This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services 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 Urquidi, V. Articles by Goodison, S. Search for Related Content PubMed PubMed Citation Articles by Urquidi, V. Articles by Goodison, S.

Contrasting Expression of Thrombospondin-1 and Osteopontin Correlates with Absence or Presence of Metastatic Phenotype in an Isogenic Model of Spontaneous Human Breast Cancer Metastasis

University of California, San Diego Cancer Center and Department of Pathology, University of California, San Diego, La Jolla, California 92093 [V. U., D. S., K. K., D. A., D. T., S. G.], and Cranfield BioMedical Centre, Institute of Bioscience and Technology, Cranfield, University, Bedfordshire, MK43 0AL, United Kingdom [A. C. W.]

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
Knowledge of the molecular mechanisms involved in metastatic spread is needed to facilitate advances in prognostic evaluation for individual patients and in the design of therapeutic interventions to inhibit the process. In an effort to establish a methodological framework for analysis of molecules and mechanisms involved in this complex multistep process, we have developed a well defined experimental system, in which the role of candidate genes can be screened and tested.

By serial dilution cloning of the MDA-MB-435 breast tumor cell line and screening by orthotopic implantation into the mammary fat pad of athymic mice, we have derived a pair of breast tumor cell lines (M-4A4 and NM-2C5) that originate from the same breast tumor but have diametrically opposite metastatic capabilities.

In 74% of inoculated athymic mice, clone M-4A4 metastasized consistently to the lungs, mimicking a major dissemination route of human breast cancer. Conversely, although equally tumorigenic, clone NM-2C5 did not metastasize to any distal site. We have confirmed that the cell lines originate from a single genetic source by spectral karyotyping and evaluated the expression of a number of proteins previously implicated in cellular transformation and metastasis. The ability of M-4A4 to metastasize was not associated with increased angiogenesis, as measured by immunohistochemical microvessel density analysis. However, RNA and protein analyses revealed that two secreted proteins were differentially expressed: osteopontin expression was increased 30-fold in clone M-4A4 and thrombospondin-1 expression was increased 15-fold in clone NM-2C5. These cell lines constitute a stable and accessible model for the identification of genes involved in the multistep process of breast tumor metastasis. Manipulation of candidate genes in these cells will permit evaluation of their functional significance in the geometric progression of breast cancer.

	Introduction

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
Despite significant advances in the treatment of primary cancer, the ability to predict the metastatic behavior of a patient’s cancer, as well as to detect and eradicate such recurrences, remains the greatest clinical challenge in oncology. To make progress in this area it is essential to obtain more detailed knowledge of the molecular mechanisms involved in metastasis as a basis for novel approaches to the evaluation of individual patient prognosis and to rational therapeutic design.

The formation of a secondary tumor colony in a distal site is the culmination of a complicated series of sequential and highly selective events (1, 2, 3, 4) . To succeed in accomplishing lymphogenous or hematogenous metastasis, tumor cells must have invaded the local extracellular matrix and penetrated the vascular endothelium to gain access to the circulation for transport away from the primary site. In the next phase of the process, cells that survive the physical stresses of the circulation and surveillance by body defense mechanisms lodge into the capillary bed of a conducive tissue or organ and exit through the vessel wall. Finally, to thrive and form secondary deposits, the tumor cells must proliferate and attract a new vascular supply and other supporting cells from the host tissue. These consecutive events are dependent upon the coordinated regulation of gene expression.

This multistep nature of metastasis poses difficulties in both design and interpretation of experiments to unveil the mechanisms causing the process. Studies on excised fixed human tissues are complicated by the variance of genetic background between individuals and by the cellular heterogeneity of a complex tissue mass. Breast cancer is also a collection of distinct diseases, and it can be difficult to be certain of the histogenetic classification of the tumor in advanced cases, thereby causing an inappropriate combination of data from genetically distinct lesions. However, the major limitation of such studies is the inability to identify those cells in a tumor mass that are truly capable of metastasis. Even if derived from a single cell, an advanced carcinoma is a mixture of genotypically and phenotypically distinct cells, and only a tiny fraction of those cells may possess the ability to disseminate from the primary lesion. Furthermore, it is estimated that only 0.1% of tumor cells that do enter the circulation will form secondary deposits in a distal organ (5 , 6) . Analysis of actual metastases may not be helpful either, because these cells have proliferated in a non-breast tissue environment and therefore may display a markedly different molecular profile (7) and may not retain the ability to metastasize again.

Critical to the experimental analysis of metastasis has been the isolation of human tumor cell lines and the ability to study their behavior in vivo by inoculation into immune-compromised mice (8 , 9) . Several established human breast cancer cell lines with varying documented abilities of invasiveness and/or migration in vitro are available, and some are capable of spontaneous metastasis in vivo (10) , i.e., dissemination from growth in the mammary gland and proliferation in a distal site. However, most of these are polyclonal and composed of cell populations that are heterogeneous in metastatic phenotype, making them difficult to use as models in studies seeking to define genes causing metastasis. Several laboratories have obtained cell lines with increasing metastatic phenotype by recovering and culturing metastatic deposits derived from primary inoculations and recycling the cells through several rounds of orthotopic selection (11 , 12) . The resulting cell lines represent improved models for studying metastasis because they are mono- or oligoclonal and, therefore, of uniform phenotype. The most common difficulty with these models is the lack of a corresponding totally nonmetastatic, clonally uniform counterpart for comparison, because the selection process used for the derivation of metastatic lines cannot be used for the selection of the converse phenotype. The original cell line, from which the hypermetastatic cell line is selected, is not an appropriate counterpart for comparison because it is a heterogeneous polyclonal resource containing many clones of differing metastatic propensity.

To overcome this experimental conundrum we have developed an orthotopic model of breast metastasis that enables comparative molecular screening and functional evaluation of candidate metastasis-related genes in an isogenic background. A large panel of monoclonal tumor cell lines were derived by limiting dilution from the polyclonal breast carcinoma cell line MDA-MB-435 (13) and systematically tested for metastatic behavior in athymic mice. Clone M-4A4 was selected from the many metastatic clonal populations and is highly metastatic to the lungs. A separate clone, NM-2C5, was found to be totally nonmetastatic but equally tumorigenic in athymic mice.

