Journal: Molecular Cancer

Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

doi: 10.1186/s12943-016-0547-x

Figure Lengend Snippet: Transient overexpression of MT1-MMP in MCF-7 cells did not result in increased migration and invasion ( a ) qPCR, immunoblot, and gelatin zymography analysis of MT1-MMP mRNA, protein levels, and proMMP-2 activation ability, respectively, of MCF-7 breast cancer cells transiently transfected with MT1-MMP compared to mock transfected cells (control). Immunoblot analysis (AB6004) showed pro-, active, and degradation forms of MT1-MMP protein in MT1-MMP transfected MCF-7 cells. β-actin was used as a loading control. Gelatin zymography analysis showed that MCF-7 cells transiently transfected with MT1-MMP were capable of activating proMMP-2 after 24 h of incubation as shown by intermediate and active forms of MMP-2. b Gelatin zymography analysis of MCF-7 cells transiently transfected with MT1-MMP and incubated for 12 h with serum-free media (SF, top gel) or MMP-2 conditioned media (CM, bottom gel). Lanes 1 and 2: Controls showing proMMP-2 CM chemically activated by APMA. Lanes 4 and 6: Recombinant TIMP-2 (rTIMP-2) was added at 100 ng/ml to enhance MT1-MMP-mediated proMMP-2 activation. c Immunoblot analysis of MT1-MMP transfected cells showing phospho-ERK1/2 levels. Total ERK1/2 was used as a loading control. d Transwell migration and invasion assays of MCF-7 cells transiently transfected with MT1-MMP. Number of migrated/invaded cells were normalized to control MCF-7 cells and expressed as a mean percentage ± SEM. (ns, p > 0.05 by student’s t -test)

Article Snippet: Human MT1-MMP (sc116990), TIMP-2 (sc118083) and MMP-2 (sc321560) cDNA clones were purchased from Origene and subcloned into the vector pcDNA 3.3 (Thermo Fisher).

Techniques: Over Expression, Migration, Western Blot, Zymography, Activation Assay, Transfection, Incubation, Recombinant

Journal: Molecular Cancer

Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

doi: 10.1186/s12943-016-0547-x

Figure Lengend Snippet: MCF-7 cell lines producing high levels of MT1-MMP protein demonstrated TIMP-2-mediated proMMP-2 activation ( a ) qPCR analysis of MT1-MMP mRNA from MCF-7 MT1-MMP cells lines that stably express different levels of MT1-MMP. Different letters indicate significant differences at p ≤ 0.05 by one-way ANOVA, Tukey’s post-hoc test. b Immunoblot analysis (AB51074) showing pro-, active, and degradation forms of MT1-MMP protein in MCF-7 MT1-MMP cell lines. β-actin was used as a loading control. c Gelatin zymography analysis of MCF-7 MT1-MMP cell lines incubated for 6 or 12 h with either MMP-2 CM alone, or in combination with rTIMP-2 at 100 ng/ml, or rTIMP-2 and BB94 (10 μm). Cells were also incubated for 12 h in MMP-2 CM supplemented with TIMP-2 CM diluted 1:100 in SF media ( bottom ). Bar graph shows densitometry quantification of MMP-2 isoforms from representative zymography of MCF-7 MT1-MMP cell lines incubated with MMP-2 CM and TIMP-2 CM diluted 1:100

Article Snippet: Human MT1-MMP (sc116990), TIMP-2 (sc118083) and MMP-2 (sc321560) cDNA clones were purchased from Origene and subcloned into the vector pcDNA 3.3 (Thermo Fisher).

Techniques: Activation Assay, Stable Transfection, Western Blot, Zymography, Incubation

Journal: Molecular Cancer

Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

doi: 10.1186/s12943-016-0547-x

Figure Lengend Snippet: MCF-7 cell lines that express high levels of MT1-MMP demonstrated widespread ECM degradation (a) MCF-7, MT1-MMP cell lines, and MDA-MB 231 breast cancer cells were incubated on Alexa488 gelatin-coated coverslips for 24 h and processed for immunofluorescence to examine cytoplasmic MT1-MMP protein and ECM degradation. Representative fields of view are shown at 20× ( top panels ) and 60× ( bottom panels ) magnification. Panels are composed of an overlay showing the nuclei, F-actin, and Alexa488 gelatin signal ( top ) and the MT1-MMP signal with an inset of the Alexa488 gelatin channel ( bottom ). White arrows indicate cells that have degraded the underlying gelatin but are devoid of MT1-MMP signal. Scale bars = 100 μm. Cells in each sample positive for cytoplasmic MT1-MMP protein signal (MT1-MMP +) or devoid of underneath Alexa488 gelatin signal (Gelatin -) were quantified per 20× fields of view and are shown as mean percentage of total cells per field of view ± SEM

