TWI749460B - Isolated liver cancer stem cell and method for drug screening thereof - Google Patents

Abstract

The invention is an isolated rat liver cancer stem cell line which is named TW-1, wherein the isolated rat liver cancer stem cell line has been deposited in Food Industry Research and Development Institute (Taiwan) with the depositing number BCRC960524. The invention also provides a method for drug screening by using the isolated rat liver cancer stem cell line.

Description

Separated liver cancer stem cell strain and its method for drug screening

The invention relates to a liver cancer stem cell strain and its application, in particular to an isolated liver cancer stem cell strain and a method for drug screening.

Invasion (Innovation) and spread (Metastasis) are the two most important signs of cancer, as well as the main cause of the high mortality rate of cancer. The recurrence of cancer is also highly related to the intrahepatic metastasis of cancer cells, especially for patients with liver cancer. The metastasis of liver cancer has led to a very high mortality rate. In many countries including Taiwan, liver cancer is also one of the cancers with the highest mortality rate. In Taiwan, liver cancer has even become the second largest cause of death from the top ten cancers in the country, even if it is accepted Patients who have undergone radical hepatectomy will also face survival challenges due to loss of liver function. However, the mechanism of liver cancer metastasis and liver cancer recurrence is still unclear.

Since humans first identified cancer stem cells (CSC) in solid cancers, the theory of cancer stem cells has become an interesting research topic.

Cancer stem cells have the same self-renewal ability as ordinary stem cells, but they also have carcinogenic activity. Therefore, solid cancers are considered to be composed of a small number of cancer stem cells, and these cancer stem cells drive tumor growth, reproduction and metastasis.

The present invention mainly uses isolated cancer stem cell lines to establish a model of tumorgenesis and metastasis of liver cancer in rats, and uses this model to screen the efficacy of anti-cancer drugs.

The present invention provides an isolated rat liver cancer stem cell line named TW-1. The isolated rat liver cancer stem cell line was deposited at the Food Industry Development Institute on June 3, 108, with the deposit number: BCRC960524.

The isolated rat liver cancer stem cell line of the present invention is isolated from Fisher 344 (F344) rats with liver cancer induced by diethylnitrosamine (DEN).

In the isolated rat liver cancer stem cell line of the present invention, the isolated rat liver cancer stem cell line has at least one highly expressed drug resistance gene against an anticancer drug, wherein the at least one highly expressed drug resistance gene line Choose from Abca1, Abca12, Abcb1a, Abcb1b, Abcc3, Abcc4, Abccg1, Abcg3l3, Acyp1, Cyp20a1, Cyp2u1, Cyp3a9, Cyp4b1, Cyp51, Gsta1, Gsta2, Gsta3, Gsta4, Gsta5, Gstcd, Gstm1, Gstm1, Gstm1 The group consisting of Gstp1, Gstt2, Gstt3, Mgst1 and Mgst2.

The isolated rat liver cancer stem cell line of the present invention uses the at least one highly expressed drug resistance gene to resist at least one anticancer drug; further, the isolated rat liver cancer stem cell line of the present invention uses the at least one A highly expressed drug resistance gene against at least one liver cancer therapeutic drug; more specifically, the isolated rat liver cancer stem cell line of the present invention uses the at least one highly expressed drug resistance gene to resist sorafenib (Sorafenib). ).

The isolated rat liver cancer stem cell line of the present invention is characterized in that the isolated rat liver cancer stem cell line expresses CD133, CK-19, GSTP1, CD44, EpCAM, CD90 and ALDH biomarkers.

The present invention also provides a drug screening method, including providing an immune system defect Or an animal model with a normal immune system, and then inoculate an isolated rat liver cancer stem cell strain into the animal model, and provide at least one anti-cancer drug for treating the inoculated animal model to evaluate the at least one Anticancer drugs inhibit the pathological changes induced by the isolated rat liver cancer stem cell line in the animal model.

The present invention also provides another drug screening method, including providing an isolated rat liver cancer stem cell line, and providing at least one anti-cancer drug for treating the isolated rat liver cancer stem cell line for evaluating the at least one antibody Cancer drugs inhibit the growth of the isolated rat liver cancer stem cell line or inhibit recurrence.

