JP2009027928A - Method for evaluating cancer metastasis ability and three-dimensional double membrane structure for evaluating cancer metastasis ability - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for evaluating cancer metastasis ability, in which normal cells in vitro intervene and which is approximate in vivo. <P>SOLUTION: This method for evaluating the cancer metastasis ability is characterized by: (procedure 1) sequentially sticking fibroblasts to the lower side of the membrane of the first chamber having the fine pore membrane through which cancer cells can pass, sowing vascular endothelial cells on the upper side of the membrane, and making the sheet of the fibroblasts on the lower side of the membrane and the sheet of the vascular endothelial cells on the upper side of the membrane, respectively; (procedure 2) sowing cancer cells on the sheet of the vascular endothelial cells on the upper side of the membrane, disposing the first chamber obtained in the procedure 1 in the second chamber having a fine pore membrane through which the cancer cells disposed on the culture plate from the upper side can not pass, and culturing the cells in a culture solution in a state that the layers are overlapped at a distance; (procedure 3) taking out the overlapped first chamber, sowing the prescribed number of cells having electric resistance on the membrane of the second chamber having the fine pore membrane through which the left cancer cells can not pass; (procedure 4) culturing the second chamber; and then (procedure 5) measuring the electric resistance of the second chamber with an electric resistance-measuring instrument. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for evaluating cancer metastasis. The present invention also relates to a three-dimensional bilayer structure for evaluating cancer metastasis ability.

  Some cancer types have started to see improvements in survival rates little by little due to early detection of cancer and the development of new anticancer drugs. However, most solid cancers are still esoteric for humanity. The major cause is recurrence and metastasis due to a pathological mechanism unrelated to preventive medicine even if it is completely removed by surgery. Even though chemotherapy, radiation therapy, immunotherapy, and molecular targeted therapy are tried for this abhorrent metastasis, there are many cases of cancer death. In order to prevent and take measures against this cancer metastasis, an in vitro metastasis model that is close to the living body is a powerful weapon.

Cancer metastasis involves cancer cell withdrawal from the primary lesion and invasion of surrounding tissues, entry into the vessel, migration, implantation of distant tissue into microvessels, escape to the extravasation, and proliferation at the site of metastasis. It has been established through a complicated process.
Ikuo Saiki., 1995: Molecular mechanism of cancer metastasis and its application to therapy. Molecular Medicine, 32 (4) .372-379 Jun Murata., 1995: Cell motility promoting factor and cancer metastasis. Molecular Medicine, 32 (4) .392-397

For this reason, it is pointed out that the role of host cells that are normal cells in the metastasis process is involved more complicatedly than the cancer invasion process in the primary lesion, and in order to elucidate the metastasis mechanism, metastasis cannot be told by excluding this normal cell ing.
Brooks PC., 1996: Cell adhesion molecules in angiogenesis.Can.Met.Rev, 15.189-194

  From the above process, it is understood that cancer metastasis, in particular, a distant metastasis model cannot be established except for the intervention of a plurality of normal cells. Therefore, in vivo metastasis research in animals is the mainstream,

In vivo metastatic potential studies include subcutaneous transplantation into rats and mice, intraperitoneal transplantation, intrasplenic injection, and invasive cancer cell transfer from the tail vein. Moreover, since it is slaughtered each time, follow-up over time is difficult.
Lue JY et al., 2003: Establishment of red fluorescent protein-tagged HeLa tumor metastasis models: Determination of DsRed2 insertion effects and comparison of metastatic patterns after subcutaneous, intraperitoneal, or intravenous injection.Clin Exp Metastasis, 20.121-133

Computed tomography (CT) images of clinical metastases show circular shadows at many metastases, and lung metastases resulting from transfer from the tail vein in animal experiments are calculated and evaluated as intrapulmonary metastasis colonies.
Ikuo Saiki., 1995:. Molecular mechanism of cancer metastasis and its application to therapy. Molecular Medicine, 32 (4) .372-379

Accordingly, a first object of the present invention is to provide a method for evaluating cancer metastasis ability that is closer to a living body and that is mediated by normal cells in vitro without using animals for experiments, and the second object of the present invention. The purpose is to provide a model for in vitro cancer metastasis assessment method.

