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【摘要】
This study tests the hypothesis that invasion of partially transformed keratinocytes is initiated by diffusible, proinvasive signals provided by species-specific fibroblasts. In vitro organotypic cultures of neoplastic human oral mucosa were constructed by growing a partially transformed, nontumorigenic keratinocytic cell line isolated from a dysplastic human oral lesion (DOK-ECACC94122104) on top of various types of connective tissue equivalents. Cultured tissues were analyzed by histomorphometry (depth and area of invasion: Dinv, Ainv) and immunohistochemistry. Presence of human fibroblasts in the matrix induced a local invasion of DOK (Dinv = 95.6 ?? 7.1 µm, Ainv = 45.8 ?? 3.5%). Minimal invasion (P < 0.05) was observed when DOK grew on simple collagen matrix (Dinv = 14.1 ?? 2.1 µm, Ainv = 3.7 ?? 0.8%) or matrices containing fibroblasts from mouse (Dinv = 11.5 ?? 4.0 µm, Ainv = 4.3 ?? 1.0%) or rat (Dinv = 15.6 ?? 1.2 µm, Ainv = 6.1 ?? 0.5%). In these cultures, local invasion could be induced by the presence of human fibroblasts in a bottom layer of the collagen matrix (P < 0.05) or by conditioned medium from organotypic cultures of DOK on human fibroblast-containing matrix (P < 0.05) but not by conditioned medium from human fibroblast monocultures (P > 0.05). Deposition of human collagen IV was observed at epithelial-matrix interface only when DOK behaved invasively. In conclusion, invasion of partially transformed oral keratinocytes was triggered by keratinocyte-induced fibroblast-derived diffusible factor(s) in a species-specific manner and associated with de novo synthesis of collagen IV.
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An increasing number of reports suggests that fibroblasts from tumor stroma actively contribute to malignant progression of epithelial neoplasms.1-5 Both normal6-8 and activated carcinoma-associated fibroblasts9-12 have been shown to enhance in vitro invasiveness of human squamous cell carcinoma (SCC) cell lines. Several reports of experiments done on monolayer cultures have suggested that this effect could be attributable to diffusible factors synthesized by fibroblasts.13-15 However, it has been suggested recently that the results from studies performed on conventional, two-dimensional monolayer cell culture models are difficult to extrapolate to the in vivo situation because they do not account for the much more complex mechanisms involved in the three-dimensional process of cancer development and invasiveness.4,16,17 Thus, further experimental evidence from more complex in vitro organotypic models that closely mimic the in vivo three-dimensional tissue structure and cell-to-cell interactions is needed to identify the specific fibroblast-related factor(s) of major importance for in vivo invasiveness of SCCs. On the other hand, the previous studies have used fully transformed cell lines with an established in vivo invasive phenotype. Whether the same fibroblast-dependent mechanism(s) of invasiveness takes place at earlier stages of keratinocyte transformation, when local invasion is initiated, it is not yet known, and therefore it has been the focus of this investigation.
Another fibroblast-related factor of importance for the in vivo invasiveness of carcinoma cells seems to be the origin of fibroblasts. A role for organ specificity of fibroblasts in promoting carcinoma cell invasion has been shown.11,18-20 Several studies have reported that only a limited number of tumor cells of human origin grew in nude mice.21,22 The sensitivity of the in vivo malignancy test for human neoplastic cells, especially those at early stages of malignancy has been questioned,23 and attempts to develop functionally reliable in vivo experimental models of human tissues in mice have been done by humanizing the mice microenvironment through addition of human fibroblasts before xenotransplantation of the human epithelial cells,24 but the issue of species specificity has not been further investigated partially because of the lack of appropriate experimental models.25 The later development of heterologous organotypic models has made possible such studies by construction of models harboring cell types from different species.26
In the present study we have used such organotypic models 1) to test the hypothesis that invasion of partially transformed oral epithelial cells is triggered by diffusible, proinvasive signal(s) provided by species-specific oral fibroblasts; 2) to investigate whether the growth factors suggested by monolayer culture studies to be important for SCC invasiveness play a key role for local invasiveness of partially transformed oral keratinocytes in organotypic three-dimensional cultures; and 3) to identify specific phenotypical changes of partially transformed oral keratinocytes associated with the transition from a noninvasive to an invasive behavior.
