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.

Figure 1

Isolation and characterization of CD133⁺ progenitor cells from human renal carcinomas. A: Flow cytometric analysis showed that ∼1% of total cells extracted from renal carcinomas express CD133 but not CD34 antigen (right dot plot; left dot plot = isotypic control). B: Flow cytometric analysis of CD133 immunomagnetic sorted cells showing expression of CD133, CD44, CD29, and CD73 but not of CD14, CD45, CD34, or KDR. All cell preparations (n = 30) were examined with similar results. C: Micrograph representative of immunohistochemical detection of CD133⁺ progenitor cells within tissue sections of renal carcinomas. Cells expressing membrane CD133 are visible as single cells or as small cell aggregates near vessels (arrows). D: Micrograph representative of a section stained with the isotypic control instead of the anti-CD133 mAb. E: Micrographs representative of Pax-2 nuclear staining of isolated and cultured RC-CD133⁺ cells. F: Pax-2 mRNA expression evaluated by RT-PCR in RC-CD133⁺ cells (lane 1). CD133⁺/CD34⁺ cells from peripheral blood (lane 2) and bone marrow-derived mesenchymal stem cells (lane 4) were negative. Cloned Pax-2 was used as positive control (lane 3). Original magnifications, ×250.

Figure 2

Differentiation of tumor-derived CD133⁺ progenitor cells. Representative immunofluorescence micrographs of differentiation markers in RC-CD133⁺ cells cultured in epithelial, endothelial, adipogenic, and osteogenic differentiation medium (see Materials and Methods). Cytoplasmic expression of cytokeratin and vimentin and surface expression of E-cadherin was observed in epithelial differentiated cells, which were negative for vWF (original magnifications, ×400). vWF expression, in the classical cytoplasmic punctuate pattern, was detected in endothelial differentiated cells, which were negative for cytokeratin (original magnifications, ×400). Adipogenic and osteogenic differentiation was not observed in RC-CD133⁺ progenitor cells. Adipogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) was detected by the presence of fat droplets stained by Oil Red. Osteogenic differentiation was detected by positive staining for calcium deposits using Alizarin Red (original magnifications, ×250).

Figure 3

Endothelial differentiation of RC-CD133⁺ progenitor cells by the tumor-conditioned medium. A: Representative cytofluorimetric evaluation of endothelial marker expression by RC-CD133⁺ cells before and after culture in the presence of tumor-conditioned medium. After 10 days of culture, cells lost CD133 expression and acquired the expression of the endothelial markers CD31, KDR, VE-cadherin, and CD105. Dark areas, tested antibodies; white areas, isotypic controls. Ten cell preparations were studied with similar results. Micrograph: representative immunofluorescence staining showing the surface expression of VE-cadherin by RC-CD133⁺ cells cultured for 10 days in the presence of tumor-conditioned medium. VE-cadherin was absent in nondifferentiated cells (day 0). B and C: In vivo differentiation of RC-CD133⁺ progenitor cells in functional vessels. CD133⁺ progenitor cells (1 × 10⁶) cultured for 15 days with tumor-conditioned medium were incorporated at 4°C within Matrigel and subcutaneously injected in SCID mice (n = 8). Mice were sacrificed after 7 days, and the Matrigel plugs were excised and processed for histology and immunohistochemistry. B: Scanning electron microscopy performed on a freeze-hatched Matrigel plug, showing representative vessels containing red blood cells. C: The human nature of endothelial cells lining the vessels is indicated by positive immunohistochemical staining for human HLA class I antigen showing that vessels (arrows) formed in Matrigel derived from the implanted human progenitor cells. Original magnifications: ×1000 (B); ×400 (C).

