Overexpression of Carcinoma and Embryonic Cytotrophoblast Cell-Specific Mig-7 Induces Invasion and Vessel-Like Structure Formation
2007; Elsevier BV; Volume: 170; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2007.060969
ISSN1525-2191
AutoresAaron Petty, Kiera L. Garman, Virginia D. Winn, Celee M. Spidel, J. Suzanne Lindsey,
Tópico(s)Caveolin-1 and cellular processes
ResumoMolecular requirements for carcinoma cell interactions with the microenvironment are critical for disease progression but are poorly understood. Integrin αvβ5, which senses the extracellular matrix, is important for carcinoma cell dissemination in vivo. αvβ5 signaling induces Mig-7, a novel human gene product that is apparently carcinoma-specific. We hypothesized that Mig-7 expression facilitates tumor cell dissemination by increasing invasion and vasculogenic mimicry. Results show that embryonic cytotrophoblasts up-regulated Mig-7 expression before they acquired an invasive phenotype capable of pseudovasculogenesis. Mig-7 protein primarily co-localized with vasculogenic mimicry markers factor VIII-associated antigen, vascular endothelial-cadherin, and laminin 5 γ2 chain domain III fragment in lymph node metastases. Overexpression of Mig-7 increased γ2 chain domain III fragments known to contain epidermal growth factor (EGF)-like repeats that can activate EGF receptor. Interestingly, EGF also induced Mig-7 expression. Carcinoma cell adhesion to laminins was significantly reduced by Mig-7 expression. Remarkably, in two-dimensional and three-dimensional Matrigel cultures, Mig-7 expression caused invasion and vessel-like structures. Melanoma cells, which were previously characterized to invade aggressively and to undergo vasculogenic mimicry, expressed Mig-7. Taken together, these data suggest that Mig-7 expression allows cells to sense their environment, to invade, and to form vessel-like structures through a novel relationship with laminin 5 γ2 chain domain III fragments. Molecular requirements for carcinoma cell interactions with the microenvironment are critical for disease progression but are poorly understood. Integrin αvβ5, which senses the extracellular matrix, is important for carcinoma cell dissemination in vivo. αvβ5 signaling induces Mig-7, a novel human gene product that is apparently carcinoma-specific. We hypothesized that Mig-7 expression facilitates tumor cell dissemination by increasing invasion and vasculogenic mimicry. Results show that embryonic cytotrophoblasts up-regulated Mig-7 expression before they acquired an invasive phenotype capable of pseudovasculogenesis. Mig-7 protein primarily co-localized with vasculogenic mimicry markers factor VIII-associated antigen, vascular endothelial-cadherin, and laminin 5 γ2 chain domain III fragment in lymph node metastases. Overexpression of Mig-7 increased γ2 chain domain III fragments known to contain epidermal growth factor (EGF)-like repeats that can activate EGF receptor. Interestingly, EGF also induced Mig-7 expression. Carcinoma cell adhesion to laminins was significantly reduced by Mig-7 expression. Remarkably, in two-dimensional and three-dimensional Matrigel cultures, Mig-7 expression caused invasion and vessel-like structures. Melanoma cells, which were previously characterized to invade aggressively and to undergo vasculogenic mimicry, expressed Mig-7. Taken together, these data suggest that Mig-7 expression allows cells to sense their environment, to invade, and to form vessel-like structures through a novel relationship with laminin 5 γ2 chain domain III fragments. For more than 100 years, researchers have proposed that embryonic cytotrophoblast cells (CTBs) and cancer cells are one and the same.1Beard J Embryological aspects and etiology of carcinoma.Lancet. 