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Tumor Biology |
Departments of Anatomy and Structural Biology [J. B. W., J. S. C., J. E. S.], Pathology [J. G. J.], and the Analytical Imaging Facility (J. S. C.), Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSIntravital Imaging of MTLn3...DISCUSSIONREFERENCES |
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The most common assays for metastatic ability in vivo have been end point assays. For example, i.v. injection of tumor cells (often termed "experimental metastasis"), followed by determination of the number of metastases in a target organ such as the lung, is a simple method for evaluation of arrest and growth of tumor cells in target organs (3) . Detailed studies using this assay have demonstrated that extravasation per se tends not to be rate limiting, but that growth of metastases is inefficient (4 , 5) . However, this assay is limited by the introduction of a bolus (typically 100,000 cells) of in vitro cultivated cells in a nonphysiological manner. A more physiological approach to analysis of tumor cell metastasis makes use of the injection of tumor cells into an appropriate (orthotopic) tissue, followed by growth of a primary tumor as the source of tumor cells for metastasis. Such "spontaneous metastasis" assays are more accurate models of human disease in that they rely on growth of a primary tumor, and the tumor cells themselves then must actively leave the primary tumor and enter the vasculature (6, 7, 8) . Cell lines specifically selected for high metastatic ability through use of the experimental metastasis assay are not necessarily highly metastatic in the spontaneous metastasis assay (9) . Thus, for a detailed comparison of all of the various steps of metastasis, an assay such as the spontaneous metastasis assay must be used.
However, analysis of metastasis using the spontaneous metastasis assay typically measures only the growth of the primary tumor and the number of metastases that form in a target organ. The relative efficiencies for various steps in this assay have never been directly determined. Such an analysis is crucial because of the heterogeneity of tumors and tumor cell lines. Human primary tumors show extensive variation in all properties, ranging from growth and morphology of primary tumors through tumor cell density in the blood and then formation and growth of metastases. Similarly, tumor cell lines show broad variation in formation of a primary tumor and metastatic ability. As specific cell lines are manipulated to express particular activated or inhibitory oncoproteins, the effects on metastatic abilities will need to be interpreted in terms of the particular steps in the metastatic cascade that are affected. Growth of the primary tumor is simple to quantify and has been an extremely useful assay for determining proteins that are important for tumor formation. However, entry of tumor cells into the circulation is the critical first step in the metastatic cascade, and although it has been assayed in various ways (10, 11, 12) , it has not been observed directly.
Because for certain tumor types and conditions there are high levels of circulating tumor cells (13 , 14) , it has sometimes been assumed that entry into the circulation is not a critical step in tumor cell metastasis and that formation of metastases in target organs is rate limiting. Some studies using cell-based assays indicate that tumor cell burden in the blood can correlate with poor prognosis (15 , 16) . However, PCR or antibody-based assays for tumor cells in the blood may not show as strong a correlation (17, 18, 19, 20) . Animal studies using well-characterized cell lines and controlled conditions are needed to evaluate the role of intravasation.
In this study, we describe a straightforward procedure for comparing intravasation, extravasation, and growth in target organs during metastasis. For two mammary adenocarcinomas, the metastatic MTLn3 and poorly metastatic MTC cell lines, we use this procedure to demonstrate that intravasation is important. We then use in vivo time lapse confocal microscopy of MTLn3 and MTC primary tumors expressing GFP3 to compare the behavior of metastatic and nonmetastatic cells relative to blood vessels. We find that although both metastatic and nonmetastatic cells are motile in the primary tumor, metastatic cells are polarized toward blood vessels and nonmetastatic cells tend to fragment during intravasation. These results provide new insights into the differences between metastatic and nonmetastatic cells and have implications for the types of assays that should be developed for prognosis.
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MATERIALS AND METHODS |
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Blood Burden, Single Cells in the Lung, and Metastases.
Tumor cell blood burden was determined by placing a rat with a
6-week-old tumor under isoflurane anesthesia and removing 4 ml of blood
from the right atrium via heart puncture. The blood was then spun at
5000 rpm for 5 min, and the serum layer and buffy coat
region were plated into 
-MEM growth medium. The following day,
plates were rinsed twice with Dulbecco’s PBS (Life Technologies, Inc.)
to remove RBCs, and regular growth medium was added. After 6 days, all
clones in the dish were counted. To test cell viability in the
collection process, blood was drawn as above from noninjected rats.
