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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5930fjev1 20/11/1892    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hu, J. Articles by Verkman, A. S. Search for Related Content PubMed PubMed Citation Articles by Hu, J. Articles by Verkman, A. S.

Increased migration and metastatic potential of tumor cells expressing aquaporin water channels

Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California, USA

¹Correspondence: 1246 Health Sciences East, University of California, San Francisco, CA 94143-0521, USA. E-mail: verkman{at}itsa.ucsf.edu

SPECIFIC AIMS

Aquaporin (AQP) water channels are expressed in high-grade tumor cells of different tissue origins, without known function. We hypothesized that AQPs in tumor cells may facilitate their migration and thus their metastatic potential, which, if correct, would suggest AQP inhibition as a possible strategy for inhibition of tumor spread. Our approach was to study the migration of AQP1-expressing vs. control tumor cells in culture and in vivo models and to measure tumor growth and metastasis in vivo.

PRINCIPAL FINDINGS

1. Characterization of tumor cell lines
Two tumor cell lines were selected for the studies here (B16F10 and 4T1 cells) for their low water permeability, stable AQP1 expression after transfection, and metastatic potential in mice. Control and AQP1-transfected B16F10 and 4T1 cell lines had similar appearance by phase-contrast microscopy, and similar growth rates as assayed by tritiated thymidine incorporation and cell counting. Immunofluorescence showed plasma membrane AQP1 expression in the transfected cells. Osmotic water permeability, as measured using a calcein swelling assay, was much greater in the AQP1-expressing than the control cells.

2. Increased in vitro migration of AQP1-expressing tumor cells
In vitro analysis of tumor cell migration was done using transwell migration and wound healing assays. In the transwell migration assay, cells in medium containing 1% serum were added to the upper surface of a Boyden chamber and allowed to adhere. Migration was measured over 6 h by contacting the chamber (containing a porous membrane with 8 µm diameter pores) with medium containing 1% (control) or 10% serum. Migration was significantly greater in the AQP1-expressing cells. Cell labeling with the fluorescent cell tracker dyes CMFDA (green fluorescent) or CMRA (red fluorescent), to be used in in vivo mouse studies below, did not significantly affect migration, nor did cell transfection with a yellow fluorescent protein (YFP)-encoding plasmid. Cell adherence to filters coated with collagen, fibronectin, or laminin was not different in control vs. AQP1-tranfected cells.

A complementary would healing assay of in vitro cell migration was done in which an 300 µm-wide linear strip of cells was scraped from confluent monolayers using a pipette tip. Wound closure, as quantified from serial micrographs, was significantly accelerated in the AQP1-transfected vs. control tumor cells. Also, there was polarized expression of AQP1 at the leading edge of AQP1-expressing migrating cells. Video imaging of migrating B16F10 and 4T1 cells indicated more frequent bleb/lamella formation after AQP1 transfection and greater lamellipodial area per leading edge surface.

3. Increased extravasation and metastasis of AQP1-expressing tumor cells in vivo
Tumor cells were labeled with fixable fluorescent dyes in order to identify them after intravenous (i.v.) injection in mice. Cells were stained with the green fluorescent dye CMFDA or the red fluorescent dye CMRA, each of which is cell permeable and within cells becomes entrapped by covalent reaction with cytoplasmic proteins. The two-color cell labeling strategy for simultaneous detection of control and AQP1-expressing cells was tested using the in vitro transwell assay in which a mixture of CMRA-labeled control 4T1 cells (red) and CMFDA-labeled AQP1-expressing 4T1 cells (green) was added to the upper chamber. Fig. 1 A (left panel) shows the easily recognizable red and green adherent cells at 6 h after cell addition. More green than red cells remained after scraping the upper surface of the filter (middle panel). From counting many cells on multiple filters, the ratio of AQP1-expressing vs. control cells was significantly increased after scraping cells (Fig. 1A , right).

View larger version (31K): [in this window] [in a new window]   Figure 1. Increased lung extravasation of AQP1-expressing tumor cells after tail vein injection. A) Control (CMRA labeled, red fluorescent) and AQP1-expressing 4T1 cells (CMFDA labeled, green fluorescent) were mixed 1:1 and applied to transwell filters. The upper chamber contained 1% serum and the lower chamber contained 10% serum. Fluorescence micrographs showing red and green cells before (left) and after (middle) scraping nonmigrated cells from the upper surface of the porous membrane. Scale bar, 50 µm. Right, summary of ratios of AQP1-expressing vs. control cells that migrated in 6 h (SD, n=6, *P<0.05). B) Top, fluorescence micrograph of red CMRA-labeled AQP1-expressing 4T1 cells mixed 1:1 with green CMFDA-labeled control 4T1 cells. Scale bar, 200 µm. Middle and bottom, frozen sections of mouse lung at 6 h after tail vein injection of 1:1 cell mixtures with indicated red/green labeling. Scale bar, 50 µm. C) Tumor cell count ratios (green/red or red/green cells as indicated) and number of cells per microscope field in the injection mixture (open bars) and in lung at 10 min and 6 h after tail vein injection (SD, n=3 for 10 min group, n=6–8 for 6 h group, *P<0.05). Labeling scheme shown at the bottom, with experiments done for CMRA labeling of control and CMFDA labeling of AQP1-expressing cells, and with the labeling reversed.

