WO2018072758A1 - 一种药物筛选方法及其装置 - Google Patents

一种药物筛选方法及其装置 Download PDF

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WO2018072758A1
WO2018072758A1 PCT/CN2017/107386 CN2017107386W WO2018072758A1 WO 2018072758 A1 WO2018072758 A1 WO 2018072758A1 CN 2017107386 W CN2017107386 W CN 2017107386W WO 2018072758 A1 WO2018072758 A1 WO 2018072758A1
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drug
tumor
tumor cells
sensitivity
detecting
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PCT/CN2017/107386
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English (en)
French (fr)
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闻丹忆
张菲菲
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上海立迪生物技术股份有限公司
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Priority to AU2017346232A priority Critical patent/AU2017346232B2/en
Priority to EP17862968.9A priority patent/EP3531127A4/en
Priority to CA3055800A priority patent/CA3055800A1/en
Priority to US16/071,405 priority patent/US20190154663A1/en
Priority to KR1020197014159A priority patent/KR102487944B1/ko
Priority to CN201780003121.3A priority patent/CN108351348A/zh
Publication of WO2018072758A1 publication Critical patent/WO2018072758A1/zh
Priority to HK19101725.9A priority patent/HK1259358A1/zh
Priority to US17/339,755 priority patent/US20220099658A1/en
Priority to AU2022100027A priority patent/AU2022100027A4/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases

Definitions

  • the invention relates to the field of in vitro drug screening, in particular to a method and device for rapid drug sensitivity detection of antitumor drugs.
  • individualized precision medicine is increasingly used in clinical, especially in the field of cancer treatment.
  • Chemotherapy is currently the main treatment for patients with advanced metastatic cancer, but chemotherapy drugs lack tumor cell specificity. While killing tumor cells, they also kill a large number of bone marrow and other proliferating normal cells, and have serious adverse reactions. .
  • traditional chemotherapy methods are prone to drug resistance after multiple chemotherapy, which will affect the effect of chemotherapy. Therefore, it is more and more urgent to target individualized precision medical treatment for patients.
  • Individualized precision medicine can improve the accuracy of treatment on the one hand, and can also effectively reduce the randomness and blindness of patients' medications on the other hand, thus reducing the harm of patients' medication.
  • PDX model patient-derived xenograft model
  • the PDX model better preserves the biological characteristics of the patient's primary tumor.
  • This model is a tumor model established by directly transplanting fresh tumor tissue from patients into immunodeficient mice (usually nude or SCID mice). This model maintains similar genetic characteristics and tumor heterogeneity to patients.
  • the main difficulty in using the PDX model for individualized precision medicine is that the model completion period and the drug sensitivity test take a long time and the transplant rate of the PDX model is higher. low. For example, primary cell transplantation takes 2 to 3 months, and subsequent drug sensitivity test time also takes 3 to 4 months.
  • the PDX model requires high operational skills, and surgeons, histologists, and researchers need to work closely with the specimens obtained from the operating room. Therefore, improving the anti-tumor drug susceptibility detection method of the PDX model is an urgent problem in the field of precision medicine.
  • a method of detecting drug sensitivity of a primary tumor cell against a tumor drug comprising the steps of:
  • the primary tumor cells are from an ex vivo tumor tissue sample.
  • the ex vivo tumor sample can be obtained from a tumor patient or a PDX model constructed from tumor cells utilizing a tumor patient.
  • the subject animal is a mouse, preferably a nude mouse.
  • the detecting the sensitivity of the tumor cells to the test drug is performed in vitro.
  • test drug is administered to the test subject by oral or parenteral route.
  • the primary tumor cells are isolated tumor cells that have been digested and sorted.
  • the detecting the sensitivity of the tumor cells to the test drug is performed 5-14 days, preferably 5-7 days after administration of the test drug to the test animal.
  • the implant device preferably has a molecular weight cutoff of about 500,000
  • the tubular device of Dalton's molecule preferably a modified polyvinylidene fluoride tube, preferably has an inner diameter of about 1-2 mm.
  • kits for detecting drug sensitivity of a primary tumor cell against a tumor drug comprising an implant device having a molecular weight cutoff of about 500,000 Daltons
  • the tubular device of the molecule preferably a modified polyvinylidene fluoride tube, preferably has an inner diameter of about 1-2 mm.
  • the kit is used to practice a method of detecting the drug susceptibility of a primary tumor cell to a tumor drug according to the present invention.
  • a further aspect of the invention provides the use of the kit of the invention for detecting the susceptibility of primary tumor cells to anti-tumor drugs and for rapid screening of anti-tumor drugs.
  • Figure 1 is a schematic flow diagram of the method of the present invention.
  • Fig. 2 is a graph showing growth curves of primary gastric cancer tumor cells derived from a patient after administration of the antitumor drug Tiggio (S-1) in a mouse conventional PDX model GAPF155.
