MXPA98007590A - Immunomagnetic separation of cells, used in the identification of genes associated with formation of cancer metastases with preference for a yes - Google Patents
Immunomagnetic separation of cells, used in the identification of genes associated with formation of cancer metastases with preference for a yesInfo
- Publication number
- MXPA98007590A MXPA98007590A MXPA/A/1998/007590A MX9807590A MXPA98007590A MX PA98007590 A MXPA98007590 A MX PA98007590A MX 9807590 A MX9807590 A MX 9807590A MX PA98007590 A MXPA98007590 A MX PA98007590A
- Authority
- MX
- Mexico
- Prior art keywords
- cells
- genes
- specific
- target cells
- expression
- Prior art date
Links
- 206010027476 Metastasis Diseases 0.000 title claims description 29
- 201000011510 cancer Diseases 0.000 title description 10
- 230000015572 biosynthetic process Effects 0.000 title description 3
- 238000005755 formation reaction Methods 0.000 title description 3
- 238000000926 separation method Methods 0.000 title description 3
- 230000014509 gene expression Effects 0.000 claims abstract description 30
- 238000010367 cloning Methods 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 24
- 230000003211 malignant Effects 0.000 claims abstract description 20
- 238000009396 hybridization Methods 0.000 claims abstract description 3
- 210000004027 cells Anatomy 0.000 claims description 98
- 210000004881 tumor cells Anatomy 0.000 claims description 39
- 206010028980 Neoplasm Diseases 0.000 claims description 32
- 210000001519 tissues Anatomy 0.000 claims description 21
- 210000001185 Bone Marrow Anatomy 0.000 claims description 14
- 210000004369 Blood Anatomy 0.000 claims description 10
- 239000008280 blood Substances 0.000 claims description 10
- 210000001165 Lymph Nodes Anatomy 0.000 claims description 9
- 241001465754 Metazoa Species 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 7
- 210000000988 Bone and Bones Anatomy 0.000 claims description 5
- 108020004999 Messenger RNA Proteins 0.000 claims description 5
- 229920002106 messenger RNA Polymers 0.000 claims description 5
- 229920000160 (ribonucleotides)n+m Polymers 0.000 claims description 4
- 229920002676 Complementary DNA Polymers 0.000 claims description 4
- 210000004185 Liver Anatomy 0.000 claims description 4
- 210000004072 Lung Anatomy 0.000 claims description 4
- 230000000306 recurrent Effects 0.000 claims description 4
- 210000001175 Cerebrospinal Fluid Anatomy 0.000 claims description 3
- 229920003013 deoxyribonucleic acid Polymers 0.000 claims description 3
- 210000003169 Central Nervous System Anatomy 0.000 claims description 2
- 210000002700 Urine Anatomy 0.000 claims description 2
- 230000001413 cellular Effects 0.000 claims 2
- 210000001124 Body Fluids Anatomy 0.000 claims 1
- 206010025538 Malignant ascite Diseases 0.000 claims 1
- 206010026673 Malignant pleural effusion Diseases 0.000 claims 1
- 239000010839 body fluid Substances 0.000 claims 1
- 239000000499 gel Substances 0.000 claims 1
- 238000001415 gene therapy Methods 0.000 claims 1
- 108020004707 nucleic acids Proteins 0.000 claims 1
- 150000007523 nucleic acids Chemical class 0.000 claims 1
- 239000000725 suspension Substances 0.000 claims 1
- 206010061289 Metastatic neoplasm Diseases 0.000 abstract description 9
- 230000001394 metastastic Effects 0.000 abstract description 9
- 206010006187 Breast cancer Diseases 0.000 description 11
- 238000000338 in vitro Methods 0.000 description 7
- 210000000278 Spinal Cord Anatomy 0.000 description 5
- 239000006285 cell suspension Substances 0.000 description 4
- 210000000056 organs Anatomy 0.000 description 4
- 238000001356 surgical procedure Methods 0.000 description 4
- 210000004556 Brain Anatomy 0.000 description 3
- 210000003703 Cisterna Magna Anatomy 0.000 description 3
- 102000004965 antibodies Human genes 0.000 description 3
- 108090001123 antibodies Proteins 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000000875 corresponding Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 230000000877 morphologic Effects 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 230000036881 Clu Effects 0.000 description 2
- 206010063045 Effusion Diseases 0.000 description 2
- 206010025650 Malignant melanoma Diseases 0.000 description 2
