Constricted migration (BMES 2019)
Cancer cell metastasis is responsible for 80 percent of cancer-related death. The metastatic cells migrate through tight constricting spaces, therefore constricted migration and its enabling mechanisms have received a lot of attentions in recent years. The most famous research groups that are working in this field are Lammerding group, Discher group, Wolf and Friedl group, Konstantopoulos group, and Reinhart-king group. Except Discher group, the rest of groups have collaborations with each other to a high extent. Most of the studies in this field is focused on the effect of constricted migration on nuclear changes and underlying mechanisms. Here are some of the interesting talks that I attend in BMES:
3D Confinement in Collagen Microtracks Upregulates Motility and Cell Contractility
J Mosier1, A Rahman-Zaman1,2, F Bordeleau1,2,3, M Zanotelli1,2, J VanderBurgh1,2, BD Hoffman4, and CA Reinhart-King1,2 1Vanderbilt University, Nashville, TN, USA 2Cornell University, Ithaca, NY, USA 3Universite Laval, Quebec, Canada, 4Duke University, Durham, NC, USA
They developed a platform with varying degrees of confinement to examine their mode of migration (Mesenchymal vs. ameboid). To mimic the range of stiffness that encountered by cells in vivo; they developed a 3D platform using collagen to create microtracks of varying width (5 to 20 μm width, 15 μm height) to study how confinement alters cell speed, cell and focal adhesion (FA) geometry, substrate strain, and contractility.
Results and Discussion:
| Mesenchymal | Ameboid | |
| Confinement | Partial | Full |
| Cell speed | Lower | Higher |
| Geometry | Alongated | Rounded |
| Focal adhesion | More/smaller | Fewer/Larger |
| Substrate strains | Lower | Higher |
Notes:
Increased cell-matrix contact increased cell migration speed.
Confinement induces changes in cells geometry but not volume or area.
Cells exhibit decreased density of larger vinculin containing adhesions when fully confined.
Questions: I cannot see why partially confined migration is considered necessarily a mesenchymal, and the fully confined is considered ameboid mode. What about other parameters such as protease dependency? Why didn’t cells open their pass into collagen?
Why did they study that specific Rho (Rho activator II)? It is known that each mode of migration is dependent to specific types of Rho family.:::: Ridley, Anne J. “Rho GTPase signalling in cell migration.” Current opinion in cell biology 36 (2015): 103-112.
Figure 1: (A) Migration speed of control and Rho + partially and fully confined cells, (B) area and (C) density of vinculin-containing adhesions in full or partial confinement, (D) fluorescent images of vinculin adhesions in full or partial confinement, (E) images of fluorescent beads when the cell is absent (blue), and when the cell is present (red).
Vimentin Intermediate Filaments Protect the Structural Integrity of the Nucleus and Suppress Nuclear Damage Caused by Large Deformations
Amir Vahabikashi 1, Alison E Patteson 2, Katarzyna Pogoda 3,4, Stephen A Adam 1, Anne Goldman 1, Robert Goldman 1, and Paul Janmey 3,5
1 Department of Cell and Molecular Biology, Northwestern University; 2 Physics Department, Syracuse University; 3 Institute for Medicine and Engineering, University of Pennsylvania; 4 Institute of Nuclear Physics, Polish Academy of Sciences; 5 Department of Physiology, University of Pennsylvania
The mechanical properties of the nucleus are an important factor in regulating cell migration through confined spaces. At the onset of migratory behavior, cells often initiate the expression of vimentin, an intermediate filament protein which assembles into networks extending from a complex juxtanuclear cage to the cell periphery. VIFs are flexible and have “structure hardening” properties. However, the specific role of vimentin intermediate filaments (VIFs) in regulating nuclear shape and mechanics during cell motility is unknown.
Method: They used confocal and super resolution microscopy, Transwell migration assays, and atomic force microscopy (AFM) to show that VIFs regulate nuclear shape and protect against nuclear damage when migrating through constricting spaces.