  • Figure 3 is a graph showing the pharmacodynamic data of the anti-tumor drug Tiggio (S-1) against gastric cancer tumor cells in ex vivo tumor tissues from the mouse PDX model GAPF155 according to the method for detecting drug susceptibility according to the present invention.
  • Figure 4 is a graph showing the growth curve of primary gastric cancer tumor cells derived from a patient after administration of the antitumor drug Tiggio (S-1) in the mouse PDX model GAPF157.
  • Figure 5 is a graph showing the pharmacodynamic data of the anti-tumor drug Tiggio (S-1) against gastric cancer tumor cells in ex vivo tumor tissues from the mouse PDX model GAPF157 according to the method for detecting drug susceptibility according to the present invention.
  • Fig. 6 is a graph showing growth curves of primary gastric cancer tumor cells derived from a patient after administration of the antitumor drug Tiggio (S-1) in the mouse PDX model GAPF161.
  • Figure 7 is a graph showing the pharmacodynamic data of the anti-tumor drug Tiggio (S-1) against gastric cancer tumor cells in ex vivo tumor tissues from the mouse PDX model GAPF161 according to the drug sensitivity detecting method of the present invention.
  • Figure 8 is a primary tumor of lung adenocarcinoma from a patient by the method and kit of the present invention. The results of the drug sensitivity test performed by the cells.
  • Figure 9 is a graph showing the results of drug sensitivity detection of duodenal cancer primary tumor cells from a patient by the method and kit of the present invention.
  • the present invention relates to a method for detecting drug sensitivity of a primary tumor cell against a tumor drug, comprising the steps of:
  • Primary tumor cells are referred to herein as tumor cells isolated from ex vivo tumor tissue.
  • the primary tumor cells are isolated from tumor tissue samples of a tumor patient, including but not limited to clinically resected tumor tissue, tumor tissue biopsy samples, and the like.
  • the primary tumor cells are isolated from a PDX mouse model comprising tumor cells from a patient's tumor tissue.
  • the tumor cells may be derived from tumor tissues of various tumor types, including but not limited to tumors located in the digestive system (eg, stomach, intestine, duodenum, colon, pancreas, bile duct, anal canal, etc.) , breast, lung, liver, endocrine glands (eg adrenal gland, parathyroid gland, pituitary gland, testis, ovary, thymus, thyroid), urinary and reproductive systems (eg kidney, bladder, ovary, testis, prostate, etc.), musculoskeletal system (eg bone, smooth muscle, striated muscle, etc.), nervous system (eg brain), skin, head and neck, blood system, etc.
  • the tumor cells may be tumor cells derived from gastric cancer, duodenal cancer, and lung cancer.
  • the tumor cell can be any type of tumor cell of the tumor at the site.
  • the primary tumor cells of the invention are isolated tumor cells that have been digested and sorted.
  • the digestion is performed by removing non-tumor tissue and necrotic tissue, cutting the tumor sample into small pieces, rinsing with HBSS and collecting the tissue mass, and digesting the tumor mass using 1X collagenase at 37 ° C. 2 hours.
  • the sorting is performed by: digesting with a 1:1 dilution of serum medium, collecting the cell suspension over a 70 ⁇ m screen; centrifuging at 1000 rpm for 3 minutes to remove the supernatant, using 10 ml containing 1% FBS Resuspend the cells in PBS, wash, and adjust the cell density to 1 ⁇ 10 8 /ml; add CD45 cell sorting magnetic beads and fibroblast sorting magnetic beads at a concentration of 20 ⁇ l/10 7 cells, incubate for 30 min at room temperature; The cells were washed with 1% FBS in PBS and resuspended in 2 ml of 1% FBS in PBS.
  • the magnetic beads were mounted on a magnet, and the magnetic beads were rinsed with PBS containing 1% FBS; the cells were added to the washed magnetic column, and the liquid was drained, and the column was washed twice with 3 ml of PBS containing 1% FBS.
  • the collected liquid was collected; the collected liquid was centrifuged at 1000 rpm for 3 minutes to remove the supernatant, the cell culture solution was resuspended, counted, and the cell density was adjusted to 1-10 ⁇ 10 5 /ml.
  • PDX mouse models Methods of constructing PDX mouse models are known in the art. See, for example, “Melanoma patient-derived xenografts accurately model the disease and develop good enough to guide treatment decisions" (Only, melanoma patient-derived xenografts accurately mimic disease and progress quickly enough to guide treatment decisions), Oncotarget, Vol.5 , No. 20, Berglind O.
  • the drug to be tested according to the present invention may be a known antitumor drug or a combination thereof, a new antitumor substance or a combination thereof, or a new combination of known antitumor drugs.
  • the drug to be tested may be used in the form of a solid, a semi-solid or a liquid, and the drug to be tested may be administered at a desired frequency of administration as needed.
