Rapid Vessel Regression, Protease Inhibition, and Stromal Normalization upon Short-Term Vascular Endothelial Growth Factor Receptor 2 Inhibition in Skin Carcinoma Heterotransplants
2005; Elsevier BV; Volume: 167; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)61226-6
ISSN1525-2191
AutoresDaniel W. Miller, Silvia Vosseler, Nicolae Mirancea, Daniel J. Hicklin, Peter Böhlen, Hans E. Völcker, Frank G. Holz, Norbert E. Fusenig,
Tópico(s)Vascular Tumors and Angiosarcomas
ResumoVascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis, and blockade of VEGF receptor 2 (VEGFR-2), with the monoclonal antibody DC101, inhibits angiogenesis and tumor growth. To examine the short-term effects of DC101, we surface transplanted the squamous cell carcinoma cell line A5-RT3 onto nude mice. After short-term treatment with DC101, we observed rapid reduction in vascularization and reversion of the tumor phenotype. Beginning 24 hours after treatment, VEGFR-2 inhibition resulted in decreased vessel density within the tenascin-c-staining tumor-associated stroma and reduced endothelial cell proliferation. Stromal expression of matrix metalloproteinase-9 and -13 was drastically reduced 96 hours after VEGFR-2 inhibition as detected by in situ hybridization and in situ zymography. Moreover, the morphology of the tumor-stroma border changed from a highly invasive carcinoma to a well-demarcated, premalignant phenotype. The latter was characterized by the appearance of a regular basement membrane in immunostaining and ultrastructural analyses. These findings suggest that VEGFR-2 inhibition by DC101 evokes very rapid reduction of preformed vessels and decreases both stromal protease expression and gelatinolytic activity, resulting in the modulation of the tumor-stroma border zone and reversion of the tumor phenotype. Thus, short-term inhibition of VEGF signaling results in complex stromal alterations with crucial consequences for the tumor phenotype. Vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis, and blockade of VEGF receptor 2 (VEGFR-2), with the monoclonal antibody DC101, inhibits angiogenesis and tumor growth. To examine the short-term effects of DC101, we surface transplanted the squamous cell carcinoma cell line A5-RT3 onto nude mice. After short-term treatment with DC101, we observed rapid reduction in vascularization and reversion of the tumor phenotype. Beginning 24 hours after treatment, VEGFR-2 inhibition resulted in decreased vessel density within the tenascin-c-staining tumor-associated stroma and reduced endothelial cell proliferation. Stromal expression of matrix metalloproteinase-9 and -13 was drastically reduced 96 hours after VEGFR-2 inhibition as detected by in situ hybridization and in situ zymography. Moreover, the morphology of the tumor-stroma border changed from a highly invasive carcinoma to a well-demarcated, premalignant phenotype. The latter was characterized by the appearance of a regular basement membrane in immunostaining and ultrastructural analyses. These findings suggest that VEGFR-2 inhibition by DC101 evokes very rapid reduction of preformed vessels and decreases both stromal protease expression and gelatinolytic activity, resulting in the modulation of the tumor-stroma border zone and reversion of the tumor phenotype. Thus, short-term inhibition of VEGF signaling results in complex stromal alterations with crucial consequences for the tumor phenotype. The formation of new vessels from pre-existing ones, termed angiogenesis, occurs physiologically in the reproductive cycle, wound healing, and ocular maturation as well as in a number of pathologies including cancer, age-related macular degeneration, and diabetic retinopathy.1Folkman J Angiogenesis in cancer, vascular, rheumatoid and other disease.Nat Med. 1995; 1: 27-31Crossref PubMed Scopus (7188) Google Scholar, 2Jain RK Carmeliet PF Vessels of death or life.Sci Am. 2001; 285: 38-45Crossref PubMed Scopus (40) Google Scholar Better understanding of angiogenesis and its mechanisms will optimize current therapies directed at treating these diseases and will provide new therapeutic targets directed against them.3Ferrara N VEGF and the quest for tumour angiogenesis factors.Nat Rev Cancer. 