1902; 1: 1758-1761Abstract Scopus (32) Google Scholar, 2Soundararajan R Rao AJ Trophoblast 'pseudo-tumorigenesis': significance and contributory factors.Reprod Biol Endocrinol. 2004; 2: 15Crossref PubMed Scopus (153) Google Scholar Although they may not be the exact same cells, embryonic CTBs are similar to cancer cells. They invade maternal tissues from the site of embryo implantation during placenta development. In addition, CTBs remodel maternal vasculature, a process known as vascular mimicry or pseudovasculogenesis, creating leaky vessels3Red-Horse K Zhou Y Genbacev O Prakobphol A Foulk R McMaster MT Fisher SJ Trophoblast differentiation during embryo implantation and formation of the maternal-fetal interface.J Clin Invest. 2004; 114: 744-754Crossref PubMed Scopus (618) Google Scholar similar to those found in tumors.4Hashizume H Baluk P Morikawa S McLean JW Thurston G Roberge S Jain RK McDonald DM Openings between defective endothelial cells explain tumor vessel leakiness.Am J Pathol. 2000; 156: 1363-1380Abstract Full Text Full Text PDF PubMed Scopus (1316) Google Scholar Invasion of these two cell types is initiated by receptor tyrosine kinase (RTK) and αvβ5 signaling cross talk.5Brooks PC Klemke RL Schon S Lewis JM Schwartz MA Cheresh DA Insulin-like growth factor receptor cooperates with integrin alpha v beta 5 to promote tumor cell dissemination in vivo.J Clin Invest. 1997; 99: 1390-1398Crossref PubMed Scopus (155) Google Scholar, 6Crouch S Spidel CS Lindsey JS HGF and ligation of αvβ5 integrin induce a novel, cancer cell-specific gene expression required for cell scattering.Exp Cell Res. 2004; 292: 274-287Crossref PubMed Scopus (32) Google Scholar, 7Klemke RL Yebra M Bayna EM Cheresh DA Receptor tyrosine kinase signaling required for integrin alpha v beta 5-directed cell motility but not adhesion on vitronectin.J Cell Biol. 1994; 127: 859-866Crossref PubMed Scopus (248) Google Scholar, 8Zhou Y Fisher SJ Janatpour M Genbacev O Dejana E Wheelock M Damsky CH Human cytotrophoblasts adopt a vascular phenotype as they differentiate.J Clin Invest. 1997; 99: 2139-2151Crossref PubMed Scopus (822) Google Scholar Data suggest that highly invasive cancer cells can form conduits in tumors, a process some researchers call vasculogenic mimicry.9Seftor REB Seftor EA Koshikawa N Meltzer PS Gardner LMG Bilban M Stetler-Stevenson WG Quanranta V Hendrix MJC Cooperative interactions of laminin 5 γ2 chain, matrix metalloproteinase-2, and membrane type-1 matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma.Cancer Res. 2001; 61: 6322-6327PubMed Google Scholar, 10Hess AR Postovit L-M Margaryan NV Seftor EA Schneider GB Seftor REB Nickoloff BJ Hendrix MJC Focal adhesion kinase promotes the aggressive melanoma phenotype.Cancer Res. 2005; 65: 9851-9860Crossref PubMed Scopus (126) Google Scholar, 11van der Schaft DWJ Hillen F Pauwels P Kirschmann DA Castermans K oude Egbrink MGA Tran MGB Sciot R Hauben E Hogendoorn PCW Delattre O Maxwell PH Hendrix MJC Griffioen AW Tumor cell plasticity in Ewing sarcoma, an alternative circulatory system stimulated by hypoxia.Cancer Res. 2005; 65: 11520-11528Crossref PubMed Scopus (176) Google Scholar Previous publications have described tumor cells lining the lumen of irregular and leaky tumor vessels (see review12McDonald DM Munn L Jain RK Vasculogenic mimicry: how convincing, how novel, and how significant?.Am J Pathol. 