Background
Tumor microenvironment has become the focus of intensive research as a potential target for cancer therapy [
1]. In the normal epithelium, parenchymal cells and stromal components are physically separated by a basement membrane. The transition from normal epithelium to invasive carcinoma is preceded by, or is concomitant with, the activation of local host stroma [
2]. Invasion occurs in close cooperation with stromal cells and the transformed epithelium [
2,
3]. Malignant progression impairs the integrity of the basement membrane resulting in the deterioration of its organized structure. Invasive tumor cells lose their epithelial characteristics and acquire metastatic phenotype. In this process a vast number of macromolecules are produced by stromal cells capable of influencing the microenvironment [
4].
Fibroblasts are characteristic cell types in the microenvironment playing a prominent role in the pathology of solid tumors [
5]. Cancer-associated fibroblasts (CAFs) within the reactive stroma express elevated amounts of extracellular matrix (ECM) proteins, proteases. These matrix metalloproteinases (MMPs) play an important role in the degradation of the basement membrane and stromal ECM initiating the invasion of malignant tumors [
6,
7]. During this process newly synthesized ECM proteins serve as a scaffold for motile tumor cells to move along, as well as providing structural support for angiogenesis [
5].
To obtain invasive phenotype, cervical carcinoma cells may utilize stromal MMPs. MMP-7 and MMP-9 expressions can be induced in cancer cells, augmented by tumor-stromal cell interaction and possibly mediated by membrane-anchored and/or soluble factors [
8]. Their expression has been shown to correlate with the amount of the hyaluronan (HA) receptor CD44 noted in low grade squamous cell carcinoma (SCC) [
9,
10].
Growth signals generated by the stromal cells of the tumor are mediated to the cancer cells by integrins. These cell surface receptors are indispensable for the cross-talk between cancer cells and tumorous stroma [
4,
11]. Integrin-mediated communication is pivotal in cell survival, proliferation, migration and tumor invasion [
12].
In addition to thrombocytes, fibroblasts are the major sources of TGF-β1, a cytokine that is one of the most important regulators of ECM. TGF-β1 has a strategic role in the regulation of assembly and remodeling of extracellular matrix during cancer progression. Furthermore, the growth inhibitory action exerted by TGF-β1 on epithelial cells disappears after malignant transformation, which together with the process of EMT, changes the status of the growth factor from inhibitor to tumor promoter [
13]. The TGF-β1 molecule is synthesized as an inactive multidomain complex and its activation occurs through multiple extracellular mechanisms that may involve proteases, thrombospondin-1 and integrins [
14,
15]. This implies that the interplay between tumor cells and fibroblasts can modulate the effect of growth factors that in turn exert their modified action on the tumor tissue [
4,
16].
In the current work we investigated the course of matrix remodeling in cervical SCC by studying the molecular components listed above. To this end, we completed an immunohistochemical analysis of paraffin-embedded cervical SCC sections and established co-culture models between normal or tumorous cervical fibroblasts and CSCC7 HPV-positive cervix SCC cells.
Methods
Tissues, cell lines and materials
A tissue microarray (TMA) was generated from 27 normal and 29 cancerous, formalin fixed and paraffin embedded tissue blocks taken from the vaginal portions of cervices removed by radical Wertheim hysterectomy (Table
1). All cases had previously been analyzed for HPV genotypes [
17]. Tissue blocks were collected from the 1
st Department of Obstetrics and Gynecology of Semmelweis University, with permission and seal from the Semmelweis University Regional and Institutional Committee of Science and Research Ethics (TUKEB permit number: 95/1999). Representative normal and tumorous areas on hematoxylin- and eosin-stained (HE) sections (identified by an independent pathologist) were excised. Tissue cores corresponding to the marked areas were used to assemble a TMA block which was then sliced, counterstained with hematoxilin and immunostained.
