WO2023045494A1 - 一种药物的筛选方法以及三维肿瘤切片模型的培养方法 - Google Patents

一种药物的筛选方法以及三维肿瘤切片模型的培养方法 Download PDF

Info

Publication number
WO2023045494A1
WO2023045494A1 PCT/CN2022/104365 CN2022104365W WO2023045494A1 WO 2023045494 A1 WO2023045494 A1 WO 2023045494A1 CN 2022104365 W CN2022104365 W CN 2022104365W WO 2023045494 A1 WO2023045494 A1 WO 2023045494A1
Authority
WO
WIPO (PCT)
Prior art keywords
dimensional
tumor
drug
culture
slice model
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2022/104365
Other languages
English (en)
French (fr)
Inventor
邓初夏
刘子铭
邢富强
王冠宇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Southern University of Science and Technology
University of Macau
Original Assignee
Southern University of Science and Technology
University of Macau
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Southern University of Science and Technology, University of Macau filed Critical Southern University of Science and Technology
Publication of WO2023045494A1 publication Critical patent/WO2023045494A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • 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
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present disclosure relates to the technical field of biomedicine, in particular, to a method for screening drugs and a method for culturing three-dimensional tumor slice models.
  • Precision medicine is an approach to tailor interventions that take into account the influence of an individual's genetics, environment, and lifestyle exposures.
  • drug discovery models used in personalized medicine for cancer treatment have many defects. Human cell lines cultured in 2D can easily lose their original functions, and their morphology, biological function, genetics, and other aspects are very different from human physiology. And dye labeling ignores cellular dynamics and metabolic changes in the drug-treated microenvironment, making it difficult to quantitatively assess drug sensitivity and efficiency in tumor fragments. Therefore, many biological tests to assess the safety and efficacy of drug candidates must be performed in animals. Such tests on whole animals are costly, leading to higher healthcare costs.
  • the existing technology cannot more intuitively obtain the information of the culture model from the molecular level, and the cells need to be terminated before the detailed identification and analysis of the cells can be carried out, and it is impossible to evaluate and screen drugs in a real-time, intuitive and effective manner.
  • the present disclosure provides a drug screening method, which includes the following steps: performing three-dimensional culture on a three-dimensional tumor slice model; wherein, the preparation method of the three-dimensional tumor slice model includes: slicing tumor tissue labeled with an apoptosis reporter substance processing to obtain the three-dimensional tumor slice model;
  • the three-dimensional tumor slice model in the three-dimensional culture is processed with the candidate drug, and the candidate drug is screened based on the signal of the apoptotic cell reporter substance.
  • the thickness of the three-dimensional tumor slice is 200-300 ⁇ m.
  • the sectioning method is as follows: the tumor tissue is wrapped with gel and sectioned.
  • the conditions of the three-dimensional culture are as follows: three-dimensional tumor slices are cultured using an insert cell culture dish, and the culture medium used contains fetal bovine serum with a volume fraction of 15% to 20% and 40 to 60 ⁇ g /ml gentamicin.
  • the apoptosis reporter substance includes: a sensor for detecting apoptosis.
  • the senor comprises a caspase-3 sensor.
  • the apoptosis reporter substance includes: SCAT3 or zipGFP.
  • the method for obtaining the tumor tissue labeled with the apoptosis reporter substance is as follows: the cells labeled with the apoptosis reporter substance or their culture are introduced into the subcutaneous tissue of an animal or an animal model to form the tumor tissue.
  • the screening method before using the three-dimensional tumor slice model to screen candidate drugs, the screening method further includes: using a tumor cell model labeled with an apoptosis reporter substance to pre-screen the drug to obtain the candidate drug .
  • the screening method further includes: performing three-dimensional culture on clinical tumor slice samples, and adding candidate drugs or drugs screened by the three-dimensional tumor slice model to the three-dimensional culture culture.
  • the clinical tumor slice samples are processed, and drugs are screened based on the signals of endogenous fluorescent substances in the clinical tumor slice samples.
  • the endogenous fluorescent substance includes: at least one of lipofuscin, flavin, aromatic amino acid and NADH.
  • the fluorescence detection conditions of the endogenous fluorescent substance are as follows: the excitation wavelength of single-photon is 488-570nm, and the excitation wavelength of two-photon is 970-1140nm , The emission wavelength is 550-700nm.
  • the drug candidates include anticancer drugs.
  • the anticancer drug includes any one of an immune checkpoint inhibitor and an antitumor drug
  • the tumor comprises any one of breast cancer, colon cancer, ovarian cancer, uterine cancer, liver cancer, gastric cancer, lung cancer, and nasopharyngeal cancer.
  • the present disclosure also provides a method for culturing a three-dimensional tumor slice model, which includes: performing three-dimensional culture on the three-dimensional tumor slice model; wherein, the method for preparing the three-dimensional tumor slice model includes: adding a tumor labeled with an apoptosis reporter substance The tissue is sliced to obtain the three-dimensional tumor slice model.
  • the thickness of the three-dimensional tumor slice model is 200-300 ⁇ m.
  • the sectioning method is as follows: the tumor tissue is wrapped with gel and sectioned.
  • the conditions of the three-dimensional culture are as follows: the three-dimensional tumor slice model is three-dimensionally cultured using an inserted cell culture dish, and the culture medium used contains fetal bovine serum with a volume fraction of 15% to 20% and 40% to 60 ⁇ g/ml gentamicin.
  • the present disclosure also provides a method for screening drugs, which is characterized in that it includes the following steps: performing three-dimensional culture on clinical tumor slice samples, processing candidate drugs on the clinical tumor slice samples in the three-dimensional culture, and based on the clinical tumor slice The signals of endogenous fluorescent substances in the samples are further screened for drugs.
  • the present disclosure also provides a method for real-time prediction of drug response, which is characterized in that it includes: performing three-dimensional culture on a three-dimensional tumor slice model; wherein, the preparation method of the three-dimensional tumor slice model includes: adding a cell apoptosis reporter substance labeled with slice the tumor tissue to obtain the three-dimensional tumor slice model;
  • the three-dimensional tumor slice model in the three-dimensional culture is processed by the candidate drug, and the response of the candidate drug is predicted in real time based on the signal of the cell apoptosis reporter substance.
  • the present disclosure also provides a drug screening system, the screening system comprising:
  • the three-dimensional tumor slice model is obtained by slicing tumor tissue labeled with a cell apoptosis reporter substance
  • the screening system uses the candidate drug to process the three-dimensionally cultured three-dimensional tumor slice model, and screens the candidate drug based on the signal of the apoptotic cell reporter substance.
  • the thickness of the three-dimensional tumor slice model is 200-300 ⁇ m.
  • the sectioning method is as follows: the tumor tissue is wrapped with gel and sectioned.
  • the conditions of the three-dimensional culture are as follows: the three-dimensional tumor slice model is three-dimensionally cultured using an inserted cell culture dish, and the culture medium used contains fetal bovine serum with a volume fraction of 15% to 20% and 40% to 60 ⁇ g/ml gentamicin.
  • the apoptosis reporter substance includes: a sensor for detecting apoptosis.
  • the senor comprises a caspase-3 sensor.
  • Figure 1 is a schematic diagram of the preparation of a three-dimensional tumor slice model and the drug screening process
  • Figure 2 is a schematic diagram of the process of applying the three-dimensional tumor slice model and the clinical sample slice model to drug screening;
  • Figure 3 is a three-dimensional tumor slice model that completely retains the structure and tumor markers of the original tumor sample; among them, (A) morphological observation of tumor growth in the three-dimensional tumor slice model of the tumor formed by nude mouse mammary gland B477-GFP cells; (B) Detecting the viability of tumor cells formed by nude mouse mammary gland B477-GFP cells in the three-dimensional tumor slice model; (C, D) Histopathological detection of biomarkers during the culture process of the genetically engineered mouse tumor three-dimensional tumor slice model, blue is Hematoxylin nuclear counterstaining, brown is DAB positive protein, ⁇ H2AX is a DNA double-strand break marker; Desmin is a regulator of sarcomere structure; E-cadherin is a calcium-dependent cell-cell adhesion molecule; Vemintin maintains cell integrity and The position of the anchor organelle in the cytoplasm; CK7 is a commonly used marker in tumors of epithelial origin; (E) Quant
  • Figure 4 shows that the three-dimensional tumor sheet culture model can maintain the cell bank and immune components of its original tumor; among them, (A-D) histological analysis of the immune biomarker changes in the genetically engineered mouse tumor with immune competence in the three-dimensional tumor sheet model; Blue is hematoxylin stained nucleus, brown is DAB positive protein; CD3e/CD8a is T lymphocyte marker; CD11b is macrophage and microglia marker; CD45 is T, NK, dendritic and lymphokine Activated killer (LAK) cell marker; F4/80 is a mouse macrophage marker; PD-1 is an immune receptor expressed by T cells; PD-L1 is myeloid, lymphoid, normal epithelial cells or PD-1 ligands expressed by cancer cells; (E) RNA-seq analysis of expression levels of immune-related genes; ssGSEA represents up- or down-regulated levels of gene datasets within a sample; (F) T-cell and B-cell immune marker gene expression RNA-
  • Figure 5 is the real-time prediction of the sensitivity of three-dimensional tumor slices to drugs based on FRET technology; wherein, (A) observe the dynamic changes of 10 ⁇ M doxorubicin-induced apoptosis of MDA-MB-231-C3 cells within 48 hours; CFP (ex: 430nm/em: 480nm) and YFP (ex: 430nm/em: 520); (B) Quantitative analysis of FRET changes in single cells; (C) Quantitative analysis of the number of adriamycin-induced apoptosis in MDA-MB-231-C3 cells and The total number of cells; where, the green line and the blue line represent the total number of cells and the number of apoptotic cells, respectively; (D) Apoptosis induced by cisplatin/doxorubicin in the three-dimensional tumor slice model; MDA-MB-231-C3 tumor slices were treated for six days; (E) quantitative analysis panel (D) FRET ratio
  • Figure 6 is a quantitative evaluation of the efficacy of three-dimensional tumor slices on drugs by MTT method; among them, (A) observe the changes of 100 ⁇ M cisplatin-treated or untreated B477-GFP breast tumor slices within 0 to 7 days by observing GFP fluorescence and MTT staining ; Scale bar is 1mm; (B) MTT method to evaluate drug efficacy in panel (A); (C) FRET fluorescence (d0-6) and MTT staining (d6) observation of MDA-MB-231 treated with 1 ⁇ M bortezomib for six days - C3 tumor slice; (D) FRET ratio (d0-6) and MTT assay (d6) to assess drug sensitivity in panel (C); scale bar is 2 mm; (E, F) by PI, FRET ratio and MTT assay Determination of the drug sensitivity of MDA-MB-231-C3 tumor slices treated with different doses of cisplatin for 6 days; the scale bar is 1 mm; (G
  • Figure 7 shows the real-time prediction of the sensitivity of various cancer samples from mice to drugs based on FRET technology and the MTT endpoint method to predict the curative effect of surgically resected tumors on three-dimensional tumor sheet culture; among them, (A) screening based on FRET technology 3D tumor slice anticancer drugs.
  • B Representative FRET images of three-dimensional tumor slices treated with drugs; scale bar is 1 mm;
  • C MTT staining of human treated with drugs for 4 days Three-dimensional tumor slices of colon and breast; the scale bar is 1 mm;
  • D-F The drug response of three-dimensional tumor slices of human colon treated with drugs for 4 days was detected by MTT method; among them, D (colon case 1), E (colon case 2), F ( Colon case 3); ceritinib (2.5 ⁇ M), daunorubicin hydrochloride (2.5 ⁇ M), osimertinib (5 ⁇ M), and the concentration of other drugs used in patient colon samples were all 20 ⁇ M;
  • G,H MTT method to detect the drug response of three-dimensional tumor slices of breast cancer patients treated with drugs for 4 days;
  • G breast case 1
  • H breast case 2
  • Mitoxantrone 5
