Compensation mechanism in tumor cell migration methods
Aim. Evidence-backed execution summary for Compensation mechanism in tumor cell migration methods from Compensation mechanism in tumor cell migration.
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
Biological model pending
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Inhibition of collagenolysis by protease inhibitor cocktail
reagent used in the protocol.
- Use
- Pericellular degradation of native or denatured collagens can be directly or indirectly provided by proteases from different classes, including MMPs, serine proteases (e.g., plasmin and urokinase-type plasminogen activator [uPA]), as well as cysteine and aspartic proteases (e.g., cathepsins) (;;; ). In addition t...
Inhibition of collagenolysis by protease inhibitor cocktail
reagent used in the protocol.
- Use
- Protease expression in HT-1080/MT1 cells and inhibition of collagenolysis. (A) mRNA expression of MMPs, ADAMs, cathepsins, and serine proteases detected by RT-PCR. Asterisks, proteases cleaving native type I collagen. (B) Degradation of 3D fibrillar collagen by HT1080/MT1 cells (500,000 cells) layered on top of a 1-...
Inhibition of collagenolysis by protease inhibitor cocktail
reagent used in the protocol.
- Use
- Guidelines for the clinical use of marimastat were from British Biotech Inc. (1998).
Inhibition of collagenolysis by protease inhibitor cocktail
reagent used in the protocol.
- Use
- Highest nontoxic concentration (for details see Materials and methods).
Inhibition of collagenolysis by protease inhibitor cocktail
reagent used in the protocol.
- Use
- Unexpectedly, although inhibition of collagenolysis by protease inhibitor cocktail was near complete, the migration efficiency of HT-1080/MT1 cells within 3D collagen lattices was barely reduced ( ). Persistent migration was monitored by sensitive real-time analysis of time-dependent population speed ( A) and the me...
Inhibition of collagenolysis by protease inhibitor cocktail
reagent used in the protocol.
- Use
- Sustained migration in the presence of protease inhibitor cocktail. (A) Cell tracking analysis of the steady-state population speed ± SD and (B) mean speed for each individual cell (time, 20 h; n = 3 experiments, 120 cells).
Induced amoeboid migration in vitro
reagent used in the protocol.
- Use
- As it became apparent from the video recordings (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200209006/DC1 ), the presence of protease inhibitor cocktail changed several aspects of migratory behavior in HT-1080/MT1 cells ( ). Whereas spontaneously moving cells maintained a spindle-shaped, elongated...
Induced amoeboid migration in vitro
reagent used in the protocol.
- Use
- Transition of spindle-shaped (mesenchymal) to more spherical (amoeboid) migration in HT1080/MT1 and MDA-MB-231 cells in the presence of protease inhibitor cocktail. (A) Conversion of elongated (left) toward spherical shape (right) in HT1080/MT1 cells, and (B) higher magnification of an amoeboid migrating cell in the...
Inhibition of collagenolysis by protease inhibitor cocktail
Sustained migration in the presence of protease inhibitor cocktail. (A) Cell tracking analysis of the steady-state population speed ± SD and (B) mean speed for each individual cell (time, 20 h; n = 3 experiments, 120 cells).
- Use
- Sustained migration in the presence of protease inhibitor cocktail. (A) Cell tracking analysis of the steady-state population speed ± SD and (B) mean speed for each individual cell (time, 20 h; n = 3 experiments, 120 cells).
Induced amoeboid migration in vitro
As it became apparent from the video recordings (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200209006/DC1 ), the presence of protease inhibitor cocktail changed several aspects of migratory behavior in HT-1080/MT1 cells ( ). Whereas spontaneously moving cells maintained a spindle-shaped, elongated...
- Use
- As it became apparent from the video recordings (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200209006/DC1 ), the presence of protease inhibitor cocktail changed several aspects of migratory behavior in HT-1080/MT1 cells ( ). Whereas spontaneously moving cells maintained a spindle-shaped, elongated...
