JPH04504049A - Native-state methods and systems for measuring tissue viability and proliferative capacity in vitro - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Measuring, autochthonous methods and systems Field to which the invention belongs The present invention uses an in vitro culture system to cultivate cells of large tissues, especially tumor tissues. Methods for measuring proliferative capacity and cell viability, and proliferative capacity and viability of cultured cells The present invention relates to a method for determining the efficacy of antitumor drugs on cancer and viability.

Background of the invention Cancer is a disease that involves inappropriate cell sites. The ecology of degenerative growth in cancer compared to normal to understand the tissue and its proliferative potential as a basis for cancer prediction and treatment. There is a great need for realistic models for use in the information provided.

Currently, thymidine labeling index (TLI), flowing blood of cells estimated to be in S phase Spherometry, or the nuclear antigen K1 found in at least some proliferating cell types The results obtained even using the tumor growth method by measuring -67 The results show that the higher the S phase fraction, the lower the predictability. Clinical research using the 几I method Study identified a small group of node-negative women with pectoral muscle disease who had a recurrence rate of 48%. and successful treatment decisions (Meyer et al., 51:1879-8 9 (1983). Therefore, such methods include cancer prediction, treatment and tumor development. There is great potential value in terms of ecology when measuring proliferative capacity.

Equally important, however, is that the measurement of TLI is is impractical and unphysiological. Chest tumor analysis It has to be done within approximately two hours, which makes it possible to carry out this measurement at a central laboratory. It's preventing things.

In general, phosphor I is measured in salt solutions under very high atmospheric pressure and Allow the gin to penetrate. Under these conditions, tumors develop after 2-3 hours. It is important to note that cells that have lost their viability and are often able to cycle cannot be measured. Must be. The reason is that the measurement time is longer than the generation time of asynchronous cells within the tumor. This is because the length is also significantly shorter. For other tumor types, surgically obtained There is little information on measuring the proliferative potential of books.

On the other hand, flow cytometry provides a rapid measurement of cell cycle dynamics and Although it is possible to evaluate numerosity as well, it has the following technical problems. (i) Mechanical or require enzymatic dissociation into a single cell suspension, resulting in loss of capacity found in the tissue structure and potential of one or more specific populations of cells. Selective losses occur. All heterogeneous cellular data of an individual tumor, including spatial histology. A complete evaluation of the type is clearly important in developing predictive testing methods.

(i) Flow cytometry is used for S-phase diploid cells and aneuploid quiescent or non-viable cells. The cells cannot be clearly distinguished.

This is an important issue. The reason, according to research, is that tumors rich in aneuploid cells Subpopulations of tumor cells were identified by dye-exclusion analysis (anal ysis) showed that the majority were non-viable (iii) The S-phase fraction of a ploid tumor is contaminated with non-proliferative and non-tumor cells, so it is Calculated measurements appear to be underestimated. Obtained from primary chest force lucitima In addition, the invasive ability of diploid cells in vitro has been clearly demonstrated (Smith et al., Proc, Natl, Acad, Sci, USA, 82: 1805-9 (1985)).

Nuclear antigen K1-67 appears to appear when breast cancer cells proliferate (McGu rrin et al., Cancer, 59: 1744-50 (1987)), Its relevance to other tissue types is not yet known.

Perhaps most importantly, these techniques at some point (flow cytometry, K1 -67), or after a very short labeling time (TLI), to measure cells in the S phase. Is Rukoto. Therefore, these measurement methods provide a more accurate assessment of tumor growth potential. It also prevents the evaluation of the total cell viable fraction of the tumor, which can adequately reflect the measurements. It is.

Importantly, both of the above-mentioned methods are suitable for use in close proximity to normal tissue, especially tumor tissue. It cannot be applied to systematically measure the proliferation ability compared to the Additionally, the method has been systematically applied to the comparative measurement of tumor growth in the presence of anti-proliferative therapeutic agents. However, the present invention aims to improve the cell proliferation capacity and cell proliferation capacity of cells obtained from patient biopsy material. An improved in vitro analysis system for measuring cell viability is provided. Main departure Measurements of cell proliferation and viability performed by and accurately predict overall invasiveness.

The present invention also provides tumor-specific effects of drugs on cell proliferation and/or viability. It also contemplates a method for measuring. This method is performed for each tumor tissue sample and normal tissue sample. tissue culture in separate vessels as a first part and a second part. be. A normal tissue sample is from tissue similar to the stem cells of a tumor. tissue culture The cultured sample is exposed to one or more concentrations of the agent whose effect is to be evaluated.

