Published OnlineFirst June 13, 2011; DOI: 10.1158/0008-5472.CAN-11-0241
Tumor and Stem Cell Biology
Microvesicles Released from Human Renal Cancer Stem Cells Stimulate Angiogenesis and Formation of Lung Premetastatic Niche
Cristina Grange1, Marta Tapparo1, Federica Collino1, Loriana Vitillo1, Christian Damasco1, Maria Chiara Deregibus1, Ciro Tetta2, Benedetta Bussolati1, and Giovanni Camussi1
Abstract
Recent studies suggest that tumor-derived microvesicles (MV) act as a vehicle for exchange of genetic information between tumor and stromal cells, engendering a favorable microenvironment for cancer develop- ment. Within the tumor mass, all cell types may contribute to MV shedding, but specific contributions to tumor progression have yet to be established. Here we report that a subset of tumor-initiating cells expressing the mesenchymal stem cell marker CD105 in human renal cell carcinoma releases MVs that trigger angiogenesis and promote the formation of a premetastatic niche. MVs derived only from CD105-positive cancer stem cells conferred an activated angiogenic phenotype to normal human endothelial cells, stimulating their growth and vessel formation after in vivo implantation in immunocompromised severe combined immunodeficient (SCID) mice. Furthermore, treating SCID mice with MVs shed from CD105-positive cells greatly enhanced lung metastases induced by i.v. injection of renal carcinoma cells. Molecular characterization of CD105-positive MVs defines a set of proangiogenic mRNAs and microRNAs implicated in tumor progression and metastases. Our results define a specific source of cancer stem cell–derived MVs that contribute to triggering the angiogenic switch and coordinating metastatic diffusion during tumor progression. Cancer Res; 71(15); 5346–56. !2011 AACR.
Introduction
Recent studies showed that exosomes/microvesicles (MV) released by cells act as mediator of intercellular commu- nications (1–3). Tumor cells produce large amount of MVs that may enter in the circulation and in other biological fluids (4, 5). It has been suggested that MVs, due to their pleiotropic effect, could be involved in cancer development, progression, and formation of the premetastatic niche (6). MVs contain mRNAs, microRNAs (miRNA), and proteins that could be transferred to target cells inducing epigenetic changes (7–10). Moreover, tumor-derived MVs may trans- port to neighboring cells, the products of oncogenes (11). Emerging evidence suggests that, in cancer patients, circu- lating miRNAs are stable in blood, probably due to their incorporation in exosomes/microvesicles, allowing their use as novel diagnostic markers (12).
Authors’ Affiliations: 1Department of Internal Medicine, Molecular Bio- technology Center and Center for Experimental Medicine (CeRMS), Uni- versity of Torino, Turin, Italy; and 2Fresenius Medical Care, Bad Homburg, Germany
Note: Supplementary data for this article are available at Cancer Research Online (http://clincancerres.aacrjournals.org/).
Corresponding Author: Giovanni Camussi, Dipartimento di Medicina Interna, Corso Dogliotti 14, 10126, Turin, Italy. Phone: 39-011-6336708; Fax: 39-011-6631184; E-mail: giovanni.camussi@unito.it
doi: 10.1158/0008-5472.CAN-11-0241
!2011 American Association for Cancer Research.
5346 Cancer Res; 71(15) August 1, 2011
It is generally recognized that tumors contain a hetero- geneous population of cells with different proliferation and differentiation potential. The majority of cells that form tumors are designated to differentiate and ultimately to stop dividing. At variance, a minor population of cells, defined as cancer stem cells or tumor-initiating cells, possess self- renewal capability and can induce tumors in immunocom- promised animals (13). Recently, we identified in human renal cell carcinoma a subset of tumor-initiating cells expressing the mesenchymal stem cell marker CD105 that display stem cell properties, such as clonogenic ability, expression of Nestin, Nanog, and Oct3-4 stem cell markers, and lack of epithelial differentiation markers (14). This CD105þ population has the capacity to generate epithelial and endothelial cells and serially transplantable tumors in vivo (14).
Previous studies showed that normal stem cells are an abundant source of MVs that may act as paracrine mediators by a horizontal transfer of genetic information (7, 8, 15).
The aim of the present study was to evaluate whether MVs released by CD105þ cancer stem cells of renal carcinomas may modify tumor microenvironment by triggering angiogenesis and may favor the formation of a premetastatic niche.