Cultured MTLn3-GFP and MTC-GFP cells were removed from growth dishes
using trypsin/EDTA. Ten, 100, and 1000 tumor cells were added to 1 ml
of blood and 1 ml of medium, respectively. The mixtures were then
centrifuged, plated, and counted as described above for blood samples
derived from rats bearing tumors. There was approximately 50% recovery
of tumor cells from mixtures with blood as compared with mixtures with
growth medium for both cell lines.
For visualization of single cells near the surface of the lungs, the lungs were removed after blood removal and euthanization of the rat. The lungs were then placed in matTek dishes (MatTek Corporation, Ashland, MA) with 1 ml of L15 medium (Life Technologies, Inc.) to keep them moist. Ten fields from each side of both major lobes were then visualized using a x60 objective on a Nikon inverted microscope. All whole single cells visible in these fields were counted. Tumor cells were shown to be countable by confirming their GFP fluorescence in the fluorescein channel and lack of fluorescence in the rhodamine channel of the Bio-Rad MRC-600 confocal microscope. A field is the 1.1-mm diameter visible area through the microscope oculars.
For measurement of metastases, excised lungs were placed in 3.7% formaldehyde, mounted in paraffin, sectioned, and stained with H&E. Slices were viewed using a x20 objective, and all visible metastases in a section containing more than five cells were counted.
Imaging of Living Tumors and Their Vasculature.
Tumor imaging was performed as described previously (21)
.
Briefly, 1 x 106 cells were
injected under the second nipple anterior from the tail of a Fischer
344 rat and allowed to grow for 2.5 weeks. After 2.5 weeks, the rat was
placed under isoflurane anesthesia and the tumor was exposed using a
simple skin flap surgery, with as little disruption of the surrounding
vasculature as possible. The animal was then placed onto a Bio-Rad
MRC-600 confocal microscope, using a x20 objective and imaged in time
lapse, with a single image being taken every minute. On average, about
three different fields of each tumor were imaged for 20–30 min each.
For visualizing vasculature, 200 µl of rhodamine-dextran (2 M dalton; Sigma Chemical Co.) at 20 mg/ml in Dulbecco’s PBS was injected into the tail vein of the rat after anesthesia, but before surgery. The vasculature in the tumor was then visualized using the rhodamine channel of the Bio-Rad confocal.
Quantification of Images.
Time lapse movies were reconstructed using NIH Image. Movies then were
viewed and evaluated for the following criteria: (a) cell
extension and retraction; (b) cell locomotion
(21)
; (c) cell orientation to the vasculature;
and (d) host cell locomotion. Fields are defined as the
visible area for each optical condition and are 2.4 x 105 square microns, unless indicated otherwise.
For statistical purposes, an experiment is defined as a single tumor,
and data are reported as the percentage of time lapse sequences for
which a recorded event happens during an experiment. The velocity of
cell locomotion was determined by the distance the cell moved between
image frames.
Percent orientation in Table 2
was determined as the percentage of
blood vessels in an imaging field with four or more directly adjacent
cells polarized toward the vessel. Percent orientation was corrected
for randomly polarized fields of cells by subtracting from the above
value the percentage of blood vessels with both adjacent and
nonadjacent cells polarized toward the vessel.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSIntravital Imaging of MTLn3...DISCUSSIONREFERENCES |
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Cells were injected into the mammary fat pads of Fisher 344 rats and
allowed to grow for 6 weeks. To determine blood burden before
filtration by any capillary bed, but with minimal perturbation of the
primary tumor, the rats were anesthetized, and 4 ml of blood was
withdrawn from the right atrium of the heart. The cells in the serum
and buffy coat were plated in growth medium and allowed to grow for 1
week. Tumor cell colonies were identified by cell morphology and GFP
fluorescence. The blood of rats carrying MTLn3 primary tumors formed
about 23 colonies/4 ml of blood, which was significantly more than the
blood burden of <1 colony/4 ml for rats carrying MTC primary tumors
(P < 0.002; Table 1
, column 2). Control experiments using defined numbers of cells mixed
with blood showed a 50% plating efficiency for both cell lines. Thus,
there was a significantly larger number of viable tumor cells present
in the blood of rats carrying MTLn3 tumors compared with the blood of
rats carrying MTC tumors. This difference was not due to larger MTLn3
tumors (Table 1
, column 1). MTC tumors tended to be larger than MTLn3
tumors.