2002; 2: 795-803Crossref PubMed Scopus (1258) Google Scholar, 4Alitalo K Carmeliet P Molecular mechanisms of lymphangiogenesis in health and disease.Cancer Cell. 2002; 1: 219-227Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar The work described here analyzed the immediate effects of inhibition of vascular endothelial growth factor (VEGF) signaling on vascular regression and normalization of the tumor-stromal interface. The study of new vessel formation is dependent on the existence of adequate model systems for angiogenic related diseases. Current in vitro cancer models are only partially capable of mimicking the complex interaction between tumor cells, vasculature, and stromal elements that occur in vivo.5Fusenig NE Skobe M Vosseler S Hansen M Lederle W Airola K Tomakidi P Stark HJ Steinbauer H Mirancea N Boukamp P Breitkreutz D Tissue models to study tumor-stroma interactions.in: Muschel RJFJ Proteases and Their Inhibitors in Cancer Metastasis. Kluwer Academic Publishers, Dordrecht2002: 205-223Crossref Google Scholar To better understand the complex interplay between these compartments, we have previously developed an in vivo assay of tumor invasion with the aid of matrix-inserted surface transplants.5Fusenig NE Skobe M Vosseler S Hansen M Lederle W Airola K Tomakidi P Stark HJ Steinbauer H Mirancea N Boukamp P Breitkreutz D Tissue models to study tumor-stroma interactions.in: Muschel RJFJ Proteases and Their Inhibitors in Cancer Metastasis. Kluwer Academic Publishers, Dordrecht2002: 205-223Crossref Google Scholar This assay involves the growth of a cell monolayer on a collagen gel, which is grafted within a silicon chamber onto the back muscle fascia of a nude mouse, resulting in the growth of a stratified epithelium that allows for the study of tumor-stromal interactions, including angiogenesis, at different stages in a polarized manner.5Fusenig NE Skobe M Vosseler S Hansen M Lederle W Airola K Tomakidi P Stark HJ Steinbauer H Mirancea N Boukamp P Breitkreutz D Tissue models to study tumor-stroma interactions.in: Muschel RJFJ Proteases and Their Inhibitors in Cancer Metastasis. Kluwer Academic Publishers, Dordrecht2002: 205-223Crossref Google Scholar, 6Mueller MM Fusenig NE Friends or foes—bipolar effects of the tumour stroma in cancer.Nat Rev Cancer. 2004; 4: 839-849Crossref PubMed Scopus (1454) Google Scholar, 7Fusenig NE Boukamp P Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin keratinocytes.Mol Carcinog. 1998; 23: 144-158Crossref PubMed Scopus (229) Google Scholar Although initially separated by the interposed collagen gel, transplanted cells rapidly stimulate the formation of granulation tissue, including vascular sprouting, from the host side. On replacement of the interposed collagen matrix by the newly formed granulation tissue, tumor invasion commences in malignant transplants, whereas normal and benign cells remain as an intact stratified surface epithelia inducing only transient angiogenesis.6Mueller MM Fusenig NE Friends or foes—bipolar effects of the tumour stroma in cancer.Nat Rev Cancer. 2004; 4: 839-849Crossref PubMed Scopus (1454) Google Scholar, 8Skobe M Rockwell P Goldstein N Vosseler S Fusenig NE Halting angiogenesis suppresses carcinoma cell invasion.Nat Med. 1997; 3: 1222-1227Crossref PubMed Scopus (407) Google Scholar Furthermore, we have successfully used this assay to selectively manipulate numerous components of the tumor-stromal system for the better understanding of their role in angiogenesis and tumor growth.8Skobe M Rockwell P Goldstein N Vosseler S Fusenig NE Halting angiogenesis suppresses carcinoma cell invasion.Nat Med. 1997; 3: 1222-1227Crossref PubMed Scopus (407) Google Scholar, 9Mueller MM Fusenig NE Tumor-stroma interactions directing phenotype and progression of epithelial skin tumor cells.Differentiation. 