- Wild type (WT) and vimentin knock out (KO) MEFs were originally obtained from mouse embryos and immortalized. For VIF cage and nuclear shape studies, cultured mouse embryonic fibroblasts (mEFs) on glass cover slips were fixed and stained for vimentin and lamin A.
- For Transwell migration assays, cells were seeded on polycarbonate Transwell membranes. After 18 h cells were fixed and stained for vimentin, DNA (Hoechst), DNA double strand breaks (ƔH2AX), or lamin A/B. Migration rate was determined as the ratio of the number of cells on the bottom of the membrane to the sum of cells on the top and bottom.
- AFM indentations were done over the nucleus of single.
Results and Discussion:
VIFs protect cell and nucleus against compressive forces, therefore:
Depletion of VIFs perturbs nuclear shape/volume and cell spreading/volume.
Loss of VIFs increases nuclear rupture in 2D and 3D. Loss of VIFs increases nuclear bleb and DNA damage.
VIFs hinder migration in pores and limit nuclear deformation.
Migration through pores increases cell aspect ratio. Loss of VIFs helps cells to preserve the aspect ratio for a longer time.
Paper: Patteson, Alison E., Amir Vahabikashi, Katarzyna Pogoda, Stephen A. Adam, Anne Goldman, Robert Goldman, and Paul Janmey. “Vimentin protects the structural integrity of the nucleus and suppresses nuclear damage caused by large deformations.” bioRxiv (2019): 566174.
Electric Field Modulates Cell Phenotype And Migration In A Confined Environment
Soontorn Tuntithavornwat1, Chao Wang1, Konstantinos Konstantopoulos1
1Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore MD, 21218, USA
Endogenous direct current (DC) electric fields are observed as a result of asymmetric distribution of ion fluxes across the cell membrane and along the intra and extracellular environment. Past attempts at studying the influences of DC electric fields on cell migration and have been performed on conventional two-dimensional (2D) surfaces. However, the effects of DC electric fields on cellular morphology and migratory phenotypes in confinement remains to be elucidated.
Application: Understanding how cancer cells migrate through physiologically relevant confined spaces under the influence of electric fields could potentially offer insights into the development of novel therapeutic strategies to control cell migration and ultimately inhibit cancer metastasis.
Methods: A polydimethylsiloxane-based microfluidic device was fabricated using photolithography techniques (WxH=3 μm x 10 μm), and Ag/AgCl electrodes embedded in agarose were used to apply DC electric fields across the microfluidic device.
Results and Discussion: In the absence of DC electric field, HT1080 fibrosarcoma cells possessed higher motility in comparison with the conventional 2D surface platform (Fig. 1D) suggesting that cells may utilize the different mechanism to migrate through the confined track.
In the presence of physiological level of the electric field (0.5 V/cm), fibrosarcoma migrated toward the cathode (Fig. 1C), while MDA-MB-231 breast cancer migrated toward the anode regardless of earlier established polarization
Unlike a conventional 2D surface, we found that the electric field (0.5 V/cm) did not alter the cell motility, but still control the direction of cell migration which can be explained by altered cell morphologies.
Electrical stimulation increases the use of a bleb-based migration mode, induce nuclear blebbing and number of focal adhesions.
EF facilitate ROCK inhibited cell migration and bypass myosin II activity.
Nuclear rupture at sites of high curvature compromises retention of DNA repair factors
Irena L. Ivanovska1,2*, Yuntao Xia1,2*, Kuangzheng Zhu1,2, Charlotte R. Pfeifer1,2, Sangkyun Cho1,2, Roger A. Greenberg1,4, and Dennis E. Discher1,2,3
1Physical Sciences Oncology Center at Penn; 2Molecular and Cell Biophysics Lab; 3Graduate Group, Department
of Physics and Astronomy; 4Cancer Biology, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, PA.