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, column 3). In addition, for rats carrying MTLn3 tumors, comparing
blood burden and fluorescent cells in the lungs in each animal there
was a significant correlation (correlation coefficient, 0.86;
P < 0.0004). However, there were no
correlations with the size of the primary tumor (correlation
coefficient, 0.23- 0.5; P > 0.05).
To evaluate metastases in the lungs of animals, sections of lungs were
stained with H&E and evaluated for metastases. Rats carrying MTLn3
tumors had significantly more metastases than rats carrying MTC tumors
(P < 0.003; Table 1
, column 4). In addition,
for rats carrying MTLn3 tumors there was a significant correlation
between blood burden and metastases (correlation coefficient, 0.96;
P < 0.0001).
In summary, from this analysis there was a clear correlation between tumor cell density in the blood and either numbers of single tumor cells in the lungs or metastases. This correlation was present both when comparing metastatic and nonmetastatic cell lines (MTLn3 and MTC) and when comparing MTLn3 tumors on the level of individual animals. To our knowledge, this is the first time that such a comparison has been performed on a single animal basis. The presence of a correlation between blood burden and both single cells, as well as metastases in the lungs, indicates that entry into the blood is important for metastasis of cells derived from the 13762 NF mammary adenocarcinoma. These data suggest that a more detailed analysis of cell behavior in the primary tumor is important for determining the properties that enable MTLn3 cells to gain access to the circulation. To perform such studies, we made use of the GFP expression of these cell lines to perform intravital imaging of the primary tumors.
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Intravital Imaging of MTLn3 and MTC Primary Tumors |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSIntravital Imaging of MTLn3...DISCUSSIONREFERENCES |
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, MTLn3-GFP cells (green) are elongated and
polarized (arrows) toward the vessel (red),
whereas cells away from the vessel are rounded (arrowheads).
Fig. 1B
shows MTC-GFP cells (green) that are
elongated throughout the field of view, with cell elongation occurring
independent of the vessel positions. Overall, for MTLn3 tumors, 57 +/-
6% of all vessels had polarized cells near them, compared with 24 +/-
4% for vessels in MTC-GFP tumors (Table 2
, column 1). This suggests that there is a vessel-mediated effect on
MTLn3 cells that results in orientation toward the vessels.
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, column 2). Fig. 2
shows the protrusive activity of an MTC-GFP tumor cell. The cell
extends a small protrusion (Fig. 2B)
, which then enlarges
(Fig. 2C)
. The movement of the leading edge of this cell was
3.8 µm/min during maximum rate of extension, consistent with
the average rate of movement seen for MTLn3 and MTC cells (see below).
For MTLn3-GFP cells, the most obvious shape changes occur at the
periphery of vessels where the cells are elongated, whereas for MTC
cells protrusion and locomotion occur randomly relative to vessels.
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moves on a linear path at a speed of 3.6 µm/min. This type of
movement is seen in 17% of all MTLn3 tumors and 10% of MTC tumors
(Table 2
, column 3). The MTC-GFP cell shown in Fig. 4
moves at a rate of 3.6 µm/min. The average velocities of movement of
MTLn3 (3.4 µm/min, 12 cells) and MTC (3.7 µm/min, 7 cells) were
similar.
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. In Fig. 5
, a fragment is seen attached to a cell extension
(A), then breaks off and moves away from the cell
(B and C). The initial rate of protrusion of the
extension was 2.8 µm/min, but the fragment speed of movement is
11–15 µm/min. This was observed in 32% of MTC-GFP tumors compared
with 6% of MTLn3-GFP tumors (Table 2
, column 4). In a number of cases,
the fragmentation was seen to occur in blood vessels. The higher
velocity of the fragment is consistent with passive flow in the blood
because the velocity is too high for cell locomotion (maximum about 4
µm/min) and close to the velocity of blood flow in small vessels.
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. It is likely that these are host immune system cells for the
following reasons: (a) the cells are not fluorescent;
(b) the cells are smaller than the tumor cells; and
(c) the speed of movement is relatively high (on the order
of 10 µm/min). Although host cells were observed at similar
frequencies in MTLn3 and MTC tumors (28% and 23%, respectively; Table 3
, column 1), there was a significant difference in the number of host
cells observed. For MTLn3-GFP tumors, there was an average of 11 host
cells observed per field, compared with 2 host cells/field for MTC-GFP
tumors (Table 3
, column 2). The increased numbers of host cells may aid
in either polarizing the tumor cells toward blood vessels or in
generating pores through which tumor cells can intravasate.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSIntravital Imaging of MTLn3...DISCUSSIONREFERENCES |
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Steady-State Analysis of Metastasis.
