WO2019133767A1 - Procédé de diagnostic in vitro de prédiction de l'efficacité d'un médicament - Google Patents

Procédé de diagnostic in vitro de prédiction de l'efficacité d'un médicament Download PDF

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WO2019133767A1
WO2019133767A1 PCT/US2018/067780 US2018067780W WO2019133767A1 WO 2019133767 A1 WO2019133767 A1 WO 2019133767A1 US 2018067780 W US2018067780 W US 2018067780W WO 2019133767 A1 WO2019133767 A1 WO 2019133767A1
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organoid
cells
drug
cell
organoids
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Jia-Ming Chang
Wei-Wei Chen
Wei-Hsuan SUN
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Development Center For Biotechnology
Dcb-Usa Llc
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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
    • G01N33/5082—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
    • 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
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/28—Vascular endothelial cells
    • 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
    • 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
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the present invention relates generally to diagnostic or assay methods for predicting drug efficacies or effects.
  • a 3D organoid is a mimic of a miniaturized organ produced in vitro in 3D.
  • 3D organoids are typically derived from one or a few cells from a tissue, embryonic stem cells, or induced pluripotent stem cells, which can propagate and self-organize in 3D culture due to their self-renewal and differentiation capacities.
  • 3D organoid model can be used to establish structures and functions of specific tissues. Because its structure and pattern closely resemble a real tissue, a 3D organoid model can provide proper cell-cell and cell-cell matrix interactions, thereby compensating for the shortcomings of conventional 2D in vitro cell culture systems, which lack tumor heterogeneity and molecular diversity, cellular heterogeneity, tumor microenvironment and tissue characteristics seen in patients in the clinics.
  • An in vitro model based on 3D organoid can be used in anti-cancer drug development/testing and can also provide timely analysis data for screening therapeutics against recurrent tumors.
  • Embodiments of the invention relate to 3D organoid systems for diagnosis or assay of drug efficacies.
  • One aspect of the invention relates to 3D organoids for diagnosis or assays.
  • a 3D organoid in accordance with one embodiments of the invention may be constructed from a tumor cell.
  • the tumor cell in the 3D organoids may be from a cell line, from circulating tumor cells isolated from a patient, or from a tumor tissue.
  • the 3D organoids may be constructed using an aqueous gel material and an adhesion molecule.
  • the aqueous gel material may be hydrogel.
  • the adhesion molecule may be ICAM-l or galectin-3.
  • the 3D organoid may be constructed by forming a scaffold with an aqueous gel material, followed by adding a mixture of the tumor cell and an adhesion molecule.
  • a method in accordance with one embodiment of the invention comprises adding a drug to the 3D organoids and observing an effect of the drug on cells in the 3D organoids.
  • the effect of the drug is analyzed with an imaging system to analyze expression levels of a gene or a protein.
  • the effect of the drug is analyzed using fluorescence labeling, luminescence, or bright light.
  • FIG. 1 shows improvements of 3D organoids cultures by adding adhesion molecules.
  • Adhesion molecule glycosylcholine
  • the 3D organoids are about 50% larger in sizes.
  • NCI-H727 cells form ribbon-shaped structures in the presence of adhesion molecule.
  • FIG. 2 shows effects of different hydrogels prepared with different collagen/PEG ratios on organoids formation.
  • Collagen PEG ratios of 1 :4, 1 :6, and 1 :8 can better support cell mass (organoid) formations.
  • Gal-3 adheresion molecules
  • the formation of 3D organoids is further enhanced.
  • endothelial cells EC
  • FIGs. 3A-3C show differential gene expressions in 2D cell culture versus 3D organoids.
  • FIG. 3A shows the expression of PIK3CA in HCC827, NCI-H727, A549, and H1975 cancer cells in both the 2D culture system and 3D organoids.
  • FIG. 3B shows the expression of EGFR in HCC827, NCI-H727, A549, and H1975 cancer cells in both the 2D culture system and 3D organoids.
  • FIG. 3C shows the expression of KRAS in HCC827, NCI- H727, A549, and H1975 cancer cells in both the 2D culture system and 3D organoids.
  • the expression levels of EGFR, PIK3CA and KRAS are relatively low in 3D organoid culture, as compared with those in the 2D culture system.
  • FIGs. 4A-4C show different effects of drug treatments in 2D cell cultures versus
  • FIG. 4A shows that the gene expression levels of PIK3CA were increased in response to Pexidartinib (PLX3397; a CSF1R inhibitor) treatment in the HCC827 and NCI- H727 cells in both the 2D cell culture systems and 3D organoids. Lower levels of expression were observed in 3D organoid culture, as compared with those in the 2D culture. PLX3397 treatment did not show dose-dependent effects in A549 and H1975 cells.