Article Snippet: Human MT1-MMP (sc116990), TIMP-2 (sc118083) and MMP-2 (sc321560) cDNA clones were purchased from Origene and subcloned into the vector pcDNA 3.3 (Thermo Fisher).

Techniques: Incubation, Immunofluorescence

Journal: Molecular Cancer

Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

doi: 10.1186/s12943-016-0547-x

Figure Lengend Snippet: Low MT1-MMP/high TIMP-2 was optimal to promote migration and ERK activation in MCF-7 cells. a ERK activation in MCF-7 and MT1-MMP cells after incubation for 12 h (top) or 15 min ( bottom ) in media containing 10 % FBS or different dilutions of TIMP-2 or ALA + TIMP-2 CM in SF media. b Scratch closure migration assay of MCF-7 MT1-MMP cell lines monitored for 3 days. Shown are representative 10× fields of view. The white dotted lines indicate the initial scratch size; red dotted lines indicate the scratch size at the respective day. Scale bars = 100 μm. Line graph on the right shows scratch closure quantification that demonstrates significantly increased migratory potential of C3 cells. c Transwell migration assays of MCF-7 MT1-MMP cell lines incubated for 48 h in TIMP-2, or ALA + TIMP-2 CM diluted 1:100 ( top ), or ALA + TIMP-2 CM in increasing dilutions ( bottom ). d ( top ) qPCR analysis showing MT1-MMP mRNA from two cell lines derived from MT1-MMP C3 cells that stably express an shRNA construct targeting MT1-MMP, and one cell line stably expressing a control scrambled shRNA construct. Different letters indicate significant differences at p ≤ 0.05 by one-way ANOVA, Tukey’s post-hoc test. Individual student’s t-tests comparing MCF-7 cells against the C3 SH 1 cell line is also shown. ( bottom ) Transwell migration assay of MT1-MMP C3 cell lines incubated for 48 h in either TIMP-2 or ALA + TIMP-2 CM diluted 1:10, or ALA + TIMP2/U0126 (10 μm)

Article Snippet: Human MT1-MMP (sc116990), TIMP-2 (sc118083) and MMP-2 (sc321560) cDNA clones were purchased from Origene and subcloned into the vector pcDNA 3.3 (Thermo Fisher).

Techniques: Migration, Activation Assay, Incubation, Derivative Assay, Stable Transfection, shRNA, Construct, Expressing, Transwell Migration Assay

Journal: Molecular Cancer

Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

doi: 10.1186/s12943-016-0547-x

Figure Lengend Snippet: Low levels of MT1-MMP expression increased survivability of MCF-7 breast cancer cells to serum-free stress. a Viability of MCF-7 MT1-MMP cell lines during incubation in media containing 10 % FBS ( top ) or serum free media ( bottom ) measured daily for 7 days using Celltiter96®. b Viability of MCF-7 MT1-MMP cell lines and C3 cell line variants during incubation in SF media measured every 3 days for 9 days. Immunoblot analysis ( bottom ) shows PH3 protein levels collected from MCF-7 MT1-MMP cell lines and C3 cell line variants incubated in SF media for 6 days. β-actin was used as a loading control. Bar graph ( right ) shows densitometry analysis of PH3 levels. c Viability of MCF-7 MT1-MMP cell lines was measured after incubation for 6 days in SF media containing increasing concentrations of U0126, BB94, a furin inhibitor or an AKT inhibitor. Black asterisks show statistically significant differences between the initial and day 6 viability within cell lines, and also differences between the day 6 viability of MCF-7 cells compared to MT1-MMP expressing cell lines. Red asterisks indicate significant differences ( p ≤ 0.05) within cell lines between the day 6 viability in SF media compared to the viability after 6 days of incubation with the different concentrations of the inhibitors

Article Snippet: Human MT1-MMP (sc116990), TIMP-2 (sc118083) and MMP-2 (sc321560) cDNA clones were purchased from Origene and subcloned into the vector pcDNA 3.3 (Thermo Fisher).