- 229920001850 Nucleic acid sequence Polymers 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 229960000070 antineoplastic Monoclonal antibodies Drugs 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 201000011231 colorectal cancer Diseases 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 229960000060 monoclonal antibodies Drugs 0.000 description 2
- 102000005614 monoclonal antibodies Human genes 0.000 description 2
- 108010045030 monoclonal antibodies Proteins 0.000 description 2
- 201000008968 osteosarcoma Diseases 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000001105 regulatory Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 210000000683 Abdominal Cavity Anatomy 0.000 description 1
- 210000000577 Adipose Tissue Anatomy 0.000 description 1
- 210000004204 Blood Vessels Anatomy 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 210000002808 Connective Tissue Anatomy 0.000 description 1
- 210000002889 Endothelial Cells Anatomy 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 210000002744 Extracellular Matrix Anatomy 0.000 description 1
- 210000000936 Intestines Anatomy 0.000 description 1
- 102100006037 MUC1 Human genes 0.000 description 1
- 101700052761 MUC1 Proteins 0.000 description 1
- 206010027452 Metastases to bone Diseases 0.000 description 1
- 206010051662 Metastases to bone marrow Diseases 0.000 description 1
- 206010027457 Metastases to liver Diseases 0.000 description 1
- 206010027459 Metastases to lymph node Diseases 0.000 description 1
- 101700064921 NO93 Proteins 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 210000003281 Pleural Cavity Anatomy 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 210000003491 Skin Anatomy 0.000 description 1
- 210000002536 Stromal Cells Anatomy 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000038129 antigens Human genes 0.000 description 1
- 108091007172 antigens Proteins 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000012568 clinical material Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002596 correlated Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000002255 enzymatic Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000003394 haemopoietic Effects 0.000 description 1
- 230000002489 hematologic Effects 0.000 description 1
- 210000000548 hind-foot Anatomy 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000009114 investigational therapy Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000002595 magnetic resonance imaging Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000003170 nutritional factors Nutrition 0.000 description 1
- 102000025475 oncoproteins Human genes 0.000 description 1
- 108091008124 oncoproteins Proteins 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N oxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000003389 potentiating Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 200000000025 progressive disease Diseases 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- YEENEYXBHNNNGV-XEHWZWQGSA-M sodium;3-acetamido-5-[acetyl(methyl)amino]-2,4,6-triiodobenzoate;(2R,3R,4S,5S,6R)-2-[(2R,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound [Na+].CC(=O)N(C)C1=C(I)C(NC(C)=O)=C(I)C(C([O-])=O)=C1I.O[C@H]1[C@H](O)[C@@H](CO)O[C@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 YEENEYXBHNNNGV-XEHWZWQGSA-M 0.000 description 1
- 230000004083 survival Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 102000003995 transcription factors Human genes 0.000 description 1
- 108090000464 transcription factors Proteins 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 230000002861 ventricular Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Abstract
A method is described for identifying genes with site-specific or site-specific expression in target cells that are initially detected and isolated by repeated immunomagnetic procedures. The purified target cells are then exposed to known cloning procedures. Preferred target cells are malignant cells, for example, metastatic cells. The means of gene cloning may include differential display or substratum hybridization approaches
Description
SEPARATION HFIUNOMAGNKI CA CELLS, USED IN THE IDENTIFICATION OF GENES ASSOCIATED WITH FORMATION OF CANCER METASTASIS WITH PREFERENCE FOR A SITE.