In order to achieve the first object, the method for evaluating cancer metastasis according to the present invention according to claim 1, that is, (Procedure 1) is a membrane of a first chamber having a pore membrane through which cancer cells can pass. Affixing fibroblasts to the lower surface, and then seeding vascular endothelial cells on the upper surface of the membrane, creating a sheet of fibroblasts on the lower surface of the membrane and creating a sheet of vascular endothelial cells on the upper surface of the membrane,
(Procedure 2) Next, a porous membrane in which cancer cells of a predetermined number of cells are seeded on a sheet of vascular endothelial cells on the upper surface of the membrane and then placed in a culture plate, through which cancer cells cannot pass The first chamber obtained in the procedure 1 is inserted and placed in the second chamber having the above from above, and statically cultured for a predetermined time in a state of being layered at intervals.
(Procedure 3) Thereafter, the stacked first chamber is taken out, and a predetermined number of cells having electrical resistance are seeded on the membrane in the second chamber of the porous membrane where the remaining cancer cells cannot pass,
(Procedure 4) Then, static culture of the second chamber obtained in Procedure 3 for a certain period of time,
(Procedure 5) Subsequently, a cancer characterized by sequentially measuring the electrical resistance (Transepithelial electrical resistance: abbreviated as TEER) value of the second chamber obtained in Procedure 4 with an electrical resistance measuring instrument. This is achieved by the metastatic ability evaluation method.
In the present specification, the pore size of the “pore membrane through which cancer cells can pass” is 5.0 to 12.0 micrometers, although it varies depending on the type of cancer cells. That is, since the size and deformability of the cells differ depending on the cancer cell, the pore size differs. Further, the pore size of the “porous membrane through which cancer cells cannot pass” is approximately 4.0 micrometers or less, and preferably around 0.4 micrometers.

The second object is to produce a sheet of fibroblasts on the lower surface of the three-dimensional bilayer structure for the cancer metastasis evaluation method according to the present invention as defined in claim 2, that is, a membrane having pores. A first chamber having a pore membrane through which cancer cells can pass, in which a vascular endothelial cell sheet is prepared on the upper surface of the membrane, and a predetermined number of cancer cells are seeded on the vascular endothelial cell sheet. And a second chamber having a porous membrane through which cancer cells cannot pass, and a culture plate containing a culture solution, the second chamber being disposed in the culture plate, The third chamber for evaluating cancer metastasis capacity, wherein the first chamber is placed in an overlapping manner in the two chambers from above, and the culture plate is spaced from the second chamber. Achieved by original double membrane structure Is done.
In this specification, the interval between the pore membrane of the first chamber and the pore membrane of the second chamber is about 0.8 to 1.2 millimeters, and the interval between the pore membrane of the second chamber and the culture plate is 0.8 to 1.2 millimeters.

  An in vitro model according to the present invention that can evaluate the proliferative activity of cancer cells by separating, disrupting, or migrating between cell adhesions of vascular endothelial cells and other normal cells that are three-dimensionally constructed by cancer cells in an in vitro system According to the report, it is natural that prediction of distant metastasis can be easily conducted in vivo, in addition to the problems of experimental convenience, animal welfare, and economic effects. This is expected to make rapid progress toward further elucidation of the biological mechanism of distant metastasis and clinical treatment of metastasis prevention.

  According to the cancer relocation evaluation method according to the present invention, the evaluation time can be greatly shortened. Therefore, in the development of new drugs, the cancer metastasis evaluation method and the bilayer structure according to the present invention can be used as an evaluation by a large-scale and short-time experiment performed before an animal experiment.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1. Materials and methods [Cell lines and culture]
The cell lines used in this experiment are 3 cell lines derived from normal cells derived from GP8, a rat vascular endothelial cell, NIH3T3, a mouse fibroblast, and MDCK, a canine renal epithelial cell having electrical resistance. is there. As cell lines derived from cancer cells, there are two cell lines, SW480 (derived from human colon cancer) and SW620 (derived from human colon cancer). A cancer cell line HeLa (derived from human cervical cancer) was employed as a positive control. A total of 6 cell lines were used.