Material and Methods
Cell culture
Human umbilical vein endothelial cells (HUVEC) were obtained and characterized as previously described (8). CD105þ cancer stem cells, 3 deriving CD105þ clones,
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Cancer Research

Published OnlineFirst June 13, 2011; DOI: 10.1158/0008-5472.CAN-11-0241
CD105 tumor cells, and unsorted tumor cells were previously isolated and characterized (14). Briefly, cell suspension obtained from 5 specimens of renal carcinomas of patients undergoing radical nephrectomy with informed consent were either used to generate unsorted tumor cells or sorted by anti- CD105 magnetic beads (MACS system; Miltenyi Biotec; ref. 14). To avoid the presence of nonneoplastic contaminating cells, CD105þ cancer stem cells either were grown in expansion medium without serum (14) or were cloned. Three clones originating from 3 different renal cell carcinomas were used. The CD105 population could not generate clones. The CD105þ clones and the total CD105þ cell population were negative for the endothelial or hematopoietic markers CD31, VEGF receptor (VEGFR) 2, and CD45. In addition, they showed cancer stem cells properties as expression of stem cell markers and lack of differentiative markers, ability to grow in spheres, and the ability to initiate tumors and generate serially trans- plantable tumors with a number of cells as few as 100 cells per mouse (Supplementary Table S1). All cell types were thawed, used within 2 months, and the phenotype was characterized by fluorescence-activated cell-sorting (FACS) analysis and immunofluorescence immediately before the generation of MVs. The previously described (16) K1 renal tumor cell line was thawed and characterized by FACS immediately before their use for metastases generation.
Isolation and characterization of MVs
MVs were obtained from cell supernatants by ultracentri- fugation as previously described (8). The protein content of MV preparations was quantified by Bradford method (Bio-Rad). In selected experiments, MVs were labeled with the red PKH26 dye (Sigma). The mean diameter of MVs and zeta potential were determined using a Malvern dynamic light-scattering spectrophotometer (Malvern Zetasizer 3000HS) and by transmission electron microscopy (17). Cyto- fluorimetric analysis was carried out as described (17), using the following fluorescein isothiocyanate (FITC)- or phycoer- ythrin (PE)-conjugated antibodies: CD44 (Dakocytomation), CD73, and CD29 (BD Biosciences), CD105, a5-integrin, a6- integrin, and HLA class I (BioLegend). FITC or PE mouse isotypic IgG (Dakocytomation) were used as controls. Beads of different sizes (1, 2, and 4 mm; Invitrogen) were used as size markers. In selected experiments, CD105þ MVs derived from cloned CD105þ cancer stem cells were treated with 1 U/mL RNase (Ambion) for 3 hours at 37C (RNase CD105þ MV; refs. 9, 10). After RNase treatment, the reaction was stopped by the addition of 10 U/mL RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation. The efficacy of RNase treat- ment was evaluated by MV RNA analyses by Agilent 2100 bioanalyzer (Agilent Technologies) and by 0.6% agarose gel electrophoresis.
mRNA analysis
RNA from MVs was isolated using the RNAqueous Micro Kit (Ambion). RNA was quantified spectrophotometrically (Nano- drop ND-1000), and its quality was assessed by Agilent 2100 Bioanalyzer. mRNA expression levels were analyzed using the RT2 Profiler PCR array system (SABiosciences-
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Renal Cancer Stem Cell Microvesicles
Qiagen) to profile 84 genes involved in angiogenesis by real-time (RT-PCR). A pool of RNA from 4 MV preparations (400 ng CD105þ or CD105 MVs) was retrotranscribed and run on 7900HT RT-PCR instrument (Applied Biosystems). Raw Ct values were calculated using SDS software (version 2.3), using automatic baseline and threshold. Quantitative RT-PCR (qRT-PCR) validation of gene array data was carried out using SYBR green technique (Supplementary Material).
miRNA analysis
RNA was isolated using the mirVana miRNA Isolation Kit (Ambion). TaqMan MicroRNA Assay Human Panel Early Access kit (Applied Biosystems) was employed to profile 365 mature miRNAs by qRT-PCR. Sixty nanograms of RNA from CD105þ or CD105 MVs was analyzed. Raw Ct values were calculated using the SDS software. miRNAs with raw Ct values greater than 35 in both preparations were not included in the analysis, as they were considered nonspecific (18, 19). Using filtering criteria, 82 and 87 miRNAs present in CD105þ and CD105 MVs, respectively, were included in the analysis. As the small nucleolar RNAs (snoRNAs; internal controls) were undetectable in MV preparations, endogenous control was calculated using the mean value of 4 of the most stable miRNAs between CD105þ MVs and CD105 MVs (hsa-miR- 181b, -27a, -484, and -324-3p; refs. 20, 21). Relative quantifica- tion (RQ) was obtained using the equation 2ðDDCtÞ (where DDCt is the difference between DCt CD105þ MVs and DCt CD105 MVs; DCt 1⁄4 mean Ct miRNA  mean Ct of endogen- ous control). To confirm some miRNAs identified by micro- array analysis, qRT-PCR, using SYBR green technique, was carried out (Supplementary Material).
Gene targets analysis
The software TargetScan (http://www.targetscan.org) was employed to predict genes target for upregulated miRNAs in CD105þ MVs. To define a core list, genes that were target of at least 5 miRNAs were selected. This group of genes was searched for GO (Gene Ontology) term enrichment, using the GO annotations (http://www.geneontology.org). We used Fisher’s exact test to evaluate GO keywords overrepresenta- tion. A P value of more than 104 was considered as statis- tically significant for GO terms overrepresentation.