Next, the sample exposed to this drug is treated with a proliferation marker (DNA synthesis marker). culture (tissue culture) for a predetermined period of time. Then import the marker Measure the percentage of tumor cells and compare the results obtained from similar normal cells and tumor cells. Tumor specificity of drugs on cell viability and/or proliferation ability by comparing measure effectiveness.

Detailed description of the invention The present invention relates to cell viability and/or expansion in their native environment, i.e., parts of tissues. A method of measuring the effect of a drug on reproductive performance is provided. This kind of “drug” sensitivity When performing sex determinations, tumor cells are treated with one or more predetermined doses of one or more predetermined dose levels. Exposure to the drug and its effect using multiple containers and homogeneous specimens of the same explanted tissue Measured by culturing each individual. One of the homogeneous samples was used as a control. Quantitatively measure cell viability and proliferation without exposure to drugs . Each other homogeneous specimen was exposed to the drug at different dosage levels and then each Using protein synthesis markers and/or DNA synthesis markers to determine viability and quantitatively measure the ability to grow and proliferate. That is, the number of viable cells in each tissue sample is , depending on the number of cells that have metabolically incorporated protein synthesis markers into their cellular proteins. Show. Similarly, the number of cells that proliferate is determined by the number of cells that metabolize DNA synthesis markers into cellular DNA. It is indicated by the number of cells taken up.

Metabolic markers of cellular protein synthesis are well known in the art, and include For example, 35S-methionine, +4C-alanine, '4[-glycine, +4cmg Radioactive elements such as rutamic acid, +4cm proline, 3H-leucine, H-serine, etc. There are amino acids labeled with.

Metabolic markers of cellular DNA synthesis are also well known in the art, e.g. , 3H-deoxythymidine, 3H-deoxyadenine, 3H-deoxyguanine There are nucleotides labeled with radioactive elements such as 3H-deoxycytosine.

The various types of tissues to which the present invention can be applied include normal cells as well as primary cells. tumors, including solid tumors (carcinoma and Sarco 7). ), etc. are included. Various types of cameras to which the analysis method of the present invention can be applied Rutinomas (undifferentiated variants of glandular, scaly, and various carcinomas) include the following: including; for example, adrenal glands, bladder, chest, colon, kidneys, lungs, ovaries, pancreas, Prostate, thyroid, upper respiratory tract (head and neck), uterus (body and neck), bile ducts, villi Cancer, esophagus, liver, parathyroid gland, rectum, salivary glands, small intestine, stomach, lightning, tongue and urethra. . Various types of sarcomas and other tumors to which the present invention can be applied include, for example: For example, widespread lymphoma, Ewing tumor, Hodgkin's disease ()Iodgkin's disease), melanoma (black color and melanin deficiency), multiple myeloma, renal bud Cell tumor (Wi1m's tumor), neuroblastoma, nodular lymphoma, lateral stranguloma , angiosarcoma, brain tumor (glioma), chondroma, dysgerminoma, fibrosarcoma, smooth muscle These include sarcoma, liposarcoma, osteoblastoma, mesothelioma, osteosarcoma, retinoblastoma, and thymoma.

Generally, the tissue whose cell survival and/or proliferative properties are to be measured is septic explanted by a surgical procedure, and the portion is preferably about 0.5 to about 10 cnt. The range is about 1.0 to about 8. oant, more preferably 1.0 to 2.0 cnt Divide into quantity intercepts.

When analyzing tumors, separate analysis allows testing of many parts of the tumor. is important. The reason is that tumors are highly heterogeneous.

After cubing, the explant is usually divided into at least approximately 6 pieces to ensure a uniform specimen. do. Usually one of them is not exposed to or in contact with the drug being tested. control. The tissue is then homogenized to maintain its three-dimensional integrity. Specimens are tissue cultured on hydrous extracellular matrix-containing gels.

Gel with IJ hooks (extracellular gel with quality level suitable for tissue culture) is well known in the art, and the U.S. patent of Yannas et al.! 4,06 It is described in No. 0.081, and the product name is Ge1foan+ (upjohn). It is sold from.

Various liquid tissue culture nutrient media capable of sustaining the growth of tissue cells are well known in the art. It is publicly known. Media that can be used include insulin, transferrin, Contains additives such as selenium, estradiol, etc., contains serum, or does not contain serum. It's something that doesn't exist.