Table 1
Patient data to TMA
1
| 37 | II/A | Adenosquamous carcinoma | - |
2
| 51 | I/B | Squamous cell carcinoma | - |
3
| 42 | II/A | Squamous cell carcinoma | - |
4
| 48 | II/B | Squamous cell carcinoma | HPV18 |
5
| 39 | II/B | Squamous cell carcinoma | HPV 16, 18 |
6
| 41 | II/A | Squamous cell carcinoma | HPV16 |
7
| 38 | II/B | Squamous cell carcinoma | HPV16 |
8
| 59 | II/B | Squamous cell carcinoma | HPV16 |
9
| 39 | II/B | Squamous cell carcinoma | HPV16 |
10
| 39 | I/B | Squamous cell carcinoma | HPV16 |
11
| 71 | II/A | Clear cell carcinoma | HPV16 |
12
| 51 | II/A | Squamous cell carcinoma | HPV16 |
13
| 44 | II/B | Squamous cell carcinoma | HPV16 |
14
| 42 | II/A | Squamous cell carcinoma | HPV16 |
15
| 45 | II/B | Squamous cell carcinoma | HPV16 |
16
| 32 | I/B | Squamous cell carcinoma | HPV16 |
17
| 55 | II/B | Squamous cell carcinoma | - |
18
| 56 | II/B | Squamous cell carcinoma | HPV16 |
19
| 57 | II/B | Squamous cell carcinoma | HPV16 |
20
| 44 | II/B | Squamous cell carcinoma | HPV16 |
21
| 35 | I/B | Squamous cell carcinoma | HPV16 |
22
| 66 | II/B | Squamous cell carcinoma | HPV16 |
23
| 57 | II/A | Squamous cell carcinoma | HPV16 |
24
| 38 | II/B | Squamous cell carcinoma | HPV16 |
25
| 40 | II/B | Squamous cell carcinoma | HPV16 |
26
| 52 | II/B | Squamous cell carcinoma | HPV16 |
27
| 62 | II/B | Squamous cell carcinoma | HPV16 |
28
| 57 | II/B | Squamous cell carcinoma | HPV16 |
29
| 54 | II/B | Squamous cell carcinoma | HPV16 |
Fresh surgical specimens obtained from radical Wertheim hysterectomy were sent for routine pathology service to the 1
st Department of Pathology and Experimental Cancer Research from the Maternity Private Department of the Kútvölgyi Klinikal Block of Semmelweis University. Fibroblasts from normal and tumorous regions of uterine cervix not used for diagnosis were obtained from explant cultures. The surgical material was collected and used based on approval by the Semmelweis University Regional and Institutional Committee of Science and Research Ethics (TUKEB permit number: 95/1999). CSCC7 HPV16 positive cervical cancer cells, derived from a case of planocellular cervical cancer, were the gift of G. Gorter from Leiden University [
18]. These cells exhibit a clear epithelial morphology and form nests when grown in monoculture. They are positive for pan-cytokeratin but negative for vimentin. In contrast, fibroblasts are vimentin positive, pan-cytokeratin negative cells displaying spindle-like morphology, with elongated, oval nuclei.
Materials and consumables used in cell cultures and the general chemicals used for the experiments were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), SARSTEDT AG&Co (Nümbrecht, Germany) and Merck (Darmstadt, F. R. Germany).
Tissue microarray, histochemistry, immunohistochemistry and immunocytochemistry
TMA slides were immunostained for α-smooth muscle actin (SMA), laminin-1, laminin-5 and fibronectin. Subsequently, they were scanned with a Scan Scope CS2 (Aperio Technologies Inc., Vista, CA, USA) and analyzed by MAN-0023 Color Deconvolution Algorithm Positive Pixel Count Analysis software (Aperio Technologies Inc.). Staining intensities were measured only in stromal areas, and percentage of positive and negative pixels were evaluated. Immunocyto- and immunohistochemistry procedures followed standard protocols [
19]. Antibodies used are listed in Table
2. Cell nuclei were counterstained with TOTO-3 (Invitrogen by Life Technologies Co., Carlsbad, California, USA). Images were taken using MRC-1024 confocal laser scanning microscope (Bio-Rad Laboratories GmbH, Münich, Germany).