2000; 156: 383-388Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). These tumor cells that form vessel-like structures and CTBs can masquerade as endothelial cells by expressing vascular endothelial (VE)-cadherin and factor VIII-associated antigen (FVIII assoc:ag; also known as von Willebrand factor), as well as other endothelial markers.10Hess AR Postovit L-M Margaryan NV Seftor EA Schneider GB Seftor REB Nickoloff BJ Hendrix MJC Focal adhesion kinase promotes the aggressive melanoma phenotype.Cancer Res. 2005; 65: 9851-9860Crossref PubMed Scopus (126) Google Scholar, 11van der Schaft DWJ Hillen F Pauwels P Kirschmann DA Castermans K oude Egbrink MGA Tran MGB Sciot R Hauben E Hogendoorn PCW Delattre O Maxwell PH Hendrix MJC Griffioen AW Tumor cell plasticity in Ewing sarcoma, an alternative circulatory system stimulated by hypoxia.Cancer Res. 2005; 65: 11520-11528Crossref PubMed Scopus (176) Google Scholar, 13Folberg R Hendrix MJC Maniotis AJ Vasculogenic mimicry and tumor angiogenesis.Am J Pathol. 2000; 156: 361-381Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar, 14Hess AR Seftor EA Gardner LMG Carles-Kinch K Schneider GB Seftor REB Kinch MS Hendrix MJC Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: role of epithelial cell kinase (Eck/EphA2).Cancer Res. 2001; 61: 3250-3255PubMed Google Scholar Laminin 5 γ2 chain promigratory fragments contribute to cell motility, invasion,15Hintermann E Quaranta V Epithelial cell motility on laminin-5: regulation by matrix assembly, proteolysis, integrins and erbB receptors.Matrix Biol. 2004; 23: 75-85Crossref PubMed Scopus (82) Google Scholar, 16Hornebeck W Maquart FX Proteolyzed matrix as a template for the regulation of tumor progression.Biomed Pharmacother. 2003; 57: 223-230Crossref PubMed Scopus (51) Google Scholar, 17Miyazaki K Kikkawa Y Nakamura A Yasumitsu H Umeda M A large cell-adhesive scatter factor secreted by human gastric carcinoma cells.Proc Natl Acad Sci USA. 1993; 90: 11767-11771Crossref PubMed Scopus (151) Google Scholar, 18Pirilä E Sharabi A Salo T Quaranta V Tu H Heljasvaara R Koshikawa N Sorsa T Maisi P Matrix metalloproteinases process the laminin-5 [gamma]2-chain and regulate epithelial cell migration.Biochem Biophys Res Commun. 2003; 303: 1012-1017Crossref PubMed Scopus (85) Google Scholar, 19Pyke C Romer J Kallunki P Lund LR Ralfkiaer F Dano K Tryggvason K The gamma 2 chain of kalinin/laminin 5 is preferentially expressed in invading malignant cells in human cancers.Am J Pathol. 1994; 145: 782-791PubMed Google Scholar and vessel-like structure formation by tumor cells.9Seftor REB Seftor EA Koshikawa N Meltzer PS Gardner LMG Bilban M Stetler-Stevenson WG Quanranta V Hendrix MJC Cooperative interactions of laminin 5 γ2 chain, matrix metalloproteinase-2, and membrane type-1 matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma.Cancer Res. 2001; 61: 6322-6327PubMed Google Scholar Invasive melanoma cells capable of forming vessels express significantly higher levels of laminin 5, a basement membrane component that is a heterotrimer of α3, β3, and γ2 chains. Matrix metalloproteinase-2 (MMP-2) and membrane type 1 (MT-1) MMP cooperate to cleave γ2 chain into fragments that cause melanoma cell invasion leading to vasculogenic mimicry.9Seftor REB Seftor EA Koshikawa N Meltzer PS Gardner LMG Bilban M Stetler-Stevenson WG Quanranta V Hendrix MJC Cooperative interactions of laminin 5 γ2 chain, matrix metalloproteinase-2, and membrane type-1 matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma.Cancer Res. 2001; 61: 6322-6327PubMed Google Scholar In vivo, predominantly tumor cells rather than endothelial cells form vessels in the interior, more hypoxic regions of tumors, and are thought to provide new blood flow to the tumor causing renewed growth and dissemination.20Hendrix MJC Seftor EA Kirschmann DA Quaranta V Seftor REB Remodeling of the microenvironment by aggressive melanoma tumor cells.Ann NY Acad Sci. 2003; 995: 151-161Crossref PubMed Scopus (102) Google Scholar This formation of tumor cell-lined vessels seems to be resistant to