DESCRIPTION OF THE INVENTION
The aim of the invention is to provide a novel approach for detecting novel genes with site-specific expression patterns in tumor cells found in different tissues. It is well known that many types of cancer cells show typical patterns of spread, and that metastasis preferentially appears in certain tissues or organs, often in an orderly manner. Thus, breast cancer metastasis usually develops first in the axillary lymph nodes, while bone marrow / bone metastases represent the first site and the most common (50%) of distant dissemination. Of the other tissues, the liver, lungs and central nervous system (up to 20%) become the hosts of breast cancer metastases. Similarly, prostate cancer results in skeletal metastasis; colon cancer spreads in lymph nodes and liver; osteosarcoma to the lungs; and malignant melanoma to lymph nodes, liver, lungs and brain. Very little is known about the factors that determine such cancer dissemination with preference for tissues, but specific characteristics of the tumor cells are certainly involved in allowing, for example, the tumor cells to lodge in the target organ, to move and that they invade the host tissue, which respond to local growth factors, inducing angiogenesis that reacts to them in an unknown manner. These characteristics must be associated with the expression of specific proteins that are expressed by known or unknown genes. Therefore, it would be of great importance to identify such genes for the understanding of the mechanisms of metastasis and in this way provide new clues or clues for diagnosis and therapy. Various methods have been used in the investigation of genes that are expressed in high amounts in certain cell populations but not in others, which include procedures such as cloning of subtractive hybridization and the use of the differential display approach. Most of the cloning projects have involved the use of in vitro cell lines or clones as initial material. However, it is known that cell lines can differ significantly from the tumor cells from which they originated and that in vitro culture conditions can produce up-regulation or down-regulation of the expression of the genes involved to decide the capacity of the cells to invade extracellular matrices and stromal tissue as well as their total metastatic capacity.
A logical alternative to cell lines for comparing the expression of transcripts of genes or proteins of interest would be the use of tumor tissue samples from primary tumors and patient metastases. However, such an approach does not include several possibilities for error and also presents technical difficulties. When tumor cells of different patients are used, the expression of characteristic genes of each individual must be subtracted before the patterns of gene expression related to the study objective are compared. This adds immense complexity to such gene cloning. Therefore, it would be advantageous to be able to compare expression patterns in cancer cells obtained from different tumor manifestations located at different sites in an individual. This may be possible by collecting tumor tissue from both the primary tumor and from overt metastasis detected and removed in surgery, and / or by surgery or biopsy of recurrent tissue, or secondary tumors in patients with progressive disease who have or have not received other modalities. of treatment after primary surgery. In any gene cloning project, it is important to work with populations of target cells as pure as possible, trying to avoid irrelevant signals from non-target cells that confuse the expression patterns to be compared. In samples of solid tumors it is difficult to obtain, as well as in surgical samples and biopsies the tumor cells will be mixed with normal fibrous tissue, including stromal and endothelial cells, these conventional methods of preparation of woven can not be removed satisfactorily. In haematological cancers, malignant cells share determinants of a corresponding subpopulation of normal cells, preventing the separation of the two cell types. In attempts to identify genes that are involved in early stages of ttimor spread, it would be important to obtain tumor cells of relevant sites when the size of secondary tumors is small, or if possible even of subclinical tumor cell foci. An example would be malignant cells present in blood or bone marrow before conventional diagnostic measurements can demonstrate solid manifestation of metastasis. Another example would be cancer cells present in cerebrospinal fluid, in urine or in effusions in the pleural and abdominal cavities before conventional morphological procedures can detect such cells. In addition, in primary surgery or if metastatic spread is suspected, lymph nodes are often removed because they are enlarged. However, a morphological examination can still be negative in cases where a limited amount of tumor cells may be present. In all of these examples, the present invention describes a means for detecting and selecting target tumor cells for gene cloning purposes. In addition to using cancer cells obtained from different tissues