Mechanisms of induced nonproteolytic migration
Consistent with impaired collagenolysis, amoeboid moving HT-1080/MT1 cells did not cause structural remodeling of collagen fibers (Video 6, available online at http://www.jcb.org/cgi/content/full/jcb.200209006/DC1 ). Induced protease-independent migration resulted from adaptation and alignment of the cell body along...
- Use
- Consistent with impaired collagenolysis, amoeboid moving HT-1080/MT1 cells did not cause structural remodeling of collagen fibers (Video 6, available online at http://www.jcb.org/cgi/content/full/jcb.200209006/DC1 ). Induced protease-independent migration resulted from adaptation and alignment of the cell body along...
Mesenchymal-amoeboid transition in vivo
Although collagen lattices provide a complex 3D ECM scaffold and a barrier for moving cells, a putatively different spacing and molecular composition of life connective tissue may impose additional physical and molecular constraints, putatively yielding in distinct migration mechanisms. Therefore, the in vivo migrat...
- Use
- Although collagen lattices provide a complex 3D ECM scaffold and a barrier for moving cells, a putatively different spacing and molecular composition of life connective tissue may impose additional physical and molecular constraints, putatively yielding in distinct migration mechanisms. Therefore, the in vivo migrat...
Materials and methods
Subconfluent HT-1080 fibrosarcoma cells stably transfected with MT1-MMP ( ) or MDA-MB-231 breast carcinoma cells (; ) were detached by EDTA (2 mM), washed, incorporated into 3-D collagen lattices (1.67 mg/ml; native dermal bovine type I collagen; Vitrogen; Cohesion Inc.), and monitored by time-lapse video microscop...
- Use
- Subconfluent HT-1080 fibrosarcoma cells stably transfected with MT1-MMP ( ) or MDA-MB-231 breast carcinoma cells (; ) were detached by EDTA (2 mM), washed, incorporated into 3-D collagen lattices (1.67 mg/ml; native dermal bovine type I collagen; Vitrogen; Cohesion Inc.), and monitored by time-lapse video microscop...
Materials and methods
For 3D time-lapse confocal microscopy (Leica-SP2 system), cells within the lattice were labeled by calcein-AM (1 µM), scanned at 2.5-min time intervals for simultaneous fluorescence and backscatter signal, and reconstructed ( ). MT1-MMP and β1 integrins were detected by rabbit AB815 (Chemicon), secondary r...
- Use
- For 3D time-lapse confocal microscopy (Leica-SP2 system), cells within the lattice were labeled by calcein-AM (1 µM), scanned at 2.5-min time intervals for simultaneous fluorescence and backscatter signal, and reconstructed ( ). MT1-MMP and β1 integrins were detected by rabbit AB815 (Chemicon), secondary r...
Computer-assisted cell tracking and reconstruction of cell morphology
Locomotor parameters were obtained by computer-assisted cell tracking and reconstruction of the x and y coordinates of the cell paths ( ). The population speed was calculated from each 15-min tracking interval for all cells divided by the number of cells. Single cell speed represents the total length of the path div...
- Use
- Locomotor parameters were obtained by computer-assisted cell tracking and reconstruction of the x and y coordinates of the cell paths ( ). The population speed was calculated from each 15-min tracking interval for all cells divided by the number of cells. Single cell speed represents the total length of the path div...
Intravital multiphoton microscopy
Reconstruction of HT1080/MT1 cell positioning and ECM structure were monitored by multiphoton microscopy ( ) of the mouse dermis in adult C57BL/6 mice, using a novel modification of a bone marrow intravital video microscopy model ( ). The epidermis and upper dermis, including hair follicles of the frontoparietal sca...
- Use
- Reconstruction of HT1080/MT1 cell positioning and ECM structure were monitored by multiphoton microscopy ( ) of the mouse dermis in adult C57BL/6 mice, using a novel modification of a bone marrow intravital video microscopy model ( ). The epidermis and upper dermis, including hair follicles of the frontoparietal sca...
Before you run
What should be confirmed before execution?
First confirmation
Species or subject information is missing.