antiangiogenic therapies.21van der Schaft DWJ Seftor REB Seftor EA Hess AR Gruman LM Kirschmann DA Yokoyama Y Griffioen AW Hendrix MJC Effects of angiogenesis inhibitors on vascular network formation by human endothelial and melanoma cells.J Natl Cancer Inst. 2004; 96: 1473-1477Crossref PubMed Scopus (192) Google Scholar In addition, invading tumor cells are resistant to current therapies.22Condeelis J Singer RH Segall JE The great escape: when cancer cells hijack the genes for chemotaxis and motility.Annu Rev Cell Dev Biol. 2005; 21: 695-718Crossref PubMed Scopus (281) Google Scholar Because no specific marker expressed by tumor cells that invade or mimic endothelial cells has previously been discovered, these cells can evade detection. Understanding the regulation of tumor cell invasion and vessel formation is therefore of considerable importance for development of more efficient and effective targeted tumor cell detection as well as therapies. Mig-7 is a cysteine-rich protein found in carcinoma cell membrane and cytoplasm protein lysate fractions by immunoblotting. Expression of Mig-7 seems to be restricted to carcinoma cells and putatively to early placenta, based on homology with expressed sequence tags (ESTs) isolated from this tissue. Human malignant tumors, blood, and metastatic sites from more than 200 cancer patients express Mig-7 regardless of tissue origin. Notably, Mig-7 is not detected in 25 different normal human tissues or in blood from normal patients.6Crouch S Spidel CS Lindsey JS HGF and ligation of αvβ5 integrin induce a novel, cancer cell-specific gene expression required for cell scattering.Exp Cell Res. 2004; 292: 274-287Crossref PubMed Scopus (32) Google Scholar, 23Phillips TM Lindsey JS Carcinoma cell-specific Mig-7: a new potential marker for circulating and migrating cancer cells.Oncol Rep. 2005; 13: 37-44PubMed Google Scholar Signaling that initiates CTBs and tumor cell invasion from RTK c-Met, the hepatocyte growth factor/scatter factor (HGF/SF) receptor, and αvβ5 integrin induces Mig-7 expression.6Crouch S Spidel CS Lindsey JS HGF and ligation of αvβ5 integrin induce a novel, cancer cell-specific gene expression required for cell scattering.Exp Cell Res. 2004; 292: 274-287Crossref PubMed Scopus (32) Google Scholar, 8Zhou Y Fisher SJ Janatpour M Genbacev O Dejana E Wheelock M Damsky CH Human cytotrophoblasts adopt a vascular phenotype as they differentiate.J Clin Invest. 1997; 99: 2139-2151Crossref PubMed Scopus (822) Google Scholar, 24Dokras A Gardner LMG Seftor EA Hendrix MJC Regulation of human cytotrophoblast morphogenesis hepatocyte growth factor/scatter factor.Biol Reprod. 2001; 65: 1278-1288Crossref PubMed Scopus (27) Google Scholar Based on these data, we hypothesized that Mig-7 expression plays a role in behaviors in common between CTBs and carcinoma cells, namely invasion and vascular cell mimicry. To test this hypothesis, Mig-7 expression by human CTBs was examined in vitro and in vivo. Tumor microenvironment extracellular matrix (ECM) and its growth factors important for these behaviors were used with endogenous Mig-7 and short interferring RNA (siRNA) stable knockdown cell lines, as well as overexpressed Mig-7 in two-dimensional and three-dimensional cultures, to determine Mig-7 biological relevance. Immunohistochemistry (IHC) of lymph nodes from our nude mouse model of tumor cell invasion determined localization of Mig-7 expression and topographical relationships in situ with VE-cadherin, laminin 5 γ2 chain domain III fragments, and FVIII assoc:ag. Adhesion assays were also used to elucidate mechanisms by which Mig-7 expression contributes to tumor cell invasion and its potential role in vessel-like structure formation. Human CTBs were isolated from second