Title: CD133+ Renal Progenitor Cells Contribute to Tumor Angiogenesis
Abstract: In the present study, we tested the hypothesis that resident progenitor cells may contribute to tumor vascularization and growth. CD133+ cells were isolated from 30 human renal carcinomas and characterized as renal resident progenitor cells on the basis of the expression of renal embryonic and mesenchymal stem cell markers. CD133+ progenitors differentiated into endothelial and epithelial cells as the normal CD133+ counterpart present in renal tissue. In the presence of tumor-derived growth factors, these cells were committed to differentiate into endothelial cells able to form vessels in vivo in SCID mice. Undifferentiated CD133+ progenitors were unable to form tumors when transplanted alone in SCID mice. When co-transplanted with renal carcinoma cells, CD133+ progenitors significantly enhanced tumor development and growth. This effect was not attributable to the tumorigenic nature of CD133+ progenitor cells because the same results were obtained with CD133+ cells from normal kidney. CD133+ progenitors contributed to tumor vascularization as the majority of neoformed vessels present within the transplanted tumors were of human origin and derived from the co-transplanted CD133+ progenitors. In conclusion, these results indicate the presence of a renal progenitor cell population in renal carcinomas that may differentiate in endothelial cells and favor vascularization and tumor growth. In the present study, we tested the hypothesis that resident progenitor cells may contribute to tumor vascularization and growth. CD133+ cells were isolated from 30 human renal carcinomas and characterized as renal resident progenitor cells on the basis of the expression of renal embryonic and mesenchymal stem cell markers. CD133+ progenitors differentiated into endothelial and epithelial cells as the normal CD133+ counterpart present in renal tissue. In the presence of tumor-derived growth factors, these cells were committed to differentiate into endothelial cells able to form vessels in vivo in SCID mice. Undifferentiated CD133+ progenitors were unable to form tumors when transplanted alone in SCID mice. When co-transplanted with renal carcinoma cells, CD133+ progenitors significantly enhanced tumor development and growth. This effect was not attributable to the tumorigenic nature of CD133+ progenitor cells because the same results were obtained with CD133+ cells from normal kidney. CD133+ progenitors contributed to tumor vascularization as the majority of neoformed vessels present within the transplanted tumors were of human origin and derived from the co-transplanted CD133+ progenitors. In conclusion, these results indicate the presence of a renal progenitor cell population in renal carcinomas that may differentiate in endothelial cells and favor vascularization and tumor growth. 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The possibility that tissue-resident normal stem cells may contribute to tumor vascularization and growth has not so far been investigated. We recently identified a population of CD133+ resident renal progenitor cells in adult normal human kidney.18Bussolati B Bruno S Grange C Buttiglieri S Deregibus MC Cantino D Camussi G Isolation of renal progenitor cells from adult human kidney.Am J Pathol. 