identification of a tumor-initiating stem cell population in human renal carcinomas

The FASEB Journal • Research Communication

Identification of a tumor-initiating stem cell populationin human renal carcinomas

Benedetta Bussolati,1 Stefania Bruno,1 Cristina Grange, Ugo Ferrando,and Giovanni Camussi2

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

ABSTRACT The purpose of the present study was tosearch for the presence of a tumor-initiating stem cellpopulation in renal carcinomas. Based on the recentidentification of mesenchymal stem cells in normalkidneys, we sorted cells expressing the mesenchymalstem cell marker CD105 from 5 human renal carcino-mas. Because the CD105� but not the CD105� popu-lation showed enhanced tumorigenicity when injectedin severely compromised immunodeficient (SCID)mice, we cloned and characterized CD105� cells andevaluated their stemness, differentiative ability, andserial tumor generation. Characterization of the pheno-type of CD105� clones revealed several stem cellproperties: 1) clonogenic ability, 2) expression of nes-tin, Nanog, Oct4 stem cell markers, and lack of differ-entiative epithelial markers, 3) ability to grow in non-adhesive spheroids, 4) in vitro differentiation intoepithelial and endothelial cell types, and 5) generationin vivo of serially transplantable carcinomas containingan undifferentiated CD105� tumorigenic and a differ-entiated CD105� nontumorigenic population. In addi-tion, some vessels present in carcinomas generatedfrom CD105� clones were of human origin, suggestingthe capability of tumor-initiating stem cells to in vivodifferentiate also in endothelial cells. In conclusion, wedemonstrate that CD105� cells and clones derivedfrom renal carcinomas were enriched in tumor-initiat-ing cells with stem characteristics.—Bussolati, B.,Bruno, S., Grange, C., Ferrando, U., Camussi, G.Identification of a tumor-initiating stem cell populationin human renal carcinomas. FASEB J. 22, 3696–3705(2008)

Key Words: angiogenesis � CD105 � tumorigenesis � kidney tu-mor � progenitors � mesenchymal stem cells

Emerging evidence showed that the capacity of atumor to grow and propagate resides in a small popu-lation of tumor cells, termed cancer stem cells ortumor-initiating cells (1). In particular, a tumor-initiat-ing population able to sustain and maintain the tumorwas identified in several solid tumors, such as breast,brain, colon, pancreatic, prostate, and ovary cancers aswell as in melanomas (2–10). Although the specificmarkers may differ from one tumor to another, tumor-

initiating cells are characterized by their ability to formnew serially transplantable tumors in immunodeficientmice and to display stem/progenitor cell propertiessuch as ability for self-renewal and capacity to reestab-lish tumor heterogeneity (11). The identification of thetumor-initiating stem cell population has a great impactin the understanding of tumor biology as well as intumor therapy. However, no study has addressed thepossible presence of tumor stem cells in renal carcino-mas.

Renal carcinoma is a common form of urologictumor, representing 3% of total human malignancies,with a high metastatic index at the diagnosis and a highrate of relapse (12). Renal carcinoma is resistant toradiation and chemotherapies, and at the momentsurgery is the only curative option (12). Thus, newdrugs that interfere with the tumor cell biology areneeded (13). The aim of the present study was toidentify a tumor-initiating stem cell population in renalcarcinomas.

In the search for a tumor stem cell, researchers haveexploited the similarities between normal and tumorstem cells of the same tissue, because many of themolecules expressed by normal stem cells have beenfound in their malignant counterparts (14).

We have identified in normal renal tissue a popula-tion of CD133� stem cells able to differentiate in vitroand in vivo into endothelial and epithelial cells (15).However, CD133� renal tumor-derived progenitor cellswere not tumorigenic in vivo but rather supportedangiogenesis and tumor growth in the presence oftumor cells (16). Recently, stem cells with mesenchymalcharacteristics have been identified within the adultkidney (17–20). In the present study, we investigatedwhether a mesenchymal stem cell population waspresent within renal carcinomas and whether this pop-ulation exhibited tumor-initiating properties.