Confirm before execution
Equipment is listed but no product mappings are linked.
Confirm before execution
This page is backed by a publishable Replication Data Ledger package with zero critical source-verification issues.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
Inhibition of collagenolysis by protease inhibitor cocktail
Pericellular degradation of native or denatured collagens can be directly or indirectly provided by proteases from different classes, including MMPs, serine proteases (e.g., plasmin and urokinase-type plasminogen activator [uPA]), as well as cysteine and aspartic proteases (e.g., cathepsins) (;;; ). In addition to MT1-MMP, many of these proteases, including up to five other collagenases toward native type collagen ( A, asterisks), were expressed by HT-1080/MT1 cells, as detected by RT-PCR. Because collagenolytic redundancy was anticipated, we used a cocktail of broad-spectrum protease inhibitors to simultaneously target a wide spectrum of endoproteolytic activity ( ). To maintain sufficient inhibitory activity within the matrix, the employed concentrations of protease inhibitors were orders of magnitudes higher than the known maximum inhibitory values, unless cell viability was dos...
Induced amoeboid migration in vitro
Transition of spindle-shaped (mesenchymal) to more spherical (amoeboid) migration in HT1080/MT1 and MDA-MB-231 cells in the presence of protease inhibitor cocktail. (A) Conversion of elongated (left) toward spherical shape (right) in HT1080/MT1 cells, and (B) higher magnification of an amoeboid migrating cell in the presence of inhibitor cocktail. Time in B, 117 min. (C) Median elongation (calculated from length divided by width) in the absence and presence of protease inhibitor cocktail ( n = 3; 170 cells; ***, P < 0.0001). (D) Inhibition of collagen degradation by MDA-MB-231 cells by protease inhibitor cocktail ( n = 3; P < 0.05, unpaired two-tailed t test). (E) Conversion from spindle shaped (left) to more spherical morphology (right), and (F) reduced median elongation in the presence of protease inhibitors in MDA-MB-231 cells ( n = 3; 200 cells; ***, P < 0.0001). (G) Frequency of...
Mechanisms of induced nonproteolytic migration
Cellular mechanism of nonproteolytic movement within 3D collagen matrix and related changes in β 1 integrin, MT1-MMP, and F-actin distribution in HT-1080/MT1 cells. (A) Induced amoeboid migration lacking fiber degradation. Alignment of cell body along a fiber strand (white arrowheads) and intact individual collagen fiber at its original position after cell detachment (black arrowhead). (B) Migratory alignment of the cell depicted in A along the preexisting fiber scaffold. The outline of the cell edge at 2.5-min time intervals (blue lines) was superimposed onto the 3D reconstruction of the transmigrated matrix structure. Bright pixels indicate colocalization of cell boundary and fibers (arrowheads). (C) Migration through a narrow gap bordered by fibers (black arrowhead) resulting in morphological adaptation and the formation of a constriction ring. (D) Reduced F-actin and β1...
Mesenchymal-amoeboid transition in vivo
Although collagen lattices provide a complex 3D ECM scaffold and a barrier for moving cells, a putatively different spacing and molecular composition of life connective tissue may impose additional physical and molecular constraints, putatively yielding in distinct migration mechanisms. Therefore, the in vivo migration of HT-1080/MT1 cells within the mouse dermis was investigated by multiphoton microscopy. 3 h after the injection of HT-1080/MT1 cells into the loose connective tissue of the mouse dermis ( A), both nontreated control cells (green) and cells pretreated with protease inhibitor cocktail (red) were detected at and passively scattered around the injection site ( A, asterisk), or located within multicellular cords ( A, black arrowheads). In wash-out experiments in vitro, preincubation of cells with protease inhibitor cocktail for 6 h resulted in stable amoeboid movement for a...