2005; 166: 545-555Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar These cells were capable of expansion, self-renewal, and differentiation into endothelial or epithelial cells both in vitro and in vivo. In the present study, we investigated the possibility that renal resident progenitor cells may contribute to vascularization, and thus growth, of renal tumors. To test this hypothesis, we investigated the presence of CD133+ progenitor cells in 30 human renal tumors, and after purification and characterization we evaluated their angiogenic potential. Moreover, we studied whether co-transplantation of resident CD133+ progenitor cells with renal carcinoma cells promoted tumor development, angiogenesis, and growth in SCID mice. We also evaluated the tumorigenic ability of CD133+ progenitor cells isolated from tumors in respect to the normal counterpart isolated from normal renal tissue. Studies were performed on 30 renal carcinomas (histological types: 23 clear-cell, four papillary, one chromophobe, and two undetermined renal carcinomas). Patients' ages ranged from 29 to 82 years, and 65% were male. Tumor specimens were finely minced with scissors and then digested by incubation for 1 hour at 37°C in Dulbecco's modified Eagle's medium (DMEM) containing collagenase II (Sigma Chemical Co., St. Louis, MO). After washing in medium plus 10% fetal calf serum (FCS) (Life Technologies, Inc., Grand Island, NY), 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 fluorescence-activated cell sorting analysis for quantification of CD133+ cells. CD133+ cells were isolated by magnetic cell sorting, using the MACS system (Miltenyi Biotec, Auburn, CA).18Bussolati B Bruno S Grange C Buttiglieri S Deregibus MC Cantino D Camussi G Isolation of renal progenitor cells from adult human kidney.Am J Pathol. 2005; 166: 545-555Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar CD133+ cells were plated onto fibronectin in the presence of a nondifferentiating expansion medium, consisting of 60% DMEM LG (Invitrogen, Paisley, UK), 40% MCDB-201, with 1× insulin-transferrin-selenium, 1× linoleic acid 2-phosphate, 10−9 mol/L dexamethasone, 10−4 mol/L ascorbic acid 2-phosphate, 100 U of penicillin, 1000 U of streptomycin, 10 ng/ml epidermal growth factor, and 10 ng/ml platelet-derived growth factor-BB (all from Sigma) and 2% FCS (EuroClone, Wetherby, UK).19Jiang Y Jahagirdar BN Reinhardt RL Schwartz RE Keene CD Ortiz-Gonzalez XR Reyes M Lenvik T Lund T Blackstad M Du J Aldrich S Lisberg A Low WC Largaespada DA Verfaillie CM Pluripotency of mesenchymal stem cells derived from adult marrow.Nature. 2002; 418: 41-49Crossref PubMed Scopus (5254) Google Scholar For cell cloning, single cells were deposited in 96-well plates in the presence of the nondifferentiating expansion medium. The CD133− renal tumor cell fraction, used as control, was plated and maintained in DMEM with 10% FCS. CD133-positive cells from normal renal tissue were obtained as previously described.18Bussolati B Bruno S Grange C Buttiglieri S Deregibus MC Cantino D Camussi G Isolation of renal progenitor cells from adult human kidney.Am J Pathol. 2005; 166: 545-555Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar The K1 renal cancer cell line was previously established and characterized.20Bussolati B Russo S Deambrosis I Cantaluppi V Volpe A Ferrando U Camussi G Expression of CD154 on renal cell carcinomas and effect on cell proliferation, motility and platelet-activating factor synthesis.Int J Cancer. 2002; 100: 654-661Crossref PubMed Scopus (47) Google Scholar Epithelial differentiation was obtained in the presence of fibroblast growth factor-4 (10 ng/ml) and hepatocyte growth factor (20 ng/ml) (Sigma).19Jiang Y Jahagirdar BN Reinhardt RL Schwartz RE Keene CD Ortiz-Gonzalez XR Reyes M Lenvik T Lund T Blackstad M Du J Aldrich S Lisberg A Low WC Largaespada DA Verfaillie CM Pluripotency of mesenchymal stem cells derived from adult marrow.Nature. 