1 These authors contributed equally to this work.2 Correspondence: Cattedra di Nefrologia, Dipartimento di

Medicina Interna, Ospedale Maggiore S. Giovanni Battista,Corso Dogliotti 14, 10126 Turin, Italy. E-mail: [email protected]

doi: 10.1096/fj.08-102590

3696 0892-6638/08/0022-3696 © FASEB

MATERIALS AND METHODS

Isolation and in vitro expansion of CD105� cells and clonesfrom renal tumor specimens

Studies were performed on specimens of renal carcinomasobtained from patients undergoing radical nephrectomy.Specimens were finely minced with scissors and then digestedby incubation for 1 h at 37°C in Dulbecco modified Eaglemedium (DMEM) containing collagenase II (Sigma-AldrichCorp., St Louis, MO, USA). After washings in medium plus10% fetal calf serum (FCS) (Gibco, Grand Island, NY, USA),the cell suspension was forced through a graded series ofmeshes to separate the cell components from stroma andaggregates. Aliquots of the cell suspension were subjected toFACS analysis for quantification of CD105� cells. Cells wereisolated, using anti-CD105 Ab coupled to magnetic beads, bymagnetic cell sorting using the magnetic-activated cell sorting(MACS) system (Miltenyi Biotec, Auburn, CA, USA) from 5renal carcinomas (histological types: 3 clear-cell type and 2undifferentiated carcinomas). Briefly, cells were labeled withthe anti-CD105 mAb for 20 min, then washed twice andresuspended in MACS buffer (PBS without Ca2� and Mg2�,supplemented with 1% BSA and 5 mM EDTA) at a concen-tration of 2 � 107 cells/80 �l. After washings, cells wereseparated on a magnetic stainless steel wool column (MiltenyiBiotec), according to the manufacturer’s recommendations.CD105-positive cells were plated in the presence of theexpansion medium, a modification of that described formultipotent adult progenitor cells (21), consisting of DMEMLG (Invitrogen, Paisly, UK), with insulin-transferrin-sele-nium, 10�9 M dexametasone, 100 U penicillin, 1000 Ustreptomycin, 10 ng/ml epidermal growth factor (EGF) (allfrom Sigma-Aldrich) and 5% FCS (EuroClone, Wetherby,UK). For cell cloning, single cells were deposited in 96-wellplates in presence of the expansion medium. CD105� renaltumor cells were plated and maintained in DMEM with 10%FCS. All clones were used for the in vivo experiments betweenthe second and the fifth passage. The clones were kept inculture in adhesion in the expansion medium for more than50 passages without phenotypic changes.

Growth kinetics

Growth curves describing culture kinetics were generated asdescribed previously (18). The growth area occupied by clonecultures, corresponding to 25 cm2, was assumed as 1 as amatter 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 growtharea was 3 (i.e., 3 times the growth area occupied by a primaryculture). At the end of the second passage, the split ratio atpassage 2 (1:3) was multiplied by the cumulative growth areaat passage 1 (3�3�9). This procedure was repeated for eachpassage, providing a theoretical growth curve that is directlyproportional to the cell number.

Sphere culture conditions

To evaluate the ability of clones to growth in nonadhesivecondition as floating spheres, cells were plated at 1 � 103

cells/ml in serum-free DMEM-F12 (Cambrex BioScience,Venviers, Belgium), supplemented with 10 ng/ml basic fibro-blast growth factor (bFGF), 20 ng/ml EGF, 5 �g/ml insulin,and 0.4% bovine serum albumin (all from Sigma), as de-scribed (22). Spheres were enzymatically dissociated every7–10 days by incubation in a nonenzimatic cell dissociationsolution (Sigma) for 2 min at 37°C and passaged at 1 � 103.

In addition, to evaluate the clonal sphere formation, sphereswere dissociated as described above and 100 cells were platedin a 96-well culture plate to obtain a single cell per well in 200�l of growth medium; 25 �l of medium per well were addedevery 5 days. The number of clonal spheres for each 96-wellculture plate were evaluated after 14 days of culture andexpressed as percentage of spheres.