We have used a steady-state analysis of tumor cell distributions in
individual animals to develop a general method for comparing
intravasation with growth in the lungs during metastasis. We have found
that although the primary tumor sizes formed are similar, rats carrying
the more metastatic MTLn3 tumors have about 90 times more cells in the
blood than rats carrying MTC tumors. Although previous studies had
demonstrated that MTC cells injected i.v. form few metastases in the
lungs (27)
, this is the first demonstration that MTC cells
are also unable to effectively enter the circulation.
There are two limitations in our estimate of tumor cell density in the blood. First, because we are using colony counts, each clump of tumor cells would be counted as a single colony-forming unit, or cell (10 , 12 , 14) . Second, the cells must be able to grow in vitro. Given that these tumor cell lines grow well in vitro before injection into the animal to form a tumor, the latter limitation is not significant. Reconstruction experiments in vitro indicate that exposure to blood per se produces a roughly 50% reduction in plating efficiency for both cell lines. Thus, our estimate of tumor cell density in the blood may be an underestimate of the true number.
To determine whether metastasis of the MTLn3 cells could be dependent on intravasation as well, we compared the tumor cell density in the blood with single cells and metastases in the lungs for each rat carrying MTLn3 tumors. We found a significant correlation between blood density and single cells or metastases in the lungs. This result suggests that entry into or survival in the vasculature is an inefficient step for MTLn3 cells as well as for MTC cells. In addition, for animals carrying MTLn3 tumors, the number of lung metastases is less than would be expected given the single cell density in the lungs. This indicates that growth of metastases in the lungs is also inefficient, as has been well recognized (28, 29, 30, 31, 32) .
In Vivo Imaging of the Primary Tumor.
Given the results above indicating that intravasation is an important
difference between metastatic and nonmetastatic tumors, a more detailed
analysis of the cell behavior in the primary tumor becomes important. A
number of researchers have made use of GFP (5
, 21
, 33, 34, 35)
or lacZ (36, 37, 38, 39, 40)
expression to increase the sensitivity of
observations of disseminated tumor cells in target organs. We made use
of techniques developed to follow individual tumor cells in primary
tumors with an intact blood supply. By using a confocal scanning
microscope to image cells that stably express green fluorescent
protein, we are able to follow cell movements over 30 min. These
studies provide the first comparison of in vivo cell
behavior at the primary tumor with metastatic ability.
There are three major differences between the MTLn3 and MTC primary tumors: increased cell orientation toward blood vessels in MTLn3 tumors, increased fragmentation of cells in MTC tumors, and increased numbers of putative host cells in MTLn3 tumors. There are not as dramatic differences in protrusive activity and random cell translocation between these tumors.
Increased cell orientation of MTLn3 cells toward blood vessels could increase the efficiency with which they can then intravasate. The increased orientation could result in directed movement into the blood vessel and more cells moving into the blood. The orientation could be induced by chemoattractant diffusing from the blood vessel. Growth factors, including EGF or platelet-derived growth factor, are present in platelets and smooth muscle cells (e.g., see Refs. 41, 42, 43, 44 ). Release of growth factors from these cells or endothelial cells could provide a gradient that would produce a chemotactic response. MTLn3 cells are chemotactic toward EGF in vitro, whereas MTC cells are not (45) . MTLn3 cells express more EGF receptors than MTC cells (46) , and expression of the EGF receptor in MTC cells increases chemotactic responses to EGF and metastatic ability (47 , 48) . Expression of the EGF receptor and other EGF receptor homologues, such as ErbB2, have been correlated with poor prognosis. It is possible that chemotactic signaling mediated by the EGF receptor is important in enhancing metastatic capability in addition to the well characterized effects of EGF receptor signaling on mitogenesis.
The increased fragmentation of MTC cells compared with MTLn3 cells provides an additional mechanism by which viable cell number in the blood might be low for MTC cells. In some cases, we have identified fragmentation occurring during intravasation. If the shear forces in the blood vessels are causing MTC cells to fragment as they enter the blood vessel, that event will eliminate the possibility of metastasis. The increased susceptibility to fragmentation of MTC cells could reflect a lack of orientation toward the blood vessel and slow traversal of the endothelium due to a reduced chemotactic response, as suggested above. Morris et al. (49) have reported fragmentation of tumor cells injected i.v. and then viewed during extravasation into microvascular beds. At the extravasation stage of metastasis, fragmentation of both metastatic and nonmetastatic cells may occur if cells do not rapidly polarize and exit the blood vessel.