  • test drug is administered to the test animal by oral administration, parenteral administration (for example, by intravenous, intramuscular, subcutaneous injection or intravenous infusion), topical administration, inhalation administration, Transdermal administration is carried out, for example, by dermal patches, implants, suppositories, and the like. Those skilled in the art will select the desired route of administration as desired.
  • detecting the sensitivity of the tumor cells to the test drug is performed 5-14 days, preferably 5-7 days after administration of the test drug to the test animal.
  • the sensitivity of detecting tumor cells to the test drug in the present invention can be carried out by a method for drug sensitivity detection in vitro, including but not limited to ATP assay, MTT assay, Brdu labeling method, Ki67 staining method and the like.
  • the implant device is a tubular implant device, preferably a molecule that retains a molecular weight of about 500,000 Daltons.
  • the tubular implant device is a modified polyvinylidene fluoride tube that retains molecules having a molecular weight of about 500,000 Daltons, preferably the modified polyvinylidene fluoride tube has an inner diameter of about 1-2 mm.
  • the implant device can be implanted subcutaneously in the subject animal. Those skilled in the art will appreciate that suitable implantation methods known in the art can be selected as desired.
  • kits comprising the implant device of the invention.
  • the kit comprises a modified polyvinylidene fluoride tube having an inner diameter of 1-2 mm, which intercepts molecules having a molecular weight of 500,000 Daltons.
  • the kit further comprises an insert page containing instructions for use of the kit.
  • the method of the invention enables primary tumor cells, in particular isolated tumor cells that have been digested and/or sorted, to survive in an implant device implanted in an experimental animal (eg, a mouse) and to use the animal as a nutrient Providing that the primary tumor cells can grow in the in vivo environment of the test animal, and can be obtained quickly and efficiently by detecting the growth state, the apoptosis state, and the degree of differentiation of the cells in vitro after administration of the test drug. Drug sensitivity test results. It has been verified that this method has a very high correlation with the drug susceptibility results obtained by traditional PDX.
  • the invention can combine the in vivo and in vitro experimental techniques to carry out rapid and efficient anti-tumor drug evaluation, greatly shortening the time required for the pharmacodynamic experiment of the traditional PDX model, and saving the cost of the clinical experiment.
  • the invention has the advantages of rapid, convenient operation, low cost, strong repeatability and easy to be popularized, in particular, rapid and accurate detection of the sensitivity of tumor cells to anti-tumor drugs.
  • mice Female Nu/Nu mice (purchased from Beijing Vital Lihua Experimental Animal Technology Co., Ltd.) from 4-5 weeks old were ordered from the supplier and raised in the SPF animal room. The animals were kept at least for three days before the start of the experiment.
  • Freshly collected clinical tumor surgical specimens or mouse PDX samples containing patient tumor cells are taken in transport preservation tubes, transported on ice, and shipped to the laboratory in the shortest possible time.
  • the tumor samples were cut into small pieces of 1-3 cubic millimeters, washed with HBSS and collected for tissue block, centrifuged at 1000 rpm for 3 minutes to remove supernatant, using 1X collagenase (purchased from 1X collagenase) Gibco), digesting the tumor mass at 37 ° C for 1-2 hours;
  • the cells were suspended by 1:1 dilution with serum medium (purchased from Life Technologies) and the cell suspension was collected through a 70 ⁇ m sieve;
  • the supernatant was removed by centrifugation at 1000 rpm for 3 minutes, and the cells were resuspended in 10 ml of PBS containing 1% fetal bovine serum (FBS) and washed, and the cells were resuspended in PBS containing 1% FBS, counted, and the cell density was adjusted to 1 ⁇ . 10 8 /ml;
  • FBS fetal bovine serum
  • CD45 cell sorting magnetic beads purchased from Miltenyi Biotec
  • fibroblast sorting magnetic beads purchased from Miltenyi Biotec
  • the cells were washed with PBS containing 1% FBS and resuspended in 2 ml of 1% FBS in PBS.
  • the magnetic beads were mounted on the magnet, and the magnetic beads were rinsed with PBS containing 1% FBS;
  • the cells were added to the washed magnetic column, and the liquid was drained.
  • the column was washed twice with 3 ml of PBS containing 1% FBS to collect the liquid under flow;
  • the collected liquid was centrifuged at 1000 rpm for 3 minutes to remove the supernatant, and the cell culture medium PC-1 (purchased from Lonza) was resuspended, counted, and the cell density was adjusted to 1-10 ⁇ 10 5 /ml.
  • the implant device i.e., a modified polyvinylidene fluoride tube having a diameter of 1 mm
  • the implant device i.e., a modified polyvinylidene fluoride tube having a diameter of 1 mm
  • pretreatment such as soaking, rinsing, and autoclaving of ethanol. Cut each tube about 2 cm in length, rinse 3-5 times with PBS, and blow off the liquid;
  • the modified polyvinylidene fluoride tube was inoculated subcutaneously into the back of the experimental mice with a tumor puncture needle, and the administration group and the control group were set, and the wound was adhered with medical glue, and the administration treatment of the mice was performed separately;
  • mice Five to fourteen days after the administration, the mice were sacrificed, and the modified polyvinylidene fluoride tube was taken out to measure the cell viability by quantitatively measuring ATP by the CellTiter-Glo luminescence method.