In vitro cell differentiation

To evaluate cell differentiation, clones were incubated indifferentiating media. Epithelial differentiation was obtainedin the presence of RPMI plus 10% FCS, without the additionof growth factors (23). Endothelial differentiation was ob-tained culturing the cells in EBM medium (Cambrex Bio-Science) with vascular endothelial growth factor (VEGF) (10ng/ml) (Sigma) and 10% FCS on endothelial cell attachmentfactor (Sigma) (15). Osteogenic and adipogenic differentia-tion were induced and assessed as described (24).

In vivo angiogenic and tumorigenic potential of renaltumor-initiating cells

To evaluate the tumorigenic potential, CD105� and CD105�

cells (1�106 to 1�102 cells) were collected and implantedsubcutaneously into SCID mice (Charles River, Jackson Lab-oratories, Bar Harbor, ME, USA). Cells cultured in adhesionin the presence of the expansion medium and harvestedusing trypsin-EDTA, or sorted cells, were washed with PBS,counted in a microcytometer chamber, and resuspended in150 �l DMEM. Cells were chilled on ice and injected subcu-taneously into the left back of SCID mice via a 26-gaugeneedle using a 1 ml syringe. Typically, CD105� and CD105�

cells were injected into the left and right sides of the sameanimal. In selected experiments, cells were added to 150 �l ofMatrigel at 4°C. After 1 to 8 wk, mice were sacrificed, andtumors recovered and processed for histology. In addition,tumors were digested in collagenase II and the recovered cellsprocessed to selection of CD105� cells. CD105� cells werecharacterized by cytofluorimetric analysis or immediatelyinjected to generate serial tumors.

Immunofluorescence

Cytofluorimetric analysis was performed using the followingantibodies, all fluorescein isothiocyanate (FITC) or phyco-erythrin (PE) conjugated: anti-CD24, anti-CD44, anti-CD31,anti-CD146/Muc-18, anti-CD90, anti-CD73, anti-CD29, andanti-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-matchedFITC- or PE-conjugated control mouse G (IgG) were fromDako. Cells were incubated for 30 min at 4°C with theappropriate antibody or with the irrelevant control in PBScontaining 2% heat-inactivated human serum. Cells wereanalyzed on a FACScan (Becton Dickinson, Franklin Lakes,NJ, USA). Ten thousand cells were analyzed at each experi-mental point.

Indirect immunofluorescence was performed on cells cul-tured on chamber slides or on cryostatic or paraffin-embed-ded samples of tumors recovered from SCID mice. Cells werefixed in 3.5% paraformaldehyde containing 2% sucrose and,when needed, permeabilized with Hepes-Triton X-100 buffer.The following antibodies were used: anti-von WillebrandFactor (vWF) (Dako) and anti-pan-cytokeratin (CK) Abs(Biomeda, Foster City, CA, USA), anti-CK7 mAb (VentanaMedical Systems, Illkirch, France), anti-vimentin (Sigma),

3697CD105� TUMOR-INITIATING CELLS IN RENAL CARCINOMAS

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 forHLA class I polyclonal Ab (BioLegend, San Diego, CA, USA),polyclonal anti-mouse �2-microglobulin Ab (Santa Cruz Bio-technology, 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 minin 1mM citrate buffer, pH 6. Alexa Fluor 488 or Texas Redgoat anti-rabbit IgG and Alexa Fluor 488 or Texas Redanti-mouse IgG (all from Molecular Probes, Eugene, OR,USA) were used as secondary antibody. Confocal microscopyanalysis was performed using a Zeiss LSM 5 Pascal modelconfocal microscope (Carl Zeiss, Oberkochen, Germany).Hoechst 33258 dye (Sigma) was added for nuclear staining.

Immunohistochemistry

Sections from paraffin-embedded blocks of human tumorsobtained in SCID mice were collected onto poly-l-lysine-coated slides and stained using the following antibodies:rabbit anti-CK, anti-mouse �2-microglobulin, or rabbit anti-HLA class I Ab, anti-EMA mAb (clone E29), and anti-vimentinmAb (clone R9) (all from Dako). Endogenous peroxidaseactivity was blocked with 6% H2O2 for 8 min at roomtemperature. Primary antibodies were applied to slides over-night or for 1 h at 4°C. Horseradish peroxidase-labeledanti-rabbit or anti-mouse Envision polymers (Dako) wereincubated for 1 h 30 min. The reaction product was devel-oped using 3,3-diaminobenzidine. Omission of the primaryantibody or substitution with an unrelated rabbit serum ormouse IgG served as negative control.

