Classification Method

Introduction

In the development and selection of anti-cancer therapeutics the specificity of the agents is an important parameter to minimize side effects for the patient. 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. Whereas two-dimensional assays cannot reproduce the complexity and heterogeneity of a cancer in vivo and often fail to predict the effects of compounds added to the system for later in-vivo application, 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). The effect of additional cell types, such as stromal cells, and the resulting heterotypic cell-cell crosstalk can be investigated in heterotypic 3-D cell cultures. Moreover, 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

2- D systems (Friedrich et al., 2009; Lee et al., 2013). In particular, modifications in gene expression were detected for genes encoding signal transduction proteins (Lovitt et al, 2014). Tumor spheroids reflect more closely the clinical expression profiles than 2-D systems. Therefore, 3-D cell systems are being explored for creating a more relevant model of tumors (Herrmann et al., 2008; Hickman et al., 2014; Ivascu and Kubbles, 2006).

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). As shown in Fig. 1, poorly vascularized areas in solid tumors are characterized by irregular tissue architecture and proliferation as well as oxygen/nutrient gradients. In multicellular tumor spheroids large proliferating cancer cells are typically arranged in a concentric manner in the periphery and smaller non-proliferating cells in deeper regions. Depending on the microtissue size, the cancer cell type, and the culture condition, a necrotic core emerges.

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). In particular 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).

It is an object of the present invention to avoid at least some disadvantages of the current 3-D cell systems. It is further an object of the present invention to provide a three-dimensional multicellular spheroid for drug testing, selection and characterization which enables the parallel assessment of safety and efficacy of drug candidates and is compatible with high- throughput screening technologies.

It is another object of the present invention to provide a three-dimensional multicellular spheroid model system offering the possibility of discriminating of at least two cell types within a heterotypic microtissue model and to compare and classify the effects of compounds tested on the entire system specifically for each cell type.

It is yet another object of the present invention to provide a three-dimensional multicellular spheroid model system offering the possibility of specifically discriminating between nonproliferative stromal cells and the proliferative cancer cells, contributing to cell-based approaches for the discovery of effective anti-cancer drugs with reduced toxicity on non- dividing cells.

It is yet another object of the present invention to provide a three-dimensional multicellular spheroid model system offering the possibility of specifically discriminating between two different cell populations in a single spheroid to test the toxicological impact of compound metabolites combing hepatocytes and a second non-proliferative reporter gene expressing cell type.

It is yet another object of the present invention to provide a three-dimensional multicellular spheroid model system offering the possibility to use the non-proliferative transgenic cell population as an internal standard for quality control of a drug testing method. This is especially important if spheroids are generated from highly heterogenic cell sources such as primary tumor material. Summary of the Invention

These objects are met with methods and means according to the independent claims of the present invention. The dependent claims are related to preferred embodiments. It is to be understood that value ranges delimited by numerical values are to be understood to include the said delimiting values.

Embodiments of the invention

Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts or structural features of the devices or compositions described or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include singular and/or plural referents unless the context clearly dictates otherwise. Further, in the claims, the word "comprising" does not exclude other elements or steps. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values. It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done.

According to one aspect, 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. According to another embodiment of the present invention, 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. As used herein, 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.

According to another embodiment of the present invention, 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.

In this embodiment, 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.

According to a preferred embodiment of the present invention, such three-dimensional spheroid as described above has been subjected to transfection, wherein said transfection is a transient transfection.

According to a particularly preferred embodiment of the present invention, said transient transfection is performed by means of electroporation.

According to another particularly preferred embodiment of the present invention, said transient transfection is performed by means of nucleofection using a Nucleofector™.

As used herein, the term 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, Nucleofector™ technology, electroporation, microinjection, "gene gun" technologies or viral vector-based transfer (Kim and Eberwine, 2010).

In stable transfection, 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.

In 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. Thus transient transfectants will resemble more closely untreated cells in their biochemical, physiological and behavioral characteristics. Unlike in most stable transfection procedures, 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. Furthermore, many primary cell types, such as neuronal cells or hepatocytes, are hardly or not at all amenable to stable transfection, but can be transiently transfected with high efficiency, particularly by use of Nucleofector™ 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.

The various 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. According to one embodiment of the present invention, said transient transfection yields an efficiency of at least 50% of transfected cells.

According to a further aspect of the present invention, 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.

