The present invention concerns a method for evaluating the metastatic tendency of tumor cells, and a kit for use in said method.
Metastasis is a multifactorial process by which tumor cells escape from the primary tumor, disseminate through blood and lymph vessels, evade host immune defence, and home to specific target organs where they extravasate and re-colonize. Metastatic cells must encounter and cross the basement membrane during the extravasation of the blood vessels. The metastatic process is usually described as a three-step model, wherein metastatic cells must first adhere to the basement membrane, then digest via proteolytic enzymes the basal lamina, and finally migrate through the vessel wall.
Evaluating the metastatic potential of a specific tumor is essential for diagnostic and prognostic purposes, for determining optimal course of treatment, as well as for various research purposes such as developing therapeutical methods. and medicaments for the treatment of metastasis.
Correlation between various biomolecules and the metastatic tendency of tumor cells has been previously described. There has been a disclosure of association of increased basement membrane invasiveness, which is the first step in the process of metastasis, with the absence of estrogen receptor and expression of vimentin in a human breast cancer cell line (Thompson et al, J. of Cellular Physiol., 150:534-544 (1992)). A correlation between the invasive potential of various human breast cancer cell lines and binding and degradation of hyaluronan has also been described (Cultry et al., J. of Cellular Physiol., 160:275-286 (1994)). Metastatic potential of various tumor cell lines has also been associated with amplification in the expression of various proto-oncogenes, such as the HER-2/neu proto-oncogene which is amplified in 25% of human primary breast cancer and its amplification is associated with shorter time to relapse and shorter overall survival (Slamon et al., Science 244:707-712 (1989)).
Although currently change in the level of some of the above biomolecules, especially amplification of the HER-2/neu proto-oncogenes, is used for prognostic purposes, the prognostic use of expression or amplification of this gene has been quite controversial (Press, M. F. et al., Cancer Res, 53.4960-4970 (1993)). The prognostic factors currently used to assess the breast tumor state are: tumor size, histological grade, steroid hormone receptor status, DNA ploidity, proliferative index, cathepsin D and analysis of growth factor receptors such as epidermal growth factor receptor.
There is a great need today for additional, accurate prognostic factors for tumor cells which will be easy to identify and which are in good correlation to the metastatic tendency of those cells.
The thrombin receptor (ThR) has been cloned and identified as a member of the seven trans-membrane domain super-family of G-protein coupled receptors. It is activated by cleavage of the Arg41-Ser42 residues of the extracellular N-terminus part of the receptor by the protease thrombin. This cleavage exposes the ligand of the thrombin receptor which is an integral part of the receptor itself. Thus, the ThR serves as a classical substrate for protease, rather than the traditional ligand-receptor complex, during the course of cellular activation (Vu, et al., Cell, 64:1057-1068, (1991)). Recently, there has been a report that W256 carcinoma and mouse melanoma tumor cells contain functional thrombin receptors (Wojtukiewicz, et al., Cancer Res., 55:698-704 (1995)), however, only the existence and function of the thrombin receptor has been assessed in this publication and there has been no determination of the level of its expression, nor its connection to the malignancy of the tumor.
The present invention is based on the surprising finding that there exists a direct correlation between the level of ThR expression in tumor cells and their metastatic tendencies, so that high levels of ThR expression are evident in aggressive metastatic tumor cells, low to moderate levels of expression can be detected in medium metastatic tumor cells and essentially no detectable expression of ThR is evident in non-metastatic tumor cells.
The above surprising finding enables to evaluate the metastatic tendency of tumor cells by determining the level of ThR expression therein.
Thus, the present invention provides a method for evaluating the metastatic tendency of tumor cells comprising:
(a) determining a test level of a cellular parameter in said cells said level of parameter being:
(aa) the level of thrombin receptor (ThR), or
(ab) the level of expression of a gene coding for ThR;
(b) comparing said test level with a control level, being a level of a corresponding parameter obtained from a cell having a known metastatic tendency, a high or low test level indicating a high or low metastatic tendency, respectively.