The observation of fragmentation of poorly metastatic cells has important implications for biochemical or molecular-based assays of blood. Given that cell fragments would contain tumor protein and DNA, immunological assays for tumor proteins or PCR-based assays for tumor DNA would be positive, although the fragments would be unable to metastasize. In that event, PCR- or protein-based assays of blood samples might not be useful for predicting metastases. A cell-based assay that evaluates the size of cells may be a more powerful predictor of poor prognosis.
An unexpected dividend of the GFP-based imaging was the ability to image motile nonfluorescent cells against the background of fluorescent cells. We have observed small, rapidly moving cells that are likely to be host immune cells. H&E sections of the primary tumor confirm that host immune cells are present in the primary tumor (data not shown), supporting this interpretation. Increased numbers of immune system cells could contribute to increased metastasis by production of chemotactic factors or degradation of extracellular matrix barriers (e.g., see Refs. 50, 51, 52, 53, 54 ).
Breast cancer both in humans and in animal models including the MTLn3 cells (21 , 55) spreads to lymph nodes, indicating that the lymphatic circulation can also be important for tumor cell metastasis. In our in vivo studies, in cases in which tumor cells are oriented or exiting the tumor in the absence of a labeled blood vessel, it is possible that the cells are interacting with lymphatics. Comparing the in vivo behavior of tumor cells around lymphatics with their behavior around blood vessels awaits development of an appropriate in vivo marker for lymphatics.
In summary, we have demonstrated a method for providing a more detailed analysis of metastatic ability of tumor cells. The method is straightforward and should be valuable in determining the contributions to metastasis of specific proteins expressed in tumor cells. In addition, our in vivo analysis of tumor cell behavior has indicated that cell orientation toward blood vessels may be a characteristic of metastatic cells and that poorly metastatic cells can fragment during intravasation. These distinctions may prove important in choosing appropriate assays for predicting disease progression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Department of
Defense Army Breast Cancer Program [DAMD17-94-J-4314 (to J. E. S.)
and DAMD17-96-1-6129 (to J. S. C.)] and a New York State Department
of Health Empire Award (J. S. C.). J. E. S. is supported by an
Established Scientist Award from the New York City affiliate of the
American Heart Association. 
2 To whom requests for reprints should be
addressed, at Department of Anatomy and Structural Biology, Albert
Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461.
Phone: (718) 430-4237; Fax: (718) 430-8996; E-mail: segall{at}aecom.yu.edu
3 The abbreviations used are: GFP, green
fluorescent protein; EGF, epidermal growth factor.
4 http://www.aecom.yu.edu/asb/segall/segall.htm.
Received 8/27/99. Accepted 3/14/00.
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 H. Yamaguchi, M. Lorenz, S. Kempiak, C. Sarmiento, S. Coniglio, M. Symons, J. Segall, R. Eddy, H. Miki, T. Takenawa, et al. Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin J. Cell Biol., January 31, 2005; 168(3): 441 - 452. [Abstract] [Full Text] [PDF]
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 L. Xu and X. Deng Tobacco-specific Nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Induces Phosphorylation of {micro}- and m-Calpain in Association with Increased Secretion, Cell Migration, and Invasion J. Biol. Chem., December 17, 2004; 279(51): 53683 - 53690. [Abstract] [Full Text] [PDF]
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W. Wang, S. Goswami, K. Lapidus, A. L. Wells, J. B. Wyckoff, E. Sahai, R. H. Singer, J. E. Segall, and J. S. Condeelis Identification and Testing of a Gene Expression Signature of Invasive Carcinoma Cells within Primary Mammary Tumors Cancer Res., December 1, 2004; 64(23): 8585 - 8594. [Abstract] [Full Text] [PDF]
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 S. Paris and R. Sesboue Metastasis models: the green fluorescent revolution? Carcinogenesis, December 1, 2004; 25(12): 2285 - 2292. [Abstract] [Full Text] [PDF]
