TUMOR CELL XENOGRAFT MODEL IN ZEBRAFISH, AND METHODS OF CONSTRUCTING AND USING THE SAME

Abstract

A tumor cell xenograft model in zebrafish, and methods of constructing and using the same. Primary cells dissociated from the tumor tissue of a patient are transplanted into zebrafish, so as to obtain a patient-derived tumor xenograft model. The tumor xenograft model retains the pathological features of human gastric cancer tissues in clinic, has higher clinical relevance, and can be used in the systematic research into the mechanisms underlying the proliferation, metastasis, spread and drug resistance of tumors, and to screen effective drugs for tumor treatments.

Claims

1. A patient-derived tumor cell xenograft model comprising a zebrafish embryo transplanted with primary single cells isolated and cultured from tumor tissue derived from a patient.

2. The model according to claim 1, wherein the transplantation is carried out within 24-72 hours after fertilization.

3. The model according to claim 1, wherein the transplantation site is in the yolk sac of the embryos.

4. The model according to claim 1, wherein the primary single cells are stained.

5. The model according to claim 1, wherein the tumor is selected from gastric cancer and lung cancer.

6. (canceled)

7. A method for studying the mechanism underlying the proliferation, metastasis, spread or drug resistance of tumors, or in screening effective therapeutic drugs for tumors using the model according to claim 1, the method comprising the steps of: determining the highest drug concentration within the safe range of a tumor drug candidate for the embryos; treating the zebrafish embryos with the drug candidate having a drug concentration in the safe range, and taking the solvent for dissolving the drug candidate as a control and undergoing the same process; and performing qualitative analysis or/and quantitative analysis of the proliferation and spread of the patient-derived cells in the zebrafish embryos under a fluorescence microscope.

8. The method according to claim 7, wherein for the quantitative analysis, the antitumor effect is calculated according to the following formula:
the tumor inhibition rate of the drug=(the fluorescence intensity of the treatment group/the fluorescence intensity of the control group)*100%.

9. The method according to claim 7, wherein the treatment with the drug candidate is continued for 2 to 5 days.

10. The method according to claim 7, wherein the tumor is selected from gastric cancer and the drug is selected from 5-fluorouracil.

11. The method according to claim 7, wherein the tumor is selected from lung cancer and the drug is one selected from gefitinib, cis-cisplatin or docetaxel, or a combination of two thereof.

12. A method for constructing a patient-derived tumor cell xenograft model in zebrafish, comprising the steps of: (1) dissociating a patient-derived clinical surgical specimen of tumor tissue into primary single cells; (2) staining the primary cells obtained by dissociation; and (3) injecting the primary single cells obtained in the step (2) into the yolk sac of zebrafish embryos.

13. The construction method according to claim 12, wherein the dissociation in the step (1) comprises aseptically cleaning the sample in physiological saline, then cutting into small pieces in a phosphate buffer solution, trypsinizing to complete dissociation, and centrifuging to remove trypsin; for the staining in the step (2), the dye used is CM-Dil, the dye concentration is 1-5 g/ml, the staining time is 1-10 hours, the dye is removed after staining, and the cells are washed with a phosphate buffer solution and resuspended to a cell density of 510.sup.3-510.sup.5 cell/; and the injection in the step (3) comprises: immobilizing the zebrafish embryos 36-60 hours after fertilization, and injecting 10-30 nl of the primary single cells obtained in the step (2) into the yolk sac of the zebrafish embryos using a microinjector under a stereoscope.

14. The construction method according to claim 12, further comprising, after the step (3), a step of performing qualitative analysis or/and quantitative analysis under a fluorescence microscope.

15. The construction method according to claim 12, wherein the metastasis and spread of the fluorescent cells in the zebrafish are observed under a fluorescence microscope after the zebrafish embryos are anesthetized with tricaine within 1-7 days after the cells are xenografted.

16. The model according to claim 2, wherein the transplantation is carried out within 36-60 hours after fertilization.

17. The model according to claim 2, wherein the transplantation is carried out within 48 hours after fertilization.

18. The model according to claim 4, wherein the primary cells are stained with CM-Dil.

19. The use according to claim 9, wherein the treatment with the drug candidate is continued for at least 3 days.

20. The construction method according to claim 13, wherein the injection in the step (3) comprises: immobilizing the zebrafish embryos 36-60 hours after fertilization, and injecting at least 20 nl of the primary single cells obtained in the step (2) into the yolk sac of the zebrafish embryos using a microinjector under a stereoscope.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 shows the phenotype of zebrafish embryos after patient-derived primary gastric cancer cells are injected in Example 1.