The medium found to be particularly suitable for the present invention is the Eagle Minimum, typically tested cells. The tissue is incubated for at least 2 to 5 days, preferably about 3 to 5 days, before exposure to the agent to be tested. Culture the tissue for 4 days. This culture typically involves culturing tissue samples in a humidified atmosphere. Perform the test at a temperature that corresponds to the body temperature of the animal from which it was collected, e.g., 37°C for large tissue samples. .

Exposure of cells to drugs for the purpose of determining viability and/or proliferation ability The process involves treating tissue samples with protein synthesis markers and/or DNA synthesis markers. Do it before you do it. This treatment involves treating a tissue sample with a predetermined amount of drug (predetermined concentration). , incubating for a predetermined period of time (predetermined time), and then injecting the tissue sample with the drug. The sample tissue can be separated from the drug and preferably washed to remove any residual drug from the sample tissue. preferable.

In this specification, "dose level of exposure to a drug" refers to a dosage level at which the drug concentration is quantitatively adjusted ( (e.g., μl) and exposure time (e.g., hours or minutes). Usually drug concentration and the exposure time, the dose level of drug exposure achieved in vivo is comparable to that achieved in vitro. Calculations are made from pharmacological data that can be simulated. Analysis of the invention Required dose level of drug exposure when measuring drug susceptibility according to the law Regarding the known anticancer drugs to be tested with the system of the present invention, the clinically achieved drug It has been found that up to 5% to 10% of the dose level exposed to

The drug susceptibility measurements described above involve the measurement of a given drug against specific human tumors obtained from explanted cells. Enables measurement of "drug sensitivity index" indicating antitumor activity for a given drug It can be done by method.

This method involves measuring drug sensitivity at multiple dose levels over many logarithmic ranges. and then use the results of these measurements to determine the survival rate (%) Analytical values obtained from drug-exposed versus non-drug-exposed controls This includes plotting the dose level of exposure to the drug on a curve. Next The “drug susceptibility index” for a given drug is defined as the highest clinically achievable index for that drug. Measure the area under the curve up to a defined upper limit that correlates with the dose level of exposure to the drug. Quantitate by

The susceptibility index obtained by the above method is This shows the anti-tumor activity of the drug to a high degree, and even a low sensitivity index shows high anti-tumor activity. Shows tumor activity.

After culturing the cells in tissue culture (approximately 1-4 days) in the presence of the agent to be tested, the sample is determined to be proliferative. Treated in the presence of a Ka marker and a viability marker.

Cells from tumor samples are exposed to drugs and labeled with proliferative and viability markers. After treatment and subsequent tissue culture, the sample is typically treated with a Fix in a suitable tissue fixative, embed in paraffin, etc., and fix with microtin (mic section on rotine). Viability marker and/or proliferative marker contains a radioactive label, the sections are then labeled with NTB (Kodak). Treated with nuclear-track emulsion and developed do.

The sections are then analyzed to determine the presence of viability or proliferative markers. Calculate the percentage of cells that are positive. Using radioactive labels and nuclear trace emulsions In this case, this analysis can be performed with a polarizing microscope. Preferred embodiment here is a computer-linked video that can digitize the analyzed images. The enlarged image can be digitized by processing it through a camera.

As is clear from what has been described so far, the in vitro analysis system of the present invention is useful for in vitro prediction of clinical response to cancer chemotherapy, as well as for clinical trials. It can be used to screen for new anticancer drugs. For example, certain When treating a specific patient with cancer, the extraneous tissue obtained from a biopsy of this specific cancer Explanted cells can be analyzed according to the techniques of the present invention and used for chemotherapy for certain cancers. Measuring drug sensitivity for multiple different anti-cancer drugs with potential clinical efficacy can do. After determining the relative drug sensitivity index of the various drugs being tested, These susceptibility indices can be used to predict the most promising effective agents for use in chemotherapy. It can be used for various selections. The reserve of this technology, both historical and predictive. found between in vitro prediction and in vivo response in clinical trials. The correlation is impressively high, reaching 100%.

In another aspect, the invention contemplates a method of determining the "growth factor index" of a tumor. . The tumor growth factor index is related to the in vivo extent and stage of the tumor, i.e. related to the in vivo invasiveness of carcinomas, particularly breast and ovarian carcinomas.

