The Chemokine Receptor CXCR4 and the Metalloproteinase MT1-MMP Are Mutually Required during Melanoma Metastasis to Lungs
2009; Elsevier BV; Volume: 174; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2009.080636
ISSN1525-2191
AutoresRubén A. Bartolomé, Sergio Ferreiro, María E. Miquilena-Colina, Lorena Martínez‐Prats, María Luisa Soto‐Montenegro, David García‐Bernal, J.J. Vaquero, Reuven Agami, Rafaël Delgado, Manuel Desco, Paloma Sánchez‐Mateos, Joaquı́n Teixidó,
Tópico(s)Immunotherapy and Immune Responses
ResumoMelanoma is the most aggressive skin cancer once metastasis begins; therefore, it is important to characterize the molecular players involved in melanoma dissemination. The chemokine receptor CXCR4 and the membrane-bound metalloproteinase MT1-MMP are expressed on melanoma cells and represent candidate molecules for the control of metastasis. Using human melanoma transfectants that either overexpress or silence CXCR4 or MT1-MMP, or that have a combination of overexpression and interference of these proteins, we show that CXCR4 and MT1-MMP coordinate their activities at different steps along melanoma cell metastasis into the lungs. Results from in vivo xenograft mouse models of melanoma lung colonization and mice survival and short-term, homing nested polymerase chain reaction experiments from lung samples indicated that CXCR4 is required at early phases of melanoma cell arrival in the lungs. In contrast, MT1-MMP is not needed for these initial steps but promotes subsequent invasion and dissemination of the tumor with CXCR4. Investigation of potential cross talk between CXCR4 and MT1-MMP revealed that MT1-MMP accumulates intracellularly after melanoma cell stimulation with the CXCR4 ligand CXCL12, and that this process involves the activation of the Rac-Erk1/2 pathway. Subsequent to cell contact with specific basement membrane proteins, MT1-MMP redistributes to the cell membrane in a phosphatidylinositol 3-kinase-dependent manner. These results suggest that combination therapies that target CXCR4 and MT1-MMP should improve the limitations of the current therapies for metastatic melanoma. Melanoma is the most aggressive skin cancer once metastasis begins; therefore, it is important to characterize the molecular players involved in melanoma dissemination. The chemokine receptor CXCR4 and the membrane-bound metalloproteinase MT1-MMP are expressed on melanoma cells and represent candidate molecules for the control of metastasis. Using human melanoma transfectants that either overexpress or silence CXCR4 or MT1-MMP, or that have a combination of overexpression and interference of these proteins, we show that CXCR4 and MT1-MMP coordinate their activities at different steps along melanoma cell metastasis into the lungs. Results from in vivo xenograft mouse models of melanoma lung colonization and mice survival and short-term, homing nested polymerase chain reaction experiments from lung samples indicated that CXCR4 is required at early phases of melanoma cell arrival in the lungs. In contrast, MT1-MMP is not needed for these initial steps but promotes subsequent invasion and dissemination of the tumor with CXCR4. Investigation of potential cross talk between CXCR4 and MT1-MMP revealed that MT1-MMP accumulates intracellularly after melanoma cell stimulation with the CXCR4 ligand CXCL12, and that this process involves the activation of the Rac-Erk1/2 pathway. Subsequent to cell contact with specific basement membrane proteins, MT1-MMP redistributes to the cell membrane in a phosphatidylinositol 3-kinase-dependent manner. These results suggest that combination therapies that target CXCR4 and MT1-MMP should improve the limitations of the current therapies for metastatic melanoma. Trafficking of cancer cells from primary tumor sites via intravasation into blood circulation and later extravasation to colonize distant organs requires tightly regulated directional cues and cell migration and extracellular matrix (ECM) degradation that are mediated by chemokines, growth factors, integrins, and metalloproteinases.1Chambers AF Groom AC MacDonald IC Dissemination and growth of cancer cells in metastatic sites.Nat Rev Cancer. 