Internalization of MVs
HUVEC labeled with carboxyfluoroscein succinimidyl ester (CSFE Vybrant CFDA SE Cell Tracer Kit; Molecular Probe) were incubated for 1 hour at 37C with PKH26-labeled CD105þ and CD105 MVs, and after washing they were analyzed by confocal microscopy (LSM 5 Pascal; Carl Zeiss International; ref. 17). Hoechst 33258 dye (Sigma) was added for nuclear staining.
In vitro angiogenesis assay
In vitro formation of capillary-like structures was done on
growth factor–reduced Matrigel (BD Biosciences; ref. 8). HUVECs (3  104 cells per well) were seeded onto Matri- gel-coated wells in RPMI þ 5% fetal calf serum (FCS) with or without 30 mg/mL MVs. Cell organization onto Matrigel was
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microscopically recorded after 16 hours. Data were expressed as the mean  SD of tubule length in arbitrary units per field.
Invasion, apoptosis, and adhesion assays
The effect of CD105þ MVs, RNase CD105þ MVs, CD105 MVs, and unsorted tumor MVs on Matrigel invasion and apoptosis resistance of HUVECs and on adhesion of K1 tumor cells to HUVEC were evaluated. Invasion was evaluated in 24- well cell culture inserts (BD Biosciences) with a porous membrane (8.0-mm pore size) precoated with 100 mg Matrigel per well as described (21). Total area of invaded Matrigel (magnification  100) was evaluated by MicroImage analysis system (Cast Imaging srl). Apoptosis was carried out using terminal deoxynucleotidyl transferase–mediated dUTP nick
end labeling (TUNEL) assay (ApopTag Fluorescein Direct In Situ Apoptosis; Millipore). Adhesion assay was carried out on HUVEC monolayer pretreated for 24 hours at 37C in RPMI þ 5% FCS with or without MVs. Renal K1 tumor cells (5  105 per well), labeled with CSFE, were added to the endothelial monolayer. The adhesion assay in static conditions was evaluated after 6 hours. After washings, cells adherent to HUVECs were counted by fluorescence microscopy (magnifi- cation  200) in 10 fields and expressed as mean  SD of cells per field.
In vivo angiogenesis
Animal experiments were carried out according to the
guidelines for the care and use of research animals and
ABC
D
10 α5-integrin
100 101 102 103 104 CD44
HLA class I
100 101 102 103 104 α65-integrin
100 101 102 103 104 α6-integrin
100 101 102 103 104 CD105
0 101 102 103 104 100 101 102 103 104
1μm
100 101 102 103 104 1 2 3 FSC-H 100 10 10 10 104
100 101 102 103 104 CD44
100 101 102 103 104 α6-integrin
CD 105
Figure 1. Characterization of MVs. Representative micrographs of transmission electron microscopy of CD105þ MVs (A) and CD105 MVs (B) showing a spheroid shape (original magnification 10,000; bar, 100 nm). CD105 MVs display the same morphology and size (not shown). C, representative FACS analyses of CD105þ MVs showing the size (with 1, 2, and 4-mm beads used as internal size standards) and the expression of CD105, a6-integrin, CD44, a5- integrin, and HLA class I (thick lines) surface molecules. In the CD105, a6-integrin, CD44, and a5-integrin experiments, the Kolmogrov–Smirnov statistical analysis between relevant antibodies and the isotypic control was significant (P < 0.001). No significant expression of HLA class I was observed. D, representative cytofluorimetric analyses of CD105 MVs showing the expression of a6-integrin, CD44, a5-integrin (the Kolmogrov–Smirnov statistical analysis between relevant antibodies and the isotypic control was significant: P < 0.001). CD105 was negative. Dotted lines indicate the isotopic controls. MV preparations derived from 3 CD105þ clones, 5 CD105þ uncloned cancer stem cells, and 5 CD105 tumor cells were analyzed with similar results.
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0
Counts
40 80 120 160 200
0 40
Counts
80 120 160 200
Counts
0 40 80 120 160 200
Counts
0 40 80 120 160 200
0
Counts
40 80 120 160 200
0 40
Counts
80 120 160 200
0
Counts
40 80 120 160 200
0 40
Counts
80 120 160 200
Counts
0 40 80 120 160 200
SSC-H 100 101 102
103 104

Published OnlineFirst June 13, 2011; DOI: 10.1158/0008-5472.CAN-11-0241
were approved by the local Ethics Committee. HUVEC, pres- timulated with or without 70 mg MVs, were implanted subcutaneously into severe combined immunodeficient (SCID) mice (Charles River) within Matrigel (22). At day 10, mice were sacrificed and the Matrigel plug was recovered. Angiogenesis was calculated as the mean  SD of the number of vessels with red cells inside per total area of hematoxylin and eosin–stained sections. Immunohistochemistry was car- ried out using anti-HLA class I (Santa Cruz Biotechnology) and anti-von Willebrand factor (vWF; Dakocytomation) antibody.