Table 2
Antibodies used in the present study
Primary antibodies
|
Host
|
Manufacturer*
|
Cat. no.
|
Dilution IHC/ IF
|
Dilution WB/DB
|
β-Actin | Rabbit polycolnal IgG | Cell Signaling | 4967 | - | 1:1000 |
Laminin | Rabbit polycolnal IgG | Dako | Z0097 | 1:200 | - |
Laminin α1 (H-300) | Rabbit polycolnal IgG | Santa Cruz | sc-5582 | - | 1:200 |
Laminin β1 (H-300) | Rabbit polycolnal IgG | Santa Cruz | sc-5583 | 1:50 | 1:200 |
Laminin 5 | Rabbit polycolnal IgG | Abcam | Ab14509 | 1:100 | - |
Laminin-5 (P3E4) | Mouse monoclonal IgG1 | Santa Cruz | sc-13587 | 1:50 | 1:200 |
Fibronectin | Rabbit polycolnal IgG | Dako | A0245 | 1:100 | - |
Fibronectin (IST-9) | Mouse monoclonal IgG1 | Santa Cruz | sc-59826 | 1:40 | 1:500 |
Smoth Muscle Actin colone 1A4 | Mouse monoclonal IgG1 | Dako | M0851 | 1:200 | - |
Thrombospondin 1 (A6.1) | Mouse monoclonal IgG1 | Santa Cruz | sc-59887 | - | 1:250 |
Perlecan | Mouse monoclonal IgG1 | Zymed | 13-4400 | - | 1:500 |
Collagen I | Rabbit polycolnal IgG | Abcam | ab34710 | - | 1:1000 |
Collagen III | Rabbit polycolnal IgG | Abcam | Ab7778 | - | 1:1000 |
Anti-Human Collagen IV | Mouse monoclonal IgG1 | DakoCytomation | M 0785 | - | 1:200 |
Anti-Human MMP-7 | Mouse monoclonal IgG1K | Chemicon | MAB3315 | - | 1:500 |
TIMP1 (D10E6) | Rabbit monoclonal IgG | Cell Signaling | 8946 | - | 1:1000 |
TIMP3 (D74B10) | Rabbit monoclonal IgG | Cell Signaling | 5673 | - | 1:1000 |
Integrin α4 | Rabbit polycolnal IgG | Cell Signaling | 4600 | - | 1:1000 |
Integrin α5 | Rabbit polycolnal IgG | Cell Signaling | 4705 | - | 1:1000 |
Integrin α6 | Rabbit polycolnal IgG | Cell Signaling | 3750 | - | 1:750 |
Integrin αV | Rabbit polycolnal IgG | Cell Signaling | 4711 | - | 1:1000 |
Integrin β1 | Rabbit polycolnal IgG | Cell Signaling | 4706 | - | 1:1000 |
Integrin β3 | Rabbit polycolnal IgG | Cell Signaling | 4702 | - | 1:100 |
Integrin β4 | Rabbit polycolnal IgG | Cell Signaling | 4707 | - | 1:1000 |
CD44 | Mouse monoclonal IgG2a | Antibodies online | ABIN96695 | 1:100 | 1:500 |
CD151 (PETA-3) | Mouse monoclonal IgG2b | Novocastra™ Leica Biosystems | NCL-CD151 | 1:50 | - |
Secondary antibodies
|
Host
|
Manufacturer*
|
Cat. no.