Growth factor indices are measured by tissue culturing samples of tumor cells as described above. Ru. Subsequently, the cells are treated with one of the aforementioned DNA synthesis markers, e.g. Treat with proliferation markers such as H-deoxythymidine. Place the treated cells in Samples with cellular DNA that have been cultured for a specified period of time and then incorporate proliferation markers Measure the percentage of cells. This percentage (%) is a growth factor index specific to that tumor. Show the number.

In another aspect, the present invention provides an in vitro method for determining cell viability and/or proliferation ability as described above. It is intended as a measurement kit. This kit includes at least one analysis. to test its effectiveness in terms of viability and/or proliferation ability. Examples of reagents and viable hebuki are for the purpose of illustrating the present invention only, and are not intended for use by others. It is not intended to limit the invention unless otherwise specified.

1. Cell proliferation ability Specimens of various normal tissues and tumor tissues were collected as described in the paper in parentheses below. were explanted from human patients (Freeman et al., cited herein). All references are incorporated herein by reference. To explain briefly After the tissue was surgically removed, it was cut into sections with a diameter of 1 to 2 mrn and cut into pig skin. (Gelfoaa+.

of the above-mentioned hydrous extracellular mal-IJ wax-containing flexible gel prepared from on top to form a three-dimensional culture. Eagle's Salts, Glutamine, Fetal Bovine Serum Eig containing 10% non-essential amino acids and the antibiotics Garamycin and Claforan Add the minimum essential medium (MEM) to the culture without covering the top of the gel. The culture was maintained at 37°C in a carbon dioxide incubator, and the explant tissue specimens were grown. I made it possible to do so.

After 10 to 12 days of culturing, the cells in the three-dimensional culture that can grow were cultured for 3 days. -Thymidine and 3H-deoxyuridine (2 μCi each; I Ci = 3 Labeled (V Escio et al.'s paper, Proc, Natl, Acad, Sci.

” Fat, 84:5029-33 (1987)). Cellular DNA replicates within tissues. It is labeled when it is inside the cell. . After 4 days of labeling, cultures were incubated with phosphate buffer. Washed with normal saline, placed in a histology capsule, and fixed in 10% formalin. . This culture was then dehydrated, embedded in paraffin, and sectioned using standard methods. and placed the section on top of a mounting glass.

Next, paraffin was removed from the sections on the mounting glass, and Kodak NTB-2 was placed in the dark. After being coated with an emulsion, prepared for radiophotography, and exposed for 5 days, the imaged. After development and rinsing, sections were stained with hematoxylin and eosin. did.

Next, the proportion (%) of cells that performed DNA synthesis in various tissue cultures was measured. Sections were analyzed by determining the For this measurement, an incidental illumination polarized illumination system was used. (epi-illumination polarization light Nikon or Olympus polarizing microscope camera equipped with used. Replicating cells are silver grains that appear bright green without incidental illumination and polarization systems. This was confirmed by the presence of the child on the surface of the cell nucleus. These silver particles are radioactive DN It was produced by the exposure of the NTB-2 emulsion to A. With the above method, the tumor Obtain histological radiographs showing the proliferative potential of cells in tumor explants. I can do it.

Most tumors cultured in autochthonous systems have at least some areas with high cellularity. showed proliferative capacity and was heterogeneous among tumors with respect to proliferative capacity. Tumor tested Tumors include tumors of the colon, ovaries, bladder, kidneys, brain, and parotid glands, and small cell lung tumors. Ewing sarcoma and Ewing sarcoma. In all cases correspond to the original organization Three-dimensional tissue organization was maintained throughout the culture period. With incidental illumination polarized light microscopy, Radioactively labeled proliferating cells can be detected at a high level. This microscope is Scattering of incident polarized light increases the amount of radiographically sensitive silver particles detected.

The growth potential of metastatic colorectal tumors showed a high degree of labeling in culture. observe More than 90% of the cells in cultured preparations were metastatic to this relative undifferentiated colon metastasis to the liver. proliferated during the labeling period. The growth potential of small cell lung tumors is determined by the oat cell type ( It was shown that two main types of cell types (oat cell type) were maintained. child The typical small cell and more elongated spindle-shaped cell types each have a high cell proliferation rate. It had a high level of breeding.