2002; 2: 563-572Crossref PubMed Scopus (3007) Google Scholar Solid tumor cells express chemokine receptors that provide guidance to these cells to organs where their ligands are expressed, constituting a homing model resembling the one used by immune cells to exert their immune surveillance functions.2Balkwill F Cancer and the chemokine network.Nat Rev Cancer. 2004; 4: 540-550Crossref PubMed Scopus (1884) Google ScholarCXCR4 is a chemokine receptor expressed by tumor cells in melanoma, breast, prostate, and colon cancer.3Müller A Homey B Soto H Ge N Catron D Buchanan ME McClanahan T Murphy E Yuan W Wagner SN Barrera JL Mohar A Verastegui E Zlotnik A Involvement of chemokine receptors in breast cancer metastasis.Nature. 2001; 410: 50-56Crossref PubMed Scopus (4410) Google Scholar, 4Robledo MM Bartolome RA Longo N Rodriguez-Frade JM Mellado M Longo I van Muijen GN Sanchez-Mateos P Teixido J Expression of functional chemokine receptors CXCR3 and CXCR4 on human melanoma cells.J Biol Chem. 2001; 276: 45098-45105Crossref PubMed Scopus (209) Google Scholar, 5Murakami T Maki W Cardones AR Fang H Tun Kyi A Nestle FO Hwang ST Expression of CXC chemokine receptor-4 enhances the pulmonary metastatic potential of murine B16 melanoma cells.Cancer Res. 2002; 62: 7328-7334PubMed Google Scholar, 6Taichman RS Cooper C Keller ET Pienta KJ Taichman NS McCauley LK Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone.Cancer Res. 2002; 62: 1832-1837PubMed Google Scholar Its ligand CXCL12 (also called SDF-1) is expressed in lymph nodes, lungs, bone marrow, and liver.3Müller A Homey B Soto H Ge N Catron D Buchanan ME McClanahan T Murphy E Yuan W Wagner SN Barrera JL Mohar A Verastegui E Zlotnik A Involvement of chemokine receptors in breast cancer metastasis.Nature. 2001; 410: 50-56Crossref PubMed Scopus (4410) Google Scholar The importance of the CXCR4/CXCL12 axis in cancer is exemplified by the fact that blocking CXCR4 function leads to inhibition of metastasis in in vivo mouse models of breast carcinoma and pancreatic cancer.3Müller A Homey B Soto H Ge N Catron D Buchanan ME McClanahan T Murphy E Yuan W Wagner SN Barrera JL Mohar A Verastegui E Zlotnik A Involvement of chemokine receptors in breast cancer metastasis.Nature. 2001; 410: 50-56Crossref PubMed Scopus (4410) Google Scholar, 7Smith MC Luker KE Garbow JR Prior JL Jackson E Piwnica-Worms D Luker GD CXCR4 regulates growth of both primary and metastatic breast cancer.Cancer Res. 2004; 64: 8604-8612Crossref PubMed Scopus (603) Google Scholar, 8Saur D Seidler B Schneider G Algul H Beck R Senekowitsch-Schmidtke R Schwaiger M Schmid RM CXCR4 expression increases liver and lung metastasis in a mouse model of pancreatic cancer.Gastroenterology. 2005; 129: 1237-1250Abstract Full Text Full Text PDF PubMed Scopus (171) Google ScholarMelanoma incidence has been steadily growing in western populations. Although melanoma only accounts for less than 5% of skin cancers, current therapies are primarily refractory for metastatic melanoma. Therefore, melanoma is responsible for 80% of deaths from skin cancers.9Miller AJ Mihm Jr, MC Melanoma.N Engl J Med. 2006; 355: 51-65Crossref PubMed Scopus (1148) Google Scholar Expression of CXCR4 in human melanoma has been detected in the vertical growth phase and on regional lymph nodes and correlated with poor prognosis and increased mortality.10Longo-Imedio MI Longo N Trevino I Lazaro P Sanchez-Mateos P Clinical significance of CXCR3 and CXCR4 expression in primary melanoma.Int J Cancer. 