JPH0728757B2 - Anticancer drug sensitivity test method - Google Patents

Description

TECHNICAL FIELD The present invention relates to an anticancer drug sensitivity test method, and
The present invention relates to a method for culturing cancer cells in vitro to test the sensitivity of the cancer cells to an anticancer agent.

[Prior Art] To test the effect, or sensitivity, of an anticancer agent, a clinical test method in which an anticancer agent is administered to a cancer patient and a change in cancer tissue is diagnosed and tested, an animal in which a human cancer tissue is transplanted is administered an anticancer agent Experimental method, and after contacting the cancer cells removed from the cancer patient with an anticancer agent, the cancer cells are cultured with an appropriate substrate, or the anticancer agent is contacted during the culture, and the cancer after a certain period of time There are cell culture test methods and the like for measuring the degree of cell proliferation.

At the initial stage of the development of an anticancer drug, clinical test methods cannot be applied suddenly, so first of all, after sufficiently confirming the anticancer effect of the anticancer drug on the cancer cells, that is, the sensitivity of the anticancer drug, by the cell culture test method or the animal test method. , It is usual to confirm the final effect by clinical test methods. The animal experiment method has problems that it is difficult to control experimental animals such as nude mice, the success rate of transplantation of human cancer cells is low, and it takes a long time to confirm the experimental results. Has the advantage that it can be carried out with relatively simple equipment and work, and results are obtained quickly. Therefore, in the development of an anticancer drug, the anticancer drug susceptibility test by the cell culture test method plays an extremely important role.

Conventionally, as a typical cell culture test method,
There is a method called HTCA (Human Tumor Clonogenic Assay) method. This method is to prepare a single cell dispersion liquid from a cancer tissue taken out of a living body, contact an anticancer agent with this, and then cultivate the cancer cells in a soft agar medium. The sensitivity of the anticancer drug is tested by measuring the number of colonies and evaluating the inhibition rate of colony formation by the anticancer drug. The number of colonies is calculated visually or using a colony counter. In addition, there is a method of culturing using a monolayer culturing method and using an isotope method or a DNA measuring method to check the influence of an anticancer agent on cancer cells.

[Problems to be Solved by the Invention]

However, the HTCA method, which is a typical anticancer drug sensitivity test method in the cell culture test method, has the following problems.

First, the culture method using soft agar medium has an advantage of suppressing the proliferation of fibroblasts, but a large number of cells are required due to the low colony formation rate of cancer cells, and as a result, a certain amount of cells are required. Fewer tests can be performed on the harvested cells.
Moreover, there is a problem that the types of cancer cells that can be cultured are limited. That is, the types and properties of cancer cells differ variously due to differences in affected tissues and the like, and among these various types of cancer cells, the number that can be cultured in an agar medium is considerably limited.

Therefore, the present inventors, in a system of primary culture using human cancer cells, as a result of a study on a new anti-cancer agent sensitivity test in which soft agar medium was changed to a collagen gel substrate, the collagen gel culture method was found to grow in soft agar medium. It was found that even human cancer cells that do not grow can be stably grown (Hizuka et al. 6
See “Tissue Culture Research”, Vol. 6, No. 1, 1987).
As a result, the application range of the cell culture test method could be broadened significantly. It was also found that collagen gel has an advantage that the required number of cells is small and the test results can be obtained efficiently in a short time because the culture of cancer cells can be performed more efficiently than in the soft agar medium.

When collagen gel is used as the substrate, the method of measuring the proliferation degree is generally performed by measuring the DNA amount of cells grown in the collagen gel and calculating the proliferation degree of cancer cells.

However, when culturing cancer cells on a collagen gel matrix,
Depending on the type of cancer tissue, there arises a problem that fibroblasts contained in the cancer tissue grow at the same time as the cancer cells.
Since fibroblasts contain DNA like cancer cells, the above-described method for measuring the proliferation of cancer cells by quantifying DNA cannot be applied. Therefore, in this case, the formed colonies are measured by visual inspection or using a colony counter. However, when the measurement is performed by visual observation using a stereoscopic microscope, it takes a great deal of effort to pick up and measure only the cancer cells, and variations occur due to the measurer, which makes accurate measurement difficult. Further, it is practically impossible to clearly identify and measure cancer cells and fibroblasts with a normal colony counter. Many of the human cancer tissues obtained from clinical materials are
Since fibroblast proliferation occurs at the same time as cancer cells, it was extremely difficult to experience the sensitivity of an anticancer drug to cancer cells cultured on a collagen gel substrate unless the cancer cells and fibroblasts were separated. . Conventionally, there are cis-oxyproline method, D-valine method, citrulline method and the like as methods for suppressing fibroblast proliferation without suppressing cancer cell proliferation, but all are of a very limited type. It can be applied only to the fibroblasts, and is difficult to put into practical use.