The term xe2x80x9cmetastatic tendencyxe2x80x9d refers to the expressed metastatic capacity of tumors which have already begun their metastatic spread and begun recolonization in target organs, as well as to the future metastatic potential of tumors which are still in the initial pre-metastatic stage and which may metastasize in the future. A high metastatic tendency refers to a situation where the tumor cells metastasize rapidly and colonialize in many target sites. Moderate metastatic tendency refers to tumor cells which metastasize slowly, and colonialize only at a few target sites, and low metastatic tendency refers to tumor cells which virtually do not metastasize and are confined to their original site.
The tumor cells which metastatic tendency is evaluated can be any type of tumor cells, especially those types which feature varying levels of metastatic tendencies such as breast cancer, testicle cancer, melanoma, epithelial carcinoma, colon carcinoma, ovarian carcinoma, cervical carcinoma, as well as various types of sarcoma.
The level of ThR can be determined either by assaying the level expression of the gene coding for the thrombin receptor, i.e. by determining the mRNA level, or by determining directly the level of the thrombin receptor present either on the membranes of the cells or within the cytoplasm.
The levels of the mRNA can be determined. for example, by separating the mRNA molecules, obtained from the assayed cells on an electrophoretogram (Northern blot) and then identifying the separated thrombin mRNA by hybridization with suitable probes which carry detectable labels; by in situ hybridization to the mRNA, present in the isolated tumor cell or present in isolated tissue obtained from the tumor, with suitable probes carrying detectable labels; or by RT-PCR amplification using suitable primers.
The ThR level may be determined for receptors present on the membrane of the tumor cells and/or present within the cell. The determination can be carried out, for example, by using antibodies capable of recognizing the thrombin receptor (either in its membranal or cytoplasmal forms, or in both forms) in one of the state-of-the-art immunoassays, or by determining the level of binding of a labeled ligand to the thrombin receptor.
Once the level of the thrombin receptor is determined, either through mRNA or through protein determination, it should be compared to the corresponding level of either mRNA or thrombin receptor of other cells which metastatic tendency is a priori known, which cells may also be non-tumor normal cells, or tumor cells from established cell lines known to have a high, low, or moderate metastatic tendency.
In practice and during clinical use there will be no need to calibrate de novo each ThR level obtained since it will already be known by the practitioner, due to prior experience. which specific color pattern of an in situ hybridization, unique pattern of band formation in Western or Northern blot, or specific amount of mRNA amplified using RT-PCR, indicates that the ThR level is high and thus that the tumor cell features a high metastatic tendency. It is also possible to compare the levels obtained from the tested tumor cells to two corresponding control levels: one obtained from highly metastatic cell lines, and the other obtained from low metastatic cell lines to determine more precisely whether the tested level is high, low or intermediate.
Cells, which after being compared as specified above show a high level of ThR, are those which feature a high metastatic tendency; cells which show a low or almost no level of ThR are those which have low metastatic tendency; and cells which show intermediate levels are those which are usually moderately metastatic.
The present invention also concerns a kit for use in the above method.
Where the determination of the level of ThR is carried out by determining the level of mRNA, the kit may comprise labeled probes and reagents suitable for in situ hybridization, labeled probes and reagents for carrying out Northern blot, or reagents and primers necessary for RT-PCR.
Where the determination of the ThR is carried out by determining the level of the thrombin receptor, the kit may comprise antibodies capable of specifically binding to the ThR, either when present on the membranal surfaces of the tumor cells or when present within the cells, together with reagents for carrying out a suitable immunoassay. Alternatively, the kit for determining the ThR protein level may be based on a ligand-binding assay and in that case the kit should comprise a labeled ligand, which can be for example the synthetic peptide corresponding to residues Ser42-Lys51 of the native thrombin receptor (the so called xe2x80x9cinner ligandxe2x80x9d).
The invention will now be further illustrated with reference to some specific non-limiting drawings and embodiments.