Techniques: Expressing, Incubation, Western Blot

Journal: Molecular Cancer

Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

doi: 10.1186/s12943-016-0547-x

Figure Lengend Snippet: MT1-MMP activity is inversely correlated to the migratory potential of MCF-7 breast cancer cells ( a ) MCF-7 MT1-MMP cell lines stably expressing zsGreen were seeded on Alexa594-gelatin coated coverlips in media containing 0.1 % DMSO (control) or 10 μm BB94 and incubated in a live imaging chamber. Each sample was imaged at the same five stage positions every 10 mins for 20 h to visualize zsGreen cell movement and associated ECM degradation (Additional files 3, 4, 5 and 6). Shown are stills of the Alexa594 gelatin channel and an overlay including the zsGreen cells at time 0 and 20 h post-seeding of the control sample (BB94 not shown). Scale bars = 100 μm. b Time-lapse videos from ( a ) were analyzed using the ADAPT plugin for ImageJ and all individual cells tracked from each cell line were examined and grouped according to their migration distance from initial point of tracking. c Percentage of cells per field of view from each cell line that degraded the underlying AlexaFluour594-gelatin at five different time points

Article Snippet: Human MT1-MMP (sc116990), TIMP-2 (sc118083) and MMP-2 (sc321560) cDNA clones were purchased from Origene and subcloned into the vector pcDNA 3.3 (Thermo Fisher).

Techniques: Activity Assay, Stable Transfection, Expressing, Incubation, Imaging, Migration

Journal: Molecular Cancer

Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

doi: 10.1186/s12943-016-0547-x

Figure Lengend Snippet: MT1-MMP expression does not correlate with increased migratory potential of breast cancer cells. a qPCR analysis of MT1-MMP mRNA levels from MCF-7, MDA-MB 231, and HS578t breast cancer cells. b Immunoblot (AB51074) and reverse zymography analysis comparing MT1-MMP, phospho-ERK and TIMP-2 protein levels between MCF-7, MDA-MB 231, and HS578t breast cancer cells. β-actin and total ERK1/2 were used as loading controls. c Gelatin zymography analysis of MCF-7, MDA-MB 231, and HS578t breast cancer cells incubated in SF media for 12 h. Lane 1 shows proMMP-2 CM activated by MCF-7 C2 cells treated with TIMP-2 CM diluted 1:100 to show proMMP-2 activation as a result of TIMP-2/MT1-MMP. d Transwell migration assay of MCF-7, MDA-MB 231, and HS578t breast cancer cells incubated in SF media for 24 h

Article Snippet: Human MT1-MMP (sc116990), TIMP-2 (sc118083) and MMP-2 (sc321560) cDNA clones were purchased from Origene and subcloned into the vector pcDNA 3.3 (Thermo Fisher).

Techniques: Expressing, Western Blot, Zymography, Incubation, Activation Assay, Transwell Migration Assay

Journal: Molecular Cancer

Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

doi: 10.1186/s12943-016-0547-x

Figure Lengend Snippet: Overexpression of MT1-MMP in MDA-MB 231 cells negatively affected migration and viability ( a ) Immunoblot analysis (AB6004) showing pro-, active, and degradation forms of MT1-MMP in MDA-MB 231 breast cancer cells and three MDA-MB 231 cell lines expressing different levels of MT1-MMP . β-actin was used as a loading control. b Transwell migration assay of MDA-MB 231 MT1-MMP cells incubated in SF media for 12 h. c Viability of MDA-MB 231 MT1-MMP cell lines during incubation in media containing 10 % FBS ( top ) or serum free media ( bottom ) measured daily for 7 days using Celltiter96®

Article Snippet: Human MT1-MMP (sc116990), TIMP-2 (sc118083) and MMP-2 (sc321560) cDNA clones were purchased from Origene and subcloned into the vector pcDNA 3.3 (Thermo Fisher).