In this report we describe characterization of this paired cell line model, comparing their cellular and histopathological tumor morphology, in vitro and in vivo growth rates, and molecular profiling to date. We have demonstrated that the cell lines originate from the same genetic source by spectral karyotyping, and we report data on the expression of a number of gene products previously implicated in transformation and metastasis by RNA and immunochemical analyses. The majority of such targeted comparative analyses revealed equal levels of gene expression in both clonal cell lines, further supporting the tightly controlled nature of the experimental system. However, two major differences were revealed by multiple analyses. The expression of the secreted proteins TSP-1² and OPN was found to correlate with the nonmetastatic and the metastatic phenotypes, respectively. Verification of the differential expression of these genes in cultured cells and in xenograft tissues was achieved using quantitative RNA and protein analyses.

The derivation of this pair of breast tumor cell lines provides a powerful experimental system for controlled evaluation of molecular mechanisms involved in metastasis. The information already obtained indicates that it will greatly facilitate the identification of genetic and biochemical differences between metastatic and nonmetastatic neoplastic breast epithelia. Manipulation of candidate genes and subsequent analysis of upstream and downstream effects will then permit evaluation of their functional significance in this clinically important process.

	Materials and Methods

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
Isolation of Clonal Cell Lines and Culture Conditions.
Clonal sublines of the metastatic, polyclonal MDA-MB-435 human breast carcinoma line (10 , 13) were isolated by the limiting dilution technique, with direct microscopic monitoring of monocellular origin. Monoclonal cell lines were propagated in RPMI 1640 supplemented with 10% newborn calf serum (Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere of 5% CO₂-95% air. Cells were harvested by washing the monolayer with PBS and briefly incubating with 0.25% trypsin/0.02% EDTA. The detached cells were washed by centrifugation and resuspended in RPMI 1640 media and counted before passaging or inoculation. Two selected clones of nonmetastatic and metastatic phenotypes (NM-2C5 and M-4A4, respectively) were propagated for further investigation. Subsequent analyses were performed on cultures passaged no more than 10 times from frozen stock vials designated passage 1 at the time of in vivo inoculation. Cultures were tested and declared free of Mycoplasma and common murine pathogens.

Tumorigenicity and Metastasis Formation in Vivo.
Female athymic mice (MF1Nu strain) were housed in an isolation suite for the duration of the experiments. The tumorigenicity and spontaneous metastatic capability of the cell lines were determined by injection into the mammary fat pad. One million cells in 0.05 ml of a 1:1 mixture of RPMI 1640 medium and ECM gel (Sigma Chemical Co., St. Louis, MO) were inoculated into the anesthetized mouse. Animals were monitored every 2 days for up to 5 months for tumor growth and general health. The rate of primary tumor growth of the clones was determined by plotting the means of two orthogonal diameters of the tumors, measured at 7-day intervals. Animals were sacrificed and autopsied at 3–6 months postinoculation, unless moribund earlier. Metastasis formation was assessed by macroscopic observation of all major organs for secondary tumors and confirmed by histological examination of organs and lymph nodes. Tissue samples harvested for histological analysis were either fixed and embedded in paraffin wax or snap-frozen in liquid nitrogen.

Histological Evaluation.
Tissues recovered after autopsies of inoculated animals were fixed in 4% paraformaldehyde for 24 h and stored in 70% ethanol. Paraffin embedding, sectioning, and staining were performed using standard protocols. To confirm the presence or absence of internal lung and lymph node metastases, sections were cut at 50-µm intervals throughout each of the tissue samples, nine sections per organ. These sections were stained with H&E, and organs from 10 animals that were inoculated with each cell line were evaluated for the presence of microscopic metastasis by three independent pathologists.

Spectral Karyotyping.
Metaphase spreads were prepared according to standard procedures. Briefly, cells in log phase culture were treated with colcemid (0.3 µg/ml final concentration; Sigma) for 2 h before harvest by trypsinization. Centrifuged cells were exposed to hypotonic shock with 75 mM KCl for 20 min at 37°C and fixed in 3:1 methanol/glacial acetic acid solution. Cell suspensions were dropped onto slides, air-dried, and aged at room temperature for several days. Probe hybridization and image capture/analysis (14 , 15) were performed according to the manufacturer’s instructions (Applied Spectral Imaging, Carlsbad, CA). Probes are created using purified, single-chromosome templates, and PCR amplified using degenerate oligo-primers and incorporating three fluorochromes and two haptens. The specific combination of these five labels results in a unique spectral signature for each chromosome. Indirect detection of haptens was performed using Cy5-conjugated avidin and Cy5.5-conjugated antimouse IgG (Rockland, Gilbertsville, PA). Slides were counterstained with 4',6-diamidino-2-phenylindole and mounted with Prolong antifade solution (Molecular Probes, Eugene, OR). Spectral imaging was achieved using a SpectraCube system (Applied Spectral Imaging) mounted on a Zeiss Axiophot microscope, viewed through a x63 oil-immersion plan/apo objective illuminated by a xenon lamp (Opti Quip, Highland Mills, NY). Chromosome classification was performed with SkyView software (Applied Spectral Imaging). 4',6-Diamidino-2-phenylindole banding was captured separately and inverted for alignment with spectral representations using SkyView software.

RT-PCR.
Cultured cells were grown to 75% confluence before extraction of RNA. Frozen tissues recovered from nude mice were sectioned on a cryostat, and 100 sections (5 µm) were used for RNA extraction. Total RNA was isolated using an RNeasy kit (Qiagen, Valencia, CA), and mRNA was purified with Oligotex (Qiagen), treated with DNase I, and reverse transcribed using Moloney murine leukemia virus reverse transcriptase with a combination of oligodeoxythymidylic acid and random decamers (Ambion, Austin, TX). The resulting cDNA was used as a template for PCR using gene-specific primers. Hot-start PCR conditions were performed, and cycling conditions were adjusted for primer pair characteristics and estimated transcript abundance. Amplification products were resolved by agarose gel electrophoresis. For verification of specificity, products were recovered from gels and sequenced directly using the amplification primers.