  • Figure 8 shows the drug response of three-dimensional tumor slices to PD-1/PD-L1 immune checkpoint inhibitors;
  • A PD-1 antibody induces cell death in three-dimensional tumor slices of mice, and mice Smad4 co/co ; Pten co/ co ; Alb-Cre (SPC) tumor slices (658-607) were treated with control IgG or ⁇ PD-1 antibody for 6 days, and the cell viability of dimension tumor slices was analyzed by MTT method;
  • B PD-L1 antibody induced three-dimensional tumor slices of mice Cell death; MTT method to analyze the viability of mouse SPC tumor sections (658-607) treated with IgG or ⁇ PD-L1 for 6 days; compare with the level of PD-L1IHC% (n/a: no data for PD-L1IHC) ;
  • C Histochemical analysis of PD-L1 levels in fresh tumor samples in (B);
  • D PD-L1 IHC staining of surgically resected tumors from patients;
  • E PD-L1 antibody
  • FIG. 9 shows that lipofuscin (lipofuscin), as an unlabeled autofluorescent molecule, appears red fluorescence enhancement in tumor samples undergoing chemotherapy and immunotherapy; among them, (A) the fluorescence emission spectrum of lipofuscin; (B) two-photon image observation at 25 ⁇ M Cisplatin, 2.5 ⁇ g/mL ⁇ PD-1 or 2.5 ⁇ g ⁇ PD-L1 antibody treatment lipofuscin fluorescence enhancement in three-dimensional tumor slices of mice with GFP, GFP (green, such as @960nm) and lipofuscin (red, such as @1040nm );
  • Figure 10 shows the method of PI staining and MTT staining to confirm the occurrence of cell death in the mouse mammary three-dimensional tumor slice sample after chemotherapy and immunotherapy; among them, (A) the left column shows the observation of NADH (blue, ex.@740nm) by two-photon fluorescence imaging , propidium iodide PI (red, ex.@1040nm) and the second harmonic generation imaging of collagen (green, ex.@1040nm); the right column is the second harmonic imaging of collagen (blue, ex.@ 890nm), two-photon fluorescence imaging of flavins (green, ex.@890nm) and propidium iodide PI (red, ex.@1040nm); this set of images was evaluated by propidium iodide (PI) staining with 25 ⁇ M Cancer cell death in three-dimensional breast tumor slices of genetically engineered mice treated with cisplatin, 2.5 ⁇ g/mL ⁇ PD-1 or 2.5 ⁇ g
  • Figure 11 shows the real-time monitoring of the response of three-dimensional tumor slices to chemotherapy and immune checkpoint inhibitor treatment based on label-free fluorescence imaging technology; where (A) 25 ⁇ M cisplatin, 2.5 ⁇ g/mL ⁇ PD-1 or 2.5 ⁇ g/mL ⁇ PD-L1 treatment4 Two-photon autofluorescence images of collagen collagen, lipofuscin and flavin in tumor slices of genetically engineered mice on day 1; the scale bar is 24 ⁇ m; (B) three-dimensional tumor slices of genetically engineered mice treated with drugs for 0-5 days Two-photon autofluorescence enhanced imaging of lipofuscin; the scale bar is 24 ⁇ m; (C) quantitative analysis of lipofuscin autofluorescence intensity in three-dimensional tumor slices of genetically engineered mice treated with drugs; (D, E) 10 ⁇ g/mL ⁇ PD-1 or 10 ⁇ g Two-photon autofluorescence imaging and lipofuscin intensity assessment of lipofuscin in 3D tumor slices of human na
  • cell viability refers to the percentage of viable cells among total cells.
  • apoptosis refers to the autonomous and orderly death of cells controlled by genes in order to maintain a stable internal environment, that is, the process of actively ending life automatically determined by genes, so it is often called programmed cell death.
  • high-throughput screening refers to experimental methods based on molecular and cellular levels, using microplates (such as microwell plates of different densities) as experimental tool carriers, and performing experiments with automated operating systems
  • microplates such as microwell plates of different densities
  • the process uses sensitive and fast detection devices to detect tens of millions of samples at the same time, collect experimental data and digital analysis and processing, and use the corresponding database to support the operation of the technical system.
  • One embodiment of the present disclosure provides a drug screening method, which includes the following steps: performing three-dimensional culture on the three-dimensional tumor slice model; wherein, the preparation method of the three-dimensional tumor slice model includes: the The tumor tissue is sliced to obtain the three-dimensional tumor slice model; then, the three-dimensional tumor slice model in three-dimensional culture is processed with the candidate drug, and the candidate drug is screened based on the signal of the apoptosis reporter substance, as shown in FIG. 1 .
  • processing the three-dimensional tumor slice model in three-dimensional culture with a candidate drug may refer to: contacting the drug with the three-dimensional tumor slice model, for example, by adding the drug to the three-dimensional culture medium.
  • the three-dimensional tumor slice model established in the present disclosure can accurately preserve the cell bank and immune components of the original tumor.
  • the screening approach described above allows time-course and quantitative assessment of cell death, enabling testing of individualized drug responses and immune checkpoint blockade therapies against surgically resected cancers.
  • stromal cells, immune cells, and cancer cells are contained in the three-dimensional tumor slice model; 2) all resected tumor samples can be used for drug evaluation; 3) It is a rapid and low-cost in vitro system.
  • the screening or evaluation process can be completed within 4-7 days, while PDO takes more than two weeks.
  • the thickness of the three-dimensional tumor slice is 200-300 ⁇ m, such as 210-290 ⁇ m, 230-280 ⁇ m or 240-260 ⁇ m, such as 200 ⁇ m, 220 ⁇ m, 240 ⁇ m, 260 ⁇ m, 280 ⁇ m or 300 ⁇ m.
  • the thickness of the three-dimensional tumor slice within the scope of the present disclosure can improve the efficiency and quality of the slice, and keep the microenvironment of the original tumor intact. At the same time, the effectiveness of detecting endogenous fluorescent signals is guaranteed, which is conducive to maintaining the validity and stability of the detection results.
  • the thickness of three-dimensional tumor slices below this range may lead to the efficiency and quality of slices, and damage the microenvironment of the original tumor. If the thickness is higher than this range, the detection of endogenous fluorescent signals may be reduced, which is not conducive to maintaining the validity and stability of detection results. sex.
  • the sectioning method is as follows: the tumor tissue is wrapped with agarose gel and sectioned, and the steps are as follows: the tumor is collected and placed in a pre-cooled refrigerator. DMEM culture medium, containing 10% FBS and 1% penicillin/streptomycin (Gibco); use low-melting point agarose gel to wrap the tumor tissue and slice it; tumor slices (200-300 ⁇ m) are 2-6 hours after operation A Leica VT1200S (Leica Biosystems Nussloch GmbH, Germany) was used for vibrating sections under sterile PBS conditions.
  • the three-dimensional culture conditions can refer to existing published methods and materials, as long as the three-dimensional tumor slice model can grow in a three-dimensional manner.
  • the conditions of the three-dimensional culture are as follows: three-dimensional tumor slices are cultured using an insert cell culture dish, and the culture medium used contains fetal bovine serum with a volume fraction of 15% to 20% and 40 to 60 ⁇ g/ml Gentamicin, the volume fraction of fetal bovine serum is for example 16% ⁇ 19%, 17% ⁇ 18% or 17.5% ⁇ 18%, such as can be 15%, 16%, 17%, 18%, 19% or 20% %.
  • the final concentration of gentamicin is, for example, 43-58 ⁇ g/ml, 45-55 ⁇ g/ml or 47-52 ⁇ g/ml, such as 40 ⁇ g/ml, 45 ⁇ g/ml, 50 ⁇ g/ml, 55 ⁇ g/ml or 60 ⁇ g/ml.
  • the conditions of the three-dimensional culture are as follows: Precool A (rat tail collagen I), B (10X Ham's F-12) and C (sterile buffer solution, 2.2g NaHCO 3 in 100ml 0.05N NaOH and 200mM HEPES) according to the ratio of 8:1:1 preparation. Take the prepared 100 ⁇ L gel solution and spread it on the membrane (membrane pore size 0.4mm) in the Millicell insert cell culture inner dish (PIHP01250) with a diameter of 12mm, and put it in a 37°C incubator for 20-30 minutes until the gel Freeze completely. Then, the tumor slice was carefully placed on the surface of the gel. Add another 100 ⁇ L of gel solution to cover the tumor piece, and place it in a 37° C.
  • the culture medium was Ham's F12 containing 20% fetal calf serum and 50 ⁇ g/ml gentamicin. Finally, the three-dimensional tumor slices were placed in an incubator with 5% carbon dioxide and 37°C and the culture medium was changed every 4 days.
  • the culture conditions of the disclosed three-dimensional tumor sheets preserve important features of the original tumor, such as cancer heterogeneity and structure, immune components, and stable gene expression.
  • the present disclosure does not limit the type of the apoptosis reporter substance, as long as it satisfies the ability to detect apoptosis.
  • it may include: a sensor for detecting apoptosis.
  • the apoptosis reporter substance includes a sensor for detecting apoptosis, optionally a caspase-3 sensor Sensor C3.
  • a caspase-3 sensor (Sensor C3).
  • sensor C3 Traditionally, assessment of drug response has relied on destructive fixation and toxic dye labeling, which are destructive and toxic to cells, respectively, with valuable cytological and metabolic information being discarded.
  • the present disclosure established a FRET-based sensor Sensor C3 as a three-dimensional tumor slice model of apoptosis reporter to screen multiple anticancer drugs. This is achieved by inhibiting cell growth and inducing apoptosis. This approach should be applicable to large-scale drug screens to narrow down potential drug candidates for further analysis.
  • the apoptosis reporter substances may also include but not limited to SCAT3 and zipGFP.
  • the method for obtaining the tumor tissue labeled with the apoptosis reporter substance is as follows: the cells labeled with the apoptosis reporter substance or their culture are introduced into the subcutaneous tissue of an animal or an animal model to form Tumor tissue labeled with an apoptosis reporter.
  • the screening method further includes performing at least one of histochemical analysis, nucleic acid sequence analysis, chemotherapy result analysis, and immune efficacy analysis on the three-dimensional tumor slice model.
  • the screening method before using a three-dimensional tumor slice model to screen candidate drugs, the screening method further includes: using a tumor cell model labeled with a cell apoptosis reporter to pre-screen the drug to obtain the candidate drug.
  • the screening method further includes: performing three-dimensional culture on clinical tumor slice samples, and using the candidate drug or the drug screened by the three-dimensional tumor slice model on the three-dimensional
  • the clinical tumor slice samples in culture are processed, and drugs are screened based on the signals of endogenous fluorescent substances in the clinical tumor slice samples, as shown in FIG. 2 .
  • endogenous fluorophores may provide valuable alternatives to transgenic fluorescent labels.
  • Significant progress has been made in label-free imaging techniques in recent years, such as visualization of the tumor microenvironment, membrane potential, iron-binding transferrin in unlabeled intact breast cancer cells and tumor xenografts, cell cycle status, and cellular metabolic responses .
  • This disclosure firstly applies label-free fluorescence imaging technology to time-course imaging of three-dimensional tumor slices, and the results show the potential of label-free fluorescence imaging in pharmacodynamic visualization of three-dimensional tumor slices.
  • Excitation methods include one-photon excitation and two-photon excitation.
  • the endogenous fluorescent substances include: at least one of lipofuscin, NADH and flavins.
  • the endogenous fluorescent substance is optionally lipofuscin, and it is believed, without being bound by theory, that endogenous fluorescent substances within the scope of the present disclosure, such as lipofuscin, have low background values and are less susceptible to other metabolite state changes in intensity Or optical parameters, the endogenous fluorescent substances disclosed in the present disclosure, such as lipofuscin, are relatively specific to cell apoptosis, and can reflect cell apoptosis more accurately and effectively.
  • the fluorescence detection conditions of the endogenous fluorescent substance are as follows: the excitation wavelength of single-photon is 488-570nm, the excitation wavelength of two-photon is 970-1140nm, and the emission The wavelength is 550-700nm.
  • Lipofuscin can be excited by one-photon or two-photon excitation. No matter which excitation method is used, the emission band is 550-700nm, and it can be detected within this range.
  • the slicing method of the clinical tumor slice sample can be the same as the slicing method of the three-dimensional tumor slice model in the foregoing embodiment, and will not be repeated here.
  • the candidate drugs include anticancer drugs, and optionally, the anticancer drugs include any one of immune checkpoint inhibitors and antitumor drugs.