2005; 117: 861-865Crossref PubMed Scopus (62) Google Scholar, 11Scala S Ottaiano A Ascierto PA Cavalli M Simeone E Giuliano P Napolitano M Franco R Botti G Castello G Expression of CXCR4 predicts poor prognosis in patients with malignant melanoma.Clin Cancer Res. 2005; 11: 1835-1841Crossref PubMed Scopus (259) Google Scholar We previously demonstrated that CXCL12 stimulates in vitro melanoma cell invasion involving Vav-Rho GTPase activation, as well as activation of the metalloproteinase MT1-MMP/MMP-2 ECM-degrading system.12Bartolomé RA Galvez BG Longo N Baleux F Van Muijen GN Sanchez-Mateos P Arroyo AG Teixido J Stromal cell-derived factor-1alpha promotes melanoma cell invasion across basement membranes involving stimulation of membrane-type 1 matrix metalloproteinase and Rho GTPase activities.Cancer Res. 2004; 64: 2534-2543Crossref PubMed Scopus (128) Google Scholar, 13Bartolomé RA Molina-Ortiz I Samaniego R Sanchez-Mateos P Bustelo XR Teixido J Activation of Vav/Rho GTPase signaling by CXCL12 controls membrane-type matrix metalloproteinase-dependent melanoma cell invasion.Cancer Res. 2006; 66: 248-258Crossref PubMed Scopus (106) Google ScholarMT1-MMP is a key component of the pericellular proteolysis machinery involved in degradation of gelatin, laminin, and fibrillar collagens and is an activator of pro-MMP-2 in coordination with TIMP-2.14Seiki M The cell surface: the stage for matrix metalloproteinase regulation of migration.Curr Opin Cell Biol. 2002; 14: 624-632Crossref PubMed Scopus (190) Google Scholar, 15Sternlicht MD Werb Z How matrix metalloproteinases regulate cell behavior.Annu Rev Cell Dev Biol. 2001; 17: 463-516Crossref PubMed Scopus (3196) Google Scholar Accordingly, its cell membrane expression must be tightly controlled to avoid excessive ECM pericellular degradation. Furthermore, MT1-MMP proteolytic activity controls cell adhesion and growth.14Seiki M The cell surface: the stage for matrix metalloproteinase regulation of migration.Curr Opin Cell Biol. 2002; 14: 624-632Crossref PubMed Scopus (190) Google Scholar, 15Sternlicht MD Werb Z How matrix metalloproteinases regulate cell behavior.Annu Rev Cell Dev Biol. 2001; 17: 463-516Crossref PubMed Scopus (3196) Google Scholar MT1-MMP is expressed on melanoma, and breast and lung cancer, and its expression often correlates with tumor invasiveness across tissue barriers.16Sato H Takino T Okada Y Cao J Shinagawa A Yamamoto E Seiki M A matrix metalloproteinase expressed on the surface of invasive tumour cells.Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar, 17Kurschat P Zigrino P Nischt R Breitkopf K Steurer P Klein CE Krieg T Mauch C Tissue inhibitor of matrix metalloproteinase-2 regulates matrix metalloproteinase-2 activation by modulation of membrane-type 1 matrix metalloproteinase activity in high and low invasive melanoma cell lines.J Biol Chem. 1999; 274: 21056-21062Crossref PubMed Scopus (148) Google Scholar, 18Sabeh F Ota I Holmbeck K Birkedal-Hansen H Soloway P Balbin M Lopez-Otin C Shapiro S Inada M Krane S Allen E Chung D Weiss SJ Tumor cell traffic through the extracellular matrix is controlled by the membrane-anchored collagenase MT1-MMP.J Cell Biol. 2004; 167: 769-781Crossref PubMed Scopus (479) Google Scholar, 19Iida J Wilhelmson KL Price MA Wilson CM Pei D Furcht LT McCarthy JB Membrane type-1 matrix metalloproteinase promotes human melanoma invasion and growth.J Invest Dermatol. 