Therefore, the present invention takes advantage of a collagen gel matrix capable of culturing cancer cells satisfactorily and, even when fibroblast proliferation, which is a defect of the collagen gel matrix, occurs, changes in proliferation of only cancer cells are suppressed. It is an object of the present invention to provide an anticancer drug susceptibility test method that can be easily and reliably measured.

[Means for Solving the Problems]

In order to solve the above problems, the present invention proliferates cancer cells using a collagen gel as a substrate, and uses an image analyzer as a means for measuring the degree of proliferation of the cancer cells, and an image signal of a sample obtained by imaging. An anti-cancer drug susceptibility test method for testing the susceptibility of an anti-cancer drug by selectively extracting image signals of cancer cells and colonies thereof from the cells and measuring the proliferation rate of the cancer cells from the obtained image information of the cancer cells and colonies To do.

According to one aspect of the present invention, the degree of proliferation of cancer cells is measured by selectively extracting the image signals of the cancer cells and colonies thereof from the image signals of the sample obtained by imaging. The estimated volume value of the cancer cell and its colony is calculated from the image signal of the cancer cell and its colony.

According to another aspect of the present invention, the degree of proliferation of cancer cells can be measured by emphasizing the concentration characteristic of an object from the image signal of a sample obtained by imaging by theoretical calculation. The image signals of the cancer cells and colonies thereof are selectively extracted by a method of extracting only the image of the object according to a certain reference value regarding the density from the emphasized image, and the selectively extracted cancer cells and The number of cancer cells and colonies is calculated from the image signal of the colonies.

The collagen serving as a substrate may be any collagen having a gelling ability, but type I collagen having high gel strength is preferable. Also, because optical measurement is performed,
In the gelled state, it is preferable that it has sufficient transmittance for light having a wavelength used for measurement and is optically homogeneous. Further, those which do not cause optical deterioration with time during culture are desirable.

In addition, the step of culturing the cancer cells with a substrate made of collagen gel is performed according to various tissue culture methods using conventional collagen gel. The timing of contacting the cancer drug with the cancer cells is
For example, before seeding cancer cells (primary cells) collected from a patient on a collagen gel matrix, before subculturing primary cancer cells and then seeding on a collagen gel matrix, or
There is no particular limitation as long as it is set appropriately such as immediately after seeding on the collagen gel substrate or after culturing for several days.

An image analyzer is used as a means for measuring changes in the growth of cancer cells. The purpose of an image analysis device is to connect to an optical device such as a microscope, convert the image signal obtained from it into numerical image information, and then perform arithmetic processing to clarify information about the object. Used in. To this end, the image analysis apparatus is provided with an image processing mechanism such as a microcomputer, an image storage mechanism such as a fixed disk device, an output mechanism such as a monitor television or a video printer that outputs analyzed image information.

As the microscope, a microscope similar to a conventional microscope for measuring cancer cells by visual inspection can be used. By appropriately selecting the conditions such as the magnification of the microscope, that is, the size of the visual field, the depth of focus, the subject distance, the mounting structure of the culture container, and the illumination device, it is possible to improve the measurement capability or the measurement accuracy. An input device such as a TV camera is attached to the microscope in order to input a sample image signal to the image analysis device.

Although it is possible to manually set the culture sample at the observation position of the microscope, if the continuous transport mechanism that can continuously send and take out the culture sample to the observation position is provided, the efficiency of measurement will be improved. be able to.