  • FIG. 4B shows that the gene expression levels of EGFR were increased in response to Pexidartinib treatment in the HCC827 and NCI-H727 cells in the 2D cell culture systems, but barely in 3D organoids.
  • FIG. 4C shows that the gene expression levels of KRAS were increased in response to Pexidartinib treatment in the HCC827 and NCI-H727 cells in the 2D cell culture systems, but only in HCC827 cells in the 3D organoids. PLX3397 treatment did not show dose-dependent effects in A549 and H1975 cells.
  • FIGs. 5A-5E show effects of various therapeutic agents in the 2D cell cultures or 3D organoids. Different therapeutics have different effects with different tumor cells.
  • FIG. 5 A shows the effects on NCI-H727 lung cancer cells in 2D cell culture and 3D organoids.
  • FIG. 5B shows the effects on HCC-827 lung cancer cells in 2D cell culture and 3D organoids.
  • FIG. 5C shows the effects on NCI-H460 lung cancer cells in 2D cell culture and 3D organoids.
  • FIG. 5D shows the effects on NCI-H1975 lung cancer cells in 2D cell culture and 3D organoids.
  • FIG. 5E shows the effects on colorectal cancer cells (HCT-116 and HT-29)in 2D cell culture and 3D organoids.
  • the 2D cell culture systems and the 3D organoid systems show different results.
  • Pacitaxel, Gefitinib, and Erlotinib are shown to be effective against HCC-827 lung cancer cells in 2D cell culture systems, whereas these same drugs are not effective in the 3D organoid systems.
  • the 3D organoid system shows that only Afatinib is effective against HCC-827. This inconsistency is shown to be due to the inaccurate results from the 2D cell culture system.
  • FIG. 6 shows results of validation of 3D organoid systems using an in vivo animal model.
  • Pacitaxel at 5 mpk or 20 mpk was not effective in inhibiting tumor growth.
  • Afatinib at 5 mpk or 20 mpk are very effective in inhibiting tumor growth.
  • FIG. 7 shows results of 3D organoids established with patient-derive xenograft.
  • FIG. 8 shows 3D organoid models containing blood vessel cells in three separate experiments (test 1, test 2, and test 3).
  • Embodiments of the invention relate to 3D organoid systems for diagnosis or assay of drug efficacies. Embodiments of the invention also relate to methods for establishing 3D organoid systems and methods for using the 3D organoids to assay drug treatment effects.
  • 3D organoid tumor culture can be used to establish structure and functions of tissues.
  • the structure and function of a 3D organoid are highly similar to an actual tissue. They can provide cell-cell interactions, as well as cell-matrix interactions. Therefore, 3D organoid models can overcome shortcomings of in vitro 2D cell cultures, which often cannot present clinically observed tumor heterogeneity and molecular diversity, cell heterogeneity, tumor environments, and tissue characteristics. ETsing 3D organoid models, one can provide timely information for anti-tumor drug testing and can provide drug screening information for treating tumor recurrence.
  • a 3D organoid in accordance with one embodiments of the invention may be constructed from a tumor cell.
  • the tumor cells in the 3D organoids may be from a cell line, from circulating tumor cells isolated from a patient, or from a tumor tissue.
  • the 3D organoid may be constructed using an aqueous gel material and an adhesion molecule.
  • the aqueous gel material may be hydrogel.
  • the 3D organoids of the invention may be constructed by forming a scaffold with an aqueous gel material, followed by adding a mixture of the tumor cell and an adhesion molecule.
  • adhesion molecules may be any suitable adhesion molecules known in the art, such as ICAM-l and/or galectin-3.
  • 3D organoids of the invention may be used to assay drug efficacies.
  • a method for drug assay in accordance with one embodiment of the invention comprises adding a drug to a 3D organoid and observing an effect of the drug on cells in the 3D organoid.
  • the effects of the drug may be analyzed with any suitable methods, for example using an imaging system, to analyze expression levels of a gene or a protein.
  • an imaging system the effects of a drug may be analyzed using a microscope and fluorescence labeling, luminescence, or white light.
  • 3D organoid models may be established using tumor cells from a patient, for example from a tumor tissue or from circulating tumor cells (CTC) isolated form liquid biopsy.
  • CTC circulating tumor cells
  • organoid systems may be established using cells having the same genetic background as the tumors of interest in patients.
  • Several 3D organoid system or tumor models have been established.
  • the 3D organoids may be established in 24-well or 96- well plates. Then, the drug at various concentrations may be added to each well. One would then monitor the drug effects on the cells, for example by monitoring an enzyme or a marker. As an example, one may monitor ATPase activities as an indication of cell activities.
  • RNA FISH single-molecule RNA FISH
  • bioimagina to monitor and quantify the expression levels of the gene. Quantitative information from such assays may be used to derive IC50 values or to obtain z-score from various drugs.