Quantitative PCR Analysis.
OPN and TSP-1 mRNA transcripts in cultured cells and in primary xenograft tumor tissues recovered from athymic mice were quantified (16) using the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA). Total RNA was isolated using the RNeasy kit (Qiagen), and DNA was removed by digestion with RNase-free DNase A (Ambion). cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase and an oligodeoxythymidylic acid primer (Ambion). PCR was performed using the SYBR Green PCR Master Mix kit containing SYBR green I dye, AmpliTaq Gold DNA Polymerase, deoxynucleotide triphosphates with dUTP, passive reference, and optimized buffer components (PE Applied Biosystems). PCR primers were designed against the 3'-UTR of the human target genes using MacVector software (Oxford Molecular, Beaverton, OR) and checked for the absence of potential binding to mouse homologue sequences. All primers were used at a final concentration of 100 nM and 1 µl of cDNA dilution was added in 25 µl PCR reactions. No-template controls were included for each target. Thermocycling was initiated with a 10 min, 95°C enzyme activation step followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. All reactions were done in triplicate, and each reaction was gel-verified to contain a single product of the correct size. Data analysis was performed using the relative standard curve method as outlined by the manufacturer (PE Applied Biosystems) and as described previously (17 , 18) . The mean glyceraldehyde-3-phosphate dehydrogenase concentration (primer set supplied by PE Applied Biosystems) was determined once for each cDNA sample and used to normalize expression of all other genes tested in the same sample. The relative difference in expression was recorded as the ratio of normalized target concentrations for the same cDNA dilution. Primer sequences used were: OPN (GenBank accession no. AF052124), forward primer 5'-TGAGAGCAATGAGCATTCCGATG, reverse primer 5'-CAGGGAGTTTCCATGAAGCCAC; TSP-1 (GenBank accession no. X14787), forward 5'-AACAACCCCACACCCCAGTTTG, reverse 5'-TTGAAGCAGGCATCAGTCAC.

Immunocytochemistry and Immunohistochemistry.
Cultured cells were grown on glass disks in the appropriate medium before fixation in cold methanol (10 min, 4°C). Primary xenograft tumor tissue was either frozen in liquid nitrogen or fixed in formalin and embedded in paraffin. Cryostat sections (5 µm) were air dried and subsequently fixed for 20 min in 4% formaldehyde on Superfrost slides (Fisher Scientific, Pittsburgh, PA). Paraffin-embedded tissues were dewaxed with xylenes, followed by a 100%, 95%, 80% ethanol series and rehydrated in PBS. Standard immunohistochemical procedures were performed using hematoxylin counterstaining. Sections incubated with secondary antibodies alone served as negative controls.

Antibodies.
TSP-1 and focal adhesion kinase antibodies were obtained from Transduction Laboratories (Lexington, KY). OPN was from Chemicon (Temecula, CA). CD44 mAb Hermes-3 (19) against an epitope encoded by CD44 exon 5 was a kind gift from Dr. E. C. Butcher, Department of Pathology, Stanford University (Stanford, CA). CD44 mAb 2F10 (R & D Systems, Inc., Minneapolis, MN) against an epitope encoded by CD44 exon 11 (CD44v6) was used. ER- F-10, Ras, and p53 were from Santa Cruz Biotechnology (Santa Cruz, CA). MMP-2, MMP-9, and TIMP-2 were from Oncogene Research Products (Cambridge, MA). Maspin was obtained from PharMingen (Franklin Lakes, NJ). Paxillin was from (Sigma Chemical Co.). The phosphatase and tonsin homologue antibody was a generous gift from DNAX, Palo Alto, CA. The NG2 proteoglycan antibody was a generous gift from Dr. W. B. Stallcup, The Burnham Institute, La Jolla, CA. The Nm23-H1 antibody was a generous gift from Dr. C. Chang, University of California, San Diego School of Medicine, La Jolla, CA.

Southern Blot Detection of CD44 Transcripts.
RNA extraction and cDNA synthesis are described above. cDNA was used as a template for CD44-specific PCR using primers which anneal to CD44 exon 3 and exon 18 respectively (20) , yielding amplification products that include the variant exon sequences present in CD44 transcripts. Thirty PCR cycles consisting of 94°C for 30 s, 55°C for 1 min, and 72°C for 2 min were performed after a "hot start" procedure in 50-µl reactions. PCR products (5 µl) were separated by agarose gel electrophoresis and transferred to a Hybond N+ nylon membrane (Amersham Biosciences, Piscataway, NJ). The membrane was hybridized with probes to the standard region (CD44s) and to exon 11 (v6) of the variant region (20 , 21) . Exon-specific nucleic acid probes were synthesized by PCR amplification of a CD44 genomic clone (22) . Probes were gel purified and labeled directly using the ECL nucleic acid labeling kit (Amersham Biosciences). Hybridization and detection were performed according to the ECL manfacturer’s instructions.

Northern Blot Analysis.
Total RNA was isolated as described above and mRNA was purified with Oligotex resin (Qiagen). RNA was treated with DNase I (Ambion). Northern blotting was performed using 1.0% agarose/glyoxal gels and blotting overnight onto NorthernStar nylon membrane (Ambion) in diethyl pyrocarbonate-treated 10x SSC. Oligonucleotide primers were designed using sequences obtained from the National Center for Biotechnology Information GenBank database and gene-specific probes created by PCR using cell line cDNA as template. Products of expected size were gel purified and sequenced: TSP-1 (accession no. X14787) forward primer, 5'-AACAACCCCACACCCCAGTTTG; reverse primer, 5'-TTGAAGCAGGCATCAGTCAC; OPN (accession no. AF052124) forward primer, 5'-TGAGAGCAATGAGCATTCCGATG; reverse primer, CAGGGAGTTTCCATGAAGCCAC. Probe (25 ng) was labeled using the Decaprime kit (Ambion) and [-³²P]dCTP (Amersham Biosciences). Hybridization was performed for 4–12 h at 68°C in NorthernMax hybridization buffer (Ambion). Images were obtained by use of a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and quantitative values were obtained using ImageQuant software (Molecular Dynamics).

Western Blot Analysis.
Cultured cell lines were grown to 75% confluency, harvested, and lysed for protein extraction according to established protocols. Protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) and adjusted to 1 mg/ml by dilution with PBS. For secreted-protein analysis, serum-free culture medium was removed after 24 h incubation and centrifuged (600 x g for 10 min) to remove any cellular material; then, the supernatant was frozen and stored at -70°C. After 100 times concentration through Ultrafree Centrifugal filters (Millipore, Bedford, MA), soluble protein was measured as described above. Aliquots of standardized samples (5 µg of total protein in 10 µl of buffer) and molecular weight standards were boiled for 5 min before separation by 4–12% SDS-PAGE under reducing conditions. The resolved proteins were electroblotted on to an Immobilon-P membrane (Millipore, Bedford, MA) using transfer buffer (48 mM Tris, 39 mM glycine, 0.1% SDS, 20% methanol, pH 9.2). Nonspecific reactions were blocked by incubation with Tris-buffered saline containing 5% skimmed milk for 1 h, before the membrane was incubated overnight at 4°C with primary antibody. Reactivity was determined by the addition of peroxidase-conjugated secondary antibody at room temperature, and the signals were visualized using the ECL detection system (Amersham Biosciences).

