Background
The progression from normal tissue to a malignant tumor is driven by the acquisition of genetic and epigenetic changes together with a selection of the cells with an advantage in proliferation and survival [
1]. Tumor microenvironments, composed by non-neoplastic cells, can also induce transcriptional reprogramming in neoplastic cells by the secretion of factors like TGF-β and PDGF [
2], hormones or hypoxic stress [
3]
. The final outcome is the coexistence in a given tumor of phenotypically different subpopulations or subclones of tumor cells (intratumoral heterogeneity).
Neoplastic cell subpopulations can interact with non-neoplastic elements of the tumor microenvironment and use them for their advantage [
4]. In addition, different cell subpopulations within a tumor can interact with each other as in any ecological niche [
5], either by competing for common resources [
6] or by cooperating for mutual benefit [
7,
8]. In this context, interclonal cooperativity can occur, defined as the state in which two or more neoplastic clones display a more malignant phenotype in coexistence than in isolation [
9,
10]. Thus, two neoplastic clones - of which one, or both, is not intrinsically invasive and/or metastatic- can interact when they are in proximity to one another in order to become invasive and metastatic.
In a previous study [
11], we have characterized clonal subpopulations derived from the PC-3 prostate cancer cell line in which one subpopulation displayed features suggestive of enrichment for CSCs, including high tumorigenic and metastatic potentials, and a second subpopulation was depleted of CSCs and was poorly tumorigenic and metastatic (non-CSC subpopulation). In this model, the CSC-enriched subpopulation shows a strong epithelial phenotype, while, in contrast, the non-CSC subpopulation shows a strong and stable mesenchymal phenotype. We found that the non-CSC subpopulation enhanced the metastatic potential of the CSC-enriched subpopulation [
11], thus providing experimental support to the hypothesis of cooperative interactions among CSC and non-CSC tumor cell subpopulations displaying distinct phenotypes [
7,
12] with the result of enhanced metastatic dissemination of the overall tumor. Our preliminary evidence also suggested that such cooperation was at least partially mediated by diffusible factors in our cellular models [
11]. Here we report that the matricellular protein SPARC is the major diffusible factor produced by the PC-3S non-CSC clonal subpopulation that mediates the enhanced invasiveness and metastatic dissemination of the CSC-rich PC-3M subpopulation of the PC-3 prostate cancer cell line.
Discussion
Neoplastic cellular subpopulations displaying distinct phenotypes can interact among them in different ways [
31], including competition [
6,
32] or cooperation [
33]. In a prostate cancer cell model previously characterized by us [
11], two subpopulations interact in a cooperative manner, such that one subpopulation enhances the metastatic dissemination potential of the other. In this model, a clonal tumor cell subpopulation displays non-CSC characteristics, including poor anchorage-independent growth
in vitro and limited tumorigenic or metastatic potential
in vivo, whereas a second subpopulation is enriched in CSCs as inferred from vigorous anchorage-independent growth and tumor formation and lung colonization potential
in vivo. We had previously found that the non-CSC subpopulation enhances the ability of the CSC subpopulation for metastatic dissemination, and produced preliminary evidence that this activity involved, at least in part, diffusible factors secreted by S cells [
11]. In the present study, we have confirmed and extended our original observations, including the induction of epithelial-mesenchymal transition in the CSC-enriched epithelial PC-3M subpopulation upon culture with conditioned medium from the non-CSC PC-3S subpopulation, and the dependence of this effect on specific signaling pathways.
