Patent Application
20080138805
(1) Field of the Invention
The present invention generally relates to the characterization of motile cells and invasive cells of tumors. More specifically, the invention is directed to methods of isolating motile cells, in particular invasive cells, and the characterization of gene expression in those cells.
(2) Description of the Related Art
Understanding how cancer cells spread from the primary tumor is important for improving diagnostic, prognostic and therapeutic approaches that allow control of cancer metastasis. Alterations in gene expression along with protein activation by cancer cells leads to transformation, proliferation, invasion, intravasation, dissemination in blood or lymphatic vessels and eventually growth of distant metastases. In order for a tumor cell to become metastatic, it must be able to survive in the circulation and respond appropriately to new environments. This includes being able to migrate both within and beyond the primary tumor, in and out of blood and lymph vessels, and to utilize growth factors available at the site of metastasis for attachment and growth (Lin and Van Golen, 2004).
We have studied the motility-associated behavior of metastatic and non-metastatic mammary tumor cell lines by intravital imaging within primary tumors (Farina et al., 1998a; Wang et al., 2002; Wyckoff et al., 2000a). These studies have shown that the metastatic cells migrate to blood vessels and intravasate in a series of steps that involve active cell motility and may involve chemotaxis (Wang et al., 2002; Wyckoff et al., 2000a; Condeelis and Segall, 2003).
Many of the formative steps that determine the invasive and metastatic potential of carcinoma cells occur within the primary tumor. Much evidence suggests that the progress of cells from normal to invasive and then to metastatic involves progressive transformation through multiple genetic alterations selected by the tumor microenvironment (Hanahan and Weinberg, 2000). To identify the steps in progression and the genes involved in metastasis, recent emphasis has been on the use of molecular arrays to identify expression signatures in whole tumors with differing metastatic potential (Liotta and Kohn, 2001). A well recognized problem here is that primary tumors show extensive variation in properties with different regions of the tumor having different growth, histology, and metastatic potential and where only a small subset of cells within the parental tumor population may be capable of metastasizing (Fidler and Kripke, 1977). The array data derived from whole tumors results inevitably in averaging of the expression of different cell types from all of these diverse regions. The expression signature of invasive tumor cells, arguably the population essential for metastasis, may be masked or even lost because of the contribution of surrounding cells which represent the bulk of the tumor mass. Even so, recent studies of expression profiling of primary tumors suggest that the metastatic potential of tumors is encoded in the bulk of a primary tumor, thus challenging the notion that metastases arise from rare cells within a primary tumor acquired late during tumor progression (Ramaswamy et al., 2003).
This leaves us with a conundrum concerning the contribution of rare cells to the metastatic phenotype. The relative contribution of subpopulations of cells to the invasive and metastatic phenotype of primary tumors has not been assessed due to the difficulty in isolating phenotypically distinct cell populations from whole tumors. In addition, the metastatic cascade has been studied most heavily at the level of extravasation and beyond using experimental metastasis models removing the primary tumor from scrutiny. Thus, the microenvironment of the primary tumor that contributes to invasion and intravasation, and the process of selection of metastatic cells, has not been studied directly (Chambers et al., 2002).
In this context it has become important to develop technologies to separate pure populations of invasive cancer cells for gene expression studies. To this end, the development of Laser Capture Microdissection (LCM) has been an important advance (Bonner et al., 1997). However, the identification of cells within the tumor relies on morphology within fixed tissue making uncertain the identity of the collected cells and their behavior within the tumor before fixation. Alternative approaches involve the collection of cells from metastatic tumors and their expansion in culture (Clark et al., 2000; Kang et al., 2003; Ree et al., 2002). The pitfall of these approaches is that during culturing, the gene expression patterns may change to represent the in vitro culture conditions which are likely to be irrelevant to invasion in vivo.
Accordingly, the inventor has developed methods of isolating motile cells from animal tissues, and the use of those methods to isolate metastatic cells from cancerous tissue and quantify expression of various genes in those cells.
Thus, in some embodiments, the invention is directed to methods of isolating motile cells of interest from an animal tissue, where the animal tissue comprises the motile cells of interest and other motile cells. The methods comprise obtaining a microneedle or capillary filled with a porous matrix comprising a chemotactic factor; inserting the microneedle or capillary into the tissue for a time sufficient for the motile cells of interest to migrate into the porous matrix; expelling the porous matrix with motile cells from the microneedle or capillary; combining the porous matrix with microbeads, where the microbeads comprise a binding partner to a surface marker present on the other motile cells but not the motile cells of interest; and removing the microbeads.
In other embodiments, the invention is directed to methods of determining mRNA or protein expression of a gene in motile cells of interest from an animal tissue. The methods comprise isolating the motile cells of interest by the method described above, then extracting the mRNA or protein from the cells of interest, then determining mRNA or protein expression in the extraction of the cells of interest.
The invention is also directed to methods of determining whether a cancer in a tissue of a mammal is likely to metastasize. The methods comprise obtaining a microneedle or capillary filled with a porous matrix comprising a chemotactic factor; inserting the microneedle into the cancer for a time sufficient for motile cells to migrate into the porous matrix; expelling the porous matrix with motile cells from the microneedle; combining the porous matrix with microbeads, where the microbeads comprise a binding partner to a surface marker present on macrophages from the tissue; removing the microbeads; and quantifying the motile cells, where the presence of more motile cells than from the tissue when noncancerous or when comprising a non-metastatic cancer indicates that the cancer in the tissue of the mammal is likely to metastasize.
In further embodiments, the invention is directed to methods of inhibiting metastasis of a cancer in a tissue of a mammal. The methods comprise enhancing ZBP-1 activity in the tissue.
The invention is additionally directed to methods of inhibiting metastasis of a cancer in a tissue of a mammal. The methods comprise reducing the presence or activity of a protein in the tissue, where the protein is selected from the group consisting of Arp2/3 p16 subunit, Arp2/3 p21 subunit, alpha subunit of capping protein, beta subunit of capping protein, cofilin, WAVE3, ROCK1, ROCK2, LIMK 1, PKCζ, LIM-kinase, PAK, type II alpha isoform of PI4, 5 kinase, mena, tropomyosin, calpain, gelsolin-like protein (CAPG), zyxin, vinculin, and integrin 1.
The invention is further directed to methods of determining resistance of a motile cancer cell population in an animal tissue to a chemotherapeutic agent. The methods comprise obtaining the motile cancer cell population by the method described above; contacting the motile cancer cell population with the chemotherapeutic agent at a concentration and for a time sufficient to cause apoptosis in cancer cells susceptible to the chemotherapeutic agent; and determining apoptosis in the motile cancer cell population. In these embodiments, less apoptosis in the motile cancer cell population indicates that the motile cancer cell population is resistant to the chemotherapeutic agent.
The present invention is based on the development of methods of isolating motile cells, especially motile (metastatic) cancer cells from animal tissues, and the use of those methods to quantify expression of various genes in those motile cells.
Thus, in some embodiments, the invention is directed to methods of isolating motile cells of interest from an animal tissue, where the animal tissue comprises the motile cells of interest and other motile cells. The methods comprise obtaining a microneedle or capillary filled with a porous matrix comprising a chemotactic factor; inserting the microneedle or capillary into the tissue for a time sufficient for the motile cells of interest to migrate into the porous matrix; expelling the porous matrix with motile cells from the microneedle or capillary; combining the porous matrix with microbeads, where the microbeads comprise a binding partner to a surface marker present on the other motile cells but not the motile cells of interest; and removing the microbeads. Some preferred embodiments of these methods are described in Wang et al., 2003.
These methods can be used with tissue from any animal. Preferably, the animal is a vertebrate, more preferably a mammal, for example a rodent or a human.
Any tissue in the animal can be utilized in these methods, where the tissue has motile cells that are directed toward a chemotactic factor. Preferably, the issue is cancerous, since the isolation of motile cells from cancerous tissue is particularly useful, e.g., for determining the metastatic potential of the cancer. A non-limiting example of a tissue useful for these methods is mammary tissue. See examples.
The methods can be used with tissue in culture, tissue taken from a biopsy, or directly on tissue in a living mammal.
These methods are not narrowly limited to the use of any particular porous matrix. The matrix must only allow motile cells in the tissue to move through the matrix in response to the chemotactic factor. In preferred embodiments, the matrix is matrigel, since that matrix is similar chemically to vertebrate extracellular matrix.
The methods are also not limited to any particular microneedle or capillary; the microneedle or capillary must only be of sufficient bore to be capable of being filled with the porous matrix and to allow the motile cells to move into the matrix in response to the chemotactic factor. In some preferred embodiments, a microneedle is used; a preferred bore is 33-gauge.
Any binding partner capable of binding to the other motile cells but not the motile cells of interest, and capable of being bound (either covalently or noncovalently) to a microbead can be used. Nonlimiting examples include aptamers or, preferably, antibodies or antibody fragments, where the binding site is preferably specific for a cell surface marker present on the surface of the other motile cells but not the motile cells of interest. For example, where the motile cells of interest are carcinoma cells and the other motile cells are macrophages, a preferred microbead has antibodies specific for CD11b, which is present on the surface of macrophages but not carcinoma cells. See Wang et al., 2003. The skilled artisan could formulate a binding partner for any particular motile cell of interest/other motile cell combination without undue experimentation.
