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. Author manuscript; available in PMC: 2013 Jun 12.
Published in final edited form as: Mol Cancer Res. 2010 Nov;8(11):1489–1500. doi: 10.1158/1541-7786.MCR-10-0060

Caveolin-1 modulates the ability of Ewing's sarcoma to metastasize

Miguel Sáinz-Jaspeado 1,†, Laura Lagares-Tena 1,†, Jaime Lasheras 1,#, Fariba Navid 2, Carlos Rodriguez-Galindo 2, Silvia Mateo-Lozano 3, Vicente Notario 4, Xavier Sanjuan 1, Xavier Garcia del Muro 1, Àngels Fabra 1, Oscar M Tirado 1,*
PMCID: PMC3679656  NIHMSID: NIHMS480220  PMID: 21106507

Abstract

Metastasis is the final stage of tumor progression and is thought to be responsible for up to 90% of deaths associated with solid tumors. Caveolin-1 (CAV1) regulates multiple cancer-associated processes related to malignant tumor progression. In the present study we tested the hypothesis that CAV1 modulates the metastatic ability of ESFT cells. First, we analyzed the expression of CAV1 by immunostaining a tissue microarray containing 43 paraffin-embedded ESFT tumors with known EWS translocations. Even though no evidence was found for a significant association between CAV1 expression and stage, size or tumor site, all metastatic samples (10/10) had significantly high CAV1 expression, suggesting that high CAV1 content could positively contribute to enhance ESFT metastasis. To determine the effect of CAV1 on the migratory and invasive capabilities of ESFT cells, we knocked down CAV1 expression in TC252 and A673 cells by stably transfecting a previously validated shRNA construct. In vitro, migration and invasion assays showed that, for both cell lines CAV1 knocked-down cells migrated and invaded significantly less (p≤0.01) than control cells. Moreover, control A673 cells introduced into Balb/c nude mice by tail vein injection strongly colonized the lungs. In contrast, animals injected with CAV1 knocked-down cells showed either no incidence of metastasis or developed lung metastases after a significant delay (P<0.0001). Finally, we show that the molecular mechanisms by which CAV1 carries out its key role in regulating ESFT metastasis involve MMP production and activation as well as the control of the expression of SPARC, a known determinant of lung colonization.

Introduction

Metastasis is the final stage in tumor progression and is thought to be responsible for up to 90% of deaths associated with solid tumors. This multifaceted process consists of a series of steps whereby cancer cells enter the circulation, disseminate to distal capillary beds, enter a parenchyma by extravasation, adapt to the new host microenvironment, and eventually grow into lethal tumor colonies in distal organs (1). Much has been learned about the processes that initiate and sustain general tumor growth; however, the mechanisms that enable metastasis remain largely unknown.

The Ewing's sarcoma family of tumors (ESFT) includes aggressive bone-associated malignancies that affect the pediatric population. ESFT are characterized by early metastases, and metastatic spread is commonly hematogenous. Nearly all ESFT patients already have micrometastases at diagnosis, resulting in a >95% relapse rate when treated locally and a 40% relapse rate after systemic chemotherapy. Therefore, knowledge of key regulators of circulating or dormant metastatic tumor cells after traditional therapy is needed. Most ESFT harbor a reciprocal translocation, t(11;22)(q24;q12), which links a strong transcriptional activation domain from EWS to the ETS DNA-binding domain of FLI-1 (2). The EWS/FLI-1 fusion is required for Ewing's sarcoma oncogenesis, as inhibition of its function results in the loss of transformation of ESFT cells (3–5). Thus, enhancing our current knowledge on EWS/FLI-1 function is critical to understand ESFT development.

Caveolin-1 (CAV1) was previously identified as a metastasis-associated gene that is a transcriptional target of EWS/FLI-1 as well as an important determinant of ESFT malignant phenotype, tumorigenicity and resistance to chemotherapy-induced apoptosis of ESFT cells (6–7). CAV1 is a multifunctional scaffolding protein with multiple binding partners that associates with cell surface caveolae. CAV1 regulates multiple cancer-associated processes including cellular transformation, tumor growth, cell death and survival, multidrug resistance, angiogenesis, cell migration and metastasis (8). Clinical studies have revealed that CAV1 upregulation is associated with poor prognosis and the occurrence of metastasis in several human cancers (9). Moreover, it was recently reported that CAV1 regulates cell migration and invasion in human breast cancer cells (10).