2004; 122: 167-176Crossref PubMed Scopus (60) Google Scholar MT1-MMP and MMP-2 are found in malignant melanoma often associated to the invading tumor front,20Nakahara H Howard L Thompson EW Sato H Seiki M Yeh Y Chen WT Transmembrane/cytoplasmic domain-mediated membrane type 1-matrix metalloprotease docking to invadopodia is required for cell invasion.Proc Natl Acad Sci USA. 1997; 94: 7959-7964Crossref PubMed Scopus (360) Google Scholar, 21Hofmann UB Westphal JR Zendman AJ Becker JC Ruiter DJ van Muijen GN Expression and activation of matrix metalloproteinase-2 (MMP-2) and its co-localization with membrane-type 1 matrix metalloproteinase (MT1-MMP) correlate with melanoma progression.J Pathol. 2000; 191: 245-256Crossref PubMed Scopus (145) Google Scholar suggesting that their proteolytic activity could be involved in melanoma cell dissemination.Whereas the above data support an important role for CXCR4 and MT1-MMP in melanoma metastasis, the potential functional relationships and mechanistic coordination of these molecules in lung metastasis, as well as their roles at different steps of melanoma cell homing into lungs, have not been evaluated. In the present study we have generated stable transfectants of the highly metastatic human melanoma cell line BLM expressing combinations of overexpression and silencing of CXCR4 and MT1-MMP to investigate whether these proteins establish coordinated activities during in vivo melanoma metastasis. The results reveal that CXCR4 and MT1-MMP need each other's activities during melanoma metastasis into lungs and provide mechanistic characterization of molecular cross-talks between these proteins. The data suggest that combinatorial therapies against these proteins might provide beneficial advantages to inhibit melanoma metastasis.Materials and MethodsCells, Antibodies, and ReagentsBLM human melanoma cells were cultured as previously described.4Robledo MM Bartolome RA Longo N Rodriguez-Frade JM Mellado M Longo I van Muijen GN Sanchez-Mateos P Teixido J Expression of functional chemokine receptors CXCR3 and CXCR4 on human melanoma cells.J Biol Chem. 2001; 276: 45098-45105Crossref PubMed Scopus (209) Google Scholar Anti-CXCR4 antibodies were from R&D Systems (Minneapolis, MN), anti-MT1-MMP LEM-2/15 mAb was a gift from Dr. Alicia G. Arroyo (Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain), and control P3X63 and antibodies to β1 and α4 integrins (Lia 1/2.1 and HP1/2, respectively) were from Dr. Francisco Sánchez-Madrid (Hospital de la Princesa, Madrid, Spain). Anti-Rac1 antibodies were from BD Biosciences (San Diego, CA), anti-RhoA from Santa Cruz Biotechnology (Santa Cruz, CA), anti-β-actin from Sigma-Aldrich (St. Louis, MO), anti-GFP from Molecular Probes (Eugene, OR), and anti-Erk1/2, anti-phospho-Erk1/2, anti-Akt, and anti-phospho-Akt (Ser473) antibodies were from Cell Signaling Technology (Danvers, MA). Type IV collagen and LY294002 were from Sigma-Aldrich Co., and U0126 from Calbiochem (Darmstadt, Germany). We used a laminin 1-rich extract from Sigma-Aldrich (catalog no. L2020) as source for laminin. Transforming growth factor-β1 and epidermal growth factor were obtained from R&D Systems.Vectors, siRNA, Transfections, and Retroviral Gene TransferThe pEGFP-C1 vector or GFP-fused forms of wild-type or activated V12-Rac1 were gifts from Dr. Francisco Sanchez-Madrid and were transfected into BLM cells following the described method.13Bartolomé RA Molina-Ortiz I Samaniego R Sanchez-Mateos P Bustelo XR Teixido J Activation of Vav/Rho GTPase signaling by CXCL12 controls membrane-type matrix metalloproteinase-dependent melanoma cell invasion.Cancer Res. 2006; 66: 248-258Crossref PubMed Scopus (106) Google Scholar siRNA for Rac1 has been earlier reported22García-Bernal D Wright N Sotillo-Mallo E Nombela-Arrieta C Stein JV Bustelo XR Teixido J Vav1 and Rac control chemokine-promoted T lymphocyte adhesion mediated by the integrin {alpha}4{beta}1.Mol