2002; 70: 486-497Crossref PubMed Scopus (154) Google Scholar, 10Boukamp P Petrussevska RT Breitkreutz D Hornung J Markham A Fusenig NE Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3440) Google Scholar, 11Boukamp P Stanbridge EJ Foo DY Cerutti PA Fusenig NE c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo but lacks correlation with malignancy.Cancer Res. 1990; 50: 2840-2847PubMed Google Scholar, 12Bajou K Noel A Gerard RD Masson V Brunner N Holst-Hansen C Skobe M Fusenig NE Carmeliet P Collen D Foidart JM Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization.Nat Med. 1998; 4: 923-928Crossref PubMed Scopus (613) Google Scholar, 13Bajou K Masson V Gerard RD Schmitt PM Albert V Praus M Lund LR Frandsen TL Brunner N Dano K Fusenig NE Weidle U Carmeliet G Loskutoff D Collen D Carmeliet P Foidart JM Noel A The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin. Implications for antiangiogenic strategies.J Cell Biol. 2001; 152: 777-784Crossref PubMed Scopus (293) Google Scholar, 14Skobe M Fusenig NE Tumorigenic conversion of immortal human keratinocytes through stromal cell activation.Proc Natl Acad Sci USA. 1998; 95: 1050-1055Crossref PubMed Scopus (196) Google Scholar, 15Bleuel K Popp S Fusenig NE Stanbridge EJ Boukamp P Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization.Proc Natl Acad Sci USA. 1999; 96: 2065-2070Crossref PubMed Scopus (106) Google Scholar, 16Javaherian A Vaccariello M Fusenig NE Garlick JA Normal keratinocytes suppress early stages of neoplastic progression in stratified epithelium.Cancer Res. 1998; 58: 2200-2208PubMed Google Scholar Among other components, we have also studied the role of VEGF in this system. VEGF is considered to be a key regulatory molecule in angiogenesis in which it induces vascular growth and permeability while acting as a survival factor for newly formed vessels.17Benjamin LE Golijanin D Itin A Pode D Keshet E Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal.J Clin Invest. 1999; 103: 159-165Crossref PubMed Scopus (1045) Google Scholar One of its receptors, VEGFR-2 is the major mediator of VEGF's mitogenic and permeability enhancing effects in endothelial cells.3Ferrara N VEGF and the quest for tumour angiogenesis factors.Nat Rev Cancer. 2002; 2: 795-803Crossref PubMed Scopus (1258) Google Scholar, 18Carmeliet P Mechanisms of angiogenesis and arteriogenesis.Nat Med. 2000; 6: 389-395Crossref PubMed Scopus (3432) Google Scholar By blocking signaling of VEGFR-2 with the antibody DC101,19Witte L Hicklin DJ Zhu Z Pytowski B Kotanides H Rockwell P Bohlen P Monoclonal antibodies targeting the VEGF receptor-2 (Flk1/KDR) as an anti-angiogenic therapeutic strategy.Cancer Metastasis Rev. 1998; 17: 155-161Crossref PubMed Scopus (302) Google Scholar we have demonstrated inhibition of tumor vascularization and abrogation of tumor invasion using this assay.8Skobe M Rockwell P Goldstein N Vosseler S Fusenig NE Halting angiogenesis suppresses carcinoma cell invasion.Nat Med. 1997; 3: 1222-1227Crossref PubMed Scopus (407) Google Scholar Systemic and chronic administration of DC101 to animals carrying surface transplants of the highly aggressive and metastasizing human squamous cell carcinoma cell line A-5RT3 resulted in reversion of the tumor phenotype with a normalized tumor-stroma border including a well-demarcated basement membrane.20Mueller MM Peter W Mappes M Huelsen A Steinbauer H Boukamp P Vaccariello M Garlick J Fusenig NE Tumor progression of skin carcinoma cells in vivo promoted by clonal selection, mutagenesis, and autocrine growth regulation by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.Am J Pathol. 2001; 159: 1567-1579Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 21Vosseler S Mirancea N Bohlen P Mueller MM Fusenig NE Angiogenesis inhibition by VEGFR-2 blockade reduces stromal MMP expression, normalizes stromal tissue and reverts tumor phenotype.Cancer Res. 2005; 65: 1294-1305Crossref PubMed Scopus (111) Google Scholar These initial experiments examined long-term effects of multiple DC101 treatments on tumor phenotype and raised numerous questions about which mechanisms were responsible for the effects of VEGFR-2 inhibition on tumor-stromal interactions. An important question was whether DC101-induced changes in the tumor stroma were due to chronic treatment or if they could be observed as immediate effects of limited treatments, whose mechanisms of action could be studied. The study described here examined the