[0034] FIG. 2 is a diagram showing the anticancer effects of 5-FU evaluated in two 5-FU non-sensitive patients #1 and #2-derived gastric cancer cell xenograft models in zebrafish in Example 2 of the present invention.

[0035] FIG. 3 is a diagram showing the anticancer effects of 5-FU evaluated in two 5-FU sensitive patients #3 and #4-derived gastric cancer cell xenograft models in zebrafish in Example 2 of the present invention.

[0036] FIG. 4 is a diagram showing the anticancer effects of 5-FU evaluated in two human gastric cancer cell line xenograft models in zebrafish.

[0037] FIG. 5 shows the phenotype of a patient-derived gastric cancer cell xenograft model in zebrafish treated with curcumin in Example 4 of the present invention.

[0038] FIG. 6 is a diagram showing the anticancer effect of curcumin evaluated in a patient-derived gastric cancer cell xenograft model in zebrafish in Example 4 of the present invention.

[0039] FIG. 7 shows the phenotype of zebrafish embryos after patient-derived primary lung cancer cells are injected in Example 5.

[0040] FIG. 8 is a diagram showing the anticancer effect of cisplatin in combination with docetaxel evaluated in patients *1 and *2-derived lung cancer cell xenograft models in zebrafish in Example 6 of the present invention.

[0041] FIG. 9 is a diagram showing the anticancer effect of cisplatin in combination with docetaxel evaluated in patients *3 and *4-derived lung cancer cell xenograft models in zebrafish in Example 6 of the present invention.

[0042] FIG. 10 is a diagram showing the anticancer effect of gefitinib evaluated in patients *5 and *6-derived lung cancer cell xenograft models in zebrafish in Example 7 of the present invention.

[0043] FIG. 11 is a diagram showing the anticancer effect of gefitinib evaluated in patients *7 and *8-derived lung cancer cell xenograft models in zebrafish in Example 7 of the present invention.

[0044] FIG. 12 is a diagram showing the anticancer effects of gefitinib evaluated in two human lung cancer cell line xenograft models in zebrafish in Example 8.

DETAILED DESCRIPTION

[0045] The present invention is described in detail below with reference to the embodiments and the accompanying drawings, but the following examples should not be construed as limiting the scope of the present invention.

[0046] The methods given in examples below are all conventional methods, unless it is otherwise stated. The experimental method can also reflect the difference in accuracy between the patient-derived gastric or long cancer xenograft (PDX) model and the human gastric or lung cancer cell line xenograft model in guiding the personalized medication of patients with gastric or lung cancer in clinic.

Example 1: Construction of a Patient-Derived Gastric Cancer Cell Xenograft Model in Zebrafish of the Present Invention

[0047] 1. Isolation of Primary Cells from Gastric Cancer Tissue

[0048] The patient-derived clinical tissue biopsy that was a surgical specimen of gastric cancer was placed in physiological saline. The blood clot, necrotic tissue, fat and connective tissues on the surface of the tumor tissue were removed under aseptic conditions. The tissue was cut by an ophthalmic scissor after sterilization, and washed 2 times with sterile phosphate buffer (pH 7.4). A small amount of phosphate buffer was added, and the tissue was repeatedly cut with an ophthalmic scissor until the tissue became a paste and was about 1 mm.sup.3. 0.25% trypsin was added and the tissue was digested at 37 C. for 10 minutes. After the tissue mass was observed to be completely dissociated, centrifugation was performed to remove trypsin. The cells were re-suspended in RPMI-1640 medium containing 10% FBS (fetal calf serum).

[0049] 2. Staining of Primary Cells

[0050] The primary cells obtained by dissociation were stained with CM-Dil, where the final dye concentration was 2 g/ml, and the staining time was 1 hour. The dye was removed by centrifugation, and the cells were washed with a phosphate buffer and re-suspended to a cell density of 110.sup.4/l.

[0051] 3. Cell Transplantation

[0052] The stained cells were filled into a microinjector. The zebrafish embryos were immobilized 48 hours postfertilization, and 20 nl of the primary cells obtained in the step (2) into the yolk sac of zebrafish embryos using the microinjector under a stereoscope.