Biol Cell. 2005; 16: 3223-3235Crossref PubMed Scopus (83) Google Scholar and was transfected into BLM cells according to the published procedure.13Bartolomé RA Molina-Ortiz I Samaniego R Sanchez-Mateos P Bustelo XR Teixido J Activation of Vav/Rho GTPase signaling by CXCL12 controls membrane-type matrix metalloproteinase-dependent melanoma cell invasion.Cancer Res. 2006; 66: 248-258Crossref PubMed Scopus (106) Google Scholar CXCR4 and MT1-MMP shRNA vectors were based on 19-mer sequences (5′-GCAGTCCATGTCATCTACA-3′, for CXCR4; 5′-TTGGCAGCCTCTCACTACT-3′, for MT1-MMP), which constitute targets for siRNA-mediated interference.13Bartolomé RA Molina-Ortiz I Samaniego R Sanchez-Mateos P Bustelo XR Teixido J Activation of Vav/Rho GTPase signaling by CXCL12 controls membrane-type matrix metalloproteinase-dependent melanoma cell invasion.Cancer Res. 2006; 66: 248-258Crossref PubMed Scopus (106) Google Scholar Synthetic oligonucleotides (64-mer) that include these 19-mer sequences were synthesized, annealed, and ligated into pSuper vector as described.23Brummelkamp TR Bernards R Agami R A system for stable expression of short interfering RNAs in mammalian cells.Science. 2002; 296: 550-553Crossref PubMed Scopus (3945) Google Scholar pSuper cassettes coding CXCR4 or MT1-MMP shRNAs were cloned into pRETRO-Hygro vector,24Voorhoeve PM Agami R The tumor-suppressive functions of the human INK4A locus.Cancer Cell. 2003; 4: 311-319Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar generating pRETRO-Super-CXCR4 and pRETRO-Super-MT1-MMP. cDNAs for CXCR4 and MT1-MMP (from Dr. José Miguel Rodríguez-Frade, Centro Nacional de Biotecnología, Madrid, Spain, and Dr. Alicia G. Arroyo, respectively) were cloned into pRetro-Blast retroviral vector24Voorhoeve PM Agami R The tumor-suppressive functions of the human INK4A locus.Cancer Cell. 2003; 4: 311-319Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar for overexpression of CXCR4 and MT1-MMP (pRetro-CXCR4 and pRetro-MT1-MMP, respectively). Retroviral particles were obtained by co-transfecting these vectors with plasmids coding for retroviral proteins gag.pol (pNGVL-MLV-gag-pol)25Yang S Delgado R King SR Woffendin C Barker CS Yang ZY Xu L Nolan GP Nabel GJ Generation of retroviral vector for clinical studies using transient transfection.Hum Gene Ther. 1999; 10: 123-132Crossref PubMed Scopus (110) Google Scholar and the vesicular stomatitis virus envelope glycoprotein (pNGVL-VSV-G),26Mañes S del Real G Lacalle RA Lucas P Gomez-Mouton C Sanchez-Palomino S Delgado R Alcami J Mira E Martinez AC Membrane raft microdomains mediate lateral assemblies required for HIV-1 infection.EMBO Rep. 2000; 1: 190-196Crossref PubMed Scopus (315) Google Scholar into 293FT packaging cells using lipofectamine (Invitrogen Corp., Carlsbad, CA). Conditioned media containing viral particles were used to infect BLM cells, which were selected with blasticidin (Invitrogen Corp.) or hygromicin (Calbiochem), obtaining sublines with interference (CXCR4lo and MT1lo), overexpression (CXCR4hi and MT1hi), or simultaneous overexpression and interference of CXCR4 and MT1-MMP (CXCR4hiMT1lo and CXCR4loMT1hi). As a control, we infected BLM cells with empty pRETRO-containing viral particles to generate the mock subline.Flow Cytometry, Western Blotting, and ZymographyMelanoma cells were incubated with primary antibodies, followed by incubation with fluorescein isothiocyanate-conjugated secondary antibodies (DAKO A/S, Copenhagen, Denmark) and analysis in an Epics XL cytofluorometer (Beckman Coulter, Fullerton, CA). P3X63 was used as control primary antibody. For Western blotting, cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins transferred to membranes that were incubated with primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Proteins were visualized using SuperSignal chemiluminescent substrate (Pierce, Rockford, IL). For zymography we used the described method.13Bartolomé RA Molina-Ortiz I Samaniego R Sanchez-Mateos P Bustelo XR Teixido J Activation of Vav/Rho GTPase signaling by CXCL12 controls membrane-type matrix metalloproteinase-dependent melanoma cell invasion.Cancer Res. 2006; 66: 248-258Crossref PubMed Scopus (106) Google Scholar Briefly, cells were lysed in Laemmli buffer, and samples resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels embedded with 1 mg/ml of fibrinogen (Calbiochem-Novabiochem Co., Darmstadt, Germany). Gels were rinsed in 2.5% Triton X-100, followed by incubation in 50 mmol/L Tris-HCl, pH 7.5, 10 mmol/L CaCl2, and 200 mmol/L NaCl. Gels were stained with Coomassie Blue, and areas of gelatinolytic activity were visualized as transparent bands.Proliferation and Cell Cycle AssaysCells (1.2 × 105) were cultured in Dulbecco's modified Eagle's medium/fetal bovine serum 1% with or without CXCL12 (R&D Systems) (200 ng/ml), and after detachment, cells were counted in a Neubauer chamber. For cell cycle assays, cells were pulsed with 5′-bromodeoxyuridine (BrdU) (Sigma-Aldrich Co) (15 μmol/L for 1 hour) and cultured for different times. Thereafter, cells were detached, fixed with 70% ethanol, permeabilized with 0.5% Triton X-100 in 2 mol/L HCl, and washed with 0.1 mol/L sodium tetraborate. Finally, cells were incubated with fluorescein isothiocyanate-conjugated anti-BrdU antibodies (BD Biosciences), followed by analysis in a cytofluorometer. Cell cycle time was estimated as the interval necessary to have a ratio of BrdU-labeled cells in S phase to BrdU-labeled cells in G2 phase equal as in 0 hours time.Invasion and Adhesion AssaysInvasions across Matrigel were done as previously established.12Bartolomé RA Galvez BG Longo N Baleux F Van Muijen GN Sanchez-Mateos P Arroyo AG Teixido J Stromal cell-derived factor-1alpha promotes melanoma cell invasion across basement membranes involving stimulation of membrane-type 1 matrix metalloproteinase and Rho GTPase activities.Cancer Res. 2004; 64: 2534-2543Crossref PubMed Scopus (128) Google Scholar Briefly, cells (3.5 × 104) were loaded on the upper compartments of invasion chambers coated with Matrigel (BD Biosciences, Bedford, MA). The lower compartments were filled with invasion medium with or without CXCL12. Invasive cells were fixed, stained, and counted under a microscope. For adhesion to Matrigel, cells (6 × 105) were labeled for 20 minutes at 37°C with 2′,7′-bis(carboxyethyl)-5(6′)-carboxyfluorescein-acetoxymethyl ester (Molecular Probes), and after pre-incubation with antibodies, cells in triplicates (3 × 104) were loaded on 96-well dishes (Costar, Cambridge, MA) coated with Matrigel (1 μg/ml). Cells were allowed to adhere for 10 minutes at 37°C and, on washing to remove unbound cells, adhesion was quantified using a fluorescence analyzer (Polarstar Galaxy, Offenburg, Germany).Animal StudiesBALB/c SCID mice (Harlan, Indianapolis, IN) bred and maintained under specific pathogen-free conditions were used for xenograft studies. The Consejo Superior de Investigaciones Cientificas Ethics Committee (Madrid, Spain) approved the protocols used for experiments with mice. Mice were injected subcutaneously in the lateral thoracic wall or intravenously into the tail vein with 2 × 106 cells resuspended in 0.2 ml of phosphate-buffered saline (PBS). Mice were daily inspected for local tumor growth and general condition, and were sacrificed when clear signs of respiratory stress were noted, or when subcutaneous tumors reached volumes of 2.5 cm3. For nested reverse transcriptase-polymerase chain reaction (RT-PCR), portions of lungs from mice