We have applied a comparative proteomics approach in an effort to identify proteins differentially secreted by S (non-CSC)
vs. M (CSC) cells as candidates to explain the observed paracrine effects. Immunodepletion, specific transcript knockdown and complementation experiments have led us to conclude that, of the secreted proteins with the strongest differential secretion between S and M cells, the matricellular protein SPARC, abundantly secreted by S cells, explains most of the pro-invasive effects of S conditioned medium on M cells. Knock down of SPARC abrogated not only the pro-invasive activity of the conditioned medium from the non-CSC S subpopulation on the CSC-enriched M cells, but also the overall pro-invasive and pro-metastatic activities observed upon co-culture
in vitro or co-inoculation
in vivo. Of note, conditioned medium from non-CSC S cells required specific signaling pathways for the enhancement of the invasive behavior of M cells, including PI3K, MAPK, NF-κB and non-receptor tyrosine kinase pathways, which are also involved in cellular responses elicited by SPARC [
34]. The observed induction by SPARC in M cells of the MAPK and PI3K-AKT signaling pathways further supports the role played by these pathways in our model. This underlines the relevance of paracrine interactions between tumor cell subpopulations displaying non-CSC and CSC properties to modulate the phenotypic outcomes of the tumor. Other laboratories performing unbiased secretome analyses have shown that SPARC also mediates cooperation between tumor cell subpopulations with different invasive potentials in ovarian and bladder cancer models [
17,
35].
Although our knockdown and complementation experiments strongly support that SPARC is indeed the key factor mediating invasive and metastatic cooperation in our interacting CSC
vs. non-CSC neoplastic subpopulation model, other molecules produced by neoplastic or non-neoplastic cells, such as PAI-1, could also participate in this process. In this regard, we observed that knockdown of SPARC in S cells was accompanied with a decrease in PAI-1 expression. It is known that the expression of PAI-1 is modulated by changes in the expression levels of SPARC [
26]. Our subsequent immunodepletion experiments suggest that PAI-1 may not play a major role in the enhanced invasiveness of M cells by S-CM. However, additional experiments may be required to completely rule out the involvement of PAI-1 in the cooperative interaction between our tumor cell subpopulations, albeit secondary to modulation of SPARC levels.
SPARC, also known as osteonectin or BM-40, is a matricellular glycoprotein expressed in a variety of tissues during development and tissue repair and remodeling that regulates extracellular matrix deposition and cell-matrix interactions [
36]. SPARC has been shown to regulate cell proliferation, cell rounding, cell adhesion, angiogenesis, extracellular matrix remodeling and tumorigenesis, and to be involved in epithelial-mesenchymal transition. In cancer, SPARC has been reported to exert apparently contrasting activities [
37‐
39], either stimulating or inhibiting cell migration and invasion, promoting or reducing tumor growth and metastatic dissemination, sustaining cell survival or causing apoptosis, senescence and sensitization to genotoxic drugs. This suggests that the outcomes of activities mediated by SPARC may be context-dependent. Relevant variables include the source of SPARC (tumoral, stromal, or hematopoietic cells) with differences in glycosylation or peptide fragment patterns that result in functional differences, activities mediated by intracellular
vs. extracellular SPARC, or the status of pathways regulated by p53 or PTEN [
34]. The contrasting activities displayed by SPARC in different normal and neoplastic cell types are reflected in its varying status in different tumors [
40]. Thus, SPARC expression is elevated in many tumor types, including prostate cancer [
27], and it has been found to enhance the migration and invasiveness of prostate cancer cells [
41,
42]. In contrast, other studies have reported downregulation of SPARC in several tumor types, often in association with promoter hypermethylation, and its expression levels to be negatively correlated with tumor stage, therapeutic response or patient outcome. The importance of the interactions between neoplastic cells and tumoral environment is highlighted by the associations of stromal expression of SPARC with tumor progression and patient outcome [
43].
We have found that both primary prostate cancer samples associated with metastasis and those without metastatic association express SPARC in their stromal components at variable levels that do not correlate with metastatic status. However, our immunohistochemical analysis has revealed that only those primary tumors associated with metastasis express significant levels of SPARC in their epithelial tumoral components. The origin of this SPARC in the neoplastic component is supported by laser microdissection and RNA quantification experiments that allow us to distinguish the epithelial-tumoral
vs. stromal origin of SPARC and show that SPARC immunostaining in the epithelial tumoral component correlates with the expression of relatively high mRNA levels in the tumoral component. The observed staining pattern was generally cytoplasmic, perinuclear or juxtamembrane dots, but also a diffuse nuclear staining in a significant proportion of cells in over half of the metastatic primary tumor samples analyzed. A nuclear localization of SPARC has long been noted [
44,
45] and, although the potential functions of this nuclear form of SPARC are unknown, it has been linked to cellular proliferation [
44]. The observed enhanced epithelial/stromal ratios of SPARC expression levels in metastasis-associated primary tumors did not result in increased overall SPARC transcript levels in the same samples or in plasma SPARC protein levels. These observations, along with those from our
in vitro and mouse xenograft cell models, underscore the importance of tumor-produced SPARC over that produced by stromal cells in conferring a metastatic phenotype to prostate cancer tumor cells.