【关键词】 species-specific fibroblasts required triggering invasiveness partially transformed keratinocytes
Materials and Methods
Cell Lines
Partially transformed human oral keratinocytes (DOK cell line, accession number 94122104) were obtained from The European Collection of Cell Cultures (Salisbury, Wiltshire, UK)27 and routinely grown in Dulbecco??s modified Eagle??s medium (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum, 20 µg/ml L-glutamine, and 5 µg/ml hydrocortisone (all from Sigma). Primary normal human oral fibroblasts were isolated from six biopsies of human buccal mucosa, after surgical removal of wisdom teeth as previously described.28 The study, approved by the Ethics Committee of the University of Bergen, included clinically healthy donors only after informed consent. Research was performed at the Department of Odontology, Oral Pathology and Forensic Odontology, Faculty of Dentistry, University of Bergen, Bergen, Norway. Normal mouse oral fibroblasts were isolated from the buccal mucosa of six B6D2F mice (Jackson Laboratory, Bar Harbor, ME), and normal rat oral fibroblasts were isolated from the buccal mucosa of six BD IX rats (Charles River Laboratories, France) following the same procedure. Animal care was in accordance to national legislation and institutional guidelines. Primary human fibroblasts were routinely grown in minimum essential medium Eagle (Sigma). Primary mouse and rat fibroblasts were grown in Dulbecco??s modified Eagle??s medium supplemented with 10% fetal calf serum, 20 µg/ml L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B (all from Sigma). DOK in passage 29 and primary fibroblasts in early passages (2 to 4) were used in the study.
Organotypic Cell Culture
In vitro organotypic cell cultures of neoplastic oral mucosa were developed by seeding the DOK cells on top of connective tissue equivalents, as previously described.29 The study design implied construction of various connective tissue equivalents: simple collagen type I matrix (Figure 1 , panel 1); primary human oral fibroblast-containing matrix (Figure 1 , panel 2); primary mouse oral fibroblast-containing matrix (Figure 1 , panel 3); primary rat oral fibroblast-containing matrix (Figure 1 , panel 3); sandwich matrices, in which an intermediate layer (200 µl per culture) of either simple collagen matrix (Figure 1 , panel 4) or of mouse fibroblast-containing matrix (Figure 1 , panel 5) separated the epithelial compartment (0.5 x 106 DOK cells per culture) from the human fibroblast-containing matrix (500 µl per culture). Conditioned medium obtained from either monolayer cultures of human fibroblasts (Figure 1 , panels 6 and 7) or parallel organotypic cultures of DOK on human fibroblast-containing matrix (Figure 1 , panels 6 and 7) was mixed 1:1 vol with fresh culture medium and added to some of the organotypic cultures of DOK on top of either simple collagen matrix (Figure 1 , panel 6) or mouse fibroblast-containing matrix (Figure 1 , panel 7). Recombinant human growth factors and cytokines (hepatocyte growth factor, HGF; granulocyte-macrophage colony stimulation factor, GM-CSF; stem cell factor, SCF; epidermal growth factor, EGF; keratinocyte growth factor, KGF; transforming growth factor-, TGF-; interleukin 1, IL-1; all from Sigma) were tested alone or in various combinations by adding them to the culture media, in a concentration range from 0.1 to 100 ng/ml, at the time of tissue lifting at the liquid-air interface (day 3 of co-culture) (Figure 1 , panels 8 and 9). The cultures were grown for 10 days in serum-free FAD medium (3 vol Dulbecco??s modified Eagle??s medium to 1 vol Ham??s F-12) supplemented with 0.4 µg/ml hydrocortisone, 5 µg/ml insulin, 20 µg/ml transferrin, 50 µg/ml L-ascorbic acid, 1 mg/ml linoleic acid-albumin, 20 µg/ml L-glutamine (all from Sigma). The cultured tissues were harvested at day 10 of co-culture. One half of each culture was snap-frozen in isopentane prechilled in liquid nitrogen, and the other half was fixed in 4% buffered formalin, pH 7.15, and embedded in paraffin. Experiments were run six times, in duplicates.