RESULTS

Isolation and characterization of CD105� cells andclones from renal carcinomas

Based on the recent identification of mesenchymalstem cells in normal kidneys (17–19), we sorted cellsexpressing the mesenchymal stem cell marker CD105from 5 human renal carcinomas. The frequency ofCD105� cells in renal carcinomas was 8.06 � 3.3%(n�10). To test the hypothesis whether a small popu-lation in renal carcinomas is responsible for tumorgeneration, we evaluated the tumor-initiating ability ofCD105� and CD105� cells after xenograft in SCIDmice. Before injection, isolated CD105� and CD105�

cell populations were cultured in adhesion overnightand analyzed by FACS. The CD105� population con-tained 50.07 � 4.3% CD31� cells and 5.5 � 1.4%CD45� cells. This population did not express EMA. Atvariance, the CD105� cells expressed EMA at 98.5 �0.5%. As shown in Table 1, the CD105� populationinduced tumors with 100% incidence, whereas theCD105� population induced tumors with only 10%incidence.

As CD105 marker is also expressed by endothelialcells, fibroblasts, and tumor-infiltrating macrophages,to avoid the presence of non-neoplastic contaminatingcells, we cloned the CD105� cells after immunomag-netic separation in an expansion medium, a modifica-tion of that described for multipotent adult progenitor

cells (21). CD105� cells showed a clonal ability around1/15,000. Twelve clones originating from differentrenal cell carcinomas expressing CD105 in 100% ofcells were expanded and characterized (Fig. 1). TheCD105� clones were negative for the endothelial orhematopoietic markers CD31, VEGF receptor 2, andCD45. Moreover, CD105 clones expressed several mark-ers characteristic of mesenchymal stem cells, such asCD44, CD90, CD146, CD73, and CD29 and vimentin(Figs. 1 and 2). Moreover, they expressed the stem cellmarkers nestin, Nanog, Musashi, and Oct4 as well as therenal embryonic marker Pax2 and were negative for theepithelial marker pan-CK (Fig. 2A), for the CD133 andCD24 antigens (Fig. 1), known to be expressed onnormal renal progenitors (15, 25), and for the endo-thelial marker vWF (Fig. 2A). All 12 clones (4 derivedfrom clear-cell carcinomas and 8 from undifferentiatedcarcinomas) showed the same phenotype. Typically,the clones were cultured in adhesion in the expansionmedium for more than 50 passages without phenotypicchanges. Figure 2B shows the growth rate of 4 differentclones in the first 100 days of culture.

When plated in sphere-generating medium, theCD105� clones were able to grow in nonadhesivecondition and to generate spheres (Fig. 2C). Primaryspheres, enzymatically digested after 7–10 days andreplated as single-cell suspension, generated secondpassage spheres. To evaluate whether the ability to formspheres was maintained, cells were serially passagedusing this procedure and propagated as spheres for 10passages (approximate growth rate of the clones inspheres: 1 cell doubling every 30 h). To exclude thatspheres were derived from cell aggregates, the sphereswere cloned by plating 1 single cell per well into 96-wellculture plates. The clonal ability was 42 � 7%, indicat-ing that �1 in 2.4 cells is a sphere-generating cell.

In vitro epithelial and endothelial differentiation

CD105� clones were plated in epithelial- and endothe-lial-differentiating medium to evaluate their possibledifferentiative ability. Two weeks after culture in epi-thelial differentiating medium, almost all cells acquiredthe expression of CK—in particular, they were positivefor CK7—and maintained vimentin expression but lostthe stem cell marker nestin, indicating an epithelialdifferentiation (Fig. 3A). When cultured in endothelialdifferentiating medium, cells from the same clonesacquired an endothelial phenotype after 2 wk of cul-ture. Cells acquired expression of the endothelial mark-

TABLE 1. Tumor generation from CD105� cells and clonesfrom renal carcinoma

TypeUncloned cells

(1�106)

Clones

1 � 105 1 � 104 1 � 102

CD105� 10/10 25/28 8/10 5/6CD105� 1/10 — — —

3698 Vol. 22 October 2008 BUSSOLATI ET AL.The FASEB Journal

ers vWF, KDR, VEGFR3, and CD31, which were nega-tive in undifferentiated cells, maintained CD105 andCD146 expression, but were negative for CK (Fig. 3B,C). In addition, cells lost the mesenchymal markersCD73 and vimentin. No ability to differentiate intoadipogenic or osteogenic cells was found when cellswere cultured in the appropriate differentiating media.Bone-marrow-derived cells were used as positive controlfor these differentiative conditions (not shown).