Materials and methods
Subconfluent HT-1080 fibrosarcoma cells stably transfected with MT1-MMP ( ) or MDA-MB-231 breast carcinoma cells (; ) were detached by EDTA (2 mM), washed, incorporated into 3-D collagen lattices (1.67 mg/ml; native dermal bovine type I collagen; Vitrogen; Cohesion Inc.), and monitored by time-lapse video microscopy (; ). The collagen was resistant to trypsin and sensitive to degradation by MT1-MMP (not depicted), confirming its native state. HT-1080/MT1 cells served as model for high constitutive collagenase expression and activity, allowing a robust visualization of surface MT1-MMP and fiber breakdown in the process of migration. In control experiments, HT1080/ neo cells as well as HT-1080 wild-type cells were used for collagen degradation and migration experiments yielding similar results, including MAT (not depicted). For inhibition studies, blocking anti-β1 integrin...
Intravital multiphoton microscopy
Reconstruction of HT1080/MT1 cell positioning and ECM structure were monitored by multiphoton microscopy ( ) of the mouse dermis in adult C57BL/6 mice, using a novel modification of a bone marrow intravital video microscopy model ( ). The epidermis and upper dermis, including hair follicles of the frontoparietal scalp, were removed by careful separation from the underlying connective tissue using microscissors. A fixation ring was inserted into the incision to spread the skin, and Hepes-buffered medium containing 10% fetal bovine serum was applied. Subconfluent HT-1080/MT1 cells were preincubated for 3-4 h in protease inhibitor cocktail (each 20 µM BB-2516, pepstatin A, E-64, 0.7 µM aprotinin, 2 µM leupeptin) or medium alone. Control cells and inhibitor cocktail-treated cells were labeled with calcein-AM (green) or TRITC (red), respectively, washed twice, su...
Measurement outputs
What raw and processed outputs should exist?
Pericellular degradation of native or denatured collagens can be directly or indirectly provided by proteases from different classes, including MMPs, serine proteases (e.g., pla...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Protease expression in HT-1080/MT1 cells and inhibition of collagenolysis. (A) mRNA expression of MMPs, ADAMs, cathepsins, and serine proteases detected by RT-PCR. Asterisks, pr...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Unexpectedly, although inhibition of collagenolysis by protease inhibitor cocktail was near complete, the migration efficiency of HT-1080/MT1 cells within 3D collagen lattices w...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Sustained migration in the presence of protease inhibitor cocktail. (A) Cell tracking analysis of the steady-state population speed ± SD and (B) mean speed for each individ...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
As it became apparent from the video recordings (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200209006/DC1 ), the presence of protease inhibitor cocktail changed several aspects of migratory behavior in HT-1080/MT1 cells ( ).
from paperScoring or quantification
Quantify the primary readouts for this experiment: Pericellular degradation of native or denatured collagens can be directly or indirectly provided by proteases from different classes, including MMPs, serine proteases (e.g., pla...; Protease expression in HT-1080/MT1 cells and inhibition of collagenolysis. (A) mRNA expression of MMPs, ADAMs, cathepsins, and serine proteases detected by RT-PCR. Asterisks, pr...; Unexpectedly, although inhibition of collagenolysis by protease inhibitor cocktail was near complete, the migration efficiency of HT-1080/MT1 cells within 3D collagen lattices w...; Sustained migration in the presence of protease inhibitor cocktail. (A) Cell tracking analysis of the steady-state population speed ± SD and (B) mean speed for each individ....
from paperStatistical comparison
As it became apparent from the video recordings (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200209006/DC1 ), the presence of protease inhibitor cocktail chang...; Inhibition of constitutive proteolysis, instead of causing the cells to become "trapped" within the fibrillar network, induced a program of morphodynamic and molecul...; Locomotor parameters were obtained by computer-assisted cell tracking and reconstruction of the x and y coordinates of the cell paths ( ). The population speed was calculated fr...