The two types of cancer cell lines used in this in vitro metastasis experiment were selected from colon cancer cell lines established from different sites in the same patient. That is, SW480 is a cancer cell line established from the primary lesion of colon cancer, and SW620 is a cancer cell line established from the lymph node metastasis of the same patient. SW480 is a low metastasis cell line and SW620 is a high metastasis cell line.
Hewitt RE et al., 2000: Validation of a model of colon cancer progression. J Pathol, 192.446-454

All cell lines in this experiment were subcultured and the experimental medium was Dulbecco's Modified Eagle's Medium (DMEM, abbreviation: Sigma ™) plus 10% fetal calf serum (FBS, abbreviation: GIBCO ™) Using the solution (medium), the cells were subcultured in a 25 cm ² cell culture flask (Cornig) in a 5% CO ₂ incubator at 37 ° C. In GP8, G418 300 μg / ml was added to the medium and subcultured. On the third day, the medium was washed with phosphate buffered saline (abbreviated as PBS), and the cells were detached with 0.05% trypsin-EDTA (GIBCO (trademark)) and subcultured. The cell number was adjusted with 0.05% trypsin-EDTA, then shaken so as to become single cells, and the number of viable cells was counted with a hemocytometer by trypan blue staining (trypan blue discharge test). It was confirmed that mycoplasma infection was non-infected and was used for the experiment.

[material]
The modeling of the three-dimensional double membrane structure chamber was devised so that two porous membrane cylindrical chambers were combined in a 12-well culture plate. The experimental materials used this time are introduced.

  FIG. 1 is a photograph of Transwell (registered trademark) 3422 and 3460 chambers used in this example. The 3422 chamber is a polycarbonate membrane (showing white) and has a pore size of 8.0 micrometers (upper left in Fig. 1). The 3460 chamber is a polyester membrane (shows white) and has a pore size of 0.4 micrometers (upper and right in Fig. 1). The 3422 chamber can be inserted into the 3460 chamber precisely as shown in the picture (lower part of Fig. 1).

  Two types of chambers for the production were (1) a chamber of Transwell 3422 (registered trademark) (made by Corning) having a membrane (polycarbonate membrane) having a diameter of 6.5 millimeters and a pore size of 8.0 micrometers (2) ) A chamber of Transwell (registered trademark) insert 3460 (Corning) having a membrane (polyester membrane) having a diameter of 12.0 mm and a pore size of 0.4 micrometers was used. The 3422 chamber is housed in a 24-well culture plate and the 3460 chamber is housed in a 12-well culture plate. The 3422 chamber overlaid the 3460 chamber in the 12-well culture plate and was properly placed. When the 3422 chamber was overlaid on the 3460 chamber, the distance between the two membranes was about 1.0 millimeter. The distance between the 3460 chamber membrane and the bottom of the 12-well culture plate was 1.0 millimeter. The 3422 chamber is an example of the “first chamber” in the present invention, the 3460 chamber is an example of the “second chamber” in the present invention, and the 12-well culture plate is an implementation of the “culture plate” in the present invention. It is an example.

  FIG. 2 is a schematic diagram showing a structure based on a three-dimensional two-film structure chamber model used in this example. Schematic cross section showing a state in which the 3422 chamber (first chamber 1) overlaid on the 3460 chamber (second chamber 2) is stored in a 12-well culture plate (culture plate 3) in this example. FIG.

  The 100 mm cell culture dish (Falkon) was used for attaching cells to the lower surface of the membrane of the 3422 chamber 1c, and the 35 mm cell culture dish (Falcon) was used for morphological observation with a microscope.

  Fluorescent staining solution for identification of cell colonies during culture in which other types of cells intervene (coexist) can be stained with live cells, and Rhodamine123 (Sigma (trademark)) with low toxicity is dissolved in DMEM + 10% FBS. used. The inverted microscope used Olympus CK 40 (manufactured by Olympus), was subjected to fluorescence excitation with Olympus U-RFLT 50 (manufactured by Olympus), and cell colonies were observed. The photo was taken with Olympus PM10SP (Olympus).

TEER value measurement was performed using Recorder Output EVOM (trademark) (manufactured by World Precision Instruments) and ENDOHM-12 (manufactured by World Precision Instruments). The unit of TEER value is Ω · cm ² . The measurement method followed the manual. FIG. 3 is a schematic view showing the structure of the electrical resistance measuring instrument used in this embodiment.

[Method]
In basic experiments 1 and 2, one sample (n = 1) was used, and in basic experiment 3, the experiment of the present invention was performed with three samples (n = 3).