In the present invention, the TV camera of the image analysis device captures a grayscale image of the culture sample. The imaged sample image may be only an image of cancer cells and colonies thereof (hereinafter collectively referred to as cancer cells), or may be a mixture of these images and the above-described image of fibroblasts. The image analyzer removes unnecessary images such as fibroblasts other than cancer cells from the sample image. To separate and remove the image of the cancer cells and the other unnecessary images, the grayscale and shape separation functions of the image analysis device are used. That is, cancer cells form a mass, that is, a colony, which has a certain size, shade and contour, in which a large number of cells gather, whereas, for example, fibroblasts, as the name implies, are thin fibrous or The cells are branched, and cancer cells and fibroblasts can be clearly distinguished by the difference in shape and image density. Therefore, the image analysis apparatus recognizes only images that satisfy certain conditions such as shape, area, density and color difference as cancer cells, and recognizes images that do not satisfy the above conditions as unnecessary images such as fibroblasts. As a result, images other than the cancer cell image can be removed and only the remaining cancer cell image can be extracted. If the shape of each image shown in the image of the cancer cells is arithmetically processed and evaluated, the change in the proliferation of the cancer cells, that is, the proliferation degree can be measured.

To evaluate the sensitivity of a carcinostatic agent, for example, cancer cells separately contacted with one carcinostatic agent or two or more different carcinostatic agents and control cancer cells not contacted with a carcinostatic agent are cultured under the same conditions, The change in the proliferation of cancer cells measured by the image analysis device may be compared.

In order to measure the growth rate of cancer cells from the cancer cell images selectively extracted as described above, there are, for example, the following two methods. The first is to measure the number of colonies from the cancer cell image, and the second is to measure the volume of colonies from the cancer cell image.

According to the first method, for example, the number of colonies obtained by culturing the cancer cells that have been contacted with the anticancer agent and the number of colonies obtained by culturing the control cancer cells that have not been exposed to the anticancer agent are measured by automatic measurement and compared. To do.

Further, according to the second method, for example, the volume of colonies produced by culturing cancer cells contacted with an anticancer agent and the volume of colonies produced by culturing control cancer cells not contacted with an anticancer agent are calculated and compared. To do. Here, the volume of the colony is calculated, for example, as described below, but is not limited thereto.

[Action]

By culturing the cancer cells on a collagen gel matrix,
The types of cancer cells that can be cultured increase, the range of application of the cell culture test method expands, and cancer cells proliferate well, so that test results can be obtained reliably and quickly.

By analyzing and measuring the amount of colony formation of cancer cells with an image analyzer, it is possible to eliminate the influence of fibroblasts that proliferate at the same time as the cancer cells. That is, in the image of the cultured sample, the cancer cell image forms a massive and dark image, and the fibroblasts form a thin fibrous and pale image. Have a clear difference. Since the image analysis device can extract only the image of the shape and density according to the certain conditions from the input image, the cancer cell image and the fibroblast image are separated and only the cancer cell image is extracted. You can In addition to fibroblasts, an image of an unwanted object having a different shade or contour than a cancer cell can be separated from the cancer cell image. By measuring the amount of formed cancer cell colonies from the extracted cancer cell image, it is possible to measure the proliferation change of only cancer cells that do not contain fibroblasts.

Compared with, for example, the method of macroscopically measuring under a microscope, the measurement with an image analyzer can measure complex quantities in a much shorter time, more efficiently, and with the skill of the measurer. Since the measurement can be performed under certain conditions while avoiding the variation of results and the possibility of erroneous measurement, the test results are stable.

Moreover, since it is a non-destructive measurement method, it is possible to measure the same sample over time, and when measuring the amount of change in an object, that is, cancer cells, it is possible to easily realize measurement accuracy that cannot be obtained by the destructive measurement method. . Further, the steps required for the analysis can be automated in the image analysis apparatus, and by linking with the continuous measurement mechanism, the measurement time and labor can be saved and rationalized.

When the proliferation degree of cancer cells is measured by counting the number of colonies from the selectively extracted cancer cell images, it is possible to accurately measure with an inexpensive image analysis device when there is no contact between colonies.

In addition, if the proliferation of cancer cells is measured by measuring the volume of the colonies from the selectively extracted cancer cell images, the number of colonies can be measured even if the colonies are in contact with each other or overlap. More accurate measurement is possible compared to.

〔Example〕

Next, specific examples of the present invention will be described in detail, but the present invention is not limited to the following examples.

