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Comparative Study
. 2011 Nov 23;31(47):17250-9.
doi: 10.1523/JNEUROSCI.3938-11.2011.

"V体育ios版" Hydrodynamic cellular volume changes enable glioma cell invasion

Stacey Watkins  1 , Harald Sontheimer
Affiliations
Comparative Study

Hydrodynamic cellular volume changes enable glioma cell invasion

Stacey Watkins et al. J Neurosci. .

Abstract

Malignant gliomas are highly invasive brain tumors that currently lack effective treatment. Unlike other cancers, gliomas do not metastasize via the vasculature but invade surrounding brain solely along extracellular routes, primarily moving along the vasculature and nerve tracts. This study uses several model systems to visualize and quantitatively assess cell volume changes of human glioma cells invading within the brain's extracellular space of C. B. 17 severe combined immunodeficient (scid) mice and tumor cells invading in a modified Boyden chamber using three-dimensional multiphoton and confocal time-lapse microscopy. Regardless of model system used to quantitatively assess volume changes, invading glioma cells maximally decreased their volume by 30-35%, a value that was independent of barrier and cell size VSports手机版. Through osmotic challenges, we demonstrate that the observed cellular volume changes during invasion represent the smallest achievable cell volume and require glioma cells to release all free unbound cytoplasmic water. Water osmotically follows the release of Cl(-) through ion channels and cotransporters and blockade of Cl(-) flux inhibits both volume changes and cell invasion. Hence, invading glioma cells use hydrodynamic volume changes to meet the spatial constraints imposed within the brain, using essentially all free, unbound cytoplasmic water to maximally alter their volume as they invade. .