inoculated with GFP-expressing melanoma transfectants were lysed in Tri-Reagent (Sigma-Aldrich Co.), and RNA was extracted and reverse-transcribed. PCR was performed using TaqDNA polymerase (Invitrogen Corp.) and outer primers for GFP: 5′-GACTGGGTGCTCAGGTAGTG-3′ and 5′-GTAAACGGCCACAAGTTCAG-3′. A 1-μl PCR product was further amplified using inner primers for GFP: 5′-TCGTGACCACCCTGACCTAC-3′ and 5′-TCACCTTGATGCCGTTCTTC-3′. The first PCR profile was 35 cycles of 45-second denaturation at 94°C, 45-second annealing at 52°C, and 1-minute polymerization at 72°C. The second PCR was performed in the same way, except that only 30 cycles were performed and annealing was executed at 56°C. For positron emission tomography (PET) analyses, mice were anesthetized with isoflurane, followed by administration of [18F]-fluorodeoxyglucose (624 ± 49 μCi) via the tail vein before analyses. Mice were scanned in a dedicated small animal PET scanner (rPET; Suinsa, Madrid, Spain), and tomographic images reconstructed using a three-dimensional FBP algorithm creating 55 55 × 55 images. Voxel size was 0.81 × 0.81 × 0.81 mm3, and the spatial resolution was 1.65 mm full width at half maximum isotropic. The energy window was 400 to 700 keV, and decay and dead-time corrections were applied. For computed tomography (CT) studies, mice were scanned using a CT scanner (HQT-Y15D, Suinsa) for small animals. CT acquisition was performed as follows: one bed position, 200 μA, 50 kV, 200 μm pixel size, and 360 projections. PET and CT images were co-registered using a marker-based rigid registration algorithm to obtain an initial realignment followed by a refinement step based on a mutual information registration algorithm.Statistical AnalysesData were analyzed by one-way analysis of variance followed by Tukey-Kramer multiple comparisons. In both analyses, the minimum acceptable level of significance was P < 0.05.ResultsIn Vivo Melanoma Metastasis Requires CXCR4 and MT1-MMP Coordinated ActivitiesTo investigate if functional coordination between CXCR4 and MT1-MMP is needed during melanoma cell metastasis, we made BLM melanoma stable transfectants with combined overexpression and silencing of these proteins to be subsequently tested in in vivo models of metastasis. To generate these transfectants, we used pRetro-Super and pRetro retroviral vectors as templates for stably silencing or for overexpression of CXCR4 and MT1-MMP, respectively (see Materials and Methods). After antibiotic selection, total transfectant populations were used for all experiments without subsequent cloning. Flow cytometry and Western blotting analyses of single CXCR4 or MT1-MMP transfectants revealed that CXCR4lo and MT1lo cells displayed minimal levels of CXCR4 and MT1-MMP expression, whereas CXCR4hi and MT1hi transfectants showed threefold and twofold higher expression of these proteins than mock counterparts, respectively (Figure 1, A and B). Accordingly, CXCR4hiMT1lo and CXCR4loMT1hi double transfectants displayed overexpression and silencing of CXCR4 or MT1-MMP.To in vitro characterize the invasive properties of transfectants, we subjected them to invasion assays across Matrigel, a basement membrane matrix extract rich in laminin, type IV collagen, heparan sulfate proteoglycans, and growth factors, which constitutes a valuable in vitro basis for assessing the invasive potential of tumor cells.27Albini A Iwamoto Y Kleinman HK Martin GR Aaronson SA Kozlowski JM McEwan RN A rapid in vitro assay for quantitating the invasive potential of tumor cells.Cancer Res. 1987; 47: 3239-3245PubMed Google Scholar CXCR4hi and MT1hi cells showed increased CXCL12-promoted invasion compared with mock counterparts, whereas CXCR4lo and MT1lo knockdown cells barely invaded (Figure 1C). Interestingly, CXCR4hiMT1lo