or organs in patients, the invention also describes another way to obtain metastatic human tumor cells for use in research for genes with site-specific expression. The cells of several human tumors have been grown in vitro or in immunodeficient animals in vivo, and such cells have been used to establish experimental models of metastasis or models in which cells can grow orthotopically, i.e., in clinically relevant tissues; malignant melanoma on the skin; Osteosarcoma in bone; colorectal cancer in the wall of the intestine; breast cancer in the primary fat pad, etc. In the experimental models of metastasis, different patterns of tumor dissemination can be observed based on the cell line, the route of cell injection and the type of host used. Commonly, the patterns of metastasis simulate those of the corresponding tumor type in the clinical setting. By using such models it is also possible to obtain tumor cells from metastatic sites that are usually not accessible in patients, such as the spinal cord and brain tissue. Again, for purposes of gene cloning, it would be important to select human tumor cells from animal cells to avoid problems with genes expressed in normal host cells. Previously it has been impossible to carry out experiments of cloning of significant genes on specimens of solid tumors and metastases and on malignant cells in blood and bone marrow in order to identify genes with site-specific expression. This is because a considerable fraction of solid tumors and metastases are not malignant cells but connective tissue that supports and grows between malignant cells, and which also contain blood vessels that provide the necessary supply of nutritional factors and oxygen to the tumor . It has not been considered possible to properly separate tumor cells from normal cells without including an intermediate stage of cell culture in vitro, which involves manipulations that eliminate normal cells. However, such an in vitro culture would result in a selection of subpopulation of tumor cells in an environment very different from the in vivo situation, thereby reducing significant changes in gene expression patterns. Therefore, for the purpose of identifying genes associated with the metastatic process, the culture of tumor cells from solid metastases can not be used. Furthermore, it has not been considered of interest to those familiar with the gene cloning technique to compare gene expression patterns in malignant cells in untreated solid and metastatic primary tumors.
In addition, in samples of blood tumor cells and bone marrow, if all are present, they constitute a very small fraction of the total number of nucleated cells, and malignant cells in blood and bone marrow have not been considered of interest for attempts of gene cloning. This is because tumor cells can not be adequately separated from normal cells, and importantly also because people familiar with the gene cloning technique have not expected such malignant cells to be sufficiently different in patterns. of gene expression compared to the original tumors. Cancer cells isolated from human tumors that produce metastases to different tissues of clinically relevant target organs in immunodeficient animals have not been used in attempts to identify genes associated with tumor with site-specific expression, simply because such human tumor models are very rare, but also importantly due to the lack of methods to obtain pure populations of cancer cells. The aim of the present invention, therefore, is to provide a method by which target cells can be separated from a population of cells in order to identify the gene sequences of the target cells in a specific cell population environment.
This object is obtained by the present invention characterized by the appended claims. Since the aim of the invention is to identify genes specifically expressed during the early stages of tumor dissemination, solid tumors and metastases must be small in size, and malignant cells in blood and bone marrow must represent what is termed a disease. micrometastatic, that is, a limited amount of tumor cells which are present. If possible, such tumor cells should be collected even in cases where the cells can not be detected by conventional morphological examinations or by diagnostic procedures such as radiology or magnetic resonance imaging. Obviously, since such cells have not been recognizable, they have not been of interest for gene cloning purposes. The use of immunogenic techniques allows the isolation of malignant cells even when they are present in low amounts. Several methods to amplify DNA and RNA sequences make the cloning of genes possible for such low amounts of malignant cells on the condition that this cell population is sufficiently pure. The possible selection of target tumor cells can be obtained by the use of techniques known per se, as described in patent application PCT / NO93 / 00136 (WO 94/07139) and in PCT / NO95 / 00052. Since in the present case the final purity of the target cell population is important, the immunomagnetic selection processes can be performed more than once, or they can be