[0053] 4. Observation Under Fluorescence Microscope

[0054] Within 7 days after the injection, the growth, metastasis and spread of the cells derived from the patient in the zebrafish were observed under a fluorescence microscopy and photographed.

[0055] As shown in FIG. 1, the patient-derived gastric cancer cells display a proliferative and spreading phenotype in zebrafish embryos. 4 days after the injection, it can be seen that the patient-derived gastric cancer cells had spread to the abdomen and the head. 7 days after the injection, it can be seen that the patient-derived gastric cancer cells had spread to the tail and the brain of the zebrafish embryos.

Example 2: Evaluation of the Clinical Anticancer Effect of 5-FU with 4 Patient-Derived Xenograft Zebrafish Models

[0056] 1. Determination of Safe Dose

[0057] Two days after fertilization, the zebrafish embryos were treated (soaked) with various concentrations of 5-FU for three days, and the highest 5-FU concentration within the safety range for embryos was determined to be 4000 nM.

[0058] 2. Drug Treatment of Zebrafish Embryos

[0059] The 4000-M and 400 M 5-FU were used to soak the zebrafish embryo model injected with the primary gastric cancer cells derived from different patients prepared by the method of Example 1 for three days, and 0.1% DMSO was used as a solvent control.

[0060] 3. Observation of Antitumor Effect Under Fluorescence Microscope

[0061] The proliferation and spread of red patient-derived cells in the zebrafish embryos were observed after treatment. The red cells were photographed under a fluorescence microscope, and the red fluorescence intensity was quantified by Image Pro Plus software to calculate the anti-tumor effect of 5-FU by a formula: Tumor inhibition rate of the drug=(the fluorescence intensity of the treatment group/the fluorescence intensity of the control group)*100% (see FIGS. 2 and 3).

[0062] The results with the patient-derived gastric cancer xenograft model in zebrafish show that at day 7 after the injection, the gastric cancer cells derived from #1 and #2 patients with gastric cancer are not sensitive to 5-FU, and there is no obvious tumor inhibition effect; and the clinical effect of 5-FU in the treatment of the two patients with gastric cancer is not significant either. The gastric cancer cells derived from #3 and #4 patients with gastric cancer are sensitive to 5-FU, and the tumor proliferation is significantly inhibited; and the clinical symptoms of these two gastric cancer patients were significantly improved after treatment with 5-FU.

[0063] Therefore, the drug evaluation results with the patient-derived gastric cancer xenograft models in zebrafish are highly correlated with the clinical outcomes.

Example 3: Evaluation of the Anticancer Effects of 5-FU with Two Human Gastric Cancer Cell Line (SGC-7901 and AGS) Xenograft Models in Zebrafish

[0064] 1. Drug Treatment of Zebrafish Embryos

[0065] 4000 M and 400 M5-FU were used to soak the zebrafish embryos injected with gastric cancer cell lines (SGC-7901 and AGS) (constructed as in Example 1) for three consecutive days, and 0.1% DMSO was used as a solvent control.

[0066] 2. Observation of Antitumor Effect Under Fluorescence Microscope

[0067] The proliferation and spread of red patient-derived cells in the zebrafish embryos were observed after treatment. The red cells were photographed under a fluorescence microscope, and the red fluorescence intensity was quantified by Image Pro Plus software to calculate the anti-tumor effect of 5-FU by a formula: Tumor inhibition rate of the drug=(the fluorescence intensity of the treatment group/the fluorescence intensity of the control group)*100% (see FIG. 4).

[0068] The results with human gastric cancer cell line xenograft models in zebrafish show that both gastric cancer cell lines are sensitive to 5-FU at day 7 after injection. Therefore, the clinical anti-gastric cancer effects of the drug 5-FU evaluated with the xenograft zebrafish models established with the cell lines differ from the actual clinical efficacy, and cannot be used to guide the clinical medication of gastric cancer.

Example 4: Evaluation of the Anti-Gastric Cancer Effect of Curcumin with Patient-Derived Gastric Cancer Xenograft Animal Models

[0069] 1. Four groups, each group having four randomly selected zebrafish embryos that were two days old after fertilization, were placed in a solution of curcumin containing 0.1% DMSO. The curcumin concentrations were 10 M, 30 M, 50 M, and 70 M, respectively, and the treatment was continued for 3 days. The death of the zebrafish embryos was observed, and the highest concentration of curcumin in the safe range for the embryos was determined to be 50 M.