  • the tumor includes any one of breast cancer, colon cancer, ovarian cancer, uterine cancer, liver cancer, gastric cancer, lung cancer and nasopharyngeal cancer.
  • An embodiment of the present disclosure provides a method for cultivating a three-dimensional tumor slice model, which includes: performing three-dimensional culture on the three-dimensional tumor slice model; The tumor tissue is sliced to obtain the three-dimensional tumor slice model.
  • An embodiment of the present disclosure also provides a method for screening drugs, which includes the following steps: performing three-dimensional culture on clinical tumor slice samples, processing candidate drugs on the clinical tumor slice samples in the three-dimensional culture, and based on the clinical tumor slice samples The signal of the endogenous fluorescent substance in further screens the drug.
  • the embodiment of the present disclosure also provides a method for real-time prediction of drug response, which includes: performing three-dimensional culture on the three-dimensional tumor slice model; wherein, the preparation method of the three-dimensional tumor slice model includes: adding the cell apoptosis reporter substance labeled with The tumor tissue is sliced to obtain the three-dimensional tumor slice model.
  • the three-dimensional tumor slice model in the three-dimensional culture is processed by the candidate drug, and the response of the candidate drug is predicted in real time based on the signal of the cell apoptosis reporter substance.
  • the preparation method of the three-dimensional tumor slice model, the conditions of three-dimensional culture and the detection method of the signal can all be the same as any of the foregoing embodiments. , and will not be repeated here.
  • 3D tumor slice demonstrated its strong potential for testing compounds or antibodies directly on patient-derived tumor tissue in a relatively short time, not only for targeted chemotherapy but also for immunotherapy.
  • 3D tumor slices can accelerate drug discovery, improve cancer treatment by providing accurate clinical guidance, and thus, may improve individualized therapy for individual patients when standard clinical options are faced Exhausting dilemma.
  • the disclosure establishes a three-dimensional tumor slice model culture method, combined with label-free technology and/or time-lapse imaging method of apoptosis reporter substances, realizes high-throughput screening of drugs, and obtains specific cancer samples within a week highly effective drugs.
  • the culture of three-dimensional tumor slice models completely preserved the immune components of the original tumor, which made it possible to successfully implement immune checkpoint blockade experiments with immune checkpoint inhibitors.
  • This technology provides an inexpensive, fast and simple platform for anticancer drug discovery, accelerating precision anticancer therapy.
  • Three-dimensional tumor slice models are able to maintain the cellular repertoire and immune components of their original tumors.
  • Three-dimensional tumor slice models were prepared as follows: Fresh tumors were embedded in low-melting point agarose and sliced at a thickness of 300 ⁇ m with a vibrating microtome. Then, the tumor slices were placed on the air-liquid interface system for culture, and the time-course images of the slices were captured from the original tumor before culture (referred to as day 0, denoted as D0) to the 7th day in culture (denoted as D7) , using a Leica M165FC fluorescence stereomicroscope.
  • the culture conditions of the three-dimensional tumor slices are: pre-cooled A (rat tail collagen I), B (10X Ham's F-12) and C (sterile buffer solution, 2.2g NaHCO 3 in 100ml 0.05N NaOH and 200mM HEPES (4-Hydroxyethylpiperazineethanesulfonic acid)) was prepared according to the ratio of 8:1:1 (volume ratio). Take the prepared 100 ⁇ L gel solution and spread it on the membrane (membrane pore size 0.4mm) in the Millicell insert cell culture inner dish (PIHP01250) with a diameter of 12mm, and put it in a 37°C incubator for 20-30 minutes until the gel Freeze completely.
  • A rat tail collagen I
  • B X Ham's F-12
  • C sterile buffer solution, 2.2g NaHCO 3 in 100ml 0.05N NaOH and 200mM HEPES (4-Hydroxyethylpiperazineethanesulfonic acid)
  • the tumor slice was carefully placed on the surface of the gel.
  • the culture medium was Ham's F12 containing 20% fetal calf serum and 50 ⁇ g/ml gentamicin.
  • the three-dimensional slices were placed in an incubator with 5% carbon dioxide and 37°C and the culture medium was changed every 4 days.
  • the tumor slices from the mouse mammary gland tumor B477-GFP became larger within 7 days, and the GFP signal became stronger (Fig. 3A).
  • the model was validated using various methods as follows.
  • the cell viability i.e., the percentage of viable cells in the total cells
  • the cell viability was monitored by counting viable cells, and it was found that the total cell number gradually increased, while the cell viability remained at >80% within 10 days ( Figure 3, B ).
  • E-cadherin epithelial cadherin
  • vimentin markers of epithelial and mesenchymal cells, respectively
  • CK7 a protein that maintains the tumor parenchyma
  • RNA-sequencing results showed stable expression of immune genes in 3D tumor slices within 8 days (E-F in Figure 4). Pathways of primary tumors before culture D0 and immediately after culture D1 to D8 by calculating the Pearson correlation coefficients of macrophages, neutrophils, monocytes, NK cells, T cells and B cells from D1 to D8 and D0 Gene expression levels were quantified and the results showed high correlation (G in Figure 4). Taken together, the results demonstrate that 3D tumor sheets maintain the cellular repertoire and immune components of their original tumors.
  • caspase-3 caspase-3 reporter sensor Sensor C3 expressing CFP-Asp-Glu-Val-Asp(DEVD)-YFP fusion protein was introduced into cancer cell lines.
  • the amino acid sequence DEVD is the cleavage site of caspase-3.
  • Cells expressing the sensor C3 can transfer electronic energy from CFP to YFP, causing the cells to fluoresce green, known as fluorescence resonance energy transfer (FRET) in live sensor cells.
  • FRET fluorescence resonance energy transfer
  • caspase-3 when caspase-3 is activated, it cleaves DEVD between the fluorescent proteins, eliminating energy transfer and causing the cell to fluoresce blue.
  • sensor C3 allowed us to monitor the dynamics of caspase-3 during apoptosis in living cells (Fig. 5, A–C).
  • Time-course studies demonstrate that C3-labeled cancer cells are more robust in time-lapse assessments of drug efficacy compared to traditional antibody-based caspase-3 activation assays or apoptosis staining methods such as the TUNEL assay, which only Endpoints can be monitored and early drug response of cultures cannot be detected.
  • this example uses GFP-labeled cells, C3-labeled cells and non-fluorescent-labeled cells in three-dimensional tumor slices to test the drug response.
  • cell viability of fluorescent samples was measured in cisplatin-treated B477-GFP breast tumor sections (A-B in Figure 6). An increase in GFP signal was found within 7 days during the three-dimensional tumor slice, and this increase was prevented after cisplatin treatment, such as tumor slice size, GFP intensity, etc. These phenomena were consistent with the cell viability determined by the MTT assay.
  • MDA-MB-231-C3 tumor slices were next treated with 1 ⁇ M bortezomib (drug), and drug responses were measured in a time series study. It was found that the FRET ratio decreased in a stepwise manner during bortezomib treatment (C-D in Figure 6). On day 6, the cell viability in the MDA-MB-231-C3 tumor slices treated with bortezomib was measured by MTT method (D in FIG. 6 ).
  • three-dimensional tumor slices of MDA-MB-231-C3 treated with increasing doses of cisplatin for 6 days also found a decrease in FRET ratio and enhanced staining of PI, which is excluded from living cells with intact cell membranes but can penetrate Permeabilizes dead or damaged cells and binds to DNA and RNA by intercalating between their bases (E-F in Figure 6).
  • the cell drug screening model mediated by the sensor C3 can be used to screen drugs against a certain cancer and identify a small number of anticancer drugs for further evaluation.
  • 166 candidate compounds were screened from the library (Table 1), most of which were approved by the FDA for clinical treatment of various cancers, and used MDA-MB-231-C3, Hct116-C3, PANC1-C3, A549-C3 Drug response was tested with HepG2-C3 cell line.
  • IC50 50% cell death
  • the present embodiment first uses the concentration of all drugs as 20 ⁇ M to treat the cell line for 0-96 hours, so that in 5 Rapid tests were performed in several cell lines (Table 2 and Table 3).
  • % of apoptotic cells is the percentage of apoptotic cells, which is the percentage of apoptotic cells/cell number at each time point; % of cell number is the percentage of cell number at each time point/the number of cells on 0d, after same.
  • the aim is to quickly assess the efficacy of these drugs against the tested cancer cells and to select the best drug candidates for further testing in 3D tumor slice models.
  • the 3D tumor slice model was then used to evaluate the drug efficacy of patient-derived tumor slices.
  • Tumor slices extracted from patient colon and breast cancer samples were treated with drugs that showed strong apoptosis-inducing ability in C3-labeled colon and breast cancer cells (Fig. 7, C-H). Changes in cell viability in response to drug treatment were examined in individual cancer samples after 4 days of treatment.
  • colon cancer colon case 1
  • tegafur ftorafur
  • daunorubicin hydrochloride osimertinib
  • Figure 7, D cisplatin, doxorubicin , irinotecan, neratinib, regorafenib and daunorubicin hydrochloride all significantly inhibited cell viability
  • Figure 6, E cisplatin, doxorubicin , irinotecan, neratinib, regorafenib and daunorubicin hydrochloride all significantly inhibited cell viability
  • Colon case 3 samples showed strong sensitivity to adrucil, doxorubicin, neratinib, ceritinib, daunorubicin hydrochloride, and osimertinib, resulting in cell viability ⁇ doxorubicin ( F) in Figure 7.
  • This experiment used an IgG antibody as a treatment control.
  • liver cancer sections from genetically engineered mouse models except for three liver cancer tumors (597-T1, T2, and T3), ⁇ PD-1 (A in Figure 8) or ⁇ PD-L1 (B in Figure 8) antibodies
  • ⁇ PD-1 A in Figure 8
  • ⁇ PD-L1 B in Figure 8
  • Histochemical staining with PD-L1 antibody showed that the expression levels of PD-L1 in these tumors ranged from 17% to 71% (C in Figure 8).
  • Example 5 Real-time monitoring of sensitive responses of three-dimensional tumor slices to chemotherapy and immunotherapy drugs based on label-free fluorescence imaging technology.
  • label-free optical methods allow rapid, noninvasive and time-course monitoring of tissue morphology and metabolic state, which has important applicability in clinical and/or commercial endeavors.
  • the kinetic characteristics of the three-dimensional tumor slice model during drug treatment were evaluated using a Nikon A1R Ti2-E multiphoton inverted microscope.
  • flavin as an indicator of cellular oxidative metabolism
  • the red 600nm autofluorescence of lipofuscin (Lipofuscin) was selectively excited at 1040nm (spectral verification of A in Figure 9).
  • mice tumor slices with GFP were prepared, and it was confirmed that the fluorescence intensity of lipofuscin increased and the number of GFP-positive cells decreased during the three-dimensional tumor slice treatment (B in FIG. 9 ).
  • PI staining (A in FIG. 10 ) and MTT assay (B, C in FIG. 10 ) were performed and confirmed that all three drug-induced increases in fluorescence were associated with cell death.
  • lipofuscin fluorescence intensity was also significantly increased in patient-derived colon cancer 3D tumor slices after 7 days of treatment with 10 ⁇ g/mL ⁇ PD-L1 antibody .
  • nasopharyngeal carcinoma samples nasopharyngeal carcinoma 3D tumor slices from two patients showed different responses to immune antibody treatment.
  • a slight increase in lipofuscin fluorescence intensity was found in the 3D tumor slices of ⁇ PD1/ ⁇ PD-L1 antibody-treated patient 11 (colon case 11).
  • the disclosure provides a method for screening drugs and a method for culturing three-dimensional tumor slice models.
  • the disclosure combines label-free technology and/or time-lapse imaging of apoptosis reporter substances to achieve high-throughput screening of drugs, and Get highly effective drugs for specific cancer samples within a week.
  • the culture of three-dimensional tumor slice models completely preserved the immune components of the original tumor, which made it possible to successfully implement immune checkpoint blockade experiments with immune checkpoint inhibitors.
  • This technology provides an inexpensive, fast and simple platform for anticancer drug discovery, accelerates precision anticancer therapy, and has excellent industrial practicality.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • General Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