  • the percentage of tumor cell viability proliferation (t/c%) in the drug-administered group was calculated, and the drug efficacy of the corresponding drug was evaluated.
  • Fig. 1 is a flow chart showing the method for rapid drug efficacy detection of the antitumor drug of the present invention.
  • the isolated tumor tissue is obtained, then subjected to digestion and sorting, and the isolated tumor cells are obtained, and the tumor cells are loaded into an implantation device, inoculated into an animal (for example, a mouse), and the drug to be tested is administered.
  • the implant device was removed 5-7 days after the mouse, and the tumor cell viability test was performed.
  • Figures 2-7 show a comparison of the traditional PDX efficacy test method with the drug efficacy test method of the present invention.
  • the mouse PDX model GAPF155 was obtained by implanting primary gastric cancer tumor cells from patients into mice.
  • Patient-derived gastric cancer cells were obtained from GAPF155 after 2 months of rearing sample.
  • the sensitivity of gastric cancer cells in GAPF155 to S-1 was examined using the conventional PDX method and the method of the present invention, respectively (Fig. 2 and Fig. 3). The results all showed that the tumor cells were sensitive to S-1.
  • the mouse PDX model GAPF157 was obtained by implanting primary gastric cancer tumor cells from patients into mice. After 2 months of feeding, patient-derived gastric cancer cell samples were obtained from GAPF157. The sensitivity of gastric cancer cells in GAPF157 to S-1 was examined using the conventional PDX method and the method of the present invention, respectively (Fig. 4 and Fig. 5). The results all showed that the tumor cells were sensitive to S-1.
  • the mouse PDX model GAPF161 was obtained by implanting primary gastric cancer tumor cells from patients into mice. After 2 months of feeding, a patient-derived gastric cancer cell sample was obtained from GAPF161. The sensitivity of gastric cancer cells in GAPF161 to S-1 was examined using the conventional PDX method and the method of the present invention, respectively (Fig. 6 and Fig. 7). The results all showed that the tumor cells were not sensitive to S-1.