Microvessel Density.
Three formalin-fixed, paraffin-embedded specimens of primary tumors were randomly selected from both NM-2C5 and M-4A4 resources, and several sections were prepared from each for immunostaining. The CD31 antibody (murine monoclonal antibody; DAKO, Carpinteria, CA) was used to identify capillary-sized vessels. Standard avidin-biotin complex peroxidase immunohistochemical staining was performed on 5-µm-thick sections from paraffin-embedded, formalin-fixed tissue. A negative control staining without primary antibody was performed. The microvessel counting procedure as described by Weidner (23) was used. The areas with the greatest density of CD31-positive endothelial cells were designated "hot spots." The whole section was scanned at low power (x40) to identify the best fields for counting. Counting was performed on three separate fields within a hot spot at x200 magnification using a Nikon E600 microscope. Each stained endothelial cell or cell cluster was counted as one microvessel, and the presence of a lumen was not required (see Fig. 5 ). If two or more CD31-positive foci appeared to belong to a single continuous vessel, this was counted as one microvessel. Specimen section edges and areas of necrosis or sclerosis were ignored. The MVD count was defined as the sum of the three field counts within the hot spot. Sections were analyzed by two independent observers (D. S., S. G.) without knowing prior results or the identity of the samples. Averages of counts were calculated, and the Wilcox rank sum test was used to compare medians.

View larger version (81K): [in this window] [in a new window]   Fig. 5. Representative immunohistochemical staining of NM-2C5 and M-4A4 breast cells growing in the mammary fat pad of athymic mice. Tissue sections were stained with hematoxylin and CD31 immunostaining to reveal endothelium. Left, M-4A4; right, NM-2C5. Original magnification x40.

	Results

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

View larger version (81K): [in this window] [in a new window]   Fig. 1. Cell morphology of nonmetastatic (NM-2C5, left) and metastatic (M-4A4, right) cultured cells. Cells were seeded at passage 5 and imaged, without fixing, using differential interference microscopy (x20 magnification).

View larger version (19K): [in this window] [in a new window]   Fig. 2. Growth rates of cultured NM-2C5 and M-4A4 clonal breast carcinoma cell lines. Cell numbers were determined by counting live cells harvested from separate culture flasks on a hemocytometer at 24-h intervals.

View larger version (127K): [in this window] [in a new window]   Fig. 3. Representative histological sections of M-4A4 (upper left) and NM-2C5 tumors (upper right) resulting from inoculation into the mammary fat pad of athymic mice. The tumors are poorly differentiated adenocarcinomas showing classical features of an infiltrating lobular carcinoma of the breast including Indian-file arrangement of tumor cells (arrows, upper right; magnification x200). In the upper left panel, tumor cells are seen enmeshed within a fibrin embolus in the lumen of a peripheral blood vessel (arrow), and cross-sectional profiles of normal mouse breast ducts are seen in the adjacent fat pad (*; magnification x200). An example of pulmonary metastasis (lower right; magnification x100) and a tumor cell embolus (lower left; arrow) are shown in the cortical sinus of a draining lymph node in an M-4A4 tumor-bearing mouse. Magnification x400.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Spectral karyotyping of the NM-2C5 and M-4A4 cell lines. A, a representative metaphase cell (actually two overlapping cells) of NM-2C5 depicted as a 24-color spectral image of the spectral karyotyping hybridization. Arrows indicate three of the multiple marker chromosomes. B, representative metaphase cell chromosome complement after spectral karyotyping classification. Numbers alongside the derivative chromosomes indicate the origin of the translocated material. Arrows indicate marker chromosomes: yellow and green arrows indicate derivative chromosomes common to both cell lines, and the white arrow indicates a derivative chromosome observed only in nonmetastatic NM-2C5 cells.

Targeted Molecular Analyses.
Having evaluated some characteristics of the two cell lines and their associated tumors, we then investigated and compared the expression of a number of genes previously implicated in tumorigenesis and/or metastasis. Expression analyses were performed using immunohistochemistry, Western blotting, Northern blotting, and/or PCR (Table 2) . Targets were selected based on a reported role in transformation, angiogenesis, extracellular matrix degradation, or metastasis. A majority of genes evaluated were found to be either absent or present in both cell lines, and when present, they were expressed at similar levels. Table 2 lists the genes evaluated and indicates their presence or absence. Both breast cell lines were negative for ER- isoform expression. However, the ER-ß form did appear to be present at very low levels, detected only by PCR amplification. ER--positive MCF-7 and ER-ß-positive Du-145 cells were used as positive controls (data not shown).

View larger version (57K): [in this window] [in a new window]   Fig. 6. Comparative CD44 expression analyses. CD44 protein (left, upper and lower) expression was evaluated by Western blot analysis. Lysates (5 µg of protein/well) from cultured cells (upper) and from recovered primary xenograft tissue (lower) of NM-2C5 (2) and M-4A4 (4) were incubated with antibodies to epitopes encoded by CD44 variant exon 11 (Exon 11) and exon 3 (Standard). Molecular mass (kDa) of prestained protein markers is indicated to the left of each panel. CD44 transcripts were evaluated by Southern hybridization of RT-PCR products obtained from amplification of NM-2C5 (2) and M-4A4 (4) mRNA, recovered from cultured cells (upper right) and primary xenograft tissue (lower right). The blot was hybridized with probes specifically annealing to exon 11 (Exon 11) and exon 5 (Standard) of the CD44 gene. Molecular mass (bp) of nucleic acid markers is indicated to the right of each panel.

View larger version (82K): [in this window] [in a new window]   Fig. 7. Differential expression of TSP-1 and OPN in M-4A4 and NM-2C5 cells. A, protein levels in serum-free CM from cultured cells. a: the higher expression level of TSP-1 in NM-2C5 was clearly noticeable by Coomassie Blue staining of an SDS-PAGE gel. Note that TSP-1 expression in M-4A4 decreases as cell density increases. b: confirmation of the identity of TSP-1 and of its differential expression was obtained by Western blot using anti-TSP-1 antibodies. c: Western blot with OPN specific antibody, showing a substantially higher expression in M-4A4. B, Northern analysis reveals that the level of TSP-1 mRNA is higher in NM-2C5 (a), and the level of OPN mRNA is higher in M-4A4 (b).

