y., Lund, T., Blackstad, M., Jiang, Y., Luttun, A., Rosenberg, M. E. (2006) Isolation and characterization of kidney-derived stem cells. J. Am. Soc. Nephrol. 17,3028-3040[Abstract/Free Full Text]
  18. Da Silva Meirelles, L., Chagastelles, P. C., Nardi, N. B. (2006) Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J. Cell Sci. 119,2204-2213[Abstract/Free Full Text]
  19. Plotkin, M. D., Goligorsky, M. S. (2006) Mesenchymal cells from adult kidney support angiogenesis and differentiate into multiple interstitial cell types including erythropoietin-producing fibroblasts. Am. J. Physiol. Renal Physiol. 291,F902-F912[Abstract/Free Full Text]
  20. Humphreys, B. D., Bonventre, J. V. (2008) Mesenchymal stem cells in acute kidney injury. Annu. Rev. Med. 59,311-325[CrossRef][Medline]
  21. Jiang, Y., Jahagirdar, B. N., Reinhardt, R. L., Schwartz, R. E., Keene, C. D., Ortiz-Gonzalez, X. R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M., Du, J., Aldrich, S., Lisberg, A., Low, W. C., Largaespada, D. A., Verfaillie, C. M. (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418,41-49[CrossRef][Medline]
  22. Ponti, D., Costa, A., Zaffarono, N., Pratesi, G., Petrangolini, G., Cordini, D., Pilotti, S., Pierotti, M. A., Daidone, M. G. (2005) Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res. 65,5506-5511[Abstract/Free Full Text]
  23. Dontu, G., Abdallah, W. M., Foley, J. M., Jackson, K. W., Clarke, M. F., Kawamura, M. J., Wicha, M. S. (2003) In vitro propagation and trascriptional profiling of human mammary stem progenitors cells. Genes Dev. 17,1253-1270[Abstract/Free Full Text]
  24. Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D., Moorman, M. A., Simonetti, D. W., Craig, S., Marshak, D. R. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284,143-147[Abstract/Free Full Text]
  25. Sagrinati, C., Netti, G. S., Mazzinghi, B., Lazzeri, E., Liotta, F., Frosali, F., Ronconi, E., Meini, C., Gacci, M., Squecco, R., Carini, M., Gesualdo, L., Francini, F., Maggi, E., Annunziato, F., Lasagni, L., Serio, M., Romagnani, S., Romagnani, P. (2006) Isolation and characterization of multipotent progenitor cells from the Bowman’s capsule of adult human kidneys. J. Am. Soc. Nephrol. 17,2443-2456[Abstract/Free Full Text]
  26. Khoury, J. D., Abrahams, N. A., Levin, H. S., MacLennan, G. T. (2002) The utility of epithelial membrane antigen and vimentin in the diagnosis of chromophobe renal cell carcinoma. Ann. Diagn. Pathol. 6,154-158[CrossRef][Medline]
  27. Liu, L., Qian, J., Singh, H., Meiers, I., Zhou, X., Bostwick, D. G. (2007) Arch immunohistochemical analysis of chromophobe renal cell carcinoma, renal oncocytoma, and clear cell carcinoma: an optimal and practical panel for differential diagnosis. Pathol. Lab. Med. 131,1290-1297
  28. Cantley, L. G. (2005) Adult stem cells in the repair of the injured renal tubule. Nat. Clin. Pract. Nephrol. 1,22-32[CrossRef][Medline]
  29. Dressler, G. R., Douglass, E. C. (1992) Pax-2 is a DNA-binding protein expressed in embryonic kidney and Wilms tumor. Proc. Natl. Acad. Sci. U. S. A. 89,1179-1183[Abstract/Free Full Text]
  30. Dekel, B., Metsuyanim, S., Schmidt-Ott, K. M., Fridman, E., Jacob-Hirsch, J., Simon, A., Pinthus, J., Mor, Y., Barasch, J., Amariglio, N., Reisner, Y., Kaminski, N., Rechavi, G. (2006) Multiple imprinted and stemness genes provide a link between normal and tumor progenitor cells of the developing human kidney. Cancer Res. 66,6040-6049[Abstract/Free Full Text]