performed as a combination of positive selection (with monoclonal antibodies that recognize the target cell) as negative (with antibodies that bind to unwanted cells). These techniques have been successfully used to isolate target cells from blood, bone marrow, malignant effusions and cell suspensions alone prepared from tumor tissue of primary and lymph node tumors and other metastases. Similarly, selection of human malignant cells from normal stromal or hematopoietic cells in animal hosts has also been demonstrated, even in cases where the tumor cells reside in bone, bone marrow, cerebrospinal fluid or brain tissue. Since one goal would be to look for genes with products involved in the early stages of metastasis formation, it can happen that only a relatively small amount of tumor cells are obtained, the purity of which is particularly important. With this point of view, it is possible to obtain a better purified target population of patients and animal hosts compared to another known technique, which provides unique possibilities for cloning of genes with site-specific expression. The next step of the invention involves the use of known methods of gene cloning, such as the use of the differential display procedure first described by Liang, A. and Pardee A.B. (Science, Vol. 257, 967-971, 1992). In this method, polymerase chain reactions are performed with enzymes and primers (primers) that provide reverse transcription and random amplification of transcripts of genes present in the target cells. The resulting DNA fragments in the cell populations to be compared subsequently are studied in a sequencing gel and site-specific fragments are extracted and sequenced. Subsequently, the gene fragments of interest can be further studied, including in examining their expression patterns in material similar to that used for cloning. Again, it is very important to have access to purified tumor cell populations without intervening non-target and irrelevant cells. The possibility of using human tumor cells isolated from models of metastases in immunodeficient animals is advantageous because the models provide a reliable and continuous source of target tumor cells for extended uses. It is also important that the target cells, either from patients or from animal models, be isolated and treated for DNA and RNA studies directly and very quickly to avoid unwanted alterations in the expression of the gene which are not related to the objective of identifying genes with site-specific expression.
The different methods used to clone new genes have their inherent limitations. Differential display methods based on polymerase chain reaction techniques may suffer from problems related to representativeness and reproducibility. With our approach, we have consistently included stages that aim to minimize such problems. This has been achieved mainly through the use of the immunosphere selection technique which makes it possible to specifically study biologically representative cells, not only for the first stage of differential display, but also in stages where the candidate genes are examined for levels of expression in relevant cells and tissues.
Example 1:
Tumor cells of primary tumor and axillary lymph nodes of a patient with breast cancer were prepared by physical and enzymatic methods to obtain a single cell suspension. A bone marrow sample (50 ml) was aspirated and a peripheral blood sample obtained by venous puncture was obtained, and the mononuclear cells were both isolated on Lymphoprep (Nycomed Pharma, Oslo, Norway). The cell suspensions were independently incubated with the monoclonal antibodies MOC-31 and BM7, which recognize a panepithelial antigen and the MUC-1 gene product, respectively. After washing and incubation, Dynabeads M-450 SAM SD (Dynal, Oslo, Norway) were added, which bind to the Fc region of the primary antibodies. The tumor cells with the bound antibody complex Dynabeads M-450 SAM SD can subsequently be isolated from normal mononuclear cells by the use of a potent magnet. The nature of the selected cells is microscopically confirmed. From different cell populations, it is extracted in RNA and the material is subjected to a differential display cloning procedure (Liang and Pardee, 1992), in which partial cDNA sequences of mRNA subpopulations obtained by reverse transcription are amplified. , by means of a polymerase chain reaction. Comparison of cDNA fragments from various populations in tumor cells was compared in a sequencing gene, and fragments expressed specifically in cells obtained at one of the sites were extracted and sequenced. Among a number of gene sequences of interest with specific expression either in isolated tumor cells, with our magnetic immunosphere technology, from bone marrow cells or in the tumor, immunomagnetically isolated from lymph node metastases, we have found a single transcription factor related to the cell cycle, an oncogene product, in addition to the genes not yet identified. The expression of two sequences of genes identified in biologically relevant model systems and in clinical material is currently being analyzed.