  • the pharmacodynamic data obtained by conventional PDX and by the method of the present invention showed that S-1 is excellent for tumor cells in the GAPF155 model (Figs. 2 and 3) and tumor cells in the GAPF157 model (Figs. 4 and 5). Antitumor activity, while S-1 had no significant antitumor activity against tumor cells in the GAPF161 model ( Figures 6 and 7).
  • the results obtained by the conventional PDX method and by the method of the present invention are uniform.
  • the susceptibility of a patient-derived primary lung adenocarcinoma tumor cell to a tumor drug is detected by the drug susceptibility detecting method of the present invention.
  • the experimental results showed that the paclitaxel + cisplatin regimen showed strong antitumor activity against the tumor samples of the patient (Fig. 8).
  • Drug screening is performed according to the methods of the invention. The results showed that the patient responded well to gemcitabine in the treatment of pancreatic cancer (Fig. 9).
  • the doctor selected the gemcitabine + cisplatin treatment program, and the patient's condition was controlled.
  • Table 1 below shows that the inventors have screened a variety of primary tumor cells from patients by the method of the present invention:

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Abstract

一种抗肿瘤药物快速药效测试方法，包括以下步骤： (1)将原代肿瘤细胞置于植入装置中； (2)将包含原代肿瘤细胞的植入装置植入受试动物体内； (3)将待测药物施用于所述受试动物； (4)检测肿瘤细胞对待测药物的敏感性。

Description

一种药物筛选方法及其装置
本申请要求2016年10月21日提交的中国专利申请第201610918458.4号的优先权，通过援引将其全文内容并入本申请。
技术领域
本发明涉及离体药物筛选领域，尤其涉及一种抗肿瘤药物快速药敏检测方法及其装置。
背景技术
目前，个体化精准医疗被越来越多的应用在临床上，尤其是肿瘤治疗领域。化疗是目前对晚期转移性癌症患者的一种主要治疗方式，但化疗药物均缺乏肿瘤细胞特异性，在杀伤肿瘤细胞的同时，也杀伤大量骨髓及其他增殖旺盛正常细胞，有较严重的不良反应。同时传统的化疗方法在进行多次化疗后容易产生耐药性，这会影响化疗效果。由此针对患者个体化精准医疗需求越来越迫切。个体化精准医疗一方面提高治疗的精确性，另一方面也可以有效地降低患者用药的随机性和盲目性，从而减轻患者用药的伤害。近年来，一种患者来源的异种移植物模型(patient-derived xenograft model，PDX模型)广泛应用于精准医疗中。北美已有多家大型临床医疗中心通过建立PDX模型来进行临床前药物研发和药物筛选以及指导癌症患者的个性化精准用药。
PDX模型较好地保持了患者原发肿瘤的生物学特性。该模型是将患者新鲜的肿瘤组织直接移植到免疫缺陷小鼠(常用裸鼠或SCID小鼠)体内而建立的肿瘤模型，此类模型保持了与患者相似的遗传特性和肿瘤异质性。目前，运用PDX模型进行个体化精准医疗的主要难点在于模型建成周期与药敏测试花费时间长以及PDX模型的移植成瘤率较 低。例如原代细胞移植就需要2～3个月，而随后药物敏感性测试时间也需要3～4个月。另外，PDX模型的操作技术要求很高，标本从手术室获得后需要外科医生、组织学家和研究者密切配合。因此，对PDX模型的抗肿瘤药物药敏检测方法进行改进是精准医疗领域中亟需解决的问题。
发明内容
为了克服以上传统PDX模型药敏测试存在的技术问题，发明人开发了一种快速精准检测抗肿瘤药物的药物敏感性的方法。
在本发明的一个方面提供了一种检测原代肿瘤细胞对抗肿瘤药物的药物敏感性的方法，包括以下步骤：