提供了一种药物的筛选方法以及三维肿瘤切片模型的培养方法，涉及生物医药领域。建立了一种三维肿瘤切片模型的培养方法，结合无标记技术和/或细胞凋亡报告物质的延时成像方法，实现了对药物的高通量筛选，并在一周内取得针对特定癌症样本的高效药物。三维肿瘤切片模型的培养完整保留了原始肿瘤的免疫成分，这使得用免疫检查点抑制剂成功实现免疫检查点阻断试验成为可能。该技术为抗癌药物发现提供了一个廉价、快速和简单的平台，加速了精准抗癌治疗。

Description

一种药物的筛选方法以及三维肿瘤切片模型的培养方法
相关申请的交叉引用
本公开要求于2021年09月23日提交中国专利局的申请号为“CN 202111114318.9”名称为“一种药物的筛选方法以及三维肿瘤切片模型的培养方法”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及生物医药技术领域，具体而言，涉及一种药物的筛选方法以及三维肿瘤切片模型的培养方法。
背景技术
精准医学是一种考虑个人基因、环境和生活方式暴露的影响来定制干预措施的方法。以往用于癌症治疗的个性化医疗的药物发现模型存在很多缺陷，2D培养的人细胞系很容易失去其原始功能，并且其形态、生物功能、遗传等方面的表现和人体生理差异极大，固定和染料标记忽略了药物治疗微环境中的细胞动力学和代谢变化，难以定量评估肿瘤碎片中的药物敏感性和效率。因此，许多评估候选药物安全性和有效性的生物学测试必须在动物体内进行。对整只动物进行的此类测试的成本高昂，导致医疗保健成本更高。
个体化治疗的成功需要准确的预测以确定药物敏感性。而准确的预测需要与其原始条件最相似的微环境中生长的肿瘤样本。目前，基于三维培养条件的细胞培养模型和类器官培养模型得到了医药行业的关注。采用三维培养条件取代普通培养条件应用于培养筛药模型，其能够在一定程度上保留类器官或细胞的部分生理特征，但同时也存在着很多缺陷。
比如，现有技术无法更加直观地从分子层面获取培养模型的信息，且需要终止试验后，才能对细胞进行详细的鉴定和分析，无法实时且直观有效地对药物进行评估和筛选。
例如现有专利利用受试者自己的血清或体液用于3D培养条件下培养受试者的组织或癌细胞，使得细胞或组织在更加真实地微环境的生长。然而，该方法存在一定的局限，患者可提供的血清或体液的量是有限的，且过度的提取患者的血清或体液会对患者的身心造成一定程度的伤害，不利于患者的治疗和恢复。此外，该方法也是基于3D培养细胞或组织的状态进行观察，无法更加直观地从分子层面获取细胞或组织的信息，需要在试验结束后，才能进一步进行分析鉴定。
尽管针对如何真实模拟地癌症患者的肿瘤微环境的问题做出了许多努力，但在创建适用于来自不同个体的所有类型肿瘤的体外环境方面仍然存在挑战。三维细胞培养模型以及类器官培养模型对于药物治疗以及新药开发仍然存在着的敏感性和反应性不足的缺点，且筛药过程需要耗费大量的时间，无法在短时间内实现地对药物的实时评估和筛选。
发明内容
本公开提供一种药物的筛选方法，其包括以下步骤：对三维肿瘤切片模型进行三维培养；其中，所述三维肿瘤切片模型的制备方法包括：将标记有细胞凋亡报告物质的肿瘤组织进行切片处理，获得所述三维肿瘤切片模型；
采用候选药物对三维培养中的三维肿瘤切片模型进行处理，基于所述凋亡细胞报告物质的信号筛选候选药物。
在一些实施方式中，所述三维肿瘤切片的厚度为200～300μm。
在一些实施方式中，所述切片处理的方法如下：采用凝胶对肿瘤组织进行包裹后切片。
在一些实施方式中，所述三维培养的条件如下：采用插入式细胞培养皿对三维肿瘤切片进行三维培养，采用的培养液中包含体积分数为15％～20％的胎牛血清以及40～60μg/ml庆大霉素。
在一些实施方式中，所述细胞凋亡报告物质包括：用于检测细胞凋亡的传感器。
在一些实施方式中，所述传感器包括caspase-3传感器。
在一些实施方式中，所述细胞凋亡报告物质包括：SCAT3或zipGFP。
在一些实施方式中，标记有细胞凋亡报告物质的肿瘤组织的获取方法如下：将所述标记有细胞凋亡报告物质的细胞或其培养物导入动物或动物模型的皮下组织，以形成所述肿瘤组织。
在一些实施方式中，在采用三维肿瘤切片模型对候选药物进行筛选前，所述筛选方法还包括：采用标记有细胞凋亡报告物质的肿瘤细胞模型对药物进行预筛选，以获得所述候选药物。
在一些实施方式中，在采用三维肿瘤切片模型进行药物筛选后，所述筛选方法还包括：对临床肿瘤切片样本进行三维培养，将候选药物或经三维肿瘤切片模型筛选后的药物对三维培养中的临床肿瘤切片样本进行处理，基于临床肿瘤切片样本中的内源性荧光物的信号筛选药物。
在一些实施方式中，所述内源性荧光物包括：脂褐素、黄素、芳香族氨基酸和NADH中的至少一种。
在一些实施方式中，当所述内源性荧光物为脂褐素时，内源性荧光物的荧光检测条件如下：单光子的激发波长为488～570nm，双光子的激发波长为970～1140nm，发射波长为550～700nm。
在一些实施方式中，所述候选药物包括抗癌药物。
在一些实施方式中，所述抗癌药物包括免疫检查点抑制剂和抗肿瘤药物中的任意一种；
在一些实施方式中，所述肿瘤包括乳腺癌、结肠癌、卵巢癌、子宫癌、肝癌、胃癌、肺癌和鼻咽癌中的任意一种。
本公开还提供了一种三维肿瘤切片模型的培养方法，其包括：对三维肿瘤切片模型进行三维培养；其中，所述三维肿瘤切片模型的制备方法包括：将标记有细胞凋亡报告物质的肿瘤组织进行切片处理，获得所述三维肿瘤切片模型。
在一些实施方式中，所述三维肿瘤切片模型的厚度为200～300μm。
在一些实施方式中，所述切片处理的方法如下：采用凝胶对肿瘤组织进行包裹后切片。
在一些实施方式中，所述三维培养的条件如下：采用插入式细胞培养皿对三维肿瘤切片模型进行三维培养，采用的培养液中包含体积分数为15％～20％的胎牛血清以及40～60μg/ml庆大霉素。
本公开还提供一种用于筛选药物的方法，其特征在于，其包括以下步骤：对临床肿瘤切片样本进行三维培养，将候选药物对三维培养中的临床肿瘤切片样本进行处理，基于临床肿瘤切片样本中的内源性荧光物的信号进一步筛选药物。
本公开还提供一种实时预测药物反应的方法，其特征在于，其包括：对三维肿瘤切片模型进行三维培养；其中，所述三维肿瘤切片模型的制备方法包括：将标记有细胞凋亡报告物质的肿瘤组织进行切片处理，获得所述三维肿瘤切片模型；
采用候选药物对三维培养中的三维肿瘤切片模型进行处理，基于所述细胞凋亡报告物质的信号实时预测候选药物的反应。
本公开还提供一种药物的筛选体系，所述筛选体系包括：
经三维培养的三维肿瘤切片模型；
候选药物；
所述三维肿瘤切片模型通过将标记有细胞凋亡报告物质的肿瘤组织进行切片处理获得；
所述筛选体系采用所述候选药物对经三维培养的三维肿瘤切片模型进行处理，基于所述凋亡细胞报告物质的信号筛选候选药物。
在一些实施方式中，所述三维肿瘤切片模型的厚度为200～300μm。
在一些实施方式中，所述切片处理的方法如下：采用凝胶对肿瘤组织进行包裹后切片。
在一些实施方式中，所述三维培养的条件如下：采用插入式细胞培养皿对三维肿瘤切片模型进行三维培养，采用的培养液中包含体积分数为15％～20％的胎牛血清以及40～60μg/ml庆大霉素。
在一些实施方式中，所述细胞凋亡报告物质包括：用于检测细胞凋亡的传感器。
在一些实施方式中，所述传感器包括caspase-3传感器。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为三维肿瘤切片模型的制备以及筛药流程示意图；
图2为三维肿瘤切片模型以及临床样本切片模型应用于筛药的流程示意图；
图3为三维肿瘤切片模型完整保留了原始肿瘤样本的结构和肿瘤标志物；其中，(A)形态观察裸鼠乳腺B477-GFP细胞形成的肿瘤在三维肿瘤切片模型中的肿瘤生长；(B)检测裸鼠乳腺B477-GFP细胞形成的肿瘤在三维肿瘤切片模型中肿瘤细胞的活力；(C，D)组织病例学检测基因工程小鼠肿瘤三维肿瘤切片模型培养过程的生物标志物，蓝色为苏木精核复染，棕色为DAB阳性蛋白，γH2AX是DNA双链断裂标记；Desmin是调节肌节结构；E-cadherin是一种钙依赖性细胞-细胞粘附分子；Vemintin维持细胞完整性并锚定细胞器在细胞质中的位置；CK7是上皮性来源的肿瘤常用的标记物；(E)定量分析C、D中三维肿瘤切片模型中生物标志物；Caspase-3没有被激活，因此没有计算；抗体染色的组织载玻片的5个数字图像用于计算每个样品的平均值；
图4为三维肿瘤片培养模型能够维持其原始肿瘤的细胞库和免疫成分；其中，(A-D)组织学分析带有免疫能力的基因工程小鼠肿瘤在三维肿瘤片模型中免疫生物标志物变化；蓝色为苏木精染核，棕色为DAB阳性蛋白；CD3e/CD8a为T淋巴细胞标志物；CD11b为巨噬细胞和小胶质细胞标记物；CD45为T、NK、树突状和淋巴因子激活的杀伤(LAK)细胞标志物；F4/80为小鼠巨噬细胞标记物；PD-1为一种由T细胞表达的免疫受体；PD-L1为髓系、淋巴系、正常上皮细胞或癌细胞表达的PD-1的配体；(E)RNA序列分析免疫相关基因的表达水平；ssGSEA代表样本内基因数据集的水平上调或下调；(F)T细胞和B细胞免疫标记基因表达的RNA序列分析；其中，TPM用于估计转录本或基因表达水平；(G)通过计算D1至D8的T细胞和B细胞的Pearson相关系数，与D0比较基因表达水平；
图5为基于FRET技术实时预测三维肿瘤片对药物的敏感性；其中，(A)观察10μM阿霉素诱导MDA-MB-231-C3细胞凋亡在48小时内的动态变化；CFP(ex:430nm/em：480nm)与YFP(ex:430nm/em：520)；(B)量化分析单个细胞的FRET变化；(C)量化分析阿霉素诱导MDA-MB-231-C3细胞凋亡数量和细胞总数；其中，绿线和蓝线分别表示总细胞和凋亡细胞数；(D)顺铂/阿霉素在三维肿瘤片模型中诱导的细胞凋亡；用100μM顺铂或10μM阿霉素对MDA-MB-231-C3肿瘤片处理六天；(E)量化分析面板(D)实验中FRET比率；(F)不同剂量顺铂处理MDA-MB-231-C3三维肿瘤片模型中细胞凋亡的动态过程；(G)量化面板(F)实验中三维肿瘤片的FRET比率；(H)量化分析100μM顺铂处理六天后MDA-MB-231-C3三维肿瘤片的面积；
图6为通过MTT法定量评估三维肿瘤片对药物的疗效；其中，(A)通过观察GFP荧光和MTT染色观察100μM顺铂处理或未处理的B477-GFP乳腺肿瘤切片在0～7天内的变化；比例尺为1mm；(B)MTT法评估面板(A)中的药物功效；(C)FRET荧光(d0-6)和MTT染色(d6)观察1μM硼替佐米处理六天的MDA-MB-231-C3肿瘤片；(D)FRET比率(d0-6)和MTT测定(d6)评估面板(C)中的药物敏感性；比例尺为2毫米；(E，F)通过PI、FRET比率和MTT法测定不同剂量顺铂处理6天的MDA-MB-231-C3肿瘤片的药物敏感性；比例尺为1毫米；(G)PI染色和MTT法观察顺铂处理6天的B477三维肿瘤片；比例尺为0.8mm；(H)MTT法测定(G)中的药效；(I)MTT法评估顺铂处理5天的PDX来源的结肠肿瘤片对药物敏感性；
图7为基于FRET技术实时预测小鼠来源的多种癌症样本对药物敏感性及MTT终点法预测病人手术切除的肿 瘤在三维肿瘤片培养后对药物的疗效；其中，(A)基于FRET技术筛选三维肿瘤片抗肿瘤药物。药物分别处理MDA-MB-231-C3和HepG2-C3三维肿瘤片；(B)药物处理的三维肿瘤片后的代表性FRET图像；比例尺为1毫米；(C)MTT染色药物处理4天的人结肠和乳腺三维肿瘤片；比例尺为1毫米；(D-F)MTT法检测药物处理4天的人结肠三维肿瘤片的药物反应；其中，D(结肠病例1)、E(结肠病例2)、F(结肠病例3)；色瑞替尼(2.5μM)、盐酸柔红霉素(2.5μM)、奥希替尼(5μM)，其余用于病人结肠样本的药物浓度均为20μM；(G,H)MTT法检测药物处理4天的病人乳腺癌三维肿瘤片的药物反应；G(乳房病例1)，H(乳房病例2)；除米托蒽醌Mitoxantrone(5μM)和来那替尼Neratinib(10μM)外，用于病人乳腺样本的药物浓度为20μM；
图8为三维肿瘤片对PD-1/PD-L1免疫检查点抑制剂的药物反应；(A)PD-1抗体诱导小鼠三维肿瘤片细胞死亡，将小鼠Smad4 co/co；Pten co/co；Alb-Cre(SPC)肿瘤切片(658-607)用对照IgG或αPD-1抗体处理6天后，MTT法分析维肿瘤片的细胞活力；(B)PD-L1抗体诱导小鼠三维肿瘤片细胞死亡；MTT法分析对照IgG或αPD-L1处理6天后的小鼠SPC肿瘤切片(658-607)的活力；与PD-L1IHC％的水平做对应比较(n/a：PD-L1IHC无数据)；(C)组化分析(B)中新鲜肿瘤样本的PD-L1水平；(D)病人手术切除肿瘤的PD-L1IHC染色；(E)PD-L1抗体诱导病人三维肿瘤片的细胞死亡；MTT法分析对照IgG1或αPD-L1(durvalumab)处理7天后的人结肠癌(结肠病例4-结肠病例7)肿瘤片；(F)PD-1抗体诱导人结肠癌细胞死亡；MTT法分析对照IgG4或αPD-1抗体处理7天后的人结肠肿瘤片(结肠病例7-结肠病例9)；(G)MTT法测定PD-1抗体联合IL-2处理6天小鼠MK 3941三维肿瘤片的免疫治疗反应；
图9为脂褐素(lipofuscin)作为无标记的自发荧光分子在化疗和免疫治疗的肿瘤样本出现红色荧光增强；其中，(A)脂褐素的荧光发射光谱；(B)双光子影像观察25μM顺铂、2.5μg/mLαPD-1或2.5μgαPD-L1抗体处理带GFP的小鼠三维肿瘤片中脂褐素荧光增强现象，GFP(绿色，例如@960nm)和脂褐质(红色，例如@1040nm)；