  • 3D organoids may be used to establish drug dosage relationship.
  • cancer cells are inoculated in a 24-well or 96-well plate. Once 3D organoid models are established from the cancer cells.
  • a drug to be tested may be added to the 3D organoid system at different concentrations (e.g., 10 nM - 1,000 nM). After incubation with the drug, total RNA of the cells may be extracted to generate cDNA (e.g., using PCR). Then, cDNA may be used in qPCR to monitor specific marker expressions under the action of the drug.
  • 3 genes are known to be associated with lung cancers. Based on these 3 genes, one can find corresponding cell lines to generate 3D organoids. After establishment of the 3D organoids, one can use them to assess the effects of target therapies on these 3 gene expression.
  • Example 1 Galectin-3 improves 3D tumor organoid formation
  • Two different lung cancer cell lines HCC-827 and NCI-H727) from in vitro cultures are used to establish 2D cell cultures and 3D organoid cell cultures, respectively. Briefly, 1 xlO 5 cells/mL lung cancer cells are added into each well of a 96-well low-attach plate, wherein each well also contains a specific concentration of an adhesion molecule (e.g., galectin-3). The cells are cultured to form 3D organoids. The growth and size changes of the 3D organoids can be monitored using a microscope.
  • 3D organoids are well established in wells containing the adhesion molecule (galectin-3, Gal-3) at concentrations of 0.3 pg/mL, 0.6 pg/mL, 1.25 pg/mL, and 2.5 pg/mL.
  • the adhesion molecule galectin-3, Gal-3
  • the 3D organoids are about 50% larger in sizes.
  • NCI-H727 cells form ribbon-shaped structures in the presence of adhesion molecule.
  • Example 2 Hydrogel and Galectin-3 can stimulate the formation of blood vessel 3D
  • Hydrogels can be prepared with different ratios of collagen and PEG, such as
  • 1 :2, 1 :4, 1 :6, and 1 :8 (collagen : PEG).
  • 3 mg/ml type I collagen and 300 mg/ml PEG (e.g., 7500 MW) are added into a cell culture medium (containing 10% bovine serum or 5% human serum).
  • This solution is mixed well with a reconstitution solution consisting of acetic acid and 10X MEM cell culture media, and then allowed to stand in a cell culture incubator for 20 minutes or longer to form a hydrogel.
  • hydrogels prepared with collagen PEG ratios of 1 :4, 1 :6, and 1 :8 can better support cell mass (organoid) formations.
  • Gal-3 adhesion molecules
  • the formation of 3D organoids is further enhanced.
  • endothelial cells EC
  • formation of blood vessel is observable.
  • Example 3-1 Genes express differently in 2D cell culture systems as compared with 3D organoid systems
  • qPCR Real-time Quantitative Polymerase Chain Reaction
  • FIG. 3 The expression levels of PIK3CA, EGFR, and KRAS from these analyses are shown in FIG. 3. As shown in FIG. 3, in most lung cancer cell lines, the expression levels of EGFR, PIK3CA and KRAS are relatively lower in the 3D organoid culture, as compared with those in the 2D culture system.
  • Example 3-2 PLX3397 (CSF1R inhibitor) treatment effects
  • 2D cell culture plate 4 human lung cancer cell lines (1 xlO 5 cell/mL) separately in a 24-well plate. Incubate the plate under the conditions of 5% CO2’ 37°C overnight. [0040] Confirm that the cells have completely covered the wells under microscope, and then add the prepapred solution of PLX3397 at 10, 100, and 1,000 nM, respetively, into the wells. Then, incubate the plate under the conditions of 5% C0 2 , 37°C for 24 hours.
  • 3D organoid culture plate 4 human lung cancer cell lines (6 xlO 5 cell/mL) separately in a low-attachment 24-well plate. Incubate the plate under the conditions of 5% CO2, 37°C until the 7th day. On the 7th day, add the prepapred solution of PLX3397 at 10, 100, and 1,000 nM, respetively, into the wells. Then, incubate the plate under the conditions of 5% CO2, 37°C for 24 hours.
  • FIG. 4A shows that the gene expression levels of PIK3CA were increased in response to Pexidartinib (PLX3397; a CSF1R inhibitor) treatment in the HCC827 and NCI-H727 cells in both the 2D cell culture systems and 3D organoids. Lower levels of expression were observed in 3D organoid culture, as compared with those in the 2D culture. PLX3397 treatment did not show dose-dependent effects in A549 and H1975 cells.
  • FIG. 4B shows that the gene expression levels of EGFR were increased in response to Pexidartinib treatment in the HCC827 and NCI-H727 cells in the 2D cell culture systems, but barely in 3D organoids.