Figure 1 shows the time course and results of liver tumor induction in Fisher 344 (F344) rats in the embodiment of the present invention.

Figure 2 shows the results of gene microarray analysis of liver tumor cells and general liver cells in the embodiment of the present invention.

Figure 3 shows the steps of liver tumor cell transplantation in the embodiment of the present invention.

Figure 4 shows the screening and isolation of liver tumor cells in an embodiment of the present invention. Figure 4A shows the steps of screening and isolation of liver tumor cells; Figure 4B shows the type of isolated cell lines, the left image is the TW-1 cell line, and the right image is the liver tumor cell line (Hepatic Tumor Cells; HTC).

Figure 5 shows the cell identification of the isolated tumor cell line of the present invention. Figure 5A shows the Western blot method identification results of TW-1 cell line and HTC cell line. Figure 5B shows the results of gene microarray analysis of TW-1 cell line and HTC cell line. Figure 5C shows tumor regeneration Time course of the test; Figure 5D shows the results of the tumor regeneration test.

Figure 6 shows the results of the drug screening experiment according to the embodiment of the present invention. Figure 6A shows the results of in vitro drug screening for Sorafenib; Figure 6B shows the results of microarray analysis of the expression of drug resistance genes after Sorafenib treated TW-1 cell lines ; Figure 6C shows the results of Sorafenib in vivo drug screening; Figure 6D shows the results of Sorafenib treatment of TW-1 cell line metastasis.

Figure 7 shows the time course and results of the immune evasion test according to the embodiment of the present invention. Figure 7A shows the immune evasion test results of the orthotopic model of cancer in situ; Figure 7B shows the immune evasion test results of the tumor metastasis model (metastasis model).

1. Induction, isolation and identification of liver tumor cells

1a. Selection and grouping of experimental animals

In the embodiment of the present invention, the experimental animals used are 6-week-old wild-type Fisher 344 (F344) male mice (Wild Fisher 344 (F344)) with normal immune function and 3-week-old BALB/cAnN with immune system deficiency. Cg- Foxnl ^nu /CrlNarl male mice (both purchased from the National Laboratory Animal Center) were reared for one week and began to experiment after adapting to the environment.

The experimental animals are kept in standard plastic rat and mouse cages. The ambient temperature is maintained at 25±2°C. The light cycle is 12 hours. During the experiment, feed and drinking water are provided with free intake. The experimental animals are divided into a control group and an experimental group. 6 in each group.

1b. Induce liver tumorigenesis in Fisher 344 (F344) rats

In an embodiment of the present invention, the selected liver tumor inducer is diethylnitrosamine (DEN), and is configured with phosphate buffer solution (PBS) to a target concentration.

As shown in Figure 1, the experimental group of Fisher 344 (F344) rats were given DEN by intraperitoneal injection. A total of 4 doses were given. /Kg body weight), 160mg/kg (mg/kg body weight), 160mg/kg (mg/kg body weight), 160mg/kg (mg/kg body weight), after completing the 4 doses, these rats After 48 weeks of rearing, they were sacrificed, and liver tumor tissues were taken out for transplantation into BALB/cAnN.Cg- Foxnl ^nu /CrlNarl mice to further analyze the degree of liver tumor formation.

The control group was given phosphate buffered solution (PBS) by intraperitoneal injection.

1c. Transplantation, purification and separation of liver tumor cells

As shown in Figure 2, the liver tumor tissue of Fisher 344 (F344) rats induced by DEN was processed into tiny masses and transplanted into the experimental group BALB/cAnN.Cg- Foxnl ^nu /CrlNarl by subcutaneous embedding. Mice, and observe for 8 weeks.

The control group was divided into a positive control group and a negative control group. The positive control group was subcutaneously injected with human liver cancer cells HepG2, and the negative control group was subcutaneously injected with phosphate buffered solution (PBS).

The experimental results showed that the rat liver tumor tissue from number II grew most obviously in immunodeficient mice, so the liver tumor tissue number II and the liver tissue of the control group were subjected to gene microarray analysis, as shown in Figure 3 The analysis results showed that compared with normal liver tissues, the expression levels of most liver cancer genes (EpCAM, CK-19/Krt19, GSTP-1) in liver tumor tissues numbered II were significantly increased, but the interesting thing is that the number The genetic characteristics of some stem cells were also found in the liver tumor tissue of II (for example: CD133/Prom1, CD90, CD44, etc.).

