WO2017081260A1 - Procédé multiparamétrique, basé sur des sphéroïdes tridimensionnels de type multicellulaire, de classification de composés - Google Patents

Procédé multiparamétrique, basé sur des sphéroïdes tridimensionnels de type multicellulaire, de classification de composés Download PDF

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WO2017081260A1
WO2017081260A1 PCT/EP2016/077445 EP2016077445W WO2017081260A1 WO 2017081260 A1 WO2017081260 A1 WO 2017081260A1 EP 2016077445 W EP2016077445 W EP 2016077445W WO 2017081260 A1 WO2017081260 A1 WO 2017081260A1
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cells
cell
type
spheroid
dimensional
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Zoe WEYDERT
Jens M. KELM
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Insphero Ag
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • C—CHEMISTRY; METALLURGY
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    • 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
    • G01N33/5011—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
    • 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
    • G01N33/5044—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 involving specific cell types
    • G01N33/5067—Liver 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
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    • 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
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    • C12N2510/00—Genetically modified 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
    • 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

  • Drug discovery concepts mainly focus on efficacy and potency as their primary end points using either biochemical or simple monolayer-based cell culture screening concepts. However, the closer the native tumor microenvironment can be resembled, the higher the predictive power of the screening campaign.
  • the novel multi-parametric screening concept based on 3-D spheroids with incorporated sensor cells according to the present invention serves to discriminate either specific from non-specific drug response or detect metabolic-mediated cytotxicity.
  • Three-dimensional cell culture models have become increasingly popular and are thought to be a more accurate physiological representation of the in vivo situation as compared to cells grown on plastic surface in two-dimensional monolayers, where many cellular characteristics are impaired due to artificial conditions.
  • Three-dimensional model systems better reflect the histological, biological and molecular characteristics of, e.g., primary tumors with its tissue- specific architecture.
  • 3-D cell cultures are expected to have a significant impact on, e.g., the success of drug development programs as a selective bridge between simple monolayer cell cultures and in-vivo studies (Hickman et al., 2014; Lovitt et al., 2014).
  • additional cell types such as stromal cells
  • the resulting heterotypic cell-cell crosstalk can be investigated in heterotypic 3-D cell cultures.
  • cells which do proliferate in monolayer cultures do not proliferate in 3-D cultures enabling prolonged stable expression of a transient transduced reporter genes.
  • Gene expression profiles in 3-D cultures significantly differ from expression profiles seen in
  • 3- D culture systems can be grouped into different formats: scaffold- free multicellular aggregates, cells cultured on inserts, or scaffold-based 3-D culture systems (Kimlin et al, 2013).
  • the 3-D culture systems based on scaffold- free multicellular aggregates or spheroids are also referred to as "microtissues”.
  • Multicellular tumor spheroids resemble intervascular tumor microregions or micrometastases with respect to the tissue architecture, the volume growth kinetics, and the micromilieu (Friedrich et al., 2009; Mueller- Klieser, 2000).
  • poorly vascularized areas in solid tumors are characterized by irregular tissue architecture and proliferation as well as oxygen/nutrient gradients.
  • the cell-cell communication between cancer and stromal cells is known to promote cancer development, progression and metastasis (Kimlin et al., 2013; Zhang and Huang, 2011).
  • cytokines and growth factors secreted by cells from the tumor microenvironment have a profound effect on cancer cells (Zhang and Huang, 2011).
  • the co-cultivation of tumor and stromal cells in 3-D spheroids represents a simple approach to mimic cell-cell and cell- matrix interactions as seen in vivo (Kimlin et al., 2013).
  • the present invention relates to a three-dimensional spheroid comprising at least two types of cells, wherein the cells of at least one of said cell types have been subjected to transfection with a heterologous gene.
  • the three-dimensional spheroid is comprising at least one type of tumor cells and at least one type of non-proliferating cells, wherein the cells of at least one type of said non-proliferating cells have been subjected to transfection with a heterologous gene.
  • the term spheroid refers to microtissues of cells growing and/or interacting within their surroundings in all three dimensions in an artificially-created environment.
  • Such microtissues can comprise a plurality of homotypic or heterotypic cells, preferably mammalian cells, more preferably human cells.
  • Such 3-D cell cultures more closely resemble the in vivo surroundings of the cells as compared to 2-D cell cultures.
  • Spheroids provide a more accurate model system for cellular, physiological and/or pharmaceutical studies than cells grown in conventional two-dimensional cultures.
  • the three-dimensional spheroid comprises two types of non-proliferating cells wherein said two types of non-proliferating cells are hepatocytes and non-hepatic cells, respectively, and wherein the non-hepatic cells have been subjected to transfection with a heterologous gene.
  • the hepatocytes can be either of primary origin or a cell line, e.g., HepaRG cells.
  • the hepatocytes can also be selected from the group consisting of embryonic- derived stem cells, induced pluripotent stem cells (IPS), and Lgr-5 -positive hepatocyte stem cells.
  • such three-dimensional spheroid as described above has been subjected to transfection, wherein said transfection is a transient transfection.
  • said transient transfection is performed by means of electroporation.
  • said transient transfection is performed by means of nucleofection using a NucleofectorTM.
  • transfection means the introduction of foreign DNA into the nucleus of eukaryotic cells, or of RNA into eukaryotic cells.