As used herein, “antibody” includes the well-known naturally occurring immunoglobulin molecules as well as fragments thereof that comprise a typical immunoglobulin antigen binding site (e.g., Fab or Fab2). The antibodies can be from a polyclonal, monoclonal, or recombinant source, and can be of any vertebrate (e.g., mouse, chicken, rabbit, goat or human), or of a mixture of vertebrates (e.g., humanized mouse).
These methods are also not narrowly limited to any particular microbeads for binding the other motile cells. For example, the microbeads can be heavy particles that are pelleted under centrifugal conditions that do not pellet the motile cells of interest. Alternatively, the microbeads can be buoyant particles that are not pelleted under centrifugal conditions that pellet the motile cells of interest. In preferred embodiments, the microbeads are colloidal super-paramagnetic beads as described in Wang et al., 2003.
The chemotactic factor can be any factor capable of attracting the motile cells of interest. Where the motile cells of interest are cancer cells, a preferred chemotactic factor is an epidermal growth factor.
Although the other motile cells in the examples herein and in Wang et al., 2003 are substantially macrophages, it is anticipated that other normal stromal cells such as fibroblasts or eosinophils may be predominant in other applications, e.g., where the cancer is in tissues other than mammary tissue. It is believed that the skilled artisan could easily identify binding partners that are effective for removal of any other motile cells without undue experimentation.
The motile cells of interest for these methods are not limited to cancer cells, and can be normal stromal cells such as macrophages. Additionally, the other motile cells (such as macrophages where the motile cells of interest are cancer cells) can be retained and further analyzed, since they are generally isolated in essentially pure form on the microbeads. The further analysis can include, e.g., quantitation of the cells, or analysis of mRNA or protein expression.
These methods are generally useful for isolating live motile cells of interest in highly enriched form, such that culture of the cells, and/or further analysis, can be performed. For example, the cells can be quantified, in order to approximate the number of motile cells of interest present in a given amount of tissue, or to compare the amount of motile cells of interest to the amount of the other motile cells.
In some preferred embodiments, mRNA or protein expression of at least one gene is determined in the motile cells of interest. See Example 2, where mRNA expression of various genes is quantified in the motile cells of interest (carcinoma cells) and compared with expression of the same genes in other carcinoma cells in the same tissue.
As shown in Example 2, motile breast carcinoma cells have significantly higher mRNA expression of Arp2/3 p16 subunit, Arp2/3 p21 subunit, alpha subunit of capping protein, beta subunit of capping protein, cofilin, WAVE3, ROCK1, ROCK2, LIMK 1, PKCζ, LIM-kinase, PAK, type II alpha isoform of PI4, 5 kinase, mena, tropomyosin, calpain, gelsolin-like protein (CAPG), zyxin, vinculin, integrin β1, tight junction protein 2, member Ras oncogene family, and epidermal growth factor receptor than nonmotile carcinoma cells from the same tissue, indicating involvement of these genes in the metastatic phenotype. Additionally, mRNA expression of ZBP-1, collagen type III α1, G-protein coupled receptor 26, and fibroblast growth factor receptor 1 is significantly reduced in motile breast carcinoma cells when compared to the nonmotile carcinoma cells, indicating a role of these proteins in regulation of metastasis. Additionally, when ZBP-1 is overexpressed in a carcinoma cell line, motility of the cells is greatly reduced (Example 2), further establishing the role of ZBP-1 in metastasis regulation. Thus, determination of protein, or, preferably, mRNA expression of any of those genes, especially ZBP-1 is particularly desirable.
As shown in Table 2 and the accompanying discussion in Example 2, motile cancer cells have a characteristic pattern of downregulation of collagen type III α1, G-protein coupled receptor 26, ZBP-1, and fibroblast growth factor receptor 1, and upregulation of Arp2/3 p16 subunit, tight junction protein 2, member Ras oncogene family, and epidermal growth factor receptor. Thus, it is also preferred that protein or, especially, mRNA expression is determined in at least two, and preferably all, of those genes.
When analysis of mRNA or protein expression of more than one gene is desired, microarray technology can be employed. This well-established technology can analyze mRNA or protein expression of many thousands of genes at once, allowing comparison of expression of, e.g., an entire genome between motile and non-motile cells.
These methods are capable of isolating a few hundred motile cells from a tissue. This typically provides 20-50 ng of total RNA, which is insufficient for array analysis. Therefore, the mRNA from these cells is preferably amplified prior to the determination of expression of the genes. Preferably, the amplification is by reverse transcription and cDNA amplification. A preferred method is the SMART PCR cDNA amplification method (ClonTech Laboratories). See Wang et al., 2003.
The motile cells of interest can also be tested for resistance to chemotherapeutic agents. See Example 1.
In other embodiments, the invention is directed to methods of determining mRNA or protein expression of a gene in motile cells of interest from an animal tissue. The methods comprise isolating the motile cells of interest by the method described above, then extracting the mRNA or protein from the cells of interest, then determining mRNA or protein expression in the extraction of the cells of interest. Preferably, mRNA or protein expression of more than one gene is determined, for example using a microarray by known methods.
When mRNA expression is determined using these methods, the mRNA is preferably extracted and amplified in the motile cells of interest, then mRNA expression of the gene(s) are determined from the amplified mRNA. As described above, the mRNA in these methods is preferably amplified by reverse transcription and cDNA amplification.
In these methods, the animal is preferably a vertebrate; more preferably the animal is a mammal, such as a rodent or a human.
These methods are particularly useful for analysis of motile cells of interest in cancerous tissue, for example carcinoma tissue, such as breast cancer in mammary tissue. See Example 2. As with the methods described above, these methods can be used with tissue in culture, tissue taken from a biopsy, or directly on tissue in a living mammal.
As discussed above, preferred genes for determination of protein or mRNA expression are Arp2/3 p16 subunit, Arp2/3 p21 subunit, alpha subunit of capping protein, beta subunit of capping protein, cofilin, WAVE3, ROCK1, ROCK2, LIMK 1, PKCζ, LIM-kinase PAK, type II alpha isoform of PI4, 5 kinase, mena, tropomyosin, calpain, gelsolin-like protein (CAPG), zyxin, vinculin, integrin β1, collagen type III α1, G-protein coupled receptor 26, ZBP-1, fibroblast growth factor receptor 1, tight junction protein 2, member Ras oncogene family, and epidermal growth factor receptor. In particular, mRNA expression of the group collagen type III α1, G-protein coupled receptor 26, ZBP-1, fibroblast growth factor receptor 1, Arp2/3 p16 subunit, tight junction protein 2, member Ras oncogene family, and epidermal growth factor receptor is desirable to identify a characteristic signature of metastasis.
The present invention is also directed to methods of determining whether a cancer in a tissue of a mammal is likely to metastasize. The method comprises obtaining a microneedle or capillary filled with a porous matrix comprising a chemotactic factor; inserting the microneedle into the cancer for a time sufficient for motile cells to migrate into the porous matrix; expelling the porous matrix with motile cells from the microneedle or capillary; combining the porous matrix with microbeads, where the microbeads comprise a binding partner to a surface marker present on macrophages from the tissue; removing the microbeads; and quantifying the motile cells, where the presence of more motile cells than from the tissue when noncancerous or when comprising a non-metastatic cancer indicates that the cancer in the tissue of the mammal is likely to metastasize. Since the motile cell isolation method isolates metastatic cells from cancerous tissue, the presence of more motile cells from a cancerous tissue than from a normal tissue establishes that the cancerous tissue as metastatic potential. These methods are useful for analyzing potentially metastatic cancer in any tissue. In some preferred embodiments, the tissue is mammary tissue, since breast carcinoma is often metastatic.
These methods can be used with any animal. Preferably, the animal is a mammal, such as a rodent or a human.
As established in Wang et al., 2003, and Example 2, where the cancer is a carcinoma, and in particular a breast cancer, common other motile cells in these methods are macrophages. In those cases, a preferred binding partner is an antibody is specific for CD11b. Additionally, where the cancer is a carcinoma, a preferred chemotactic factor is an epidermal growth factor.
The motile cells resulting from these methods can be quantified by any known method. Preferred methods include the use of a fluorescence-activated cell sorter, after labeling the cells with a fluorescent marker by known methods. Alternatively, the motile cells may be quantified by simple microscopic observation, e.g., with a hemocytometer.
As described above, the microneedle or capillary is a preferably a microneedle, and the porous matrix preferably comprises matrigel.