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Figures

Figure 1.
Figure 1.
Invading/migrating glioma cells from mice bearing xenograft tumors undergo dynamic volume changes. A, Low-power image (100×) of D54-EGFP tumor mass (green) associated with vasculature (red) as viewed through a cranial window. Shown is a 3D reconstruction of 350 sections taken 400 nm apart. Scale bar, 100 μm. B, High-power image (600×) of individual tumor cells (green) invading and migrating away from the main tumor mass. Vasculature is red. Shown is a two-dimensional reconstruction of 90 sections taken 400 nm apart. Scale bar, 50 μm. C, Volume changes of the two invading cells depicted in B over a 4 h time course. Every 15 min, Z-stacks consisting of 80–110 optical sections 400 nm apart were acquired. In all images, the vasculature was highlighted (red) by rhodamine dextran administered by tail vein injection.
Figure 2.
Figure 2.
Invasion in brain slices is associated with cell volume changes. A, 3D reconstructions of various time points during ex vivo invasion/migration assay (Time 0, 120, and 240 min) highlighting shape and volume changes occurring. Every 15 min, 600× Z-stacks of 50–150 sections taken 800 nm apart were acquired. Scale bar, 20 μm. B, Volume measurements of single glioma cell invading along the vasculature highlighted in A (arrowhead). C, Volume reconstructions of various time points during in situ invasion/migration assay (Time 0, 120, and 240 min) highlighting shape and volume changes occurring. Every 15 min, 600× Z-stacks of 50–150 sections taken 800 nm apart were acquired. Scale bar, 20 μm. D, Volume measurements of single glioma cell invading and migrating along the vasculature highlighted in C. E, Comparison of cellular volume changes occurring during glioma invasion/migration in various model systems using D54-EGFP and U251-EGFP cells.
Figure 3.
Figure 3.
Modified Transwell assay allowing real-time confocal imaging with defined barrier. A, Schematic of barrier used to image cells actively invading through pores of various sizes on the membrane of a Transwell insert. 1, Water-immersion objective (60×); 2, vitronectin (3 μg/ml); 3, Transwell invasion/migrating assay insert with pores of various size (3.0 μm, 5.0 μm, or 8.0 μm); 4, human glioma cells (D54-EGFP or U251-EGFP); 5, chemoattractant (10 ng/ml EGF in 17% serum-containing media); 6, migration assay buffer; 7, 3% agar; 8, vacuum grease. Temperature and pH were maintained at physiologic conditions and cells were imaged using a laser scanning confocal microscope. B, Confocal image of invading D54-EGFP cells (green) traversing 8.0 μm × 15.0 μm pores present on the membrane of a Transwell invasion/migration insert. Image represents a snapshot of cells midway through the process of invasion with the cell body present on either side of the membrane. Scale bar, 50 μm. C, Volume reconstructions of an individual glioma cell that successfully migrated from one side of the membrane, through the pore, to the other side of the membrane. Green, red, and blue lines represent the x-, y-, and z-axes, respectively. Image reconstructions were made from Z-stacks of 80 sections taken 800 nm apart and acquired every 20 min.
Figure 4.
Figure 4.
Comparable cellular volume changes occur during in vitro invasion/migration assay. A, Cell volume measurements of cells that successfully traversed 8.0 μm pores were obtained every 15–30 min for each cell examined. Each cell volume was normalized to its minimum cell volume measured and set to time 0. Minimum cell volumes were observed when the cell had traversed halfway through the pore forming a characteristic dumbbell shape (inset; scale bar, 15 μm). B, Percentage of cell volume located on either side of the membrane of Transwell invasion/migration insert at the cell's minimum volume measured. The initial plane of the membrane facing the surface the cells were initially seeded onto (initial side) was used as the reference value. p = 0.7972, two-tailed unpaired t test. C, Comparison of total volume change of cells that invaded and migrated through 8.0 μm pores (invading) versus noninvading and nonmigrating cells (sedentary). p = 0.0001; two-tailed unpaired t test. D, Fluctuations of volumes exhibited by sedentary glioma cells. Each cell volume was normalized to its median cell volume. E, A linear correlation exists between the total volume change and the maximum total volume measured in cells migrating through an 8.0 μm pore, y = 0.3365x − 111.93, R2 = 0.7366, R = 0.8583. F, Total volume change occurring in glioma cells during the process of invasion and migration is independent of the size of the barrier. p = 0.8633, two-tailed unpaired t test.
Figure 5.
Figure 5.
Minimal achievable cell volume. A, Representative images of glioma cells at various osmolarities (300, 450, and 1200 mOsm). Green, red, and blue lines represent the x-, y-, and z-axes, respectively. B, Actual normalized mean total cell volumes measured after hyperosmotic challenge compared to the theoretical expected for cells behaving like a perfect osmometer.
Figure 6.
Figure 6.
Chloride flux is required for volume change occurring during glioma cell invasion. A, Mixture of chloride channels and transport inhibitors (200 μm NPPB, 250 μm Cd2+, and 40 μm DIOA) significantly reduced glioma invasion through Transwell filters (*p = 0.0223, two-tailed unpaired t test). B, Fluctuations in volume exhibited by glioma cells exposed to the mixture of chloride channel and cotransporter inhibitors. Each cell volume measurement is normalized to its median cell volume. C, In vitro invasion/migration assay comparing cellular volume changes in invading (Invading) cells, noninvading and nonmigrating cells (Sedentary), and glioma cells that initiated the process of invasion and migration but were exposed to a mixture of drugs that inhibit the movement of chloride ions through chloride channels and cotransporters (+ Inhibitors). #p < 0.001, Tukey–Kramer multiple-comparisons test. D, Representative 3D reconstructions from in situ invasion/migration assay demonstrating the lack of cellular shape and volume changes that occur in the presence of chloride channel and cotransporter inhibitors. Green, D54-EGFP cells; red, vasculature. Scale bar, 20 μm. E, In situ invasion/migration assay comparing cellular volume changes in invading and migrating cells (Invading), noninvading and nonmigrating cells (Sedentary), and glioma cells that initiated the process of invasion and migration but were subsequently exposed to the mixture of drugs (+ Inhibitors). #p < 0.001, **p < 0.01; Tukey–Kramer multiple-comparisons test.
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