  31. Lazzeri, E., Crescioli, C., Ronconi, E., Mazzinghi, B., Sagrinati, C., Netti, G. S., Angelotti, M. L., Parente, E., Ballerini, L., Cosmi, L., Maggi, L., Gesualdo, L., Rotondi, M., Annunziato, F., Maggi, E., Lasagni, L., Serio, M., Romagnani, S., Vannelli, G. B., Romagnani, P. (2007) Regenerative potential of embryonic renal multipotent progenitors in acute renal failure. J. Am. Soc. Nephrol. 18,3128-3138[Abstract/Free Full Text]
  32. Aractingi, S., Kanitakis, J., Euvaard, S., Le Danff, C., Peguillet, I., Khosrotehrani, K., Lantz, O., Carosella, E. D. (2005) Skin carcinoma arising from donor cells in a kidney transplant recipient. Cancer Res. 65,1755-1760[Abstract/Free Full Text]
  33. Barozzi, P., Luppi, M., Facchetti, F., Mecucci, C., Alù, M., Sarid, R., Rasini, V., Ravazzini, L., Rossi, E., Festa, S., Crescenzi, B., Wolf, D. G., Schulz, T. F., Torelli, G. (2003) Post-transplant Kaposi sarcoma originates from the seeding of donor-derived progenitors. Nat. Med. 9,554-561[CrossRef][Medline]
  34. Houghton, J., Stoicov, C., Nomura, S., Rogers, A. B., Carlson, J., Li, H., Cai, X., Fox, J. G., Goldenring, J. R., Wang, T. C. (2004) Gastric cancer originating from bone marrow-derived cells. Science 306,1568-1571[Abstract/Free Full Text]
  35. Galiè, M., Konstantinidou, G., Peroni, D., Scambi, I., Marchini, C., Lisi, V., Krampera, M., Magnani, P., Merigo, F., Montani, M., Boschi, F., Marzola, P., Orrù, R., Farace, P., Sbarbati, A., Amici, A. (2008) Mesenchymal stem cells share molecular signature with mesenchymal tumor cells and favor early tumor growth in syngeneic mice. Oncogene 27,2542-2551[CrossRef][Medline]
  36. Kurose, K., Gilley, K., Matsumoto, S., Watson, P. H., Zhou, X. P., Eng, C. (2002) Frequent somatic mutations in PTEN and TP53 are mutually exclusive in the stroma of breast carcinomas. Nat. Genet. 32,355-357[CrossRef][Medline]
  37. Hu, M., Yao, J., Cai, L., Bachman, K. E., van den Brule, F., Velculescu, V., Polyak, K. (2005) Distinct epigenetic changes in the stromal cells of breast cancers. Nat. Genet. 37,899-905[CrossRef][Medline]
  38. Fiegl, H., Millinger, S., Goebel, G., Muller-Holzner, E., Marth, C., Laird, P. W., Laird, P. W., Widschwendter, M. (2006) Breast cancer DNA methylation profiles in cancer cells and tumor stroma: association with HER-2/neu status in primary breast cancer. Cancer Res. 66,29-33[Abstract/Free Full Text]
  39. Pelham, R. J., Rodgers, L., Hall, I., Lucito, R., Nguyen, K. C., Navin, N., Hicks, J., Mu, D., Powers, S., Wigler, M., Botstein, D. (2006) Identification of alterations in DNA copy number in host stromal cells during tumor progression. Proc. Natl. Acad. Sci. U. S. A. 103,19848-19853[Abstract/Free Full Text]
  40. Li, H., Fan, X., Kovi, R. C., Jo, Y., Moquin, B., Konz, R., Stoicov, C., Kurt-Jones, E., Grossman, S. R., Lyle, S., Rogers, A. B., Montrose, M., Houghton, J. (2007) Spontaneous expression of embryonic factors and p53 point mutations in aged mesenchymal stem cells: a model of age-related tumorigenesis in mice. Cancer Res. 67,10889-10898[Abstract/Free Full Text]
  41. Bussolati, B., Grange, C., Sapino, A., Camussi, G. (2008) Endothelial cell differentiation of human breast tumor stem/progenitor cells. [E-pub ahead of print] J. Cell Mol. Med. Apr. 9




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