2002; 418: 41-49Crossref PubMed Scopus (5254) Google Scholar Endothelial differentiation was obtained by culturing the cells in EBM medium (Cambrex Bio Science, Baltimore, MD) with vascular endothelial growth factor (10 ng/ml) (Sigma) and 10% FCS on endothelial cell attachment factor (Sigma).21Rafii S Lyden D Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration.Nat Med. 2003; 9: 702-712Crossref PubMed Scopus (1467) Google Scholar For osteogenic differentiation, cells were incubated in α-minimal essential medium (α-MEM) supplemented with 10% FCS, 10% horse serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 12 mmol/L l-glutamine, 20 mmol/L β-glycerol phosphate (Sigma), 50 ng/ml thyroxine (Sigma), 1 nmol/L dexamethasone (Sigma), and 0.5 μmol/L ascorbate 2-phosphate (Sigma).22Pittenger MF Mackay AM Beck SC Jaiswal RK Douglas R Mosca JD Moorman MA Simonetti DW Craig S Marshak DR Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (18433) Google Scholar The media was changed two times per week for 3 weeks. The cells were fixed with 10% formalin for 20 minutes at room temperature and stained with Alizarin Red, pH 4.1 (Sigma) for 20 minutes at room temperature. For adipogenic differentiation, the cultures were incubated in α-MEM supplemented with 10% FCS, 10% HS, 100 U/ml penicillin, 100 μg/ml streptomycin, 12 mmol/L l-glutamine, 5 μg/ml insulin (Sigma), 50 μmol/L indomethacin (Sigma), 1 × 10−6 mol/L dexamethasone, and 0.5 μmol/L 3-isobutyl-1-methylxanthine (Sigma).22Pittenger MF Mackay AM Beck SC Jaiswal RK Douglas R Mosca JD Moorman MA Simonetti DW Craig S Marshak DR Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (18433) Google Scholar The medium was changed two times per week for 3 weeks. The cells were fixed with 10% formalin for 20 minutes at room temperature and stained with 0.5% Oil Red O (Sigma) in methanol (Sigma) for 20 minutes at room temperature. For experiments using the tumor cell-conditioned medium, K1 tumor cells were grown until subconfluence in a T75 flask and incubated overnight with 10 ml of DMEM plus 0.1% BSA. The conditioned medium was then removed and added, at 10% concentration, to DMEM plus 10% FCS for CD133+ cell stimulation. CD133+ cells cultured in DMEM plus 10% FCS in the absence of tumor-derived conditioned medium were unable to grow and died after 2 to 3 days. Cytofluorimetric analysis was performed using the following antibodies, all fluorescein isothiocyanate or phycoerythrin conjugated: anti-CD133 monoclonal antibody (mAb) (Miltenyi Biotec), anti-CD44, anti-CD31, anti-CD29, and anti-CD105 mAbs (DakoCytomation, Copenhagen, Denmark), anti-KDR mAb (R&D Systems, Minneapolis, MN); anti-CD34, anti-CD45, anti-CD73, anti-CD90, anti-CD117, and anti-vascular endothelial (VE)-cadherin mAbs (Becton, Dickinson and Company, San Jose, CA). Isotype-matched fluorescein isothiocyanate- or phycoerythrin-conjugated control mouse IgG was from DakoCytomation. For cytofluorimetric analysis, cells were incubated for 30 minutes at 4°C with the appropriate antibody (Ab) or with the irrelevant control in phosphate-buffered saline (PBS) containing 2% heat-inactivated human serum. Where a second step reagent was needed, cells were stained by the addition of fluorescein isothiocyanate-conjugated goat anti-mouse or anti-rabbit IgG and incubated for further 30 minutes at 4°C. Cells were analyzed on a FACScan (Becton, Dickinson and Company). Ten thousand cells were analyzed in each experimental point. Indirect immunofluorescence was performed on cells cultured on chamber slides, fixed in 4% paraformaldehyde containing 2% sucrose and, when needed, permeabilized with HEPES-Triton X-100 buffer.18Bussolati B Bruno S Grange C Buttiglieri S Deregibus MC Cantino D Camussi G Isolation of renal progenitor cells from adult human kidney.Am J Pathol. 