from paperReporting output
Report representative outputs alongside summary comparisons for Pericellular degradation of native or denatured collagens can be directly or indirectly provided by proteases from different classes, including MMPs, serine proteases (e.g., pla..., Protease expression in HT-1080/MT1 cells and inhibition of collagenolysis. (A) mRNA expression of MMPs, ADAMs, cathepsins, and serine proteases detected by RT-PCR. Asterisks, pr..., Unexpectedly, although inhibition of collagenolysis by protease inhibitor cocktail was near complete, the migration efficiency of HT-1080/MT1 cells within 3D collagen lattices w..., Sustained migration in the presence of protease inhibitor cocktail. (A) Cell tracking analysis of the steady-state population speed ± SD and (B) mean speed for each individ....
inferred from protocolStructured statistical methods
As it became apparent from the video recordings (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200209006/DC1 ), the presence of protease inhibitor cocktail chang...; Inhibition of constitutive proteolysis, instead of causing the cells to become "trapped" within the fibrillar network, induced a program of morphodynamic and molecul...; Locomotor parameters were obtained by computer-assisted cell tracking and reconstruction of the x and y coordinates of the cell paths ( ). The population speed was calculated fr...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (6)
Pericellular degradation of native or denatured collagens can be directly or indirectly provided by proteases from different classes, including MMPs, serine proteases (e.g., plasmin and urokinase-type plasminogen activator [uPA]), as well as cysteine and aspartic proteases (e.g., cathepsins) (;;; ). In addition to MT1-MMP, many of these proteases, including up to five other collagenases toward native type collagen ( A, asterisks), were expressed by HT-1080/MT1 cells, as detected by RT-PCR. Because collagenolytic redundancy was anticipated, we used a cocktail of broad-spectrum protease inhibitors to simultaneously target a wide spectrum of endoproteolytic activity ( ). To maintain sufficient inhibitory activity within the matrix, the employed concentrations of protease inhibitors were orders of magnitudes higher than the known maximum inhibitory values, unless cell viability was dose limiting ( ). Because most of the proteases detectable by RT-PCR escape visualization by zymography (,; ), inhibition of cell-dependent collagenolysis in situ was measured using the fibrillar collagen migration substrate as read-out. On a qualitative basis, structural breakdown of the matrix fib...
Transition of spindle-shaped (mesenchymal) to more spherical (amoeboid) migration in HT1080/MT1 and MDA-MB-231 cells in the presence of protease inhibitor cocktail. (A) Conversion of elongated (left) toward spherical shape (right) in HT1080/MT1 cells, and (B) higher magnification of an amoeboid migrating cell in the presence of inhibitor cocktail. Time in B, 117 min. (C) Median elongation (calculated from length divided by width) in the absence and presence of protease inhibitor cocktail ( n = 3; 170 cells; ***, P < 0.0001). (D) Inhibition of collagen degradation by MDA-MB-231 cells by protease inhibitor cocktail ( n = 3; P < 0.05, unpaired two-tailed t test). (E) Conversion from spindle shaped (left) to more spherical morphology (right), and (F) reduced median elongation in the presence of protease inhibitors in MDA-MB-231 cells ( n = 3; 200 cells; ***, P < 0.0001). (G) Frequency of mesenchymal and amoeboid shape in actually migrating cells in the absence (▪) and presence (□) of protease inhibitor cocktail (HT-1080/MT1 cells, n = 3, 100 cells; **, P < 0.001 for difference to untreated control; two-tailed t test for independent means). Cells of indeterminate morpholo...
Cellular mechanism of nonproteolytic movement within 3D collagen matrix and related changes in β 1 integrin, MT1-MMP, and F-actin distribution in HT-1080/MT1 cells. (A) Induced amoeboid migration lacking fiber degradation. Alignment of cell body along a fiber strand (white arrowheads) and intact individual collagen fiber at its original position after cell detachment (black arrowhead). (B) Migratory alignment of the cell depicted in A along the preexisting fiber scaffold. The outline of the cell edge at 2.5-min time intervals (blue lines) was superimposed onto the 3D reconstruction of the transmigrated matrix structure. Bright pixels indicate colocalization of cell boundary and fibers (arrowheads). (C) Migration through a narrow gap bordered by fibers (black arrowhead) resulting in morphological adaptation and the formation of a constriction ring. (D) Reduced F-actin and β1 integrin focalization at fiber binding sites in an amoeboid HT1080/MT1 cell, compared with F and Fig. 1 C. Because of constriction caused by a perpendicular collagen fiber, this cell contains a lobulated main body. Black arrowhead, uropod. (E) Loss of clustered MT1-MMP and β1 integrins from int...