Figure 1. Study design. Partially transformed human oral keratinocytic cell line (DOK) was organotypically grown on top of various connective tissue equivalents to investigate whether its growth pattern and invasive phenotype could be modulated by underlying mesenchymal stroma. The flow chart (1 to 9) of various models used in the study is presented.
Histomorphometry
Tissue sections (5 µm) from paraffin-embedded specimens, stained with hematoxylin and eosin, were morphometrically analyzed by a computer-based optical image analyzer (analySIS 11.0; Pro Soft Imaging System, GmbH, Munster, Germany). An arbitrary straight line was drawn between the epithelial compartment and the connective tissue compartment through the upper remnants of the collagen gel. Epithelial thickness (ET) was measured from that line to the surface of the epithelium. The degree of local invasiveness was determined by measuring two parameters: the depth of epithelial invasion (Dinv) and the area of epithelial nests that grew invasive in the collagen matrix (Ainv). Dinv was measured from the arbitrary line to the deepest pick of epithelial cells that migrated into the collagen matrix (two picks were measured per each field). Ainv was determined as percentage of the matrix area that was invaded by epithelial cells in a standard square of 30,000 µm2, measured in the matrix layer immediately under epithelium, starting from the arbitrary line described above. All measurements were done in six different fields per slide, situated 200 µm apart, at x100 magnification.
Immunohistochemistry
Immunohistochemical staining was performed using the Autostainer universal staining system (DAKO-USA, Carpinteria, CA) as previously described.29 Five-µm formalin-fixed, paraffin-embedded sections were stained for Ki67 (MIB-1clone, IgG1, titration 1:50; DAKO, Glostrup, Denmark), -smooth muscle actin (-SMA, clone 1A4, IgG2a, titration 1:25; DAKO), and collagen IV (CIV221 clone, IgG1, titration 1:25; DAKO). Fresh-frozen, acetone-fixed samples were stained for c-met (NCL-cMET, IgM, titration 1:50; Novocastra Laboratories Ltd., Newcastle on Tyne, UK). Cell proliferation index (PI) was determined as percentage of Ki67-expressing cells among all cells of the epithelial compartment per microscopic field. At least 500 cells were counted per field in six fields per slide situated 200 µm apart, at x200 magnification.
Statistical Analysis
Wilcoxon paired test was used with a level of significance set at 5% (SPSS 11.0; SPSS, Chicago, IL).
Results
Fibroblast-Derived Diffusible Factors Support Epithelial Growth of Neoplastic Epithelium Reconstructed in Vitro from Partially Transformed Keratinocytes
When grown organotypically, DOK formed a stratified squamous epithelium with a disorganized stratification, cellular atypia, and hyperchromatic nuclei (Figure 2, ACD) . Epithelial thickness (ET = 61.1 ?? 4.6 µm) and cell proliferation index (PI = 15.1 ?? 0.4%) of the epithelium formed when DOK were grown on top of simple collagen matrices were significantly lower (P < 0.05) than the epithelium formed when DOK were grown on top of either human (ET = 97.3 ?? 6.7 µm, PI = 21.2 ?? 1.2%), mouse (ET = 84.6 ?? 3.6 µm, PI = 19.9 ?? 1.4%), or rat (ET = 81.1 ?? 6.7 µm, PI = 18.1 ?? 1.8%) fibroblast-containing matrices (Table 1) . Diffusible factors from the bottom layer of human fibroblast-containing matrix of sandwich models supported the growth of DOK cells through the layer of simple collagen gel in a similar manner (P > 0.05) as the fibroblast-containing matrix in direct contact with DOK cells (Table 1 and Figure 2, C and E ). Addition of conditioned media from monolayer cultures of human fibroblasts or from parallel organotypic co-cultures of DOK on human fibroblast-containing matrix induced an increase in ET and PI of DOK epithelium cultured on simple collagen gels that became comparable to DOK epithelium formed on human fibroblast-containing matrices (P > 0.05) (Table 1) . Furthermore, addition of 10 ng/ml of TGF- or of a cocktail of human growth factors and cytokines (10 ng/ml each of HGF, GM-CSF, SCF, EGF, KGF, TGF-, and IL-1) increased ET and PI of DOK epithelium grown on top of simple collagen matrices up to values comparable to the DOK epithelium grown on human fibroblast-containing matrices (P > 0.05) (Table 1) .