(1)将原代肿瘤细胞置于植入装置中；
(2)将包含所述原代肿瘤细胞的植入装置植入受试动物体内；
(3)将待测药物施用于所述受试动物；
(4)检测肿瘤细胞对待测药物的敏感性。
在一个实施方案中，所述原代肿瘤细胞来自离体肿瘤组织样品。所述的离体肿瘤样品可以获得自肿瘤患者，或者获得自利用肿瘤患者的肿瘤细胞构建的PDX模型。
在一个实施方案中，所述的受试动物为小鼠，优选裸小鼠。
在一个实施方案中，所述检测肿瘤细胞对待测药物的敏感性在体外进行。
在一个实施方案中，所述待测药物通过口服或胃肠外途径施用于所述受试动物。
在一个实施方案中，所述原代肿瘤细胞是经过消化和分选的分离的肿瘤细胞。
在一个实施方案中，所述检测肿瘤细胞对待测药物的敏感性在将待测药物施用于受试动物后5-14天，优选5-7天进行。
在一个实施方案中，所述植入装置优选是截留分子量约为500,000 道尔顿的分子的管状装置，优选改良聚偏氟乙烯管，其内径优选约为1-2mm。
本发明的另一方面提供了一种用于检测原代肿瘤细胞对抗肿瘤药物的药物敏感性的试剂盒，其包含一种植入装置，所述植入装置是截留分子量约为500,000道尔顿的分子的管状装置，优选改良聚偏氟乙烯管，其内径优选约为1-2mm。在一个实施方案中，所述试剂盒用于实施本发明所述检测原代肿瘤细胞对抗肿瘤药物的药物敏感性的方法。
本发明的又一方面提供了本发明的试剂盒在检测原代肿瘤细胞对于抗肿瘤药物的药物敏感性以及快速筛选抗肿瘤药物中的用途。
附图说明
图1为本发明方法的流程示意图。
图2为在小鼠传统PDX模型GAPF155中，给予抗肿瘤药物替吉奥(S-1)后患者来源的原代胃癌肿瘤细胞的生长曲线图。
图3为根据本发明的药敏检测方法，抗肿瘤药物替吉奥(S-1)对于来自小鼠PDX模型GAPF155的离体肿瘤组织中的胃癌肿瘤细胞的药效数据图。
图4为小鼠PDX模型GAPF157中，给予抗肿瘤药物替吉奥(S-1)后患者来源的原代胃癌肿瘤细胞的生长曲线图。
图5为根据本发明的药敏检测方法，抗肿瘤药物替吉奥(S-1)对于来自小鼠PDX模型GAPF157的离体肿瘤组织中的胃癌肿瘤细胞的药效数据图。
图6为小鼠PDX模型GAPF161中，给予抗肿瘤药物替吉奥(S-1)后患者来源的原代胃癌肿瘤细胞的生长曲线图。
图7为根据本发明的药敏检测方法，抗肿瘤药物替吉奥(S-1)对于来自小鼠PDX模型GAPF161的离体肿瘤组织中的胃癌肿瘤细胞的药效数据图。
图8为通过本发明的方法和试剂盒对来自患者的肺腺癌原代肿瘤 细胞进行药物敏感性检测的结果。
图9为通过本发明的方法和试剂盒对来自患者的十二指肠癌原代肿瘤细胞进行药物敏感性检测的结果。
发明详述
本发明涉及检测原代肿瘤细胞对抗肿瘤药物的药物敏感性的方法，其包括以下步骤：
(1)将原代肿瘤细胞置于植入装置中；
(2)将包含所述原代肿瘤细胞的植入装置植入受试动物体内；
(3)将待测药物施用于所述受试动物；
(4)检测肿瘤细胞对待测药物的敏感性。
原代肿瘤细胞在本文中指分离自离体肿瘤组织的肿瘤细胞。根据一个实施方案，所述的原代肿瘤细胞分离自肿瘤患者的肿瘤组织样品，包括但不限于临床切除的肿瘤组织，肿瘤组织活检样品等。根据另一个实施方案，所述的原代肿瘤细胞分离自PDX小鼠模型，所述PDX小鼠模型包含来自患者肿瘤组织的肿瘤细胞。
所述的肿瘤细胞可以来源于多种肿瘤类型的肿瘤组织，包括但不限于位于以下部位的肿瘤：消化道系统(例如胃，肠，十二指肠，结肠，胰腺，胆管，肛管等)、乳腺，肺，肝，内分泌腺(例如肾上腺、甲状旁腺、脑垂体、睾丸、卵巢、胸腺、甲状腺)，泌尿和生殖系统(例如肾，膀胱，卵巢，睾丸，前列腺等)，骨骼肌肉系统(例如骨，平滑肌，横纹肌等)，神经系统(例如脑)，皮肤，头颈部，血液系统等。例如所述的肿瘤细胞可为来源于胃癌，十二指肠癌，肺癌的肿瘤细胞。所述的肿瘤细胞可以是该部位肿瘤的任何类型的肿瘤细胞。
在一个实施方案中，本发明的原代肿瘤细胞是经过消化和分选的分离的肿瘤细胞。在一个实施方案中，所述的消化通过如下方法进行：除去非肿瘤组织和坏死组织，将肿瘤样品切成小块，用HBSS冲洗并收集组织块，使用1X胶原酶37℃消化肿瘤块1-2小时。在一个实施方 案中，所述的分选通过如下方法进行：用血清培养基1:1稀释终止消化，过70μm筛网收集细胞悬液；1000rpm离心3分钟去除上清，使用10ml含1％FBS的PBS重悬细胞，清洗，并调整细胞密度至1×108/ml；以20μl/107细胞的浓度加入CD45细胞分选磁珠和成纤维细胞分选磁珠，室温孵育30min；加入含1％FBS的PBS清洗细胞，并用2ml 1％FBS的PBS重悬。将磁珠装在磁铁上，用含1％FBS的PBS对磁珠进行润洗；将细胞加入润洗过的磁柱中，待液体流尽，用3ml含1％FBS的PBS清洗两次柱子，收集流下的液体；收集的液体1000rpm离心3分钟去除上清，细胞培养液重悬，计数，调整细胞密度至1-10×105/ml。