|
Dilution IHC/IF
|
Dilution WB/DB
|
Anti-mouse Ig/HRP | Goat polyclonal | DakoCytomation | P0447 | - | 1:2000 |
Anti-rabbit Ig/HRP | Goat polyclonal | DakoCytomation | P0448 | - | 1:2000 |
Alexa Fluor® 647 anti-mouse IgG (H + L) | Donkey polyclonal | Invitrogen | A31571 | 1:200 | - |
Alexa Fluor® 488 anti-mouse IgG (H + L) | Donkey polyclonal | Invitrogen | A21202 | 1:200 | - |
Alexa Fluor® 647 anti-rabbit IgG (H + L) | Goat polyclonal | Invitrogen | A21244 | 1:200 | - |
Alexa Fluor® 568 anti-rabbit IgG (H + L) | Goat polyclonal | Invitrogene | A11011 | 1:200 | - |
Generation of cell cultures
Tumorous and normal areas of surgical specimens taken from the same patient and not used for diagnosis were excised and cut into small pieces and placed into six-well tissue culture dishes containing Cytogen Amnio Grow Plus medium (CytoGen GmbH, Sinn, Germany), optimized for development of primary cell culture. Fibroblasts were allowed to grow till the third passage and were then routinely transferred into DMEM-low glucose medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin. Normal fibroblasts are hereinafter referred to as NF, fibroblasts derived from tumorous areas as TF. Purity of the fibroblast cultures was tested by means of vimentin and citokeratin immunostaining. On average, <3% of epithelial contamination was found in the established fibroblast cultures. CSCC7 cells were routinely cultured in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/mL penicillin and 100 μg/mL streptomycin. All cell lines were cultured in a humidified 95% air/5% CO2 incubator at 37°C. The cell lines used were between the 8th and 12th passage, within which period both fibroblasts and CSCC7 cells proved to be stable.
Co-culture systems
Two models were used to study the interaction between fibroblasts and tumor cells. Direct co-cultivation allowed physical contact between cells, whereas in indirect co-cultures cells were separated by a transwell insert with a 0.45 μm pore size, allowing only molecular communication.
NF, TF, and CSCC7 cells were seeded in culture dishes 10 cm in diameter alone or in direct co-cultures with 5×105 cells/dish density in a 1:1 (v/v) mixture of DMEM-low glucose and RPMI-1640 supplemented with 5% FBS. Seventy two hours after seeding, the FBS content was reduced to 0.3% and cells were incubated for 24 h. Conditioned culture media (CCMs) and cell layers (CLs) were then harvested and frozen for further protein analyses. Samples from direct co-cultures are indicated as NF + CSCC7 and TF + CSCC7.
Indirect co-cultures were set up as follows: fibroblasts were seeded in 6-well plates (Corning Incorporated Life Sciences, Acton, MA, USA) at a density of 2.5×10
4 cells/well in DMEM-low glucose with 10% FBS. CSCC7 cells were placed in Transwell® polyester membrane inserts (Corning) with 0.45 μm filter pore size at a density of 5×10
4 cells/insert in RPMI-1640 with 10% FBS. Forty-eight hours after seeding CSCC7 containing inserts were placed in fibroblast containing wells followed by addition of 1:1 (v/v) mixture of DMEM-low glucose and RPMI-1640 supplemented with 5% FBS to the indirect co-culture and to control cells growing alone. Seventy-two hours later the FBS content was reduced to 0.3% for another 24-h incubation period. CCMs and CLs were then collected and frozen for subsequent dot blot, gelatin zymography and ELISA assays. This treatment of the cells minimized the potential disturbing effects of serum metalloproteinases [
20].
NF/CSCC7 and
TF/CSCC7 indicate fibroblast samples isolated from the bottom of the filter compartment, whereas NF/
CSCC7 and TF/
CSCC7 designate tumor cell samples isolated from the inserts of indirect co-culturing plates.