early effects of VEGFR-2 inhibition on tumor phenotype by using the surface transplant model described above. Beginning 3 hours after systemic administration of the VEGFR-2 blocking antibody DC101, vascular density, endothelial proliferation, protease expression, and tumor-stromal interactions were analyzed until 96 hours after initial DC101 treatment for the response to VEGFR-2 inhibition. The highly malignant tumorigenic clone (A-5RT3) was derived from the immortalized human keratinocyte cell line HaCaT10Boukamp P Petrussevska RT Breitkreutz D Hornung J Markham A Fusenig NE Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3440) Google Scholar after transfection with the c-Ha-ras oncogene and recultivation of heterotransplants in nude mice, as described previously.7Fusenig NE Boukamp P Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin keratinocytes.Mol Carcinog. 1998; 23: 144-158Crossref PubMed Scopus (229) Google Scholar, 11Boukamp P Stanbridge EJ Foo DY Cerutti PA Fusenig NE c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo but lacks correlation with malignancy.Cancer Res. 1990; 50: 2840-2847PubMed Google Scholar, 20Mueller MM Peter W Mappes M Huelsen A Steinbauer H Boukamp P Vaccariello M Garlick J Fusenig NE Tumor progression of skin carcinoma cells in vivo promoted by clonal selection, mutagenesis, and autocrine growth regulation by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.Am J Pathol. 2001; 159: 1567-1579Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar All cells were grown in enriched minimum essential medium (4×) supplemented with 5% fetal calf serum and 200 μg/ml geneticin as described previously.20Mueller MM Peter W Mappes M Huelsen A Steinbauer H Boukamp P Vaccariello M Garlick J Fusenig NE Tumor progression of skin carcinoma cells in vivo promoted by clonal selection, mutagenesis, and autocrine growth regulation by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.Am J Pathol. 2001; 159: 1567-1579Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar Cells were transplanted onto the dorsal muscle fascia of 7- to 9-week-old nude mice (Swiss/c nu/nu back crosses) as monolayer cultures growing on collagen type 1 gels using a silicone chamber device, as described in detail.5Fusenig NE Skobe M Vosseler S Hansen M Lederle W Airola K Tomakidi P Stark HJ Steinbauer H Mirancea N Boukamp P Breitkreutz D Tissue models to study tumor-stroma interactions.in: Muschel RJFJ Proteases and Their Inhibitors in Cancer Metastasis. Kluwer Academic Publishers, Dordrecht2002: 205-223Crossref Google Scholar, 8Skobe M Rockwell P Goldstein N Vosseler S Fusenig NE Halting angiogenesis suppresses carcinoma cell invasion.Nat Med. 1997; 3: 1222-1227Crossref PubMed Scopus (407) Google Scholar Transplants were dissected en bloc, embedded in Tissue-Tek (Miles Laboratories, Elkhart, IN), and frozen in liquid nitrogen vapor for preparation of cryostat sections. For labeling of proliferating cells, mice received tail vein injections of 5-bromodeoxyuridine (BrdU) and 2-deoxycytidine (65 mmol/L each) in 0.9% NaCl (100 μl) 1.5 hours before being sacrificed. The in vivo anti-angiogenic activity of the VEGFR-2 neutralizing antibody DC101 was tested in mice carrying transplants of the highly malignant keratinocyte clone HaCaT-ras A-5RT3 starting 18 days after transplantation,20Mueller MM Peter W Mappes M Huelsen A Steinbauer H Boukamp P Vaccariello M Garlick J Fusenig NE Tumor progression of skin carcinoma cells in vivo promoted by clonal selection, mutagenesis, and autocrine growth regulation by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.Am J Pathol. 2001; 159: 1567-1579Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar when invasive tumor tissues had formed.20Mueller MM Peter W Mappes M Huelsen A Steinbauer H Boukamp P Vaccariello M Garlick J Fusenig NE Tumor progression of skin carcinoma cells in vivo promoted by clonal selection, mutagenesis, and autocrine growth regulation by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.Am J Pathol. 