(Experiment 1) -Comparison between collagen gel culture and other culture methods-First, experiments confirmed that the collagen gel substrate used in the present invention is more suitable for culturing human cancer cells than conventional soft agar medium. It was

Table 1 below shows the results of culturing various human cancer cells in a collagen gel matrix and a conventional soft agar medium.

In the table, the soft agar medium used was the culture method used in the conventional HTCA method. After culturing, the culture sample was observed, and those having more than 20 colonies of cancer cells were evaluated as successful culture. The numerical value in each column indicates the number of successes / the number of tests. The number of seeded cells is 5 × 10 ⁴ collagen gel matrix / m 2.
The soft agar medium was 5 × 10 ⁵ cells / ml.

As can be seen from the above results, it was demonstrated that by using a collagen gel substrate, far more kinds of cancer cells can be cultured as compared with conventional soft agar medium and the like.

(Example 1) The anticancer agent sensitivity test of the present invention was conducted by the following procedure to compare the effects of a plurality of anticancer agents.

-Culture of cancer cells-First, the cancer cells to be subjected to image analysis were cultured.

As the cancer cells, human cancer cells isolated from human colon cancer of nude mouse transplantation system by a stepwise enzyme treatment method using collagenase and pronase were embedded in collagen gel and cultured.

The embedding method is Cell Matrix Type IA (0.3%
Acid-soluble collagen solution: 1 volume of 10-fold concentrated Ham's F12 medium (without NaHCO ₃ ) to 8 volumes of Nitta Gelatin Co., Ltd., reconstitution buffer (260 mM-NaHCO ₃ and 200 mM-HEPE)
1 volume of S-containing 50 mM-NaOH solution) and 1 volume of FBS (fetal bovine serum) were added, and the human cancer cells obtained by the enzyme treatment were added thereto, mixed well, and stored in ice.
This collagen mixture was dispensed in 1 ml portions into a Petri dish having a diameter of 35 mm. The cell number was 5 × 10 ⁴ cells / ml. By heating this at 37 ° C. in a CO ₂ incubator, a collagen gel matrix containing human cancer cells was prepared.

1m of culture solution containing anti-cancer agent on the obtained collagen gel substrate
The human cancer cells were brought into contact with the anticancer drug by overlaying the cells with each other at 37 ° C. for 24 hours in a CO ₂ incubator. Then, the culture medium containing the carcinostatic agent was removed by suction, and then 2 ml of the culture medium containing no carcinostatic agent was added and shaken in a CO ₂ incubator to wash the collagen gel matrix. By repeating this operation every 10 minutes for a total of 10 times, the anticancer agent was washed off from the collagen gel matrix. As a control, a culture solution containing no anticancer drug was used, and the human cancer cells were not contacted with the anticancer drug.

Then, add 2 ml of the culture solution and keep it in a CO ₂ incubator at 37 ℃.
Culture was performed for 10 days. The medium was replaced every 1 to 2 days. After culturing, a collagen gel substrate containing cells was fixed with 10% formalin to prepare a sample.

The culture medium used above was DME + 10% FBS + Insulin.
(1 μg / ml) + EGF medium (10 ng / ml). Here, DME: Dulbecco's modified Eagle medium (Dulbecco's Modifi
ed Eagle's medium, Nissui Pharmaceutical Co., Ltd. FBS: Fetal bovine serum (Fetal Bovine Serum, Gibco) Insulin: Insulin (Sigma) EGF: Epidermal Growth Factor (Collaborative) Using.

Further, as an anticancer agent, mitomycin (MMC: manufactured by Kyowa Hakko Kogyo Co., Ltd.), fluorouracil (5-FU: manufactured by Kyowa Hakko Kogyo Co., Ltd.), vindesine sulfate (VDS: manufactured by Shionogi Pharmaceutical Co., Ltd.), etoposide ( VP-16: Nippon Kayaku Co., Ltd.)
And 5 types of cisplatin (CDDP: manufactured by Bristol-Myers Co., Ltd.) were used.

As the contact concentration of the anticancer drug to cells, 1/10 of the maximum blood concentration of each drug used in clinical use (concentration in tumor tissue) was used. That is, MMC = 0.1 μg / ml, 5-FU = 1.0 μg / ml, V
DS = 0.005 μg / ml, VP-16 = 0.1 μg / ml, CDDP = 0.2 μg / m
It was l.