Proliferation assay
The principles of sulforhodamine B (SRB) colorimetric assay were described earlier [
21]. This protocol was used in the current study with the following modifications. Fibroblasts or CSCC7 cells were seeded in 96 well plates at densities of 2.5×10
3 or 3.5×10
3 cells/well in 200 μL complete growth medium. All experimental conditions were run in 8 or 16 parallel samples. After counting, viable cells were let to seed and attach. Zero time point was considered three hours later after all cells were attached. SRB measurements were carried out at the time points of 0, 24, 48, 72 and 96 h. Cells were originally grown in the presence of 5% FBS, but to observe the potential negative effects of serum starvation applied in the last 24 h of the co-culture experiments, the FBS concentration was decreased to 0.3% 24 h before harvesting the cells. To mimic the effects of co-cultivation on cell proliferation, fibroblasts were allowed to grow with CCM of tumor cells, and the latter with CCM of fibroblasts. Specifically, the culture medium contained 50% regular and 50% conditioned medium that was conditioned for 48 h and sterile filtered. The incubation mixture was replaced every 24 h. To control these assays, cells were grown in DMEM-low glucose and RPMI-1640 mixed in 1:1 (v/v) ratio and supplemented with 5% FBS.
Chemotaxis assay
Chemotaxis assays were performed in Boyden chambers as previously described [
21]. The following materials were used as chemoattractants in separate assays: tissue culture medium with 10% FBS, medium conditioned by the two types of fibroblasts (NF and TF), fibronectin (from human plasma, Sigma, 25 μg/mL), and laminin-1 (from Engelbreth-Holm-Swarm murine sarcoma basement membrane, Sigma) diluted in serum-free medium to 25 μg/mL. The cells were treated with 10 μg/mL mitomycin C (Sigma) for five minutes in order to inhibit proliferation [
22]. Two days after mitomycin C treatment, 5×10
4 CSCC7 cells were seeded into the upper chambers of a 48-well Micro Chemotaxis Chamber (Neuro Probe, Gaithersburg, MD, USA) with medium containing 10% FBS and migration was allowed for 24 h. Cell migration toward each chemoattractant was measured in triplicate samples. Migrated cells were stained with toluidine blue with 3 random fields per well. Accordingly, 9 random fields per each chemoattractant were counted.
Protein expression and activity measurements
Western and dot blot
For Western blot, cells were grown as indicated above in the co-culture system. CCMs were collected and cells were extracted by lysis buffer containing 20 mM HEPES pH 7.8, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 1% (v/v) Nonidet P40 and protease inhibitory cocktail, and then cells were homogenized. Protein concentrations were determined by the method of Bradford [
23], using Ultroscpec-2000 UV/VIS Spectrophotometer (Hoefer Pharmacia Biotech Inc, San Francisco, CA, USA). Isolated proteins were run on Western blot or loaded onto dot blot as described previously [
19,
24]. An amount of 20 μL of each sample was loaded per lane. Lysates from indirect co-cultures were quantified and 15 μg total protein of each sample was loaded per lane. Western blot was normalized to β-actin. To prepare dot blots 200 μL CCM per well was blotted onto a nylon membrane by Minifold-Vacuum-Filtration system SRC-96 (Schleicher&Schuell, Dassel, W. Germany), then subjected to immunoassays. Results were normalized to Ponceau S staining. Primary and secondary antibodies are listed in Table
2.
Caseinase and gelatinase zymogram analysis
For caseinase and gelatinase zymogram analysis, 20 μL CCM was used, and normal human serum as control [
19,
25]. Protease activities were visualized by Coomassie Blue staining (Bio-Rad Laboratories GmbH). Densitometry was carried out on a Kodak Image Station 4000MM Digital Imaging System using Kodak Molecular Imaging Software v. 4.0.3 (Eastman Kodak Company, Rochester, NY, USA).
ELISA
CXCL12/SDF-1 and TGF-β1 levels were quantified in mono and co-cultures with the human CXCL12/SDF-1α and TGF-β1 ELISA Kit (R&D Systems, Minneapolis, MN, USA) using 100 μL CCM per sample. As the kit measures active TGF-β1, latent TGF-β complexes were activated before immunodetection as suggested by the manufacturer. ELISA plates were read at 570 nm with Labsystem Multiskan MS 352 (Labsystems, Finland) plate reader.