2005; 166: 545-555Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar The following antibodies were used: rabbit anti-von Willebrand factor (vWF), mouse anti-E cadherin, and mouse anti-VE cadherin (DakoCytomation), rabbit anti-pan-cytokeratin (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-vimentin (Sigma), and rabbit anti-Pax2 (Covance, Princeton, NJ). Control mouse, rabbit, or goat nonimmune immunoglobulins were used as controls. Fluorescein isothiocyanate-conjugated anti-mouse or anti-rabbit IgG (DakoCytomation) was used as secondary antibodies. Sections from paraffin-embedded blocks of human renal carcinomas or Matrigel plugs were collected onto poly-l-lysine-coated slides. Endogenous peroxidase activity was blocked with 6% H2O2 for 8 minutes at room temperature. Anti-CD133 mAb (Miltenyi), anti-mouse β2-microglobulin, or anti-HLA class I polyclonal Ab (Santa Cruz Biotechnology) or anti-vWF polyclonal antibody or anti-fluorescein/Oregon Green polyclonal Ab (Molecular Probes, Leiden, The Netherlands), used to detect cells labeled with fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CSFE; Molecular Probes),23Garton HJL Schoenwolf GC Improving the efficiency of fluorescent labelling for histological tracking of cells in early mammalian and avian embryos.Anat Rec. 1996; 244: 112-117Crossref PubMed Scopus (18) Google Scholar were applied to slides overnight at 4°C. Horseradish peroxidase-labeled anti-rabbit or anti-mouse Envision polymers (DakoCytomation) were incubated for 30 minutes. The reaction product was developed using 3,3-diaminobenzidine. Omission of the primary Ab or substitution with an unrelated rabbit serum or mouse IgG served as negative control. The percentage of vessels positive for mouse β2-microglobulin or human HLA class I was counted in four nonsequential sections for each experiment at ×100 magnification. For confocal microscopy analysis, sections from cryostatic or paraffin-embedded sample of Matrigel plugs were stained for CD133 antigen, HLA class I polyclonal or HLA class I mAb (BioLegend, San Diego, CA), polyclonal anti-mouse β2-microglobulin Ab (Santa Cruz Biotechnology), anti-fluorescein/Oregon Green polyclonal Ab, and anti-human CD31 (Becton, Dickinson and Company) or anti-mouse CD31 (Abcam, Cambridge, UK) mAbs. Alexa Fluor 488 or Texas Red goat anti-rabbit IgG and Alexa Fluor 488 or Texas Red anti-mouse IgG (all from Molecular Probes) were used as secondary antibody. Confocal microscopy analysis was performed using a Zeiss LSM 5 Pascal model confocal microscope (Carl Zeiss Int., Oberkochen, Germany). Hoechst 33258 dye (Sigma) was added for nuclear staining. Fluorescence in situ hybridization was performed using the Vysion kit for the detection of human chromosome 17 (Vysis Inc., Downers Grove, IL). SG CEP 17 DNA probe hybridizes to the centromere (band region 17p11.1-q11.1, locus D17Z1) of human chromosome 17. In situ hybridization was performed on 5-μm sections according to the manufacturer's guidelines. In brief, the sections were deparaffinized, dehydrated in 100% ethanol, and dried at 45 to 50°C for 2 to 5 minutes. Slides were then subjected to protease digestion for 10 to 20 minutes at 38°C, denatured (72°C for 5 minutes), and hybridized (37°C) with prewarmed probes (CEP17 Spectrum Green; Vysis Inc.) overnight (16 to 18 hours) in HYBrite hybridization system (Vysis Inc.). They were then washed with posthybridization wash buffer (2× standard saline citrate, 0.3% Nonidet P-40) at 72°C and counterstained with 4,6-diamindino-2-phenylindole, mounted, maintained in darkness for 15 minutes at 4°C, and then observed with a confocal microscope. Total RNA was extracted from cells with guanidinium thiocyanate/phenol/chloroform, precipitated with isopropyl alcohol, and washed with 70% ethanol. Two μg of RNA were reverse-transcribed using oligo(dT) primers and 15 U of reverse transcriptase enzyme (Eppendorf, Hamburg, Germany). Two μl of cDNA were amplified with forward (5′-ATGGATATGCACTGCAAAGCAGA-3′) and reverse (5′-CGTGCTGGGAACAATGGTGTG-3′) primers, with TaqDNA polymerase (Invitrogen), as described.24Buttiglieri S Deregibus MC Bravo S Cassoni P Chiarle R Bussolati B Camussi G Role of Pax2 in apoptosis