In vivo metastasis
SCID mice were injected intravenously daily for 5 days with
70 mg of MVs in 100 mL PBS. On day 5, mice received an i.v. injection into the tail vein of 6  105 renal K1 tumor cells. Mice were sacrificed after 5 weeks, and organs (lung, spleen, liver, and kidney) were collected for histology. Lung metastases were counted in 5 nonsequential serial sections; results were expressed as mean  SD of metastasis per lung (23). On day 5, a total of 8 mice treated with CD105þ MVs, CD105 MVs, and PBS (vehicle) were sacrificed and their lungs processed for histology, RNA extraction, and murine endothelial cells sorting using magnetic beads anti-CD146 (MACS system; Supplementary Fig. S1 and Supplementary Material). Immu- nohistochemistry was carried out using matrix metallopro- teinases (MMP) anti-MMP2 and MMP9 (Santa Cruz Biotechnology) antibodies. Cytofluorimetric analysis on lung
Figure 2. Characterization of MV RNAs. A, representative bioanalyzer profile of the RNAs contained in CD105þ MVs derived from CD105þ clones and in CD105 MVs showing that the ribosomal subunits 28S and 18S were absent or barely detectable. B, representative bioanalyzer profile of small RNAs was obtained using RNA subtypes present in CD105þ MVs and CD105. Three different samples tested in triplicate were analyzed with similar results. C, GO enrichment analysis of target genes of at least 5 upregulated miRNAs in CD105þ MVs. Fisher’s exact test to evaluate GO keywords overrepresentation was used. A P < 104 was considered statistically significant for GO terms. Overrepresented biological processes are grouped according to their common ancestor.
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Renal Cancer Stem Cell Microvesicles
endothelial cells was carried out using anti-CD31, anti-CD146 (BD Biosciences), anti-CD45 (Miltenyi), anti-VEGFR1 (R&D), and anti-a6-integrin (Biolegend) monoclonal antibodies. qRT- PCR for murine MMP9, MMP2, and VEGF was carried out using SYBR green technique on total lung tissues and endothe- lial cell fractions (Supplementary Material).
Statistical methods
Differences were determined by Student’s t test or by ANOVA followed by the Newman–Keuls multicomparison test when appropriate. A value of P < 0.05 was considered significant.
Results
Characterization of MVs shed by CD105þ renal cancer stem cells
MVs released from CD105þ cancer stem cells (n 1⁄4 5) and deriving clones (n 1⁄4 3) were compared with MVs released from CD105 tumor cells (n 1⁄4 5). MVs generated by CD105þ cancer stem cells and derived clones and by the CD105 tumor cells had the same morphology and size, ranging from 10 to 100 nm as determined by zeta-size analysis and electron microscopy (Fig. 1A and B). Moreover, they showed the same zeta potential of 22.4  3.5 mV. By cytofluorimetric analysis, MVs were detected below the forward scatter signal corre- sponding to 1-mm beads. The main difference between MVs
A [FU] 5
CD105+ MV
[FU]
2
CD105– MV
B [FU] 20
0
CD105+ MV
[FU] 20
0
CD105– MV
80 150 [nt]
C
Metabolic process (23.5%) Transcription (14.7)
Regulation of biological process (11.8%) Nucleic acid binding (8.8%) Developmental process (8.8%)
Transcription regulator activity (5.9%) Cell adhesion (5.9%)
Regulation of cell proliferation (5.9%) Cellular component (5.9%)
Other (5.9%)
0
152025303540455055 [s]
0
152025303540455055 [s]
4
20 40
80
150
[nt]
4
20 40
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Table 1. miRNAs differentially expressed in CD105þ MVs with respect to CD105 MVs
Downregulated miRNA
Upregulated
RQ miRNA RQ
hsa-miR-142-5p RNU6B hsa-miR-15a hsa-miR-129 hsa-miR-101 hsa-miR-296 hsa-miR-145 hsa-miR-361 hsa-miR-23b hsa-miR-23a hsa-miR-100 hsa-miR-99b hsa-miR-324-5p hsa-miR-30a-5p hsa-let-7b hsa-miR-7 hsa-miR-15b hsa-miR-27b hsa-let-7f hsa-miR-615 hsa-miR-218 hsa-miR-328 hsa-miR-10a hsa-miR-222 hsa-let-7a hsa-miR-342 hsa-miR-125a hsa-miR-572 hsa-miR-149 hsa-miR-30d hsa-let-7c hsa-miR-451 hsa-miR-25
0.0006 hsa-miR-200c 133.9685
0.0040 hsa-miR-146a 0.0162 hsa-miR-184 0.01657 hsa-miR-335 0.0396 hsa-miR-646 0.0651 hsa-miR-449b 0.0694 hsa-miR-650 0.0874 hsa-miR-141 0.1088 hsa-miR-183 0.1385 hsa-miR-19b 0.1470 hsa-miR-29c 0.1985 hsa-miR-182 0.2096 hsa-miR-19a 0.2379 hsa-miR-92 0.2441 hsa-miR-301 0.2555 hsa-miR-151 0.2622 hsa-miR-130b 0.2986 hsa-miR-29a 0.3062 hsa-miR-22 0.3205 hsa-miR-186 0.3264 hsa-let-7g 0.3407 hsa-miR-140 0.3594 hsa-miR-486 0.3697 hsa-miR-26b 0.3741
0.3881 0.3922 0.3965 0.4018 0.4114 0.4418 0.4593 0.4798
68.6291 34.3860 34.2195 30.2686 19.5724 12.9308
9.2135 7.3653 4.2390 3.4791 3.1749 3.1010 3.0903 2.8064 2.7697 2.6884 2.3097 2.1927 2.1565 2.1313 2.1239 2.1210 2.0601
NOTE: RQ was obtained using the equation 2ðDDCtÞ (where DDCt is the difference between DCt CD105þ MVs and DCt CD105 MVs; DCt 1⁄4 mean Ct miRNA  mean Ct of endo- genous control).