1) Basic experiment 1 (Rhodamine 123 discharge time)
In order to identify cancer cells in a culture mediated by MDCK, the six types of cells in column 0014 were cultured in a 35 mm cell culture dish, and the time for complete disappearance of Rhodamine 123, a fluorescent dye, was examined. In the method, Rhodamine 123 was added dropwise to the culture medium, and the cells were cultured and stained for 30 minutes in the dark. After 30 minutes, this specimen was washed twice with PBS, Rhodamine 123 was removed, and the medium was replaced with a new medium. Observation was carried out with a fluorescence microscope until time. The time during which the fluorescence was completely invisible with a fluorescence microscope was defined as the Rhodamine 123 excretion time of each cell.

2) Basic experiment 2 (Effect of MDCK on cancer cell colony formation)
Cancer cells will be mediated by MDCK. Therefore, the influence of MDCK on cancer cell colony formation was examined. MDCK and cancer cells HeLa were examined in a 35 mm cell culture dish.

Specimens seeded with 1 × 10 ⁴ , 5 × 10 ³ , 1 × 10 ³ , 5 × 10 ² , 1 × 10 ² HeLa on the culture dish (HeLa alone), and the presence of HeLa in the above number of cells after 24 hours Two types of specimens (HeLa + MDCK example) seeded with MDCK in the lower cell culture dish were prepared, Rhodamine 123 staining was performed on the seventh day of culture, and the morphology was observed with an optical microscope and a fluorescence microscope, and compared.

3) Basic Experiment 3 (Influence of NIH3T3 and GP8 on TECK value of MDCK)
The relationship between the cell dynamics (behavior) of NIH3T3 and GP8 laid on the lower surface 1c and upper surface 1b of the 3422 chamber and MDCK having electrical resistance was examined.

First, 1 × 10 ⁵ NIH3T3 or GP8 was seeded on the upper surface 1b of the 3422 chamber, this chamber was overlaid in the 3460 chamber of the 12-well culture plate (culture plate 3), and the 3422 chamber was removed after 2 days. On the upper surface 2b of the 3460 chamber, 2 × 10 ⁵ MDCKs were seeded and statically cultured for 7 days. Meanwhile, the culture medium was exchanged twice. Seven days after MDCK seeding, TEER values in 3460 chambers were measured, and cell behavior of NIH3T3 and GP8 mediated by MDCK was observed by Rhodamine123 staining.

4) Experiment of the present invention (change in TEER value and observation of cancer cell colonies in a three-dimensional double membrane chamber model)
4A and 4B are schematic diagrams showing an example procedure of a cancer metastasis evaluation method modeled on the present experiment according to the present invention. Moreover, it is the schematic which shows the preparation procedure of the three-dimensional double membrane structure chamber structure which concerns on this invention. Procedures 1 to 7 show the order of the experiments over time. After step 7, the 3460 chamber was removed and the electrical resistance of the 3460 chamber was measured and evaluated. In addition, cancer cell colonies were observed.

Step 1
Remove the 3422 chamber from the 24-well culture plate, place the chamber upside down on a 100 mm cell culture dish, and drop NIH3T3 (1 × 10 ⁵ pieces: 50 microliters) on the bottom of the 3422 chamber membrane on the top, NIH3T3 was attached to the lower surface of the 3422 chamber membrane by culturing for 4 hours.

Step 2
This 3422 chamber was returned to the 24-well culture plate containing the culture solution as it was, and left for 24 hours. Next, GP8 was seeded on the upper surface of the membrane of the 3422 chamber (2 × 10 ⁵ cells: 100 microliters).

Step 3
After 72 hours of static culture, NIH3T3 sheets (membrane lower surface) and GP8 sheets (membrane upper surface) in a state where cells were in close contact were prepared so that the membrane in the 3422 chamber was sandwiched up and down.

Step 4
The adjusted number of cancer cells was seeded on the upper surface of the 3422 chamber of the 24-well culture plate.

Step 5
Immediately after that, the two chambers were overlaid by transferring and inserting the 3422 chamber into the 3460 chamber in the 12-well culture plate containing the culture solution.
The culture was allowed to stand for 48 hours in a layered state.

Step 6
Thereafter, the 3422 chamber was removed from the 3460 chamber, 2 × 10 ⁵ MDCK having electric resistance were seeded on the upper surface of the membrane of the remaining 3460 chamber, and a culture solution was added.