[0070] 2. The zebrafish embryos that have been injected with the patient-derived primary gastric cancer cells were placed in 10 M and 50 M aqueous solutions of curcumin containing 0.1% DMSO as described in the step 1, for three consecutive days. The zebrafish embryos that have been injected with humanized gastric cancer primary cells were placed in an aqueous 0.1% DMSO solution for three consecutive days and taken as a solvent control group.

[0071] 3. The proliferation and spread of patient-derived gastric cancer cells in the zebrafish embryos were observed after treatment. In this example, the gastric cells are proliferated in situ. The human-derived gastric cells are photographed under a fluorescence microscope (FIG. 5), the fluorescence intensity is quantified using Image Pro Plus software, and the antitumor effect of curcumin is calculated (FIG. 6).

Example 5: Construction of a Patient-Derived Lung Cancer Cell Xenograft Model in Zebrafish of the Present Invention

[0072] 1. Isolation of Primary Cells from Lung Cancer Tissue

[0073] The patient-derived clinical tissue biopsy that was a surgical specimen of lung cancer was placed in physiological saline. The blood clot, necrotic tissue, fat and connective tissues on the surface of the tumor tissue were removed under aseptic conditions. The tissue was cut by an ophthalmic scissor after sterilization, and washed 2 times with sterile phosphate buffer (pH 7.4). A small amount of phosphate buffer was added, and the tissue was repeatedly cut with an ophthalmic scissor until the tissue became a paste and was about 1 mm.sup.3 in size. 0.25% trypsin was added and the tissue was digested at 37 C. for 10 minutes. After the tissue mass was observed to be completely dissociated, centrifugation was performed to remove trypsin. The cells were resuspended in RPMI-1640 medium containing 10% FBS (fetal calf serum).

[0074] 2. Staining of Primary Cells

[0075] The primary cells obtained by dissociation were stained with CM-Dil, where the final dye concentration was 2 g/ml, and the staining time was 1 hour. The dye was removed by centrifugation, and the cells were washed with a phosphate buffer and re-suspended to a cell density of 110.sup.4/l.

[0076] 3. Cell Transplantation

[0077] The stained cells were filled into a microinjector. The zebrafish embryos were immobilized 48 hours after fertilization, and 20 nl of the primary cells obtained in the step (2) into the yolk sac of zebrafish embryos using the microinjector under a stereoscope.

[0078] 4. Observation Under Fluorescence Microscope

[0079] Within 4 days after the injection, the growth, metastasis and spread of the cells derived from the patient in the zebrafish were observed under a fluorescence microscopy and photographed.

[0080] As shown in FIG. 7, the patient-derived lung cancer cells display a proliferative and spreading phenotype in zebrafish embryos. 4 days after the injection, it can be seen that the patient-derived lung cancer cells had spread to the abdomen and the head.

Example 6: Evaluation of the Clinical Anticancer Effect of Docetaxel+Cisplatin with 4 Patient-Derived Lung Cancer Xenograft Models in Zebrafish. Determination of Safe Dose

[0081] Two days after fertilization, the zebrafish embryos were treated (soaked) respectively with various concentrations of cisplatin and docetaxel for three days, and the highest cisplatin concentration is 40 M within the safety range for embryos was determined to be 40 M and the docetaxel concentration was 10 M.

[0082] 2. Drug Treatment of Zebrafish Embryos

[0083] The 40-M cisplatin+10-M docetaxel and 4-M cisplatin+1-M docetaxel were used to soak the zebrafish embryo model injected with the primary lung cancer cells derived from different patients prepared by the method of Example 1 for three consecutive days, and 0.1% DMSO was used as a solvent control. The zebrafish embryo models with the same patient-derived lung cancer primary cells were treated with two different concentrations of the agents.

[0084] 3. Observation of Antitumor Effect Under Fluorescence Microscope

[0085] The proliferation and spread of red patient-derived cells in the zebrafish embryos were observed after treatment. The red cells were photographed under a fluorescence microscope, and the red fluorescence intensity was quantified by Image Pro Plus software to calculate the anti-tumor effect of the drug by a formula: Tumor inhibition rate of the drug=(the fluorescence intensity of the treatment group/the fluorescence intensity of the control group)*100% (see FIGS. 8 and 9).