A better understanding of the complex biology of ESFT may lead to the successful development of biologically targeted therapies. As the regulatory pathways responsible for transformation, growth, and metastasis of ESFT become well defined the efficacy of therapeutic intervention will improve. Therefore, because the possible involvement of CAV1 in the ability of ESFT to metastasize has not been established to date, we tested the hypothesis that CAV1 modulates the metastatic ability of ESFT cells. Our results demonstrate that CAV1 controls migration and invasion in ESFT cells in culture by mechanisms involving the production and activation of metalloproteinases as well as lung colonization in nude mice by regulating SPARC expression levels. These data conclusively demonstrates that CAV1 plays a key role in ESFT metastasis.

Results

First, we analyzed the expression of CAV1 by immunostaining a tissue microarray (TMA) containing 43 paraffin-embedded ESFT tumors with known EWS translocations (11). As expected and in agreement with previous studies, about 85% of the patients expressed CAV1. Clinicopathological analyses revealed that CAV1 overexpression correlated significantly with metastasis and poor prognosis in several human cancers, including prostate, breast and lung cancer (12). Although no evidence was found for a significant association between CAV1 expression and stage, size or tumor site, all metastatic samples (10/10) had significantly high CAV1 expression (Fig. 1), suggesting that high CAV1 content could positively contribute to enhance ESFT metastasis.

Figure 1.

Figure 1

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A. Summary of TMA data with regard to CAV1 expression (negative vs. positive) and clinical features. B. Example of negative and positive CAV1 staining of a human ESFT patient sample from the TMA (magnification 40×, insert 400×).

In order to metastasize, tumor cells need to migrate from the primary site (13). To determine the effect of CAV1 on the migratory and invasive capabilities of ESFT cells, we knocked down CAV1 expression in TC252 and A673 cells (Fig. 2A) by stably transfecting a previously validated shRNA construct (6–7). EWS/FLI-1 protein levels were unaffected by downregulation of CAV1 (Supplementary Fig. 1), further demonstrating the specificity of the shRNA construct. In vitro, wound healing (Fig. 2B) and transwell migration assays (Fig. 2C) showed that in both cell lines, CAV1 knocked-down cells migrated significantly less (p≤0.01) than mock- and vector-transfected cells. These results are consistent with previous reports that demonstrate the key role of CAV1 regulating the migratory capability of selected metastatic cell lines (14–15).

Figure 2. CAV1 downregulation reduces the migration of ESFT cells.

Figure 2

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A. Immunoblot showing substantially reduced CAV1 levels in individual clones of A673 and TC252 cells stably expressing CAV1 shRNA compared with untransfected and vector-transfected cells. ß-Actin was the loading control. B. Wound healing assay carried out with cells derived from the TC252 CAV1 silencing model. Phase-contrast microscope images (100×) were taken at the indicated times after monolayer scratching (left panel); quantification of wound healing assays with A673- and TC252-derived cells are shown (right panel) whith vertical bars representing mean values of gap closure from three independent experiments, each done in triplicate; bars, SD. *, P ≤ 0.01. C. Photomicrographs (40×) showing differences in transwell migration among the cells indicated (Left panel); quantification of transwell cell migration is shown (right panel), with vertical bars representing mean percentages of the number of migrated cells from three independent experiments each done in triplicate; bars, SD. *, P ≤ 0.01.