Tumor-initiating ability of the CD105� population

Four CD105� cell clones (2 clones derived from renalclear-cell carcinomas and 2 from undifferentiated car-cinomas) were injected subcutaneously in SCID mice.All clones were tumorigenic. Moreover, we found thatas few as 102 CD105� cells from clones were able togenerate tumors (Table 1). Then clones were used togenerate serially transplantable tumors (Table 2). Fromprimary tumors formed in SCID mice by CD105�

clones, we found the presence of CD105� and CD105�

cells, indicating that CD105� cells were able to gener-ate tumor heterogeneity (Fig. 4). We therefore sortedthe CD105� cells and compared the tumorigenic activ-ity of the CD105� cells vs. CD105� cells (Table 2).

CD105� cells gave raise to secondary and tertiarytumors, whereas CD105� cells did not. Moreover, theCD105� cells originated from serially transplanted tu-mors maintained the same phenotype of the primaryclones (not shown).

Analysis of tumors generated by CD105� tumor-initiating cells

After 1 wk from the injection of CD105� clones withinMatrigel, a vascular network of human vessels, as de-tected by the expression of human HLA class I antigen(Fig. 5A, and inset) and CD31 (not shown), was ob-served. The vessels were connected with the mousevasculature, as containing blood erythrocytes. Only afew clusters of epithelial cells were seen. After 3 wk,tumors grew in multiple epithelial nodules containingseveral vessels (Fig. 5B). By immunohistochemistry, thetumors generated by CD105� cell clones expressed CK,vimentin, and EMA (Fig. 5C–F), as described for renalcarcinomas (26, 27). No difference was observedamong tumors originated from different CD105�

clones, nor in different tumor passages. The tumorgrowth was also observed when tumor clones wereinjected subcutaneously in the absence of Matrigel. The

Figure 1. Characterization of CD105� clonesfrom renal carcinomas by cytofluorimetric anal-ysis. Representative FACS analysis of CD105�

cell clones (at the second passage) showing theexpression of the mesenchymal stem cell mark-ers CD105, CD146, CD90, CD73, CD29, andCD44 but not of CD24 and CD133. The darklines indicate the specific antibody; the dottedlines indicate the isotypic control. Similarmarker expression was detected for all 12clones.


Figure 2. Immunofluorescence analysis, growth curves, and sphere formation ofCD105� clones. A) Representative immunofluorescence expression by CD105� cellclones 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 �650. Nuclei were counterstained with Hoechst dye. Similar markerexpression 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 areaoccupied by a confluent primary culture, arbitrarily set to 1 (see Materials andMethods). C) Micrograph representative of spheres generated by cell culture of aCD105� clone (C2) in sphere medium containing EGF and FGF. Original view �200.Similar sphere-generating ability was tested for clones D2, F9, and B5.

Figure 3. Epithelial and endothelial differen-tiation of CD105� clones cultured in differen-tiating media. Cells from CD105� clones culturedin epithelial or endothelial differentiating me-dium for 2 wk acquired the expression of dif-ferentiative markers and lost stem cell markers.A) Representative micrographs showing the ex-pression of the epithelial markers CK and CK7.Cells maintained vimentin expression but lostthe stem cell marker nestin. B) Representativemicrographs showing the expression by immu-nofluorescence analysis of the endothelialmarker vWF and the loss of CK and vimentin.Nuclei were counterstained with Hoechst dye.Independent experiments using 8 different cellclones were performed with similar results. C)Representative FACS analysis of the acquire-ment by the B5 cell clone of the endothelialmarkers CD31, KDR, and VEGFR3 after 14 daysof culture (t�14) in respect undifferentiatedCD105� cells (t�0). CD105 was maintained,whereas the mesenchymal marker CD73 waslost. The dark lines indicate the specific anti-body; the dotted lines indicate the isotypiccontrol. Original view �650 (A, B).