Figure 2. Effects of human fibroblast-derived diffusible factors on growth and invasiveness of in vitro neoplastic oral epithelium reconstructed from partially transformed keratinocytes. Neoplastic oral epithelium was reconstructed by growing DOK cells on top of simple collagen gel (A), human fibroblast-containing matrix (B), or on sandwich models with a bottom layer of human fibroblast-containing matrix and an intermediate layer of simple collagen gel (C). Conditioned medium from parallel organotypic co-cultures of DOK on human fibroblast-containing matrices was added at day 3 of co-culture (D). H&E staining is shown (ACD). E: Epithelial growth (total epithelial thickness and cell proliferation index) and invasion (depth and area of invasion) were assessed. Data represent mean ?? SEM of six different experiments. ?
【参考文献】
Tlsty TD, Hein PW: Know thy neighbor: stromal cells can contribute oncogenic signals. Curr Opin Genet Dev 2001, 11:54-59
De Wever O, Mareel M: Role of tissue stroma in cancer cell invasion. J Pathol 2003, 200:429-447
Bhowmick NA, Neilson EG, Moses HL: Stromal fibroblasts in cancer initiation and progression. Nature 2004, 432:332-337
Beacham DA, Cukierman E: Stromagenesis: the changing face of fibroblastic microenvironments during tumor progression. Semin Cancer Biol 2005, 15:329-341
Mueller MM, Fusenig NE: Friends or foes??bipolar effects of the tumour stroma in cancer. Nat Rev Cancer 2004, 4:839-849
Matsumoto K, Horikoshi M, Rikimaru K, Enomoto S: A study of an in vitro model for invasion of oral squamous cell carcinoma. J Oral Pathol Med 1989, 18:498-501
Yamada S, Toda S, Shin T, Sugihara H: Effects of stromal fibroblasts and fat cells and an environmental factor air exposure on invasion of laryngeal carcinoma (HEp-2) cells in a collagen gel invasion assay system. Arch Otolaryngol Head Neck Surg 1999, 125:424-431
Costea DE, Johannessen AC, Vintermyr OK: Fibroblast control on epithelial differentiation is gradually lost during in vitro tumor progression. Differentiation 2005, 73:134-141
Atula S, Grenman R, Syrjanen S: Fibroblasts can modulate the phenotype of malignant epithelial cells in vitro. Exp Cell Res 1997, 235:180-187
Berndt A, Hyckel P, Konneker A, Kosmehl H: 3-Dimensional in vitro invasion model for oral squamous epithelial carcinomas. Evaluation of tumor and stromal cell properties as well as extracellular matrix.] Mund Kiefer Gesichtschir 1998, 2:256-260
Shekhar MP, Werdell J, Santner SJ, Pauley RJ, Tait L: Breast stroma plays a dominant regulatory role in breast epithelial growth and differentiation: implications for tumor development and progression. Cancer Res 2001, 61:1320-1326
Olumi AF, Grossfeld GD, Hayward SW, Carroll PR, Tlsty TD, Cunha GR: Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res 1999, 59:5002-5011
Sugiura T, Shirasuna K, Hayashido Y, Sakai T, Matsuya T: Effects of human fibroblasts on invasiveness of oral cancer cells in vitro: isolation of a chemotactic factor from human fibroblasts. Int J Cancer 1996, 68:774-781
Matsumoto K, Nakamura T, Kramer RH: Hepatocyte growth factor/scatter factor induces tyrosine phosphorylation of focal adhesion kinase (p125FAK) and promotes migration and invasion by oral squamous cell carcinoma cells. J Biol Chem 1994, 269:31807-31813