As established in Example 2, enhancing ZBP-1 activity in a cancerous tissue decreases the metastatic potential in that tissue. Also, since collagen type III α1, G-protein coupled receptor 26, and fibroblast growth factor receptor 1 are characteristically decreased in metastatic cells, decreasing the expression or activity of those proteins would also be expected to decrease the metastatic potential of cancer cells. Thus, the present invention is further directed to methods of inhibiting metastasis of a cancer in a tissue of a mammal. The methods comprise enhancing collagen type III α1, G-protein coupled receptor 26, fibroblast growth factor receptor 1, or especially ZBP-1 activity in the tissue. It is anticipated that these methods are particularly useful for treatment of breast cancer.
In some embodiments of these methods, the collagen type III α1, G-protein coupled receptor 26, ZBP-1, or fibroblast growth factor receptor 1 activity is enhanced by transfecting the tissue with a vector comprising a collagen type III α1, G-protein coupled receptor 26, ZBP-1, or fibroblast growth factor receptor 1 transgene, where the collagen type III α1, G-protein coupled receptor 26, ZBP-1, or fibroblast growth factor receptor 1 transgene is translated from the vector in the tissue. Such methods, and vectors for executing those methods, are well known in the art, and can be established by a skilled artisan without undue experimentation.
In other embodiments, the collagen type III α1, G-protein coupled receptor 26, ZBP-1, or fibroblast growth factor receptor 1 activity is enhanced by adding a pharmaceutical composition of collagen type III α1, G-protein coupled receptor 26, ZBP-1, or fibroblast growth factor receptor 1 protein to the tissue. Preferably, the pharmaceutical composition comprises an agent to enhance penetration of the collagen type III α1, G-protein coupled receptor 26, ZBP-1, or fibroblast growth factor receptor 1 protein into the cell, such as liposomes, etc., the use of which are well known in the art.
Example 2 also establishes that several genes are upregulated in metastatic tissue. It is therefore anticipated that metastasis can be inhibited by reducing the activity of these genes in a cancer having metastatic potential. Thus, the invention is additionally directed to methods of inhibiting metastasis of a cancer in a tissue of a mammal. The methods comprise reducing the presence or activity of a protein in the tissue, where the protein is a protein whose expression is upregulated in metastatic cells. Examples of such proteins are Arp2/3 p16 subunit, Arp2/3 p21 subunit, alpha subunit of capping protein, beta subunit of capping protein, cofilin, WAVE3, ROCK1, ROCK2, LIMK 1, PKCζ, LIM-kinase, PAK, type II alpha isoform of PI4, 5 kinase, mena, tropomyosin, calpain, gelsolin-like protein (CAPG), zyxin, vinculin, integrin β1, tight junction protein 2, member Ras oncogene family, and epidermal growth factor receptor.
The presence of any of these proteins can be reduced without undue experimentation by addition of an antisense molecule, a ribozyme, or an RNAi molecule to the tissue, where the antisense molecule, ribozyme or RNAi molecule specifically inhibits expression of the protein. In these embodiments, the antisense molecule, ribozyme, or RNAi molecule can be comprised of nucleic acid (e.g., DNA or RNA) or nucleic acid mimetics (e.g., phosphorothionate mimetics) as are known in the art. Methods for treating tissue with these compositions are also known in the art. In some embodiments, the antisense molecule, ribozyme or RNAi molecule can be added directly to the cancerous tissue in a pharmaceutical composition that preferably comprises an excipient that enhances penetration of the antisense molecule, ribozyme or RNAi molecule into the cells of the tissue. In other embodiments, the antisense molecule, ribozyme or RNAi is expressed from a vector that is transfected into the cancerous tissue. Such vectors are known in the art, and these embodiments can be developed for any of the subject proteins without undue experimentation.
In other embodiments, the presence or activity of the protein is reduced by addition of an antibody or aptamer to the tissue, wherein the antibody or aptamer specifically binds and reduces the activity of the protein in the tissue. The antibody or aptamer can be added directly to the tissue, preferably in a pharmaceutical composition comprising an agent that enhances penetration of the antibody or aptamer into the tissue. Alternatively, the antibody or aptamer can be encoded on a vector that is used to transfect the cancerous tissue.
Aptamers are single stranded oligonucleotides or oligonucleotide analogs that bind to a particular target molecule, such as a protein or a small molecule (e.g., a steroid or a drug, etc.). Thus, aptamers are the oligonucleotide analogy to antibodies. However, aptamers are smaller than antibodies, generally in the range of 50-100 nt. Their binding is highly dependent on the secondary structure formed by the aptamer oligonucleotide. Both RNA and single stranded DNA (or analog), aptamers are known.
Aptamers that bind to virtually any particular target can be selected by using an iterative process called SELEX, which stands for Systematic Evolution of Ligands by EXponential enrichment. Several variations of SELEX have been developed which improve the process and allow its use under particular circumstances. See the references cited in PCT/US04/15752, all of which are incorporated by reference.
The invention is further directed to methods of determining resistance of a motile cancer cell population in an animal tissue to a chemotherapeutic agent. The methods comprise obtaining the motile cancer cell population by the methods described above; contacting the motile cancer cell population with the chemotherapeutic agent at a concentration and for a time sufficient to cause apoptosis in cancer cells susceptible to the chemotherapeutic agent; and determining apoptosis in the motile cancer cell population. In these embodiments, less apoptosis in the motile cancer cell population indicates that the motile cancer cell population is resistant to the chemotherapeutic agent. See Example 1 for some preferred embodiments of these methods.
Examples of chemotherapeutic agents that can be utilized in these embodiments are doxorobucin, cisplatin, or etoposide.
Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims, which follow the examples.
A novel observation resulting from intravital imaging of these tumors is the dramatic fragmentation of carcinoma cells when in contact with blood vessels in non-metastatic tumors (Wyckoff et al., 2000a) compared with the ability of carcinoma cells in metastatic tumors to enter blood vessels as intact whole cells. This suggests a survival advantage for metastatic cells during migration and intravasation.
In the current study we have collected a migratory population of carcinoma cells by chemotaxis to EGF containing microneedles held in the primary tumor. The collected cells were subjected to microarray analysis for differential gene expression. The results show that anti-apoptotic genes are up regulated and pro-apoptotic genes are down regulated coordinately in the migratory subpopulation. Induction of apoptosis by doxorubicin, cisplatin and etoposide in these cells demonstrates that they exhibit a lower drug induced apoptotic index and lower cell death as compared to carcinoma cells of the whole tumor. Our study indicates, for the first time, the capability of using a rat allograft model for evaluating the apoptotic status of a migratory subpopulation of tumor cells and the ability to study their resistance to chemotherapeutic agents directly. In addition, these results indicate that tumor cells that are chemotactic and migratory in response to EGF in the primary tumor have a survival advantage over stationary tumor cells.
Recently we have shown that microarray based gene expression studies can be successfully performed on cells collected by chemotaxis into microneedles held in the primary tumor (Wang et al., 2003). In the current example we have combined this method with the analysis of pro- and anti-apoptosis gene expression to determine if migratory cells in the primary tumor have a survival advantage over that of sedentary carcinoma cells within the same tumor. In addition, anticancer drugs designed against the proliferative property of cancer cells were used to investigate if the migratory cells respond equally to the antiproliferative drugs compared to their non-migratory counterparts.
Needle collection and FACS sorting of primary tumor cells. We used MTLn3-derived mammary tumors in rats (Farina et al., 1998a), and the microneedle collection method described previously (Wyckoff et al., 2000b; Wang et al., 2003), to study the gene expression pattern of invasive subpopulation of carcinoma cells within live primary tumors. Briefly, the invasive cells were collected from MTLn3 tumor using microneedles containing EGF. Macrophages were removed from this population by using MACS CD11b Microbeads (Miltenyi Biotec) as described before (Wang et al., 2003). The residual carcinoma cells were lysed for RNA extraction. To isolate the general population of carcinoma cells from primary tumor, a small piece tumor was minced, and filtered twice through a nylon-filter to obtain a single cell suspension. FACS sorting was performed on the resulting single cell suspensions based on their GFP expression in tumor cells using a Becton Dickinson (San Jose, Calif.) FACSVantage cell sorter. GFP-positive tumor cells were collected and lysed directly for RNA extraction. All the procedures were done on ice or 4° C.
RNA extraction and amplification. RNA extraction was performed using the RNeasy kit (QIAGEN), as per manufacturer's protocol and eluted with 30 μl RNase-free water. The total RNA was reverse-transcribed and amplified directly using the SMART PCR cDNA synthesis kit (Clontech, Palo Alto, Calif.) as described previously (Wang et al., 2003).
Use of pooled reference RNA as control. An equal quantity of reference RNA (pooled RNA from rat liver, spleen, brain and kidney, 4:2:1:1, Ambion Tex.) was used as a control in all our microarray experiments, which allowed us to use one of the channels as a hybridization control for all the spots on the microarray. The use of pooled reference RNA from the same species as the MTLn3 cells allowed the same interspecies cross hybridization as the background, allowing us to use Mouse cDNA microarrays for our experiments. The pooled reference RNA covers a very broad range of gene expression and is routinely used as controls in cDNA microarray studies (Zhao et al., 2002).