There is consistent experimental evidence that the extracellular matrix remodeling, antiangiogenic and antiproliferative effects mediated by non-tumoral SPARC, produced by stromal fibroblasts or endothelial or hematopoietic cells, constitutes a barrier to tumor progression, invasion and metastasis in mouse models of lung, ovarian, bladder, prostate or pancreas cancers [
43,
46‐
50]. Such a conclusion is in line with a potentially more general anti-metastatic function of tumoral stroma, as highlighted by recent observations [
51,
52] that are prompting a revision of prior notions on the role of the tumor microenvironment in modulating the metastatic behavior of cancer cells [
53]. In parallel, there is also substantial evidence that SPARC endogenously produced by cancer cells favors their invasive, survival and tumorigenic properties [
24,
54‐
62]. The relevance in human tumor progression of a cell-autonomous pro-metastatic function of SPARC is supported by its expression by the cancerous components of metastatic primary tumor samples of glioblastoma, melanoma and prostate cancer, but not in samples from non-metastatic primary tumors [
18,
40,
55,
61].
Few experimental models have simultaneously addressed the role of both cancerous and non-cancerous SPARC in tumor progression. A TRAMP mouse model crossed to SPARC
-/- mice [
50] concluded that both cancerous and non-cancerous SPARC exert tumor suppressor functions. Our results, however, suggest a different perspective in which (1) a relevant factor may be the interaction between SPARC-producing cancerous subpopulations, which are non-metastatic in our model (S cells), with SPARC-responding cancerous subpopulations (M cells in our model) to enhance the invasive and metastatic behavior of the latter; and (2) the ratio of cancerous to non-cancerous stromal SPARC expression levels, rather than overall tumoral or stromal-only expression, may be a significant determinant of metastatic behavior, as suggested by our immunohistochemical analysis and transcript quantification of metastatic and non-metastatic primary prostate cancer samples. A pro-metastatic function of SPARC endogenously produced by neoplastic cells may be attributed to its increased levels and a paracrine function in metastasis-promoting neoplastic cells and possibly also to differences in one or more properties of SPARC produced by non-neoplastic stromal cells, as suggested by its prominent nuclear localization in a significant proportion of metastatic primary tumors. There is evidence for differential postranslational processing of SPARC in different cell types, potentially leading to differential functions [
40], and it remains to be explored if this applies to non-neoplastic
vs. neoplastic or non-metastatic
vs. metastatic cancer cells.
In addition to our own previous observations [
11], other studies have provided examples of cooperation between heterogeneous tumor cell subpopulations that leads to enhanced metastatic behaviors of tumors [
8,
33,
63,
64]. One of these studies has identified non-cancerous fibronectin as a key extracellular protein to support the enhanced invasiveness prompted by cooperating neoplastic cell subpopulations [
63]. Our current study is the first to identify a specific extracellular matrix remodeling factor produced by a non-CSC neoplastic cell subpopulation as responsible for instigating the invasion and metastatic behavior of a second subpopulation displaying CSC properties. Matricellular proteins, including osteopontin, tenascin or SPARC, are gaining increasing attention for their roles in shaping local or metastatic niches to either support or prevent the growth and colonization potentials of tumor cells [
38,
65,
66]. The observations described here indicate that SPARC can also participate in paracrine interactions between tumor cell subpopulations and influence their metastatic potential.