Figure 4

Role of tumor-derived CD133⁺ progenitor cells in growth and vascularization of transplanted tumors. Tumor-derived CD133⁺ progenitor cells (1 × 10⁴ cells) were co-transplanted with K1 renal carcinoma cells (1 × 10⁶ cells) in a ratio 1:100 within diluted Matrigel by subcutaneous injection in the left abdominal region of SCID mice. In the right abdominal region, the K1 renal carcinoma cells alone (1 × 10⁶ cells) were injected. A: Representative micrograph showing the macroscopic appearance of tumors formed in experiments of co-transplantation of K1 and CD133⁺ stem cells (left circle). The left inset shows the low-power microscopic examination of the tissue stained with H&E. No tumor developed when K1 cells were transplanted alone (right circle and inset). B and C: Representative micrographs of a section of a tumor induced by co-transplantation of K1 and CD133⁺ progenitor cells stained by immunoperoxidase with mouse β₂-microglobulin Ab. Numerous neoformed vessels derived from mouse were detectable around the tumor and very few within (arrow). D–F: Representative micrographs of a tumor section stained by immunoperoxidase with anti-human HLA class I antigen Ab. HLA class I antigen was expressed by tumor cells and endothelial cells lining capillaries both in the periphery (arrows; D and E, inset) and within the tumor (arrows, D–F) indicating an human origin of the positive vessels. G: Representative micrograph of a tumor section stained with anti-vWF Ab, showing an extensive vascular network within the tumor. Data are representative of 10 mice per experimental group. H and I: Representative micrographs of a section of a tumor induced by transplantation of K1 cells alone stained by immunoperoxidase with anti-human HLA class I antigen Ab. HLA class I antigen was expressed by tumor cells but not by endothelial cells lining capillaries (I, arrow). J and K: Representative micrographs of a tumor section stained by immunoperoxidase with mouse β₂-microglobulin Ab. Vessels derived from mouse were stained around and within the tumor (K, arrows). L: Representative micrographs showing co-localization of mouse β₂-microglobulin (red) and mouse CD31 (green) within the implanted K1 tumor seen by confocal microscopy. Data are representative of the two mice that developed K1 tumor. Original magnifications: ×40 (A, B, D); ×150 (C, H, I); ×400 (E, G, J, K); ×600 (E, inset; F); ×630 (L).

Figure 5

Contribution of tumor-derived CD133⁺ progenitor cells to vessels formed within the co-implanted K1 renal carcinoma cells. A and B: Representative micrographs showing co-localization of human HLA class I and human CD31 in vessels surrounding (A) or within (B) the implanted tumor seen by confocal microscopy. B, inset: Representative micrographs showing co-localization of human CD133 and human CD31 in a vessel within the implanted tumor. C: Representative micrographs showing the absence of co-localization of mouse β₂-microglobulin and human CD31 in vessels within the implanted tumor seen by confocal microscopy. D: Representative micrographs showing co-localization of mouse β₂-microglobulin and mouse CD31 in a vessel within the implanted tumor. Few cells of murine origin were detected around the vessel. E–G: Representative micrographs showing that positivity for human chromosome 17 is detectable in cells underlining vessels (V) as well as in isolated cells within the implanted tumor (E) by fluorescence in situ hybridization analysis. The red autofluorescence of erythrocytes was exploited to show a patent vessel (V). Chromosome 17 was used to identify the injected progenitors because deletion of this chromosome was previously found in K1 renal cells. F: Murine surrounding tissues and vessels (V) were negative. G: Human chromosome X, which identifies all human cells, was positive in tumor and vessels. Data are representative of six experiments with similar results. Original magnifications, ×600.

Figure 6

In vivo endothelial differentiation of tumor-derived CD133⁺ progenitor cells co-implanted with K1 renal carcinoma cells. CD133⁺ progenitor cells were labeled with the fluorescent dye CSFE before the co-transplantation experiments and 2 weeks after the tumors were digested, and the recovered cells were analyzed for the expression of endothelial markers. A: Representative cytofluorimetric analysis of CSFA fluorescent cells. B–D: Expression by CSFE-positive cells of CD31, CD105, and KDR that were negative in nondifferentiated CD133⁺ progenitor cells. E and F: Representative micrographs showing positive cells in some vessels within and around the tumor by immunohistochemical staining of CSFE. G: Representative micrographs showing co-localization of human CD31- and CSFE-positive cells in a vessel (arrow) but not in isolated cells detected within the tumor (arrowheads). Data are representative of four experiments with similar results. Original magnifications: ×40 (E); ×250 (F); ×400 (F, inset); ×600 (G).