human nature of the tumors was shown by the expres-sion of HLA class I antigen, and not mouse �2 micro-globulin (Fig. 6). We also analyzed whether vesselsdeveloped within the tumor may derive from the trans-planted tumor-initiating cells. We found that the ma-jority of vessels detected around and within the tumorwere of murine origin because they coexpressed CD31and the mouse �2 microglobulin, whereas some of theintratumor vessels were of human origin, as detected byHLA class I and human CD31 expression (Fig. 6).These results suggest that renal tumor-initiating cellsare able to differentiate into different cell types of thetumor, including epithelial and endothelial cells. Toevaluate whether tumors derived from CD105� clonesof different types of carcinomas could recapitulate thetumor subtype of origin, 3 animals per group wereinjected with 2 clones from undifferentiated renalcarcinomas and 2 clones from clear-cell carcinomas,respectively. The animals were sacrificed after 6–8 wk

to compare the morphology and immunohistochemis-try with that of the patients’ tumors. As shown in Fig. 7,the CD105� clones from undifferentiated tumorsshowed morphological aspects comparable to that ofthe tumor of origin. The CD105� clones derived fromclear-cell carcinomas showed the typical clear-cell as-pects only in focal areas, maintaining an undifferenti-ated aspect in most of the tumor possibly because of thehigh proliferation of the tumor. By immunohistochem-istry, tumors derived from different CD105� clonesexpressed EMA, low molecular weight CK, and vimen-tin as the tumor of origin. Tumors derived from clonesof undifferentiated tumors were PAS negative, whereasfocal areas of PAS-positive cells were detectable intumors derived from clones of clear-cell carcinomas(Table 3).

DISCUSSION

In the present study, we found a tumor-initiating cellCD105� population present in human renal carcino-mas.

The identified CD105� cells presented the character-istic of tumor stem cells previously described for cancerstem cells present in other tumor types (11). In partic-ular, the CD105� cells 1) were clonogenic, 2) expressedstem cell markers and lacked differentiative markers, 3)could differentiate in vitro into epithelial and endothe-lial cell types, and 4) could generate in vivo seriallytransplantable tumors. These tumors, despite beingderived from clones expressing mesenchymal markers,were epithelial carcinomas as the tumor of origin andwere characterized by the maintenance of a CD105�

tumorigenic population and by the presence of anontumorigenic differentiated CD105� population.

Different stem cell populations have been identifiedin the normal and neoplastic kidney. In rodents, thepresence of stem cells was identified on the base ofbromodeoxyuridine retention, of stem cell marker ex-pression (Sca-1), on the ability to extrude Hoechst dye

TABLE 2. Tumor generation in serial passages from CD105�

cell clones

Clone

Passage

1 2 3

D2 3/4CD105� 3/3 3/3CD105� 0/3 0/3

F9 3/4CD105� 3/3 3/3CD105� 0/3 0/3

C2 10/10CD105� 4/4 3/3CD105� 0/4 0/3

B5 9/10CD105� 3/3 3/3CD105� 0/3 0/3

Four different CD105� cell clones were used to generate serialtumors by subcutaneous injection of 1 � 105 cells in SCID mice.Secondary and tertiary tumors were subsequently generated fromCD105� but not CD105� cells isolated from tumors of the previouspassage.

Figure 4. Serial tumor generation fromCD105� cell clones. Tumors generated in SCIDmice by CD105� clones (primary tumor) weredigested, and cells recovered and processed bycytofluorimetric analysis. CD105� and CD105�

cells were obtained. CD105� cells were sorted,recovered, and injected to obtain serial tumors.


(so-called side population), or on stringent cultureconditions (for review, see ref. 28). In particular, mul-tipotent mesenchymal stem cells were identified inboth the tubular and glomerular components of thenephron (17–19). In humans, we found a rare popula-tion of CD133� cells that lacked the expression of

hematopoietic markers (CD34 and CD45) and ex-pressed some MSC markers, such as CD29, CD90,CD44, and CD73. Moreover, they expressed Pax-2, anembryonic renal marker (29), suggesting their renalorigin. These cells may undergo epithelial and endo-thelial differentiation both in vitro and in vivo (15).