[0086] The results with the patient-derived lung cancer xenograft model in zebrafish show that at day 7 after the injection, the lung cancer cells derived from *1 and *2 patients with lung cancer are not sensitive to the combination of cisplatin+docetaxel, and there is no obvious tumor inhibition effect; and the clinical effect of cisplatin+docetaxel in the treatment of the two patients with lung cancer is not significant either. The lung cancer cells derived from *3 and *4 patients with lung cancer are sensitive to the combination of cisplatin+docetaxel, and the tumor proliferation is significantly inhibited; and the clinical symptoms of these two lung cancer patients were significantly improved after treatment with the two drugs in combination.

[0087] Therefore, the drug evaluation results with the patient-derived lung cancer xenograft models in zebrafish are highly correlated with the clinical outcomes. The experimental results of multiple groups also confirm this conclusion.

Example 7: Evaluation of the Clinical Anticancer Effect of Gefitinib with 4 Patient-Derived Lung Cancer Cell Xenograft Models in Zebrafish

[0088] 1. Determination of Safe Dose

[0089] Two days after fertilization, the zebrafish embryos were treated (soaked) with various concentrations of gefitinib for three consecutive days, and the highest gefitinib concentration within the safety range for embryos was determined to be 50 M.

[0090] 2. Drug Treatment of Zebrafish Embryos

[0091] The 50-M and 5-M gefitinib were used to soak the zebrafish embryo model injected with the patient-derived primary lung cancer cells prepared by the method of Example 1 for three consecutive days, and 0.1% DMSO was used as a solvent control. The zebrafish embryo models with the same patient-derived lung cancer primary cells were treated with two different concentrations of the agents.

[0092] 3. Observation of Antitumor Effect Under Fluorescence Microscope

[0093] The proliferation and spread of red patient-derived cells in the zebrafish embryos were observed after treatment. The red cells were photographed under a fluorescence microscope, and the red fluorescence intensity was quantified by Image Pro Plus software to calculate the anti-tumor effect of gefitinib by a formula: Tumor inhibition rate of the drug=(the fluorescence intensity of the treatment group/the fluorescence intensity of the control group)*100% (see FIGS. 10 and 11).

[0094] The results with the patient-derived lung cancer xenograft model in zebrafish show that at day 7 after the injection, the lung cancer cells derived from *5 and *6 patients with lung cancer are sensitive to gefitinib, there is obvious tumor inhibition effect, and the tumor proliferation is obviously inhibited; and the clinical effect of gefitinib in the treatment of the two patients with lung cancer is also significant. The lung cancer cells derived from *7 and *8 patients with lung cancer are not sensitive to gefitinib; and the clinical symptoms of these two lung cancer patients are not significantly improved after treatment with the two drugs in combination.

[0095] Therefore, the drug evaluation results with the patient-derived lung cancer xenograft models in zebrafish are highly correlated with the clinical outcomes. The experimental results of multiple groups also confirm this conclusion.

Example 8: Evaluation of the Anticancer Effects of Gefitinib with Two Human Lung Cancer Cell Line (A549 and HCC827) Xenograft Models in Zebrafish

[0096] 1. Drug Treatment of Zebrafish Embryos 50 M gefitinib and 5 M gefitinib were used to soak zebrafish embryos (constructed in accordance with the method of Example 1) that have been injected with lung cancer cell lines (A549 and HCC827) for three consecutive days and 0.1% DMSO was used as a solvent control.

[0097] 2. Observation of Antitumor Effect Under Fluorescence Microscope

[0098] The proliferation and spread of red patient-derived cells in the zebrafish embryos were observed after treatment. The red cells were photographed under a fluorescence microscope, and the red fluorescence intensity was quantified by Image Pro Plus software to calculate the anti-tumor effect of gefitinib by a formula: Tumor inhibition rate of the drug=(the fluorescence intensity of the treatment group/the fluorescence intensity of the control group)*100% (see FIG. 12).

[0099] The results with human lung cancer cell line xenograft models in zebrafish show that both lung cancer cell lines are sensitive to gefitinib at day 7 after injection. Therefore, the clinical anti-lung cancer effects of the drug gefitinib evaluated with the xenograft zebrafish models established with the cell lines differ from the actual clinical efficacy, and cannot be used to guide the clinical medication of lung cancer.