trimester placentas under institutional review board approval as described previously.25Fisher SJ Cui TY Zhang L Hartman L Grahl K Zhang GY Tarpey J Damsky CH Adhesive and degradative properties of human placental cytotrophoblast cells in vitro.J Cell Biol. 1989; 109: 891-902Crossref PubMed Scopus (402) Google Scholar CTBs were cultured on an ECM (Matrigel; BD Biosciences, San Jose, CA), which initiates their differentiation along the invasive pathway.26Librach CL Werb Z Fitzgerald ML Chiu K Corwin NM Esteves RA Grobelny D Galardy R Damsky CH Fisher SJ 92-kD type IV collagenase mediates invasion of human cytotrophoblasts.J Cell Biol. 1991; 113: 437-449Crossref PubMed Scopus (644) Google Scholar HEC1A, RL95-2 (RL95) endometrial carcinoma, and HT29 colon carcinoma cell lines (all from American Type Culture Collection, Rockville, MD) were cultured as described previously.6Crouch S Spidel CS Lindsey JS HGF and ligation of αvβ5 integrin induce a novel, cancer cell-specific gene expression required for cell scattering.Exp Cell Res. 2004; 292: 274-287Crossref PubMed Scopus (32) Google Scholar, 23Phillips TM Lindsey JS Carcinoma cell-specific Mig-7: a new potential marker for circulating and migrating cancer cells.Oncol Rep. 2005; 13: 37-44PubMed Google Scholar Epidermal growth factor (EGF; Calbiochem, La Jolla, CA) or HGF/SF (R&D Systems, Minneapolis, MN) treatments (each at 20 ng/ml) were incubated for the indicated time with RL95 cells that had been serum-starved for 12 hours. Melanoma cell lines, MUM-2B, MUM-2C, C918, C8161, and A375P, were maintained in RPMI 1640 medium (Life Technologies, Inc., Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (Life Technologies, Inc., Invitrogen), 20 mmol/L HEPES, and 0.1% gentamicin sulfate. Cultures, invasive phenotypes, and ability to undergo vasculogenic mimicry are well characterized for melanoma cell lines.9Seftor REB Seftor EA Koshikawa N Meltzer PS Gardner LMG Bilban M Stetler-Stevenson WG Quanranta V Hendrix MJC Cooperative interactions of laminin 5 γ2 chain, matrix metalloproteinase-2, and membrane type-1 matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma.Cancer Res. 2001; 61: 6322-6327PubMed Google Scholar, 10Hess AR Postovit L-M Margaryan NV Seftor EA Schneider GB Seftor REB Nickoloff BJ Hendrix MJC Focal adhesion kinase promotes the aggressive melanoma phenotype.Cancer Res. 2005; 65: 9851-9860Crossref PubMed Scopus (126) Google Scholar, 14Hess AR Seftor EA Gardner LMG Carles-Kinch K Schneider GB Seftor REB Kinch MS Hendrix MJC Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: role of epithelial cell kinase (Eck/EphA2).Cancer Res. 2001; 61: 3250-3255PubMed Google Scholar, 20Hendrix MJC Seftor EA Kirschmann DA Quaranta V Seftor REB Remodeling of the microenvironment by aggressive melanoma tumor cells.Ann NY Acad Sci. 2003; 995: 151-161Crossref PubMed Scopus (102) Google Scholar, 27Hess AR Seftor EA Seftor REB Hendrix MJC Phosphoinositide 3-kinase regulates membrane type 1-matrix metalloproteinase (MMP) and MMP-2 activity during melanoma cell vasculogenic mimicry.Cancer Res. 2003; 63: 4757-4762PubMed Google Scholar, 28Seftor REB Seftor EA Hendrix MJC Molecular roles for integrins in human melanoma invasion.Cancer Metastasis Rev. 1999; 18: 359-375Crossref PubMed Scopus (117) Google Scholar All cultures were maintained in a humidified incubator at 37°C in 5% CO2 air. For two-dimensional cultures, HT29, HEC1A, or RL95 cells were plated on Matrigel containing growth factors (GF+ Matrigel) or growth factor-reduced Matrigel (GFR Matrigel, >10 mg/ml lots; BD Biosciences) ∼0.2 mm thick from 80% confluent cultures for indicated times before collecting protein lysates. Three-dimensional cultures were performed using 50-μl domes of GF+ Matrigel (no dilution) allowed to polymerize for 30 minutes at 37°C in a humidified, 5% CO2 incubator. Cells were removed nonenzymatically from their plates [2 mmol/L ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid] and 2 μl of single-cell suspensions (7.5 × 104) were injected into the dome of Matrigel. Images of two-dimensional and three-dimensional cultures were taken using a Nikon Diaphot inverted microscope (Nikon, Tokyo, Japan) and Retiga 2000R digital charge-coupled device camera with QCapture 5.1 software (QImaging, Burnaby, BC, Canada). The nude mouse model was used as described previously23Phillips TM Lindsey JS Carcinoma cell-specific Mig-7: a new potential marker for circulating and migrating cancer cells.Oncol Rep. 2005; 13: 37-44PubMed Google Scholar under Institutional Animal Care and Use Committee approval. Briefly, 1 × 105 HEC1A or RL95 viable cells in 250 μl of media were combined with 250 μl of GF+ Matrigel and injected subcutaneously into the dorsal neck region of nu/nu athymic mice. Negative controls were mice injected with Matrigel alone (ie, no cells). Five animals per group were injected. After 6 weeks, animals were euthanized. Brachial and axillary lymph nodes were harvested, flash-frozen in OCT (Miles Laboratories, Elkhart, IN), and stored at −80°C until cryosectioned. Selection of transfected pooled clones (n = 3 each construct), as well as expression of 3XFLAG CMV Mig-7, were described previously.6Crouch S Spidel CS Lindsey JS HGF and ligation of αvβ5 integrin induce a novel, cancer cell-specific gene expression required for cell scattering.Exp Cell Res. 2004; 292: 274-287Crossref PubMed Scopus (32) Google Scholar Endogenous Mig-7 expression was reduced with siRNA. In brief, three Mig-7-specific siRNAs sequences (Table 1) were designed by Ambion (Austin, TX) with BamHI and HindIII restriction enzyme sites at each 5′ and 3′, respectively, for directional cloning. No significant homology was found to any human sequence other than Mig-7 (accession no. DQ080207). Double-stranded DNA oligonucleotides that encode each Mig-7 siRNA were synthesized by Invitrogen (Carlsbad, CA), resuspended, quantified, annealed, cut with BamHI and HindIII restriction enzymes, and ligated into the pSilencer vector (pSilencer 3.1-H1 neo) cut with the same enzymes according to the manufacturer's instructions (Ambion). After one optimal concentration of G418 for RL95 cell killing was determined, RL95 cells were transfected in triplicate for each construct at a ratio of 1 μg of plasmid to 3 μl of FuGene 6 (Roche, Indianapolis, IN). After G418 selection at 600 μg/ml, levels of Mig-7 protein expression were determined for each pooled transfected cell line by immunoblotting and densitometry using β-tubulin as a normalizing gene expression.Table 1siRNA Sequences Specific to Mig-7Sequence anti-sense-loop-senseLocation in Mig-7 sequence (accession: DQ080207) in base pairsPooled clones number5′-AAAGTTTCATTCTTCGACTTCAAGAGAGTCGAAGAAATGAAACTTT-3′379 to 3981–3A5′-AGATTTCCTGTGATTTAAGTTCAAGAGACTTAAATCACAGGAAATCT-3′728 to 7462–3B5′-CATGATCTGGATTTGAATCTTCAAGAGAGATTCAAATCCAGATCATG-3′1275 to 12933–1A Open table in a new tab Detection of Mig-7 protein was performed using Mig-7-specific affinity-purified antibody produced in rabbits immunized with KLH-conjugated Mig-7 peptide (MAASRCSGL) representing the first nine amino acids of Mig-7 protein, as previously described.6Crouch S Spidel CS Lindsey JS HGF and ligation of αvβ5 integrin induce a novel, cancer cell-specific gene expression required for cell scattering.Exp Cell Res. 2004; 292: 274-287Crossref PubMed Scopus (32) Google Scholar In brief, cryostat sections (10 μm) of fresh-frozen lymph node from five HEC1A-, four RL95-, and five control (Matrigel-alone)-injected