Having determined the differential expression of TSP-1 and OPN in M-4A4 and NM-2C5 cultured cells, we examined the expression of TSP-1 and OPN transcripts in primary xenograft tissue recovered from nude mice. Real-time quantitative PCR analysis of three primary tumors originating from each clone revealed that OPN and TSP-1 transcripts were also differentially expressed in vivo. Real-time quantitative PCR results (normalized against average values for the housekeeping gene GAPDH) revealed that TSP-1 mRNA was 22-fold higher in NM-2C5 tissue relative to M-4A4, and OPN mRNA was 21-fold higher in M-4A4 tissue relative to NM-2C5. Real-time PCR evaluation of TSP-1 and OPN expression in cultured cells revealed 33-fold and 88-fold differential expression, respectively (lower fold differences revealed using Northern and Western analyses were presumably underestimated by solid-phase filter analyses). Immunochemical analysis of xenograft tissue revealed that both OPN and TSP-1 were expressed, primarily accumulating in the extracellular matrix, but truly quantitative protein analysis was unattainable because both are secreted proteins. We used the cytogenetic evaluation of the cell lines (Fig. 4) to estimate gene dosage and to assess the gross integrity of the chromosomal structure at the OPN and TSP-1 loci. OPN is a single-copy gene located on chromosome 4, and both NM-2C5 and M-4A4 cell lines were found to contain two copies of this chromosome of normal constitution. TSP-1 is also a single-copy gene, located on chromosome 15 at position 15q15. Chromosome 15 does show some rearrangement in our cell lines (Fig. 4) , but this does not appear to affect the TSP-1 gene. Two chromosomes 15' with structural integrity at 15q15 were always present in both cell lines. Furthermore, the chromosome 15 translocation prevalent in NM-2C5 cells has a breakpoint mapped as t(12;15)(q22;q26.1), and the t(8;15) translocation common to both cell lines is a t(8;15)(q24;q21), as determined by G-banding and FISH (data not shown).

	Discussion

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
The experimental approach adopted in this work is based on the knowledge that advanced tumors are highly heterogeneous (34 , 35) and that clones that are capable of completing the multistep process of metastasis from the primary lesion may be only a tiny fraction of the total tumor cell population. The evolution of a neoplastic cell to an invasive and then a metastatic cell depends upon a transient period of genomic instability and/or the ability to rapidly adjust to changing local microenvironments during its dissemination, with appropriate adaptive shifts in gene expression patterns. Earlier studies on the continuity of metastatic capabilities of clonal tumor cell populations cultured over several generations in vitro (34 , 36) repeatedly showed that this pattern of behavior is retained and heritably transferred within a cell population, but that it can drift in degree, or even be extinguished, over time. It is, therefore, probably programmed by genetic mechanisms that remain stable over sufficient periods of time to make them amenable to analysis but are not necessarily constant or predictable. In this work we have taken special precautions to avoid the tendency for clonal drift in metastatic capability over many cell generations described by Poste, Nicholson, and others (36, 37, 38) . This trend was restricted in our experimental system by limiting clonal expansion to less than 10 passages in vitro for all cell line analyses. However, because of the orthotopic inoculation used in our model, the cell lines experienced extended passage in vivo of many tens of passages, yet this did not result in phenotypic drift with regard to metastatic potential, suggesting a high level of genomic stability.

Because of the method of derivation of the majority of cell line models capable of spontaneous metastasis, no completely nonmetastatic counterpart has been isolated for direct comparative evaluation. In contrast, our goal was to isolate a matched pair of clonal cell lines with diametrically opposite metastatic capabilities, derived from the same tumor source and, therefore, originating from a common genetic progenitor. It was reasoned that this experimental design would help to narrow the search for differentially expressed genes relevant to metastasis. Another feature of our approach is the use of the immune-compromised mouse to inform us of the metastatic behavior of inoculated human breast cells. The inoculum was already monoclonal; therefore, no recovery or reculturing of cells was necessary or desired. The human polyclonal parent MDA-MB-435 cell line, the source for the derivation of this metastasis model, was known to be metastatic; thus, the probability of isolating a metastatic clone was high. However, the isolation of a nonmetastatic clone was much less certain because of the difficulty of confirming a negative result. Hence, the method used to derive a cell line metastasis model such as the M-4A4 and NM-2C5 pair requires a major investment of effort. However, this effort can yield a valuable investigative resource with regard to biological relevance, because the cell lines of opposing metastatic phenotype have emerged through natural selection within the original human tumor and have not been genetically manipulated by the investigator or selected by passage through a murine host.

Our approach to the investigation of the molecular basis of metastasis uses these paired cell lines as a starting point, focusing directly on the differences between defined metastatic and nonmetastatic monoclonal cell populations. We considered commencing with analysis of recovered human primary and metastatic tumor xenograft tissues resulting from inoculation of these lines but decided that this could lead to interpretive difficulties, because of the obligatory mix of human tumor and murine host cells. We reasoned that an initial differential observation in the pure cell populations followed by human-specific verification of any promising candidates in the primary xenograft material constitutes a rational approach. Initially, we targeted specific molecules for study in this model, selected on the basis of reported roles in transformation, angiogenesis, extracellular matrix degradation, or metastasis. The oncogene products c-myc and Ras were equally present in both transformed cell lines. The levels of these proteins were far greater than those observed in nontransformed cells, indicative of overexpression and/or alteration of protein half-life, most often caused by genetic mutation of these genes (39 , 40) . The cell adhesion molecule E-cadherin and the cytokeratins are specifically expressed in epithelial tissue, and in tumor cells, the loss of E-cadherin expression parallels tumor progression toward a malignant invasive state and is correlated with a loss of the epithelial phenotype (41) . These molecules were absent from both of the clonal breast cell lines, suggesting some degree of loss of differentiation. Their absence may also be linked to the negative ER status of both cell lines, inasmuch as there are reports that the expression of these genes in breast epithelia is regulated by estrogens (42) . In any case, the data indicate that these classes of molecules are not actively involved in generating the differences between the metastatic phenotypes of the two clones.

Angiogenesis, the process by which new vasculature is formed from preexisting vessels, is an essential component to primary tumor growth and distant metastasis. The degree of tumor vascularization is a result of pro- and antiangiogenic factors originating from both the tumor cells and those of the host (43) . Analysis of the recovered xenografts did not reveal any significant difference in MVD between the M-4A4 and NM-2C5 cell line-derived tumors, suggesting that the ability of M-4A4 to metastasize is not associated with increased angiogenesis. However, further work is required to evaluate the constitution of the neovasculature as recent reports have indicated that it is not only microvessel density but the size and the composition of the vasculature that may influence the passage of cells into the circulation (44) .