图10为PI染色和MTT染色方法确认化疗和免疫治疗后在小鼠乳腺三维肿瘤片样本出现细胞死亡；其中，(A)左列为双光子荧光成像观察NADH(蓝色，ex.@740nm)、碘化丙啶PI(红色，ex.@1040nm)和collagen的二次谐波生成成像(绿色，ex.@1040纳米)；右列为collagen的二次谐波成像(蓝色，ex.@890nm)、flavins的双光子荧光成像(绿色，ex.@890nm)和碘化丙啶PI(红色，ex.@1040纳米)；这组图像通过碘化丙啶(PI)染色评估了用25μM顺铂、2.5μg/mLαPD-1或2.5μg/mLαPD-L1处理的基因工程小鼠乳腺三维肿瘤片中的癌细胞死亡；(B)MTT法染色25μM顺铂、2.5μg/mLαPD-1或2.5μg/mLαPD-L1处理的三维肿瘤片；(C)MTT法测定顺铂处理的小鼠乳腺三维肿瘤片中的细胞活力；
图11为基于无标记荧光成像技术实时监测三维肿瘤片对化疗和免疫检查点抑制剂治疗的反应；其中，(A)25μM顺铂、2.5μg/mLαPD-1或2.5μg/mLαPD-L1处理4天的基因工程小鼠肿瘤片中胶原蛋白collagen、脂褐素lipofuscin和黄素flavin的双光子自发荧光影像；比例尺为24μm；(B)药物处理0-5天的基因工程小鼠三维肿瘤片中脂褐素的双光子自发荧光增强成像；比例尺为24μm；(C)量化分析药物处理的基因工程小鼠三维肿瘤片中脂褐素自发荧光强度；(D，E)10μg/mLαPD-1或10μg/mLαPD-L1处理三天的人鼻咽癌三维肿瘤片中脂褐素的双光子自发荧光成像和脂褐素强度评估；比例尺为24μm；(F，G)10μg/mLαPD-1或10μg/mLαPD-L1处理7天的人结肠癌切片(结肠病例10)中脂褐素的双光子自发荧光成像和脂褐素强度评估。
具体实施方式
术语定义
如本文所用，术语“细胞活力”是指总细胞中活细胞所占的百分比。
如果本所所用，术语“细胞凋亡”是指为维持内环境稳定，由基因控制的细胞自主的有序的死亡，即主动的由基因决定的自动结束生命的过程,所以也常常被称为程序化细胞死亡。
如果本所所用，术语“高通量筛选”是指以分子水平和细胞水平的实验方法为基础，以微板(诸如不同密度的微孔平板)形式作为实验工具载体，以自动化操作系统执行试验过程，以灵敏快速的检测装置在同一时间检测数以千万的样品、采集实验数据和数字化分析处理，并以得到的相应数据库支持运转的技术体系。
为使本公开实施方式和实施例的目的、技术方案和优点更加清楚，下面将对本公开实施方式和实施例中的技术方案进行清楚、完整地描述。实施方式和实施例中未注明条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本公开一实施方式提供了一种药物的筛选方法，其包括以下步骤：对三维肿瘤切片模型进行三维培养；其中，所述三维肿瘤切片模型的制备方法包括：将标记有细胞凋亡报告物质的肿瘤组织进行切片处理，获得所述三维肿瘤切片模型；然后，采用候选药物对三维培养中的三维肿瘤切片模型进行处理，基于所述细胞凋亡报告物质的信号筛选候选药物，可参照图1。
本文中“采用候选药物对三维培养中的三维肿瘤切片模型进行处理”可以指：将药物与三维肿瘤切片模型接触，例如可以通过在三维培养的培养基中添加药物的手段。本公开建立的三维肿瘤切片模型可以准确保留原始肿瘤的细胞库和免疫成分。
上述筛选方法允许对细胞死亡进行时间进程和定量评估，能够测试针对手术切除的癌症的个体化药物反应和免疫检查点阻断疗法。与其他离体系统(如PDO)相比，具有几个主要优点：1)三维肿瘤切片模型中含有基质细胞、免疫细胞和癌细胞；2)所有切除的肿瘤样本均可用于药物评价；3)它是一种快速且低成本的体外系统。事实上，筛选或评估过程可以在4-7天内完成，而PDO需要两周以上的时间。这些特性使三维肿瘤切片成为精准肿瘤学领域的生理相关模型，用于为每个癌症患者选择最佳和最有效的药物。
在可选的实施方案中，所述三维肿瘤切片的厚度为200～300μm，例如可以为210～290μm、230～280μm或240～260μm，诸如可以为200μm、220μm、240μm、260μm、280μm或300μm。三维肿瘤切片的厚度在本公开的范围内可以提高切片的效率和质量，完好地保持原初肿瘤的微环境。同时保证检测内源性荧光信号的有效性，有利于 保持检测结果的有效性和稳定性。三维肿瘤切片的厚度低于该范围可能导致切片的效率和质量，破坏原初肿瘤的微环境，高于该厚度范围可能导致检测内源性荧光信号的降低，不利于保持检测结果的有效性和稳定性。
可选地，所述切片处理的方法如下：采用琼脂糖凝胶对肿瘤组织进行包裹后切片，步骤如下：肿瘤被收集并放入预冷的。DMEM培养液，含10％FBS和1％青霉素/链霉素(Gibco)；采用低熔点琼脂糖凝胶对肿瘤组织进行包裹后切片；肿瘤切片(200～300μm)是在手术后2-6小时内使用莱卡VT1200S(Leica Biosystems Nussloch GmbH,德国)在无菌PBS条件下做震动切片。
在可选的实施方式中，三维培养条件可参照现有公开的方法和材料，只要能够满足使三维肿瘤切片模型呈空间立体方式生长即可。
可选地，所述三维培养的条件如下：采用插入式细胞培养皿对三维肿瘤切片进行三维培养，采用的培养液中包含体积分数为15％～20％的胎牛血清以及40～60μg/ml庆大霉素，胎牛血清的体积分数例如为16％～19％、17％～18％或17.5％～18％，诸如可为15％、16％、17％、18％、19％或20％。庆大霉素的终浓度例如43～58μg/ml、45～55μg/ml或47～52μg/ml，诸如可以为40μg/ml、45μg/ml、50μg/ml、55μg/ml或60μg/ml。
可选地，所述三维培养的条件如下：将冰上预冷A(鼠尾胶原I)，B(10X Ham’s F-12)和C(无菌缓冲液，2.2g NaHCO 3在100ml 0.05N NaOH和200mM HEPES)按照8：1：1比例配制。取配置的100μL凝胶溶液平铺到直径为12mm的Millicell插入式细胞培养内皿(PIHP01250)中的膜上(膜孔径0.4mm)，放入37℃孵化器中保温20-30分钟，直到凝胶完全凝固。然后，将肿瘤切片小心地放置在凝胶表面。再加100μL凝胶溶液覆盖到肿瘤片上，放入37℃孵化器中保温20-30分钟，直到凝胶完全凝固。最后在插入式细胞培养外皿中加入400μL培养液。培养液是Ham’s F12包含20％的胎牛血清和50μg/毫升庆大霉素。最后，三维肿瘤切片放置在5％的二氧化碳和37℃的培养箱中培育并且每4天更改一次培养液。
本公开的三维肿瘤片的培养条件保留了原始肿瘤的重要特征，例如癌症异质性和结构、免疫成分和稳定的基因表达。
在可选实施方式中，本公开不对所述细胞凋亡报告物质的种类进行限定，只要满足能够检测细胞凋亡的作用即可。在一些实施方式中，其可以包括：用于检测细胞凋亡的传感器。
在可选的实施方式中，所述细胞凋亡报告物质包括用于检测细胞凋亡的传感器，可选地为胱天蛋白酶-3传感器Sensor C3。
可选地为胱天蛋白酶-3传感器(Sensor C3)。传统上，评估药物反应依赖于破坏性固定和有毒染料标记，它们分别对细胞具有破坏性和毒性，有价值的细胞学和代谢信息被丢弃。为了克服与缺乏有效的预测性生物标志物或药物发现报告基因相关的挑战，本公开建立了基于FRET的传感器Sensor C3作为细胞凋亡报告物质的三维肿瘤切片模型，以筛选多种抗癌药物。这是通过抑制细胞生长和诱导细胞凋亡来实现的。这种方法应该适用于大规模的药物筛选，以缩小潜在的候选药物进行进一步分析。
在其他实施方式中，细胞凋亡报告物质还可以包括但不限于SCAT3和zipGFP。
在可选的实施方式中，标记有细胞凋亡报告物质的肿瘤组织的获取方法如下：将所述标记有细胞凋亡报告物质的细胞或其培养物导入动物或动物模型的皮下组织，以形成标记有细胞凋亡报告物质的肿瘤组织。
在一些实施方式中，筛选方法还包括对三维肿瘤切片模型进行组化分析、核酸序列分析、化疗结果分析以及免疫疗效分析中的至少一种。
在可选的实施方式中，在采用三维肿瘤切片模型对候选药物进行筛选前，所述筛选方法还包括：采用标记有细胞凋亡报告物质的肿瘤细胞模型对药物进行预筛选，以获得所述候选药物。
在可选的实施方式中，在采用三维肿瘤切片模型进行药物筛选后，所述筛选方法还包括：对临床肿瘤切片样本进行三维培养，将候选药物或经三维肿瘤切片模型筛选后的药物对三维培养中的临床肿瘤切片样本进行处理，基于临床肿瘤切片样本中的内源性荧光物的信号筛选药物，可参照图2。
在一些实施方式中，除了转基因荧光标记，内源性荧光物(内在代谢荧光团)也可以提供有价值的替代品。近年来，无标记成像技术取得了显着进展，例如肿瘤微环境的可视化、膜电位、未标记的完整乳腺癌细胞和肿瘤异种移植物中的铁结合转铁蛋白、细胞周期状态和细胞代谢反应。本公开首次将无标记荧光成像技术应用于三维肿瘤片的时程成像，结果表明，无标记荧光成像在三维肿瘤片药效学可视化中的潜力。
肿瘤组织中存在很多内源性荧光物，这些内源性荧光物能够被特定波长的激发光激发，发射特定波长段的荧光信号，而这些信号最终可以实现对应分子的无标记成像。激发的方式包括单光子激发和双光子激发。
可选地，所述内源性荧光物包括：脂褐素、NADH和黄素类(Flavins)中的至少一种。内源性荧光物可选地为脂褐素，据信不受理论的约束，本公开范围内的内源性荧光物诸如脂褐素的背景值较低，不容易受到其它代谢物状态改变强度或者光学参数，本公开的内源性荧光物诸如脂褐素对于细胞凋亡比较具有专一性，能够更加准确有效地反应细胞凋亡。
可选地，当所述内源性荧光物为脂褐素时，内源性荧光物的荧光检测条件如下：单光子的激发波长为488～570nm，双光子的激发波长为970～1140nm，发射波长为550～700nm。脂褐素可以采用单光子激活或双光子激发，无论是采用哪种激发方式，发射波段均为550～700nm，在该区间内都可以侦测。
在可选的实施方式中，临床肿瘤切片样本的切片方法可以同前述实施方式中三维肿瘤切片模型的切片方式，在此不再赘述。
可选地，所述候选药物包括抗癌药物，可选地，所述抗癌药物包括免疫检查点抑制剂和抗肿瘤药物中的任意一种。可选地，所述肿瘤包括乳腺癌、结肠癌、卵巢癌、子宫癌、肝癌、胃癌、肺癌和鼻咽癌中的任意一种。
本公开实施方式提供了一种三维肿瘤切片模型的培养方法，其包括：对三维肿瘤切片模型进行三维培养；其中，所述三维肿瘤切片模型的制备方法包括：将标记有细胞凋亡报告物质的肿瘤组织进行切片处理，获得所述三维肿瘤 切片模型。
可以理解的是，三维肿瘤切片模型、其制备及其培养方法同前述任意实施方式所述，不再赘述。
本公开实施方式还提供了一种用于筛选药物的方法，其包括以下步骤：对临床肿瘤切片样本进行三维培养，将候选药物对三维培养中的临床肿瘤切片样本进行处理，基于临床肿瘤切片样本中的内源性荧光物的信号进一步筛选药物。
本公开实施方式还提供了一种实时预测药物反应的方法，其包括：对三维肿瘤切片模型进行三维培养；其中，所述三维肿瘤切片模型的制备方法包括：将标记有细胞凋亡报告物质的肿瘤组织进行切片处理，获得所述三维肿瘤切片模型。采用候选药物对三维培养中的三维肿瘤切片模型进行处理，基于所述细胞凋亡报告物质的信号实时预测候选药物的反应。
可以理解的是，在用于筛选药物的方法以及实时预测药物反应的方法的实施方式中，三维肿瘤切片模型的制备方法、三维培养的条件以及信号的检测方法均可以同前述任意的实施方式所述，在此不再赘述。
总之，三维肿瘤切片模型(三维肿瘤片)展示了其在相对较短的时间内直接在患者来源的肿瘤组织上测试化合物或抗体的强大潜力，不仅适用于靶向化疗，也适用于免疫治疗。凭借转基因报告系统和无标记代谢成像技术，三维肿瘤片可以加速药物发现，通过提供准确的临床指导来改善癌症治疗，因此，当标准临床选择时，可能会改善个体患者的个性化治疗所面临的筋疲力尽的困境。
本公开建立了三维肿瘤切片模型的培养方法，结合无标记技术和/或细胞凋亡报告物质的延时成像方法，实现了对药物的高通量筛选，并在一周内取得针对特定癌症样本的高效药物。三维肿瘤切片模型的培养完整保留了原始肿瘤的免疫成分，这使得用免疫检查点抑制剂成功实现免疫检查点阻断试验成为可能。该技术为抗癌药物发现提供了一个廉价、快速和简单的平台，加速了精准抗癌治疗。
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1
三维肿瘤切片模型能够维持其原始肿瘤的细胞库和免疫成分。
三维肿瘤切片模型的制备方法如下：将新鲜肿瘤嵌入低熔点琼脂糖并用振动切片机以300μm的厚度切片。然后，将肿瘤切片放置在气液界面系统上进行培养，并从培养前的原始肿瘤(指第0天，记为D0)到培养中的第7天(计为D7)捕获切片的时程图像，使用Leica M165FC荧光体视显微镜。
其中，三维肿瘤切片的培养条件为：将冰上预冷A(鼠尾胶原I)，B(10X Ham’s F-12)和C(无菌缓冲液，2.2g NaHCO 3在100ml 0.05N NaOH和200mM HEPES(4-羟乙基哌嗪乙磺酸))按照8：1：1(体积比)比例配制。取配置的100μL凝胶溶液平铺到直径为12mm的Millicell插入式细胞培养内皿(PIHP01250)中的膜上(膜孔径0.4mm)，放入37℃孵化器中保温20-30分钟，直到凝胶完全凝固。然后，将肿瘤切片小心地放置在凝胶表面。再加100μL凝胶溶液覆盖到肿瘤片上，放入37℃孵化器中保温20-30分钟，直到凝胶完全凝固。最后在插入式细胞培养外皿中加入400μL培养液。培养液是Ham’s F12包含20％的胎牛血清和50μg/毫升庆大霉素。最后，三维切片放置在5％的二氧化碳和37℃的培养箱中培育并且每4天更改一次培养液。
在三维肿瘤片的培养条件下，来自小鼠乳腺肿瘤B477-GFP的肿瘤切片在7天内变大，GFP信号变强(图3中A)。为了进一步确定三维肿瘤片是否可以模拟肿瘤生长，使用各种方法验证了该模型如下。
首先，通过计数活细胞来监测三维肿瘤片的细胞活力(即总细胞中活细胞所占的百分比)，发现总细胞数逐渐增加，而细胞活力在10天内保持在>80％(图3中B)。