Uchida D, Kawamata H, Omotehara F, Nakashiro K, Kimura-Yanagawa T, Hino S, Begum NM, Hoque MO, Yoshida H, Sato M, Fujimori T: Role of HGF/c-met system in invasion and metastasis of oral squamous cell carcinoma cells in vitro and its clinical significance. Int J Cancer 2001, 93:489-496
De Wever O, Nguyen QD, Van Hoorde L, Bracke M, Bruyneel E, Gespach C, Mareel M: Tenascin-C and SF/HGF produced by myofibroblasts in vitro provide convergent pro-invasive signals to human colon cancer cells through RhoA and Rac. FASEB J 2004, 18:1016-1018
Alt-Holland A, Zhang W, Margulis A, Garlick JA: Microenvironmental control of premalignant disease: the role of intercellular adhesion in the progression of squamous cell carcinoma. Semin Cancer Biol 2005, 15:84-96
Fabra A, Nakajima M, Bucana CD, Fidler IJ: Modulation of the invasive phenotype of human colon carcinoma cells by organ specific fibroblasts of nude mice. Differentiation 1992, 52:101-110
Kawai K, Iwashita T, Murakami H, Hiraiwa N, Yoshiki A, Kusakabe M, Ono K, Iida K, Nakayama A, Takahashi M: Tissue-specific carcinogenesis in transgenic mice expressing the RET proto-oncogene with a multiple endocrine neoplasia type 2A mutation. Cancer Res 2000, 60:5254-5260
Hsieh JT, Wu HC, Gleave ME, von Eschenbach AC, Chung LW: Autocrine regulation of prostate-specific antigen gene expression in a human prostatic cancer (LNCaP) subline. Cancer Res 1993, 53:2852-2857
Chang SE: In vitro transformation of human epithelial cells. Biochim Biophys Acta 1986, 823:161-194
Mehta RR, Graves JM, Hart GD, Shilkaitis A, Das Gupta TK: Growth and metastasis of human breast carcinomas with Matrigel in athymic mice. Breast Cancer Res Treat 1993, 25:65-71
Fusenig NE, Breitkreutz D, Dzarlieva RT, Boukamp P, Bohnert A, Tilgen W: Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo. J Invest Dermatol 1983, 81:168s-175s
Kuperwasser C, Chavarria T, Wu M, Magrane G, Gray JW, Carey L, Richardson A, Weinberg RA: Reconstruction of functionally normal and malignant human breast tissues in mice. Proc Natl Acad Sci USA 2004, 101:4966-4971
Mueller MM, Fusenig NE: Tumor-stroma interactions directing phenotype and progression of epithelial skin tumor cells. Differentiation 2002, 70:486-497
Stark HJ, Szabowski A, Fusenig NE, Maas-Szabowski N: Organotypic cocultures as skin equivalents: a complex and sophisticated in vitro system. Biol Proc Online 2004, 6:55-60
Chang SE, Foster S, Betts D, Marnock WE: DOK, a cell line established from human dysplastic oral mucosa, shows a partially transformed non-malignant phenotype. Int J Cancer 1992, 52:896-902
Costea DE, Dimba EA, Loro LL, Vintermyr OK, Johannessen AC: Proliferation and Differentiation in Organotypic Serum Free Cultures of Normal Human Oral Mucosa. 2002Presented at the 8th International Congress on Oral Cancer, Rio de Janeiro, Brazil
Costea DE, Loro LL, Dimba EA, Vintermyr OK, Johannessen AC: Crucial effects of fibroblasts and keratinocyte growth factor on morphogenesis of reconstituted human oral epithelium. J Invest Dermatol 2003, 121:1479-1486