The invasion of cancer cells into the surrounding host stroma is also a crucial step in the process of metastasis (45) . The MMP family is a major component of extracellular matrix degradation and remodeling and could, therefore, have a role in enhancing the dissemination of cells from a primary tumor (46) . To date, we have examined the expression of the collagenases MMP-2 and MMP-9 and their inhibitors TIMP-1 and -2. Analysis of these secreted proteins revealed no differences in protein levels in the CM recovered from the cells in culture (Table 2) . The enzymatic activity of the MMP-2 and MMP-9 secreted proteins was also equal, as tested by gelatin zymography (data not shown). We are currently examining the expression of other members of the MMP family in this experimental system.

Changes in the expression profile of CD44 variant isoforms have been observed in many carcinomas, including those of the breast (26 , 47) , and much interest and investigative effort has centered on the possible involvement of CD44 isoforms in tumor metastasis (48) . Gunthert et al. (27) demonstrated that an antibody that inhibited metastasis of a rat pancreatic adenocarcinoma cell line specifically recognized an epitope encoded by CD44 exon v6 (exon 11) of this gene. Transfection of a nonmetastatic cell clone from the same rat pancreatic cancer, with a construct containing this exon, induced it to become metastatic (49 , 50) . Furthermore, two other groups provided evidence indicating that, in experiments with cultured human melanoma (51) and lymphoma cell lines (52) , there appeared to be a correlation between raised CD44 expression and metastatic capability. However, there is also evidence that CD44 function is not always necessary for metastatic capability. A murine lymphoma cell line with a CD44 gene double knockout was reported to retain its local invasiveness and metastatic potential despite losing all hyaluronan-binding ability (53) . Analysis of our human breast metastasis model revealed multiple isoforms, including the metastasis-implicated CD44v6 isoforms, but at equivalent levels in both clones. In this breast metastasis model, specific CD44 isoform expression does not appear to be sufficient to confer metastatic capability.

The two genes that have been identified thus far in our current work as being significantly differentially expressed in our experimental model have each been implicated previously in tumorigenesis and metastasis. TSP-1 is a homotrimeric multidomain glycoprotein with disulfide-linked subunits of molecular weight M_r 150,000 and is encoded by a gene mapped to human chromosome 15. The protein is synthesized and secreted by numerous cell types including platelets, endothelial cells, macrophages, fibroblasts, vascular smooth muscle cells, type II pneumocytes, and keratinocytes (reviewed in Refs. 54 and 55 ). Because of its interaction with a wide variety of proteins, TSP-1 has been implicated in a number of biological processes including coagulation, cell adhesion, modulation of cell-cell and cell-matrix interactions, control of tumor growth and metastasis, and angiogenesis (55) . The effect of TSP-1 expression on tumor growth and metastasis has been examined in vivo by orthotopic inoculation of TSP-1-overexpressing MDA-MB-435 cells. A dose-dependent inhibition of spontaneous pulmonary metastases was reported, but this was paralleled by an inhibition of primary tumor growth (56) . TSP-1 has also been implicated in the phenomenon of concomitant tumor resistance. This refers to the ability of some large primary tumors to hold smaller, distant tumors in check, preventing their progressive growth. Volpert et al. (57) demonstrated this phenomenon using tumors formed by the human fibrosarcoma line HT1080 in nude mice and obtained data indicating that it was caused by secretion of TSP-1 by the primary tumor. However, some of the properties attributed to TSP-1 are conflicting: TSP-1 can be both adhesive and antiadhesive in vitro, can stimulate or inhibit angiogenesis, and can inhibit or enhance proteolytic enzyme activity. The composition of the matrix, the availability of cytokines and proteases, and the expression of various receptors in a given cellular environment might explain these opposing functions. Our findings are in agreement with TSP-1 playing a role in the reduction of metastatic potential, but it does not appear to be through inhibition of angiogenesis per se, as judged by microvessel density evaluation, in the NM-2C5 xenografts.

A wide variety of transformed cells in culture express much higher levels of OPN than do their normal counterparts (58 , 59) , and a series of functional studies links OPN causally to tumor progression. Transfection of antisense OPN constructs or OPN-targeting ribozymes into transformed tumorigenic cells can result in impaired anchorage-independent growth, as well as diminished tumor-forming ability in vivo (31 , 32 , 60) . Furthermore, transfection of OPN expression constructs into cells producing noninvasive mammary epithelial tumors has been reported to convert them to a malignant phenotype (33 , 58) . OPN is also typically overexpressed in human tumors (61 , 62) , including breast cancer (63) ; and among cell lines derived from human breast tumors, the highest levels of expression are found in the most metastatic (64 , 65) . Consistent with overexpression of OPN in tumors, the concentration of OPN in the blood of patients with metastatic cancer is substantially elevated in comparison with the low levels that normally circulate. Higher OPN levels in patients with metastatic breast cancer may be associated with an increased number of involved sites and decreased survival (66) . There may also be a link between OPN function and CD44 expression. OPN binds to naturally expressed and stably transfected variant CD44 isoforms in a manner that is specific, dose-dependent, and inhibitable by anti-CD44 antibodies. The CD44-OPN interaction can mediate chemotaxis or attachment, depending on presentation of OPN in soluble or immobilized form (67) . We have shown that multiple CD44 isoforms exist in both the NM-2C5 and M-4A4 cell lines, and although CD44 does not itself appear to be sufficient for metastasis, it may be necessary for OPN function.

Relatively few studies have produced functional data demonstrating the role of candidate genes in the regulation of human breast tumor cell metastasis, but some of the most interesting candidates have been identified using the MDA-MB-435 breast tumor cell line (68, 69, 70) . Accordingly, we tested the expression of KAI1 and KISS-1, molecules that have both been reported to suppress metastasis in MDA-MB-435 cells (68 , 71) and in other experimental systems (70 , 72) . By RNA analysis, KAI1 expression was not detected in either the NM-2C5 or M-4A4 clonal cell lines. Conversely, KISS-1 mRNA was found to be expressed in both cell lines at equal and relatively high levels. The metastasis suppressor gene Nm-23-H1 (73) has also been found to quantitatively inhibit MDA-MB-435 metastasis (74) , but the Nm23 protein product was found to be equally expressed in both NM-2C5 and M-4A4 cell lines, as evidenced by Western blot analysis.