derived from CD105þ cancer stem cells and CD105 tumor cells was the expression of CD105 present only on MVs derived from CD105þ cells (CD105þ MVs) but not on those derived from CD105 cells (CD105 MVs). Both CD105þ and CD105 MVs expressed CD44 and adhesion molecules such as a5- and a6-integrins (Fig. 1C and D) as the cells of origins, whereas CD29 was barely detectable in CD105þ MVs and negative in CD105 MVs (not shown). Both MV types did not express HLA class I (Fig. 1) and CD73 (not shown).
Characterization of RNAs shuttled by MVs
We carried out a bioanalyzer profile of total RNA present in CD105þ MVs from cloned cancer stem cell preparations and CD105 MVs. Both MVs contained RNA of different size, suggesting the presence of mRNAs and of small RNAs compatible with the presence of miRNAs, whereas the ribosomal subunit 28S and 18S were barely detectable (Fig. 2A). In the CD105þ MVs, we observed an enrichment of small RNAs of the size of miRNAs (42.3%  2.5%) in comparison with CD105 MVs (20.2%  1.7%; Fig. 2B). miRNA expression by MVs shed from CD105þ and CD105 cells was then screened by qRT-PCR profiling 365 human mature miRNAs. CD105þ and CD105 MVs revealed the presence of 82 and 87 miRNAs, respectively. Twenty-four miRNAs were significantly upregulated in CD105þ MVs with respect to CD105 MVs, whereas 33 miRNAs were signifi- cantly downregulated (Table 1). To confirm data obtained from miRNA screening, single miRNAs were selected and analyzed in 3 different preparations of CD105þ and CD105 MVs by qRT-PCR (Supplementary Table S2). To characterize the biological processes modulated by the upregulated miRNAs present in CD105þ MVs, we analyzed their target genes predicted by TargetScan algorithm, selecting those genes targeted by almost 5 miRNAs. This list counted 157 genes (Supplementary Table S3). We carried out the func- tional characterization of the gene target list searching for GO keywords enrichment and we found a strong overrepre- sentation of terms belonging to crucial biological processes such as transcription, metabolic process, nucleic acid bind- ing, cell adhesion molecules, and regulation of cell prolifera- tion (Fig. 2C and Supplementary Table S4).
Moreover, we investigated whether CD105þ MVs contained mRNAs involved in the stimulation of angiogenesis in compar- ison with CD105 MVs. mRNAs of genes involved in angiogen- esis were detected only in CD105þ MVs. In particular, they contained mRNAs for growth factors such as VEGF, fibroblast growth factors 2 (FGF2), angiopoietin1, and ephrin A3 and for MMP2 and MMP9. Each mRNA detected was confirmed on 3 different CD105þ MV preparations by using qRT-PCR.
In vitro activation of HUVEC by CD105þ MVs
To evaluate whether MVs derived from CD105þ renal cancer stem cells could be responsible for stimulating tumor angio- genesis and invasion, we compared their effects with MVs from CD105 tumor cells. We first evaluated the uptake of CD105þ and CD105 MVs labeled with PKH26 dye by HUVEC, after 1- hour incubation at 37C. HUVECs incorporated in equal man-
ner both CD105þ and CD105 MVs (Fig. 3A). 5350 Cancer Res; 71(15) August 1, 2011
CD105þ MVs from cancer stem cells and deriving clones stimulated HUVEC to organize in vitro into capillary-like structures on Matrigel. In contrast, CD105 MVs did not induce the formation of capillary-like structures. MVs derived from unsorted tumor cells also induced the formation of capillary- like structures, but the proangiogenetic effect of MVs from CD105þ sorted cells was significantly greater (Fig. 3B and C). Moreover, CD105þ MVs, but not CD105 MVs, significantly enhanced the invasion of HUVECs through Transwells coated with Matrigel, with respect to CD105 MVs as well as to MVs from unsorted tumor cells (Fig. 4A and B). CD105þ MVs also
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Renal Cancer Stem Cell Microvesicles
Figure 3. Internalization of MVs in HUVECs and in vitro angiogenic effect. A, representative confocal microscopy analysis of red-labeled MVs in HUVECs stained with CFSE (green). Seven experiments were carried out with similar results (original magnification 630). Quantitative evaluation (B) and representative micrographs (C) showing the formation of capillary-like structure formed by HUVECs seeded on Matrigel-coated plates in a serum-starved condition (RPMI) and stimulated with 30 mg/mL of CD105þ MVs from uncloned and cloned cancer stem cell preparations, RNase CD105þ MVs derived from cloned cancer stem cells, CD105 MVs, and MVs from unsorted tumor cell (TMV). Data are expressed as the mean  SD of the length of capillary-like structure after 16 hours, evaluated by the computer analysis system in arbitrary units (AU) in at least 10 different fields at 200 magnification. Four different experiments per group were carried out in duplicate. ANOVA with the Newman–Keuls multicomparison test was carried out: *, P < 0.05, CD105þ MV versus RPMI, RNase CD105þ MV, CD105 MV, and TMV; §, P < 0.05, TMV versus RPMI and CD105 MV.
