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WO2019133767A1 - A method of in vitro diagnostic for prediction of drug efficacy - Google Patents

A method of in vitro diagnostic for prediction of drug efficacy Download PDF

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Publication number
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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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
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    • 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
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    • 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
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    • 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
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    • 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

A 3D organoid is used for diagnosis or assay, wherein the 3D organoid is constructed from a tumor cell. The tumor cell in the 3D organoid is from a cell line, from circulating tumor cells isolated from a patient, or from a tumor tissue. The 3D organoid is constructed using an aqueous gel material and an adhesion molecule. The aqueous gel material is hydrogel. The adhesion molecule is ICAM-1 or galectin-3. The 3D organoid is constructed by forming a scaffold with an aqueous gel material, followed by adding a mixture of the tumor cell and an adhesion molecule.

Description

A METHOD OF IN VITRO DIAGNOSTIC FOR PREDICTION OF DRUG EFFICACY
BACKGROUND OF INVENTION Field of the Invention
[0001] The present invention relates generally to diagnostic or assay methods for predicting drug efficacies or effects.
BACKGROUND OF INVENTION
[0002] Traditional 2D cell culture in preclinical studies can provide many important drug efficacy predictions. However, in the clinic, a patient's response to drugs or the expression of genes are very different from those in the preclinical settings. Therefore, new 3D organoid systems can provide improved drug testing environments and provide cell models that mimic the clinical environment for illustration of the drug action.
[0003] 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.
[0004] 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.
SUMMARY OF THE INVENTION
[0005] 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.
[0006] One aspect of the invention relates to methods for drug efficacy or reaction assays using a 3D organoid of the invention. 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.
[0007] Other aspects of the invention will become apparent with the following detailed description and the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows improvements of 3D organoids cultures by adding adhesion molecules. Adhesion molecule (galectin-3, Gal-3) at concentrations of 0.3 pg/rnL, 0.6 pg/rnL, 1.25 pg/rnL, and 2.5 pg/mL. In the presence of the adhesion molecule, the 3D organoids are about 50% larger in sizes. In addition, NCI-H727 cells form ribbon-shaped structures in the presence of adhesion molecule. These results indicate that the adhesion molecules can support the formation of larger 3D organoids.
[0009] 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. With the addition of Gal-3 (adhesion molecules), the formation of 3D organoids is further enhanced. With the addition of endothelial cells (EC), formation of blood vessel is observable.
[0010] 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. In most lung cancer cell lines, the expression levels of EGFR, PIK3CA and KRAS are relatively low in 3D organoid culture, as compared with those in the 2D culture system.
[0011] FIGs. 4A-4C show different effects of drug treatments in 2D cell cultures versus
3D organoids. 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. PLX3397 treatment did not show dose-dependent effects in A549 and H1975 cells. 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.
[0012] 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. 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. This inconsistency is shown to be due to the inaccurate results from the 2D cell culture system.
[0013] 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. In contrast, Afatinib at 5 mpk or 20 mpk (mg/kg) are very effective in inhibiting tumor growth. These results are consistent with those observed using the 3D organoids of the invention, thereby validating the 3D organoids of the present invention for drug efficacy testing.
[0014] FIG. 7 shows results of 3D organoids established with patient-derive xenograft.
Addition of Gal-3 produced better organized tumor mass, as compared to the ones without added adhesion molecule. [0015] FIG. 8 shows 3D organoid models containing blood vessel cells in three separate experiments (test 1, test 2, and test 3).
DETAILED DESCRIPTION
[0016] 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.
[0017] 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.
[0018] 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.
[0019] Inventors of the invention found that adding adhesion molecules to the culture can greatly improve 3D organoid formation. The adhesion molecules may be any suitable adhesion molecules known in the art, such as ICAM-l and/or galectin-3.
[0020] 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. As an example, using an imaging system, the effects of a drug may be analyzed using a microscope and fluorescence labeling, luminescence, or white light. [0021] To test drug efficacies, 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. Alternatively, 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.
[0022] For drug efficacy assays, 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.
[0023] In addition, one may use optical methods to image 3D organoid cells to understand the drug effects. For example, one may use single-molecule RNA FISH to monitor a particular gene expression that is known to be impacted by the drug. One may use a bio imaging system 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.
[0024] In accordance with embodiments of the invention, 3D organoids may be used to establish drug dosage relationship. As an example, 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.
[0025] For example, 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.
[0026] Embodiments of the invention will be further illustrated with the following specific examples. One skilled in the art would appreciate that these examples are for illustration only and are not intended to limit the scope of the invention. One skilled in the art would appreciate that modifications and variations from these examples are possible without departing from the scope of the invention.
Example 1 : Galectin-3 improves 3D tumor organoid formation [0027] 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 xlO5 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.
[0028] As shown in FIG. 1, on day 4, 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. In the presence of the adhesion molecule, the 3D organoids are about 50% larger in sizes. In addition, NCI-H727 cells form ribbon-shaped structures in the presence of adhesion molecule. These results indicate that the adhesion molecules can support the formation of larger 3D organoids.
Example 2: Hydrogel and Galectin-3 can stimulate the formation of blood vessel 3D
organoids
[0029] Hydrogels can be prepared with different ratios of collagen and PEG, such as
1 :2, 1 :4, 1 :6, and 1 :8 (collagen : PEG). For example, 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.
[0030] Prepare a homogeneous mixture of 1 c 105 cells/mL tumor cells, 2>< l04 cells/mL blood vessel endothelial cells (tumor cell : blood vessel endothelial cell = 5: 1) and 1 pg/mL galectin-3. Inject this homogenous mixture into the hydrogel prepared above. Place the hydrogel in a cell culture incubator (37 °C, 5% C02) to incubate. The growth progress is monitored with an automatic multi-function optical imaging system (Cytation 5, Bio-Tek, ETSA). FIG. 2 shows the 3D organoid formation progress with time, with and without Gal-3 and with or without EC (endothelial cells).