Probe labeling and microarray hybridization. After amplification, cDNAs were purified using the QIAquick PCR Purification Kit (Qiagen) and eluted with TE buffer. Labeling was performed using Label IT® (Mirus) following the manufacturer's instructions. Briefly, labeling reactions were prepared by mixing 10× Mirus Labeling Buffer A, purified cDNA and Cy5 (or Cy3) dye. After incubating the reaction mix at 37° C. for 1 hour, the two resulting probes were purified by passing through gel filteration columns. The purified probes were then combined and concentrated using Microcon columns. The concentrated cDNA probes were denatured at 94° C., and hybridized to an arrayed slide overnight at 50° C. Details of slide washing and image collection were described in previous studies (Wang et al., 2002; Wang et al., 2003).
Quality control and data analysis for microarrays. The scanned images were analyzed using the software Genepix (Axon Instruments, Inc. CA) and an absolute intensity value was obtained for both the channels. The entire raw data set was filtered to accommodate a requirement of at least 2 good quality measurements for each triplicate experiment. Values from only the good quality measurements (where the signal strength was more than twice the standard deviation of the background plus the background) were considered for further analysis. Two types of normalization were performed routinely in tandem on all the experiments using the GeneSpring software package (Silicon Genetics, Redwood City, Calif.). First, intensity-based-normalization was performed to take into consideration the overall signal strength of both channels and normalize the signal strength between all the different chips, reducing the chance of chip-to-chip variability. Second, a reference channel-based normalization was performed which takes into consideration the reference channel (which in this case is pooled reference RNA) and normalizes the values in all the spots. This reduces the chance of spot to spot variability. The final data was a result of both these types of normalization.
Significance analysis of microarrays. In order to determine the significance of up-regulated and down-regulated genes, we performed significance analysis using the software Significance Analysis of Microarrays (SAM) (8). Briefly after normalizing the data as mentioned above the data was log transformed to Log 2 and subjected to SAM analysis. The algorithm performs a significance analysis by comparing the relative variance of the replicates between the samples. The result were determined at 5% False Discovery Rate (FDR).
Real time PCR confirmation. To verify the data obtained from microarrays, QRT-PCR analysis of selected over expressed and under expressed genes was performed by using the ABI 7900 (Applied Biosystems, Foster City, Calif.) with sequence-specific primer pairs for all genes tested (see Supplement Table 2 for primer sequences, amplicon size and annealing temperature) as described previously (Wang et al., 2002). SYBR Green was used for real-time monitoring of amplification. Results were evaluated with the ABI Prism SDS 2.0 software. All the genes tested for regulation were compared to at least two housekeeping genes (Beta actin and GAPDH).
Cell culture and apoptosis assay. The cells extruded from the needles and tumor cells FACS sorted were cultured in DMEM 20% FCS along with streptomycin and penicillin, for 16 hrs. Subsequently, the cells were challenged with either doxorobucin (17 μM) or cisplatin (50 μM) or etoposide (50 μM) for 1 hr, washed and allowed to recover for 24 hrs. The cells were then subjected to an apoptosis assay kit containing Annexin V Cy5 for staining the apoptotic cells and Propedium Iodide (PI) for staining the dead cells (BD Biosciences San Jose, Calif.). After staining the cells using the manufacturer's protocol, the cells were observed under a fluorescent microscope in the green, red and high red channel for GFP, PI and Cy5 respectively. The total number of GFP cells counted was compared to the number of PI positive and Annexin V-Cy5 positive cells.
GFP-labeled tumor cells were injected into rat mammary fat pads, and primary tumors were allowed to grow for 2-2.5 weeks. To provide insight into the pattern of gene expression associated with chemotactic and migratory carcinoma cells in vivo, we compared the gene expression profile of a subpopulation of tumor cells collected from the primary tumor by chemotaxis into a microneedle, called the invasive cells, with that of the general population of GFP-expressing tumor cells sorted from the whole primary tumor by FACS sorting (
Another category of genes found to be significantly regulated in the chemotactic and migratory population of cells in the primary tumor is that of cell motility. These genes have been explained in detail in an accompanying paper. Since there are 5 steps of the motility cycle which are coordinated to assure efficient cell motility, the up regulation of genes for major effectors in the pathways of each step predicts that the invasive cells will have a heightened migratory activity compared to carcinoma cells of the general tumor population and this is consistent with the high velocities of migration seen in tumors (Condeelis and Segall, 2003).
Regulation of pro and anti-apoptotic genes along with mechanical stability genes. Of particular relevance to survival, stress and apoptosis associated genes showed large changes in regulation (
A potential explanation for mechanical stability and survival advantage observed in invasive cells (Jolly and Morimoto, 2000; Condeelis et al., 2003) is the large relative over expression of cytokeratins by carcinoma cells and the suppression of apoptosis gene expression in metastatic tumors and cell lines (Wang et al., 2002). Keratins form the largest subfamily of intermediate filament proteins that play critical roles in the mechanical stability of epithelial cells subjected to shear forces (Coulombe and Omary, 2002). In addition, it was found that carcinoma cells in metastatic tumors and in culture express laminins and cadherins and apoptosis suppressor genes at high levels, all of which might contribute to survival during intravasation and in the circulation (Wang et al., 2002). In contrast, carcinoma cells in non-metastatic tumors and in culture express genes involved in programmed cell death at higher levels. The combination of these factors may contribute to the increased numbers of viable carcinoma cells in the circulation of metastatic tumors and to fragmentation during intravasation and cell death seen in non metastatic tumors (Wyckoff et al., 2000a; Condeelis et al., 2003).
In addition, the anti-apoptotic and pro-apoptotic genes are inversely regulated in the chemotactic and migratory population of cells in the primary tumor (
Drug resistance in invasive cells measured by apoptosis assay. The finding that the anti-apoptotic genes are up regulated in the invasive cells prompted us to study the functional importance of this finding and whether these cells indeed have a survival advantage over the resident population. We challenged the invasive cells with three most commonly used anticancer drugs, doxorobucin, cisplatin and etoposide. Previous studies have shown that these drugs to induce apoptosis in the MTLn3 cells (Huigsloot et al., 2002). We performed these studies on the invasive and general populations of cells from MTLn3-derived tumors. After treatment with the drugs the cells were allowed to recover for 24 hr. Subsequently, the apoptotic index and cell viability was measured as described in the Methods section. The results, shown in
Most of the anticancer drugs like doxorobucin, cisplatin and etoposide are designed against the proliferative cells (Awada et al., 2003) making them cytotoxic. Recently, there is an increasing effort to make cytostatic drugs, which prevent the proliferation and invasion as opposed to killing the cells. There has been a demand in the field to have a method to isolate these invasive cells and look for the effect of cytostatic drugs specifically on invasive cells. We believe that in our studies we have demonstrated a method that makes possible this analysis on migratory cells of the primary tumor.
Coordinate regulation of survival genes in the invasive cells. Previous studies have shown that the anti-apoptotic pathways are overexpressed in the metastatic cell lines (Real et al., 2002), and these cells have a survival advantage via Stat3 dependent over expression of BCL-2. In our study we find that a number of anti-apoptotic genes are upregulated. These genes belong to all three pathways, rendering a survival advantage to the cells. On one hand upregulation of the defender against death 1 (DAD1) gene indicates that the extrinsic pathway is blocked in these invasive cells. On the other hand there are signs of down regulation of the intrinsic pathway as well by the over expression of ornithine decarboxylase 1 (ODC1). Upregulation of the expression of apoptosis inhibitor 1, 4 and 5 (Api1, Api4 and Api5) genes indicate an involvement of the convergence pathway as well. Finally there is the robust over expression of the genes like immediate early response gene 3 (IER3) which is a multi-pathway regulator involving the NFκB family of transcription factors (Reed, 2003). Simultaneously a number of the pro-apoptotic were down regulated, significantly a key regulator of the intrinsic pathway APAF-1 was down-regulated in the invasive cells.
In the current study we have attempted to investigate the pathways leading to metastasis, which provides this survival advantage to these cells. In previous studies, authors have used cell lines derived from an established secondary tumor (Real et al., 2002). We on the other hand have performed a dynamic assessment of the process of metastasis and have captured the cells prior to the entry into the blood.
In our studies we have identified pathways, which get regulated in the invasive cells, which are not proliferative (
We combined chemotaxis-based cell collection and cDNA microarray technology to identify the gene expression profile of invasive carcinoma cells from primary mammary tumors in experimental animals. Expression of genes involved in cell division and survival, metabolism, signal transduction at the membrane, and cell motility were most dramatically increased in invasive cells, indicating a population that is not dividing but intensely metabolically active and motile. In particular, the genes coding for the minimum motility machine that regulates β-actin polymerization, and therefore the motility of carcinoma cells, were dramatically up regulated, while ZBP-1, which regulates the localization of β-actin, was downregulated. This pattern of expression suggested ZBP-1 is a suppressor of invasion. Overexpression of ZBP-1 suppressed chemotaxis and invasion in primary tumors and inhibited metastasis from tumors generated using intensely metastatic cell lines. We identified genes important for the invasion of tumor cells in this study. We demonstrate that the identification of these genes provides new insight for the invasion process and the regulation of invasion and demonstrate the importance of these pathways in invasion and metastasis by altering the expression of a master gene, ZBP-1.