Burns JE, Clark LJ, Yeudall WA, Mitchell R, Mackenzie K, Chang SE, Parkinson EK: The p53 status of cultured human premalignant oral keratinocytes. Br J Cancer 1994, 70:591-595
Lewis MP, Lygoe KA, Nystrom ML, Anderson WP, Speight PM, Marshall JF, Thomas GJ: Tumour-derived TGF-ß1 modulates myofibroblast differentiation and promotes HGF/SF-dependent invasion of squamous carcinoma cells. Br J Cancer 2004, 90:822-832
Breuninger H, Schaumburg-Lever G, Holzschuh J, Horny HP: Desmoplastic squamous cell carcinoma of skin and vermilion surface: a highly malignant subtype of skin cancer. Cancer 1997, 79:915-919
Hagedorn HG, Bachmeier BE, Nerlich AG: Synthesis and degradation of basement membranes and extracellular matrix and their regulation by TGF-ß in invasive carcinomas. Int J Oncol 2001, 18:669-681
Ziober BL, Silverman SS, Jr, Kramer RH: Adhesive mechanisms regulating invasion and metastasis in oral cancer. Crit Rev Oral Biol Med 2001, 12:499-510
Berndt A, Hyckel P, Konneker A, Katenkamp D, Kosmehl H: Oral squamous cell carcinoma invasion is associated with a laminin-5 matrix re-organization but independent of basement membrane and hemidesmosome formation. Clues from an in vitro invasion model. Invasion Metastasis 1997, 17:251-258
Berndt A, Borsi L, Hyckel P, Kosmehl H: Fibrillary co-deposition of laminin-5 and large unspliced tenascin-C in the invasive front of oral squamous cell carcinoma in vivo and in vitro. J Cancer Res Clin Oncol 2001, 127:286-292
Hindermann W, Berndt A, Borsi L, Luo X, Hyckel P, Katenkamp D, Kosmehl H: Synthesis and protein distribution of the unspliced large tenascin-C isoform in oral squamous cell carcinoma. J Pathol 1999, 189:475-480
Jiang X, Multhaupt H, Chan E, Schaefer L, Schaefer RM, Couchman JR: Essential contribution of tumor-derived perlecan to epidermal tumor growth and angiogenesis. J Histochem Cytochem 2004, 52:1575-1590
Ortiz-Urda S, Garcia J, Green CL, Chen L, Lin Q, Veitch DP, Sakai LY, Lee H, Marinkovich MP, Khavari PA: Type VII collagen is required for Ras-driven human epidermal tumorigenesis. Science 2005, 307:1773-1776
Laurich C, Wheeler MA, Iida J, Neudauer CL, McCarthy JB, Bullard KM: Hyaluronan mediates adhesion of metastatic colon carcinoma cells. J Surg Res 2004, 122:70-74
Andriani F, Garfield J, Fusenig NE, Garlick JA: Basement membrane proteins promote progression of intraepithelial neoplasia in 3-dimensional models of human stratified epithelium. Int J Cancer 2004, 108:348-357
Mueller MM, Fusenig NE: Constitutive expression of G-CSF and GM-CSF in human skin carcinoma cells with functional consequence for tumor progression. Int J Cancer 1999, 83:780-789
Mestas J, Hughes CC: Of mice and not men: differences between mouse and human immunology. J Immunol 2004, 172:2731-2738
DeVore DP, Houchens DP, Ovejera AA, Dill GS, Jr, Hutson TB: Collagenase inhibitors retarding invasion of a human tumor in nude mice. Exp Cell Biol 1980, 48:367-373
作者单位：From the Departments of Oral Pathology and Forensic Odontology,* Oral Surgery and Oral Medicine, and Pathology, The Gade Institute, Haukeland University Hospital, University of Bergen, Bergen, Norway