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S. Goswami, W. Wang, J. B. Wyckoff, and J. S. Condeelis Breast Cancer Cells Isolated by Chemotaxis from Primary Tumors Show Increased Survival and Resistance to Chemotherapy Cancer Res., November 1, 2004; 64(21): 7664 - 7667. [Abstract] [Full Text] [PDF]
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J. Wyckoff, W. Wang, E. Y. Lin, Y. Wang, F. Pixley, E. R. Stanley, T. Graf, J. W. Pollard, J. Segall, and J. Condeelis A Paracrine Loop between Tumor Cells and Macrophages Is Required for Tumor Cell Migration in Mammary Tumors Cancer Res., October 1, 2004; 64(19): 7022 - 7029. [Abstract] [Full Text] [PDF]
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G. Mouneimne, L. Soon, V. DesMarais, M. Sidani, X. Song, S.-C. Yip, M. Ghosh, R. Eddy, J. M. Backer, and J. Condeelis Phospholipase C and cofilin are required for carcinoma cell directionality in response to EGF stimulation J. Cell Biol., August 30, 2004; 166(5): 697 - 708. [Abstract] [Full Text] [PDF]
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Y. H. Lin, Z.-Y. Park, D. Lin, A. A. Brahmbhatt, M.-C. Rio, J. R. Yates III, and R. L. Klemke Regulation of cell migration and survival by focal adhesion targeting of Lasp-1 J. Cell Biol., May 10, 2004; 165(3): 421 - 432. [Abstract] [Full Text] [PDF]
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M. E. Mycielska and M. B. A. Djamgoz Cellular mechanisms of direct-current electric field effects: galvanotaxis and metastatic disease J. Cell Sci., May 1, 2004; 117(9): 1631 - 1639. [Abstract] [Full Text] [PDF]
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S. J. Kempiak and J. E. Segall Stimulation of Cells Using EGF-Coated Magnetic Beads Sci. Signal., February 3, 2004; 2004(218): pl1 - pl1. [Abstract] [Full Text] [PDF]
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 R. S. Muraoka, Y. Koh, L. R. Roebuck, M. E. Sanders, D. Brantley-Sieders, A. E. Gorska, H. L. Moses, and C. L. Arteaga Increased Malignancy of Neu-Induced Mammary Tumors Overexpressing Active Transforming Growth Factor {beta}1 Mol. Cell. Biol., December 1, 2003; 23(23): 8691 - 8703. [Abstract] [Full Text] [PDF]
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A. Mamoune, J.-H. Luo, D. A. Lauffenburger, and A. Wells Calpain-2 as a Target for Limiting Prostate Cancer Invasion Cancer Res., August 1, 2003; 63(15): 4632 - 4640. [Abstract] [Full Text] [PDF]
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F. Ahmed, J. Wyckoff, E. Y. Lin, W. Wang, Y. Wang, L. Hennighausen, J.-i. Miyazaki, J. Jones, J. W. Pollard, J. S. Condeelis, et al. GFP Expression in the Mammary Gland for Imaging of Mammary Tumor Cells in Transgenic Mice Cancer Res., December 15, 2002; 62(24): 7166 - 7169. [Abstract] [Full Text] [PDF]
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A. Zijlstra, R. Mellor, G. Panzarella, R. T. Aimes, J. D. Hooper, N. D. Marchenko, and J. P. Quigley A Quantitative Analysis of Rate-limiting Steps in the Metastatic Cascade Using Human-specific Real-Time Polymerase Chain Reaction Cancer Res., December 1, 2002; 62(23): 7083 - 7092. [Abstract] [Full Text] [PDF]
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W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, L. Cermak, E. P. Bottinger, R. H. Singer, J. G. White, et al. Single Cell Behavior in Metastatic Primary Mammary Tumors Correlated with Gene Expression Patterns Revealed by Molecular Profiling Cancer Res., November 1, 2002; 62(21): 6278 - 6288. [Abstract] [Full Text] [PDF]
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M. B. A. Djamgoz, M. Mycielska, Z. Madeja, S. P. Fraser, and W. Korohoda Directional movement of rat prostate cancer cells in direct-current electric field: involvement of voltagegated Na+ channel activity J. Cell Sci., March 9, 2002; 114(14): 2697 - 2705. [Abstract] [Full Text] [PDF]
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 A. D. Thor, S. M. Edgerton, S. Liu, D. H. Moore II, and D. J. Kwiatkowski Gelsolin as a Negative Prognostic Factor and Effector of Motility in erbB-2-positive Epidermal Growth Factor Receptor-positive Breast Cancers Clin. Cancer Res., August 1, 2001; 7(8): 2415 - 2424. [Abstract] [Full Text] [PDF]
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J. B. Wyckoff, J. E. Segall, and J. S. Condeelis The Collection of the Motile Population of Cells from a Living Tumor Cancer Res., October 1, 2000; 60(19): 5401 - 5404. [Abstract] [Full Text]
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