The most recent gene reported to be implicated functionally in MDA-MB-435 metastasis is a breast metastasis suppressor gene, termed BRMS-1. Based upon cytogenetic data that identified chromosome 11 as containing multiple genetic aberrations associated with breast cancer progression, Phillips et al. (75) showed that introduction of a normal chromosome 11 into MDA-MB-435 cells significantly reduced the metastatic potential of the cell line without affecting tumorigenicity. Subsequent analyses identified the location of one of the genes responsible for this effect (69) , and it was named breast metastasis suppressor-1 (BRMS-1). Stable transfection of BRMS-1 cDNA into MDA-MB-435 and MDA-MB-231 cell lines correlated with locally invasive tumor growth in athymic mice and with significant reduction of metastases to the lungs and lymph nodes (69) . However, quantitative RNA evaluation of BRMS-1 expression in NM-2C5 and M-4A4 cells revealed equal levels of expression in both cell lines; and, therefore, BRMS-1 does not appear to suppress metastasis in M-4A4 cells. Furthermore, our initial cytogenetic analysis of NM-2C5 and M-4A4 cells has revealed no genetic aberrations on the chromosome 11q arm where BRMS-1 resides. The small GTPase RhoC molecule has also recently been identified as being influential in the metastasis of the A375 series of human melanoma cell lines (76) . By cDNA array-based comparative analysis of an increasingly metastatic series of cell lines derived by reculturing of metastatic deposits, RhoC expression was identified as correlating with metastatic capability. Subsequent overexpression of RhoC or a dominant-negative RhoC in recipient A375 cells confirmed this correlation. RhoC was found to be expressed equally in both the metastatic M-4A4 and the nonmetastatic NM-2C5 cells; thus, RhoC expression was not limiting for either cell line. As is the case with many of the previously implicated metastasis-related genes, in our model RhoC expression may be permissive, or even essential for metastasis. However, our data suggest that expression of this molecule is not, in itself, enough to cause metastasis, as evidenced by the data from NM-2C5.

Perhaps the most significant experimental difficulty with cell line analyses, reemphasized by our current study, is the heterogeneity of the cultured populations. Most widely available cell lines have been isolated from advanced tumors or ascites containing millions of tumor cells, and these lines have often been excessively passaged and have passed through many laboratories, making interlaboratory comparisons difficult. Furthermore, many previous studies of metastasis-implicated genes have logically investigated cell clones that have been transfected to achieve high level expression of the exogenous gene. However, comparative metastasis analyses then have often been made with the phenotype of the polyclonal parent cell line, without determining the metastatic capability of the specific selected clone before transfection. In such circumstances, it remains possible that the phenotype displayed by the transfected clone was already present before transfection and that a difference observed between this selected clone and its polyclonal parent cell line merely reflects heterogeneity in the original population (39 , 42) .

Despite decades of research, no single consistent marker or effector of metastatic behavior has yet been identified. Given the complexity of the metastatic process during which a disseminating tumor cell must accomplish several sequential tasks and survive in many different environments, it is far more likely to be a set of genes that is responsible for manifesting the overall phenotype. Modern technological advances now permit the application of high throughput gene expression analysis, by methods such as gene-chips and spotted microarrays, to identify whether coordinated patterns of expression of clusters of genes are involved in this complex process. Current work in this laboratory is focused on applying these approaches to this tested and well characterized experimental system.

	ACKNOWLEDGMENTS

 
We are grateful for the expertise of the University of California, San Diego Histology Core facility for help with histological preparations. We also thank Carrie Viars for technical and analytical advice during cytogenetic evaluations.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at UCSD Cancer Center, 9500 Gilman Drive, La Jolla, CA, 92093-0912. Phone: (858) 822-2083; Fax: (858) 822-2084; E-mail: sgoodison{at}ucsd.edu

² The abbreviations used are: TSP-1, thrombospondin-1; OPN, osteopontin; RT-PCR, reverse transcription-PCR; ER, estrogen receptor; MMP, metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; ECL, enhanced chemiluminescence; MVD, microvessel density; FISH, fluorescence in situ hybridization; CM, conditioned medium.

Received 7/16/01; revised 10/ 8/01; accepted 10/ 8/01.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 