[0031] As shown in FIG. 2, hydrogels prepared with collagen : PEG ratios of 1 :4, 1 :6, and 1 :8 can better support cell mass (organoid) formations. With the addition of Gal-3 (adhesion molecules), the formation of 3D organoids is further enhanced. With the addition of 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
[0032] In this example, 4 different lung cancer cell lines (HCC-827, NCI-H727, A549, and H1975) from in vitro cultures are used to establish 2D cell and 3D organoid culture systems. The procedures are as follows.
[0033] Plate 1 xlO5 cell/mL lung cancer cells with specific concentrations of adhesion molecules in 96-well low-attachment plate to culture the 3D organoids. The sizes of the 3D organoids were observed and recorded under microscope (see FIG. 1).
[0034] To establish 2D cell culture model, the procedures are as follows: plate 4 human lung cancer cell lines (1 xlO5 cell/mL) separately in a 24-well plate. Incubate the plate under the conditions of 5% C02, 37°C overnight. Confirm that the cells have completely covered the wells under microscope, and then collect the cells.
[0035] To establish 3D organoid model, the procedures are as follows: plate 4 human lung cancer cell lines (6 xlO5 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, collect the 3D orgnoid cell groups.
[0036] After collecting the cells from the 2D cell culture and 3D organoid, perform total RNA extractions from these cells, respectively. Then, synthesize cDNA from the total RNA to construct the cDNA form.
[0037] Using Real-time Quantitative Polymerase Chain Reaction (qPCR) to analyze the gene expression levels for PIK3CA, EGFR, and KRAS in the cDNA from the 2D cell culture and 3D organoid.
[0038] 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
Test methods:
[0039] 2D cell culture: plate 4 human lung cancer cell lines (1 xlO5 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% C02, 37°C for 24 hours.
[0041] 3D organoid culture: plate 4 human lung cancer cell lines (6 xlO5 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.
[0042] After collecting the cells from the 2D cell culture and 3D organoid, perform total RNA extractions from these cells, respectively. Then, synthesize cDNA from the total RNA to construct the cDNA form.
[0043] Using Real-time Quantitative Polymerase Chain Reaction (qPCR) to analyze the gene expression levels for PIK3CA, EGFR, and KRAS in the cDNA from the 2D cell culture and 3D organoid. The expression levels of PIK3CA, EGFR, and KRAS after drug treatments are shown in FIGs. 4A-4C.
[0044] As shown in 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. PLX3397 treatment did not show dose-dependent effects in A549 and H1975 cells. 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.
[0045] These results clearly show that the 2D cell culture system and the 3D organoid system produce inconsistent results. As will be discussed in a later section, the results from 3D organoid system are validated with in vivo animal xenograft test, whereas the results from 2D cell culture systems are not supported by the in vivo animal model. Example 4: Assess the Therapeutic Effects of Drugs ETsing 3D Organoid Tumor Culture
System
[0046] For these tests, 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 lO4 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.
[0047] As an example of the drug efficacy test, 11 different chemotherapeutic agents, target therapeutics, and immune modulators (at 0, 0.01, 0.1, 1, and 10 nM), together with caspase 3/7 fluorescence detection kit reagents, were individually tested in the 2D cell culture and 3 -day old 3D organoid tumor systems. The reactions were allowed to proceed for 24 hours.
[0048] After 24 hours, the caspase 3/7 fluorescence yields in the 2D cell cultures and
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.
[0049] As shown in 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. This inconsistency is shown to be due to the inaccurate results from the 2D cell culture system, as in vivo animal xenograft model validates the results of the 3D organoid systems, but not the 2D cell culture system (see the later section and FIG. 6).
Example 5 : Validation of the 2D cell culture system and 3D organoid system using animal xenograft models
[0050] 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>< l06 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 mm3, the drug tests can begin.
[0051] 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.
[0052] As shown in FIG. 6, Pacitaxel at 5 mpk or 20 mpk was not effective in inhibiting tumor growth. In contrast, Afatinib at 5 mpk or 20 mpk (mg/kg) are very effective in inhibiting tumor growth. These results are consistent with those observed using the 3D organoids of the invention, thereby validating the 3D organoids of the present invention for drug efficacy testing.
[0053] Referring to FIG. 5B, 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
[0054] Inoculate colorectal cancer cells from a patient into a mouse to establish a xenograft. Take a piece of 300 mm3 colorectal cancer tissue from the xenograft and digest the tissue with collagenase NB4G (0.5 PZU/mL) for 40 minutes to separate the cells.
[0055] Wash the cells with HBSS buffer. Centrifuge and take the cell suspension for the experiments. About 5 c 104 cells were added into a hydrogel-containing cell culture medium in each well of a 96-well, and add galectin-3 (Gal-3, 1 pg/mL) to each well. Analyze the sizes of the tumor mass in the well over the next few days, using an automatic multi function optical imaging system (Cytation 5, Bio-Tek, USA). The results are shown in FIG. 7.
[0056] As shown in FIG. 7, the tumor mass grew substantially over 4 days, with or without added adhesion molecule (Gal-3). However, 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
[0057] A hydrogel prepared from collagen and PEG (MW = 7500; at a ratio of 1 :2) is placed in wells of a 96-well plate. Briefly, a cell culture medium (containing 10% bovine serum or 5% human serum), 3 mg/ml type I collagen ' 300 mg/ml PEG (7500 MW), and a reconstitution solution consisting of acetic acid and 10X MEM cell culture media are mixed well, and then the mixture is allowed to stand in a cell culture incubator for 20 minutes or longer to form the hydrogel.
[0058] Mix 1 x 105 cell/mL tumor cells, 2 x 104 cell/mL blood vessel endothelial cells
(tumor cells : blood vessel endothelial cells = 5: 1), and 1 ug/ml Gal-3. Inject the mixture into the hydrogel and incubate the plates in a cell culture incubator (37°C, 5% C02) to incubate for 2 days to allow the formation of tumor mass containing blood vessels.
[0059] Add 2ul of anti-CD3 l antibody [JC/70A] (Alexa Flour® 647) ab2l59l2 into each well of the plate from the above incubation to stain the blood vessel endothelial cell marker and allow the reaction to occur in the incubator for 60 minutes (in the dark).
[0060] Add 0.05% Triton XI 00 to permeate the cells and allow the reaction to proceed on ice for 15 minutes. Then, add 450nM DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride, D1306, Invitrogen™) staining agent to stain the cells. Allow the staining to proceed in the dark for 1 hr and place the vessel in the cell culture incubator for 12-16 hours.
[0061] Measure the fluorescence intensities for anti-CD31 and DAPI stainings using an automatic multi-function optical imaging system (Cytation 5, Bio-Tek, USA). The results are shown in FIG. 8.
[0062] As shown in FIG. 8, the procedures for 3D organoids have been repeated as test
1, test, and test 3. The images show the formation of blood vessel in the organoids. All three experiments produced consistent results.
[0063] The above examples clearly show that 3D organoids can be efficiently constructed with the help of 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. In addition, the 3D organoids can be established that include blood vessel formation to more accurately represent in vivo tumor microenvironments.
[0064] Embodiments of the invention have been illustrated with limited number of examples. However, one skilled in the art would appreciate that these specific procedures are for illustration only and other variations and modifications are possible without departing from the scope of the invention. Therefore, the invention scope should be determined by the attached claims.