使用针对E-cadherin(上皮钙黏素)和波形蛋白(Vimentin)的抗体(分别为上皮细胞和间充质细胞的标志物)以及CK7(一种维持肿瘤实质的蛋白质)的抗体染色(图3中C、E)，在长达8天的培养时间过程中也未检测到显着差异。
接下来，对三维肿瘤片和各种生物标志物(包括γH2AX、Ki67、活化的胱天蛋白酶-3(cleaved caspase-3)和结蛋白(desmin))进行组织学分析(图3中D-E)。结果表明，三维肿瘤片总体上表现出与原发性肿瘤相似的特征，除了Ki67+细胞增加，这与之前观察到的增殖细胞增加一致(图3中B)。
接下来，确定三维肿瘤片是否也可以维持原始肿瘤的肿瘤免疫微环境。因此，在三维肿瘤片中分析了免疫成分，该三维肿瘤片来自内源性乳腺肿瘤携带乳腺特异性破坏乳腺癌1号基因(Brca1，Brca1Co/Co；MMTV-Cre)的基因工程小鼠模型。通过组织病例学分析免疫标志物，包括CD3e、CD8a、CD11b、CD45、F4/80、PD-1和PD-L1(图4中A-D)。在8天的培养期间，所有这些免疫标志物在三维肿瘤片中均呈阳性。
此外，与D0(新鲜肿瘤切片)相比，没有观察到显着差异，表明免疫成分在切片培养条件下得到了很好的保存。RNA测序结果表明，在8天内，三维肿瘤片中免疫基因的稳定表达(图4中E-F)。通过计算D1至D8与D0的巨噬细胞、中性粒细胞、单核细胞、NK细胞、T细胞和B细胞的Pearson相关系数，对培养D0前、培养D1至D8后即刻的原始肿瘤的通路基因表达水平进行量化，结果显示高相关性(图4中G)。总之，结果表明三维肿瘤片保持了其原始肿瘤的细胞库和免疫成分。
定量和延时预测三维肿瘤片对体外抗癌药物的反应。
建立三维肿瘤片作为药物敏感性测试的可靠模型需要对细胞死亡进行定量测量。因此，将表达CFP-Asp-Glu-Val-Asp(DEVD)-YFP融合蛋白的胱天蛋白酶-3(caspase-3)报告传感器Sensor C3引入癌细胞系。氨基酸序列DEVD是胱天蛋白酶-3的切割位点。表达传感器C3的细胞可以将电子能量从CFP转移到YFP，导致细胞发出绿色荧光，即活传感器细胞中的荧光共振能量转移(FRET)。然而，当胱天蛋白酶-3被激活时，它会切割荧光蛋白之间的DEVD，从而消除能量转移并导致细胞发射蓝色荧光。因此，传感器C3使我们能够监测活细胞凋亡过程中胱天蛋白酶-3的动态(图5中A-C)。时间进程研究表明，与传统的基于抗体的胱天蛋白酶-3激活测定或凋亡染色方法(如TUNEL测定)相比，C3标记的癌细胞在药物疗效的延时评估中更强大，后者仅可以监测终点，不能检测培养 物的早期药物反应。
接下来，将C3标记的癌细胞进行培养，之后消化收集细胞，细胞悬浮于PBS溶液中并用注射器将1x10 6细胞注入小鼠的皮下组织，约1个月后，获得肿瘤组织，将肿瘤组织进行切片处理，获得对应的三维肿瘤片。并测试是否也可以实现三维肿瘤片中药物反应的延时测试。结果发现，顺铂(抗癌药物)或阿霉素(药物)处理三维肿瘤片以剂量依赖性方式降低导致FRET比率(图5中D-E)。另一项时间过程实验也显示FRET比率以剂量和时间依赖性方式降低(图5中F-H)。
实施例2
基于FRET延时技术和MTT终点法预测化学药物在三维肿瘤片平台中的疗效。
由于临床癌症样本缺乏荧光标志物，因此，本实施例在三维肿瘤片中使用了GFP标记细胞、C3标记的细胞和非荧光标记的细胞来测试药物反应。首先，用顺铂处理的B477-GFP乳腺肿瘤切片中测量了荧光样品的细胞活力(图6中A-B)。在三维肿瘤片期间的7天内发现GFP信号增加，并且这种增加在顺铂治疗后被阻止，如肿瘤片大小、GFP强度等，这些现象和MTT法测定的细胞活力一致。
接下来用1μM硼替佐米(药物)处理MDA-MB-231-C3肿瘤片，并在时间序列研究中测量了药物反应。发现FRET比率在硼替佐米治疗期间以梯级方式下降(图6中C-D)。在第6天，MTT法测定硼替佐米处理的MDA-MB-231-C3肿瘤片中细胞活力(图6中D)。
此外，用增加剂量的顺铂处理MDA-MB-231-C3的三维肿瘤片6天，也发现FRET比降低和PI染色增强，而PI是排除在具有完整细胞膜的活细胞之外，但可以穿透死亡或受损的细胞并通过插入它们的碱基之间来与DNA和RNA结合(图6中E-F)。
然后使用MTT法对细胞活力进行定量，结果发现细胞活力的下降类似于FRET减少的趋势。这些数据表明PI染色和MTT法分析都可以从不同药物的治疗中产生比较理想的结果。由于所有临床样本均未有标记，因此继续用PI染色和MTT法分析和测试小鼠非荧光B477和PDX来源的人结肠499/552肿瘤切片中的药物反应，发现所有肿瘤片均可用于定量测量细胞活力(图6中G-I)。
实施例3
基于FRET和延时监测技术的离体三维肿瘤片培养模型的药物筛选。
采用传感器C3介导的细胞筛药模型首先可以用来筛选针对某种癌症的药物并鉴定少量抗癌药物以供进一步评估。从文库中筛选出166种候选化合物(表1)，其中大部分被FDA批准用于各种癌症的临床治疗，并使用MDA-MB-231-C3，Hct116-C3，PANC1-C3，A549-C3和HepG2-C3细胞系测试药物反应。
表1 166种候选化合物
Figure PCTCN2022104365-appb-000001
Figure PCTCN2022104365-appb-000002
Figure PCTCN2022104365-appb-000003
由于许多药物抑制肿瘤细胞的生长，IC50(50％细胞死亡)在5-20μM之间并取决于细胞系，本实施例首先使用所有药物的浓度为20μM处理细胞系0-96小时，以便在5种细胞系中进行快速测试(表2和表3)。
表2 231C3以及HepG2-C3的测试结果
Figure PCTCN2022104365-appb-000004
Figure PCTCN2022104365-appb-000005
Figure PCTCN2022104365-appb-000006
Figure PCTCN2022104365-appb-000007
表3 PANC1-C3和A549-C3的测试结果
Figure PCTCN2022104365-appb-000008
Figure PCTCN2022104365-appb-000009
Figure PCTCN2022104365-appb-000010
表4 Hct116-C3的检测结果
Figure PCTCN2022104365-appb-000011
Figure PCTCN2022104365-appb-000012
Figure PCTCN2022104365-appb-000013
Figure PCTCN2022104365-appb-000014
备注：凋亡细胞％为凋亡细胞百分比，是每个时间点凋亡细胞/细胞数的百分比；细胞数量％为细胞数量百分比是每个时间点的细胞数量/0d的细胞数量的百分比，后同。
对于以高效率杀死癌细胞的药物，使用较低剂量和较短时间点做了进一步测试，结果表明，细胞数量逐渐减少和细胞凋亡的增加(表5～7)。
表5 检测结果
Figure PCTCN2022104365-appb-000015
Figure PCTCN2022104365-appb-000016
表6 检测结果
Figure PCTCN2022104365-appb-000017
Figure PCTCN2022104365-appb-000018
Figure PCTCN2022104365-appb-000019
表7 检测结果
Figure PCTCN2022104365-appb-000020
Figure PCTCN2022104365-appb-000021
对于不能达到IC50的药物，就不再继续在三维肿瘤片中使用。通过这种方式，旨在快速评估这些药物对所测试癌细胞的功效，并选择最佳候选药物在三维肿瘤片模型中进行进一步测试。
基于在体外传感器C3标记的癌细胞系的高通量、延时药物筛选结果，随后评估了这些高效药物在三维肿瘤片模型中的药敏试验结果。为了在三维肿瘤片平台中筛选药物，用药物来处理231-C3和HepG2-C3细胞的三维肿瘤片。结果发现，在体外诱导细胞凋亡的药物同样能够以不同的效率在三维肿瘤片模型中引起细胞凋亡(图7中A-B)。
监测患者来源的三维肿瘤片对化疗药物的反应。
接着用三维肿瘤片模型来评估患者来源的肿瘤片的药物疗效。用在C3标记的结肠癌和乳腺癌细胞中表现出很强的凋亡诱导能力的药物进行处理从病人结肠癌和乳腺癌样本中提取的肿瘤片(图7中C-H)。在4天的处理后检测了个体癌症样本对药物处理的细胞活力变化。
对于结肠癌(结肠病例1)，替加氟(ftorafur)、盐酸柔红霉素和奥希替尼显著抑制细胞活力(图7中D)，而对于结肠病例2，顺铂、多柔比星、伊立替康、来那替尼、瑞戈非尼和盐酸柔红霉素都显著抑制细胞活力(图6中E)。结肠病例3样品对adrucil、多柔比星、来那替尼、色瑞替尼、盐酸柔红霉素和奥希替尼表现出很强的敏感性，导致≤多柔比星的细胞活力(图7中F)。
然后通过MTT法测定在两个人乳腺肿瘤样本中测量药物功效，表明两者都非常敏感(图7中G-H)。尽管测试的11种药物中有9种导致乳腺癌病例1样品的细胞活力≤品的细，但只有盐酸表柔比星的作用具有统计学意义(图7中G)。对于乳腺癌病例2，10种药物(来那替尼除外)导致≤药物(来的细胞活力，其中8种(阿杜西、顺铂、多西他赛、多柔比星、盐酸表柔比星、米托蒽醌、盐酸帕博西尼和柠檬酸他莫昔芬)具有统计学意义(图7中H)。这些数据凸显了精准医疗中个性化药物测试的必要性，三维肿瘤片模型可能起到特别有用的作用。
实施例4
监测三维肿瘤片对免疫检查的抑制剂的敏感性。
为了测试三维肿瘤片的免疫治疗效果，在三维肿瘤片模型中检测了PD-1和PD-L1抗体处理原发性小鼠和人类来源的肿瘤片的药物反应。
该实验使用IgG抗体作为治疗对照。在来自基因工程小鼠模型的肝癌切片中，除了三种肝癌肿瘤(597-T1、T2和T3)外，在用αPD-1(图8中A)或αPD-L1(图8中B)抗体处理后发现细胞活力降低。PD-L1抗体组化染色显示，这些肿瘤中PD-L1的表达水平范围为17％-71％(图8中C)。这些数据表明，PD-L1在一定水平的表达，即使在大约17％的细胞水平，也可以使PD-1/PD-L1抗体对癌细胞敏感性的起到杀伤力，而即使高达约50％的细胞表达PD-L1在一些肿瘤中，例如597-T1-3和607，它们的敏感性仍然很低。这一观察结果强调了在临床治疗前进行药物敏感测试的必要性。
同时，还评估了PD1抗体或PD-L1抗体对源自病人结肠癌的三维肿瘤片的药物敏感性反应。这些肿瘤以低水平(范围为2％-11％)表达PD-L1，并且对PD-1/PD-L1抗体的敏感性也低于早期测试的小鼠肿瘤(图8中D-F)。结肠病例4以约11％的细胞表达PD-L1水平，并且比其他以较低水平表达PD-L1的癌症表现出更高的敏感性。总而言之，数据表明，尽管有一些例外，癌细胞中PD-L1的较高表达水平通常会使癌症对药物具有更高的敏感性。在此也测试了αPD-1抗体和IL-2在三维肿瘤片模型中的药物组合处理效果(图8中G)。数据表明，对源自小鼠BRCA1突变体(MK3941)的三维肿瘤片，该突变体对αPD-1抗体不敏感，而使用αPD-1和IL-2的组合治疗具有显着的统计学响应。这些实验原则上表明，可以使用三维肿瘤片系统测试各种药物组合的潜在影响。
实施例5：基于无标记荧光成像技术实时监测三维肿瘤片对化疗和免疫治疗药物的敏感反应。
与荧光染料标记相比，无标记光学方法允许对组织形态和代谢状态进行快速、无创和时程监测，这在临床和/或商业努力中具有重要的适用性。使用尼康A1R Ti2-E多光子倒置显微镜评估三维肿瘤片模型在药物处理的动力学特征。除了作为细胞氧化代谢指标的黄素外，选择性地在1040nm处激发了脂褐素(Lipofuscin)的红色600nm自发荧光(图9中A的光谱验证)。
紧接着制备了带GFP的小鼠肿瘤片，证实了三维肿瘤片治疗过程中脂褐素荧光强度的增加和GFP阳性细胞数量的减少(图9中B)。接下来，进行了PI染色(图10中A)和MTT法测定(图10中B，C)并确认所有三种药物诱导的荧光增加与细胞死亡有关性。
作为平行验证，用25μM顺铂、2.5μg/mLαPD-1或2.5μg/mLαPD-L1处理后，使用三维肿瘤片在4天内观察到小鼠肿瘤样品中脂褐素荧光强度显着增加(图11中A)。然后根据细胞中胶原蛋白(Collagen)、黄素和应激诱导的脂褐素的分子成像对非荧光对应物进行双光子显微镜检查，证实双光子荧光成像系统可以监测源自小鼠非荧光标记肿瘤的三维肿瘤片中药物诱导的细胞凋亡(图11中B-C)。在对病人来源的三维肿瘤片样品的快速演示中(图11中D-G)，在用10μg/mLαPD-L1抗体处理7天后，病人来源的结肠癌三维肿瘤片中的脂褐素荧光强度也显著增加。对于鼻咽癌样本，来自两名患者的鼻咽癌三维肿瘤片对免疫抗体治疗表现出不同的反应。在αPD1/αPD-L1抗体处理的患者11(结肠病例11)的三维肿瘤片中发现脂褐素荧光强度略有增加。相比之下，在用10μg/mLαPD1治疗3天后，来自患者12(结肠病例12)的三维肿瘤片的脂褐素荧光有明显增强。这些结果表明，脂褐素荧光可以作为一种有价值的光学报告器，用于无标记实时监测三维肿瘤片中的药物反应，并可能转化为临床实践。
以上所述仅为本公开的可选的实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性
本公开提供了一种药物的筛选方法以及三维肿瘤切片模型的培养方法，本公开结合无标记技术和/或细胞凋亡报告物质的延时成像方法，实现了对药物的高通量筛选，并在一周内取得针对特定癌症样本的高效药物。三维肿瘤切片模型的培养完整保留了原始肿瘤的免疫成分，这使得用免疫检查点抑制剂成功实现免疫检查点阻断试验成为可能。该技术为抗癌药物发现提供了一个廉价、快速和简单的平台，加速了精准抗癌治疗，具有优异的工业实用性能。

