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Interaction between prostate cancer cells and prostate fibroblasts promotes accumulation and proteolytic processing of basement membrane proteins

Tumor microenvironment or stroma has the potency to regulate the behavior of malignant cells. Fibroblast‐like cells are abundant in tumor stroma and they are also responsible for the synthesis of many extracellular matrix components. Fibroblast‐cancer cell interplay can modify the functions of both cell types.


| INTRODUCTION
Malignant tumors contain several nontransformed cell types, including endothelial cells, various inflammatory cells, and fibroblasts. 1 Cancer cells as well as all other cell types in tumors are surrounded by extracellular matrix (ECM). ECM is an insoluble scaffold for cells and at the same time a reservoir of matrix-bound growth factors, which has the ability to alter malignant cell behavior. 2 ECM in the tumor stroma is mainly synthesized by fibroblast-like cells, often by a special group of activated mesenchymal cells, called cancerassociated fibroblasts (CAFs). 3 In prostate cancer fibroblasts become activated before the actual malignant growth, at the prostate intraepithelial neoplasia stage. 4 In vitro coculture of stromal cells together with cancer cells has been used to study cell behavior in tumor-like circumstances. CAFs' contribution to the cancer cell motility has been shown to be mediated by secreted growth factors 5,6 or by CAF-modified aligned matrix. [7][8][9] Many studies have reported abnormal composition, organization, or stiffness of the ECM in malignant tumors. 4,8,[10][11][12] Tumor progression and resistance to therapy can partially be connected to altered ECM architecture or biochemical properties. [13][14][15][16] In addition, ECM can be modulated by degradation, posttranslational modification or de novo synthesis of matrix proteins. 1 Many proteases, including matrix metalloproteinases (MMPs), mediate the degradation of ECM and consequently reorganize the matrix and release matrix-derived bioactive peptides. 17 It is well acknowledged that CAFs release growth factors and bioactive protein fragments that can regulate cancer cell division and invasion. 6 Most often CAFs have been connected to the promotion of tumor growth, [7][8][9]18,19 but inhibition of tumor progression has been reported as well. 20 The aim of the present study was to determine how direct prostate cancer cell-prostate fibroblast interaction modulates tumor stroma and cell invasion. The study included "out of spheroid" invasion assays and mass spectrometry analysis of the ECM proteins.
Our results show that the interplay between prostate fibroblasts with DU145 or PC3 cells in 3D spheroid cocultures leads to remarkable changes in the composition of ECM and concomitantly to the accelerated invasion of the prostate cancer cells.

| Cell culture
The human androgen-independent prostate epithelial adenocarcinoma cell lines DU145 and PC3 (both from American Type Culture Collection) and human prostate-derived fibroblastic cells, described previously 21 were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Lonza), supplemented with 10% fetal calf serum (FCS, Biowest), 2 mM Ultraglutamate, 100 U/mL penicillin and streptomycin (Lonza). In the indicated experiments also serum-free (SF) RPMI-1640 medium or keratinocyte serum-free medium (KSFM) with 5 ng/mL human recombinant epidermal growth factor, 50 μg/mL bovine pituitary extract (Gibco), supplemented with 2 ng/mL recombinant human leukemia inhibitory factor and 2 ng/mL stem cell factor (Sigma Aldrich) was used. Cells were routinely screened with MycoAlert PLUS Mycoplasma detection kit (Lonza).

| Spheroid cultures
Spheroids were made in micro-molds according to the manu-

| Spheroid intensity profiling
The spheroid intensity profiles obtained from ImageJ were aligned by normalizing the distances from the spheroid edge by spheroid diameter. The original intensity values and spheroid diameter were determined from a single z-stack (the middle stack of all the z-stacks) of confocal images by using the plot profile tool in ImageJ. For each channel separately, the aligned intensities were normalized by the total area under the intensity curve. 15 spheroids from three independent biological replicates were analyzed.