A potential approach to determine the cellular mechanisms that contribute to invasion is to collect live cells from the primary tumor based on their ability to invade, and profile their gene expression patterns. One of the properties correlated with metastasis is chemotaxis to blood vessels (Wyckoff et al., 2000a). This cell behavior allows cells to orient and move toward blood vessels facilitating their intravasation. Based on these observations, we have developed a complementary approach to directly select for live, invasive cells from live primary tumors in intact rats using a microneedle containing a chemoattractant to mimic chemotactic signals from blood vessels and/or surrounding tissue (Wyckoff et al., 2000b). Overexpression of the EGF receptor and other family members has been correlated with poor prognosis (Nicholson et al., 2001), and therefore we have developed methods for collecting invasive tumor cells that use gradients of EGF to direct tumor cell invasion into microneedles. Gradients of EGF receptor ligands can be generated by diffusion from the blood as well as stromal cells in the tumor microenvironment (O'Sullivan et al., 1993; LeBedis et al., 2002). Thus we are using a physiologically relevant stimulus to mimic tumor cell invasion induced at the borders of tumors near blood vessels and other elements of connective tissue. We have used this method to test the hypothesis that chemotaxis to blood vessels is an important form of egress of carcinoma cells from the primary tumor. Cells have been collected from live rats with tumors that have been generated by the injection of carcinoma cells with different metastatic potential (Wyckoff et al., 2000b), and from live mice with mammary tumors derived from the expression of the PyMT oncogene (Lin et al., 2002; Lin et al., 2001; Wang et al., 2003).
In order to perform gene expression profiling using high density arrays on the few hundred cells commonly collected in microneedles, it is necessary to amplify mRNA by about 1000 fold to the amounts required for arrays. It is also necessary to have a pure cell population. Both of these conditions have been met using recently developed methods (Wang et al., 2003). RNA obtained from as few as 400 cells collected in a single microneedle from the primary tumor, when amplified as cDNA using the PCR based cDNA amplification technique (18), can be used for microarray expression analysis. We have validated this amplification method and demonstrated that it retains the original mRNA's copy abundance and complexity in the amplified product (Wang et al., 2003).
In the current study, the collection of invasive cells from the primary tumor using chemotaxis is combined with gene expression profiling using the above-described PCR based cDNA amplification techniques. This technology has allowed the characterization of gene expression patterns of invasive carcinoma cells from the primary tumor without potential artifacts that arise from the culturing of small populations of cells. We identified a group of genes that define motility pathways that are coordinately up regulated in invasive cells. These pathways may account for the enhanced migratory behavior of the collected cells. Furthermore, we tested the contribution of these pathways to invasion and metastasis by altering the expression of a master gene that regulates the expression of the common molecule on which these pathways converge.
Needle collection and FACS sorting of primary tumor cells. We used MTLn3-derived mammary tumors in rats (Farina et al., 1998b), and the microneedle collection method described previously (Wyckoff et al., 2000b; Wang et al., 2003), to study the gene expression pattern of invasive subpopulation of carcinoma cells within live primary tumors. Briefly, the invasive cells were collected from MTLn3 tumor using microneedles containing EGF. Cell collection was imaged using a multi-photon microscope as described previously (Wang et al., 2002) by inserting the bevel of a matrigel and EGF containing needle into the field of view. A 50 mm z-series consisting of 5 mm steps allows for the imaging of a large number of cells around the needle. 1/10th of the volume from each needle was used to determine the number of cells collected. From the remaining 9/10 volume from the microneedle, macrophages were removed by magnetic separation, and RNA extraction was done as previously described (Wang et al., 2003).
To isolate the general population of carcinoma cells from primary tumor, a small piece tumor was separated from the whole tumor, minced, and filtered twice through a nylon-filter to obtain a single cell suspension. FACS sorting was performed on the resulting single cell suspensions based on their GFP expression in tumor cells. GFP-positive tumor cells were collected into a tube and lysed directly for RNA extraction. All the procedures were done on ice or 4° C.
Because EGF and Matrigel are present in the needle, as a control experiment, we identified genes whose expression is altered by EGF or Matrigel application. Carcinoma cells from the primary tumor were FACS-sorted as described above. The resulting cells were split and plated on Mettek dishes covered with Matrigel (1:5) in the presence or absence of EGF (1 nM) for 4 hr at 37° C. The cells were then lysed directly on the dish for total RNA extraction.
An equal quantity of reference RNA (pooled RNA from rat liver, spleen, brain and kidney, 4:2:1:1, Ambion Tex.) was used to generate probes as a control in all our microarray experiments, which allowed us to use one of the channels as a hybridization control for all the spots on the microarray. The use of pooled reference RNA from the same species as the MTLn3 cells allowed the same interspecies cross hybridization as the background, allowing us to use mouse cDNA microarrays for our experiments. The pooled reference RNA covers a very broad range of gene expression and is routinely used as controls in cDNA microarray studies (Zhao et al., 2002).
RNA amplification, probe labeling and microarray hybridization. The RNA was then concentrated by ethanol precipitation and re-dissolved in 3.5 μl DEPC water. The total RNA was reverse-transcribed directly using the SMART PCR cDNA synthesis kit (Clontech, Palo Alto, Calif.) according to the manufacturer's protocol. After amplification, cDNAs were purified using the QIAquick PCR Purification Kit (Qiagen) and eluted with TE buffer. Labeling was performed using Label IT® (Mirus) following the manufacturer's instructions. Briefly, labeling reactions were prepared by mixing 10× Mirus Labeling Buffer A (10 μL), purified cDNA (3.5 μg), Cy5 (or Cy3) dye (5 μL) in a total volume of 100 μL. After incubating the reaction mix at 37° C. for 1 hr, the two resulting probes were purified by passing through SigmaSpin columns followed by Qiaquick columns. The purified Cy-3 and Cy-5 DNA probes were then combined and concentrated using micron YM 50 columns. Microarray analysis was performed by using cDNA microarrays made at AECOM. About 27,000 mouse genes (Incyte Genomics) were precisely spotted onto a single glass slide. Detailed descriptions of microarray hardware and procedures are available from http://129.98.70.229/. Microarray analysis was performed in three independent repeats. Details of slide hybridization, washing and image collection were described in previous studies (Wang et al., 2003; Wang et al., 2002).
Quality control and data analysis for microarrays. The scanned images were analyzed using the software Genepix (Axon Instruments, Inc. CA) and an absolute intensity value was obtained for each of the channels for the reference RNA and the RNA derived from the cells. The entire raw data set was filtered to accommodate a requirement of at least two good quality measurements for each triplicate experiment. Values from only the good quality measurements (where the signal strength was more than twice the standard deviation of the background plus the background) were considered for further analysis. Two types of normalization were performed routinely in tandem on all the experiments using the GeneSpring software package (Silicon Genetics, Redwood City, Calif.). First, intensity-based-normalization was performed which takes into consideration the overall signal strength of both channels and normalizes the signal strength between all the different chips, reducing the chance of chip-to-chip variability due to the experiment being performed on different days. Second, a reference-channel-based normalization was performed which takes into consideration the reference channel (which in this case is pooled reference RNA) and normalizes the values in all the spots. This reduces the chance of spot to spot variability. The final data was a result of both these types of normalization.
In order to determine the significance of upregulated and downregulated genes, we calculated the standard deviation of the reference channel in all of the chips and found it to be 0.18 and used 5× standard deviation as the cutoff, indicating a high level of fidelity in our data above 2-fold. Genes that were up- or down-regulated in the arrays performed on control samples (FACS sorted cells which were treated with Matrigel and EGF) were removed from the final list of genes specific to the invasive subpopulation of tumor cells.
Real time PCR confirmation. To verify the data obtained from microarrays, QRT-PCR analysis of selected overexpressed and underexpressed genes was performed by using the iCycler Apparatus (Bio-Rad) with sequence-specific primer pairs for all genes tested (see Supplementary Table 3 for primer sequences, amplicon size and Tm) as described previously (Wang et al., 2002). The SYBR Green PCR Core Reagents system (Perkin-Elmer Applied Biosystems) was used for real-time monitoring of amplification.
Plasmid construction, cell culture transfection, infection and generation of ZBP-1 stable expression cell lines. FLAG-ZBP-1 (Farina et al., 2003) was digested with BamHI/XbaI and inserted into the BamHI/XbaI sites of EGFP-C1 (Clontech). The EGFP-FLAG-ZBP-1, which encodes a fusion protein, was then isolated as Eco47III/XbaI restriction fragment, blunt ended and inserted into a filled XhoI site of pMCSVneo (Clontech). This vector contains a viral packaging signal, neomycin resistance gene, and the 5′ and 3′ long terminal repeats from the murine PCMV virus. As a result, the LTR drives high-level constitutive expression of the EGFP-FLAG-ZBP-1 gene. PHOENIX cells were cultured under standard conditions (Dal Canto et al., 1999) and were transfected with EGFP-FLAG-ZBP-1 using FUGENE (Roche). Retroviral supernatant was harvested and used to infect MTLn3 cells as previously described (Dal Canto et al., 1999). Stable MTLn3 cells were selected in the presence of neomycin.