The BALB/cAnN.Cg-Foxnl ^nu /CrlNarl mice transplanted with liver tumor tissue number II were sacrificed, and the liver tumor tissues that grew again in the BALB/cAnN.Cg- Foxnl ^{nu/CrlNarl mice were subjected to cellular} Screening and separation.

The cell screening and isolation of liver tumor tissues are shown in Figure 4A.

After the liver tumor tissue was cut into fine pieces, 0.05% collagenase was added and digested in a 37°C incubator for 1 hour, and then the isolated cells were added to DMEM/F12 medium containing antibiotics and cultured for 24 hours . After 7 days of culture, the surviving attached cells were subcultured with 0.25% trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA) aqueous solution, and the cells were collected separately using the time difference of cell suspension.

After repeating the above steps twice, observe the colony effect of the cells through a microscope, and separate the cells in the same colony. In the present invention, two groups of different cell lines are separated, named TW-1 cell line and HTC cell line, respectively. The types of TW-1 cell line and HTC cell line are shown in Figure 4B.

1d. Identification of cell lines

In order to identify the characteristics of the TW-1 cell line and the HTC cell line, the Western blot method was used to perform protein analysis on biomolecules related to liver cancer or stem cells, including CD133, CK-19, GSTP-1, CD44, EpCAM, AFP, ALB And CD34 and other proteins, the results are shown in Figure 5A.

From the results shown in Figure 5A, we can find significant differences between the two groups of cells. Compared with the HTC cell line, the TW-1 cell line not only has significantly higher performance of stem cell-related biomolecules CD133 and CD44, but also TW-1 cells. Compared with the HTC cell line, the strain also has significantly higher performance of the biomolecules CK-19, GSTP-1 and EpCAM related to liver cancer; in contrast, the HTC cell line, HTC The cell lines show the molecular markers of general liver cancer cells, such as AFP and ALB, which have significantly higher performance than the TW-1 cell line.

Furthermore, the gene microarray analysis of the TW-1 cell line and the HTC cell line found that, as shown in Figure 5B, some of the weak stem cell gene expressions found in Figure 2 can be further determined by the genes of the TW-1 cell line. It is completely seen in performance (for example: CD44). At the same time, the TW-1 cell line also expresses genes related to stem cells such as Prom1 (CD133) and CD90. Therefore, the above information preliminarily proves that the TW-1 cell line is in the tumor tissue. That group of cells with the characteristics of stem cells.

In addition, the tumor regeneration test was used to identify the characteristics of the isolated liver cancer cells. As shown in Figure 5C, the TW-1 cell line and the HTC cell line were proliferated and then the cells were injected into the immune system at ^{5×10 5 cells per site.} Defective BALB/cAnN.Cg- Foxnl ^nu /CrlNarl mice were observed for 4 weeks to confirm whether the cells would form tumors again. The result is shown in Figure 5D. Only TW-1 can re-form tumor tissues. This shows that The TW-1 cell line has strong tumor regeneration.

The identification results in Figures 4 and 5 above indicate that the TW-1 cell line should be a liver cancer stem cell line, while the HTC cell line may belong to a general liver cancer cell line.

2. Application of drug screening methods

2a. In vitro drug screening test

In this embodiment, the purpose of the in vitro drug screening method of the present invention is to screen the active ingredients of anticancer drugs on the market for the ability to inhibit the growth, recurrence or metastasis of the TW-1 cell line, and use the HTC cell line as a control Group, the active ingredient of the anticancer drug selected in this embodiment is Sorafenib.

The in vitro drug screening method of the present invention is to plant the TW-1 cell line and the HTC cell line ^{in a 96-well plate at 2×10 5} cells/well, and after the cells are attached, different concentrations of sorafenib are respectively administered (Sorafenib), the concentrations of which were 1μM, 5μM, 10μM, 20μM and 40μM, and the negative control group was phosphate buffered solution (PBS). The cells were cultured for 24 and 48 hours after administration, and the cell survival rate was observed.