As the acquisition of an invasive phenotype by cancer cells is a critical step for tumor progression (13), we also examined the effects of CAV1 downregulation on the invasive ability of CAV1 knocked-down ESFT cells derived from both cell lines. Relative to control cells, as shown in Figure 3A, downregulation of CAV1 correlated significantly (p≤0.01) with a reduction in the invasiveness of A673 and TC252 cells. Early events in cancer cell invasion and metastasis involve the proteolytic degradation of extracellular matrix components. Matrix metalloproteinases (MMPs) are zinc-containing proteolytic enzymes that cleave extracellular matrix proteins, and play important roles in tumor cell migration and invasion. Along with other family members, matrix metalloproteinase 9 and 2 (MMP9 and MMP2) are able to degrade the main components of the basement membranes and play a key role in metastasis (16). To determine whether ESFT invasion was associated with MMP activation, conditioned media from both model cell cultures were analyzed by zymography with gelatin as the substrate (Fig. 3B). Proteolytic activities of MMP9 and MMP2 were present in conditioned media from all cell lines. Interestingly, the band detected at 100 kDa corresponding to the pro-MMP9 precursor form that became active during the zymography, was found substantially decreased in all CAV1 knocked-down cell lines. Furthermore, a faster migrating gelatinolytic form of MMP2, was present only in the control cells, but was undetectable in CAV1 knocked-down cells, suggesting that CAV1 expression is required for MMP9 production and MMP2 activation. The involvement of MMPs on the invasive ability of Ewing's sarcoma cells was further confirmed by using a known MMP2/9 inhibitor {N-phenylsulfonyl-hydroxamic acid derivative [2R-[(4-biphenylsulfonyl)amino]-N-hydroxy-3-phenylpropinamide] (BiPS)}. BiPS induced a significant reduction in the invasiveness of A673 and TC252 cells (Supplementary Fig. 2). MMP9 production had been shown to be regulated at transcriptional level in other tumor types (17). To determine whether the MMP9 decrease was associated with a downregulation of its mRNA levels, we compared the expression of MMP9, MMP2 and TIMP2 in cells derived from both CAV1 silencing models by RT-PCR. As shown in Figure 3C, only MMP9 mRNA was downregulated as a consequence of CAV1 knockdown, suggesting that CAV1 regulates MMP9 expression differently from the way it affects other MMPs.

Figure 3. CAV1 downregulation reduces the invasive ability of ESFT cells.

Figure 3

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A. Invasion of cells through 8 μm Matrigel-coated polycarbonate pores was determined using Boyden chamber assays. Vertical bars represent mean percentages of invading cells from three independent experiments each done in triplicate; bars, SD. *, P ≤ 0.01. B. Gelatin zymogram measuring activation of MMP9 and MMP2 related to CAV1 expression in the same cells shown in panel A. C. MMP9, MMP2 and TIMP2 mRNA levels were analyzed by semi-quantitative RT-PCR (left panel). β-Actin was used as the loading reference. Histogram bars (right panel) represent mean densitometry values from two independent experiments; bars, SD. *, P ≤ 0.05.

Activation of the secreted pro-MMP2 is mediated by a cell-surface complex that consists of a homodimer of membrane type 1 matrix metalloproteinase (MT1-MMP) and a single molecule of tissue inhibitor of metalloproteinases 2 (TIMP2; a natural inhibitor of MMPs) (18–19). Moreover, CAV1 had been shown to be required for invadopodia formation and extracellular matrix degradation through regulation of MT1-MMP function in human breast cancer cells (20). Ewing's sarcoma cells had been shown to express MT1-MMP (21). Consequently, zymography from isolated membrane extracts (22) in the A673 model, showed membrane-bound MMP2 was mostly observed in control but barely present in low CAV1 cells (Supplementary Fig. 3) suggestive of MT1-MMP activation in control cells but not in low CAV1 cells. Furthermore, confocal microscopy analysis showed that the decrease of MT1-MMP activity correlated with a diffuse redistribution of this protein in low CAV1 cells (Fig. 4). We were unable to observe wide co-localization between CAV1 and MT1-MMP. However, in control cells these two proteins co-localized in some structures at the bottom of the cell suggestive of invadopodia (Supplementary Fig. 4).

Figure 4. Localization of CAV1 and MT1-MMP in cells derived from the A673 CAV1 silencing model.

Figure 4

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Exponentially growing, A673-derived cells were immunostained with anti-CAV1 antibody using an Alexa Fluor®488 Goat anti-mouse secondary antibody (CAV1 panels), anti-MT1-MMP antibody using an Alexa Fluor®594 Goat anti-rabbit secondary antibody (MT1-MMP panels) or stained with DAPI to identify the nuclear compartment. Merging of the three images (MERGE panels) showed the relocation of MT1-MMP from the cell membrane to a more diffusely staining compartment in CAV1 knocked-down cells (magnification 630×).