* equal contribution
The nucleus is physically linked to the cytoskeleton, adhesions, and extracellular matrix—all of which sustain forces, but their relationships to DNA damage are obscure. Nuclear rupture is one conceivable mechanism for mislocalization of DNA repair factors and a consequent excess of DNA damage.
Hypothesis: The mentioned process occurs as a result of high nuclear curvature, with rupture frequency increased by both intracellular and extracellular structural factors that include low levels of lamin A, high actomyosin stress, and stiff ECM.
Methods: Nuclei in live U2OS osteosarcoma cells were probed with atomic force microscopy (AFM) tips of either medium or high curvature (4.5-μm sphere or pyramidal tip <0.1-μm diameter) with simultaneous imaging of the mobility of fluorescently tagged factors in the nucleus or cytoplasm.
Results and Discussion:
High-curvature probes rapidly rupture nuclei, especially with low lamin A. When lamin cannot bent effectively with nuclear envelope, it detaches from the nuclear envelope.
High curvature and high pressure increase rupture probability (not just force).
NOT NEW: Mislocalization is greatly enhanced by lamin A depletion, and correlates with an increase in pannucleoplasmic foci of the DNA damage marker γH2AX.
Excess DNA damage is rescued in ruptured nuclei by cooverexpression of multiple DNA repair factors as well as by soft matrix or inhibition of actomyosin stress on the nucleus.
Stiff tumors with low lamin-A indeed exhibit increased nuclear curvature, more frequent nuclear rupture, and excess DNA damage. Additional stresses likely play a role, but the data suggest high curvature promotes nuclear rupture.
Metabolic Requirements for Cancer Cell Migration in Confined 3D Environments
Emily S. Bell1,2, Philipp Isermann1, Tulasi A. Gopalan1, Noam Zuela-Sopilniak1, Nicole Zaragoza Rodriguez2, Warren Zipfel1, Jan Lammerding1
1Cornell University, Ithaca, NY; 2The Pennsylvania State University, University Park, PA;
Their Question: what is the metabolic cost of cell migration through confined spaces?
Cell migration requires ATP-intensive processes. Therefore, metabolic strategies that best support the energetic demands of a migrating cell could be a contributing factor in the invasion, migration, and metastasis of cancer cells. Studying single cell energetics in real-time can aid in understanding how cancer cells dynamically employ metabolic strategies during migration and how this can be targeted by therapeutic intervention.
Methods: They developed an improved fluorescent biosensor (PercevalHR2) which enabled ratiometric imaging and real-time measurement of ATP:ADP in living cells, and they expressed PercevalHR2 in cancer cell lines and imaged these cells during migration through confined space in a 3D microfluidic device.
They applied a panel of inhibitors impacting ATP production and metabolic signaling and measured their effect on confined migration and cellular force generation.
Results and Discussion:
- Experiments with cancer cells expressing the PercevalHR2 biosensor revealed that cells with a higher ATP:ADP ratio exhibited faster migration through confined spaces. T
- Treating cells with the mitochondrial-targeting drug Metformin specifically impaired confined migration, without decreasing cell speed through wider channels (Fig. 1). This was accompanied by decreased force generation.
- Treatment with 2-deoxyglucose (2DG) to inhibit glycolytic ATP production did not impede migration through the microfluidics devices, despite diminishing ATP levels and activating AMPK to a greater extent than Metformin.
(These results indicate that the physical environment of the cell alters the metabolic requirements for cell migration, and that the source of ATP production, rather than the ATP levels along, can modulate tumor cell migration through confined environments.)
- Both 2DG and Metformin severely inhibited proliferation, supporting that the metabolic phenotypes that fuel highly invasive cell behaviors are likely to be distinct from the pathways characterized to promote tumor growth.
(Successful cancer therapy may require distinct strategies to target both the metabolic pathways supporting tumor growth and the pathways that enhance invasion and metastasis.)
Important point:
Considering the fact that different systems with different sizes were used with different groups, a fair comparison cannot be achieved between these results.