2001; 159: 1567-1579Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar Mice received intraperitoneal injections of the monoclonal antibody DC101 [800 μg per mouse in 150 μl of phosphate-buffered saline (PBS)] or PBS alone at 0 hours and 48 hours after initial injection. Transplants were dissected at 3, 6, 24, 48, 72, and 96 hours after initial injection with all animals after the 48-hour time point receiving a second DC101 injection at 48 hours. The experiment was performed with three animals per time point and repeated two times. All animal experiments were performed in compliance with the relevant laws and institutional guidelines with permission of the Regierungspräsidium Karlsruhe, dated 7.4.1999 and 11.3.2003 (AZ 35-9185.81/G-16/03). The rat monoclonal antibody DC101 to mouse VEGFR-2 (flk-1) was obtained from ImClone Systems Inc. (New York, NY) and described in detail previously.19Witte L Hicklin DJ Zhu Z Pytowski B Kotanides H Rockwell P Bohlen P Monoclonal antibodies targeting the VEGF receptor-2 (Flk1/KDR) as an anti-angiogenic therapeutic strategy.Cancer Metastasis Rev. 1998; 17: 155-161Crossref PubMed Scopus (302) Google Scholar Rat monoclonal antibody against mouse CD31 was obtained from BD PharMingen (Heidelberg, Germany), guinea pig polyclonal pan-keratin anti-serum from Progen (Heidelberg, Germany), sheep polyclonal antibody against BrdU from NatuTec (Frankfurt, Germany), rabbit polyclonal antibody against tenascin-c from Telios Pharmaceuticals (San Diego, CA), rabbit polyclonal antibody against mouse collagen type IV was from Novotec (Lyon, France), rat monoclonal antibody against mouse neutrophil granulocytes from Serotec (Düsseldorf, Germany), and sheep polyclonal antibody against mouse matrix metalloproteinase (MMP)-9 was a gift from Prof. Gillian Murphy (University of Cambridge, Cambridge, UK). Secondary antibodies were obtained from Dianova (Hamburg, Germany) and Hoechst 33258 bisbenzimide for nuclear staining from Sigma-Aldrich (Taufkirchen, Germany). Mouse cDNA encoding MMP-9 was from Novus Molecular Inc. (San Diego, CA) and mouse MMP-13 cDNA was a gift from Dr. Peter Angel (Deutsches Krebsforschungszentrum, Heidelberg, Germany). For immunofluorescence staining, frozen sections were fixed for 5 minutes in 80% methanol at 4°C and 2 minutes in acetone at −20°C, and rehydrated in PBS. For BrdU localization in DNA, sections were additionally denatured in 2 mol/L HCl for 10 minutes at room temperature and washed (3 × 10 minutes). Primary antibodies were incubated in 12% bovine serum albumin/PBS at room temperature for 2 hours or 4°C overnight. After washing (3 × 10 minutes) sections were incubated with appropriate secondary antibodies together with 5 μg/ml Hoechst bisbenzimide for staining of cell nuclei. Before embedding in Permafluor (Immunotech, Marseille, France) sections were washed again (3 × 10 minutes) in PBS. Detection of apoptotic cells was performed using the In Situ Cell Death Detection kit (Roche, Mannheim, Germany) following the manufacturer's protocol. Stained sections were examined and photographed with an Olympus AX-70 microscope (Olympus, Hamburg, Germany) fitted with epifluorescence optics and connected to a PC using Analysis Imaging Software (Soft Imaging Systems GmbH, Münster, Germany). The heterotransplant assay used here, involves the growth of a tumor cell monolayer on a collagen gel, which is grafted within a silicon chamber onto a nude mouse. In this model system, stratified tumor epithelium forms from the cell monolayer. This, in turn, induces murine stroma formation from the host toward the stratified cell layer. Subsequently, tumor cells then invade the stroma allowing for the analysis of tumor-stromal interactions in a polarized manner.5Fusenig NE Skobe M Vosseler S Hansen M Lederle W Airola K Tomakidi P Stark HJ Steinbauer H Mirancea N Boukamp P Breitkreutz D Tissue models to study tumor-stroma interactions.in: Muschel RJFJ Proteases and Their Inhibitors in Cancer Metastasis. Kluwer Academic Publishers, Dordrecht2002: 205-223Crossref Google Scholar, 6Mueller MM Fusenig NE Friends or foes—bipolar effects of the tumour stroma in cancer.Nat Rev Cancer. 2004; 4: 839-849Crossref PubMed Scopus (1454) Google Scholar, 7Fusenig NE Boukamp P Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin keratinocytes.Mol Carcinog. 