Based on the in vitro results, we asked whether downregulation of CAV1 in ESFT cells may affect their metastatic potential in an experimental metastasis assay in vivo. The cell lines derived from the A673 CAV1 silencing model were injected into the tail vein of Balb/c nude mice. Animals were sacrificed after manifestation of morbidity. In agreement with a previous report using A673 cells as a model of metastasis (23), our parental and vector-transfected A673 cells strongly colonized the lungs. On the contrary, animals injected with CAV1 knocked-down cells either showed essentially no incidence of metastasis to any organ, or showed lung colonization only after a significant delay. Consequently, there were highly significant (P=0.0001) differences in survival among the experimental group of mice (Fig. 5A). Macroscopically, metastatic foci were quantified showing significant differences between control and low CAV1 cell-derived lungs (Fig. 5B–C). Histological analysis of paraffin-embedded lungs clearly evidenced several lung metastases in mice injected with control cells in comparison with the lungs of animals injected with A/ShCAV1-5 and A/ShCAV1-9 cells (Fig. 5D). Although previous studies with A673 cells also reported metastasis to sites other than the lungs, only lung metastases developed in our experimental system. This may be explained by their use of a different immunodeficient mouse strain which has been recently shown to allow ESFT cells to colonize other organs in addition to the lungs (24). Immunohistochemical analyses of paraffin-embedded metastatic lung tumors showed either diminished or no detectable CAV1 expression in A/ShCAV1-derived lung tumors from mice sacrificed throughout the experiment compared with the highly positive staining of parental or control–derived lung tumors from mice sacrificed during the third week (Fig. 5E). In agreement with the in vitro results MMP9 expression appeared mostly in controls. These results further demonstrated the validity of the shRNA construct used in the present study and strongly support the hypothesis that CAV1 is necessary for ESFT cells to metastasize.

Figure 5. CAV1 downregulation reduces metastasis in nude mice.

Figure 5

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A. Survival of mice bearing ESFT cells using a mouse lung metastasis model. Kaplan-Meier survival curves of nude mice bearing tumors initiated by injection of mock transfected A673 cells (A673 Mock), control vector transfected cells (A673/ShC), or CAV1 knocked-down (A/ShCAV1-5 and A/ShCAV1-9) cells (n=10 for each cell line). B. Quantification of lung metastases derived from Control (A673 Mock and A673/ShC) and CAV1 knocked-down (ShCAV1) cells (A/ShCAV1-5 and A/ShCAV1-9) C. Representative lungs excised from animals injected with A673-derived cells. Control mice were sacrificed during the third week whereas animals injected with cells expressing shCAV1s were sacrificed during the eighth week. D. Hemathoxylin and Eosin (H&E) from corresponding paraffin-embedded lungs (200×). E. CAV1 and MMP9 staining of metastasis on lung sections from mice injected with control vector transfected A673 cells (A673/ShC) sacrificed on day 24 and ShCAV1 transfected cells (A/ShCAV1) sacrificed on day 60 (magnification 100×).

The ability of ESFT cells to enter into the bloodstream (intravasation) and to exit from capillary beds into the parenchyma of an organ (extravasation) may be explained by the regulation that CAV1 exerts over MMPs. However, the ability of these cells to colonize the lungs may also be driven by the so-called metastasis virulence genes (25). Among a subset of these genes we focused in SPARC (secreted protein acidic and rich in cysteine), a matricellular glycoprotein that mediates interactions between cells and their microenviroment as both CAV1 and SPARC have been found to be determinants of metastasis in basal-like breast carcinomas and melanomas (26–27). In addition, neither the expression of SPARC in ESFT nor the mechanistic interaction between the SPARC and CAV1 proteins has been reported to date. Therefore, we tested whether SPARC mRNA and protein expression was affected by CAV1 knockdown. Results showed that indeed SPARC mRNA and protein levels were downregulated in CAV1 knockdown cells (Fig. 6A–B) suggesting that CAV1 regulates SPARC expression at transcriptional level. SPARC has been found to induce migration and invasion in different types of cancer (28). Consequently, in order to explore whether SPARC collaborated with CAV1 inducing such processes, we knocked down SPARC in A673 cells by stably transfecting a validated (38) specific shRNA construct (Fig. 6C) without affecting CAV1 expression (Supplementary Fig. 5) and analyzed its effects on migration, invasion and lung colonization. Even though wound healing (Fig. 6D), transwell migration (Fig. 6E) and invasion assays (Fig. 6F) showed that SPARC knocked-down cells migrated and invaded significantly less (p≤0.05) than control cells, the effect of SPARC downregulation did not recapitulated the extent of the inhibitory effects induced by CAV1 knockdown. Conditioned media from SPARC knocked-down cell cultures were analyzed by zymography (Fig. 6G). Similarly to what happened in CAV1 knocked-down cells, MMP2 activation was undetectable in SPARC knocked-down cells. In contrast, SPARC downregulation did not provoke a decrease of MMP9 suggesting that, SPARC knocked-down cells invaded better than CAV1 knocked-down cells in part because MMP9 was unaffected and thus remained active despite the low cellular levels of SPARC. Lastly, experimental metastasis assays showed that in contrast to control cells, SPARC knocked-down cells were unable to colonize the lungs (Fig. 6H) suggesting that SPARC is necessary for lung metastasis by Ewing's sarcoma cells.