1. Tarin D. Clinical and experimental studies on the biology of metastasis. Biochim. Biophys. Acta, 780: 227-235, 1985.[Medline]
2. Tarin D., Matsumura Y. Recent advances in the study of tumour invasion and metastasis. J. Clin. Pathol., 47: 385-390, 1994.[Free Full Text]
3. Fidler I. J. Host and tumour factors in cancer metastasis. Eur. J. Clin. Invest., 20: 481-486, 1990.[Medline]
4. Fidler I. J., Kumar R., Bielenberg D. R., Ellis L. M. Molecular determinants of angiogenesis in cancer metastasis. Cancer J. Sci. Am., 4 (Suppl 1): S58-S66, 1998.
5. Tarin D., Price J. E., Kettlewell M. G., Souter R. G., Vass A. C., Crossley B. Mechanisms of human tumor metastasis studied in patients with peritoneovenous shunts. Cancer Res., 44: 3584-3592, 1984.[Abstract/Free Full Text]
6. Fidler I. J. Metastasis: quantitative analysis of distribution and fate of tumor emboli labeled with 125 I-5-iodo-2'-deoxyuridine. J. Natl. Cancer Inst., 45: 773-782, 1970.
7. Radinsky R. Molecular mechanisms for organ-specific colon carcinoma metastasis. Eur. J. Cancer, 31A: 1091-1095, 1995.[CrossRef]
8. Fidler I. J. Rationale and methods for the use of nude mice to study the biology and therapy of human cancer metastasis. Cancer Metastasis Rev., 5: 29-49, 1986.[CrossRef][Medline]
9. Fidler I. J. Orthotopic implantation of human colon carcinomas into nude mice provides a valuable model for the biology and therapy of metastasis. Cancer Metastasis Rev., 10: 229-243, 1991.[CrossRef][Medline]
10. Price J. E., Polyzos A., Zhang R. D., Daniels L. M. Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice. Cancer Res., 50: 717-721, 1990.[Abstract/Free Full Text]
11. Bruns C. J., Harbison M. T., Kuniyasu H., Eue I., Fidler I. J. In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using orthotopic implantation in nude mice. Neoplasia, 1: 50-62, 1999.[CrossRef][Medline]
12. Gehlsen K. R., Davis G. E., Sriramarao P. Integrin expression in human melanoma cells with differing invasive and metastatic properties. Clin. Exp. Metastasis, 10: 111-120, 1992.[Medline]
13. Cailleau R., Olive M., Cruciger Q. V. Long-term human breast carcinoma cell lines of metastatic origin: preliminary characterization. In Vitro, 14: 911-915, 1978.[Medline]
14. Schrock E., du Manoir S., Veldman T., Schoell B., Wienberg J., Ferguson-Smith M. A., Ning Y., Ledbetter D. H., Bar-Am I., Soenksen D., Garini Y., Ried T. Multicolor spectral karyotyping of human chromosomes. Science (Wash. DC), 273: 494-497, 1996.[Abstract]
15. Macville M., Veldman T., Padilla-Nash H., Wangsa D., O’Brien P., Schrock E., Ried T. Spectral karyotyping, a 24-colour FISH technique for the identification of chromosomal rearrangements. Histochem. Cell Biol., 108: 299-305, 1997.[CrossRef][Medline]
16. Heid C. A., Stevens J., Livak K. J., Williams P. M. Real time quantitative PCR. Genome Res., 6: 986-994, 1996.[Abstract/Free Full Text]
17. Schmittgen T. D., Zakrajsek B. A., Mills A. G., Gorn V., Singer M. J., Reed M. W. Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal. Biochem., 285: 194-204, 2000.[CrossRef][Medline]
18. Rajeevan M. S., Vernon S. D., Taysavang N., Unger E. R. Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR. J. Mol. Diagn., 3: 26-31, 2001.[Abstract/Free Full Text]
19. Jalkanen S., Jalkanen M., Bargatze R., Tammi M., Butcher E. C. Biochemical properties of glycoproteins involved in lymphocyte recognition of high endothelial venules in man. J. Immunol., 141: 1615-1623, 1988.[Abstract]
20. Aogi K., Kitahara K., Urquidi V., Tarin D., Goodison S. Comparison of telomerase and CD44 expression as diagnostic tumor markers in lesions of the thyroid. Clin. Cancer Res., 5: 2790-2797, 1999.[Abstract/Free Full Text]
21. Woodman A. C., Goodison S., Drake M., Noble J., Tarin D. Noninvasive diagnosis of bladder carcinoma by enzyme-linked immunosorbent assay detection of CD44 isoforms in exfoliated urothelia. Clin. Cancer Res., 6: 2381-2392, 2000.[Abstract/Free Full Text]
22. Goodison S., Yoshida K., Churchman M., Tarin D. Multiple intron retention occurs in tumor cell CD44 mRNA processing. Am. J. Pathol., 153: 1221-1228, 1998.[Abstract/Free Full Text]
23. Weidner N. Current pathologic methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors. Breast Cancer Res. Treat., 36: 169-180, 1995.[CrossRef][Medline]
24. Ogawa Y., Chung Y. S., Nakata B., Takatsuka S., Maeda K., Sawada T., Kato Y., Yoshikawa K., Sakurai M., Sowa M. Microvessel quantitation in invasive breast cancer by staining for factor VIII-related antigen. Br. J. Cancer., 71: 1297-1301, 1995.[Medline]
25. Goodison S., Yoshida K., Sugino T., Woodman A., Gorham H., Bolodeoku J., Kaufmann M., Tarin D. Rapid analysis of distinctive CD44 RNA splicing preferences that characterize colonic tumors. Cancer Res., 57: 3140-3144, 1997.[Abstract/Free Full Text]
26. Goodison S., Tarin D. Clinical implications of anomalous CD44 gene expression in neoplasia. Front. Biosci., 3: E89-E109, 1998.
27. Gunthert U., Hofmann M., Rudy W., Reber S., Zoller M., Haussmann I., Matzku S., Wenzel A., Ponta H., Herrlich P. A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell, 65: 13-24, 1991.[CrossRef][Medline]
28. Rudy W., Hofmann M., Schwartz-Albiez R., Zoller M., Heider K. H., Ponta H., Herrlich P. The two major CD44 proteins expressed on a metastatic rat tumor cell line are derived from different splice variants: each one individually suffices to confer metastatic behavior. Cancer Res., 53: 1262-1268, 1993.[Abstract/Free Full Text]
29. Lackner C., Moser R., Bauernhofer T., Wilders-Truschnig M., Samonigg H., Berghold A., Zatloukal K. Soluble CD44 v5 and v6 in serum of patients with breast cancer. Correlation with expression of CD44 v5 and v6 variants in primary tumors and location of distant metastasis. Breast Cancer Res. Treat., 47: 29-40, 1998.[CrossRef][Medline]
30. Martin S., Jansen F., Bokelmann J., Kolb H. Soluble CD44 splice variants in metastasizing human breast cancer. Int. J. Cancer, 74: 443-445, 1997.[CrossRef][Medline]
31. Gardner H. A., Berse B., Senger D. R. Specific reduction in osteopontin synthesis by antisense RNA inhibits the tumorigenicity of transformed Rat1 fibroblasts. Oncogene, 9: 2321-2326, 1994.[Medline]
32. Su L., Mukherjee A. B., Mukherjee B. B. Expression of antisense osteopontin RNA inhibits tumor promoter-induced neoplastic transformation of mouse JB6 epidermal cells. Oncogene, 10: 2163-2169, 1995.[Medline]
33. Oates A. J., Barraclough R., Rudland P. S. The identification of osteopontin as a metastasis-related gene product in a rodent mammary tumour model. Oncogene, 13: 97-104, 1996.[Medline]
34. Fidler I. J., Kripke M. L. Metastasis results from preexisting variant cells within a malignant tumor. Science (Wash. DC)., 197: 893-895, 1977.[Abstract/Free Full Text]
35. Fidler I. J. Tumor heterogeneity and the biology of cancer invasion and metastasis. Cancer Res., 38: 2651-2660, 1978.[Abstract/Free Full Text]
36. Poste G., Fidler I. J. The pathogenesis of cancer metastasis. Nature (Lond.), 283: 139-146, 1980.[CrossRef][Medline]
37. Nicolson G. L., Poste G. Tumor cell diversity and host responses in cancer metastasis—part I—properties of metastatic cells. Curr. Probl. Cancer, 7: 1-83, 1982.[Medline]
38. Nicolson G. L., Winkelhake J. L., Nussey A. C. An approach to studying the cellular properties associated with metastasis: some