nude mice on Superfrost plus slides were washed two times in Dulbecco's phosphate-buffered saline (D-PBS; Hyclone, Logan, UT) and then treated with 50 mmol/L NH4CL (Sigma, St. Louis, MO) for 10 minutes at room temperature. Slides were washed two times in D-PBS and then permeabilized with 0.01% digitonin (Aldrich Chemical Co., Milwaukee, WI) in phosphate-buffered saline (PBS) at room temperature for 30 minutes. After washing two times in D-PBS, slides were blocked in 10% horse serum (Life Technologies, Inc.) in D-PBS for 30 minutes at room temperature. Primary antibodies, polyclonal rabbit, anti-human Mig-7, monoclonal mouse, human-specific anti-γ2 domain III (clone D4B5; Chemicon, Temecula, CA), polyclonal rabbit anti-VE-cadherin (Cayman Chemical, Ann Arbor, MI), and polyclonal rabbit, FVIII assoc:ag (DAKO, Carpinteria, CA) were diluted 1:50 and incubated on the tissue for 2 hours at room temperature and overnight at 4°C. Slides were washed two times in D-PBS then incubated for 20 minutes in 3% H2O2 in methanol. Slides were washed two times in D-PBS and then incubated for 30 minutes in 1:200 dilution of goat anti-rabbit IgG-horseradish peroxidase (HRP) or goat anti-mouse IgG-HRP labeled antibody (Santa Cruz Biotechnology, Santa Cruz, CA) relevant to the primary antibody used in D-PBS containing 0.5% bovine serum albumin (catalog no. BP1600-100; Fisher Scientific, Fair Lawn, NJ). Slides were washed two times in D-PBS and then developed using 3,3′-diaminobenzidine (DAB) substrate kit (Vector Laboratories, Burlingame, CA) to detect HRP-labeled secondary antibodies until brown specific staining was detected by microscopy (less than 3 minutes). After washing in water for 5 minutes, slides were counterstained in Hematoxylin QS (Vector Laboratories). Slides were dehydrated in two incubations for 3 minutes each of 75, 95, and 100% ethanol, air-dried, and mounted in DPX mounting medium (BDH Laboratory Supplies, Poole, UK). Controls included sections without primary or secondary antibodies, as well as staining of normal (vehicle-injected animal) lymph node sections with each primary antibody. Ten percent formalin-fixed, paraffin-embedded 5-μm sections were used for basal plate placenta analyses. After deparaffinization with xylene for 10 minutes, rehydration through 100, 95, and 70% ethanol, and antigen retrieval for 2 seconds on ice in a microwave oven, these sections were processed as described above to detect Mig-7 or cytokeratin 7 (CK7). Mouse anti-human antibody for CK7 (DAKO) was used at 1:100. Images from low (×40) to high (×1000) magnifications were taken on a Nikon Microphot microscope with a Retiga 2000R digital charge-coupled device camera and analyzed with the QCapture 5.1 software program (both QImaging) and Canvas 8.0 (Deneba Systems Inc.). Measurement of vessels in lymph nodes invaded by HEC1A and RL95 cells was performed with the QCapture software program calibrated with a micrometer. At least four different tumors from four mice injected with each cell line (HEC1A or RL95) or Matrigel alone were analyzed. Western blot analyses were performed as described previously with the following modifications.6Crouch S Spidel CS Lindsey JS HGF and ligation of αvβ5 integrin induce a novel, cancer cell-specific gene expression required for cell scattering.Exp Cell Res. 2004; 292: 274-287Crossref PubMed Scopus (32) Google Scholar Cells grown on plastic or two-dimensional Matrigel (10 mg/ml lot) were homogenized in lysis buffer [2% sodium dodecyl sulfate, 100 mmol/L dithiothreitol, 0.01% bromphenol blue, 60 mmol/L Tris, 10% glycerol, 2× protease inhibitor (Complete; Roche)] and quantitated using RC/DC protein assay (Bio-Rad, Hercules, CA). Equal amounts of protein were loaded onto a 