Step 7
The cells were statically cultured for 7 days for preparation of MDCK cell adhesion (MDCK sheet) and formation of cancer cell colonies. Meanwhile, 1/2 exchange of the culture solution in the 3460 chamber was performed twice.

  On this 7th day, the TEER value of the 3460 chamber was measured using the electrical resistance measuring instrument shown in FIG. Furthermore, cancer cell colonies were observed by Rhodamine123 staining.

[Statistical processing]
Statistical analysis software was Stat.View Ver.4.0, and statistical processing was performed by t-test. Statistical significance was P <0.05.

[result]
・ Basic experiment 1 (Rhodamine 123 discharge time)
Table 1 shows the Rhodamine 123 fluorescent dye excretion time of the six types of cell lines of 0014 column used in the basic experiment 1. The vertical is the cell line type, the horizontal is the time (h) transition (○: the time when the fluorescent staining was confirmed in the cells with the fluorescence microscope, ×: the fluorescence staining could not be confirmed within the cells with the fluorescence microscope Means time).

  From this result, it was clarified that for the identification of cancer cell colonies under the intervention of MDCK, the fluorescence of cancer cell colonies can be observed by the disappearance of Rhodamine123 fluorescence of MDCK completely after 3 hours. Therefore, the subsequent cancer cell colony observation was performed 3 to 4 hours after the removal of Rhodamine123 staining.

2) Basic experiment 2 (Effect of MDCK on colony formation of cancer cells)
The number of cancer cell colonies of HeLa consisting of 10 or more cancer cell HeLa alone cases and HeLa + MDCK cases is 941 and 1099 when seeding 1 × 10 ⁴ and 303 when seeding 5 × 10 ^{3 respectively.} 366, 1 × 10 ³ seeding was 76 and 56, and 5 × 10 ² seeding was 14 and 10. In the specimens seeded with 1 × 10 ⁴ HeLa alone, cancer cell colonies were found to coalesce with each other, but in MDLa-mediated HeLa + MDCK cases, no cancer cell colonies were found to fuse. It was.

  From the above results, it was found that the cancer cell colony formation of HeLa is not affected by the number of colonies formed by MDCK, and that the colony of HeLa does not heal by MDCK and the number of cancer cell colonies can be calculated clearly .

3) Basic Experiment 3 (Influence of NIH3T3 and GP8 on TECK value of MDCK)
There was no difference in the TEER values between the 3460 chambers of the MDCK samples with electrical resistance and the NIH3T3-mediated MDCK samples dropped from the 3422 chamber. The same operation was performed with GP8. GP8 dropped from the 3422 chamber and formed Rhodamine123-stained colonies in the presence of MDCK. However, the colonies were small constituent cell colonies and dendritic morphology (cell groups that did not form colonies). This affected GP8's TECK value of 3460 chamber MDCK.

Based on these results, GP8 passes through the pore size of 8.02 micrometers in the 3422 chamber, so it was considered necessary to devise measures to prevent entry into the 3460 chamber. Before CP8 was seeded on the upper surface of the 3422 chamber membrane, NIH3T3 was laid on the lower surface of the 3422 chamber membrane, and the MDCK TEER value was 7540 ± 319.1 Ω · cm ² (average ± SE). When no cells (NIH3T3, GP8) were seeded in the 3422 chamber, the MDCK TEER value was 7679 ± 180.3 Ω · cm ² , which was not different from the above. This result showed that NIH3T3 could prevent GP8 from moving to the lower surface of the film, and the effect of GP8 on MDCK was resolved.

4) Experiment of the present invention (change in TEER value and observation of cancer cell colonies in a three-dimensional double membrane chamber model)
TEER value of MDCK-mediated 3460 chamber with electrical resistance by 2.5 × 10 ⁵ cancer cells using a 3D bilayer membrane chamber model with cells derived from NIH3T3, GP8 and MDCK normal cells Changes and cancer cell colony observation were performed.