The proliferative capacity in ovarian rutinomas is consistent with a very high index of proliferation in epithelial cells. The stromal cells remained inactive, while the stromal cells remained inactive. This histological radiation The photo shows ovarian rutinoma cells that have invaded the gel matrix that maintains their growth. It showed high proliferation ability. This invasive effect shows that in vivo, ovarian tumors frequently invade the peritoneum. It is very similar to the way it invades.

including tumors of the pancreas, bladder, kidneys, brain, and parotid glands, and Ewing sarcoma. The proliferative capacity of many of these cultured tumors is It showed an intricate line structure including

Proliferating malignant and normal cell types, e.g., breast tumor epithelial cells and normal It is important to be able to observe and distinguish stromal cells from prepared radioactive photographs. .

A further important point to note in these studies is that normal tissues are cultured and The goal is to make them multiply sufficiently. Explanted tumor tissue obtained from the chest of patient No. 431 and Nearby normal tissue was compared.

It has been found that cell proliferation of large scales exists in normal tissues. But even higher It was observed that a level of histology was maintained in normal tissue. This By using stems, tumor and normal biology, e.g. auxotrophy, growth factors, It is now possible to compare child requirements and metabolic pathways. Also, especially important The antitumor susceptibility of a potential tumor drug is determined by the sensitivity of tumor and normal tissue to that drug. It is now possible to measure by comparing responses. In this case, the implementation Cell proliferation is used as an end point, as described in Example 3.

These results demonstrate that the present invention can be used to treat all major types in relatively long-term culture. This shows that this is a general system used to measure the growth potential of large tumors.

As mentioned above, all cultures in this report were performed in vitro over relatively long periods. I did the questions for 14 days. For most tumor specimens, the culture period can be extended even further. (data not shown). More than 90% of surgically obtained specimens are cells can be cultured and analyzed for growth potential.

This autochthonous culture system, using polarized light microscopy as an auxiliary tool, has the potential to There is a high probability that cells with reproductive potential can be detected.

A video camera was attached to the microscope for image analysis of proliferating cells. next , a radioactivity photograph taken using the above method using chest force rutinoma tissue was exposed to illumination light. (Bright-field light) was not used and was examined under polarized light.

This allows only radioactive cells exposed to the silver particles in the nuclear trace emulsion to be visualized. It became. These radioactive cells brightly reflected polarized light. The obtained image, Computer-assisted image fraction that is first digitized by a digital board Corresponding to the number of cells that are labeled, i.e. bright, and subsequently analyzed by an analyzer Fas-cam of the P-3ee program, with the bright region as the region of enhanced pixels. version (The Microworks, Del Mar, CA) Calculated by running on an IBM PCXT compatible computer. enhanced The area of labeled pixels is proportional to the number of labeled cells.

Use this image analysis system to convert radioactive photographs into numbers of labeled proliferating cells. automatically analyzed. Cultured breasts by using illumination light and polarized light microscopy Labeled cells of the tumor appeared bright green. This measurement does not use illumination light. , of labeled cells by using a polarized light microscope with incidental illumination using polarized light. I visualized it. Next, images of the labeled cells are captured using a video camera and a P-3ee program. Digitized via Gram. Next, the bright region, i.e. the region of enhanced pixels, is The area was measured automatically by the Pas-Com program. Area of enhanced pixels is proportional to the number of labeled cells, allowing automatic counting of labeled proliferating cells. numbers become possible.

An important point in this culture system is the flexible extracellular matrix on which the tumor is explanted. The method is to use a gel containing J wax. Other researchers have used flexible extracellular matrices. It has been stated that culture media containing sugar are unstable for the growth and functionalization of differentiated cells. (Li et al.'s paper, Proc, Natl, Acad, Sci, ll5A , 84:136-40 (1987); Davis et al., 5science , 236:1106-9<197) H5chaefer et al., Canc. er Res,, 4th Applications, eds, Kru Se et al., (Academic, New York), pp, 367 -7H1973)).

The general principles described here can be applied to all types of large organizations; Enables the accumulation of potentially important biological and clinical predictive information. Furthermore, this It must be noted that many of these tumors have a high degree of cell proliferation ability. No. A final understanding of allowing such deregulation of proliferative capacity. should be promoted using said system, which allows us to predict tumor formation. enables a deeper understanding of the changes that occur.