From the results in Figure 6A, it can be found that the TW-1 cell line is highly sensitive to Sorafenib when the concentration of Sorafenib is lower than 10μM after 24 hours of administration. However, when the concentration of Sorafenib reaches 40μM, the TW-1 cell line has a higher drug tolerance to Sorafenib; and after the cells are cultured for 48 hours, TW-1 cells The strain's high drug tolerance to high-dose Sorafenib (Sorafenib) is more obvious.

In order to further understand the reasons for the high drug tolerance of the TW-1 cell line to high-dose Sorafenib, the TW-1 cell line and the HTC cell line were also genetically analyzed by microarray to explore the TW-1 cell line Differences between strains and HTC cell strains. From the genetic analysis of the microarray in Figure 6B, it was found that the intracellular drug resistance genes of the TW-1 cell line (e.g.: Abca1, Abca12, Abcb1a, Abcb1b, Abcc3, Abcc4, Abcg1, Abcg3l3, Acyp1, Cyp20a1, Cyp2u1, Genes such as Cyp3a9, Cyp4b1, Cyp51, Gsta1, Gsta2, Gsta3, Gsta4, Gsta5, Gstcd, Gstm1, Gstm2, Gstm4, Gsto1, Gstp1, Gstt2, Gstt3, Mgst1, Mgst2, etc.) have a significant tendency to show a large number of expressions compared with HTC cell lines This may indicate that the TW-1 cell line produces high drug tolerance to high-dose Sorafenib through the action of these drug resistance genes, which is also the reason for the poor effect of drug treatment. one.

2b. In-vivo drug screening

In this embodiment, the purpose of the in vivo drug screening method of the present invention is to screen the ability of the active ingredients of anticancer drugs on the market to the TW-1 cell line, and use BALB/cAnN.Cg- Foxnl ^nu / which is defective in the immune system. CrlNarl mice are used as experimental animal models, and the active ingredient of the anticancer drug selected in this embodiment is Sorafenib.

The in vivo drug screening method of the present invention is to transplant the TW-1 cell line into BALB/cAnN.Cg- Foxnl ^nu /CrlNarl mice with immune system deficiency to test the drug activity subcutaneously. The transplanted TW-1 cell line is measured at each site 5×10 ⁵ cells are transplanted. The groups of experimental animals were the control group, the low-dose Sorafenib group (10 mg/kg body weight) and the high-dose Sorafenib group (50 mg/kg body weight), each group 5 mice.

When the TW-1 cells at the transplantation site have grown to a measurable size (approximately 100-150mm ³ ), the drug will be administered (day 0), then every 3 days, and the measurement will continue for 30 days, every 5 days Measure once and record the tumor volume. As the feeding time increases, the tumors formed by allograft cells tend to increase. The results are shown in Figure 6C. Surprisingly, the tumor size measured by mice in the high-dose Sorafenib group was significantly higher than that in the low-dose Sorafenib group. Measured tumor size.

From the results of Figure 6A to Figure 6C, it can be found that although in vitro drug screening experiments, it can be found that 40μM Sorafenib can inhibit the growth of TW-1 cell line, but TW-1 cell line It has not been completely eliminated. After further genetic analysis of the microarray, it was found that the TW-1 cell line had an abnormally large number of drug resistance genes compared to the normal hepatocarcinoma cell line (HTC). From the in vivo drug screening experiments, it was found that the tumor size measured by mice in the high-dose Sorafenib group was significantly higher than that in the low-dose Sorafenib group. Measured tumor size. These results show that Sorafenib, the first-line liver cancer drug currently on the market, cannot effectively treat the TW-1 cell line. This also explains why the recurrence and metastasis of liver cancer are so difficult to control.

In addition, the results of the in vivo drug screening of the present invention also found that cancer cells of the TW-1 cell line metastasized to the lungs, and the degree of metastasis increased with the increase of the dose of anticancer drugs, as shown in Figure 6D It is shown that this phenomenon is presumed to be related to the increase in drug resistance genes and oncogenic genes of the TW-1 cell line, and may have immune evasion characteristics.