Although collagen lattices provide a complex 3D ECM scaffold and a barrier for moving cells, a putatively different spacing and molecular composition of life connective tissue may impose additional physical and molecular constraints, putatively yielding in distinct migration mechanisms. Therefore, the in vivo migration of HT-1080/MT1 cells within the mouse dermis was investigated by multiphoton microscopy. 3 h after the injection of HT-1080/MT1 cells into the loose connective tissue of the mouse dermis ( A), both nontreated control cells (green) and cells pretreated with protease inhibitor cocktail (red) were detected at and passively scattered around the injection site ( A, asterisk), or located within multicellular cords ( A, black arrowheads). In wash-out experiments in vitro, preincubation of cells with protease inhibitor cocktail for 6 h resulted in stable amoeboid movement for at least 10 h before reversion toward mesenchymal migration occurred (unpublished data), indicating both relatively slow turnover of the target proteases after inhibition and full reversibility of inhibitor-induced phenotypic change. In the dermis, ortotopic 3D reconstruction of the injection site 3...
Subconfluent HT-1080 fibrosarcoma cells stably transfected with MT1-MMP ( ) or MDA-MB-231 breast carcinoma cells (; ) were detached by EDTA (2 mM), washed, incorporated into 3-D collagen lattices (1.67 mg/ml; native dermal bovine type I collagen; Vitrogen; Cohesion Inc.), and monitored by time-lapse video microscopy (; ). The collagen was resistant to trypsin and sensitive to degradation by MT1-MMP (not depicted), confirming its native state. HT-1080/MT1 cells served as model for high constitutive collagenase expression and activity, allowing a robust visualization of surface MT1-MMP and fiber breakdown in the process of migration. In control experiments, HT1080/ neo cells as well as HT-1080 wild-type cells were used for collagen degradation and migration experiments yielding similar results, including MAT (not depicted). For inhibition studies, blocking anti-β1 integrin mAb 4B4 (10 µg/ml; Coulter) or protease inhibitor cocktail ( ) was added to the lattice before polymerization as well as to the supernatant. Protease inhibitor cocktail consisted of BB-2516 (marimastat; provided by British Biotech Inc.), leupeptin (Molecular Probes), pepstatin A, E-64, and apro...
Reconstruction of HT1080/MT1 cell positioning and ECM structure were monitored by multiphoton microscopy ( ) of the mouse dermis in adult C57BL/6 mice, using a novel modification of a bone marrow intravital video microscopy model ( ). The epidermis and upper dermis, including hair follicles of the frontoparietal scalp, were removed by careful separation from the underlying connective tissue using microscissors. A fixation ring was inserted into the incision to spread the skin, and Hepes-buffered medium containing 10% fetal bovine serum was applied. Subconfluent HT-1080/MT1 cells were preincubated for 3-4 h in protease inhibitor cocktail (each 20 µM BB-2516, pepstatin A, E-64, 0.7 µM aprotinin, 2 µM leupeptin) or medium alone. Control cells and inhibitor cocktail-treated cells were labeled with calcein-AM (green) or TRITC (red), respectively, washed twice, suspended together in medium (10 4 cells/3 µl each), and carefully injected into the mid-dermis over the immobilized mouse scull. After injection, a temperature of 37°C was maintained by a water circulation system through the fixation ring and the use of a tempered water immersion objective....