  • Transfection can be mediated by various methods including, but not limited to, calcium phosphate precipitation, DEAE-dextran method, the use of lipids, liposomes, cationic polymers, activated dendrimers, or magnetic beads, NucleofectorTM technology, electroporation, microinjection, "gene gun” technologies or viral vector-based transfer (Kim and Eberwine, 2010).
  • foreign DNA is delivered to the nucleus by passage through the cell and nuclear membranes, is integrated into the host genome, and is sustainably expressed.
  • transient transfection foreign DNA is delivered into the nucleus of eukaryotic cells but is not integrated into the genome, or foreign RNA is delivered into the cytosol where it is translated.
  • Gene expression is usually limited to a certain period of time in transient transfection; in proliferating cells, the transfected nucleic acid is getting diluted out over time.
  • Expression systems with episomal (extrachromosomal) replication of the transiently transfected nucleic acid have been developed to compensate for this disadvantage.
  • Transient transfection of cells for the generation of three-dimensional spheroids according to the present invention offers several advantages over stable transfection.
  • Transient transfection methods are usually less time-consuming and overall less expensive.
  • Transient transfections yield less phenotypic changes of the cells, as compared to stable transfectants with foreign nucleic acid integrated into the host genome at various and usually random positions.
  • transient transfectants will resemble more closely untreated cells in their biochemical, physiological and behavioral characteristics.
  • transient transfection does not require the addition of antibiotics to the cell culture medium which is expensive and also often has an influence on the cell physiology.
  • primary cell types such as neuronal cells or hepatocytes
  • NucleofectorTM devices are hardly or not at all amenable to stable transfection, but can be transiently transfected with high efficiency, particularly by use of NucleofectorTM devices.
  • Such primary cells are of particular interest for the generation of spheroids. Since most primary cell types do not significantly replicate in cell culture, the loss of nucleic acid delivered by transient transfection over time due to episomal segregation is less of an issue in spheroid systems using primary cells.
  • transfection methods mentioned above yield different transfection efficiencies with regard to the number of transfected cells (transfectants) per total number of cells subjected to transfection.
  • said transient transfection yields an efficiency of at least 50% of transfected cells.
  • the heterologous gene transfected into at least one cell type forming the three-dimensional spheroid is a reporter gene.
  • This reporter gene is encoding a gene product which is secreted or not secreted from the cell.
  • This reporter gene can serve, e.g., for monitoring of the survival and/or vitality of said transfected cells via expression of a reporter protein which can be qualitatively or quantitatively measured.
  • heterologous gene refers to a gene, fragment of a gene, or expression cassette enabling the cell subjected to transfection with the same to performing heterologous protein expression.
  • Heterologous protein expression refers to expression of a protein which is not normally expressed in the respective cell type, tissue or species of origin of the cell transfected, or which is normally expressed only at lower levels in the respective cell type, tissue or species of origin of the cell transfected.
  • reporter gene refers to a gene, fragment of a gene, or expression cassette, whose presence or expression can be easily detected after it has been introduced into a cell, a tissue or an organism.
  • a reporter gene indicates its presence and expression in said cell, tissue or organism.
  • expression cassette relates particularly to a nucleic acid molecule and a region of a nucleic acid molecule, respectively, containing a regulatory element or promoter being positioned in front of the coding region, a coding region and an open reading frame, respectively, as well as a transcriptional termination element lying behind the coding region.
  • the regulatory element and the promoter, respectively, residing in front of the coding region can be a constitutive, i.e., a promoter permanently activating the transcription (e.g., CMV promoter), or a regulatable promoter, i.e., a promoter which can be switched on and/or off (e.g., a tetracycline regulatable promoter).
  • the coding region of the expression cassette can be a continuous open reading frame as in the case of a cDNA having a start codon at the 5' end and a stop codon at the 3' end.
  • the coding region can be comprised of a genomic or a newly combined arrangement of coding exons and interspersed non-coding introns.
  • the coding region of the expression cassette can be comprised of several open reading frames, separated by so-called IREs (Internal Ribosome Entry Sites).
  • said reporter gene can be selected from the group comprising genes encoding and expressing Luciferase, NanoLUC luciferase, green fluorescence protein (gfp), red fluorescence protein (rfp), secreated alkaline phosphatase (seap), beta-galactosidase (lacZ), beta-glucuronidase (gus), neomycin phosphotransferase (neo), and chloramphenicol acetyltransferase (cat).
  • NanoLUC luciferase is a small (19.1 kDa), engineered enzyme originating from the deep seashrimp, Oplophorus gracilirostris (Hall et al., 2012). It uses a coelenterazine analogue called furimazine in an ATP-independent reaction to produce glow-type luminescence. Secreted NanoLUC luciferase has been shown to be stable in culture medium for more than 4 days at 37 °C. The bright luminescence and the stability of NanoLUC make it well suited for high-throughput screening applications.
  • the three-dimensional spheroid as described above is comprising at least one type of tumor cells selected from the group consisting of primary tumor cells of a patient, immortalized or transformed cells, cells of an established tumor cell line, and/or tumor cells from patient-derived xenografts (PDX).
  • Said cells of an established tumor cell line can be selected from the group comprising SKOV-3, HEY or PANC-1 cells.
  • primary cell refers to cells that were obtained by direct removal from an organism, an organ or a tissue and put in culture. Most primary cells exhibit only a limited life span in cell culture.
  • primary tumor cells refers to cells that were obtained or derived from cells that were obtained by direct removal from tumor tissue of a patient.
  • cell line refers to cells which are genetically modified in such a way that they may continue to grow permanently in cell culture under suitable culture conditions. Such cells are also called immortalized cells.