Example 2:
In this example, cells from a model for experimental metastasis of a human breast cancer were used. In athymic nude rats, the injection of human breast cancer cells MA-11 into the cisterna magna (CM) results in leptomeningeal dissemination and growth of malignant cells. In addition, MA-11 cells injected into the left ventricular (LV) ventricle form metastases in the spinal cord which results in the development of hind paw paralysis in animals after approximately 35 days. Tumor cells were obtained from both sites by shredding the host tissue and preparing single cell suspensions, and the immunosphere technique described under Example 1 was used to positively select malignant cells. Subsequently, RNA extraction of cells from these two sites was performed, or together with MA-11 cells cultured in vitro and the material was subjected in a similar manner to a differential display cloning procedure as already described. further, mRNA extracted from relevant normal tissues in the rats was included as controls. It should be noted that the MA-11 cell line was established from micrometastatic tumor cell isolate with the immunomagnetic method of the bone marrow of a patient with stage II breast cancer. Several specific candidate fragments have been detected for the growth of cells in the leptomeninge or as metastases for the spinal cord. The analysis by sequence showed that these fragments represent both novel and known genes. Among the fragments confirmed as differentially expressed in MA-11 cells selected from metastatic cells, up to now four candidate genes have been examined in greater detail. Two of these, called LVl and LV12 are particularly interesting. LV1 shows a high selectivity of expression in tumor cells of spinal cord metastasis, whereas LV12 is downregulated in cells that grow in the leptomeningeal area. It has been found that LV1 is up-regulated in numerous cell lines that are known to have a high capacity to form experimental metastases in immunodeficient animals. In a panel of primary tumors of patients with breast cancer, the low expression of LV12 is related to the short survival of the corresponding patients. Taken together, the results show that mRNA for LV1 appears to be up-regulated in highly metastatic cells, and the level of mRNA for LV12 is inversely correlated with the progress and metastasis of breast cancer cells. Both genes are novel. In addition, a third promising candidate gene, CM13, also a novel gene upregulated in tumor cells growing in the leptomeninges. Several other candidates are still to be analyzed. Full-length cDNA cloning has been initiated for both LV1 and LV12.
Example 3:
In a similar model described in Example 2, human mammary cancer cells MT1 have given rise to growth in the leptomeninges after CM injection, whereas injected LV cells produce metastases to bone marrow and bone in the vertebrae of the spinal cord and in the long bones. Cells from these two positions, as well as cells that have grown in vitro, have been isolated and RNA from the different cell populations has been extracted. Cloning procedures similar to those described in Example 2 have been applied to these materials.
Example 4;
Tumor cells, isolated by the technique described in example 1, have been isolated from other breast cancer patients from patients with colorectal cancer where the tumor cells were isolated from primary and recurrent tumors, from lymph nodes and liver metastases, and sample of blood and bone marrow, as well as of patients with prostate cancer with cells selected from primary or recurrent tumor samples, lymph nodes in addition to blood and bone marrow samples. The isolated cells have been used for gene cloning purposes. In addition to using the material described in the examples
1-4 for gene cloning, can be used to examine patterns of expression of all gene sequences with sufficient possibilities.
Claims (11)
1. A method for identifying genes with site-specific or site-specific expression in specific target cells in a different cellular environment or not of origin, the method is characterized in that the target cells are initially detected and isolated by repeated immunomagnetic procedures in order to obtain up to 100% of specific target cells before exposing the target cells to known gene cloning procedures, where unknown genes are compared to differences in mRNA expression levels in the target cells isolated from different tissues.
2. The method according to claim 1, characterized in that the target cells used are malignant cells obtained from primary or recurrent solid tumors; and / or from metastasis of such tumors to lymph nodes; and / or blood; and / or bone marrow; and / or bone tissue; and / or liver; and / or lungs; and / or central nervous system; and / or malignant pleural effusions and ascites, urine; and / or cerebrospinal fluid; and / or other bodies in other places.
3. The method according to claims 1-2, characterized in that the malignant cells are isolated from suspensions of single cells prepared from solid tumor manifestations; and / or fractions of mononuclear cells obtained from bone marrow or blood sample; and / or of cells present in other body fluids.
4. The method according to claims 1-2, characterized in that the malignant cells used are cultured human tumor cells, 222 vitro, and / or human tumor cells growing in specific tissues in immunodeficient animals; and / or experimental human tumor metastases in such animals.