3. Immune evasion test

3a. Selection of experimental animal models and pre-experiment processing

In this experiment, Fisher 344 (F344) rats with normal immune system were used as experimental animal models, and Retrorsine was treated by intraperitoneal injection once a week at a dose of 30 mg/kg-body weight for a total of two weeks. Inhibit the cell proliferation of the liver to enhance the growth advantage of the TW-1 cell line in the microenvironment.

3b. Immune evasion test in orthotopic model of carcinoma in situ

After Fisher 344 (F344) rats were treated with Retrorsine for two weeks, the administration was stopped for one week to metabolize the remaining Retrorsine in the body, and then the TW-1 cell line was injected into the liver ^{at a rate of 2×10 7 cells per site.} And keep feeding for four weeks.

The results show that, referring to Figure 7A, the liver of the animal model of cancer in situ produced a large number of tumor cells, which indicates that the TW-1 cell line can escape the attack of immune cells and continue to proliferate.

3c. Immune evasion test of tumor metastasis model (metastasis model)

After Fisher 344 (F344) rats were treated with Retrorsine for two weeks, the administration was stopped for two weeks to metabolize the remaining Retrorsine in the body, and then the TW-1 cell line was injected into the liver ^{at a rate of 2×10 7 cells per site} , And reared for four weeks.

The results show that referring to Figure 7B, rats with a tumor metastasis pattern are not only in the liver A large number of tumor cells are produced in the viscera, and they also quickly metastasize to the lungs within four weeks. This shows that the TW-1 cell line can not only evade the attack of immune cells and continue to proliferate, but also can further metastasize to the surrounding organs.

【Biological Material Deposit】

Domestic deposit information [please note in the order of deposit institution, date and number]

The isolated rat liver cancer stem cell strain TW-1 has been deposited at the Institute of Food Industry Development, a consortium legal person. The deposit date is June 3, 108, and the deposit number is BCRC960524.

Claims (9)

An isolated rat liver cancer stem cell line named TW-1. The isolated rat liver cancer stem cell line was deposited at the Institute of Food Industry Development on June 3, 108, with the deposit number BCRC960524, of which The TW-1 cell line has drug resistance properties against Sorafenib. The rat liver cancer stem cell line as described in claim 1, which is isolated from Fisher 344 rats with liver cancer induced by diethylnitrosamine (DEN). The rat liver cancer stem cell line according to claim 1, wherein the isolated rat liver cancer stem cell line has at least one highly expressed drug resistance gene against an anticancer drug, wherein the drug resistance gene line is selected from Abca1, Abca12 , Abcb1a, Abccb1b, Abcc3, Abcc4, Abcg1, Abccg313, Acyp1, Cyp20a1, Cyp2u1, Cyp3a9, Cyp4b1, Cyp51, Gsta1, Gsta2, Gsta3, Gsta4, Gsta5, Gstcd, Gstm1, Gstm2, Gstm4, Gttt , Mgst1 and Mgst2. The rat liver cancer stem cell line according to claim 1, wherein the isolated rat liver cancer stem cell line expresses CD133, CK-19, GSTP1, CD44, EpCAM, CD90 and ALDH biomarkers. A drug screening method, comprising: (a) providing at least one anti-cancer drug to an animal model with an immune system deficiency or a normal immune system, wherein the animal model has the rat liver cancer stem cell strain as described in claim 1; and ( b) Evaluate the efficacy of the at least one anticancer drug in inhibiting the pathological changes induced by the rat liver cancer stem cell line in the animal model. The drug screening method according to claim 5, wherein the animal model is Fisher 344 rats or BALB/cAnN.Cg- Foxnl ^nu /CrlNarl mice. The drug screening method according to claim 5, wherein the lesion refers to the growth, recurrence or metastasis of liver cancer cells induced by the rat liver cancer stem cell line. A drug screening method, comprising: providing a rat liver cancer stem cell strain as described in claim 1; and providing at least one anti-cancer drug to treat the rat liver cancer stem cell strain to evaluate the at least one anti-cancer drug The effect of inhibiting the growth of the rat liver cancer stem cell line or inhibiting recurrence. The drug screening method according to claim 8, wherein the rat liver cancer stem cell line and the control cell line are isolated from Fisher 344 rats with liver cancer induced by diethylnitrosamine (DEN).

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