Microchemotaxis chamber assay. A 48-well microchemotaxis chamber (Neuroprobe) was used to study the chemotactic response to EGF, following the manufacturer's instructions and as described previously (SEGALL ET AL., 1996).
Blood burden, single cells in the lung, and metastases. MTLn3-ZBP-1 or MTLn3-GFP cells were injected into the mammary fat pads of female Fischer 344 rats. Tumor cell blood burden was determined as described previously (Wyckoff et al., 2000a). After blood removal and euthanization of the rat, the lungs were removed and the visible metastatic tumors near the surface of the lungs were counted. 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 20× objective, and all metastases in a section containing more than five cells were counted (Wyckoff et al., 2000a).
Gene expression patterns unique to invasive tumor cells. GFP-labeled tumor cells were injected into rat mammary fat pads, and primary tumors were allowed to grow for 2-2.5 weeks. To provide insight into the pattern of gene expression associated with chemotactic and invasive carcinoma cells in vivo, we compared the gene expression profile of the subpopulation of invasive tumor cells collected from the primary tumor by chemotaxis into a microneedle with that of the general population of GFP-expressing tumor cells sorted from the whole primary tumor by FACS (
The collected cells were a mixture of carcinoma cells (75%) and macrophages (25%) as shown previously (Wang et al., 2003). Macrophages were removed by binding to magnetic beads conjugated with anti-MAC-1, giving a greater than 96% pure population of carcinoma cells for analysis (Wang et al., 2003). The general population of primary tumor cells was collected by FACS sorting and plated either on matrigel or matrigel and EGF for 4 hours, the interval of time required for microneedle collection, to mimic the collection conditions prior to purification of the RNA. These controls were done to subtract patterns of gene expression resulting from stimulating cells with matrigel and EGF, and allowed identification of the gene expression signature of the invasive cells (
Differential gene expression analysis comparing the invasive and general populations of tumor cells revealed 1366 genes that were differentially expressed (Supplementary Table 4). As shown in
In order to determine the significance of changes in gene expression in each of the functional categories of the genes represented in our arrays, Chi-square or SAM analysis were performed. The functional categories of Cell Cycle, Apoptosis, Metabolism, Protein Metabolism, Cytoskeleton & ECM, Growth Factor & Signal Transduction and Nucleic Acid Chemistry were found to be statistically significant in the invasive cells by Chi-square (Zigeuner et al., 2004) or SAM analysis (Tusher et al., 2001). Random sets of equal numbers of genes did not generate the same pattern of up and down regulation indicating that the pattern was not observed by chance (P<0.05). Similarly, clustering the results from all genes of the general population in the same space of all genes on the microarray did not yield an outcome similar to the invasion signature (P>0.05). A detailed table indicating each of the functional categories and the significant analysis is given as a supplementary table (Supplementary Table 5) indicating the number of genes printed on the microarray and the number regulated in invasive cells.
It is interesting to note that the number of genes whose expression is regulated up or down in the functional category called cell cycle (
Finally, there is an increase in the number of regulated genes in the Cytoskeleton and Extracellular Matrix category (
Genes involved in invasion. In order to be collected by the microneedle, the carcinoma cells must be capable of moving toward and crawling into the extracellular matrix of the microneedle within the 4 hr. collection interval. If a cell moves 2 cell diameters during this interval to gain entry to the microneedle it would have a minimum speed of 0.2 μm/min, similar to the velocity of carcinoma cells in vitro. However, carcinoma cells move in the primary tumor at speeds up to 10× this minimum value (Condeelis and Segall, 2003) indicating that cells from hundreds of microns away from the microneedle can be recruited for collection and that the cells may penetrate the extracellular matrix in the collecting microneedle. Consistent with this prediction is the observation that carcinoma cells are found within the matrix of the collecting microneedle, indicating that cells have traveled hundreds of microns during the collection interval. This indicates speeds much greater than 0.2 μm/min in vivo.
The motility cycle of chemotactic crawling cells is composed of 5 steps; signal sensing, protrusion toward the signal source, adhesion, contraction and tail retraction (Bailly and Condeelis, 2002). As shown in Table 1 and
List of motility related genes differentially expressed in the invasive sub-population of tumor cells. Genes associated with motility are displayed in this table and the ratios on the right indicated the level of expression in the invasive compared to the general population of cells of the primary tumor.
The protrusion of a pseudopod toward the chemotactic signal initiating the motility cycle is the key step in defining the leading edge of the cell and therefore its direction during migration (Bailly and Condeelis). Protrusion is driven by actin polymerization-based pushing against the cell membrane and this requires the minimum motility machine composed of cofilin, Arp2/3 complex and capping protein acting on their common downstream effector, β-actin (Mogilner and Edelstein-Keshet, 2002). The elevated expression of any one of these three effectors is expected to significantly enhance the speed of migration of cells since doubling the amount of either Arp2/3 complex, capping protein or cofilin in the reconstituted minimum motility machine can increase protrusion rate by 10× (Loisel et al., 1999). Therefore, it is significant, as shown in
Similar increases in both the stimulatory and inhibitory parts of the capping protein pathway are upregulated in invasive carcinoma cells (
Genes coding for proteins involved in myosin mediated contraction and tail retraction (tropomyosin, ROCK1, and calpain), gelsolin-like protein (CAPG) and adhesion molecules (zyxin, vinculin, and integrin β1) are up regulated, as well (Table 1). ROCK plays a crucial role in cell adhesion and motility and is linked to pathogenesis and progression of several human tumors (Sahai and Marshall, 2003). Integrin β1 has previously been implicated in the ability of an experimentally transformed fibroblast cell line to metastasize (Brakebusch et al., 1999), and its expression is increased in upper aerodigestive tract and cervical squamous cell carcinomas (Van Waes et al., 1995).
ZBP-1 as a master gene regulating cell polarity. A gene that is strongly downregulated in invasive cells is Zip-code binding protein (ZBP-1) (Table 1 and
To test the hypothesis that ZBP-1 expression can suppress invasion, the full length ZBP-1 gene was subcloned in a pMCSVneo vector (
To investigate the chemotactic properties of the ZBP-1 overexpressing cells, two independent clones of ZBP-1 overexpressing cell lines were characterized. Chemotaxis was measured in a Boyden chamber. ZBP-1 overexpressing cells migrated through the filter in response to EGF poorly compared to the parental MTLn3 cells (
Injection of the ZBP-1 over expressing cells into the mammary fat pads of rats resulted in tumors that were less metastatic. The metastatic potential of these tumors was characterized as the number of tumor cells present in circulating blood (
Signature of invasive carcinoma cells. By comparing gene expression patterns of invasive cells to those of the general population of carcinoma cells in the same primary tumor, we were able to find patterns in the regulation of gene expression unique to the invasive subpopulation of cells. Our results indicate that the regulation of genes involved in cell division, metabolism, signal transduction at the membrane, cell survival and cell motility was most dramatically changed in invasive cells predicting a population that is neither proliferating nor apoptotic but intensely metabolically active and motile. While increased cell proliferation during tumor development has been associated with poor prognosis in patients (Evan and Vousden, 2001), the results reported both here and in previous studies (Wyckoff et al., 2000a) indicate that tumor size is neither correlated with invasion nor the ability of cells to metastasize to distant organs. In addition, invasive cells show down regulation of genes associated with apoptosis and up regulation of genes for cell survival. This is consistent with previous work where it was shown that cell survival genes were up regulated in metastatic tumors as compared to non-metastatic tumors (Wang et al., 2002) and suggests that the invasive subpopulation may contribute disproportionally to this expression profile in whole metastatic tumors.
In a previous study, the genes differentially expressed between metastatic and non-metastatic cells in culture and the tumors derived from them by orthotopic injection of the cells into the mammary gland were compared. We found that those coding for molecules involved in cell adhesion, motility, cell polarity, and signal transduction were most different. Comparing the gene expression patterns in non-metastatic tumors to metastatic tumors from the previous study (20), with the differences between the invasive cell population and general population of the same tumor defined here, we have found that a subset of genes (Table 2), maintain the same patterns of regulation in both studies. This suggests that the invasive subpopulation of cells collected from primary tumors with microneedles has enhanced an expression pattern of a subset of genes that is characteristic of the differences between metastatic and non-metastatic cell lines and tumors. This is emphasized by the fact that the invasive subpopulation of cells collected by chemotaxis into microneedles is from tumors derived from a single cell line, the MTLn3 cell line. This indicates that as the tumor progresses, highly invasive cells are selected in which a pattern of gene expression present in metastatic cells and tumors is enhanced over the pattern of expression of the cells that remain behind in the primary tumor.