  • the three-dimensional spheroid is comprising at least one type of non-proliferating cells which is selected from the group consisting of primary cells and cells of an established cell line.
  • the three-dimensional spheroid is comprising cells of established fibroblast cell lines from either animal species such as mouse NIH3T3 or human such as SV80, or primary fibroblasts and/or derivatives and/or transfectants thereof.
  • NIH3T3 cells transfected with a reporter gene and used as components of three- dimensional spheroids do not proliferate any more, thus continuing expression of the reporter protein for a long time throughout cultivation of the spheroids. Therefore, these cells are particularly well suited as model system, representing non-proliferating stroma cells in basic research, diagnostic assays, drug screening and development.
  • the three- dimensional spheroid is comprising SKOV-3 cells or HEY cells or PANC-1 cells and NIH3T3 cells, wherein the NIH3T3 cells have been subjected to transfection with a NanoLUC luciferase gene.
  • the present invention also relates to a method for production of a three-dimensional spheroid as described above comprising at least the following steps: a) Transfecting at least one type of non-proliferating cells,
  • the method for production of a three-dimensional spheroid is performed as described above, wherein transfection of at least one type of non- proliferating cells is performed in adherent culture or in suspension culture.
  • the method for production of a three-dimensional spheroid is performed as described above, wherein said type of non-proliferating cells transfected in step a) are non-hepatic cells, and wherein said cell suspension prepared in step b) comprises hepatocytes and said transfected non-proliferating cells.
  • the method for production of a three-dimensional spheroid is performed as described above, wherein the spheroid is generated by a hanging drop technique.
  • the hanging drop technique is a well-established cell culture method to produce scaffold free, three-dimensional multicellular spheroids.
  • the GravityPLUSTM 3D Culture and Assay Platform (InSphero, Schlieren, Switzerland) enables reliable, automation-compatible and affordable 3-D cell culture in hanging drops and is therefore suitable for high-throughput screening applications.
  • the present invention relates to the use of a three-dimensional spheroid as described above for the screening of potentially therapeutic agents for an effect on at least one type of tumor cell.
  • said potentially therapeutic agent can be, e.g., a natural or synthetic and/or recombinant substance, or any combination thereof; the latter would be a substance produced by recombinant expression technology or synthetic peptide synthesis, such as, e.g., a recombinant antibody, fragment or derivative thereof, fusion protein, peptide, or antibody- drug conjugate.
  • Said potentially therapeutic agent can also be a chemotherapeutic substance.
  • the agents referred to can have an effect on or influence one or several potential drug targets relating to hallmarks of cancer such as, for example, the self-sufficiency in growth signals (e.g., EGF receptor, IGF-1), insensitivity to anti-growth signals (e.g., p53, Cdc25), evasion of apoptosis (e.g., Bcl-Xl, Survivin), limitless replicative potential (e.g., Telomerase), sustained angiogenesis (e.g., VEGF), or tissue invasion and metastasis (e.g., MPPs, MAPK4).
  • Said effect which potentially therapeutic agents are screened for using a three-dimensional spheroid as described above, relates to the growth of said at least one type of tumor cell according to one embodiment of the present invention.
  • the term therapeutic window also called therapeutic index, refers to a comparison of the amount of a therapeutic agent that causes toxicity to the amount that causes the therapeutic effect.
  • a high therapeutic window or index is preferable for a drug to have a favorable safety profile.
  • the therapeutic window can be expressed as ratio of the dose that causes adverse effects at an incidence/severity not compatible with the targeted indication (e.g., toxic dose in 50% of subjects) divided by the dose that leads to the desired pharmacological effect (e.g., efficacious dose in 50%> of subjects).
  • a therapeutic index based on cytotoxicity is calculated measuring specific effects on the tumor cell population and on the non-proliferative cell, e.g. fibroblast, population within the spheroid (see Table 1).
  • CTD Cytotoxic dose IC50Fibrobiast s ; ED Effective dose (IC50rumo r ceils derived from dose response curves); TI in ⁇ : therapeutic index derived from in vitro models
  • a three-dimensional spheroid as described above also relates to the high- throughput screening of a library of potentially therapeutic agents.
  • Said library of potentially therapeutic agents is understood herein as a set of several, many or a large number of agents which are screened subsequently or in parallel for the respective effect.
  • Such libraries can be, for example, libraries of synthetic chemical compounds, libraries of complex natural compounds, libraries of extracts from plants or microorganisms, phage display libraries, yeast display libraries, or ribosomal display libraries.
  • the present invention also provides for a method for screening a library of potentially therapeutic agents comprising at least the following steps: a) Producing a three-dimensional spheroid as described above,
  • the method for screening a library of potentially therapeutic agents as described above can optionally comprise a step d) performing a multi-parametric compound classification considering at least two parameters including the in vitro-based therapeutic index.
  • multi-parametric refers to a set of measurable parameters, from at least each of the two cell populations to assess and classify the response for a substance in a biological system.
  • Said method for screening can be applied for development of a drug suited for therapy of an oncologic disease in one embodiment of the present invention.
  • the present invention provides for a method for screening as described above, wherein said spheroid comprises tumor cells, or derivatives thereof, from a patient and wherein the method is applied as a diagnostic assay in vitro for selection of chemotherapeutic drugs suitable for treatment of an oncologic disease of said patient.