| Immunofluorescence and confocal microscopy
Before spheroids were made cancer cells were stained with Cell Tracker Green (CMFDA, 5 µM, Invitrogen) and fibroblasts with Orange (CMTMR, 5 µM, Invitrogen) for 1 hour to distinct cancer cells and CAFs from each other during imaging. After staining cells were allowed to recover 30 minutes with fresh growth media, thereafter trypsinized and spheroids were prepared as explained above. Cells were let to grow for 72 hours, then fixed with 4% paraformaldehydephosphate-buffered saline (PBS), 3 hours, +4°C, stained with 4′,6-diamidino-2-phenylindole, and mounted with glycerol.

| Migration and invasion assay
Cells were seeded into micro-molds to form spheroids for 72 hours as explained above. Three different conditions were used: first, media were not changed; second media were changed daily and it contained either 50 µg/mL ascorbic acid; or contained 1 mM hydroxyurea as third option. Thereafter spheroids were transferred from molds to 96-well plate coated with Collagen I 5 µg/cm 2 o/n at +4°C and blocked with 0.1% bovine serum albumin (BSA)-PBS 1 hour at +37°C.
In migration study KSFM was added on top of the spheroid. In invasion study Collagen I gel was first added on top of the spheroid and KSFM was added into the wells after gel was properly formed.
Spheroids were imaged every 24 hours for 4 days by using IncuCyte ZOOM System. To study the effects of MMP inhibition on the cells invasion medium was supplemented with 1 µM NNGH (Sigma) or 10 µM NSC 405020 (Sigma) with respectively amount of DMSO as control.

| Western blot analysis
Cells grown as spheroids for 6 days with a daily change to fresh media (RPMI with 0% FCS, 50 µg/mL ascorbic acid) were treated with HLB as explained in the mass spectrometry section. The protein pellet was suspended into radioimmunoprecipitation assay buffer (Thermo Fisher Scientific). The 6 × sodium dodecyl sulfate (SDS) sample buffer was added and samples were boiled 5 minutes at 100°C. Protein aliquots were separated in 6% to 15% SDS-polyacrylamide gel electrophoresis gels, thereafter proteins were immobilized onto polyvinylidene difluoride-membrane. The following primary antibodies were used: laminin-5 (ab14509, Abcam); endostatin (PA1-601, Thermo Fisher Scientific); MMP-14 (AB6005, Sigma-Aldrich), and β-actin (A-1978, Sigma-Aldrich). The membranes were incubated with the primary antibodies overnight at +4°C, followed by incubation with secondary antibodies (680RD or 800CW; 926-32213, 926-32212, 926-68072, or 926-68073, LI-COR Biosciences) in 5% BSA in Tris-buffered saline with Tween-20 for 1 hour at room temperature. Odyssey CLx from Li-COR Biosciences was used to detect fluorescent bands on membrane, band intensities were analyzed with Image Studio program (LI-COR) and protein expressions were shown as relative to β-actin.

| Statistical analysis
The paired two-sample Student t tests, One way analysis of variance with Tukey or Dunnett T3 post hoc tests were performed by using IBM SPSS Statistics software version 22.  Table SI). None of the common or DU145-specific gene OJALILL ET AL.

| 717
products represented core matrisome structural proteins, while PC3 cells could produce laminin chains α3, β3, γ2, as well as HSPG2 and collagen VI (COL6A1) ( Figure 1B; Table SI). In accordance with our previous observations, 21 fibroblastic cells were responsible for the production of the structural proteins in the core matrisome ( Figure 1C; Table 1). The major components of collagenous, fibronectin-rich ECM could be detected in fibroblast spheroids, including the two α chains of fibril forming collagen I (COL1A1 and COL1A2), fibril-associated proteins, such as collagen XII (COL12A1) and decorin (DCN), tenascin C (TNC), and beaded filament-forming collagen VI (COL6A2 and COL6A3) ( Table 1). Prostate-derived fibroblasts also produced MMPs, since MMP-14 (also called membrane type 1 metalloproteinase, MT1-MMP) was recognized in the samples. Comparison of matrisome profiles in fibroblast, DU145/ fibroblast, and PC3/fibroblast spheroids revealed remarkable similarity, that is, 87 out of 107 proteins identified ( Figure 1C).
In cancer cell/fibroblast cocultures basement membrane components, such as laminins (α3, α5, β2, and β3), heparan sulfate proteoglycan (HSPG2 gene product), and collagen XVIII accumulated in a more prominent manner when compared with spheroids that contained one cell type only (Table 2). Proteoglycans agrin and syndecan 4 had enhanced expression in the cocultures, too ( Table 2).
The increased accumulation of these proteins was observed in both PC3/fibroblast and DU145/fibroblast spheroids. At the same time, the interplay between prostate cancer cells and fibroblasts led to a diminished accumulation of a number of ECM proteins, most notably fibrillin-2 (FBN2), fibulin-1 (FBLN1), and collagen XIV ( Table 2).
Staining of fibroblasts and cancer cells with distinct Cell Trackers unveiled organization of the 5 days old coculture spheroids. Confocal z-stack mode images and maximum projection were used to get an overview of the cellular localization. In the majority of DU145/fibroblast spheroids the fibroblasts surrounded the core formed by DU145 cells. In contrast, in PC3/fibroblast spheroids the two cell types were mixed, that is, the similar fibroblastic capsule was not formed and the surface of the spheroid was often covered by PC3 cells (Figure 2A,B).