Figure 5. CD105� tumor-initiatingcells formed tumors in SCID mice.A, B) Representative ematoxilinand eosin micrographs showing theorganization of CD105� cell cloneD2 (1�104 cells) injected subcuta-neously in Matrigel. Matrigel plugswere recovered after 1 or 3 wk.Several vessels connected with themouse vasculature and containingerythrocytes and small clusters oftumor cells were observed after 1 wk(A). By immunohistochemistry ves-sels were positive for the humanHLA class I antigen (A, inset). After3 wk, nodules of tumor cells werepresent (B). C–F) Representativemicrographs of tumor sectionsshowing positivity for CK (C), vi-mentin (D), and EMA (E), and anegative control section (F).CD105� cells and cell clones at different passages showed the same morphology and marker expression. Original view�100; inset, �400.

Figure 6. Vessels and tumor formation by CD105� cell clones subcutaneously injected in SCID mice. A) Representativemicrograph showing the expression of human HLA class I antigen by tumor cells. B) Representative immunofluorescencemicrographs 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 byimmunoistochemistry using an mAb that did not cross-react with the mouse CD31. D, E) Representative immunohistochemistryfor mouse �2 microglobulin showing the presence of murine vessels and isolated cells at the periphery (D) and inside (E) thetumor. F) Micrograph showing the colocalization of mouse �2 microglobulin and mouse CD31 in a peripheral vessel. Originalview �100 (A, D, E); �200 (C); �650 (B, F). Data are representative of 6 experiments with similar results.


Sagrinati et al. (25) isolated and characterized a popu-lation of CD133� CD24� cells from the Bowman’scapsule of adult human kidneys that exhibited a mul-tipotent differentiation ability.

Within renal tumors, we previously detected thepresence of CD133� renal resident stem cells that wereshown to contribute to tumor vasculogenesis and toretain the differentiative capacity of CD133� cells fromnormal renal tissue. However, these cells were nottumorigenic (16). Moreover, in the Wilms’ tumor, theidentification of multiple imprinted and stemnessgenes suggested the origin of this tumor from tumorprogenitor cells of the developing kidney (30).

In the present study, we found that the CD105�

population present in renal cell carcinomas was en-riched in tumor-initiating cells. These cells, selected for

their clonogenic ability, displayed stem cell markersand differentiative ability. In addition, CD105� cloneswere able to grow in suspension, as floating spheres, asdescribed for normal and tumor stem cells in anundifferentiated state (22, 23). Renal CD105� tumor-initiating cells expressed several mesenchymal stem cellmarkers, such as CD44, CD146, CD73, CD29, CD90,and vimentin (24). Moreover, CD105� clones ex-pressed the stem cell markers nestin, Nanog, Musashi,and Oct4. At variance with mesenchymal stem cells ofthe bone marrow (14), CD105� clones expressed therenal marker Pax2 (29) and were unable to differenti-ate into adipocytes or osteocytes.

These data may suggest that the CD105� cells repre-senting a tumor-initiating cell population originatedfrom resident renal stem cells with mesenchymal char-

Figure 7. Morphological appearance of tumorsderived from CD105� clones compared withthe original renal tumors. A, B) Representativemicrographs showing the morphological ap-pearance of an undifferentiated renal carci-noma with glandular aspects (A) and of thetumor developed in SCID mice after 6 wk fromthe subcutaneous injection of 1 � 102 cellsderiving from a CD105� clone of the sametumor (B). Similar glandular aspects are visible.C, D) Representative micrographs showing themorphological appearance of an undifferenti-ated renal carcinoma with sarcomatoid aspects(C) and similar aspects seen in the tumordeveloped in SCID mice after 6 wk from thesubcutaneous injection of 1 � 102 cells derivingfrom a CD105� clone of the same tumor (D). E,F) Representative micrographs showing themorphological appearance of a clear-cell renalcarcinoma (E) and of the tumor developed inSCID mice after 8 wk from the subcutaneousinjection of 1 � 102 cells deriving from aCD105� clone of the same tumor (E). Clear-cellchanges (arrow) and glandular features (arrow-heads) are present in focal areas (E). Originalview �200.