  • Table 1 Exemplary Work Flow of the method for multi-parametric compound classification of a library of potentially therapeutic agents
  • the following table delineates the scheme used according to the present invention for selection of suitable therapeutic agents, comprising the production of microtissues, compound testing for efficacy and safety, multi-parametric classification of potentially therapeutic agents, pathway identification and hit selection.
  • Table 2 Exemplary Work Flow of the method of hit selection
  • Step 1 Production of transiently Transient transfection does transfected, non-proliferative not interfere with genome, cells for the formation of phenotype, respectively. microtissues; Enables flexible use of nucleofection. lipofection, various reporter systems and
  • Step 2 Production of multi-cell type Co-culture model which tumor spheroids comprising a enables discrimination of non-proliferative reporter- cancer specific and harboring sensor cell and unspecific cytotoxicity.
  • PSA prostate cancer
  • the method for screening a library of potentially therapeutic agents as above comprises the use of a defined number of transfected non-proliferating cells, preferably fibroblasts, for standardization as a quality control.
  • the present invention refers to a kit comprising at least one type of non- proliferating cells which have been subjected to transfection with a heterologous gene, at least one potentially therapeutic agent, and a protocol for performing the method of screening as described above.
  • the NucleofectorTM (Lonza Cologne GmbH) technology has been used. This non-viral technology is based on a cell type specific combination of electrical parameters and solutions (Gresch et al., 2004). The method allows the introduction of foreign DNA into cells via break down areas of cell membrane through electric pulses. Cells can be efficiently trans fected by electroporation and show high viabilities.
  • the NIH3T3 cell line is a mouse embryonic fibroblast cell line established in 1962 which has become the standard fibroblast cell line.
  • the NIH3T3 cells were cultivated in DMEM medium with 10% FBS, 1% Pen/Strep and 25 mM Hepes.
  • NanoLUC luciferase In order to discriminate stromal cells from cancer cells in heterotypic microtissue models, a reporter gene was introduced into the fibroblasts in order to secrete NanoLUC luciferase.
  • the gene sequence encoding NanoLUC is designed by optimizing codon usage for expression in mammalian cells. Additionally, in order to monitor reporter expression without cell lysis, the secretion signal from human IL-6 is added to the N-terminus of NanoLUC.
  • the cells were trypsinized and an aliquot of lxl 0 6 cells was resuspended in 100 ⁇ 4DNucleofectorTM Solution. 1 ⁇ g of pNL1.3CMV[secNluc] luciferase reporter vector (Promega) was added and the suspension was transferred into a NucleocuvetteTM. The NucleocuvetteTM was placed into the 4D-NucleofectorTM X Unit. The cells were transfected with program EN- 158. After transfection the cells were plated in cell culture medium into a T-flask. For separation of dead cells, the flask was incubated overnight and vital cells were allowed to adhere to the bottom. The vital cells were subsequently used for the microtissue production process (Fig. 2).
  • Example 2 Transient transfection of primary human cells with a fluorescent
  • GFP green fluorescent protein
  • InSphero's hanging drop microtissue production platform GravityPLUS TM allowed for the formation of homo- and heterotypic scaffold- free 3-D microtissues in a 96-well plate format. After formation the microtissues were transferred into InSphero's 96-well GravityTRAPTM plate for longtime cultivation and compound treatment. For morphological investigations, microtissues are easily amenable to immunostaining.
  • the HEY cell line is a human ovarian carcinoma cell line and was derived from a xenografted tumor (HX-62). The ovarian cancer xenograft was originally grown from a peritoneal deposit of a moderately differentiated papillary cystadenocarcinoma of the ovary.
  • the cell line HEY-GFP (obtained from NIH) stably expresses GFP.
  • the HEY-GFP cells were cultivated in RPMI 1640 medium with 10 % FBS, 1 % Pen/Strep and 25 mM Hepes.
  • the SKOV-3 cell line is a human ovarian cancer cell line and has been established from a 64- years old female patient suffering from ovarian adenocarcinoma.
  • the SKOV-3 cells were cultivated in DMEM medium with 10 % FBS, 1 % Pen/Strep, 1 % NEAA and 25 mM Hepes.
  • the PANC-1 cell line is a human pancreatic cancer cell line and has been established from a 56-years old male patient suffering from pancreatic epithelioid carcinoma.
  • the PANC-1 cells were cultivated in DMEM medium with 10 % FBS, 1 % Pen/Strep and 25 mM Hepes.
  • the cells were trypsinized and a cell suspension of the desired cell density (HEY-GFP: 100 cells/drop, SKOV-3: 200 cells/drop, PANC-1 : 200 cells/drop, NIH3T3: 5,000 cells/drop) was prepared in aggregation medium (DMEM medium with 10% horse serum, 1% Pen/Strep and 25 mM Hepes).
  • a cell suspension containing both, fibroblasts and the respective cancer cells was prepared in the aggregation medium. The cell suspension was seeded into the InSphero GravityPLUSTM plate by pipetting 40 ⁇ suspension per well.
  • Culture time information will refer to the total time in culture of the cells, starting at day 0, the day of seeding the cells into the GravityPLUSTM plate.
  • microtissues were transferred three days after seeding from the GravityPLUSTM into the GravityTRAPTM plate.
  • a medium exchange with microtissue maintenance medium was performed twice a week to prevent the microtumors from nutrient starvation.
  • bright field (BF) images were taken on a regular basis.
  • the compound treatment of the microtissues was initiated at the day of the transfer from the GravityPLUSTM into the GravityTRAPTM plate. Compounds were tested within a concentration range of 2.05 pM - 20 ⁇ (11 doses, 1 :5 dilution series).