Figure 6. SPARC acts downstream CAV1 to regulate ESFT cell invasiveness.

Figure 6

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A. SPARC mRNA levels were analyzed by semi-quantitative RT-PCR in individual clones of A673 and TC252 cells stably expressing CAV1 shRNAs were compared with those in untransfected cells and in cells transfected with control vector DNA. B. SPARC protein levels in cells derived from the A673 and TC252 CAV1 silencing models. C. Immunoblot showing substantially reduced SPARC levels in individual clones of A673 cells stably expressing SPARC shRNA compared with untransfected cells and cells transfected with control vector DNA. Wherever applicable, ß-Actin was used as the loading control. D. Quantification of wound healing assays, where vertical bars represent mean values of gap closure from three independent experiments each done in triplicate; bars, SD. *, P ≤ 0.05. E. Quantification of transwell cell migration where vertical bars represent mean percentages of the number of migrated cells from three independent experiments each done in triplicate; bars, SD. *, P ≤ 0.05. F. Invasion of cells through 8 μm Matrigel-coated polycarbonate pores was determined using Boyden chamber assays. Vertical bars represent mean percentages of invading cells from three independent experiments each done in triplicate; bars, SD. *, P ≤ 0.05. G. Gelatin zymogram measuring activation of MMP9 and MMP2 related to SPARC expression in the same cells shown in panel D. H. Representative lungs excised after 15 days from mice injected with either cells transfected with control vector and from ShSPARC-transfected cells (top panel). Quantification of lung weight (bottom panel) from mice injected with Control or SPARC knocked-down cells. *, P ≤ 0.001.

Discussion

CAV1 regulates multiple cancer-associated cellular processes and its expression is a marker that predicts poor cancer patient prognosis (12). In our TMA all metastatic samples had significantly high CAV1 expression. This is in agreement with several publications reporting that certain genes expressed in primary tumors are also found in metastasis from the same patient, suggesting that the metastatic potential of human tumors is encoded in the bulk of the primary tumors (29).

Migrating cells display sequential changes in morphology characterized by protrusion of lamellipodia and filopodia at the leading edge, attachment to the substratum, forward flow of cytosol, focal adhesion loosening, and retraction of the rear of the cell (30). CAV1 drives the formation of plasma membrane caveolae and anchors them to the actin cytoskeleton, modulates cell interaction with the extracellular matrix, pulls together and regulates signaling molecules, and transports cholesterol. Through these functions, CAV1 could play an important role in cell movement through control of cell membrane composition and membrane surface expansion, polarization of signaling molecules, and/or cytoskeleton remodeling (31). Therefore, our laboratory is currently investigating whether CAV1 controls ESFT cell migration directly, acting on the migrating protrusions or indirectly behaving as functional platforms that regulate the organization, coordination and activation status of signaling complexes (receptors, transducers, and effectors) involved in cell migration.

Our data represent the first description of CAV1 regulating differently MMP9 and MMP2 to promote invasion in ESFT. The regulation of MMP expression and activity occurs at many levels, including gene transcription, post-transcriptional processing, and pro-enzyme activation (32). Our results demonstrate that CAV1 regulates MMP9 by indirectly modulating its expression at the transcriptional level, while it regulates MMP2 by promoting the activation of its pro-enzyme form. Intriguingly, CAV1 knockdown induced the downregulation of MMP9 but not MMP2 or TIMP2. Multiple cytokines and growth factors are able to induce MMP expression, although the tissue specificity of the diverse family members is mainly achieved by the combination of different transcriptional control mechanisms. The integration of multiple signaling pathways facilitates the strict spatiotemporal control of MMP transcriptional activity (33). Therefore, in our model CAV1 might be regulating specific signaling pathways that affect MMP9 expression independently of other MMPs.