resistance and proinvasive phenotype of Kaposi's sarcoma cells.J Biol Chem. 2004; 279: 4136-4143Crossref PubMed Scopus (41) Google Scholar Reactions were performed for 30 cycles at a melting temperature of 52°C and analyzed with an ethidium bromide-stained 1.5% agarose gel. Samples were postfixed in 2.5% glutaraldehyde, dehydrated in alcohol, dried, and coated with gold by sputter coating. The specimens were examined in a scanning T300 electron microscope (JEOL, Tokyo, Japan). Images were obtained via secondary electron at a working distance of 15 to 25 mm and at an accelerating voltage of 20 to 25 kV. To evaluate angiogenesis, endothelial committed tumor-derived CD133+ cells (1 × 106 cells) were implanted subcutaneously into SCID mice (Charles River, Jackson Laboratories, Bar Harbor, ME) within growth-factor depleted Matrigel basement membrane (Becton, Dickinson and Company), as described.18Bussolati B Bruno S Grange C Buttiglieri S Deregibus MC Cantino D Camussi G Isolation of renal progenitor cells from adult human kidney.Am J Pathol. 2005; 166: 545-555Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar To evaluate progenitor cell tumorigenesis, undifferentiated tumor-derived CD133+ cells (1 × 106 cells) were transplanted subcutaneously into SCID mice within Matrigel basement membrane, as described.25Bussolati B Dunk C Grohman M Kontos CD Mason J Ahmed A Vascular endothelial growth factor receptor-1 modulates vascular endothelial growth factor-mediated angiogenesis via nitric oxide.Am J Pathol. 2001; 159: 993-1008Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar Cells were harvested using trypsin-ethylenediaminetetraacetic acid, washed with PBS, counted in a microcytometer chamber, and resuspended in 250 μl of DMEM. Cells were chilled on ice, added to 250 μl of Matrigel at 4°C, and injected subcutaneously into the left back of SCID mice via a 26-gauge needle using a 1-ml syringe. To evaluate the involvement of CD133+ cells in tumor engraftment, CD133+ or CD133− human cells were transplanted subcutaneously into SCID mice in combination with renal carcinoma cells (K1) obtained from a renal clear carcinoma, at different ratio (1 × 104 CD133+cells/1 × 106 K1 cells, 1:100 ratio; or 1 × 106 CD133+ cells/1 × 104 K1 cells, 100:1 ratio) and compared with injection of K1 cells alone (1 × 104 to 1 × 106). After 7 days (angiogenesis experiments) or 3 or 8 weeks (tumor detection), mice were sacrificed and Matrigel plugs recovered and processed for histology. In selected experiments, CD133+ cells (1 × 105) were labeled with CSFE, formed tumors were digested in Matrigel-digesting solution (Becton, Dickinson and Company) plus collagenase II (Sigma), and the recovered cells were analyzed by cytofluorimetric analysis. We investigated by cytofluorimetric analysis the presence of a cell population expressing the CD133 stem cell marker in cells dissociated from tissue specimens of 30 renal carcinomas (23 clear cell, four papillary, one chromophobe, and two undetermined renal carcinomas). All carcinomas displayed a CD34−/CD133+ cell population with a percentage ranging from 0.25 to 1.87% of the total cells (Figure 1A; 0.90 ± 0.17%). This percentage was not different from that detected in normal renal tissue.18Bussolati B Bruno S Grange C Buttiglieri S Deregibus MC Cantino D Camussi G Isolation of renal progenitor cells from adult human kidney.Am J Pathol. 2005; 166: 545-555Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar No correlation among the number of CD34−/CD133+ cells and tumor stage or grade or patient age or sex was observed (not shown). CD34+/CD133+ cells were undetectable by cytometric analysis. By immunohistochemistry, CD133+ single cells or CD133+ small aggregates were found sparsely within the renal tumor tissue and in proximity of tumor vessels (Figure 1C). CD133+ cell isolates were obtained from all tumors, characterized after magnetic isolation and cultured in nondifferentiating medium.19Jiang Y Jahagirdar BN Reinhardt RL Schwartz RE Keene CD Ortiz-Gonzalez XR Reyes M Lenvik T Lund T Blackstad M Du J Aldrich S Lisberg A Low WC Largaespada DA Verfaillie CM Pluripotency of mesenchymal stem cells derived from adult marrow.Nature. 