  • the vehicle control group consisted of microtissues treated with 1% DMSO in microtissue maintenance medium. At treatment days 3 and 5, a re-dosing was performed and at day 7 the cell viability of the microtissues was measured as a final endpoint. Size and NanoLUC luciferase activity in the supernatant were measured at day 3, 5 and 7.
  • the treatment was started at the transfer day (culture time: day 3), re-dosing of the microtissues was performed 72 and 120 hours after the start of the treatment (treatment time: day 3, day 5).
  • the Dainippon SCREEN Cel iMager CC-5000 was used which allowed for scanning microtissues in the InSphero GravityTRAPTM directly.
  • the plates were scanned at treatment days 3, 5 and 7.
  • the integrated analysis software enabled automated measuring of the microtissue size, including accurate discrimination of microtissues and cell debris which might occur after compound treatment.
  • the fibroblasts were transfected with a reporter gene in order to secrete NanoLUC ® luciferase.
  • the secreted NanoLUC ® luciferase was measured 3, 5 and 7 days after the treatment started with the Nano-Glo ® Luciferase Assay (Promega) system in the culture medium.
  • the Nano-Glo ® Luciferase Assay was performed according to Promega' s manual "Nano-Glo ® Luciferase Assay System " (Promega, 2013).
  • the medium was removed from the microtissues and an aliquot of 20 ⁇ was transferred into a 96-well Half Area white plate (Greiner).
  • One volume of Nano-Glo ® Luciferase Assay Reagent equal to the volume of the sample, was added and mixed for optimal consistency. After 3 minutes of incubation time the luminescence was measured with the Tecan M200Pro (integration time: 500 ms). For analysis of the data the values from treated microtissues were normalized to the vehicle control.
  • the cell viability of the microtissues was detected by measuring the ATP content using the CellTiter-Glo ® Luminescent Cell Viability Assay (Promega).
  • the procedure for the CellTiter-Glo ® 3-D Cell Viability Assay can be summarized briefly as follows:
  • the complete culture medium was aspirated.
  • CellTiter Glo ® Reagent was mixed 1 : 1 with microtissue maintenance medium, and 40 ⁇ of the diluted reagent was added to the microtissues and mixed.
  • Microtissues and supematants were transferred into a Half Area white assay plate (Greiner). The assay plate was wrapped in aluminum foil and incubated for 20 minutes at room temperature while shaking horizontally. Luminescence was measured with the Tecan M200Pro (integration time: 500 ms). For analysis of the data the values from treated microtissues were normalized to the vehicle control.
  • Ytop is the Y value at the top plateau;
  • Hill Slope variable also called the Hill Slope coefficient, describes the steepness of the curve.
  • HEY/NIH and SKOV/NIH microtissues a homotypic ovarian model system with HEY-GFP cancer cells, referred to as HEY microtissues, was established.
  • the ovarian HEY and SKOV-3 cell lines were considered as being representative of the serous papillary (HEY) and clear cell (SKOV) histological subtypes of epithelial ovarian cancer. These cell lines are well characterized and frequently used for in vitro ovarian cancer models.
  • the cancer cell lines were initially derived from solid tumors and were therefore expected to form multicellular spheroids when cultured in hanging drops.
  • the ovarian and pancreatic co-culture models were established to mimic tumor-stroma interactions.
  • NIH3T3 fibroblasts were chosen as stromal cells. Fibroblasts represent the major cell type in cancer associated stroma in vivo, and NIH3T3 have been successfully applied in various co-culture experiments.
  • microtissue formation The accumulation of the respective cells in hanging drops resulted in microtissue formation within 3 days.
  • the spheroids were either harvested for histological analysis or transferred into a non-adhesive spheroid-specific 96-well plate for long-term culture (GravityTRAPTM, InSphero).
  • the ATP content was measured regularly (CellTiter-Glo ® , Promega) over a culture period of up to 13 days.
  • microtissue size was monitored over time by light microscope and plate scanning (Dainippon SCREEN Cel iMager, Dainippon). Characterization of internal architecture of the microtissues was achieved by IHC staining. Expression of the reporter NanoLUC ® luciferase was assessed as described in Example 1.
  • spheroids were examined by light microscopy (see Fig. 1). During the course of cultivation, heterotypic tumor spheroids exhibited a spherical geometry with a central core of non-proliferating fibroblasts and proliferating cancer cells in the periphery. In bright field images, the stromal core can be seen as a dark circular area in the center of the spheres.
  • tissue size, ATP content and morphology were analyzed over 7 days after spheroid formation and subsequent transfer of the tissues in the receiver plate.
  • the first co-culture area calculation was done on day 3, where an area of 101,117 ⁇ 5882 ⁇ 2 for SKOV/NIH microtissues and an area of 129,537 ⁇ 11 ,797 ⁇ 2 for PANC/NIH spheroids were measured.
  • the area of these cultures increased to 262,963 ⁇ 12,660 ⁇ 2 , and to 287,348 ⁇ 10,088 ⁇ 2 , respectively.
  • the viability and growth data obtained from the three heterotypic microtissue model systems supported the assumption of direct proportionality between microtissue size and intra-tissue ATP levels.