After the NIH3T3 sheet (membrane bottom surface) and GP8 sheet (membrane top surface) were prepared in the 3422 chamber, the MDCK TEER value was 7540 in the 3460 chamber (control sample (open star mark)) in which no cancer cells were seeded in the 3422 chamber. It was ± 319.1Ω · cm ² (average value ± SE). The MDCK TEER value in the 3460 chamber in the absence of any cells (NIH3T3, GP8) in the 3422 chamber was 7679 ± 180.3 Ω · cm ² , which was not significantly different from the control sample (open star mark). On the other hand, when the specimens in which cancer cells were seeded in the 3422 chamber containing NIH3T3 and GP8 were measured, the MDCK-mediated chamber (MDCK + HeLa specimen) seeded with HeLa was 3507 ± 1001Ω · cm ² , and the MDCK-mediated chamber (MDCK + SW480 specimen) seeded with SW480 was ^{7643 ± 602.0Ω · cm 2, SW620} MDCK intervening chamber (MDCK + SW620 samples) seeding was at 4417 ± 649.5Ω · cm ^2. A statistically significant difference of P <0.05 was observed between the control sample, the MDCK + HeLa sample, and the MDCK + SW620 sample. A statistically significant difference was also observed between the MDCK + SW480 specimen and the MDCK + SW620 specimen. SW620, a high metastasis cell line, showed a significantly lower MDCK TEER than SW480, a low metastasis cell line (Fig. 5).
Table 2 shows the details of the experiment similar to FIG. 5, but shows the experimental results of three samples used in the model of the present invention in detail. The table below shows the influence of each cancer cell line on the MDCK TEER value and the formation of cancer cell colonies. The electrical resistance was measured as shown in FIG. The number of cancer cell colonies was calculated by observing with a fluorescence microscope 3 to 4 hours after the removal of Rhodamine123 staining. (The control in Table 2 is the same as the control in FIG. 5 (open star mark). TEER is an electrical resistance value, the unit is Ω · cm ² : the number of colony cells is the number of colony constituting cells of one cancer cell colony. 20 to 49 pieces, 50 or more pieces are divided into two, and the number is displayed).

Cancer cell colonies were observed with an optical microscope and a fluorescence microscope on an MDCK-mediated 3460 chamber. In the light microscope, MDCK cell mass (Figure 6: red arrow) that was confused with cancer cell colonies was present due to the MDCK intervention, but with the fluorescence microscope with Rhodamine123 staining, clear cancer cell colonies could be captured (Fig. 6). 6: White arrow). As a result of the observation, the cell aggregation of HeLa and SW480 cancer cell colonies is somewhat coarse, and in HeLa, more than 50 constituent colonies and 20 or more constituent colonies are observed, and in SW480 there are no more than 50 constituent colonies, 20 There were few constituent colonies. On the other hand, the cell aggregation of SW620 cancer cell colonies was relatively dense, and there were almost no 50 or more constituent colonies, and many 20 or more constituent colonies were observed (FIG. 6). This observation was not inconsistent with the result that SW620, a high metastasis cell line, showed significantly lower MDCK TEER than SW480, a low metastasis cell line.

[Discussion]
Distant metastasis is said to be established as metastasis (colony) formation through a very complicated process after cancer cells proliferate and infiltrate at the primary lesion and invade the vessel.
Ikuo Saiki., 1995: Molecular mechanism of cancer metastasis and its application to therapy. Molecular Medicine, 32 (4) .372-379

  However, considering cancer cell dynamics from the cellular level, it is necessary for distant metastasis to have invasion (invasion) ability, migration (movement) ability, and proliferation (adaptation) ability. In addition, considering the prevention of metastasis by normal cells, cancer cells overcome and overcome the adhesion between normal cells and the substrate and the adhesion between normal cells and normal cells to overcome metastases (colony) in different tissue environments. It is to form. The inventors focused on intravascular re-entry and tissue adaptation (proliferation), which are the late processes of this distant metastasis, and three cell lines derived from normal cells (vascular endothelial cells, fibroblasts, MDCK with electrical resistance) Three-dimensional double-membrane structure, which is a model for remote transfer in vitro, by using two types of intervening pore membrane cylindrical chambers (8.0 micrometer pore membrane cylindrical type, 0.4 micrometer pore membrane cylindrical type) Invented the chamber model. The metastasis model that is the subject of this experiment is understood as an evaluation model of the late stage of metastasis mechanism (adhesion to capillary endothelium, extravasation, metastasis colonization in target organ tissue).