5. The method according to claims 1-4, characterized in that the RNA and / or DNA is extracted from isolated cells.
6. The method according to claim 5, characterized in that the extracted nucleic acids are used for gene cloning purposes.
7. The method according to the preceding claims, characterized in that the method of cloning genes is one of the approaches of differential display or subtractive hybridization, or any other procedure that can be used to identify genes with differential expression.
8. The method according to claim 7, characterized in that the amplified cDNAs obtained from malignant cells selected from different sites are studied and compared on sequencing gels, and where those with interesting patterns specific to site or preferably site are sequenced and identified .
9. The method according to claim 8, characterized in that the expression patterns of the identified gene sequences are studied on material obtained from all the relevant sites of tumor cells described according to claims 1-4.
10. The method according to the preceding claims, characterized in that the genes previously unknown, identified in preceding claims, are used for gene therapy purposes, and / or as targets for procedures that attempt to alter or inactivate the genes or their products.
11. The use of the method according to claim 1, characterized in that it is used to obtain sequences of specific genes and their expression products in target cells present in cellular environments different or not from their origin.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NO961221 | 1996-03-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA98007590A true MXPA98007590A (en) | 1999-02-24 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107828779B (en) | Prostate cancer specific exosome, lncRNA, preparation method and application thereof | |
Caldas et al. | Detection of K-ras mutations in the stool of patients with pancreatic adenocarcinoma and pancreatic ductal hyperplasia | |
EP1169479B1 (en) | Methods for detecting nucleic acids indicative of cancer | |
US6406857B1 (en) | Methods for stool sample preparation | |
CN109825586B (en) | DNA methylation qPCR kit for lung cancer detection and use method | |
US8080378B2 (en) | Method of detecting colon cancer marker | |
JP2002536635A (en) | Method for enriching or removing tumor cells from body fluids and kits suitable for such purpose | |
CN110004147B (en) | Aptamer of epithelial cell adhesion molecule EpCAM screened from human plasma and preparation method and application thereof | |
CN109097477A (en) | It is a kind of for the circRNA marker of breast cancer diagnosis and its application | |
CN109112216A (en) | The kit and method of triple qPCR detection DNA methylations | |
EP0894146B1 (en) | Immuno-magnetic cell separation used in identification of genes associated with site-preferenced cancer metastasis formation | |
Bridge et al. | Cytogenetic and molecular genetic techniques as adjunctive approaches in the diagnosis of bone and soft tissue tumors | |
CN109161543B (en) | DNA probe for enriching low-frequency DNA mutation and application thereof | |
GB2260811A (en) | Diagnosis of malignant tumours by mRNA detection | |
CN107058305A (en) | One group of nucleotide sequence and the application in EML4 ALK fusion gene quick detections | |
JP2800850B2 (en) | Methods for detecting neoplasia | |
Miyake et al. | Highly specific and sensitive detection of malignancy in urine samples from patients with urothelial cancer by CD44v8–10/CD44v10 competitive RT‐PCR | |
MXPA98007590A (en) | Immunomagnetic separation of cells, used in the identification of genes associated with formation of cancer metastases with preference for a yes | |
RU2451937C2 (en) | Diagnostic technique for breast cancer by blood plasma interleukin il-8 and/or il-18 rna level | |
Tveito et al. | Specific isolation of disseminated cancer cells: a new method permitting sensitive detection of target molecules of diagnostic and therapeutic value | |
RU98119525A (en) | IMMUNOMAGNETIC CELL SEPARATION USED FOR IDENTIFICATION OF GENES ASSOCIATED WITH SITE-PREFERRED FORMATION OF CANCER METASTASES | |
RU2665965C1 (en) | Method for screening malignant neoplasms in humans | |
Zamay et al. | Selection of DNA aptamers for breast cancer | |
RU2801094C1 (en) | Method of determining the risk group for patients with multiple myeloma complicated by plasmacytoma | |
CN117402968A (en) | Application of prostate cancer molecular marker in preparation of prostate cancer detection reagent |