The same approach was used in mice to study tumor cell extravasation, with CMRA labeling of control and CMFDA labeling of AQP1-expressing cells, and with the labeling reversed. Figure 1B shows a low magnification fluorescence micrograph of the cell suspension used for tail vein injection, with approximately equal numbers of red and green fluorescent cells. The two lower photographs show labeled tumor cells in lung at 6 h after tail vein injection, with more AQP1-expressing than control tumor cells for both labeling schemes. The data are summarized in Fig. 1C as cell count ratios and total number of cells per low power microscope field for control and AQP1-expressing 4T1 and B16F10 cells. Migration of AQP1-expressing cells was greater than that of control cells for both labeling schemes (orange bars). In control studies, lungs harvested at 10 min (instead of 6 h) showed relatively few fluorescent cells (purple bars), most representing adherent cells to lung endothelium that have not yet migrated. The cell count ratio (purple bars) in lung at 10 min was not significantly different from that in the cell suspension used for tail vein injection (open bars). When a mixture of red- and green-labeled control cells was used for tail vein injections (right panels), the cell count ratio at 6 h was similar to that at 10 min and in the original cell suspension, as expected. These data provide evidence for increased lung extravasation of AQP1-expressing tumor cells after tail vein injection.

Tumor cell metastasis and growth were also studied in vivo. In one set of experiments lung metastasis was evaluated 15 days after tail vein injection of control or AQP1-expressing 4T1 cells. Figure 2 A (left) shows a greater number of well-demarcated tumor metastases in lungs of mice injected with AQP1-expressing tumor cells. Also, in most mice receiving the AQP1-expressing cells there was evidence for tumor infiltration of alveolar walls (middle). Immunocytochemistry in Fig. 2A (right) shows AQP1 expression in alveoli (microvascular endothelia), as expected, with AQP1 expression in the 4T1-AQP1 cells but not in control 4T1 cells. The data summary in Fig. 2B shows that AQP1-expressing tumor cells produced a remarkably greater number of lung metastases, but without altered tumor colony size. Alveolar wall thickness within 50 µm of metastases was significantly greater for the AQP1-expressing tumor cells due to tumor cell invasion.

View larger version (53K): [in this window] [in a new window]   Figure 2. AQP1 expression increases lung metastasis after tail vein injection of tumor cells. A) Left and middle, hematoxylin and eosin staining of paraformaldehye-fixed, paraffin-embedded sections of mouse lung tissue 14 days after tail vein injection of 106 control or AQP1-expressing 4T1 cells. Tumor metastases are indicated by arrows. Right, AQP1 immunohistochemistry showing labeling (brown) of alveoli/vessels in both micrographs, with AQP1 also in tumor cells in the lower micrograph. B) Data summary showing number of metastases per lung, area of tumor colonies, and alveolar wall thickness within 50 µm of metastases (SE, 5 mice per group, *P<0.02). C) Tumor size at 15 and 18 days after s.c. implantation of 106 B16F10 or 2 x 105 4T1 cells (control and AQP1-expressing) (SE, 20 mice per group, differences not significant). D) Histology of peripheral tumors at 15 days after s.c. implantation of control or AQP1-expressing 4T1 cells.

In another set of experiments mice were implanted subcutaneously with control or AQP1-expressing 4T1 or B16F10 cells to study tumor growth and local invasion. Figure 2C shows that tumor growth, as assessed by tumor volume at different times after implantation, was not affected by AQP1 expression. However, finger-like projections into subcutaneous adipose tissue were often seen with AQP1-expressing but not control 4T1 cells (Fig. 2D ).

CONCLUSIONS AND SIGNIFICANCE

The data here support a novel role for AQPs in tumor cells in their migration, which occurs in metastasis during tumor cell invasion across tissue barriers such as blood vessels.

Migration of two tumor cell lines was significantly enhanced after transfection with AQP1, as assessed by both transwell and wound healing assays. AQP1 transfection did not alter tumor cell growth, proliferation, morphology, size, or substrate adherence, but as expected, increased their water permeability. The in vivo studies in mice showed a greater number of AQP1-transfected vs. control tumor cells in lung parenchyma following tail vein infusion, producing a remarkably greater number of lung metastases. The extravasation measurements were done by a ratiometric method in which mice were injected with an 1:1 mixture of control and AQP1-expressing tumor cells that were labeled with different color fluorescent dyes. This ratio counting procedure reduced data variance by controlling for intrinsic mouse-to-mouse differences, as well as variability related to the tail vein injection procedure and tissue processing.

Cell migration involves a series of cellular events including actin polymerization and depolymerization, and the generation of cell protrusions such as lamella, lamellipodia, and blebs. We propose that AQP-facilitated water permeability in cell protrusions enhances their formation and thus the rate of cell migration (Fig. 3 ). The tumor cells studied here showed AQP polarization to lamellipodia and an increased number/size of lamellipodia. Local actin depolymerization, which creates new osmoles, might provide the osmotic driving force responsible for water influx into cell protrusions, as might transmembrane solute transport and/or altered solute osmotic coefficients in submembrane cytoplasm. It is thus predicted that cell membrane water permeability at its migrating surface may be an important determinant of tumor cell migration, and so manipulation of the expression or function of tumor AQPs may alter their migration and metastatic potential.

View larger version (21K): [in this window] [in a new window]   Figure 3. Proposed model for AQP-facilitated tumor cell migration and spread. Increased cell membrane water permeability facilitates the formation of cell protrusions (lamellipodia), resulting in more rapid migration through tissues.

In summary, we found a novel role for AQP1 in tumor cell extravasation and spread that may provide a functional explanation for the expression of AQPs in many tumor types. Tumor cell water permeability and AQP expression may thus be important determinants of their migration and spread in vivo, suggesting AQPs as targets in tumor therapy.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5930fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5930fjev1 20/11/1892    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hu, J. Articles by Verkman, A. S. Search for Related Content PubMed PubMed Citation Articles by Hu, J. Articles by Verkman, A. S.