  • FIG. 4C shows that the gene expression levels of KRAS were increased in response to Pexidartinib treatment in the HCC827 and NCI-H727 cells in the 2D cell culture systems, but only in HCC827 cells in the 3D organoids. PLX3397 treatment did not show dose-dependent effects in A549 and H1975 cells.
  • 2D cell cultures and 3D organoids tumor systems were established with several strains of in vitro cultured lung cancer cells (e.g., HCC-827, NCI- H460, NCI-H727, and NCI-H1975) and colorectal cancer cells (e.g., HCT-116 and HT-29). Briefly, l x lO 4 cells/mL tumor cells are placed in each well of a 96-well low- attach plate to culture the 2D cell culture and 3D organoid tumor systems.
  • lung cancer cells e.g., HCC-827, NCI- H460, NCI-H727, and NCI-H1975
  • colorectal cancer cells e.g., HCT-116 and HT-29.
  • 3D organoids were quantified with an automatic multi-function optical imaging system (Cytation 5, Bio-Tek, EISA). The results are shown in FIGs. 5A-5E.
  • FIGs. 5A-5E different therapeutics have different effects with different tumor cells. This is known. What is notable is the inconsistency between the 2D cell culture systems and the 3D organoid systems. For example, Pacitaxel, Gefitinib, and Erlotinib are shown to be effective against HCC-827 lung cancer cells in 2D cell culture systems, whereas these same drugs are not effective in the 3D organoid systems. Instead, the 3D organoid system shows that only Afatinib is effective against HCC-827.
  • Example 5 Validation of the 2D cell culture system and 3D organoid system using animal xenograft models
  • HCC-827 lung cancer cells were injected subcutaneously to establish a xenograft models for validation of the drug efficacy test results from the 2D cell culture systems and 3D organoid systems. Briefly, 0.1 ml of HCC-827 lung cancer cells (2> ⁇ l0 6 cells/mL) was injected subcutaneously into mouse at the back. After 1 week, the tumor size was determined with a digital caliper. When the tumor grew to a size of 100 -150 mm 3 , the drug tests can begin.
  • Afatinib was given 5 times per week via an oral feeding tube, and paclitaxel was given one per week for 4 weeks. The tumor sizes and animal body weights were measured twice per week for 4 weeks. The animal test results are as shown in FIG. 6.
  • the 2D cell culture system also shows that Pacitaxel is effective. This is not validated with the in vivo xenograft model. This result proves that the conventional 2D cell culture system is not as reliable as the 3D organoid systems of the invention.
  • Example 6 Patient-derived tumor reconstruction in a 3D organoid system
  • the tumor mass grew substantially over 4 days, with or without added adhesion molecule (Gal-3).
  • the wells with Gal-3 produced better organized tumor mass, as compared to the ones without added adhesion molecule.
  • Example 7 3D organoid models containing blood vessel cells
  • a cell culture medium containing 10% bovine serum or 5% human serum
  • a reconstitution solution consisting of acetic acid and 10X MEM cell culture media
  • 1 ug/ml Gal-3 Inject the mixture into the hydrogel and incubate the plates in a cell culture incubator (37°C, 5% C0 2 ) to incubate for 2 days to allow the formation of tumor mass containing blood vessels.
  • 3D organoids can be efficiently constructed with the help of an adhesion molecule (e.g., galectin-3, ICAM-l, or a similar adhesion molecule).
  • an adhesion molecule e.g., galectin-3, ICAM-l, or a similar adhesion molecule.
  • the 3D organoids are more accurate than 2D cell cultures in representing in vivo microenvironments, and therefore, the 3D organoids can be used to accurate evaluate therapeutic effects of drugs.
  • the 3D organoids can be established that include blood vessel formation to more accurately represent in vivo tumor microenvironments.

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Abstract

La présente invention concerne un organoïde 3D utilisé pour le diagnostic ou l'essai, où l'organoïde 3D est construit à partir d'une cellule tumorale. La cellule tumorale dans l'organoïde 3D provient d'une lignée cellulaire, à partir de cellules tumorales circulantes isolées d'un patient, ou à partir d'un tissu tumoral. L'organoïde 3D est construit en utilisant un matériau de gel aqueux et une molécule d'adhésion. Le matériau de gel aqueux est un hydrogel. La molécule d'adhésion est ICAM-1 ou la galectine-3. L'organoïde 3D est construit par formation d'une charpente avec un matériau de gel aqueux, suivi de l'addition d'un mélange de la cellule tumorale et d'une molécule d'adhésion.
PCT/US2018/067780 2017-12-28 2018-12-28 Procédé de diagnostic in vitro de prédiction de l'efficacité d'un médicament WO2019133767A1 (fr)

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Citations (2)

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