Claims (14)

  1. 一种药物的筛选方法，其特征在于，其包括以下步骤：对三维肿瘤切片模型进行三维培养；其中，所述三维肿瘤切片模型的制备方法包括：将标记有细胞凋亡报告物质的肿瘤组织进行切片处理，获得所述三维肿瘤切片模型；
    采用候选药物对三维培养中的三维肿瘤切片模型进行处理，基于所述凋亡细胞报告物质的信号筛选候选药物。
  2. 根据权利要求1所述的药物的筛选方法，其特征在于，所述三维肿瘤切片模型的厚度为200～300μm；
    优选地，所述切片处理的方法如下：采用凝胶对肿瘤组织进行包裹后切片；
    优选地，所述三维培养的条件如下：采用插入式细胞培养皿对三维肿瘤切片模型进行三维培养，采用的培养液中包含体积分数为15％～20％的胎牛血清以及40～60μg/ml庆大霉素。
  3. 根据权利要求1或2所述的药物的筛选方法，其特征在于，所述细胞凋亡报告物质包括：用于检测细胞凋亡的传感器；
    优选地，所述传感器包括caspase-3传感器。
  4. 根据权利要求1～3中任一所述的药物的筛选方法，其特征在于，所述细胞凋亡报告物质包括：SCAT3或zipGFP。
  5. 根据权利要求3或4所述的药物的筛选方法，其特征在于，标记有细胞凋亡报告物质的肿瘤组织的获取方法如下：将所述标记有细胞凋亡报告物质的细胞或其培养物导入动物或动物模型的皮下组织，以形成所述肿瘤组织。
  6. 根据权利要求1～5任一项所述的药物的筛选方法，其特征在于，在采用三维肿瘤切片模型对候选药物进行筛选前，所述筛选方法还包括：采用标记有细胞凋亡报告物质的肿瘤细胞模型对药物进行预筛选，以获得所述候选药物。
  7. 根据权利要求1～6任一项所述的药物的筛选方法，其特征在于，在采用三维肿瘤切片模型进行药物筛选后，所述筛选方法还包括：对临床肿瘤切片样本进行三维培养，将候选药物或经三维肿瘤切片模型筛选后的药物对三维培养中的临床肿瘤切片样本进行处理，基于临床肿瘤切片样本中的内源性荧光物的信号筛选药物。
  8. 根据权利要求7所述的药物的筛选方法，其特征在于，所述内源性荧光物包括：脂褐素、黄素、芳香族氨基酸和NADH中的至少一种；
    优选地，当所述内源性荧光物为脂褐素时，内源性荧光物的荧光检测条件如下：单光子的激发波长为488～570nm，双光子的激发波长为970～1140nm，发射波长为550～700nm。
  9. 根据权利要求1～8任一项所述的药物的筛选方法，其特征在于，所述候选药物包括抗癌药物；
    优选地，所述抗癌药物包括免疫检查点抑制剂和抗肿瘤药物中的任意一种；
    更优选地，所述肿瘤包括乳腺癌、结肠癌、卵巢癌、子宫癌、肝癌、胃癌、肺癌和鼻咽癌中的任意一种。
  10. 一种三维肿瘤切片模型的培养方法，其特征在于，其包括：对三维肿瘤切片模型进行三维培养；其中，所述三维肿瘤切片模型的制备方法包括：将标记有细胞凋亡报告物质的肿瘤组织进行切片处理，获得所述三维肿瘤切片模型；
    优选地，所述三维肿瘤切片模型的厚度为200～300μm；
    优选地，所述切片处理的方法如下：采用凝胶对肿瘤组织进行包裹后切片；优选地，所述三维培养的条件如下：采用插入式细胞培养皿对三维肿瘤切片模型进行三维培养，采用的培养液中包含体积分数为15％～20％的胎牛血清以及40～60μg/ml庆大霉素。
  11. 一种用于筛选药物的方法，其特征在于，其包括以下步骤：对临床肿瘤切片样本进行三维培养，将候选药物对三维培养中的临床肿瘤切片样本进行处理，基于临床肿瘤切片样本中的内源性荧光物的信号进一步筛选药物。
  12. 一种实时预测药物反应的方法，其特征在于，其包括：对三维肿瘤切片模型进行三维培养；其中，所述三维肿瘤切片模型的制备方法包括：将标记有细胞凋亡报告物质的肿瘤组织进行切片处理，获得所述三维肿瘤切片模型；
    采用候选药物对三维培养中的三维肿瘤切片模型进行处理，基于所述细胞凋亡报告物质的信号实时预测候选药物的反应。
  13. 一种药物的筛选体系，其特征在于，所述筛选体系包括：
    经三维培养的三维肿瘤切片模型；
    候选药物；
    所述三维肿瘤切片模型通过将标记有细胞凋亡报告物质的肿瘤组织进行切片处理获得；
    所述筛选体系采用所述候选药物对经三维培养的三维肿瘤切片模型进行处理，基于所述凋亡细胞报告物质的信号筛选候选药物。
  14. 根据权利要求13所述的药物的筛选体系，其特征在于，所述三维肿瘤切片模型的厚度为200～300μm；
    优选地，所述切片处理的方法如下：采用凝胶对肿瘤组织进行包裹后切片；
    优选地，所述三维培养的条件如下：采用插入式细胞培养皿对三维肿瘤切片模型进行三维培养，采用的培养液中包含体积分数为15％～20％的胎牛血清以及40～60μg/ml庆大霉素；
    优选地，所述细胞凋亡报告物质包括：用于检测细胞凋亡的传感器；
    优选地，所述传感器包括caspase-3传感器。
PCT/CN2022/104365 2021-09-23 2022-07-07 一种药物的筛选方法以及三维肿瘤切片模型的培养方法 Ceased WO2023045494A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111114318.9 2021-09-23
CN202111114318.9A CN113832211A (zh) 2021-09-23 2021-09-23 一种药物的筛选方法以及三维肿瘤切片模型的培养方法