  • heterotypic microtissues (HEY/NIH, SKOV/NIH, PANC/NIH, respectively) were stained for histological characterization. Cancer and stromal cells were capable of reforming heterotypic, solid spheroids, as shown by hematoxylin and eosin staining (Fig 1). Eosin colors eosinophilic structures in various shades of red, pink and orange, whereas hematoxylin colors nuclei of cells blue. After 11 days of culture, tumor microtissues formed a necrotic area in the center of the spheres, composed of cells undergoing apoptosis and/or necrosis, most likely due to hypoxia.
  • the spheroids showed high expression of EGRF within the cancer cells (see Fig. 1, shown in black). Heterotypic tumor spheroids exhibited a spherical geometry with a central core of non-proliferating fibroblasts and proliferating cancer cells in the periphery.
  • NanoLUC ® luciferase reporter gene was introduced into the fibroblasts.
  • stromal fibroblasts Prior to spheroid formation, stromal fibroblasts were transiently transfected with CMV-driven constructs encoding secreted NanoLUC ® luciferase by using the NucleofectorTM technology. Transfected cells were referred to as NIHnanoLUC.
  • NIHnanoLUC In order to assess microtissue stability and NanoLUC expression over time, homotypic microtissues consisting of transfected stromal cells were analyzed in a first step.
  • NIHnanoLUC cells were used to produce microtissues of 3 different sizes, consisting of 5000, 2500 or 1250 cells, respectively. Tissue size, intra-tissue ATP content and NanoLUC ® expression over time were measured. The monitored ATP contents of the homotypic microtissues decreased slightly over a time period of 7 days. The decrease in ATP correlated with slightly decreasing spheroid sizes. Secreted NanoLUC ® was measured between media exchanges and the recorded relative luminescence values were calculated according to NanoLUC ® secretion per microtissue over 24 hours.
  • NanoLUC ® signal decrease over time could be observed. On the average, the signal declined by 8.19% of the respective initial values per day. Due to the dynamic characteristics of the system, subsequent NanoLUC ® data of treated microtissues were always normalized to the respective vehicle controls.
  • NIHnanoLUC cells were seeded simultaneously with cancer cells.
  • the formed spheroids were compared with respect to growth profile and ATP content over time to their non-transfected counterparts (see Fig. 2A, B, C).
  • the characterization of the microtissue models confirmed a direct proportionality between viability and growth data. Histology data indicated the analogy of microtissue architecture of intervascular tumor microregions and 3-D multicellular tumor spheroids. In addition, expression of some proteins seemed to be upregulated in heterotypic spheroids as compared to homotypic microtissues.
  • the NanoLUC ® expression profiles allowed for the assumption of the linear relationship between number of transfected stromal cells and NanoLUC luciferase signal, without being affected when co-cultivated with cancer cells. Taken together, all these data indicate stable and reproducible model systems, suitable for high-throughput screening. 5.2 Drug testing with tumor microtissues
  • the homotypic ovarian microtissue model system with HEY-GFP cancer cells (HEY) and the three heterotypic microtissue models (HEY/NIH, SKOV/NIH and PANC/NIH) were used for compound screening with potential anti-cancer agents.
  • the treatment of microtissues with compounds was initiated at the day of the transfer from the GravityPLUSTM into the GravityTRAPTM plate.
  • the size of the microtissues and the secreted NanoLUC ® luciferase was measured 3, 5 and 7 days after the treatment had started. At treatment days 3 and 5, a re- dosing was performed and at day 7 the cell viability of the microtissues was measured as a final endpoint.
  • the predictive value of phenotypic drug testing depends on how close the in vivo environment and the biomarker used to assess the clinical response can be mimicked. The closer the tissue environment and the respective disease progression can be mimicked, the better the predictive value of an assay to discover and develop new therapeutics.
  • 3D model systems can better reflect the cell composition, tissue structure, and biological characteristics of primary tumors.
  • Homo- and heterotypic ovarian and pancreatic microtissue tumor models were used for a screening of up to 40 compounds selected from the NCATS Oncology library. The compounds were chosen to target different mechanisms driving tumor cell growth and survival.
  • the goal of this study was to develop a high throughput compatible drug screening assay based on 3D multicellular spheroids from ovarian (HEY and SKOV) and pancreatic (Panc-1) cancer which enables the discrimination of tumor-specific efficacy and unspecific cytotoxicity of a drug candidate with subsequent identification of the molecular mechanism of action (MM OA).
  • MM OA molecular mechanism of action
  • the biological response measured over a 10-day drug exposure period included (i) growth kinetic (microtissue size), (ii) potency (IC50ATP 10 days) and efficacy (max. response ATP and size).
  • the biological response of the compounds was compared between the different cell culture formats tested, 2D, 3D homotypic and 3D heterotypic.
  • the comparison of IC50 values among the different ovarian cancer cell cultures showed that 21 out of the 38 compounds tested were more potent in 3D than in 2D.
  • 13 out of 20 compounds tested were more potent in 3D than in 2D.
  • most of the compounds which showed stronger potency in 3D were targeted small molecule agents. Comparing drug responses of homo- vs heterotypic ovarian tumor model systems, 3 compounds were effective only in the heterotypic model including the WNT inhibitor PNU-74654 and GABA uptake inhibitor (Gat-1) SK&F- 89976A.
  • the therapeutic index is shown as the ratio adverse effect / therapeutic effect, for acute and subchronictoxicity of the compounds.
  • Figures Figure 1 HEY, HEY/NIH, SKOV/NIH and PANC/NIH microtissues were collected and stained for histological characterization after 6 days of culture.
  • the figure shows the staining of EGFR.