1998; 23: 144-158Crossref PubMed Scopus (229) Google Scholar Areas of stromal infiltration into the tumor, the leading edge of vascular growth thought to contain the most immature vessels, were chosen for quantification. In immunostaining, the extracellular matrix (ECM) of these areas was strongly stained by antibodies to tenascin-c, a major component of the tumor stroma,22Chiquet-Ehrismann R Chiquet M Tenascins: regulation and putative functions during pathological stress.J Pathol. 2003; 200: 488-499Crossref PubMed Scopus (442) Google Scholar whereas associated vessels were strongly stained by antibodies against CD31. Photos were taken over the entire area to the edge of the stromal and tumor cell fronts from sections of two to four animals depending on the time points quantified (control 24 hours, three animals; control 48 hours; two animals; DC101 treated 3 hours, two animals; DC101 treated 24 hours, four animals; DC101 treated 48 hours, three animals; DC101 treated 72 hours, two animals; DC101 96 hours, two animals). The area of tenascin-c and CD31 staining per photograph was quantified using Analysis Imaging Software. The CD31 staining area was then divided by the tenascin-c area of each photograph to obtain a percent value. Means and standard deviations were calculated using Microsoft Excel for each time point measured. A nonpaired Mann-Whitney test was performed between controls and treated time points using SAS Stat View (SAS, Heidelberg, Germany). All column diagrams were made using Microsoft Excel (Microsoft Deutschland GmbH, Unterschleissheim, Germany). Serial sections from the same animals analyzed for tenascin-c and CD31 quantification were stained for BrdU, Hoechst, and collagen IV using the immunofluorescence techniques described above. Stromal areas were photographed as described above and endothelial nuclei (those located within collagen IV staining luminal areas) staining for Hoechst and those for BrdU were counted. A percentage of those nuclei staining positive for both BrdU and Hoechst was made, with means and standard deviations being calculated with Microsoft Excel. A nonpaired Mann-Whitney test was performed between controls and treated time points using SAS Stat View. All column diagrams were also made using Microsoft Excel. In situ hybridization was performed as described.8Skobe M Rockwell P Goldstein N Vosseler S Fusenig NE Halting angiogenesis suppresses carcinoma cell invasion.Nat Med. 1997; 3: 1222-1227Crossref PubMed Scopus (407) Google Scholar, 21Vosseler S Mirancea N Bohlen P Mueller MM Fusenig NE Angiogenesis inhibition by VEGFR-2 blockade reduces stromal MMP expression, normalizes stromal tissue and reverts tumor phenotype.Cancer Res. 2005; 65: 1294-1305Crossref PubMed Scopus (111) Google Scholar In brief, Digoxenin (DIG)-labeled RNA probes for mouse MMP-9 and MMP-13 were prepared using T7, SP6, or T3 RNA polymerase (for anti-sense and sense, respectively) according to the manufacturer's instructions (Roche, Mannheim, Germany). Cryostat sections were fixed in 4% paraformaldehyde, pretreated, hybridized, and washed at high stringency as described.23Moorman AF De Boer PA Vermeulen JL Lamers WH Practical aspects of radio-isotopic in situ hybridization on RNA.Histochem J. 1993; 25: 251-266Crossref PubMed Scopus (75) Google Scholar DIG was labeled by anti-DIG-AP (Roche) and alkaline-phosphatase reaction was detected by NBT/BCIP (Gibco-Life Technologies/Invitrogen, Eggenstein-Leopoldshafen, Germany). After DIG in situ hybridization of MMP-9 or MMP-13 counterstaining was performed by indirect immunofluorescence with antisera against pankeratin and collagen type IV. Sections were photographed with different channels being assigned different colors for further analysis using Analysis Imaging Software. Gelatinolytic activity was demonstrated in unfixed cryostat sections using DQ gelatin (EnzChek; Molecular Probes, Leiden, The Netherlands) as a substrate. Cryostat sections (7 μm) were incubated with 40 μg/ml DQ gelatin for 30 minutes at room temperature. After washing (3 × 10 minutes) sections were stained for CD31 and Hoechst using immunofluorescence techniques described