Differentially expressed genes common to invasive cells identified in this study and to metastatic tumors and cell lines identified in a previous study. Common genes regulated in a similar way in all the three samples are displayed here. Dark shading indicates overexpression and light shading represents repression. Taken together these genes outline a signature of invasion and indicate that a number of interacting pathways are involved in invasion.
Cell motility genes and their roles in cancer invasion. Chemotaxis to EGF is required for collection of cells into the microneedle because significant numbers of cells are not collected in the absence of EGF (Wyckoff et al., 2000b), and EGF-R activity is required for the collection of carcinoma cells. Therefore, the motility related genes that are differentially expressed in the invasive population may also contribute to EGF-dependent chemotaxis and enhanced migration in the primary tumor. A major result of this study is the finding that genes from the pathways associated with the minimum motility machine are greatly up regulated, predicting that protrusion velocity will be increased. Since protrusion sets cell direction and, therefore, defines chemotaxis, this step in the motility cycle may be key in determining invasive potential. Furthermore, as seen in
Our results show that cofilin, LIM-kinase 1, ROCK 1, 2 and PKCζ are all over expressed in highly invasive carcinoma cells. In previous studies, LIM-kinase 1 was shown to be over expressed in metastatic breast and prostate tumors (Davila et al., 2003; Yoshioka et al., 2003). Over expression of LIM Kinase 1 in tumor cell lines increased their motility and invasiveness in vitro (Davila et al., 2003) and in vivo (Yoshioka et al., 2003). Reduction in the expression of LIM-kinase 1 in metastatic prostate cell lines deceased invasiveness in matrigel invasion assays (Davila et al., 2003). These results are consistent with ours shown here that LIM-kinase 1 is more highly expressed in the invasive cell population.
In contrast, it has been reported that increased expression of LIM-kinase 1 in carcinoma cells significantly reduces their cell motility as the phosphorylation of cofilin by LIM-kinase 1 abolishes EGF induced actin nucleation and polymerization (Zebda et al., 2000). Our study may resolve this paradox by demonstrating that in invasive cells collected from primary tumors both the stimulatory and inhibitory pathways to LIM-kinase 1 and cofilin are over expressed together thereby increasing the steady state rate of cofilin activation in invasive carcinoma cells resulting in enhanced cell motility as predicted previously (Davila et al., 2003; Yoshioka et al., 2003; Zebda et al., 2000; Sahai et al., 2001).
ZBP-1 in metastasis. In general, cells that lack a fixed intrinsic polarity are more chemotactic to exogenous gradients presumably because there is no intrinsic polarity to be overcome by the exogenous chemotactic signal and the cell can turn in any direction to respond to a gradient (Parent and Devreotes, 1999; Iijima et al., 2002). The presence of intrinsic polarity in carcinoma cells in tumors is correlated with the stable polarization of actin polymerization at one end of the cell only, resulting in polarized locomotion. In contrast, carcinoma cells in metastatic MTLn3 tumors are unpolarized except when they are near blood vessels where they become polarized toward the blood space (Shestakova et al 1999; Wyckoff et al., 2000a). These results suggest that cells that have proceeded through the epithelial mesenchymal transition (EMT) to the point where all remnants of the intrinsic cell polarity of the original epithelium are lost, such as MTLn3 cells, are more efficient at responding to external chemotactic signals and more attracted to blood vessels in the primary tumor.
A key difference between metastatic and non-metastatic cells that may explain the inverse correlation between intrinsic cell polarity and metastasis is loss of the ability by metastatic cells to localize mRNA and proteins that define cell polarity (Shestakova et al., 1999). The mechanism relating β-actin mRNA targeting to the leading edge and intrinsic cell polarity involves the localization of β-actin nucleation to the leading edge during motility. Disruption of mRNA targeting to the leading edge using oligonucleotides that disrupt the interaction between ZBP-1 and the targeting sequence in the mRNA, the zip-code, results in delocalization of mRNA and β-actin nucleation sites, and the disruption of cell polarity (Shestakova et al., 2001). Highly metastatic cells have lost the ability to target mRNA for β-actin, which may be required to maintain a localized supply of β-actin protein to support a stable leading edge in response to the activity of the minimum motility machine. Without a stable leading edge, the intrinsic polarity of the metastatic cell is lost and cell direction is determined by signals from blood vessels, resulting in chemotaxis toward blood vessels and intravasation (Wyckoff et al., 2000a; Condeelis and Segall, 2003). Molecular profiling of MTLn3 and MTC cells and tumors using both cDNA arrays and QRT-PCR demonstrates that non-metastatic MTC cells and tumors express much higher levels of ZBP-1 than the metastatic MTLn3 cells and tumors (Wang et al., 2002). Furthermore, in the present study, invasive tumor cells isolated from primary mammary tumors using chemotaxis express much lower levels of ZBP-1 than cells that remain behind in the primary tumor even though both cell populations were derived from the same progenitor MTLn3 cells (Table 2). Furthermore, as shown in the current study, invasive carcinoma cells expressing experimentally increased levels of ZBP-1 after transfection with ZBP-1 expression vectors exhibit decreased chemotaxis, and invasion into microneedles, and the tumors made from cell grafts of these ZBP-1 expressing cells are much less metastatic by several criteria.
The results reported here indicate that ZBP-1 is a ‘metastasis repressor’ and, together with mRNA targeting status and analysis of tumor cell polarity around blood vessels discussed above, might be used in prognosis and therapy.
In view of the above, it will be seen that the several advantages of the invention are achieved and other advantages attained.
As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.
All references cited in this specification are hereby incorporated by reference. The discussion of the references herein is intended merely to summarize the assertions made by the authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.
Mus musculus TSC22-related inducible leucine zipper 1b (Tilz1b) mRNA,
Mus musculus formin binding protein 11 (FBP11) mRNA, complete cds
Mus musculus serine-threonine kinase receptor-associated protein mRNA
Mus musculus strain Swiss Webster/NIH actin-associated protein palladin
Mus musculus receptor activity modifying protein 2 mRNA, complete cds
Mus musculus mRNA for Sid6061p, complete cds
Mus musculus mRNA for phosphorylated adaptor for RNA export (PHAX
Mus musculus heat shock protein (HSPC030) mRNA, complete cds
Mus musculus brain protein 44-like protein (Brp441) mRNA, complete cds
Mus musculus protein inhibitor of activated STAT protein PIAS1 mRNA, c
Mus musculus mRNA for heterogeneous nuclear ribonucleoprotein H
Mus musculus radio-resistance/chemo-resistance/cell cycle checkpoint c
Mus musculus neuronal calcium sensor-1 (NCS-1) mRNA, complete cds
Mus musculus KOI-4 gene, partial cds
Mus musculus TBX1 protein mRNA, complete cds
Mus musculus secretory carrier membrane protein 4 mRNA, complete cd
Mus musculus cerebellar postnatal development protein-1 (Cpd1) mRNA,
Mus musculus LIM-kinase1 (Limk1) gene, complete cds; Wbscr1 (Wbscr1
Mus musculus hsp40 mRNA for heat shock protein 40, complete cds
Mus musculus mRNA for 26S proteasome non-ATPase subunit
Mus musculus mitotic checkpoint component Mad2 mRNA, complete cds
Mus musculus geminin mRNA, complete cds
Mus musculus cleavage and polyadenylation specificity factor 73 kDa sub
Mus musculus Smt3A protein mRNA, complete cds
Mus musculus mRNA for RNase 4, complete cds
Mus musculus truncated SON protein (Son) mRNA, complete cds
Mus musculus major histocompatibility complex region NG27, NG28, RP
Mus musculus insulin-like growth factor I receptor mRNA, complete cds
Mus musculus Tera (Tera) mRNA, complete cds
M. musculus mRNA for glutamyl-tRNA synthetase