For MMP2 activation TIMP2 binds to the catalytic domain of one of the MT1-MMP molecules in the dimer and to the HPX domain of pro-MMP2, thereby facilitating cleavage and activation of pro-MMP2 by the second (TIMP2-free) MT1-MMP molecule of the dimer (19). Recently, it was shown that CAV1 is required for invadopodia formation and extracellular matrix degradation in human breast cancer cells, demonstrating that CAV1 is an essential regulator of MT1-MMP function (20). Accordingly, our results strongly suggest that CAV1 mediates MMP2 activation through regulation of MT1-MMP activity. However, and contrary to our results, CAV1 has been shown to inhibit invasion and MMP expression in pancreatic cancer (34). Also, in breast cancer cell lines CAV1 has been shown to inhibit invasion, MMP activation and metastasis in vivo (35). These opposite results might be explained because ESFT is not an epithelial cancer as is the case of pancreatic and mammary tumors, indicating that depending on the tissue type CAV1 may exert opposite functions. In agreement with our results, in melanoma (another mesenchymal tumor) CAV1 promotes invasion by regulating MMPs expression (36), underscoring the importance of the tissue of origin as a determinant of protein function.

As in many other tumors, metastatic disease is a poor prognostic factor for overall survival of ESFT patients. Furthermore, this family of tumors has a great propensity to metastasize to the lungs (37). Accordingly, our experimental metastasis model showed preferential lung colonization of A673 cells in nude mice. Very recently, it has been suggested that the organ specificity of metastatic cells is determined by unique infiltrative and colonization functions which are dependent on specific gene sets (1). There is no data about genes that determine lung colonization in ESFT. Nevertheless, we focused on SPARC because it has been described as a determinant of lung colonization in metastatic breast tumors (38) and it has been shown to induce the production and activation of several MMPs (39).

Similar to MMP9, SPARC mRNA was affected by CAV1 knockdown suggesting that in ESFT expression of both genes might be regulated by CAV1. However, not all ESFT cells expressed SPARC (data not shown), this might be explained by the fact that SPARC expression in tumor cells is highly regulated by mechanisms other than transcription (39). Therefore, in some ESFT cells SPARC expression could be regulated epigenetically, posttranscriptionally or by miRNAs. In our model SPARC knockdown resulted in a significant reduction in the migratory and invasive capabilities of A673 cells. However, the effects induced by SPARC downregulation never recapitulated the extent of the inhibitory actions provoked by CAV1 knockdown suggesting that CAV1 mediates migration and invasion in part by indirectly regulating the expression of SPARC, but also by regulating other still unidentified proteins. These observations are in agreement with the situation reported in melanomas, where both proteins have been shown to promote metastatic disease (27, 36). Zymography analysis showed that as in CAV1 knockdown cells MMP2 activity was reduced as a consequence of SPARC knockdown, in contrast, MMP9 production was unaffected explaining why SPARC knockdown did not induced the same level of invasive inhibition than CAV1 knockdown. Similarly to CAV1 the activity of SPARC is context- and cell type-dependent. In fact, SPARC has shown seemingly contradictory effects on tumor progression in both clinical correlative studies and in animal models (40). Therefore, the activity of SPARC related to invasion might be different in ESFT than in other tumor types. Interestingly, compared to control cells none of SPARC knocked-down cells were able to colonize the lungs, suggesting that SPARC as an adhesion protein may be indeed necessary for ESFT cells to adhere to the lung epithelia. Alternatively, the hypothesis that SPARC plays an important role in the fusion of tumor cells with bone marrow-derived cells in circulation has been proposed a new explanation for its role in metastasis (41). Nevertheless, whether in ESFT SPARC promotes metastasis through one mechanism or the other requires further investigation.

Overall, our study demonstrates that CAV1 plays a key role in regulating the ability of ESFT cells to migrate, invade the extracellular matrix and colonize distant organs such as the lungs. This is the first study to evaluate the role of CAV1 on ESFT metastasis. Our results add relevance to the key roles that CAV1 plays in ESFT biology by controlling tumorigenicity (6), resistance to chemotherapy-induced apoptosis (7) and metastatic ability.