Techniques: Over Expression, Migration, Western Blot, Expressing, Transwell Migration Assay, Incubation

Journal: Molecular Cancer

Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

doi: 10.1186/s12943-016-0547-x

Figure Lengend Snippet: MT1-MMP overexpression in MCF-7 cells induced loss of colony organization and was inversely correlated with a protrusive morphology in 3D culture. a ( top ) MCF-7 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown is a representative field of view of each cell line at day 5, and indicated inset images which show the cell features quantified: Circular colonies ( white arrow ), disseminations around colonies ( green arrow ), or protrusions emanating from colonies ( red arrows ). Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MCF-7 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. White arrow show circular colonies. Green arrows displays single cells that disseminated from the nearby colonies and show MT1-MMP protein. Red arrow shows an F-actin protrusion emanating from a circular colony ( c ) Single cells, F-actin disseminations, F-actin protrusions, and zsGreen protrusions were quantified from 20× magnification 3D volumes acquired after MCF-7 MT1-MMP cell lines stably expressing zsGreen were embedded in Matrigel for 5 days. The 60× magnification 3D volume of MT1-MMP C3 cells shows both F-actin ( red arrow ) and zsGreen ( blue arrow ) protrusions emerging from a colony. Scale bars = 100 μm

Article Snippet: Human MT1-MMP (sc116990), TIMP-2 (sc118083) and MMP-2 (sc321560) cDNA clones were purchased from Origene and subcloned into the vector pcDNA 3.3 (Thermo Fisher).

Techniques: Over Expression, Immunofluorescence, Confocal Microscopy, Stable Transfection, Expressing

Journal: Molecular Cancer

Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

doi: 10.1186/s12943-016-0547-x

Figure Lengend Snippet: MT1-MMP overexpression inhibited the protrusive morphology of MDA-MB 231 breast cancer cells in 3D culture. a ( top ) MDA-MB 231 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown are representative fields of view of each cell line at day 5 and a respective inset. Red arrow shows a portion of the protrusive network MDA-MB 231 cells form in 3D culture. White arrows show MDA-MB 231 MT1-MMP cell colonies that have retained circularity after 5 days in 3D culture. Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MDA-MB 231 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× magnification and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. Red arrow shows protrusive MDA-MB 231 cells, whereas green arrows show circular colonies in MDA-MB 231 MT1-MMP cell lines that are positive for MT1-MMP protein signal. Scale bars = 100 μm

Article Snippet: Human MT1-MMP (sc116990), TIMP-2 (sc118083) and MMP-2 (sc321560) cDNA clones were purchased from Origene and subcloned into the vector pcDNA 3.3 (Thermo Fisher).

Techniques: Over Expression, Immunofluorescence, Confocal Microscopy

Journal: Molecular Cancer

Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

doi: 10.1186/s12943-016-0547-x

Figure Lengend Snippet: MCF-7 MT1-MMP C3 cells displayed high tumorigenic potential when implanted onto the avian embryo CAM. MCF-7 MT1-MMP cell lines and MDA-MB 231 cells stably expressing zsGreen were implanted into the CAM of day 9 ex ovo chicken embryos and visualized 8 days post –implantation using a fluorescence stereoscope to analyze tumor vascularization. Displayed are representative bright field images showing the area of implantation on the embryo, and respective fluorescent images showing the zsGreen channel. The white boxes outline the insets showing vascularization of the MT1-MMP C3 and MDA-MB 231 tumours. Bar graph shows percentage of tumours that were vascularized ( N ≥ 11). Scale bars = 2 mm

Article Snippet: Human MT1-MMP (sc116990), TIMP-2 (sc118083) and MMP-2 (sc321560) cDNA clones were purchased from Origene and subcloned into the vector pcDNA 3.3 (Thermo Fisher).