Mus musculus SIK similar protein mRNA, complete cds
Mus musculus chromosome segregation protein SmcB (SmcB) mRNA, c
Mus musculus dUB-type TGT mRNA for deubiquitinating enzyme, comple
M. musculus mRNA for fibromodulin
Mus musculus casein kinase 2 beta subunit
Mus musculus ubiquitin-specific protease
Mus musculus uroporphyrinogen III synthase gene, promoter,
Mus musculus NIMA (never in mitosis gene a)-related expressed kinase 7
indicates data missing or illegible when filed
Mus musculus centrin (Cetn2) gene, complete cds
Mus musculus Tera (Tera) mRNA, complete cds
M. musculus mRNA for ribosomal protein S5
Mus musculus Smt3A protein mRNA, complete cds
Mus musculus mRNA for 26S proteasome non-ATPase subunit
Mus musculus neuronal calcium sensor-1 (NCS-1) mRNA, complete cds
Mus musculus mRNA for mitochondrial acyl-CoA thioesterase, clone 1
Mus musculus succinyl-CoA synthetase (Sucla1) mRNA, complete cds
Mus musculus fallotein mRNA, complete cds
Mus musculus radio-resistance/chemo-resistance/cell cycle checkpoint control protei
Mus musculus 14-3-3 protein gamma mRNA, complete cds
Mus musculus elongation factor 1-beta homolog mRNA, complete cds
Mus musculus mRNA for sid2057p, complete cds
Mus musculus dUB-type TGT mRNA for deubiquitinating enzyme, complete cds
Mus musculus mRNA for Sid6061p, complete cds
Mus musculus secretory carrier membrane protein 4 mRNA, complete cds
Mus musculus Smt3A protein mRNA, complete cds
Mus musculus ASC-1 mRNA, complete cds
Mus musculus mRNA for RNase 4, complete cds
Mus musculus ubiquitin conjugating enzyme UBC9 mRNA, complete cds
Mus musculus mRNA for phosphorylated adaptor for RNA export (PHAX gene)
Mus musculus major histocompatibility complex region NG27, NG28, RPS28, NADH
Mus musculus cerebellar postnatal development protein-1 (Cpd1) mRNA, partial cds
Mus musculus LIM-kinase1 (Limk1) gene, complete cds; Wbscr1 (Wbscr1) gene, alte
Mus musculus receptor activity modifying protein 2 mRNA, complete cds
M. musculus mRNA for gas5 growth arrest specific protein
Mus musculus LNR42 mRNA, complete cds
Mus musculus strain Swiss Webster/NIH actin-associated protein palladin mRNA, pa
Mus musculus mkf-1 mRNA, complete cds
M. musculus mRNA for Pr22 protein
Mus musculus KOI-4 gene, partial cds
Mus musculus mRNA for partial LaXp180 protein
Mus musculus formin binding protein 11 (FBP11) mRNA, complete cds
Mus musculus 14-3-3 protein beta mRNA, complete cds
M. musculus mRNA for glutamyl-tRNA synthetase
Mus musculus heat shock protein (HSPC030) mRNA, complete cds
Mus musculus TCR beta locus from bases 250554 to 501917 (section 2 of 3) of the c
Mus musculus DEBT-91 mRNA, complete cds
Mus musculus mRNA for Sid6061p, complete cds
M. musculus mRNA for neuronal protein 15.6
Mus musculus drebrin E2 mRNA, complete cds
Mus musculus mRNA for mDj3, complete cds
Mus musculus mRNA for initiation factor 2-associated 67 kDa protein, complete cds
M. musculus (C57 Black/6X CBA) LAL mRNA for lysosomal acid lipase
M. musculus GAS 6 mRNA associated with growth-arrest
Mus musculus mRNA, complete cds, clone: 2-31
Mus musculus chromosome X contigB; X-linked lymphocyte regulated 5 gene, Zinc f
Mus musculus truncated SON protein (Son) mRNA, complete cds
Mus musculus acidic ribosomal phosphoprotein P1 mRNA, complete cds
Mus musculus mRNA for ribosomal protein L35a
Mus musculus ribosomal protein L23 (Rpl23) gene, complete cds
Mus musculus SIK similar protein mRNA, complete cds
Mus musculus CYP2C40 (Cyp2c40) mRNA, complete cds
M. musculus mRNA for e1 protein
Mus musculus SPARC-related protein (SRG) mRNA, complete cds
Mus musculus clone TA-9 ATP synthase b chain homolog mRNA, partial cds
Mus musculus TSC22-related inducible leucine zipper 1b (Tilz1b) mRNA, complete c
Mus musculus mRNA for sid2057p, complete cds
Mus musculus chromosome segregation protein SmcB (SmcB) mRNA, complete cds
Mus musculus brain protein 44-like protein (Brp44I) mRNA, complete cds
Mus musculus CRIPT protein mRNA, complete cds
Mus musculus SPARC-related protein (SRG) mRNA, complete cds
Mus musculus antioxidant enzyme AOE372 mRNA, complete cds
Mus musculus NADP-dependent isocitrate dehydrogenase (Idh) mRNA, complete cd
M. musculus ASF mRNA
Mus musculus X chromosome: L1cam locus
Mus musculus RW1 protein mRNA, complete cds
Mus musculus domesticus mitochondrial carrier homolog 1 isoform a mRNA, comple
Mus musculus (clone: pMAT1) mRNA, complete cds
Mus musculus mitotic checkpoint component Mad2 mRNA, complete cds
Mus musculus mRNA, complete cds, clone: 2-24
Mus musculus SIK similar protein mRNA, complete cds
Mus musculus mRNA for 26S proteasome non-ATPase subunit
Mus musculus short coiled coil protein SCOCO (Scoc) mRNA, complete cds
Mus musculus Cope1 mRNA for nonclathrin coat protein epsilon-COP, complete cds
M. musculus mRNA for neuronal protein 15.6
Mus musculus ribosomal protein L23 (Rpl23) gene, complete cds
Mus musculus BAF53a (Baf53a) mRNA, complete cds
Mus musculus ATP synthase gamma-subunit gene, nuclear gene encoding a mitoch
Mus musculus TBX1 protein mRNA, complete cds
Mus musculus mRNA for Arp2/3 complex subunit p21-Arc, complete cds
Mus musculus mRNA for heterogeneous nuclear ribonucleoprotein H
Mus musculus sodium bicarbonate cotransporter isoform 3 kNBC-3 mRNA, complete
Mus musculus mRNA similar to human Sua1, complete cds
Mus musculus geminin mRNA, complete cds
Mus musculus E2F-like transcriptional repressor protein mRNA, complete cds
Mus musculus mRNA for vinculin, partial cds
Mus musculus protein inhibitor of activated STAT protein PIAS1 mRNA, complete cd
M. musculus mRNA for GTP-binding protein
Mus musculus carboxy terminus of Hsp70-interacting protein (Chip) mRNA, complete
Mus musculus mRNA for aldolase C, partial
Mus musculus CRIPT protein mRNA, complete cds
Mus musculus mRNA for mDj8, complete cds
Mus musculus ring-box protein 1 (Rbx1) mRNA, complete cds
Mus musculus p53 apoptosis-associated target (Perp) mRNA, complete cds
Mus musculus claudin-10 mRNA, complete cds
Mus musculus protein inhibitor of nitric oxide synthase (PIN) mRNA, complete cds
Mus musculus protein kinase C inhibitor (mPKCI) mRNA, complete cds
Mus musculus RING finger protein AO7 mRNA, complete cds
M. musculus mRNA for glutamyl-tRNA synthetase
Mus musculus pre-B-cell colony-enhancing factor mRNA, complete cds
Mus musculus mACS4 mRNA for Acyl-CoA synthetase 4, complete cds
Mus musculus eosinophil secondary granule protein (mEAR-2) mRNA, complete cds
Mus musculus putative deubiquitinating enzyme UBPY (Ubpy) mRNA, complete cds
Mus musculus mRNA for Sid393p, complete cds
Mus musculus mRNA for eIF3 p66, complete cds
Mus musculus carboxy terminus of Hsp70-interacting protein (Chip) mRNA, complete
M. musculus mRNA for fibromodulin
Mus musculus mRNA for MSSP, complete cds
Mus musculus TBX1 protein mRNA, complete cds
Mus musculus BAF53a (Baf53a) mRNA, complete cds
Mus musculus PEST phosphatase interacting protein mRNA, complete cds
Mus musculus timeless homolog mRNA, complete cds
Mus musculus cyclic nucleotide phosphodiesterase (PDE1A2) mRNA, complete cds
Mus musculus mRNA, complete cds, clone: 2-68
M. musculus mRNA for GTP-binding protein
Mus musculus histone deacetylase mHDA1 mRNA, complete cds
Mus musculus MPS1 gene and mRNA, 3′end
Mus musculus short-chain dehydrogenase CRAD2 mRNA, complete cds
Mus musculus prostaglandin transporter PGT mRNA, complete cds
Mus musculus GDP-dissociation inhibitor mRNA, preferentially expressed in hematop
Mus musculus PGES mRNA for prostaglandin E synthase, complete cds
Mus musculus SOCS box-containing WD protein SWiP-2 (Swip2) mRNA, complete c
Mus musculus serine protease OMI (Omi) mRNA, complete cds
Mus musculus peptidylglycine alpha-amidating monooxygenase (PAM) mRNA, compl
Mus musculus Dkc1 gene for dyskerin, exon 1 and join CDS
Mus musculus high mobility group protein homolog HMG4 (Hmg4) mRNA, complete
Mus musculus tescalcin mRNA, complete cds
Mus musculus predicted GTP binding protein (IRG-47) mRNA, complete cds
Mus musculus E2F-like transcriptional repressor protein mRNA, complete cds
indicates data missing or illegible when filed
This application claims the benefit of U.S. Provisional Application No. 60/600,697, filed Aug. 11, 2004.
The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grants No. CA089829 and CA100324 awarded by the National Institutes of Health.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US05/27680 | 8/4/2005 | WO | 00 | 9/26/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60600697 | Aug 2004 | US |