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Compensation mechanism in tumor cell migration methods",
"description": "Evidence-backed execution summary for Compensation mechanism in tumor cell migration methods from Compensation mechanism in tumor cell migration.",
"totalTime": "PT152M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Inhibition of collagenolysis by protease inhibitor cocktail",
"text": "Pericellular degradation of native or denatured collagens can be directly or indirectly provided by proteases from different classes, including MMPs, serine proteases (e.g., plasmin and urokinase-type plasminogen activator [uPA]), as well as cysteine and aspartic proteases (e.g., cathepsins) (;;; ). In addition to MT1-MMP, many of these proteases, including up to five other collagenases toward native type collagen ( A, asterisks), were expressed by HT-1080/MT1 cells, as detected by RT-PCR. Because collagenolytic redundancy was anticipated, we used a cocktail of broad-spectrum protease inhibitors to simultaneously target a wide spectrum of endoproteolytic activity ( ). To maintain sufficient inhibitory activity within the matrix, the employed concentrations of protease inhibitors were orders of magnitudes higher than the known maximum inhibitory values, unless cell viability was dos..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Induced amoeboid migration in vitro",
"text": "Transition of spindle-shaped (mesenchymal) to more spherical (amoeboid) migration in HT1080/MT1 and MDA-MB-231 cells in the presence of protease inhibitor cocktail. (A) Conversion of elongated (left) toward spherical shape (right) in HT1080/MT1 cells, and (B) higher magnification of an amoeboid migrating cell in the presence of inhibitor cocktail. Time in B, 117 min. (C) Median elongation (calculated from length divided by width) in the absence and presence of protease inhibitor cocktail ( n = 3; 170 cells; ***, P < 0.0001). (D) Inhibition of collagen degradation by MDA-MB-231 cells by protease inhibitor cocktail ( n = 3; P < 0.05, unpaired two-tailed t test). (E) Conversion from spindle shaped (left) to more spherical morphology (right), and (F) reduced median elongation in the presence of protease inhibitors in MDA-MB-231 cells ( n = 3; 200 cells; ***, P < 0.0001). (G) Frequency of..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Mechanisms of induced nonproteolytic migration",
"text": "Cellular mechanism of nonproteolytic movement within 3D collagen matrix and related changes in β 1 integrin, MT1-MMP, and F-actin distribution in HT-1080/MT1 cells. (A) Induced amoeboid migration lacking fiber degradation. Alignment of cell body along a fiber strand (white arrowheads) and intact individual collagen fiber at its original position after cell detachment (black arrowhead). (B) Migratory alignment of the cell depicted in A along the preexisting fiber scaffold. The outline of the cell edge at 2.5-min time intervals (blue lines) was superimposed onto the 3D reconstruction of the transmigrated matrix structure. Bright pixels indicate colocalization of cell boundary and fibers (arrowheads). (C) Migration through a narrow gap bordered by fibers (black arrowhead) resulting in morphological adaptation and the formation of a constriction ring. (D) Reduced F-actin and β1..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Mesenchymal-amoeboid transition in vivo",
"text": "Although collagen lattices provide a complex 3D ECM scaffold and a barrier for moving cells, a putatively different spacing and molecular composition of life connective tissue may impose additional physical and molecular constraints, putatively yielding in distinct migration mechanisms. Therefore, the in vivo migration of HT-1080/MT1 cells within the mouse dermis was investigated by multiphoton microscopy. 3 h after the injection of HT-1080/MT1 cells into the loose connective tissue of the mouse dermis ( A), both nontreated control cells (green) and cells pretreated with protease inhibitor cocktail (red) were detected at and passively scattered around the injection site ( A, asterisk), or located within multicellular cords ( A, black arrowheads). In wash-out experiments in vitro, preincubation of cells with protease inhibitor cocktail for 6 h resulted in stable amoeboid movement for a..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Materials and methods",