Materials and methods
Cell lines
Luciferase-bearing PC-3M and PC-3S cells were clonally derived from the human cell line PC-3 [
11]. Du-145, CWR22v1 and LNCaP cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were grown at 37°C in a 5% CO
2 atmosphere in RPMI 1640 medium supplemented with 10% fetal bovine serum, L-glutamine, non-essential aminoacids, sodium pyruvate, penicillin/streptomycin (PAA Laboratories, Coelbe, Germany) and geneticin (Santa Cruz Biotechnologies, Santa Cruz, CA).
Cells (103/well) were seeded on 24-well Ultra Low Attachment culture plates (Corning Costar, Cambridge, MA) in complete culture medium containing 0.5% methyl cellulose (Sigma-Aldrich, St. Louis, MO), allowed to grow for 14 days and spheroids scored by image acquisition and spheroid area quantification with ImageJ.
In vitro invasiveness assays
Transwell-Matrigel invasion assays were performed as described [
11]. To harvest S conditioned medium (S-CM) or M conditioned medium (M-CM), cells were cultured to 70% confluence, at which time the culture medium was replaced with fresh CD-CHO medium (Invitrogen) supplemented with 8 mM glutamine (PAA). After 48 h, conditioned media were collected, centrifuged and filtered through a 0.2 μm filter (VWR Company, Darmstadt, Germany). M cells were analyzed for invasiveness by seeding with S-CM or M-CM (control) on Matrigel-hyaluronic acid-coated Transwell chambers. For co-culture experiments, M cells were loaded with Oregon Green 488 carboxy-DFFDA-SE (Invitrogen) and S cells with Far Red DDAO-SE (Invitrogen), by adding 25 μM of fluorophore to the cell suspensions for 30 min, washed with PBS, and reseeded. Fluorophore-preloaded cells were co-cultured at a 1:1 ratio on Matrigel-Transwell units and scored for invasiveness after 24 h.
SILAC labeling and sample preparation
M and S cells were cultured in L-lysine and L-arginine-depleted RPMI (Thermo Scientific, Hudson, NH) supplemented with 10% dialyzed FBS, antibiotics (PAA), and either 0.1 mg/mL 12C6- (M) or 0.1 mg/mL 13C6- (S), L-lysine and L-arginine (Thermo). The medium was replaced every 2 days, and cells routinely passaged at 80-90% confluence. After 14 days, cells were cultured for 2 days in light (M) or heavy (S) labeled medium without FBS, conditioned mediums collected, centrifuged, filtered through a 0.2 μm filter (VWR) and concentrated using Amicon centrifugal filter devices with a 3-kDa molecular weight cut-off (Millipore, Billerica, MA). Protein content was quantified (Bradford RcDc protein assay; BioRad, Hercules, CA) and 15 μg of each sample were mixed, diluted in 50 mM ammonium bicarbonate and concentrated using an Amicon centrifugal filter device with a 10-kDa molecular weight cut-off (Millipore). The <10 kDa fraction was evaporated to dryness, resuspended in 8 M urea, 50 mM ammonium bicarbonate, reduced with 50 mM dithiothreitol, alkylated with 125 mM iodoacetamide, digested with trypsin and analysed by LC-MS. The >10 kDa fraction was resuspended in loading buffer and subjected to electrophoresis on a 12.5% SDS-polyacrylamide gel.
Mass spectrometry analysis
After SDS-PAGE and Coomassie blue staining, lanes were split into 10 slices, digested with modified porcine trypsin (Promega, Madison, WI), dried, extracted with formic acid solution and analyzed on an Esquire Ultra IT mass spectrometer (Bruker, Bremen, Germany) coupled to a nano-HPLC system (Ultimate; LC Packings, Amsterdam, The Netherlands). Peptide mixtures were concentrated on a 300 mm i.d., 1 mm PepMap nanotrapping column and loaded onto a 75 mm i.d., 15 cm PepMap nanoseparation column (LC Packings). Peptides were eluted by an acetonitrile gradient (0–60% B in 150 min, where B is 80% acetonitrile, 0.1% formic acid in water; flow rate ca. 300 nL/min) through a PicoTip emitter nanospray needle (New-Objective, Woburn, MA) onto the nanospray ionization source of the IT mass spectrometer. MS/MS fragmentation (1.9 s, 100–2800 m/z) was performed on three of the most intense ions, as determined from a 1.2 s MS survey scan (310–1500 m/z), using a dynamic exclusion time of 1.2 min for precursor selection and excluding single-charged ions.