Techniques: Stable Transfection, Expressing, Fluorescence

Journal: Molecular Cancer

Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

doi: 10.1186/s12943-016-0547-x

Figure Lengend Snippet: MT1-MMP expression was inversely correlated to the extravasation efficiency of MCF-7 breast cancer cells in vivo. a Representative 3D volume views at 20× magnification of MCF-7 MT1-MMP cells stably expressing zsGreen 24 h-post intravenous injection into the chicken embryo CAM vasculature. Shown is an overlay displaying the zsGreen cells ( green ) and CAM vasculature and underlying stromal vessels labeled using lectin-rhodamine ( red ), and the isolated zsGreen channel. Scale bars = 100 μm. Bar graph shows quantification of extravasation efficiency of MCF-7 MT1-MMP cell lines 24 h post-injection. b Orthogonal views of Z-stacks acquired using confocal microscopy at 60× of MT1-MMP C1 and C3 cells 24 h post-injection showing the top of the CAM capillary bed ( top ) and the underlying stroma ( bottom ). Extravasated MT1-MMP C1 cells display loss of cell fragments ( green arrows ) and membrane blebbing ( white arrow ), whereas MT1-MMP C3 cells extravasate to below the CAM with uniform morphology ( blue arrows ) and are capable of forming invasive protrusions in the stroma ( red arrow ). Scale bars = 100 μm

Article Snippet: Human MT1-MMP (sc116990), TIMP-2 (sc118083) and MMP-2 (sc321560) cDNA clones were purchased from Origene and subcloned into the vector pcDNA 3.3 (Thermo Fisher).

Techniques: Expressing, In Vivo, Stable Transfection, Injection, Labeling, Isolation, Confocal Microscopy

Journal: Molecular Cancer

Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

doi: 10.1186/s12943-016-0547-x

Figure Lengend Snippet: Metastatic human 21 T breast cancer cells showed undetectable levels of MT1-MMP protein similar to MCF-7 C3 cells. Protein lysate from human 21 T breast cancer cell lines, which represent a progression series from atypical ductal hyperplasia (21PT-ADH), to ductal carcinoma in situ (21NT – DCIS), to invasive mammary carcinoma (21MT-1- IMC), were analyzed via immunoblot for MT1-MMP protein levels along with the MCF-7 MT1-MMP cell lines. The blots were probed with either AB6004 ( top ) or AB51074 ( bottom ) and shown as the normal exposure and as transformed versions to clearly show banding pattern. Asterisks indicate MT1-MMP isoforms (green – pro form, red- active form, orange – degradation forms). β-actin was used as a loading control

Article Snippet: Human MT1-MMP (sc116990), TIMP-2 (sc118083) and MMP-2 (sc321560) cDNA clones were purchased from Origene and subcloned into the vector pcDNA 3.3 (Thermo Fisher).

Techniques: In Situ, Western Blot, Transformation Assay

Journal: Molecular Cancer

Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

doi: 10.1186/s12943-016-0547-x

Figure Lengend Snippet: Schematic overview of MT1-MMP expression levels and associated changes in substrate degradation and cell migration in 2D culture, phenotypes in 3D culture, and tumourigenesis in vivo. Schematic representation of the findings of this study showing cell phenotypes across 2D and 3D culture platforms and in vivo. Legend describing molecular components in diagrams is shown at the top, and fold change relative to MCF-7 parental cells is in the brackets to the right of the bolded titles. MT1-MMP deficient breast cancer cells, such as MCF-7 cells, are incapable of proMMP-2 activation or ECM degradation, and show low migration and viability during serum-free incubation. These cells retain a circular morphology in 3D culture, and do not form vascularized tumours nor display high extravasation efficiency in vivo. Cells expressing high levels of MT1-MMP are capable of proMMP-2 activation and widespread ECM degradation, have increased survivability to serum-free stress, but do not demonstrate increased migration in 2D experiments. In 3D culture, these cells demonstrate a dissemination morphology and cell fragment release mediated by MT1-MMP. Despite MT1-MMP protein production and associated substrate degradation, these cells are unable to form vascularized tumours or increase their extravasation efficiency in vivo. Cells expressing low levels of MT1-MMP do not demonstrate proMMP-2 activation or widespread ECM degradation, but do show increased migratory potential, and high viability during serum-free incubation. These cells demonstrate a protrusive morphology in 3D culture, form vascularized tumours in vivo, and have significantly increased extravasation efficiency. Data figures within this study that correspond to the diagrams within this model are in red text

Article Snippet: Human MT1-MMP (sc116990), TIMP-2 (sc118083) and MMP-2 (sc321560) cDNA clones were purchased from Origene and subcloned into the vector pcDNA 3.3 (Thermo Fisher).

Techniques: Expressing, Migration, In Vivo, Activation Assay, Incubation