"text": "Subconfluent HT-1080 fibrosarcoma cells stably transfected with MT1-MMP ( ) or MDA-MB-231 breast carcinoma cells (; ) were detached by EDTA (2 mM), washed, incorporated into 3-D collagen lattices (1.67 mg/ml; native dermal bovine type I collagen; Vitrogen; Cohesion Inc.), and monitored by time-lapse video microscopy (; ). The collagen was resistant to trypsin and sensitive to degradation by MT1-MMP (not depicted), confirming its native state. HT-1080/MT1 cells served as model for high constitutive collagenase expression and activity, allowing a robust visualization of surface MT1-MMP and fiber breakdown in the process of migration. In control experiments, HT1080/ neo cells as well as HT-1080 wild-type cells were used for collagen degradation and migration experiments yielding similar results, including MAT (not depicted). For inhibition studies, blocking anti-β1 integrin..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Intravital multiphoton microscopy",
"text": "Reconstruction of HT1080/MT1 cell positioning and ECM structure were monitored by multiphoton microscopy ( ) of the mouse dermis in adult C57BL/6 mice, using a novel modification of a bone marrow intravital video microscopy model ( ). The epidermis and upper dermis, including hair follicles of the frontoparietal scalp, were removed by careful separation from the underlying connective tissue using microscissors. A fixation ring was inserted into the incision to spread the skin, and Hepes-buffered medium containing 10% fetal bovine serum was applied. Subconfluent HT-1080/MT1 cells were preincubated for 3-4 h in protease inhibitor cocktail (each 20 µM BB-2516, pepstatin A, E-64, 0.7 µM aprotinin, 2 µM leupeptin) or medium alone. Control cells and inhibitor cocktail-treated cells were labeled with calcein-AM (green) or TRITC (red), respectively, washed twice, su..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "Inhibition of collagenolysis by protease inhibitor cocktail"
},
{
"@type": "HowToTool",
"name": "Induced amoeboid migration in vitro"
},
{
"@type": "HowToTool",
"name": "Mechanisms of induced nonproteolytic migration"
},
{
"@type": "HowToTool",
"name": "Mesenchymal-amoeboid transition in vivo"
},
{
"@type": "HowToTool",
"name": "Materials and methods"
},
{
"@type": "HowToTool",
"name": "Materials and methods"
},
{
"@type": "HowToTool",
"name": "Computer-assisted cell tracking and reconstruction of cell morphology"
},
{
"@type": "HowToTool",
"name": "Intravital multiphoton microscopy"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "Inhibition of collagenolysis by protease inhibitor cocktail"
},
{
"@type": "HowToSupply",
"name": "Inhibition of collagenolysis by protease inhibitor cocktail"
},
{
"@type": "HowToSupply",
"name": "Inhibition of collagenolysis by protease inhibitor cocktail"
},
{
"@type": "HowToSupply",
"name": "Inhibition of collagenolysis by protease inhibitor cocktail"
},
{
"@type": "HowToSupply",
"name": "Inhibition of collagenolysis by protease inhibitor cocktail"
},
{
"@type": "HowToSupply",
"name": "Inhibition of collagenolysis by protease inhibitor cocktail"
},
{
"@type": "HowToSupply",
"name": "Induced amoeboid migration in vitro"
},
{
"@type": "HowToSupply",
"name": "Induced amoeboid migration in vitro"
}
],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Compensation mechanism in tumor cell migration",
"datePublished": "2003",
"author": [
{
"@type": "Person",
"name": "Katarina Wolf"
},
{
"@type": "Person",
"name": "Irina Mazo"
},
{
"@type": "Person",
"name": "Harry Leung"
},
{
"@type": "Person",
"name": "Katharina Engelke"
},
{
"@type": "Person",
"name": "Ulrich H. von Andrian"
},
{
"@type": "Person",
"name": "Elena I. Deryugina"
},
{
"@type": "Person",
"name": "Alex Y. Strongin"
},
{
"@type": "Person",
"name": "Eva-B. Bröcker"
},
{
"@type": "Person",
"name": "Peter Friedl"
}
],
"identifier": "10.1083/jcb.200209006"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Compensation mechanism in tumor cell migration methods",
"item": "https://replicatescience.com/experiments/compensation-mechanism-in-tumor-cell-migration-methods-katarina-wolf-pmc2172637/compensation-mechanism-in-tumor-cell-migration-mlph1s1n"
}
]
}
]