induced a greater apoptosis resistance in HUVECs treated with 100 ng/mL of doxorubicin (Fig. 4C). To investigate whether MV treatment could modify the adhesive property of endothelial cells, HUVECs were pretreated with different MVs and, after 6 hours, the adhesion of renal tumor cells was evaluated. CD105þ MVs significantly enhanced the adhesion of tumor cells with respect to CD105 MVs and unsorted tumor MVs (Fig. 4D). MVs from unsorted tumor cells induced inva- sion, apoptosis resistance, and tumor cells adhesion in HUVECs that were greater with respect to CD105 MVs or vehicle, suggesting that the effects observed by tumor cell– derived MVs should be ascribed to MVs released from cancer stem cells.
RNase pretreatment of CD105þ MVs significantly reduced in vitro capillary-like formation (Fig. 3B), as well as the enhanced invasion, apoptosis resistance, and adhesion prop- erties (Fig. 4), suggesting a role of RNA molecular species carried by MVs.
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In vivo effects of CD105þ MVs
To evaluate whether CD105þ MVs were able to stimulate
angiogenesis in vivo, we subcutaneously injected MV-stimu- lated HUVECs within Matrigel in SCID mice. CD105þ MVs from cloned cancer stem cell preparations stimulated the growth of HUVECs that formed dense clusters containing small vessels organized into patent capillaries connected with the murine vasculature and into large aneurismatic structures (Fig. 5A). The cells grew into Matrigel, and the vessels expressed the endothelial marker vWF and their human nature was shown by staining for HLA class I (Fig. 5B). HUVECs challenged with vehicle or CD105 MVs or RNase CD105þ MVs did not organize or proliferate into the Matrigel. MVs from unsorted tumor cells induced HUVEC proliferation and organization into small vessels, but the extent of angiogenesis was significantly lower than that induced by CD105þ MVs (Fig. 5C).
To evaluate whether CD105þ MVs contribute to establish a premetastatic niche, we intravenously injected SCID mice for
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Figure 4. Effect of MVs on endothelial cell invasion, apoptosis resistance, and tumor cell adhesion to endothelium. Quantitative evaluation (A) and representative micrographs (B) showing the invasion of Matrigel-coated Transwells by HUVECs stimulated with 30 mg/mL of MVs from uncloned and cloned cancer stem cell preparations, RNase CD105þ MVs, CD105 MVs, and TMVs. Invasion was evaluated after 24 hours. Data are expressed as the mean  SD of the area occupied by cells on total well-surface area evaluated by the computer analysis system in arbitrary units (AU) at 100 magnification. ANOVA with the Newman–Keuls multicomparison test was carried out: *, P < 0.05, CD105þ MV versus RPMI, RNase CD105þ MV, CD105 MV, and TMV; §, P < 0.05: TMV versus RPMI and CD105 MV. C, quantitative evaluation of apoptosis of HUVECs cultured in the presence of 100 ng/mL of doxorubicin plus vehicle or 30 mg/mL of MVs from uncloned and cloned cancer stem cell preparations, RNase CD105þ MVs, CD105 MVs, and TMVs. Apoptosis was evaluated by TUNEL assay after 24 hours as percentage (mean  SD of cells per field) of apoptotic cells per field. As control, cells were cultured
in endothelial basal medium (EBM) in the absence of doxorubicin. ANOVA with the Newman–Keuls multicomparison test was carried out:
*, P < 0.05, doxorubicin treatment in the presence of vehicle alone, RNase CD105þ MV, CD105 MV, and TMV induced significant apoptosis versus doxorubicin untreated (EBM); §, P < 0.05: CD105þ MV significantly inhibited apoptosis versus all other doxorubicin treatment (vehicle, RNase CD105þ MV, CD105 MV, and TMV). D, quantitative evaluation (mean  SD of cells per field) of adhesion of 5  105 K1 tumor cells labeled with CSFE to a monolayer of HUVEC unstimulated (RPMI) or stimulated with 30 mg/mL of MVs from cloned cancer stem cell preparations, RNase CD105þ MVs, CD105 MVs,
and TMVs. ANOVA with the Newman–Keuls multicomparison test was carried out: *, P < 0.05, CD105þ MV versus RPMI, RNase CD105þ MV, CD105 MV, and TMV; §, P <0.05 TMV versus RPMI and CD105 MV. For all the experimental condition, 5 different experiments were carried out in duplicate.