Claims

CLAIMS What is claimed is:
1. A SD organoid for diagnosis or assay, wherein the SD organoid is constructed from a tumor cell and an adhesion molecule.
2. The SD organoid according to Claim 1, wherein the tumor cell in the 3D organoid is from a cell line, from circulating tumor cells isolated from a patient, or from a tumor tissue.
3. The 3D organoid according to Claim 2, wherein the 3D organoid is constructed using a
hydrogel material and the adhesion molecule.
4. The 3D organoid according to Claim 3, wherein the hydrogel is prepared with PEG and
collagen at a ratio of PEG : collagen = 1-25 : 1.
5. The 3D organoid according to any one of Claims 1-3, wherein the adhesion molecule is
ICAM-1 and/or galectin-3.
6. The 3D organoid according to Claim 5, wherein the adhesion molecule is at a concentration of 1 pg/mL -2.5 ug/mL.
7. The 3D organoid according to Claim 1, wherein the 3D organoid is constructed by forming a scaffold with a hydrogel material, followed by adding a mixture of the tumor cell and an adhesion molecule.
8. The 3D organoid according to Claim 7, wherein the aqueous gel material comprises PEG and collagen, wherein a ratio of PEG : collagen is 1-25 : 1.
9. The 3D organoid according to Claim 7, wherein the adhesion molecule is at a concentration of 1 pg/mL - 2.5 ug/mL.
10. The 3D organoid according to Claim 1, further comprising blood vessels formed from
endothelial cells in the 3D organoid.
11. A method for drug efficacy or reaction assay using the 3D organoid according to Claim 1, comprising adding a drug to the 3D organoid and observing an effect of the drug on cells in the 3D organoid.
12. The method according to Claim 11, wherein the effect of the drug is analyzed with an imaging system to analyze the biochemical activity and/or the expression levels of a gene or a protein.
13. The method according to Claim 12, wherein the effect of the drug is analyzed using
fluorescence labeling, luminescence, or white light.
IB
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Citations (2)

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