-Image analysis processing-The colonies of human cancer cells that proliferated in the collagen gel matrix of the fixed sample obtained as described above and the morphology of the fibroblasts that proliferated at the same time were photographed with a TV camera through a stereoscopic microscope and obtained. Image analysis device (LUZEX III U,
The image signal was input to Nikon Corporation.

The specifications of the image analysis device are as follows.

Control processor: Intel 80386/7 32-bit processor Storage device: 40 megabytes Fixed disk device Image processing circuit: Image capacity Maximum 1024 x 1024 pixels x (8 + 1) bits Image array processor Image input device: TV camera Carnikon image pickup tube Optical Microscope image output device: Digital / analog RGB monitor Video printer The image signal input to the image analysis device is decomposed into pixels, and the light and shade corresponding to each pixel of the image is digitized,
Images of cancer cell colonies and fibroblasts were processed to separate them from the original images. In the process of separating the object image, first, the density characteristic of the object is emphasized by a logical operation. For the calculation, high frequency component extraction calculation,
A combination of density class fractionation and denoising was used. From the obtained emphasized image, only the image of the object was extracted according to a certain reference value for density. At this stage, the number of colonies containing only human cancer cells was counted by the difference in shade or the difference in change rate of shade. In the image after the image processing, only the colonies of the target cancer cells are emphasized and unnecessary images such as fibroblasts are removed, so that the number of colonies can be easily measured.

-Verification of measurement result by image processing-As described above, from the image after image processing, the result of measuring the colony of only human cancer cells and the result of performing the same measurement without performing image processing are shown. Compare.

When a formalin-fixed sample not contacted with a carcinostatic agent is viewed under a stereoscopic microscope, spherical cancer cell colonies and bipolar linear fibroblasts coexist.

In this state, the number of spherical colonies, that is, the colonies of cancer cells, was carefully measured with the naked eye, and the result was 52 cells / 1.4 mm × 1.4 mm (1 visual field). The measurement was performed under strong magnification with a stereomicroscope.

The number of colonies was 1024 / 1.4mm × 1.4mm when measured on a captured image of the same sample with a colony counter that was normally used before image processing. The exact number of colonies of cancer cells could not be counted.

Next, the measurement result of the number of colonies for the image after performing the above-described image processing (high-frequency component extraction calculation and concentration-class fraction calculation) is 51 / 1.4 mm × 1.4 mm, and the measurement result by the naked eye Almost the same result is obtained. Therefore, it was verified that the result of counting the number of colonies by image processing has sufficient accuracy. In addition, the measurement of the number of cancer cell colonies for the image after image processing is
It was much easier than visually observing a microscope and counting only the number of cancer cell colonies from the mixture of fibroblasts and the like.

-Results of colony count measurement after image analysis-Table 2 below shows the results of the image analysis and the colony count measurement as described above for the samples in contact with the respective anticancer agents. However, the number of colonies was measured under weak magnification (1 field of view 2.8 mm x 2.8 mm) in order to improve the measurement accuracy.

In the above table, (-) is the case where no anticancer drug was used at all (control), and compared with this control, the sample with a smaller number of colonies, that is, the anticancer drug of the sample in which the growth of cancer cells was suppressed, was more sensitive. Will be high.

(Experiment 2) -Comparison with conventional method-In order to confirm that the above-described test methods of the examples of the present invention show good sensitivity to an anticancer agent, the antitumor effect currently has the best clinical correlation. The results were compared with the measurement results by the nude mouse method, which is said to exist.

First, from the measurement results of the number of colonies obtained in the above Example, the percentage of the number of colonies when the anticancer agent treatment was performed to the number of colonies when the anticancer agent treatment was not performed (control) was expressed as a percentage of control ( pe
rcent of control). The result is the first
It is shown graphically in the figure.

Next, the anticancer drug sensitivity test (Comparative Example 1) using nude mice was performed as follows. "Culture of cancer cells"
Nude mouse transplantation system used in 1. Human colon cancer was cut into pieces of about 2 mm square and subcutaneously transplanted into nude mice. After that, when the estimated tumor weight reached about 100 mg, the above MMC = 3 mg / kg, 5
-FU = 50 mg / kg, VP-16 = 25 mg / kg, CDDP = 5 mg / kg or
VDS = 3 mg / kg was intraperitoneally injected into 5 nude mice per group, 4 times per week for a total of 4 times. Another group was used as a control to which no anticancer drug was administered. Tumor estimated weight was calculated from L × W ^2/2 as measured tumor major axis (L mm) and minor axis (W mm). Then, one week after the final administration, the estimated tumor weight was measured again to the average tumor weight in the case where the anticancer drug treatment was not performed (control),
The percentage of the average tumor weight when each anticancer drug treatment was performed,
It was calculated as a percent of control similar to the above. The results are shown graphically in FIG.