  • the spheroids show a high expression of EGRF within the cancer cells.
  • the IHC staining of EGFR indicated a slight upregulation of EGFR in heterotypic spheres, as compared to homotypic microtissues.
  • the heterotypic microtissues show a central core of non-proliferating fibroblasts (EGFR negative) and proliferating cancer cells (EGFR positive) in the periphery.
  • FIG. 2 Analysis of homotypic microtissues consisting of trans fected stromal cells in order to assess microtissue stability and NanoLUC expression over time. Microtissues were produced with 5000 ( ⁇ ), 2500 ( ⁇ ) and 1250 ( ⁇ ) cells respectively. After 3 days of spheroid formation, tissue size (A), intra-tissue ATP content (B) and NanoLUCsecretion (C) were monitored over 7 days. Each point represents the mean of 6 spheroids and their corresponding standard deviation.
  • FIG. 3 Primary dermal fibroblast (HDF) transiently transfected by Nucleofection with a green fluorescent protein driven by a constitutive promoter in a 3D microtissue model (HDF- GFP). Quantification of the relative fluorescence units (RLU) demonstrates stable fluorescence emission of microtissue composed of different ratios between fluorescent HDF- GFP and non-fluorescent HDF after an initial equilibration time. Using only 50% of green fluorescence cells leads to an approximate 50% reduction in signal intensity.
  • HDF Primary dermal fibroblast
  • RLU relative fluorescence units
  • Figure 4 Development over time of dose-response curves of HEY/NIH microtissues treated continuously over 7 days with the MEK inhibitor TAK-733.
  • the curves show the cancer- specific effect of the compound on the microtissues shown as tissue size at day 3, 5 and 7 as well as the unspecific cytotoxic effect of the compound shown as NanoLUC ® data at the respective time points. Tissue size and NanoLUC ® values were normalized to the respective vehicle controls.
  • Figure 5 Dose-response curves of treated HEY/NIH microtissues at day 5. The curves show the effect of the three compounds Torin-2, WAY-600 and Taxol on the tissue size at day 5, as well as the unspecific cytotoxic effect of the compounds shown as NanoLUC ® data at the same time point. Tissue size and NanoLUC ® values were normalized to the respective vehicle controls.
  • FIG. 6 Dose-response curves of HEY/NIH, SKOV/NIH and PANC/NIH microtissues treated continuously over 7 days with the ALK inhibitor TAE-684. The curves show the cancer-specific effect of the compound on the different microtissues shown as tissue sizes at day 5, as well as the unspecific cytotoxic effect of the compound shown as NanoLUC ® data at the same time point. Tissue size and NanoLUC ® values were normalized to the respective vehicle controls.
  • Figure 7 Analogy between intervascular tumor microregions and multicellular tumor spheroids. The figure illustrates the analogy between intervascular tumor microregions (a) and 3D multicellular tumor spheroids (b).
  • the pNL1.3CMV[secNluc/CMV] contains a CMV promotor, a SV40 late poly(A) region, a synthetic betalactamase (Amp 1 ) coding region and the NanoLUC ® IL6 (secNluc) reporter gene.

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Abstract

La présente invention concerne un procédé utilisant des sphéroïdes tridimensionnels comprenant au moins un type de cellules, comprenant de préférence au moins un type de cellules tumorales et au moins un type de cellules non tumorales, pour la classification de composés. Des cellules d'au moins un type desdites cellules non tumorales ont été soumises à une transfection avec un gène hétérologue et agissent comme capteur interne pour discriminer des effets spécifiques d'effets non spécifiques de médicaments. Ceci permet une classification et une sélection multiparamétriques de composés, incorporant non seulement une efficacité et une puissance mais également un indice thérapeutique généré in vitro et un impact sur la croissance tumorale. La présente invention concerne également des procédés de production desdits sphéroïdes tridimensionnels et de criblage de bibliothèques de composés dans le contexte de la mise au point de médicaments ou d'essais diagnostiques.