Protein identification and data analysis
Protein identification and quantification was performed using Protein Scape 2.1 and WARP-LC 1.2 (Bruker). Proteins were identified using Mascot (Matrix Science, London, UK) on the SwissProt human protein database. MS/MS spectra were searched with a 1.5 Da precursor mass tolerance, 0.5 Da fragment tolerance, 1 missed cleavage maximum trypsin specificity, cysteine carbamidomethylation set as fixed modification and methionine oxidation and the N-terminal and Lys and Arg SILAC labels as variable modifications. Positive identification criterion was set as an individual Mascot score for each peptide MS/MS spectrum above the homology threshold score. False positive rates for Mascot protein identification were measured by searching a randomized decoy database [
67], and estimated to be under 4%. For relative protein quantification, H/L ratios were calculated averaging the measured H/L ratio for the observed peptides, after discarding outliers. For selected proteins of interest, quantitative data obtained from the automated Protein Scape analysis were manually curated. For further protein analysis, UniProtKB (
http://www.uniprot.org) and GeneCards databases (
http://www.genecards.org) were used.
Western blotting
Samples were resuspended in Laemmli buffer (100 mM dithiothreitol, 50 mM TrisCl pH 6.8, 2% SDS, 0.1% bromophenol blue, 10% glycerol) and boiled for 5 min. Equal amounts of protein were resolved by SDS-PAGE. After electrophoresis, proteins were transferred onto fluorescent-PVDF membranes (Immobilon-FL, Millipore) for 2–4 h. Blots were washed, blocked with blocking buffer (Odyssey, LI-COR), incubated overnight with primary antibody diluted in blocking buffer-PBS (or TBS)-Tween (0.1%) (1:1), washed in PBS-T (or TBS-T) and incubated for 1 h with fluorescent secondary antibody IRDye (LI-COR Biosciences, Lincoln, NE). After final washes in PBS-T (or TBS-T), the membranes were scanned using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). Antibodies to the following antigens were used: SPARC (1:200) (H-90, Santa Cruz), PAI-1 (1:200) (C-9, Santa Cruz), AKT1 (1:100) (C-20, Santa Cruz), phospho-AKT1 (1:100) (pSer473, Santa Cruz), ERK1/2 (1:200) (H-72, Santa Cruz), phospho-ERK1/2 (1:100) (pThr177/pThr160, Santa Cruz), ILK (1:100) (E-2, Santa Cruz), phospho-ILK (1:100) (pThr173, Santa Cruz) and β-tubulin (1:2,000) (Sigma).
Lentiviral shRNA production and transduction
pLKO.1-Puro plasmids for control (shC002) and SPARC-targeting shRNAs TRCN0000008709 (sh8709) and TRCN0000008711 (sh8711) were from Sigma-Aldrich. Doxycycline-inducible SPARC-targeting V2THS_153399 (sh3399) was from Thermo. Each of these plasmids was co-transfected in HEK293T cells with pVSVG and pCMVΔR8.91 (Clontech, Mountain View, CA) using X-tremeGENE9 (Roche). Supernatants were collected for the following 48 h and filtered through 0.45 μm methylcellulose filters (Millipore). Viral particles were concentrated by ultracentrifugation on 20% sucrose gradients. Target cells were transduced in the presence of polybrene (8 μg/mL; Sigma-Aldrich), and selected with 1 μg/mL puromycin (Sigma-Aldrich) for 7 days.