2002; 418: 41-49Crossref PubMed Scopus (5254) Google Scholar All CD133+ cell isolates did not express the hemopoietic marker CD34 (Figure 1B). Moreover, selected experiments performed depleting the CD34+ cells before magnetic sorting of CD133+ cells did not affect the percentage of CD133+ cells obtained from renal carcinomas or their characteristics. CD34−/CD133+ cells derived from renal carcinomas (RC-CD133+) were characterized in comparison with circulating hematopoietic CD34+/CD133+ cells, with bone marrow-derived mesenchymal stem cells, and with CD34−/CD133+ renal progenitor cells that we previously described in normal human kidney.18Bussolati B Bruno S Grange C Buttiglieri S Deregibus MC Cantino D Camussi G Isolation of renal progenitor cells from adult human kidney.Am J Pathol. 2005; 166: 545-555Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar RC-CD133+ did not express CD45 and CD14 (Figure 1B), suggesting that they did not derive from hematopoietic CD34+/CD133+ cells. In addition, at variance with circulating endothelial progenitors, the RC-CD133+ isolates did not express the endothelial markers KDR (Figure 1B). RC-CD133+ cells expressed the mesenchymal stem cell markers CD44, CD29, and CD73,22Pittenger MF Mackay AM Beck SC Jaiswal RK Douglas R Mosca JD Moorman MA Simonetti DW Craig S Marshak DR Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (18433) Google Scholar but, at variance with bone marrow-derived mesenchymal stem cells, they did not express CD105. Moreover, bone marrow-derived mesenchymal stem cells do not usually express CD133.26Reyes M Lund T Lenvik T Aguiar D Koodie L Verfaillie CM Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells.Blood. 2001; 98: 2615-2625Crossref PubMed Scopus (1128) Google Scholar, 27Wexler SA Donaldson C Denning-Kendall P Rice C Bradley B Hows JM Adult bone marrow is a rich source of human mesenchymal stem cells but umbilical cord and mobilized adult blood are not.Br J Haematol. 2003; 121: 368-374Crossref PubMed Scopus (614) Google Scholar, 28Vogel W Grunebach F Messam CA Kanz L Brugger W Buhring HJ Heterogeneity among human bone marrow-derived mesenchymal stem cells and neural progenitor cells.Haematologica. 2003; 88: 126-133PubMed Google Scholar RC-CD133+ cells expressed the renal embryonic marker Pax-2, which is absent both in mesenchymal and hematopoietic stem cells (Figure 1, B and E) and is present in CD133+ cells derived from normal human renal tissue.18Bussolati B Bruno S Grange C Buttiglieri S Deregibus MC Cantino D Camussi G Isolation of renal progenitor cells from adult human kidney.Am J Pathol. 2005; 166: 545-555Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar No differences were observed among isolates from different histological tumor types. At variance with hematopoietic CD34+/CD133+ cells, RC-CD133+ cells grew in adhesion in nondifferentiating expansion medium for up to seven to nine passages without changes in their phenotype or marker expression. RC-CD133+ cells and clones differentiated into both endothelial and epithelial cells in the presence of specific growth factors. No differences were observed in RC-CD133+ cell lines obtained from different patients, with respect to the different age or sex. Epithelial differentiation by hepatocyte growth factor and fibroblast growth factor-4 was demonstrated by expression of cytokeratin, vimentin, E-cadherin (Figure 2), as well as of the renal markers alkaline p