Materials and Methods

Tissue samples

Forty-three tumor samples were procured from the archives of the St. Jude Children's Research Hospital (SJCRH) Pathology Department and referring institutions. A minimum of three representative cores from ESFT tumors was used to construct a tissue microarray (TMA) using a manual arrayer (Beecher Instruments, Silver Spring, MD, USA). All tumor samples were fixed in formalin, embedded in paraffin, and had not undergone decalcification. Cores from various normal paraffin-embedded tissues were scattered in each of the TMA blocks as controls. Samples from two patients in the TMA were lost during immunohistochemical procedures therefore, figure 1 only shows results from 41 patients.

Immunohistochemistry

Expression of CAV1 in human Ewing's sarcoma and lung metastasis specimens was analyzed using immunohistochemical techniques performed essentially as previously described (6–7). CAV1 was detected with a 1:2000 dilution of a rabbit polyclonal antibody (#610059, from BD Biosciences). Protein expression levels were coded as 0, 1 (low expression) or, 2 and 3 (high expression) according to the level of staining. MMP9 was detected with a 1:100 dilution of a mouse monoclonal antibody (#ab38898 from Abcam).

Cell culture and stable transfections

A673 and TC252 cell lines (gifts from Dr. Heinrik Kovar, Children's Cancer Research Institute, Kindrespitalgasse, Vienna, Austria) were cultured in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen). All cell lines were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air. Exponentially growing cells within two sequential passages were used for all experiments. Cells were transfected using Fugene (Roche) following manufacturer's protocols. Transfected cells were selected with neomycin (0.4 mg/mL, Invitrogen) (ShCAV1) and with puromycin (0.5 μg/mL, Sigma) (ShSPARC, ref 38) for 14 days, and antibiotic-resistant pools and individual colonies were isolated for further analysis and maintained in the presence of neomycin (0.2 mg/mL) and puromycin (0.1 μg/mL), respectively.

Reverse Transcription-PCR

Total RNA (3 μg), extracted using the RNeasy Mini Kit (Qiagen), was used for cDNA synthesis with SuperScript II Reverse Transcriptase (Invitrogen). Amplifications of SPARC, MMP2, MMP9, TIMP2 and β-Actin were carried out using specific primers (Supplementary Table) designed using the Oligo 6.0 software (National Bioscience). For each set of primers, the number of cycles was adjusted so that the reaction end points fell within the exponential phase of product amplification, thus providing a semi-quantitative estimate of relative mRNA abundance. Reverse transcription-PCR (RT-PCR) determinations were carried out at least twice for each relevant transcript.

Western blot

ESFT cells were lysed with RIPA buffer containing protease inhibitors (1 mM PMSF, 10 mg/ml aprotinin and 10 mg/ml leupeptin) and the lysates were centrifuged at 13,000 g, at 4°C, for 30 min. The protein content of the supernatants was determined with the BCA assay system (Pierce). Lysate aliquots (50 μg) were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes. After blocking with 5% skim milk in PBS containing 0.2% Tween-20, at room temperature, for 1 h, membranes were incubated overnight at 4°C with the appropriate primary antibody (CAV1 from BD, SPARC from Abcam and FLI-1 from Santa Cruz). Blots were then incubated at room temperature for 1 h with a HRP-conjugated secondary antibody (1/2000) and the peroxidase activity was detected by chemiluminescence (ECL, Pierce) following manufacturer's instructions. Immunodetection of β-actin was used as loading reference.

Migration assays

Wound healing assay

Cells cultured to 95% confluence were scratched with a 10-μl pipette tip. Wounds were photographed immediately and followed by imaging at later time points, ensuing wound closure using an OLYMPUS Inverted Microscope. The nude area was measured and percentage wound closure was calculated. Transwell migration assay: 2.0 × 105 cells in 100 μL serum free media were added to the top chamber of 8 μm Costar polycarbonate transwells, while 500 μL of complete medium were added to the bottom chamber. After allowing migration for various times (37 °C, 100% humidity, 5% CO2 in air), cells on the upper membrane surface were removed and migrant cells on the membrane underside were fixed using 70% ethanol, stained using 0.1% crystal violet solution (Invitrogen), and visualized and counted under the microscope. Data were presented as the average number of migrating cells in 8 high-power fields (200 ×). Each experiment was performed in triplicate, and then the data were averaged for statistical analysis.