Publications (1)

Publication Number Publication Date
WO2023045494A1 true WO2023045494A1 (zh) 2023-03-30

Family

ID=78969312

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/104365 Ceased WO2023045494A1 (zh) 2021-09-23 2022-07-07 一种药物的筛选方法以及三维肿瘤切片模型的培养方法

Country Status (2)

Country Link
CN (1) CN113832211A (zh)
WO (1) WO2023045494A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116590377A (zh) * 2023-07-17 2023-08-15 安泰康生物技术(北京)有限公司 一种患者来源器官型组织培养药物敏感性检测方法
CN118169379A (zh) * 2024-05-14 2024-06-11 北京航空航天大学 肿瘤的药敏检测方法
WO2025113423A1 (zh) * 2023-11-27 2025-06-05 创芯国际生物科技(广州)有限公司 一种类器官培养药敏检测一体化系统及方法

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113832211A (zh) * 2021-09-23 2021-12-24 澳门大学 一种药物的筛选方法以及三维肿瘤切片模型的培养方法
US12100141B2 (en) 2021-12-28 2024-09-24 International Business Machines Corporation Three-dimensional delineation of tumor boundaries via supervised machine learning algorithms and augmented reality
CN116593426B (zh) * 2022-02-07 2025-12-30 澳门大学 一种鉴定细胞凋亡和坏死的方法及其应用
CN114196620B (zh) * 2022-02-14 2022-05-20 广东省农业科学院动物科学研究所 一种母猪乳腺组织体外培养方法
CN115862890B (zh) * 2022-08-31 2023-08-11 山东第一医科大学附属肿瘤医院(山东省肿瘤防治研究院、山东省肿瘤医院) 一种抗癌药物筛选方法及系统
CN119391643B (zh) * 2024-12-31 2025-03-14 珠海澳大科技研究院 一种利用三维肿瘤片技术培养消化道肿瘤的培养基及方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1396265A (zh) * 2001-05-24 2003-02-12 香港科技大学 基于绿色荧光蛋白检测细胞凋亡的方法
CN102558310A (zh) * 2012-02-24 2012-07-11 李兵辉 实时监测蛋白水解酶活性的指示剂的制备方法及应用方法
US20200143531A1 (en) * 2015-07-20 2020-05-07 Min Xu Photon structure and chemometrics pathologic system
CN111690616A (zh) * 2020-06-18 2020-09-22 澎立生物医药技术（上海）有限公司 一种培养肿瘤组织切片的方法
CN113832211A (zh) * 2021-09-23 2021-12-24 澳门大学 一种药物的筛选方法以及三维肿瘤切片模型的培养方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1396265A (zh) * 2001-05-24 2003-02-12 香港科技大学 基于绿色荧光蛋白检测细胞凋亡的方法
CN102558310A (zh) * 2012-02-24 2012-07-11 李兵辉 实时监测蛋白水解酶活性的指示剂的制备方法及应用方法
US20200143531A1 (en) * 2015-07-20 2020-05-07 Min Xu Photon structure and chemometrics pathologic system
CN111690616A (zh) * 2020-06-18 2020-09-22 澎立生物医药技术（上海）有限公司 一种培养肿瘤组织切片的方法
CN113832211A (zh) * 2021-09-23 2021-12-24 澳门大学 一种药物的筛选方法以及三维肿瘤切片模型的培养方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
WEBER PETRA, SCHICKINGER SARAH, WAGNER MICHAEL, ANGRES BRIGITTE, BRUNS THOMAS, SCHNECKENBURGER HERBERT: "Monitoring of Apoptosis in 3D Cell Cultures by FRET and Light Sheet Fluorescence Microscopy", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 16, no. 3, 9 March 2015 (2015-03-09), pages 5375 - 5385, XP093052566, DOI: 10.3390/ijms16035375 *
XING FUQIANG, LIU YU-CHENG, HUANG SHIGAO, LYU XUEYING, SU SEK MAN, CHAN UN IN, WU PEI-CHUN, YAN YINGHAN, AI NANA, LI JIANJIE, ZHAO: "Accelerating precision anti-cancer therapy by time-lapse and label-free 3D tumor slice culture platform", THERANOSTICS, vol. 11, no. 19, 1 January 2021 (2021-01-01), AU , pages 9415 - 9430, XP093052567, ISSN: 1838-7640, DOI: 10.7150/thno.59533 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116590377A (zh) * 2023-07-17 2023-08-15 安泰康生物技术(北京)有限公司 一种患者来源器官型组织培养药物敏感性检测方法
WO2025113423A1 (zh) * 2023-11-27 2025-06-05 创芯国际生物科技(广州)有限公司 一种类器官培养药敏检测一体化系统及方法
CN118169379A (zh) * 2024-05-14 2024-06-11 北京航空航天大学 肿瘤的药敏检测方法
CN118169379B (zh) * 2024-05-14 2024-09-06 北京航空航天大学 肿瘤的药敏检测方法

Also Published As

Publication number Publication date
CN113832211A (zh) 2021-12-24

Similar Documents

Publication Publication Date Title
WO2023045494A1 (zh) 一种药物的筛选方法以及三维肿瘤切片模型的培养方法
Jacob et al. Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR T cell testing
Tebon et al. Drug screening at single-organoid resolution via bioprinting and interferometry
Pijuan et al. In vitro cell migration, invasion, and adhesion assays: from cell imaging to data analysis
van der Kuip et al. Short term culture of breast cancer tissues to study the activity of the anticancer drug taxol in an intact tumor environment
Eilenberger et al. Effect of spheroidal age on sorafenib diffusivity and toxicity in a 3D HepG2 spheroid model
Marciniak et al. Using pancreas tissue slices for in situ studies of islet of Langerhans and acinar cell biology
Benien et al. 3D tumor models: history, advances and future perspectives
Imamura et al. Comparison of 2D-and 3D-culture models as drug-testing platforms in breast cancer
Xing et al. Accelerating precision anti-cancer therapy by time-lapse and label-free 3D tumor slice culture platform
KR102255944B1 (ko) 세포 기반 약물 스크리닝 분석 방법 및 이의 용도
US20150247112A1 (en) 3D Tissue Culture Devices and Systems
CN118169379B (zh) 肿瘤的药敏检测方法
Goliwas et al. Methods to evaluate cell growth, viability, and response to treatment in a tissue engineered breast cancer model
Parker et al. A human glioblastoma organotypic slice culture model for study of tumor cell migration and patient-specific effects of anti-invasive drugs
Kjar et al. Biofunctionalized gelatin hydrogels support development and maturation of iPSC-derived cortical organoids
EP3320338B1 (en) Barrier function measurements
Augustine et al. A novel in ovo model to study cancer metastasis using chicken embryos and GFP expressing cancer cells
Vella et al. 3D cell culture models in research: applications to lung cancer pharmacology
Fedr et al. Variability of fluorescence intensity distribution measured by flow cytometry is influenced by cell size and cell cycle progression
Mohamadian Namaqi et al. Dynamic cell culture modulates colon cancer cell migration in a novel 3D cell culture system
Braun et al. Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis.
JP5800312B2 (ja) 誘導多能性幹細胞の識別方法
Tsukamoto et al. Patient-Derived Ex Vivo Cultures and Endpoint Assays with Surrogate Biomarkers in Functional Testing for Prediction of Therapeutic Response
Greier et al. Optimizing culturing conditions in patient derived 3D primary slice cultures of head and neck cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22871555

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22871555

Country of ref document: EP

Kind code of ref document: A1