above. Areas of gelatinolytic activity staining were quantified in vision fields (0.38 mm2) from sections of two to four animals per time point stained with DQ gelatin using Analysis Imaging Software. Fresh samples of A-5RT3 transplants of control and DC101-treated mice (two animals per time point) were prefixed in ice-cold 4% glutaraldehyde in 0.2 mol/L sodium cacodylate buffer, pH 7.3, for 3 hours and postfixed in 2% osmium tetroxide in 0.1 mol/L sodium cacodylate buffer for 2.5 hours at 4°C. Tissue blocs were then washed with distilled H2O, stained en bloc with 0.5% aqueous uranyl acetate overnight at 4°C, and again washed with distilled H2O. After dehydration through two graded series of ethanol and infiltration with propylene oxide, specimens were embedded in Epon 812-equivalent (glycidether 100; Serva, Heidelberg, Germany) and finally polymerized at 60°C for 48 hours. Semithin sections of 1 μm were stained with 0.1% toluidine blue for light microscopy. Ultrathin sections (50 to 90 nm) were cut by a Reichert Young ultramicrotome (Leica Microsystems Nussloch GmbH, Nussloch, Germany), counterstained with uranyl acetate, and subsequently lead citrate, and examined with a Zeiss EM10B electron microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany). Samples were cut from the top of the surface transplant containing tumor epithelium toward the underlying stroma to accurately analyze the tumor-stromal border. As shown recently, long-term application of the monoclonal antibody DC101 blocks angiogenesis, normalizes the tumor-stroma border zone, and reverts tumor phenotype.21Vosseler S Mirancea N Bohlen P Mueller MM Fusenig NE Angiogenesis inhibition by VEGFR-2 blockade reduces stromal MMP expression, normalizes stromal tissue and reverts tumor phenotype.Cancer Res. 2005; 65: 1294-1305Crossref PubMed Scopus (111) Google Scholar To better visualize and understand the altered interactions between tumor and stromal cells, early time points after inhibition of angiogenesis were analyzed using the same A-5RT3 carcinoma cells in surface transplants.5Fusenig NE Skobe M Vosseler S Hansen M Lederle W Airola K Tomakidi P Stark HJ Steinbauer H Mirancea N Boukamp P Breitkreutz D Tissue models to study tumor-stroma interactions.in: Muschel RJFJ Proteases and Their Inhibitors in Cancer Metastasis. Kluwer Academic Publishers, Dordrecht2002: 205-223Crossref Google Scholar, 8Skobe M Rockwell P Goldstein N Vosseler S Fusenig NE Halting angiogenesis suppresses carcinoma cell invasion.Nat Med. 1997; 3: 1222-1227Crossref PubMed Scopus (407) Google Scholar This assay involves the growth of a tumor cell monolayer on a collagen gel, which is grafted within a silicon chamber onto a nude mouse. In this model system, stratified tumor epithelium forms from the cell monolayer. This, in turn, induces murine stroma formation from the host toward the stratified cell layer. Subsequently, tumor cells then invade the stroma allowing for the analysis of tumor-stromal interactions in a polarized manner (Figure 1A).5Fusenig NE Skobe M Vosseler S Hansen M Lederle W Airola K Tomakidi P Stark HJ Steinbauer H Mirancea N Boukamp P Breitkreutz D Tissue models to study tumor-stroma interactions.in: Muschel RJFJ Proteases and Their Inhibitors in Cancer Metastasis. Kluwer Academic Publishers, Dordrecht2002: 205-223Crossref Google Scholar, 6Mueller MM Fusenig NE Friends or foes—bipolar effects of the tumour stroma in cancer.Nat Rev Cancer. 2004; 4: 839-849Crossref PubMed Scopus (1454) Google Scholar, 7Fusenig NE Boukamp P Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin keratinocytes.Mol Carcinog. 1998; 23: 144-158Crossref PubMed Scopus (229) Google Scholar The A-5RT3 tumor cell transplants studied here, induced rapid stromal activation with accumulation of fibroblasts and inflammatory cells and growth of new blood vessels in a directed and well-defined pattern. This was followed by tumor cell invasion and reciprocal infiltration of vascularized stromal strands into the tumor parenchyma, as visualized by differential immunostaining of tumor cells, with antibodies to keratin, and endothelial cells, with antibodies to CD31, at 24
Referência(s)