Comparing FIG. 1 showing the measurement result of the example of the present invention, which is a combination of collagen gel and image processing, with FIG. 2 showing the measurement result by the nude mouse method, it is shown that the correlation is extremely good for any anticancer agent. It was demonstrated that the method of the present invention is highly practical as an alternative method performed in vitro, ie, in vitro, to the nude mouse method performed in vivo, ie, in vivo. Moreover, the nude mouse method required a test period of one month or longer, while the examples showed that it was possible to perform a highly accurate test in a short period of about 10 days.

When measuring the degree of proliferation of cancer cells by measuring the number of colonies, the degree of proliferation of cells may deviate from the exact degree of proliferation. For example, as shown in FIG. 3, when six single cancer cells 1 are cultured in collagen gel 5 and the cell proliferation rate is uniform and small as indicated by arrow A, almost the same results are obtained. Six colonies 2 of size are formed and the colonies do not overlap each other. In this case, a result of 6 is obtained when the number of colonies is automatically measured. However, when the degree of proliferation is uneven (indicated by arrow B in FIG. 3)
Alternatively, when the degree of proliferation is large (indicated by arrow C in FIG. 3), a plurality of adjacent colonies 2 overlap each other,
Originally, multiple colonies are judged to be one by automatic measurement.
For example, in the case of the proliferation shown by the arrow B, the number of colonies is 4 in the automatic measurement, and in the case of the proliferation shown by the arrow C, the number of colonies is 2 in the automatic measurement (the exact number of colonies is 6 in both cases). ). In order to resolve such an error, the number of cells to be seeded (cell density) should be reduced (for example, 1 × 10 ⁴ cells / ml or less), or the measurement should be completed before the colonies overlap. Can be considered. However, in many primary cancer cells, colonies are not observed unless high-density culture with a cell density of 5 × 10 ⁴ cells / ml or more is performed, so that the above method cannot accurately determine the effect of the anticancer agent. In addition, since the degree of proliferation of cancer cells varies depending on the individual cancer cells, the above method makes a measurement before the difference between the control and the effect of different anticancer agents is sufficiently obtained, and the effect of the anticancer agent is also measured. Can not be grasped accurately.

As described above, if the proliferation rate of cancer cells cannot be accurately measured, inconvenience occurs in the actual anticancer drug sensitivity test.
For example, as shown in FIG. 4, six single cancer cells 1
When cultured in collagen gel 5, when not treated with a carcinostatic agent as shown by arrow D, 6 large colonies 2 are formed, but 3 of these colonies overlap or contact each other. If so, the number of colonies is 2 in the automatic measurement. On the other hand, when treated with an anticancer drug as indicated by arrow E, if 6 small colonies 2 are formed and are separated from each other, the number of colonies is 6 by automatic measurement. In general, the effect of anticancer agents (degree of growth inhibition) is The ratio must be less than 100% if the anticancer drug is effective. However, in the example of FIG. 4, the ratio is 300% [(6/2) × 100%].

When the degree of growth of cancer cells is measured by the number of colonies, it is contact and / or overlap between colonies that give such irrational results.

Therefore, in the present invention, it is preferable to measure the proliferation rate of cancer cells by measuring the colony volume. In this case, the effect of the anticancer drug (degree of growth inhibition) is If the anticancer drug is effective, the ratio is less than 100%. As seen in FIG. 5, when adjacent colonies (eg, two colonies) 2 and 2 touch each other, the volume of these colonies is the same as the total volume of the (two) separated colonies. In other words, contact has little or no effect on the total volume of multiple colonies. Also,
Even in the case of overlapping, since the volume of the overlapping portion is very small, it does not significantly affect the entire volume. Therefore, accurate measurement of cell proliferation is always possible by the colony volume, but not always by the number of colonies by automatic measurement.