5 days with 70 mg of MVs, followed by i.v. injection of 6  105 renal tumor cells. After 5 weeks, organs were recovered (liver, spleen, kidney, and lung) and the incidence of metastasis was evaluated. Metastases clearly detectable were found only in lungs (Fig. 6A). The number of metastases induced by renal tumor cells was very low in mice injected with vehicle alone or with CD105 MVs or RNase CD105þ MVs, whereas a significant increase in the number of metastases was observed in mice pretreated with CD105þ MVs or MVs from unsorted tumor cells. However, CD105þ MVs were signifi- cantly more efficient in inducing metastasis than unsorted tumor MVs (Fig. 6A). To evaluate whether the administration of MVs modify lung microenvironment, the expression of VEGFR1, VEGF, MMP9, and MMP2 was studied. By cytofluori- metric analysis, VEGFR1 expression in CD146þ-sorted lung endothelial cells was enhanced by CD105þ MVs but not by
5352 Cancer Res; 71(15) August 1, 2011
CD105 MVs (Fig. 6B). By qRT-PCR, CD105þ MVs, but not CD105 MVs, significantly enhanced MMP9 expression in total lung tissue and VEGF and MMP2 in sorted lung endothe- lial cells (Fig. 6C). The enhanced expression of MMP9 and MMP2 in lung after treatment with CD105þ MVs was con- firmed by immunohistochemistry (Fig. 6D). MMP2 staining was mainly confined to lung vessels, whereas that of MMP9 was more diffuse and the alveolar epithelial cells were positive.
Discussion
Previous studies showed an angiogenic potential of MVs derived from tumors but did not characterize the cells of origin (11, 24–26). Herein, we showed that in renal cancer, the MVs that retain the angiogenic properties were those that were derived from cancer stem cells. Indeed, MVs released
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Figure 5. In vivo angiogenesis of HUVECs stimulated with CD105þ MVs. HUVECs (1  106) treated with vehicle or 70 mg of CD105þ MVs from cloned cancer stem cell preparations, RNase CD105þ MVs, CD105 MVs, and TMVs were injected subcutaneously within Matrigel in SCID mice, and mice were sacrificed 10 days after. A, representative micrographs of hematoxylin and eosin staining of section of Matrigel showing dense cluster of cells infiltrated by small vessels and microaneurismatic structures containing erythrocytes in HUVECs stimulated with CD105þ MVs. TMVs induced only the formation of small vessels. B, representative micrograph of immunostaining for the endothelial antigen (vWF) and for HLA class I antigen (original magnification 200). C, quantitative evaluation of neo-formed vessels was expressed as the number of vessels per total area of Matrigel. Data are expressed as mean  SD of 8 individual experiments for each condition. ANOVA with the Newman–Keuls multicomparison test was carried out: *, P < 0.05, CD105þ MV versus vehicle, RNase CD105þ MV, CD105 MV, and TMV; §, P < 0.05, TMV versus vehicle, RNase CD105þ MV, and CD105 MV.
from cancer stem cells induced in vitro and in vivo angiogen- esis and favored lung metastasis. These properties were ascribed only to the MVs released from the CD105þ cell fraction, as those derived from the CD105 tumor cells were ineffective. Indeed, CD105þ MVs contained proangiogenic mRNAs and miRNAs that may be involved in tumor progres- sion and metastases.
Recently, circulating MVs were described in patients with various tumors (27–32), suggesting that they may serve as a diagnostic and prognostic tool (33–35). In the context of cancer, several studies pointed out the potential role of tumor-derived MVs in the interaction with stromal cells and in the formation of premetastatic niche (36–39). The potential of MVs to reprogram recipient cells was first established by Ratajczak and colleagues (7). Several subsequent studies indicate that mRNA delivered by MVs can be translated into the corresponding proteins by target cells (8, 9, 40).
In the present study, we investigated whether MVs derived from cancer stem cells possess biological activities that may account for the induction of a favorable environment for tumor growth and invasion. We found that MVs derived from CD105þ renal cancer stem cells differ for their content of mRNAs and miRNAs with respect to the CD105 renal cancer cell population. In particular, CD105þ MVs contained several
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proangiogenic mRNAs such as VEGF, FGF, angiopoietin1, ephrin A3, MMP2, and MMP9 that were absent in CD105 tumor MVs. The presence of the proangiogenic mRNAs cor- related with an in vitro and in vivo angiogenic effect of CD105þ MVs. The proangiogenic effect of CD105þ MVs can be ascribed to their ability to induce endothelial cell growth, organization, invasion of matrix, and resistance to apoptosis. An angiogenic effect of MVs was previously described for MVs derived from unfractionated tumor cells of lung cancer, ovarian cancer, and glioblastoma, as well as from some tumor cell lines (11, 25, 26, 33). Beside mRNAs, MVs were shown to contain and to deliver functional miRNAs to target cells (9, 20). CD105þ MVs were enriched in miRNAs with respect to the CD105 MVs. The GO analysis of predicted target genes indicated that CD105þ MVs shuttled a selected pattern of miRNAs that may modulate several biological functions rele- vant for cell growth, regulation of transcription, cell matrix adhesion, and synthesis of macromolecules. Among the miR- NAs shuttled by CD105þ MVs, we detected miR-200c, miR-92, and miR-141 that were described significantly upregulated in patients with ovarian (28, 41), colorectal (42), and prostate cancer (43), respectively. These miRNAs were suggested as marker of unfavorable prognosis (44). In addition, we detected several miRNAs such as miR-29a, miR-650, and miR-151 that were associated with tumor invasion and metastases (45–47).