构建PDX小鼠模型的方法是本领域已知的。例如参见“Melanoma patient-derived xenografts accurately model the disease and develop fast enough to guide treatment decisions(黑素瘤患者来源的异种移植物精确模拟疾病并足够快地进展从而指导治疗决定)”，Oncotarget,Vol.5,No.20,Berglind O.Einarsdottir等人，2014年9月8日公开以及“Persona lizing Cancer Treatment in the Age of Global Genomic Analyses:PALB2 Gene Mutations and the Response to DNA Damaging Agents in Pancreatic Cancer(全球基因组分析时代的个性化癌症治疗：胰腺癌中的PALB2基因突变以及对DNA破坏剂的反应)”，Molecular Cancer Therapies,首次公开日2010年12月6日；DOI:10.1158/1535-7163.MCT-10-0893,Maria C.Villarroel等人等文献。
本发明所述的待测药物可以是已知的抗肿瘤药物或其组合，新的抗肿瘤物或其组合，或者已知抗肿瘤药物的新组合。在本发明的方法中，所述的待测药物可以以固体、半固体或液体的形式使用，并可根据需要以合意的施用频率施用所述待测药物。
本发明方法中将待测药物给药受试动物可通过口服给药、胃肠外给药(如通过静脉内、肌肉内、皮下注射或静脉内输注)、局部给药、吸入给药，透皮给药例如通过皮肤贴片、植入体、栓剂等进行。本领域技术人员将根据需要选择合意的给药途径。
本发明的一个实施方案中，检测肿瘤细胞对待测药物的敏感性在将待测药物施用于受试动物后5-14天，优选5-7天进行。本发明中检测肿瘤细胞对待测药物的敏感性可通过在体外进行药敏检测的方法进行，包括但不限于ATP检测法，MTT法，Brdu标记法，Ki67染色法等。
本发明另一方面涉及一种用于本发明的方法中的植入装置。在一个实施方案中，所述植入装置是一种管状植入装置，优选其截留分子量约500,000道尔顿的分子。在一个优选实施方案中，所述管状植入装置是改良聚偏氟乙烯管，其截留分子量约500,000道尔顿的分子，优选地所述改良聚偏氟乙烯管的内径约1-2mm。本发明的一个优选实施方案中，所述植入装置可植入受试动物的皮下。本领域技术人员将理解，可根据需要选择本领域已知的适宜植入方式。
本发明另一方面还提供了一种试剂盒，其包含本发明所述的植入装置。在一个实施方案中，所述试剂盒包含内径为1-2mm的改良聚偏氟乙烯管，其截留分子量为500,000道尔顿的分子。在一个优选实施方案中，所述试剂盒还包含插入页，所述插入页包含使用该试剂盒的使用方法说明。
本发明的方法能够使得原代肿瘤细胞，尤其是经过消化和/或分选的分离的肿瘤细胞，在植入实验动物(例如小鼠)体内的植入装置中持续存活，并借助动物作为营养提供者，使得原代肿瘤细胞能够在受试动物的体内环境中生长，并可在施用待测药物后根据需要通过在体外检测细胞的生长状态、凋亡状态和分化程度等，快速高效地获得药敏检测结果。经过验证，该方法与传统PDX所得到的药敏结果有极高的相关性。本发明可以结合体内和体外实验技术进行快速高效的抗肿瘤药物评价，极大地缩短了传统PDX模型的药效实验所需的时间，节约了临床实验的成本。总之，本发明具有快速，操作便捷，费用低，可重复性强，易于推广的优点，尤其是实现了肿瘤细胞对于抗肿瘤药物的敏感性的快速且精准的检测。
具体实施方式
为了更好地说明本发明，下面参照附图予以说明。
实验动物
从供应商订购4-5周龄的雌性Nu/Nu小鼠(购自北京维通利华实验动物技术有限公司)，饲养于SPF级别动物房，实验开始前动物至少适应性饲养三天。
样本的采集
取新鲜收集的临床肿瘤手术样本或包含患者肿瘤细胞的小鼠PDX样本于运输保存管中，冰上运输，在尽可能短的时间内运至实验室。
细胞消化与分选
在生物安全柜中去除非肿瘤组织和坏死组织后，将肿瘤样品切成1-3立方毫米小块，用HBSS冲洗并收集组织块，1000rpm离心3分钟去上清，使用1X胶原酶(购自Gibco)、37℃消化肿瘤块1-2小时；
用血清培养基(购自Life Technologies)1:1稀释终止消化并过70μm筛网收集细胞悬液；
1000rpm离心3分钟去除上清，使用10ml含1％胎牛血清(FBS)的PBS重悬细胞并进行清洗后，将细胞重悬于含1％FBS的PBS，计数，并调整细胞密度至1×108/ml；
以20μl/107细胞的浓度加入商购CD45细胞分选磁珠(购自Miltenyi Biotec)和成纤维细胞分选磁珠(购自Miltenyi Biotec)，室温孵育30min；
加入含1％FBS的PBS清洗细胞，并用2ml 1％FBS的PBS重悬。同时将磁珠装在磁铁上，用含1％FBS的PBS对磁珠进行润洗；
将细胞加入润洗过的磁柱中，待液体流尽，用3ml含1％FBS的PBS清洗两次柱子，收集流下的液体；
收集的液体1000rpm离心3分钟去除上清，细胞培养液PC-1(购自Lonza)重悬，计数，调整细胞密度至1-10×105/ml。
细胞装管、接种及活力检测
在生物安全柜中取出植入装置，即直径1mm的改良聚偏氟乙烯管，所述改良聚偏氟乙烯管经过乙醇的浸泡、冲洗和高压灭菌等预处理。截取每根管子约2cm长度大小，用PBS反复冲洗3-5遍，吹掉液体；
将肿瘤细胞悬液加入改良聚偏氟乙烯管中，密封；
用肿瘤穿刺针将改良聚偏氟乙烯管接种至实验小鼠背部皮下，并设置给药组和对照组，伤口用医用胶粘合，分别进行小鼠的给药处理；