| Cancer cell-fibroblast interaction promotes proteolytic processing of basement membrane components
To confirm the mass spectrometry results we performed Western blots utilizing specific antibodies to laminin-332 (previously called laminin -5) and to endostatin, a protein derived from the C-terminal part of collagen XVIII. These analyses indicated that all three laminin subunits in laminin-332, namely, α3, β3, and γ2, were upregulated in cancer cell/fibroblast coculture spheroids ( Figure 3). Importantly, it was also possible to see lower molecular mass bands (~40 kDa), which suggested proteolytic processing of laminin ( Figure 3). Similarly, in F I G U R E 1 Cancer cells produce limited number of core matrisome proteins. A, Most of the proteins identified from DU145 and PC3 spheroid culture matrices in mass spectrometry analysis fall into the categories of ECM regulators, ECM-affiliated proteins and secreted factors. Only one protein from groups of collagens and proteoglycans was identified. B, Prostate cancer cell lines share 32 matrix proteins out of 64; DU145 cells exclusively synthesize four matrix proteins and are unable to synthesize matrix structural proteins. PC3 cells synthesize 28 proteins not identified in DU145 cells, these include laminins, HSGP2, and collagen type VI (Table SI). C, The majority of matrisome proteins are synthesized by fibroblasts. About 87 proteins were shared between fibroblasts only, DU145/fibroblasts, and PC3/fibroblasts matrisomes. ECM, extracellular matrix [Color figure can be viewed at wileyonlinelibrary.com] DU145/fibroblast spheroids collagen XVIII was intensively processed tõ 30 kDa endostatin-containing fragment ( Figure 4A,B), a common degradation product in a process also generating the classical~20 kDa endostatin. 22 The potential role of endostatin in DU145-ECM interaction was also tested using real time xCelligence technology to measure cell attachment and spreading on ECM protein fibronectincoated surfaces. Human recombinant endostatin was shown to significantly promote the adhesion of DU145 cells to fibronectin ( Figure 4C,D). To conclude these results, the spheroids contained proteolytic activity that could modify the basement membraneassociated proteins and generate their functional fragments, which also have the capability to regulate cancer cell behavior.  (Table SI). Cathepsin L has been shown to release endostatin from collagen XVIII NC1-domain. 22 Accordingly, in our coculture spheroids a selective Cathepsin L inhibitor (Z-FY-CHO) in a concentration of 50 µM significantly decreased the formation of~30 kDa endostatin-containing protein ( Figure 5).