Invasion assays

Matrigel-coated, sterile 8-μm polycarbonate filters (BD Pharmingen) were re-hydrated as described above. The lower chambers of the 24-well plate were filled with 0.5 mL of complete medium, then 0.5 mL serum-free RPMI 1640 containing 2.0 × 105 tumor cells was added to the transwell chambers, and the plate incubated at 37 °C in 5% CO2 humidified atmosphere for 48 h. Further staining and recording of results was done as mentioned above under migration assay. The specific MMP2/9 inhibitor (BiPS) was purchased from Calbiochem and used a 10 μM.

Gelatin zymography

Metalloproteinase activity was analyzed by gelatin zymography. In brief, conditioned medium was concentrated to one-third of its original volume and electrophoresed on sodium dodecyl sulfate (SDS)- polyacrylamide gels (10%) containing 120 μg/ml gelatin. After electrophoresis, the gel was incubated for 60 min in 2.5% Triton X-100 and overnight at 37°C in 50 mM Tris pH 8.0/ 5 mM CaCl2. Then, the gels were stained with 0.1% Coomassie brilliant blue (CBB) and distained with 10% isopropanol in 10% acetic acid, and the gelatinolytic activity was identified as transparent bands in the CBB-stained background.

Immunofluorescence and confocal microscopy

Cells were cultured in sterile slides (Lab-tek Chamber Slide System, NUNC) for 24 h and fixed with 4% formaldehyde, washed thrice in Dulbecco's PBS (D-PBS), permeabilized in 0.1% Triton for 2 min, blocked for 1 h in blocking buffer (10% goat serum in D-PBS), and incubated with primary antibodies overnight. Cells were then washed thrice in D-PBS for 5 min each followed by a 1 h incubation with secondary antibodies (Alexa Fluor®488 Goat anti-mouse and Alexa Fluor®594 Goat anti-rabbit, Invitrogen). Then, cells were washed twice in D-PBS for 10 min and twice in distilled water for 10 min, and mounted in ProLong ®Gold antifade reagent with DAPI (Invitrogen). Photographs were taken with a Leica TCS SP5 spectral confocal microscope (Argon, 405 Diode and DPSS561 lasers) using a lambda blue 63X 1.35 NA oil objective. Images were analyzed with the MacBiophotonics ImageJ program.

Triton X-114 Extraction

Extraction of membrane proteins was performed essentially as described previously (22). Equal numbers of cells (approximately 1 × 106) from each cell line tested were cultured in 100-mm dishes for 3 days. Cells were then washed with TBS, incubated 20 min on ice with 2.5% TX-114 detergent (Sigma) and scraped. TX-114 has the property of separating into aqueous and detergent (hydrophobic) phases at temperatures above 20 °C, and amphiphilic integral membrane proteins remain in the detergent phase. TX-114 extracts were centrifuged (13000 g for 15 min at 4 °C) to remove TX-114- insoluble material, incubated at 37 °C for 5 min and spun at 3000 g for 2 min at room temperature to separate the aqueous and the hydrophobic phases. Aliquots (100 μL) of each hydrophobic phase were stored at −80 °C.

Experimental metastasis assay

An in vivo experimental metastasis model was established by injection of 2×106 A673 cells suspended in 100 μL of saline into the tail vein of athymic nude mice (BalbCNu/Nu) from Harlan. Animal care and procedures were followed according to the Institutional Guidelines for the Care and Use of Laboratory Animals. Mice were fed under standard conditions with weight monitoring, sacrificed after manifestation of morbidity, and their lungs were removed for further analysis.

Statistical analysis

Data were analyzed for statistical significance using Student's t test and ANOVA. Survival curves were generated by Kaplan-Meier method and compared by using the log-rank test. Unless otherwise stated P ≤ 0.01 was regarded as significant.

Supplementary Material

Supplementary Materials

Acknowledgements

We acknowledge the efforts of Blanca Luena for her assistance with the in vivo metastasis experiments, Catherine Billups in providing outstanding assistance with statistics and Jemina Moreto, from Cancer Epigenetics and Biology Program, IDIBELL, for her support with confocal images acquisition and processing. LLT is funded by the Comissionat per a Universitats i Recerca (CUR) from Departament d'Innovació, Universitats i Empresa (DIUE) de la Generalitat de Catalunya i del Fons Social Europeu.This work was funded by the Fondo de Investigaciones Sanitarias-ISCIII (CP06/00151; PI080259).

Footnotes

Competing interests

The authors declare that they have no competing interests.

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Supplementary Materials

Supplementary Materials
NIHMS480220-supplement-Supplementary_Materials.pdf (701.3KB, pdf)

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