As described above, in measuring the degree of proliferation of cancer cells by image analysis, even if the degree of cell proliferation cannot be measured by measuring the number of colonies, the degree of proliferation can be measured by measuring the estimated volume value of colonies. Can measure accurately. This was confirmed by the following experiment by comparison with the measured value of the DNA amount. In this experiment, unlike the previous example, a human cancer cell line containing no fibroblast was used. Therefore, the measurement of the proliferation degree of the human cancer cell line can be accurately verified by the measurement of the DNA amount.

(Experiment 3) -Culture of cancer cells-A collagen gel-embedded culture was performed using a human lung cancer cell line in the same manner as in the control of Example 1 above, without treatment with an anticancer agent.

-Image analysis process-The above-mentioned human cancer cell line that had been subjected to collagen gel embedding culture was photographed and input as an image signal to an image analysis device in the same manner as above. The input original image was subjected to image analysis processing in the same manner as described above, and only the image of the cancer cell colony was extracted (however, in this case, since the fibroblasts are not included, the removal processing is not necessary). Next, the number of colonies was counted from this image in the same manner as described above, and at the same time, the integrated value of the volume of the solid estimated from the colony image was calculated. The estimated volume value was calculated by the following procedure.

1. A binary image that is a projected image of a colony is defined as an original image.

2. Separately store a screen that can express light and shade (storageima
ge plane).

3. Copy the original image as a density value of 1 on the accumulation screen.

4. Remove the pixels that make up the outline of the projected image from the original image.

5. A reduced projected image after removing the pixels that make up the outline is newly defined as the original image.

6. Copy the original image on the accumulation screen and add the density value 1.

7. Repeat steps 4 to 6 until the projected image disappears.
As a result, a mountain-shaped three-dimensional image (not necessarily a real image) whose bottom surface is a surface surrounded by the contour line of the first projected image is obtained.

8. The product of the density value accumulated in each pixel of the accumulation screen and the length of one side of the pixel is defined as the height.

9. Integrate the height of each pixel of the accumulated screen. This allows
The volume of the mountain-shaped stereoscopic image is obtained.

10. It is assumed that the substance of the colony is symmetrical with respect to the projected image, and the volume of the colony is obtained by doubling the integrated value. The reason for this assumption is that in three-dimensional culture, it is considered that the same shape as the laterally expanded morphology will spread vertically.

11. Output as needed.

The number of colonies, the volume of the colonies, and the DNA amount measurement results obtained from the above image analysis are summarized in Table 3 and FIG.

From the results shown in Table 3 and FIG. 6, the cells are proliferating according to the number of culture days as indicated by the DNA value. The number of counted colonies was in good agreement with the change in the amount of DNA until the 5th day of culture, but peaked at 5 to 7 days of culture and decreased thereafter. This is because, as a result of the cells proliferating, the cell density becomes high and the cells are in an overlapping state,
It seems that the accurate number of colonies is not counted. On the other hand, the colony volume correlates well with the change in the DNA value from the early stage of culture to the long-term culture, and the volumetric method is suitable for measuring the degree of proliferation of cells, particularly for measuring the degree of proliferation over a long period of time. I understand.

Moreover, there are the following problems in measuring the number of colonies. That is, in general, the degree of proliferation of cancer cells differs depending on the individual cells (see FIG. 3, arrow B).
Since the colonies 21, 22 and 23 are different in size, the number of cells contained is naturally different, but when the number of colonies is measured, it is judged that each of the colonies 21, 22 and 23 is one. On the other hand, in the colony volume, colonies 21, 22 and
23 is calculated as a different volume.

Example 2 In the “culture of cancer cells” of Example 1, cancer cells were cultured in the same manner as in Example 1 except that nude mouse transplant human lung cancer was used instead of nude mouse transplant human colon cancer. Culture was performed. Image analysis processing was performed on the obtained sample in the same manner as in Example 1 to extract only colony images.

From the obtained image, the number of colonies was measured in the same manner as in Example 1, and the integrated value of the colony volume was measured in the same manner as in Experiment 3 above.

(Comparative Example 2) In order to verify the results of Example 2, the same cells as those used in Example 2 were also used to perform a carcinogen sensitivity test by the nude mouse method at the same time as in Comparative Example 1 above. It was

Each result was calculated as a percentage of control in the same manner as above, and is shown in the graph in FIG.