给药后5-14天，处死小鼠，取出改良聚偏氟乙烯管通过CellTiter-Glo发光法通过对ATP进行定量测定来检测细胞活力。
数据分析
根据对照组肿瘤细胞的活力值，计算出给药组肿瘤细胞活力增殖百分比(t/c％)，进而评价相应药物的药效。
图1显示了本发明的抗肿瘤药物快速药效检测方法的流程图。首先，获得离体肿瘤组织，然后进行消化和分选，获得分离的肿瘤细胞，并将所述肿瘤细胞装至植入装置中，接种到动物(例如小鼠)皮下，将待测药物施用于小鼠后5-7天后取出植入装置，进行肿瘤细胞活力测试。
图2-7显示了传统的PDX药效检测法与本发明的药效检测法的比较。
实施例
实施例1
通过将来自患者的原代胃癌肿瘤细胞植入小鼠体内获得小鼠PDX模型GAPF155。饲养2个月后，从GAPF155获得患者来源的胃癌细胞 样品。分别利用传统PDX方法和本发明的方法，检测GAPF155中的胃癌细胞对于S-1的敏感性(图2和图3)。结果均显示所述肿瘤细胞对S-1敏感。
实施例2
通过将来自患者的原代胃癌肿瘤细胞植入小鼠体内获得小鼠PDX模型GAPF157。饲养2个月后，从GAPF157获得患者来源的胃癌细胞样品。分别利用传统PDX方法和本发明的方法，检测GAPF157中的胃癌细胞对于S-1的敏感性(图4和图5)。结果均显示所述肿瘤细胞对S-1敏感。
实施例3
通过将来自患者的原代胃癌肿瘤细胞植入小鼠体内获得小鼠PDX模型GAPF161。饲养2个月后，从GAPF161获得患者来源的胃癌细胞样品。分别利用传统PDX方法和本发明的方法，检测GAPF161中的胃癌细胞对于S-1的敏感性(图6和图7)。结果均显示所述肿瘤细胞对S-1不敏感。
通过传统PDX和通过本发明的方法获得的药效数据显示，S-1对于GAPF155模型中的肿瘤细胞(图2和3)和GAPF157模型中的肿瘤细胞(图4和5)均有很好的抗肿瘤活性，而S-1对于GAPF161模型中的肿瘤细胞(图6和7)无明显抗肿瘤活性。通过传统PDX方法和通过本发明的方法获得的结果是一致的。
实施例4
选择男性肺腺癌患者(60岁)。医生根据肺腺癌临床经验选择用药方案为培美曲赛(Pemetrexe)+顺铂。3个疗程9周后，患者的病情出现复发转移。经患者知情同意后，从该肺腺癌患者采集胸水获得原代肿瘤细胞。
通过本发明的药敏检测方法，检测患者来源的原代肺腺癌肿瘤细胞对抗肿瘤药物的敏感性。实验结果表明，紫杉醇+顺铂方案对该患者的肿瘤样本表现出较强的抗肿瘤活性(图8)。
调整方案后的治疗结果：
医生更换了用药方案，选择了紫杉醇+顺铂方案，用药7周，患者胸水消失，现病情稳定已出院。
实施例5
选择男性十二指肠壶腹部肿瘤患者(56岁)。经患者知情同意后，从该患者获得原代肿瘤组织。
根据本发明的方法进行药物筛选。结果显示：患者对治疗胰腺癌的吉西他滨反应良好(图9)。
医生跟据上述结果选定吉西他滨+顺铂治疗方案，患者病情得到控制。
下表1显示，本发明人通过本发明的方法对来自患者的多种原代肿瘤细胞进行了药物筛选:
表1
Figure PCTCN2017107386-appb-000001
Figure PCTCN2017107386-appb-000002
上述实施例仅仅是通过举例的方式描述。在不偏离本发明所附权利要求限定的保护范围的情况下，可有各种变体。

Claims (13)

  1. 一种检测原代肿瘤细胞对抗肿瘤药物的药物敏感性的方法，包括以下步骤：
    (1)将原代肿瘤细胞置于植入装置中；
    (2)将包含所述原代肿瘤细胞的植入装置植入受试动物体内；
    (3)将待测药物施用于所述受试动物；
    (4)检测肿瘤细胞对待测药物的敏感性。
  2. 权利要求1所述的方法，其中所述原代肿瘤细胞来自离体肿瘤组织样品。
  3. 权利要求2的方法，其中所述的离体肿瘤组织样品获得自肿瘤患者，或者获得自利用肿瘤患者的肿瘤细胞构建的PDX模型。
  4. 权利要求1至3中任一项的方法，其中所述的受试动物为小鼠，优选裸小鼠。
  5. 权利要求1至4中任一项的方法，其中检测肿瘤细胞对待测药物的敏感性在体外进行。
  6. 权利要求1至5中任一项的方法，其中所述待测药物通过口服或胃肠外途径施用于所述受试动物。
  7. 权利要求1至6中任一项的方法，其中所述原代肿瘤细胞是经过消化和/或分选的分离的肿瘤细胞。
  8. 权利要求1至7中任一项的方法，其中检测肿瘤细胞对待测药 物的敏感性在将待测药物施用于受试动物后5-14天，优选5-7天进行。
  9. 权利要求1至8中任一项的方法，其中所述植入装置是可截留分子量约500,000道尔顿的分子的管状装置，优选改良聚偏氟乙烯管，进一步优选内径约1-2mm的改良聚偏氟乙烯管。
  10. 权利要求1至9中任一项的方法，其中将所述植入装置植入受试动物的皮下。
  11. 一种用于检测原代肿瘤细胞对抗肿瘤药物的药物敏感性的试剂盒，其包含一种植入装置，所述植入装置是可截留分子量约500,000道尔顿的分子的管状装置，优选改良聚偏氟乙烯管，进一步优选内径约1-2mm的改良聚偏氟乙烯管。
  12. 权利要求11的试剂盒，其用于实施权利要求1-9的方法。
  13. 权利要求12的试剂盒在检测原代肿瘤细胞对于抗肿瘤药物的药物敏感性以及快速筛选抗肿瘤药物中的用途。
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