PCT/EP2016/077445 2015-11-11 2016-11-11 Procédé multiparamétrique, basé sur des sphéroïdes tridimensionnels de type multicellulaire, de classification de composés WO2017081260A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108486035A (zh) * 2018-03-26 2018-09-04 西北大学 一种三维类器官的液滴培养方法
WO2020104549A1 (fr) 2018-11-20 2020-05-28 Precomb Therapeutics Ag Procédé de développement de médicament combinatoire basé sur un modèle de cancer humain en 3d
WO2021016598A1 (fr) * 2019-07-25 2021-01-28 Immunowake Inc. Méthodes de mesure de la mort à médiation cellulaire par des effecteurs
WO2024010921A1 (fr) * 2022-07-08 2024-01-11 Slmp, Llc Témoins de référence de tissus humains mis au point par génie biologique et normalisés pour validation des résultats de tests d'immunohistochimie, d'hybridation in situ en fluorescence ou d'hybridation in situ en chromogène pour le dépistage du cancer

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012131000A1 (fr) * 2011-03-29 2012-10-04 Universiteit Leiden Procédé d'obtention d'un sphéroïde multicellulaire

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012131000A1 (fr) * 2011-03-29 2012-10-04 Universiteit Leiden Procédé d'obtention d'un sphéroïde multicellulaire

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
FRIEDRICH, J.; SEIDEL, C.; EBNER, R.; KUNZ-SCHUGHART, L. A.: "Spheroid-based drug screen: considerations and practical approach", NATURE PROTOCOLS, vol. 4, no. 3, 2009, pages 309 - 324
GRESCH, O.; ENGEL, F. B.; NESIC, D.; TRAN, T. T.; ENGLAND, H. M.; HICKMAN, E. S.; KORNER, I.; GAN, L.; CHEN, S.; CASTRO-OBREGON, S: "New non-viral method for gene transfer into primary cells", METHODS, vol. 33, no. 2, 2004, pages 151 - 163
HALL, M. P.; UNCH, J.; BINKOWSKI, B. F.; VALLEY, M. P.; BUTLER, B. L.; WOOD, M. G.; OTTO, P.; ZIMMERMAN, C.; VIDUGIRIS, G.; MACHLE: "Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate", ACS CHEMICAL BIOLOGY;, vol. 7, no. 11, 2012, pages 1848 - 1857
HERRMANN, R.; FAYAD, W.; SCHWARZ, S.; BERNDTSSON, M.; LINDER, S.: "Screening for compounds that induce apoptosis of cancer cells grown as multicellular spheroids", JOURNAL OF BIOMOLECULAR SCREENING, vol. 13, no. 1, 2008, pages 1 - 8
HICKMAN, J. A.; GRAESER, R.; DE HOOGT, R.; VIDIC, S.; BRITO, C.; GUTEKUNST, M.; VAN DER KUIP, H.: "Three-dimensional models of cancer for pharmacology and cancer cell biology: Capturing tumor complexity in vitro/ex vivo", BIOTECHNOLOGY JOURNAL, vol. 9, no. 9, 2014, pages 1115 - 1128
IVASCU, A.; KUBBIES, M.: "Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis", JOURNAL OF BIOMOLECULAR SCREENING, vol. 11, no. 8, 2006, pages 922 - 932
JENS KELM: "Get 3D InSightTM at SLAS2016", 23. - 27.01.2016 - SLAS 2016, 23 January 2016 (2016-01-23), XP055329783, Retrieved from the Internet <URL:http://insphero.com/company/fairs-conferences-and-events/23-27-01-2016-slas-2016> [retrieved on 20161216] *
JENS M KELM: "Studying gene function analysis in 3D tumor microtissue models", TELFORD | GENE FUNCTION ANALYSIS, 1 March 2014 (2014-03-01), XP055329574, Retrieved from the Internet <URL:http://elrig.org/downloads/ri14/Technologies-Innovations-for-Phenotypic-Screening-Presentations/Jens-Kelm.pdf> [retrieved on 20161215] *
KIM, T. K.; EBERWINE, J. H.; 2010: "Mammalian cell transfection: the present and the future", ANALYTICAL AND BIOANALYTICAL CHEMISTRY, vol. 397, no. 8, pages 3173 - 3178
KIMLIN, L. C.; CASAGRANDE, G.; VIRADOR, V. M.: "In vitro three-dimensional (3D) models in cancer research: An update", MOLECULAR CARCINOGENESIS, vol. 52, no. 3, 2013, pages 167 - 182
LEE, J. M.; MHAWECH-FAUCEGLIA, P.; LEE, N.; PARSANIAN, L. C.; LIN, Y. G.; GAYTHER, S. A.; LAWRENSON, K.: "A three-dimensional microenvironment alters protein expression and chemosensitivity of epithelial ovarian cancer cells in vitro", LABORATORY INVESTIGATION, vol. 93, no. 5, 2013, pages 528 - 542
LOVITT, C. J.; SHELPER, T. B.; AVERY, V. M.: "Advanced cell culture techniques for cancer drug discovery", BIOLOGY, vol. 3, no. 2, 2014, pages 345 - 367
MUELLER-KLIESER, W.: "Tumor biology and experimental therapeutics", CRITICAL REVIEWS IN ONCOLOGY/HEMATOLOGY;, vol. 36, no. 2, 2000, pages 123 - 139
NATASA ANASTASOV ET AL: "A 3D-microtissue-based phenotypic screening of radiation resistant tumor cells with synchronized chemotherapeutic treatment", BMC CANCER, vol. 21, no. 4, 10 June 2015 (2015-06-10), pages 588, XP055329503, DOI: 10.1200/JCO.2003.08.013 *
ZHANG, W.; HUANG, P.: "Cancer-stromal interactions: role in cell survival, metabolism and drug sensitivity", CANCER BIOLOGY & THERAPY, vol. 11, no. 2, 2011, pages 150 - 156

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108486035A (zh) * 2018-03-26 2018-09-04 西北大学 一种三维类器官的液滴培养方法
CN108486035B (zh) * 2018-03-26 2021-11-26 西北大学 一种三维类器官的液滴培养方法
WO2020104549A1 (fr) 2018-11-20 2020-05-28 Precomb Therapeutics Ag Procédé de développement de médicament combinatoire basé sur un modèle de cancer humain en 3d
CN113167788A (zh) * 2018-11-20 2021-07-23 普瑞康铂医疗公司 基于3d人癌模型的组合式药物开发方法
WO2021016598A1 (fr) * 2019-07-25 2021-01-28 Immunowake Inc. Méthodes de mesure de la mort à médiation cellulaire par des effecteurs
WO2024010921A1 (fr) * 2022-07-08 2024-01-11 Slmp, Llc Témoins de référence de tissus humains mis au point par génie biologique et normalisés pour validation des résultats de tests d'immunohistochimie, d'hybridation in situ en fluorescence ou d'hybridation in situ en chromogène pour le dépistage du cancer

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