2. Measurement of cell viability and proliferation ability in autochthonous tissue culture Tumor tissue specimens taken from patients suffering from cancer were prepared as described in Example 1. , cut into sections with a diameter of 1 mm, and place each section on a flexible gelatin I-IJ hook. , three-dimensional cultures were formed. Identical cultures were prepared for each specimen; Either 5sS-methionine or 3H-thymidine was added to 8 microcuries (μ ci) were added. Each culture contained 2 ml of medium containing the radioactive label. Ta. Labeled cultures were maintained for 4 days in advance and continuously fed with phosphate-buffered saline. Rinse with saline water (PBS) to remove excess radioactive label from each cultured specimen. , and this culture was subjected to the histological processing and radiophotographic techniques described in Example 1. Visualization processing was performed. Next, the prepared culture was transferred to the device described in Example 1. After the digitization process, analysis was performed using the analytical computer program Fas-com. analyzed. Measuring "'S-methionine taken up into cultured cells" Allows measurement of protein synthesis. Therefore, this increases cell viability. Measure. Measurement of 3■-thymidine incorporation into cultured cells can be used to measure DNA synthesis. enable.

Therefore, this measures the proliferative capacity of the cells.

Tissue specimens cultured in the presence of 3SS-methionine or 3H-thymidine remained viable. , or parts of tissue containing proliferating cells, individually or both, that are labeled with a radioactive label. I took it in. Cells that are not viable or do not proliferate should be labeled with each of these. No silver particles were present on visual inspection of the prepared specimen, and no proliferating cells were observed. It does not exist in cells either. In addition, when analyzed with an incidental illumination polarization system, this silver particle It is a bright green landmark.

The range of proliferative potential that can be measured in an in vitro autochthonous culture system depends on the extent of the tumor. and stages. The more malignant the tumor, the greater its proliferative capacity measured in vitro. It gets expensive. A range of proliferative potential exists in selected fields of view that can be observed under a microscope. Growth factor index, measured as the percentage of proliferating cells in a population of total cells (GFI). Therefore, using the measured GFI, It is possible to predict the clinical stage of progression of human cancer tissue under test. Fas-calT + Programmed analysis is a quantitative analysis that semi-automatically measures tumor growth or viability. This is an ideal and suitable means to provide 6F1 data. Ru.

Proliferative capacity analysis can be performed on many metastatic or primary and poorly or moderately differentiated cells. Average GFI for each type of tumor tested, performed on explanted tumor tissue. I got it. Metastatic tumors had an average GFI of 0.437±0.149, whereas However, the primary tumor showed a lower mean value of GFIo, 282±0.138. same Similarly, poorly differentiated tumors had an average GFI of 0.372±0.150. In contrast, tumors with appropriate differentiation had an average GFI of 0.220±0.094. there were. The results show that GFI correlates with tumor severity and clinical prediction. are doing.

Tissue specimen cultures were prepared using various tumor tissues as described in Example 2. Ta. After incubation for 4 days, multiple cultures of each tissue were treated with the drugs described below at predetermined concentrations. The cells were cultured for an additional 24 hours in the presence of .

Cultures are then washed in medium to remove excess drug and grown in the absence of drug. Cultured for 3 days to allow the cells to recover from the effects of the temporary drug, then Example 2 Labeled as described.

After labeling, the cultures were treated as described in Example 1 and incubated in the presence of drug. The degree of viability and proliferative ability of the specimens when cultured was visualized.

Cells cultured from ovarian rutinoma tissue of a patient with ovarian rutinoma was tested for cell proliferation ability by the method described above in the presence of the following drugs: was carried out. The drugs used were cisplatin <1.5 μs/ml>, 5- Fluorouracil (4 μg/ml), melphalan (10 μg/ml), metho with trexate (22.5μg/ml) or thiotepa (60ug/ml) be. As a result, due to the incorporation of both 3H-thymidine and 35S-methionine, The detectable signal generated by the test was reduced. This indicates the ability to proliferate and survive. This shows that both were inhibited. Decreased cell proliferation ability occurs when the same specimen is treated with drugs. When cultured in the absence of GFI and compared with GFI, it appears as a decrease in GFI.

Cell proliferation ability decreases to 90% when using cisplatin; 99% with Rasil, 70% with Melphalan, methotrex When Cert was used, the increase was 70%, and when Thioteba was used, the increase was 90%.

Explanted tumor tissue obtained from patients S and D with ovarian rutinoma was Cell proliferation ability was inhibited by more than 70% at 10 μg/7 ml. This tumor is The patient responded to treatment with melphalan and showed a decrease in tumor size during treatment. death Therefore, the in vivo responsiveness of tumors to drugs and the in vitro autochthonous drug response It became clear that there was a clinical correlation between sex and gender.