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Figure 6. Effect of MVs on lung metastasis formation. SCID mice (5 per group) were treated for 5 days with i.v. injections of vehicle or 70 mg of CD105þ MVs from cloned cancer stem cell preparations, RNase CD105þ MVs, CD105 MVs, or TMVs. K1 renal tumor cells (6  105) were injected intravenously on day 5, and mice were sacrificed 5 weeks later. A, quantitative evaluation of metastases carried out in 5 nonconsecutive sections of whole lungs and expressed as mean  SD per lung and representative hematoxylin and eosin–stained lung sections (original magnification 200). ANOVA with the Newman–Keuls multicomparison test was carried out, *, P < 0.05, CD105þ MV versus vehicle, RNase CD105þ MV, CD105 MV, and TMV; §, P < 0.05, TMV versus vehicle, RNase CD105þ MV, and CD105 MV. B, representative cytofluorimetric analysis of VEGFR1 expression by CD146þ-sorted lung endothelial cells obtained from mice treated for 5 days with 70 mg of CD105þ MVs (red line) or CD105 MVs (dark line) or with vehicle alone (dotted line). The percentage of positive cells was as follows: CD105þ MVs, 63%  3.1%; CD105 MVs, 36%  2.7%; vehicle, 40%  2.9%. Eight mice per group were studied with similar results. C, qRT-PCR analysis of VEGF, MMP2, and MMP9 mRNA expression in total lung and in CD146þ endothelial cells of mice treated for 5 days with 70 mg of CD105þ MVs or CD105 MVs or with vehicle alone. Data were normalized to actin mRNA and to 1 for vehicle. Eight mice per group were studied with similar results. ANOVA with the Newman–Keuls multicomparison test was carried out. *, P < 0.05, CD105þ MV versus CD105 MV. D, representative immunohistochemistry for MMP9 and MMP2 on lung sections obtained from mice treated for 5 days with 70 mg of CD105þ MVs or CD105 MVs or with vehicle alone showing MMP9 staining of vessels and alveolar epithelial cells (arrows and inset) and MMP2 staining of vessels (inset) in CD105þ MV–treated mice (original magnification: MMP9, 200; MMP2, 400; insets, 620).
Moreover, miR-19b, miR-29c, and miR-151 were observed upregulated in renal carcinomas in comparison with normal renal tissue (48) and they were significantly enriched within miRNAs present in CD105þ MVs.
It has been recently suggested that tumor-derived MVs may contribute to the formation of a premetastatic niche (37, 38).
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Herein, we showed that MVs derived from CD105þ renal cancer stem cells, but not from CD105 tumor cells, were able to significantly enhance lung metastasis formation when injected prior to a renal tumor cell line. Indeed, CD105þ MVs, but not CD105 MVs, significantly enhanced the expression of VEGFR1, VEGF, and MMP2 in CD146-sorted lung cells
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containing endothelial cells and a small population of both leukocytes and MMP9 in the whole lung. Previous studies showed that these factors are involved in the generation of lung premetastatic niche (49, 50). Our results confirm that MVs create a receptive microenvironment to coordinate metastatic diffusion (37) and identify the specific contribution of MVs derived from cancer stem cells.
A recent study indicated that tumor stem cells not only initiate tumors but may also promote metastases in virtue of their peculiar content of tumorigenic miRNAs (46). MVs may transfer products of oncogenes to bystander cells, inducing changes in their phenotype (11). The result of the present study suggests that the RNA content of MVs plays a critical role, as the RNase treatment of MVs significantly inhibited the in vitro and in particular the in vivo biological effects of CD105þ MVs. This suggests that the effects of CD105þ MVs could be, at least in part, accounted for epigenetic changes induced by transfer of mRNAs and/or miRNAs.
In conclusion, the results of the present study suggest that, in renal cancer, the MVs that favor tumor growth and invasion were those that were derived from the cancer stem cells rather than from the whole tumor cell population. These MVs by
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    Disclosure of Potential Conflicts of Interest
    No potential conflicts of interest were disclosed.
    Acknowledgments
    The technical assistance of Federica Antico and Ada Castelli is gratefully acknowledged.
    Grant Support
    This study was supported by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), project IG8912, by Italian Ministry of University and Research (MIUR) Prin08, and by Regione Piemonte, Project Oncoprot and Piattaforme Biotecno- logiche, Pi-Stem project.
    The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Microvesicles Released from Human Renal Cancer Stem Cells Stimulate Angiogenesis and Formation of Lung Premetastatic Niche
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