TABLE 3. Immunohistochemical comparison between patient tumor and tumor developed in SCIDmice by CD105� clones

Type CK-LMW (8, 18) Vimentin EMA PAS

Undifferentiated tumorsHuman tumor � � � �Xenograft clones � � � �

Clear-cell carcinomasHuman tumor � � � �Xenograft clones � � � �focal

Immunohistochemistry and PAS staining was performed on the tumor of origin and tumorxenograft in SCID mice (3 animals/group) of 2 clones from undifferentiated renal carcinomas and clearcell carcinomas. CK-LMW, low-molecular-weight CK8 and 18; EMA, antiepithelial membrane antigen;PAS, periodic acid-Schiff staining.


acteristics. The lack of CD133 and CD24 expression,which are present in adult and embryonic renal pro-genitors (15, 25, 31), by tumor-initiating CD105�

clones suggests that they are not derived from theCD133� population. Moreover, CD133� cells previ-ously isolated from renal tumors did not express CD105and were not tumorigenic (16). However, it cannot beexcluded that the CD105� stem cell population that weidentified in renal carcinomas may derive from mu-tated stromal/mesenchymal cells of the tumor or frombone-marrow-derived stem cells. Indeed, mesenchymalstem cells derived from the bone marrow have beensuggested to contribute to cancer both in human andin mouse models (32–34). Moreover, mesenchymalcells present in tumor stroma were shown to be able tofollow an autonomous fate, developing into rapidlygrowing, highly vascularized, and invasive mesenchymaltumors (35). Reports suggest the possibility that thesetumor mesenchymal cells may undergo tumorigenicphenotypical changes induced by the neoplastic micro-environment (36–38). Indeed, various studies showedchromosomal alterations in tumor stroma (39). TheCD105� tumor-initiating cells that we identified inrenal carcinomas, at variance with those derived fromthe stroma that forms mesenchymal tumors, were ableto generate carcinomas expressing an epithelial pheno-type. In vivo, CD105� cells were able to maintain thetumor-initiating CD105� population expressing stemcell properties and lacking differentiative markers.Moreover, CD105� clones were able to generate aprogeny of differentiated CD105� cells unable to gen-erate the tumor and expressing cytokeratin.

Tumor-initiating cells or “cancer stem cells” arecharacterized by their ability to display stem/progeni-tor cell properties: competence for self-renewal andcapacity to differentiate in a heterogeneous tumor cellpopulation (see refs. 1, 11). In addition, if the idea thattumor stem cells originate from mutated stem cells ofthe tissue is true, it is conceivable that tumor stem cellsmay differentiate in different lineages. This has beenshown for melanoma-derived stem/progenitor cellsthat are able to differentiate in multiple mesenchymallineages, such as adypocitic, osteocytic, and chondro-cytic lineages (10). In breast tumors, the ability ofstem/progenitor cells to differentiate in both ductal/luminal cells and myoepithelial cells has been exten-sively shown (22).

In the present study, we demonstrate that clones ofCD105� tumor stem/progenitor cells are bipotent,being able to differentiate into tumor epithelial andendothelial cells in vitro and in vivo. These results areconsistent with recent publications describing the abil-ity of mutated mesenchymal stem cells to generatetumors, tumor adipose tissue, and tumor vasculature(40) and the differentiation of breast tumor stem/progenitor cells not only into cells of the glandularepithelium but also into the endothelial lineage (41).

In conclusion, the results of the present study indi-cate 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 vivoself-maintenance and differentiative abilities and by theexpression of embryonic stem cell markers.

This work was supported by the Associazione Italiana per laRicerca sul Cancro (AIRC), Italian Ministry of University andResearch (MIUR) COFIN and MIUR ex60% grants, theItalian Ministry of Health (Ricerca Finalizzata 02), the Pro-getto S. Paolo Oncologia, and the Progetti Finalizzati RegionePiemonte, Oncoprot.

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


identification of a tumor-initiating stem cell population in human renal carcinomas

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