Ludwig et al. Have already devised an Electrical Resistance Breakdown Assay by measuring MDCK's TEER value in coexistence (mediated) culture of MDCK and malignant cells (cancer cells, etc.).
Ludwig T et al., 2002: The electrical resistance breakdown assay determines the role of proteinases in tumor cell invasion. Am J Physiol Renal Physiol, 283.319-327 Ludwig T et al., 2004: Platinum complex cytotoxicity tested by the electrical resistance breakdown assay.Cell Physio Biochem, 14.425-430 Ludwig T et al., 2005: Functional measurement of local proteolytic activity in living cells of invasive and non-invasive tumors. J Cell Physiol, 202.690-697

They created an MDCK sheet on the upper or lower surface of one type of porous membrane cylindrical chamber (0.4 micrometer pore diameter), and seeded the upper surface of the membrane with malignant cells, so that the malignant cells were malignant and sensitive to anticancer drugs. Attempts have been made to evaluate this by the variation of the MDCK TEER value. This experimental system evaluates malignancy based on changes in MDCK TEER levels by Matrix Metalloproteinases (MMPs) secreted from cancer cells, and is understood as an invasion model rather than a metastasis model.
Ludwig T et al., 2005: Functional measurement of local proteolytic activity in living cells of invasive and non-invasive tumors. J Cell Physiol, 202.690-697

  Our model is the next different normal cell where cancer cells break or break the adhesion between normal cells, move through a membrane with a pore size of 8.0 micrometers, and further release from the lower surface of the membrane It is an objective evaluation model that reduces the TECK level of MDCK by growing as a colony in the presence of MDCK, which is clearly different from the experimental system of Ludwig et al.

The structural feature of this model is the theory of cell selection and arrangement of normal cell lines. Summarizing the experimental samples used this time as an example,
(1) Rat vascular endothelium-derived cells (GP8): Arranged so as to form a sheet on the upper surface of the membrane of the 3422 chamber. This is because the late stage of metastasis, in which cancer cells adhere to and reenter vascular endothelial cells, is the initial stage of the experiment.
(2) Mouse fibroblast-derived cell (NIH3T3): Affixed to the lower surface of the 3422 chamber. The reason is significant as a feeder layer of cancer cells and vascular endothelial cells,
Nishinakamura R et al., 2006: Essential roles of Sall family genes in kidney development.J Physiol Sci, 56 (2) .131-136 Gregoire L et al., 1998: Organotypic culture of human ovarian surface epithelial cells: a potential model for ovarian carcinogenesis.In vitro Cell Dev Biol Anim, 34 (8) .636-639 Vascular endothelial cells (GP8) migrate to the lower surface It is also the purpose of preventing this. In addition, it is the fact that fibroblasts are present in the lung, liver, and bone that are metastatic sites. (3) Canine kidney epithelium-derived cells (MDCK): placed on the upper surface of the 3460 chamber. This is because MDCK has a strong tight junction between cells and can measure a stable and high TEER value which is electric resistance. Ludwig T et al., 2002: The electrical resistance breakdown assay determines the role of proteinases in tumor cell invasion.Am J Physiol Renal Physiol, 283.319-327 This is because the formation of a colony similar to the metastatic foci observed in FIG. In addition, the presence of MDCK inhibited the growth of normal cell-derived NIH3T3 (by cell-cell contact inhibition) and did not affect the electrical resistance of MDCK. From the above, a new evaluation model of the late metastasis process with a more physiological three-dimensional structure could be created.

Regarding the selection of cancer cell lines, the reason why HeLa was used as a positive control was that colony forming ability was recognized,
Lue JY et al., 2003: Establishment of red fluorescent protein-tagged HeLa tumor metastasis models: Determination of DsRed2 insertion effects and comparison of metastatic patterns after subcutaneous, intraperitoneal, or intravenous injection.Clin Exp Metastasis, 20.121-133 Similarly, the migration ability of HeLa has been confirmed using a chamber having a pore size membrane of 8.0 micrometers of Transwell (registered trademark). Zhang L et al., 2007: Effect of matrine on HeLa cell adhesion and migration.Euro J Pharmaco, 563.69-76

In addition, the co-culture of MDCK and HeLa on Transwell's 0.4 micrometer pore membrane chamber is the fact that the TEER value of MDCK decreases.
Ludwig T et al., 2005: Functional measurement of local proteolytic activity in living cells of invasive and non-invasive tumors. J Cell Physiol, 202.690-697