Production and purification of recombinant human SPARC
HEK 293T cells bearing integrated copies of a human SPARC expression vector [
68] were grown in DMEM supplemented with 10% FBS and 3 μg/mL puromycin. Confluent cells were grown in serum- and puromycin-free medium and conditioned medium harvested every 2 or 3 days for 7–8 harvests and concentrated 10-fold on 30,000 Da cut-off Amicon centrifugal filter devices (Millipore) with simultaneous exchange into starting buffer (20 mM MOPS, 200 mM LiCl, pH 6.5). Concentrates were bound to a Maxi Anion (Q) Spin Column (Thermo) equilibrated with starting buffer and eluted with 30% elution buffer (20 mM MOPS, 400 mM LiCl, pH 6.5). SPARC-containing fractions, monitored by SDS-PAGE, were concentrated and dialyzed against Hank’s balanced salt solution (HBSS).
Immunodepletion experiments
S-CM was obtained as described above and subjected to immunoprecipitation using 2 μg/mL rabbit anti-SPARC (H-90, Santa Cruz) or anti-PAI-1 (C-9, Santa Cruz) plus pre-washed protein G-Sepharose (GE Healthcare, Buckinghamsire, UK) at 4°C for 2 h. Subsequently, samples were centrifuged and supernatants collected and used in invasiveness assays. Pelleted beads were washed three times with PBS and resuspended in Laemmli buffer. Aliquots from supernatant and resuspended pelleted beads were processed for Western blotting analysis as above.
Orthotopic prostate model and bioluminescence imaging (BLI)
M and S.sh3399 cells were transduced with the pRRL-Luc-IRES-EGFP lentiviral vector [
11] for the constitutive expression of the firefly luciferase gene to generate M.Fluc and S.Fluc.sh3399 cells, respectively. M.Fluc cells (1 × 10
5), or a mix of M.Fluc (1 × 10
5) with S.Fluc.sh3399 (1 × 10
5) cells, pretreated or not
in vitro with doxycycline (1 μg/mL; Sigma-Aldrich), resuspended in 30 μL sterile PBS, were inoculated into the dorsal prostates of 6-week-old SCID-Beige mice. Doxycycline (1 mg/mL) was administered
ad libitum in drinking water containing 25 mg/mL sucrose (Sigma-Aldrich) to induce the expression of shSPARC. BLI was performed with the IVIS Spectrum Imaging System (Perkin Elmer Life Science, Boston, MA), and images and measurements were acquired and analyzed using the Living Image 4.3.1 software (Perkin Elmer). For
in vivo BLI, animals were anesthetized with 1-3% isoflurane (Abbott Laboratories, IL) and injected i.p. with 150 mg/kg of D-luciferin (Promega) in sterile PBS. For
ex vivo BLI, mice were injected i.p. with 150 mg/kg D-luciferin prior to euthanasia. Immediately postmortem, organs of interest were placed individually into separate wells with 300 μg/mL D-luciferin, imaged and quantified as above. These experiments were performed at the CIBER-BBN
In Vivo Experimental Platform.
RNA isolation, reverse transcription and real-time RT-PCR
RNA purification and reverse transcription were performed as described [
11]. Real-time quantitative PCR assays were performed on a LightCycler 480 instrument (Roche) and analyzed with the LightCycler 480 Software release 1.5.0. The Universal Probe Library system (UPL) (Roche) was used to quantify transcripts. Probes and sequences are shown in Additional file
2: Table S2. RN18S1 amplification levels were used as an internal reference, and relative transcript quantification determined by the ∆∆Cp method. Tissue samples were procured through the Hospital Clínic-IDIBAPS Biobank, a Generalitat de Catalunya authorized biobank registered at the Instituto de Salud Carlos III, and thus sample collection and processing fulfilled all ethical and legal requirements.
Tissue microdissection and transcript quantification
Eight formalin-fixed paraffine-embedded samples were used for laser microdissection, 4 from non-metastatic cases and 4 from metastatic cases. Eight-μm sections from each sample were mounted onto plastic membrane slides (Leica Microsystems, Germany), stained with hematoxylin-eosin, air dried and stored at -80°C until use. Laser microdissection was performed with the Leica LMD7000 System (Leica). Approximately 3 mm2 of either tumoral epithelium or stroma were collected separately for each sample. RNA isolation was performed with the RNeasy FFPE Kit (Qiagen) and reverse transcribed with the High-Capacity cDNA Reverse Transcription Kit (Life Technologies). cDNA was preamplified (14 cycles) with the TaqMan PreAmp Master Mix (Life Technologies). SPARC transcript levels were quantified using a TaqMan assay (Hs00234160_m1) and normalized to β-2 microglobulin (B2M) transcript levels (assay Hs00984230_m1). Real-time PCR assays were performed and analyzed as described above.