As can be seen from the results shown in FIG. 7, the result N of the nude mouse method and the result V of the colony volume measurement method have extremely good correlation with any anticancer drug. On the other hand, the result C by the colony counting method shows an abnormal value. This is because the cells used here have a high proliferation rate, and because the cells that have proliferated overlap as described above, especially when not treated with an anticancer agent (control), the measured number of colonies shows an accurate proliferation rate. Although not shown, the volume measurement value of the colony shows that the proliferation rate is accurately measured even in such a case.

That is, it was proved that the method of measuring the volume of colonies after growth is an effective method for measuring the degree of growth in the case of actively growing cells or increasing the cell density during culture.

〔The invention's effect〕

As described above, according to the anticancer drug sensitivity test method of the present invention, by using a collagen gel as a substrate for culturing cancer cells, many cancer cells that cannot be cultured in a conventional soft agar medium or the like are cultured. It becomes possible to do.

By measuring the degree of proliferation of cancer cells by image analysis, it is possible to eliminate the influence of fibroblasts that grow with the cancer cells when the cancer cells are cultured on a collagen gel substrate,
The test results can be obtained accurately and quickly.

When performing image analysis of the growth rate of cancer cells, measuring the volume of colonies enables more accurate and longer-term measurement of the growth rate than by measuring the number of colonies.

As a result, compared with the conventional test method, the applicability of the susceptibility test method suitable for the development of an anticancer agent is much wider than that of the conventional test method, and the test result can be obtained easily and quickly and accurately. Can be provided.

[Brief description of drawings]

FIG. 1 is a graph showing the sensitivity measurement result of each anticancer agent in the example according to the present invention, FIG. 2 is a graph showing the sensitivity measurement result of each anticancer agent in the comparative example, and FIG.
Explanatory diagram showing three examples of the degree of proliferation in the culture of cancer cells,
FIG. 4 is an explanatory view showing an example of the case where the anticancer agent treatment is not performed (control group) and the case where it is performed in the culture of cancer cells, and FIG. 5 is a sectional view taken along line XX in FIG. FIG. 6 is an explanatory view showing an example when colonies come into contact with each other in the culture of cancer cells, FIG. 6 is a graph showing the number of colonies and the correlation between the colony volume and the DNA amount, and FIG. 3 is a graph showing the results of measuring the sensitivity of each anticancer drug in Comparative Example 2.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP 61-164159 (JP, A) JP 61-226638 (JP, A) JP 61-248183 (JP, A) JP 62- 50606 (JP, A) Actual development Sho 62-53792 (JP, U) Actual development Sho 62-50608 (JP, U) Japan Ear Nose and Throat Science Bulletin, 94 (1), p. 16-21, 1991 Collagen Study Group Abstracts, 35th (1987), p. 61-65, 1988 Monthly Tissue Culture, 13 (2), P. 64-68, 1987 J Cell Physiol, No. Suppl 3, P.P. 105-116, 1984

Claims (2)

[Claims] 1. A method for testing the sensitivity of an anticancer agent by growing cancer cells in an environment in which a substrate is present and measuring the degree of growth of the cancer cells. An image analyzer is used as a means for measuring, and the image signal of the cancer cells and colonies thereof is selectively extracted from the image signal of the sample obtained by imaging, and the selectively extracted cancer cells and An anti-cancer drug sensitivity test method for measuring the degree of growth of cancer cells by calculating an estimated volume value of the cancer cells and the colonies from the image signal of the colonies. 2. A method for testing the sensitivity of an anticancer agent by growing cancer cells in an environment in which a substrate is present and measuring the degree of growth of the cancer cells, using a collagen gel as the substrate, and measuring the degree of growth of the cancer cells. An image analysis device is used as a measuring means, and the density characteristics of the object are emphasized by a logical operation from the image signal of the sample obtained by imaging, and the density is calculated from the obtained emphasized image. By extracting only the image of the object according to a certain reference value, the image signals of the cancer cells and colonies are selectively extracted, and the cancer signals are extracted from the image signals of the selectively extracted cancer cells and colonies. An anticancer drug susceptibility test method for measuring the degree of proliferation of cancer cells by calculating the number of cells and colonies thereof.