Statistical analysis
All experiments were performed in three independent sets, each containing at least three biological replicates. Relative gene expression results were tested for normal distribution by D’Agostino-Pearson omnibus test using GraphPad Prism 4.03 (GraphPad Software, Inc.). Significance of differences between co-culture vs. control values was evaluated by non-parametric tests (Mann–Whitney) and Student’s
t-tests depending on the distribution of data. Independent experimental sets were then compared for reproducibility. Only reproducible changes with a p < 0.05 level [
20] were considered significant. Results of SRB measurements were evaluated by χ
2 test.
Discussion
The tumor microenvironment plays a pivotal role in the behavior of cancer [
26]. To identify the characteristic changes of ECM as a result of tumor-matrix interactions we established a tissue microarray from cervical cancer specimens. One of the most striking changes noticeable after cancerous transformation was the upregulation and redistribution of laminin-1, depositing not only into basement membranes, but also into the fibrillar connective tissue. In line with this observation, SMA revealed strong stromal positivity, indicating the presence of activated myofibroblasts that are the major producers of ECM in the stroma [
27]. In support of earlier findings, laminin-5 resided in the cytoplasm of cancer cells [
28].
To obtain more information about the functions shared between fibroblasts and tumor cells, we established fibroblast cultures from tumor free cervix (normal fibroblasts) and tumorous parts of surgically removed cancerous uterine cervix (considered as tumor-associated fibroblasts). An established HPV16 positive cervical cancer cell line, CSCC7, served as the tumorous variance [
18].
When grown alone, comparison of NF and TF cells showed NFs to predominantly synthesize components of the interstitial matrix, such as fibronectin and type I and III collagens. When placed into direct co-culture, only NFs were able to stimulate cancer cells to produce TGF-β1 and MMP-7, factors needed for proliferation and local invasion [
5]. The conditioned medium of NFs but not TFs was capable of stimulating the proliferation of tumor cells.
Compared to NFs, TFs were reprogrammed to produce increased amounts of laminin-α1 and -β1. These are components of laminin-111 known to be implicated in cancer progression, which contain peptide sequences active in proliferation, angiogenesis and metastasis [
29]. Furthermore, TF synthesized a smaller amount of type IV collagen, a protein which is prerequisite for the formation of natural basement membranes. The secretion of fibrillar matrix components was found to be decreased.
Direct co-culture of tumor cells with TFs resulted in the further increase of laminin-1 and perlecan and a decrease in type IV collagen secretion. Thus, it appears that cervical cancer cells are in need of a laminin-rich fibrillar matrix for their invasion, creating an imbalance between components of basement membrane molecules. The fact that laminin-α1 and -β1 production stayed low when TFs were growing in indirect co-culture indicates that direct contact with tumor cells is needed for the enhancement of laminin synthesis. On the contrary, tumor cells were found to be capable of maintaining high laminin synthesis under such conditions, compensating for the low production of fibroblasts.
The importance of laminin-111 in the pathology of cervical cancer was underlined by the facts that laminin proved to be the most efficient chemoattractant in the Boyden chamber migration assay and that it was also produced by CSCC7 cells. These cells predominantly expressed α6β4 and α6β1 integrins, two laminin binding receptors [
30]. This observation implied that laminin-integrin interaction took place both in paracrine and autocrine fashion. In addition to laminin-111, tumor cells growing alone secreted laminin-5, perlecan, MMP-7, TIMP-1 and CD44. Although the current assay was limited to immuncytochemistry, strong expression of CD151 was found, a protein known to be involved in the stimulation of laminin receptor-associated invasion, and activation of MMPs was also noted, with both observations manifest on the surface of TF and CSCC7 [
31-
33].
These results denote that the two types of fibroblasts exhibit different actions as regards the proliferation and migration of cancer cells. NFs stimulated cell proliferation whereas TFs promoted migration. Moreover, it was found that the presence of secreted fibroblasts and matrix proteins was fundamental for the survival of tumor cells in primary tissue culture. A similar observation was published by
Maffini et al. who showed that only tumor cells injected together with activated fibroblasts are able to colonize in case of mice [
34].
