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First published online March 29, 2004
doi: 10.1242/10.1242/jcs.01035

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Secreted MMP9 promotes angiogenesis more efficiently than constitutive active MMP9 bound to the tumor cell surface

Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain

View larger version (39K): [in a new window]   Fig. 1. Partitioning of MMP9 chimeras into distinct membrane domains. (A) Schematic representation of the MMP9-GPI and the MMP9-LDL chimeras. (B) Gelatinolytic activity in membrane (M) and cytosolic (C) fractions isolated from MMP9-GPI-, MMP9-LDL-, MMP9-wt and mock-transfected MCF-7 cells. ProMMP9 (solid arrowhead) and MMP9 (open arrowhead) activity in HT-1080 conditioned medium (H). (C) Gelatinolytic activity in conditioned medium from the same cells as in B after 24 hours of serum depletion. (D) MCF-7 cells expressing all MMP9 forms were fractionated in flotation gradients and analyzed by zymography. Fractions 1 and 6 represent the top and the bottom of the gradient, respectively. The same fractions were analyzed by western blot with anti-TfR and anti-VIP21 (CAV-1) as controls for non-raft- and raft-associated proteins, respectively. (E) Confocal microscopy of MMP9-expressing cells stained with cholera toxin ß-subunit (green) and anti-MMP9 antibody (red); yellow staining indicates colocalization of the molecules. The two-color overlays show representative cells for (a) MMP9-GPI, (b) MMP9-LDL and (c) MMP9-wt (n=50). The boxed regions in these images are enlarged in d, e and f, respectively. Scale bar, 10 µm. Single-color images are available: Fig. S1, http://jcs.biologists.org/supplemental/

View larger version (48K): [in a new window]   Fig. 2. Non-raft proMMP9 is processed at the cell surface. (A) Gelatinolytic activity in MMP9-GPI and MMP9-LDL-expressing MCF-7 and DU-145 cell extracts. (B) MMP9-GPI and MMP9-LDL, partially purified from MCF-7 cell extracts, were incubated alone (–) or with 2-MG (+), then analyzed by gelatin zymography. (C) Samples in B were incubated alone or with 2-MG in the presence (+) or absence (–) of 4-aminophenylmercuric acetate (APMA) before gelatin zymography. (D) Insect cell-derived MMP9-GPI and MMP9-LDL were in vitro processed with APMA before gelatin zymography. (E) MCF-7 cells expressing MMP9-LDL were incubated with DMSO, BB-94, GM6001, aprotinin, anti-uPA or PAI-1, and cell extracts were analyzed by gelatin zymography. In all zymograms, solid and open arrowheads indicate the migration of proMMP9 and MMP9 forms, respectively. Activity is also shown in HT-1080 medium (H). (F) Zymograms in E were analyzed by densitometry and the quotient between the intensities obtained for MMP9 and proMMP9 calculated. The values represent mean±s.d. of results obtained in two independent experiments.

View larger version (70K): [in a new window]   Fig. 3. Tumor invasion is not enhanced by cell surface MMP9 activity. (A) Cell surface-associated MMP9 activity was measured using the fluorogenic substrate M-2055 in intact MCF-7 cells transfected with the MMP9 forms. The fluorescence increment in mock-transfected cells was considered as 1. Data represent mean±s.e.m. of triplicates in one representative experiment of three performed. (B) MMP9-GPI-expressing (a) and MMP9-LDL-expressing (b) MCF-7 cells were incubated with DQ-gelatin and in situ gelatin degradation analyzed by confocal microscopy. Specificity was determined by preincubation of cells with GM6001 (c). Scale bar, 10 µm. (C) Tumor cell intravasation was evaluated by quantitative PCR of human DNA purified from CAM inoculated with MMP9-expressing MCF-7 cells. Data represent the percentage of intravasated human cells in each sample (mock n=21, MMP9-wt n=18, MMP9-GPI n=27 and MMP9-LDL n=22). Mean values for mock transfected, MMP9-wt-, MMP9-GPI- and MMP9-LDL-expressing MCF-7 cells were 0.013, 0.368, 0.057 and 0.016%, respectively (**P<0.05).

View larger version (71K): [in a new window]   Fig. 4. Soluble MMP9 enhances tumor growth and angiogenesis. Female BALB/c-SCID mice received subcutaneous injections in both flanks of mock transfected or MMP9-expressing MCF-7 cells. (A) Tumor incidence was calculated as the percentage of palpable tumors per number of injection sites. (B) Tumor size was monitored weekly and tumor growth kinetics over a 14-week period was represented as mean tumor volume±s.e.m. (n=8, in all cases; **P<0.05). (C) Tumor vascular profile was evaluated by macroscopic observation (a,d,g,j), Hematoxylin-Eosin staining (b,e,h,k; magnification x400) and blood vessel staining with anti-CD31 antibody (c,f,i,l; magnificatiox200). (D) Blood vessel number was quantified in tumor sections stained with anti-CD31 (**P<0.05). (E) Vascular perimeter was estimated as the percentage of CD31-stained area per field. The value obtained in sections from mock-transfected tumors was considered as 1 (**P<0.05).

View larger version (75K): [in a new window]   Fig. 5. Analysis of MMP9 in tumor xenografts. (A) Equal protein amounts of crude tumor extract were analyzed in gelatin zymography. Solid and open arrowheads indicate migration of proMMP9 and MMP9 forms, respectively, according to the activity detected in HT-1080 medium (H). (B) Tumor sections were incubated with DQ-gelatin (a,d,g,j; green in merged images) to detect in situ gelatinolytic activity; nuclei were stained with DAPI (b,e,h,k; blue in merged images). Magnification, x400.

View larger version (33K): [in a new window]   Fig. 6. Doxycycline abolishes MMP9-wt-enhanced tumor angiogenesis. (A) Gelatinolytic activity in crude tumor extracts (left) and sera (right) from untreated (–Dox) or doxycycline-treated mice (+Dox). Solid and open arrowheads indicate migration of proMMP9 and MMP9 forms, respectively, according to the activity detected in HT-1080 medium (H). (B) MMP9 activity was measured in tumor extracts using DQ-gelatin as substrate. Values represent the mean±s.e.m. obtained in triplicates of two samples per group. The value obtained in untreated samples was considered as 100% (**P<0.05). (C) Average tumor size at 17 weeks. The difference between–Dox (n=6) and +Dox (n=8) tumors was statistically significant (**P<0.05). (D) Tumor growth kinetics in the same mice as in C, over a 17-week period. Mean values±s.e.m. are represented. (E) Anti-CD31 staining of sections from tumors in–Dox and +Dox-treated mice. Magnificatiox400. (F) Vascular perimeter estimated as the percentage of CD31-stained area. The value obtained for–Dox samples was considered as 100.

View larger version (87K): [in a new window]   Fig. 7. VEGF distribution is specifically altered in tumors expressing soluble MMP9. (A) VEGF expression was evaluated in tumor sections using GV39M antibody, which recognizes VEGF bound to VEGFR2 (b,e,h,k), and an anti-VEGF mAb, which recognizes the receptor-free form (c,f,i,l). Sections were also stained with anti-CD31 for vessel identification (a,d,g,j). Magnification, x200. (B) Sections from MMP9-wt tumors in untreated (–Dox, a-c) or doxycycline-treated (+Dox, d-f) mice were stained as in A. Magnification, x200.

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