SW480 and SW680, which were selected as experimental specimens for metastasis evaluation, are precious primary and metastatic cancer cell lines established from the same patient. In in vivo animal experiments, the low metastasis cell line SW480 did not show metastasis at any site, and the high metastasis cell line SW620 was reported to show liver metastasis, so it was used in this experiment. In this experimental result, the high metastasis cell line SW620 decreased the MDCK TEER value than the low metastasis cell line SW480.
Hewitt RE et al., 2000: Validation of a model of colon cancer progression. J Pathol, 192.446-454

For evaluation of colony-forming ability only, a soft agar colony formation method without the intervention of host cells that are normal cells is sufficient,
Zhang RD et al., 1991: Malignant potential of cells isolated from lymph node or brain metastasis of melanoma patients and implications for prognosis.Cancer Res, 51 (8). 2029-2035 metastasis model requires host cell intervention . This method breaks or collapses the cell-cell adhesion between vascular endothelial cells and fibroblasts, which are biological obstacles for cancer cells, and the colony-forming ability of free cancer cells that fall from the lower surface of the pore membrane of the chamber is MDCK This TEER value is objectively evaluated, and it can be said that it exhibits a three-dimensional structure approximating that of a living body based on the conventional transfer model (Nakayama et al 1998). Nakayama Y et al., 1998: Alterative express of the collagenase and adhesion molecules in the highly metastatic clones of human colonic cancer cell lines.Clin Exp Metastasis, 16 (5) .461-469

It is a photograph of Transwell (registered trademark) 3422 chamber and 3460 chamber used in this example. It is the schematic which shows the structure of the three-dimensional bilayer structure chamber used by a present Example. It is the schematic which shows the structure of the electrical resistance measuring device used by a present Example. It is the schematic which shows the Example procedure of the cancer metastatic ability evaluation method which concerns on this invention. It is the schematic which shows the Example procedure of the cancer metastatic ability evaluation method which concerns on this invention. It is a graph which shows the influence which it has on the electrical resistance (TEER) value of various cancer cell lines in a present Example, and cancer metastasis evaluation. It is a photograph which shows the identification of each cancer cell colony under each cancer cell culture by MDCK in a present Example. It is a photograph (* 100) of an optical microscope (left figure) and a fluorescence microscope (right figure).

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 1st chamber 1a Porous membrane 1b Membrane upper surface 1c Membrane lower surface 2 2nd chamber 2a Porous membrane 2b Membrane upper surface 2c Membrane lower surface 3 Culture plate 4 Culture solution 5 Electrode 6 Electrode 7 Wire cord 8 Wire cord

Claims (2)

(Procedure 1) Fibroblasts are attached to the lower surface of the membrane of the first chamber having a porous membrane through which cancer cells can pass, and then vascular endothelial cells are seeded on the upper surface of the membrane, and fibers are then applied to the lower surface of the membrane. Producing a sheet of blast cells and producing a sheet of vascular endothelial cells on the upper surface of the membrane,
(Procedure 2) Next, cancer cells of a predetermined number of cells are seeded on the vascular endothelial cell sheet on the upper surface of the membrane obtained in Procedure 1, and then the cancer cells placed in the culture plate pass. Placing the first chamber obtained in step 1 from above in a second chamber having a pore membrane that cannot be cultured, and culturing in a culture solution in a state of being layered at intervals,
(Procedure 3) Then, the stacked first chamber is taken out, and a predetermined number of cells having electrical resistance are seeded on the remaining membrane of the second chamber having a pore membrane through which cancer cells cannot pass. ,
(Procedure 4) Thereafter, culturing the second chamber obtained in Procedure 3 for a certain period of time,
(Procedure 5) Subsequently, the method for evaluating the ability of cancer metastasis, comprising sequentially measuring the electrical resistance value of the second chamber obtained in Procedure 4 with an electrical resistance measuring instrument. A sheet of fibroblasts is prepared on the lower surface of the membrane and a sheet of vascular endothelial cells is prepared on the upper surface of the membrane, and then cancer cells of a predetermined number of cells are seeded on the vascular endothelial cell sheet. A first chamber having a pore membrane capable of undergoing cancer treatment, a second chamber having a pore membrane through which cancer cells cannot pass, and a culture plate containing a culture solution,
A second chamber is arranged in the culture plate, and the first chamber is placed in the second chamber from above with a space from the second chamber. A three-dimensional bilayer structure for assessing cancer metastatic potential, with an interval between the plate and the second chamber.