It would seem that the differences between NFs and TFs represent the stages of fibroblast transformation from defensive to permissive cells. It can not decidedly be said that the so-called “normal fibroblasts” would indeed represent a defensive cell population. Nevertheless, the fact that they promote cell proliferation suggests that they are on the road to fall into line with supportive mechanisms of cancer. Furthermore, synthesized αv integrin is able to activate latent TGF-β1 alone or by targeting it toward MMPs [
35].
Since MMPs facilitate tumor cell invasion and metastasis, in the current study we assessed MMP activities of fibroblasts and tumor cells. Zymography revealed that three MMPs were involved in cervical cancer progression. Fibroblasts, especially TFs, secreted pro-MMP-1, responsible for the digestion of type I and type III collagens, and pro- and active MMP-2 which is implicated in the degradation of the basement membrane. In support of earlier studies, CSCC7 cells produced pro-MMP-7 [
36,
37]. MMP-7, in turn, was found to regulate the angiogenic activity of fibroblasts [
38]. Among MMPs. only MMP-2 was present in active form. Direct contact between fibroblasts and tumor cells upregulated the secretion and activation of MMP-2, but only NFs increased TIMP-1 production.
Worldwide, HPV16 and HPV18 contribute to over 70% of all cervical cancer cases [
39], therefore the HPV16 positive CSCC7 tumor cell line was selected for the current study. A number of reports propose that CD151 and integrin α6 have key roles in HPV16 infection of epithelial cells [
40-
42]. In the current study, although using immunocytochemistry alone, we found strong expression of CD151, both in fibroblasts and cancer cells. This protein is involved in the laminin-binding of various integrin pairs [
43] and in the stimulation of laminin receptor-associated invasion, by stabilizing integrins α3β1 and α6β4, which is a requirement for motility of invasive tumor cells [
44,
45]. In addition, CD151 is capable of binding and activating MMPs, including pro-MMP-7 onto the cell membrane, facilitating ECM degradation [
31-
33,
46].
We found another molecule, TSP-1, to be critical in the pathology of cervical cancers. TSP-1 is a factor participating in cell adhesion-antiadhesion [
47] and is implicated in tumor progression and angiogenesis. It upregulates integrin α6 in keratinocytes and breast cancer cells resulting in an increased level of cell adhesion and tumor cell invasion [
48]. Of interest is the rather similar behavior of this molecule in the two types of fibroblasts, which may indicate its perpetual importance in tumor-stromal interaction. We detected α4 and β1 integrins on the surface of both types of fibroblasts, an integrin pair known to bind thrombospondin-1 [
49]. In this context, this glycoprotein is capable of exerting its actions on the surface of fibroblasts. Earlier reports support the importance of thrombospondin-1 in the activation of latent TGF-β1 as well as in the stimulation of EGFR by its EGF-like domains [
50].
The efficient adhesion and directional movements of tumor cells migrating on the surface of a fibroblast layer can be explained by the ordered structure of ECM facilitated by the expression of syndecan-1 on the surface of fibroblasts [
51]. In fact, the presence of syndecan-1 is a typical feature of fibroblasts isolated from cervical cancers (unpublished result of the authors). Furthermore, migratory phenotype is attributed to the mesenchymal expression of CD44, demonstrated both on NF and TF cell surfaces [
52]. Accordingly, we were able to demonstrate that physical contact between fibroblasts and CSCC7 cells initiates tumor cells to start migrating. Soluble CD44, probably shedding due to increased MMP-7 protease activity, promotes the effect further by inhibiting adhesion of tumor cells [
53].
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AF designed and carried out the experiments, analyzed the data and contributed to drafting the manuscript. JD participated in the design of the study and performed the statistical analysis. LO created the primary cell cultures. PH contributed to drafting the manuscript. ZP and GS collected the tissue samples. KK performed the statistical analysis of tissue microarray data. SP performed the confocal microscopy and cell migration assay design. KB participated in the design of the study and draft of the manuscript. IK conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.