12% polyacrylamide gel and run at constant 200 V for 30 to 40 minutes. Gels were semidry transferred (Boekel, Feasterville, PA) to polyvinylidene fluoride membranes and blocked in Tris-buffered saline containing Tween 20 detergent (TBST; 0.1%) and 5% dry milk for 1 hour at room temperature. Endogenous or FLAG-tagged Mig-7 protein was detected using human-specific, affinity-purified Mig-7 antibody (1:2000) or the M2-peroxidase anti-FLAG antibody (1:100; Sigma), respectively. Mouse anti-β-tubulin monoclonal antibody (clone AA2; Upstate, Lake Placid, NY) or mouse anti-β-actin (clone AC-40; Sigma) served as loading controls. Antibody to laminin 5 γ2 chain domain III was described in Immunohistochemistry. After washing in TBST, a HRP-labeled secondary anti-rabbit IgG antibody at a dilution of 1:40,000 was used to detect the Mig-7 antibody or goat anti-mouse IgG-HRP-labeled antibody (Santa Cruz Biotechnology) to detect the β-tubulin, actin, or laminin 5 γ2 chain domain III antibodies. Chemiluminescence Plus Reagent (Amersham, Arlington Heights, IL) allowed detection of HRP-labeled antibodies once exposed to film. Densitometry was performed using the Bio-Rad imager and Quantity One analysis software program and comparing each protein of interest band intensity to its respective β-tubulin or actin band intensity. All raw signal intensities were corrected for background. Data analyses were performed with Prism 3.0 statistical software (GraphPad, San Diego, CA). Total RNA isolation and relative RT-PCR, including optimization of cycle number to achieve mid-linear range, were performed as described previously.6Crouch S Spidel CS Lindsey JS HGF and ligation of αvβ5 integrin induce a novel, cancer cell-specific gene expression required for cell scattering.Exp Cell Res. 2004; 292: 274-287Crossref PubMed Scopus (32) Google Scholar Placental RNA was obtained, with patient consent and institutional review board approval, as described previously.29Haimov-Kochman R Fisher SJ Winn VD Modification of the standard Trizol-based technique improves the integrity of RNA isolated from RNase-rich placental tissue.Clin Chem. 2006; 52: 159-160Crossref PubMed Scopus (23) Google Scholar PCR products were confirmed by Southern blot (Mig-7-specific cDNA probe) or by subcloning and sequencing. Northern blots were performed as described previously.6Crouch S Spidel CS Lindsey JS HGF and ligation of αvβ5 integrin induce a novel, cancer cell-specific gene expression required for cell scattering.Exp Cell Res. 2004; 292: 274-287Crossref PubMed Scopus (32) Google Scholar Reverse transcription of RNA was performed using the TaqMan Gold RT-PCR kit as described by the manufacturer (Applied Biosystems, Foster City, CA). Q-PCR, used the Applied Biosystems 9700HT sequence detection system. Each target was amplified in triplicate with a Mig-7-specific primer probe set (forward, 5′-CACCTGCCTCTGGTCGTTAGG-3′; reverse, 5′-TACTGGATTCCTCTAGCTTTGGTGTT-3′; probe, 5′-AAACTCTCAGTGATCTCT-3′) or 18S primer probe set (Applied Biosystems). Primer pairs for HLA-G and integrin α1 were as previously published.8Zhou Y Fisher SJ Janatpour M Genbacev O Dejana E Wheelock M Damsky CH Human cytotrophoblasts adopt a vascular phenotype as they differentiate.J Clin Invest. 1997; 99: 2139-2151Crossref PubMed Scopus (822) Google Scholar, 30McMaster MT Librach CL Zhou Y Lim KH Janatpour MJ DeMars R Kovats S Damsky C Fisher SJ Human placental HLA-G expression is restricted to differentiated cytotrophoblasts.J Immunol. 1995; 154: 3771-3778PubMed Google Scholar In brief, 5 μl of cDNA target was added to a 20-μl mix of 1× TaqMan universal PCR master containing Amperase UNG and 1 μl of primer/probe. Reactions were incubated at
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