| Interplay between DU145 cells and fibroblasts promotes the motility of cancer cells in 3D invasion model
In cancer the invasion of malignant cells leads to the local progression of the disease and later to the formation of metastases.
Both DU145 and PC3 are considered to represent invasive cell phenotypes. 23 Here, we established an invasion assay that measures the ability of cells to move out of the coculture spheroid. At first, prostate cancer cells or prostate-derived fibroblasts or both cell types together were allowed to form spheroids. After 72 hours in culture these spheroids were placed on collagen I coated surfaces or inside collagen I gels. Migration on collagen I or invasion through collagen I gel was followed by phase-contrast microscopy for 96 hours. Fibroblasts alone did not invade out from the spheroids ( Figure 6A-E). In this experimental setting PC3 cells in coculture with fibroblasts invaded into the collagen gel less effectively than DU145 cells ( Figure S2) and, therefore, the following experiments were performed with DU145 and DU145/fibroblast spheroids only.
In 2D conditions, cell migration out of DU145 spheroids was more efficient when compared with DU145/fibroblast spheroids ( Figure 6A).
However, invasion of cells inside 3D collagen gels was significantly faster when DU145/fibroblast spheroids were assayed and compared with spheroids solely formed by either DU145 cells or fibroblasts ( Figure 6B). When DU145 cells and fibroblasts were differentially   Figure 6C). To ensure that the difference in invasion was not affected by cell number, experiments in which hydroxyurea was used to block cell proliferation were performed. The inhibition of cell growth had no effects on the invasion ( Figure 6D).
These results suggest that in spheroids fibroblasts activate DU145 cells and initiate invasion. In the presence of ascorbic acid, the difference between DU145/fibroblast and DU145 spheroids remained the same ( Figure 6E), which suggests that triple helical collagens produced by the fibroblasts or organization of the collagenous matrix is not part of the mechanism how fibroblasts support cancer motility. DU145 cells can use direct integrin α2β1-mediated adhesion to collagen I. 24 It is also possible that basement membrane protein production induced by fibroblasts in DU145 cells has a positive effect on an invasion. However, it is not clear, whether in these conditions DU145 cells are able to take full advantage of ECM produced by fibroblasts. In addition soluble factors, for example, growth factors or MMPs, produced by fibroblasts may play part in the process. Indeed, we could detect hepatocyte growth factor (HGF) and fibroblast growth factor (FGF2) in spheroid cultures (Table 1).
To test the potential role of MMPs during the invasion process we used the two selective inhibitors, NNGH and NSC 405020, as above. Both inhibitors could significantly inhibit the migration and invasion of DU145 cells ( Figure 7A,B), but a similar phenomenon could also be seen in the absence of fibroblasts ( Figure 7A | 721 in cancer cell/fibroblast cocultures. These gene products included α3, α5, β2, β3 laminins, heparan sulfate proteoglycan (HSPG2 gene product), and collagen XVIII.
Incompletely organized, thin basement membranes as well as the down regulation of α6β4 integrin and other hemidesmosomeassociated proteins are typically seen in prostate cancer. 25,26 One earlier report suggests that the expression of LN332 related gene products (α3, β3, and γ2) is lost, whereas the corresponding messenger RNA levels have paradoxically elevated. 27 Here, our results propose that the in vivo activation of laminin genes may be due to  F I G U R E 6 Fibroblasts induce invasion capability of DU145 cancer cells in coculture. A, Representative images and quantification of migration assay. Spheroids consisting of DU145 cells or fibroblasts alone or in coculture were grown for 96 hours and placed on collagen I coated surface. The migration shown as area covered by cells was followed for 96 hours with Incucyte Zoom B, Representative images and quantification of invasion assay. Spheroids consisting of DU145 cells or fibroblasts alone or in coculture were grown for 96 hours and placed on collagen I coated surface, covered with collagen I gel and cell invasion into gel was followed for 96 hours with Incucyte Zoom. C, Representative confocal microscopy images of invasion assay at 0 and 24 hour time point. DU145 cells (green), fibroblasts (red) alone or in coculture indicate that DU145 cells move actively out from spheroids and fibroblasts tend to stay still. The treatment of cells with hydroxyl urea to inhibit cell proliferation (D) and the treatment of cells with ascorbic acid to induce the synthesis of cells own triple helical collagen (E) indicated that the observed differences were not influenced by proliferation of cells nor due to synthesis of own matrix [Color figure can be viewed at wileyonlinelibrary.com] Biobank. Maria Tuominen is acknowledged for excellent technical assistance. We also thank the Proteomics and the Cell Imaging and Cytometry core facilities, supported by Biocenter Finland.