The presence of drugs in cells cultured from tissue of patients with chest rutinoma The test was conducted as described above. As a result, the cell's ability to proliferate is expressed in the following brackets. Niadriamycin decreased to the percentage shown in 290 ng/ml (90%) , 5-fluorouracil 4 μg/ml (90%), Melphalan 1 μg/ml l (80%), methotrexate 2.25 μg/ml (70%) or Binc Restin was 23 ug/ml (70%).

Patient H. was diagnosed with thoracic rutinoma and treated with 5-fluorouracil or determined to be unresponsive to in vivo treatment with either adriamycin. Ta. as described above in the presence of either 5-fluorouracil or adriamycin. Cultures performed on sea urchins were from patients 1) and 11. Cell proliferation ability of breast rutinoma tissue The force was not blocked sufficiently (ie, more than 90%). Therefore, in There was a clinical correlation between in vivo responsiveness and reduced in vitro cell proliferation capacity.

Cultured cells from cancerous tumors taken from patients with colorectal cancer were treated with various of the above-mentioned drugs. Tests were conducted as described above in the presence of the agent.

As a result, the proliferative ability of the cells decreased to the following percentage in parentheses: 5-Flu Orouracil 4 μg/ml (90%), mitomycin C1, ug/ml (9 0%) and BCNU2ug/ml (90%).

Patients V and S were diagnosed with colorectal cancer and were treated with 5-fluorouracil. was determined to be unresponsive to in vivo treatment. In the presence of 5-fluorouracil Culture of the explanted tissue as described above was performed for colonic rutinoma of patient V, S. The cell proliferation ability of the cells was not sufficiently inhibited (ie, by more than 90%).

Again, a clinical correlation between in vivo and in vitro responsiveness was observed. .

These results indicate that if the active agent blocks the ability of cells to proliferate, it can inhibit the ability of the cells to proliferate. This study shows that it can be used to measure drug responsiveness. Cells are in proliferative state If not, tumor tissue viability as measured by 35S-methionine incorporation. can be used to demonstrate tumor drug responsiveness. In addition, the range of viability is It can be used to determine the end point for maximum response.

The foregoing description and examples are intended to illustrate the invention; It does not limit its scope. Others within the spirit and breadth of this invention It is possible to further embody the modified example, and a person skilled in the art can easily do so. It can be implemented.

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Claims (9)

[Claims] 1. A method for measuring the tumor-specific effects of drugs on proliferation and viability. hand, (a) separate the first and second portions of the tumor tissue sample and similar normal tissue sample; (b) exposing the sample to a drug; (c) treating a first portion of the sample with a DNA synthesis marker and treating a second portion of the sample with a DNA synthesis marker; (d) treating the portion with a protein synthesis marker; (d) treating the sample with a protein synthesis marker; culturing the portion for a predetermined period of time; and (e) DNA synthesis markers and proteins of the first part and the second part, respectively. Measure synthetic marker uptake and thereby assess cell proliferation and viability. A method comprising the step of measuring the tumor-specific effect of the drug. 2. The DNA synthesis marker is 3H-thymidine, and the protein synthesis marker is 2. The method according to claim 1, wherein the 35S-thymidine is 35S-thymidine. 3. Claim 1, wherein the predetermined period of time in step (d) is from 2 to about 5 days. How to put it on. 4. Before performing step (b), the first and second portions of each sample were incubated for 2 to about 6 days. 2. The method according to claim 1, which comprises interculturing. 5. A method for measuring tumor invasiveness, the method comprising: (a) culturing a sample of tumor cells; (b) treating the sample with a DNA synthesis marker; (c) treating the treated sample with a DNA synthesis marker; (d) culturing the tumor cells incorporating the DNA synthesis marker for a predetermined period of time; A method comprising measuring the percentage of cells and thereby determining the invasiveness of the tumor. 6. Claim 5, wherein the DNA synthesis marker is 3H-deoxythymidine. How to put it on. 7. cell viability and cell viability, including protein synthesis markers and DNA synthesis markers; Kit for in vitro measurement of growth and proliferation ability. 8. The protein synthesis marker is 35S-methionine, and the DNA synthesis marker The kit according to claim 7, wherein is 3H-deoxythymidine. 9. 8. The kit of claim 7, further comprising a tumor cytotoxic agent.