and CXCR4loMT1hi transfectants displayed invasion levels similar to single knockdown CXCR4lo and MT1lo cells, suggesting that CXCR4 and MT1-MMP are mutually required for invasion of melanoma cells. In addition, CXCR4hi and MT1hi transfectants displayed higher degree of migration in wound-healing assays on Matrigel layers in the presence of CXCL12 than CXCR4lo and MT1lo counterparts (see Supplemental Figure S1A at http://ajp.amjpathol.org), together indicating that in vitro invasive and migratory properties of melanoma transfectants reflect CXCR4 and MT1-MMP expression levels.CXCR4hi transfectants displayed a modest growth advantage in medium containing CXCL12, compared with CXCR4lo cells (Figure 1D). Cell cycle analyses with BrdU-pulsed cells showed that high CXCR4 expression was associated with shorter doubling times (22 hours) than CXCR4 knockdown cells (26 hours), or mock transfectants (24 hours) (Supplemental Figure S1B at http://ajp.amjpathol.org). Instead, changes in MT1-MMP expression did not influence cell cycle kinetics.To investigate if functional mutual requirement between CXCR4 and MT1-MMP observed in in vitro invasion also takes place during in vivo metastasis, we first subcutaneously injected the melanoma transfectants into SCID mice and followed the growth and dissemination of the tumors. MT1hi and CXCR4loMT1hi tumors grew significantly faster than MT1lo or CXCR4hiMT1lo ones (Figure 2A). Instead, silencing or overexpression of CXCR4 did not affect the growth rate of melanoma cells, suggesting that MT1-MMP, but not CXCR4, influences the growth of subcutaneous melanoma tumors, in agreement with the reported MT1-MMP role in cell proliferation.19Iida J Wilhelmson KL Price MA Wilson CM Pei D Furcht LT McCarthy JB Membrane type-1 matrix metalloproteinase promotes human melanoma invasion and growth.J Invest Dermatol. 2004; 122: 167-176Crossref PubMed Scopus (60) Google Scholar None of the mice injected with CXCR4lo or MT1lo transfectants had invasive tumors. These tumors remained under the skin without crossing into thoracic or abdominal cavities (not shown), and no lung metastases were detected in these mice (Figure 2B). In contrast, subcutaneously-injected CXCR4hi and MT1hi melanoma cells reached the lungs and formed metastatic nodes (one to two tumor nodes: 45% and 60% of mice, respectively). Importantly, metastasis was impaired in CXCR4hiMT1lo and CXCR4loMT1hi tumors (Figure 2B), indicating that silencing CXCR4 or MT1-MMP reversed the invasive stimulation properties provided by overexpression of the counterpart protein. The pattern of CXCR4 and MT1-MMP expression and CXCL12-promoted invasiveness across Matrigel of melanoma cells derived from subcutaneous tumors or from lung metastases was maintained with respect to the originally inoculated transfectants (see Supplemental Figure S2, A and B, at http://ajp.amjpathol.org), confirming the functional characteristics of both proteins along the metastatic process.Figure 2Growth and lung metastasis of subcutaneously-inoculated CXCR4 and MT1-MMP melanoma transfectants. Mice (n = 9) were inoculated into the lateral thoracic wall with the indicated melanoma transfectants. Plotted are the days when tumors reached 2.5 cm3, together with statistical significance (N.S., nonsignificant) (A), and percentage of mice displaying lung metastases (B). Shown is a representative result from three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)When melanoma transfectants were intravenously inoculated, we found that CXCR4lo and MT1lo mice displayed significantly longer survival than CXCR4hi and MT1hi ones, respectively (Figure 3A). Lung examinations revealed that 70 to 90% of CXCR4hi and MT
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