Immunohistochemistry
A total of 30 samples were used for immunohistochemical detection of SPARC protein, 14 from non-metastatic and 16 from metastatic cases. Two μm thick sections were mounted on xylaned glass slides (DAKO, Glostrup, Denmark) and processed for antigen retrieval in citrate buffer pH 6 for 20 minutes, incubated for 1 h with rabbit-anti-SPARC (1:300) (H-90, Santa Cruz) and reactions revealed with the Bond Polymer Refine Detection System (Leica, Wetzlar, Germany). The staining was scored as the percentage of positive cells with null, weak, moderate or strong intensities, scoring separately the epithelial and stromal compartments of each sample. Images were captured with an Olympus BX-51 microscope equipped with an Olympus DP70 camera.
Immunocytochemistry
Cells were seeded on sterile round glass coverslips, allowed to attach for 24 to 48 h, washed with PBS, fixed with cold 4% paraformaldehyde for 20 min, permeabilized with 1% Triton X-100 in PBS, blocked for 30 min with blocking buffer (3% BSA, 1% Triton X-100 in PBS), incubated with primary antibody (anti-SPARC, 1:50 in blocking buffer) for 2 h at room temperature, washed 3× with blocking buffer, and incubated for 30 min with Alexa Fluor 488-conjugated rabbit-anti-mouse antibodies (Life Technologies; 1:1,000 in blocking buffer), Alexa Fluor 555-conjugated phalloidin (Life Technologies; 1:10,000) and 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride, 4′,6-Diamidino-2-phenylindole (DAPI) dihydrochloride (Sigma; 0.1 μg/mL). After washes, samples were mounted on glass slides with Mowiol 18–88 (Sigma) and visualized under a Leica SP5 confocal microscope (Leica, Wetzlar, Germany).
Plasma SPARC quantification by ELISA (Enzyme-Linked ImmunoSorbent Assay)
Plasma samples were collected from 4 healthy controls (blood PSA <4 ng/mL) and 10 non-metastatic and 14 metastatic prostate cancer patients, using EDTA as anticoagulant. Blood from patients was extracted prior to prostatectomy. No patients or controls were under systemic treatment at the time of blood extraction. Quantification of SPARC protein levels was performed using Quantikine ELISA Human SPARC Immunoassay (R&D Systems, Abingdon, UK). Tissue and blood samples were procured through the Hospital Clínic-IDIBAPS Biobank, a Generalitat de Catalunya authorized biobank registered at the Instituto de Salud Carlos III, and thus sample collection and processing fulfilled all ethical and legal requirements.
Statistical analysis
A Student’s t-test was applied to two-way comparisons of data sets from in vitro experiments. A non-parametric Mann–Whitney test was applied to SPARC level determinations in tissues and blood and for bioluminescent data from xenograft experiments. The significance threshold was established at P < 0.05, and significance levels were schematically assigned * (0.01 ≤ P < 0.05), ** (0.001 ≤ P <0.01) or *** (P < 0.001).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
TMT and FM conceived and designed the study, analyzed the data and drafted the manuscript. FM performed most of the key in vitro experiments and patient tissue and plasma analyses. OM and TC were involved in the original conception of the study, and OM performed in vitro experiments. YF, IA and VC performed in vivo experiments. RB, AS, LS-C, LR-C and PLF participated in tissue processing, pathological diagnosis, immunohistochemichal analysis and real-time RT-PCR analysis of tissue samples. MM, LM, AA, BM, SS, KA, RB and RP provided materials and technical assistance. All authors have read and approved the manuscript.