The term heterologous gene, as used herein, 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, as used herein, 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. As used herein, the term 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. Usually by detection of its gene product, a reporter gene indicates its presence and expression in said cell, tissue or organism. The term 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. However, the coding region of the expression cassette can be comprised of several open reading frames, separated by so-called IREs (Internal Ribosome Entry Sites).

According to preferred embodiments of the present invention, 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.

According to another aspect of the present invention, 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.

As used herein, the term 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. The term primary tumor cells, as used herein, refers to cells that were obtained or derived from cells that were obtained by direct removal from tumor tissue of a patient. As used herein, the term 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.

According to another aspect of the present invention, 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. According to a preferred embodiment of the present invention, 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.

As described in detail in Example 1 and Example 5, respectively, the inventors surprisingly found that 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.

According to a particularly preferred embodiment of the present invention, 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,

b) Preparing a cell suspension comprising said at least one type of tumor cells and said at least one type of non-proliferating cells,

c) Seeding said cell suspension in a cell culture environment allowing for the formation of a spheroid,

d) Growing the spheroid in culture medium, and

e) Optionally monitoring the expression product of the transfected heterologous gene. According to a preferred embodiment, 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.

According to a preferred embodiment, 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.

According to a preferred embodiment, 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 GravityPLUS™ 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.

Furthermore, 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.

As used herein, 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 use of a three-dimensional spheroid as described above can serve for the determination of the in-vitro-based therapeutic window of an agent. As used herein, 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. During clinical development, 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). Within the in-vitro set-up, 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).

€TD.m

_,_ ^— * ' in intra

CTD: Cytotoxic dose IC50Fibrobiast _s ; ED Effective dose (IC50rumo _r ceils derived from dose response curves); TI _{in νίίη} : therapeutic index derived from in vitro models

The use of 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,

b) Incubating said spheroid with a potentially therapeutic agent, and

c) Monitoring the growth of said spheroid and/or the viability of said at least one type of tumor cell. 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.

As used herein, the term 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.

In another embodiment, 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

CaPC"4 precipitation cell types without time- consuming selection processes.

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. cancer cells

Determination of several parameters which are used for compound classification. Specific and unspecific effects can be used to determine an in vitro-based therapeutic index

Step 3 Drug supplementation

Step 4 Determination of efficacy

(max response);

Determination of potency

(IC50), only if a dose

response is being done;

Growth profiling over time;

Size (specific for cancer

cells), ATP (unspecific),

Total DNA (unspecific), total

protein (unspecific); cancer

cell specific biomarker (such

as PSA for prostate cancer

etc.); Determination of fibroblast

death, unspecific cytotoxic

effect

Step 5 Multi-parametric compound

classification on at least 2

parameters determined in

step 2

Step 6 Pathway identification by

either proteomics, ELISA,

RPPA and/or

transcriptomics, R Aseq

Step 7 Hit selection

According to a another embodiment of the present invention, 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.

Furthermore, 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.

Experiments and Figures While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. Any reference signs should not be construed as limiting the scope.

Example 1: Transient transfection of NIH3T3 cells

For transfection of stromal cells, the Nucleofector™ (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.

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.

Transfection of NIH3T3 fibroblasts in order to secrete NanoLUC luciferase using the Nucleofector™ was performed according to Lonza's manual "Amaxa™ 4D-Nucleofector™ Protocol for NIH/3T3 " (Lonza Cologne GmbH, 2010).

The cells were trypsinized and an aliquot of lxl 0 ⁶ cells was resuspended in 100 μΐ 4DNucleofector™ Solution. 1 μg of pNL1.3CMV[secNluc] luciferase reporter vector (Promega) was added and the suspension was transferred into a Nucleocuvette™. The Nucleocuvette™ was placed into the 4D-Nucleofector™ 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

intracellular reporter

Primary human dermal fibroblasts were transfected with a green fluorescent protein (GFP) expression vector by transient Nucleofection prior to microtissue production. Primary fibroblast microtissues were grown as spheroids expressing GFP. Microtissues comprising different ratios of GFP-expressing and native fibroblasts were produced, and the fluorescence intensity (RLU) was measured over 25 days. Even though a slight decrease in the intensity was observed, after 25 days a fluorescence signal was detected. The lower the ratio of GFP- expressing cells, the lower was the fluorescence intensity (Fig. 3).

Example 3: Production and cultivation of microtissues

InSphero's hanging drop microtissue production platform GravityPLUS ^™ 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 GravityTRAP™ 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.

For the microtissue production process, 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). For the production of heterotypic microtissue models, 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 GravityPLUS™ plate by pipetting 40 μΐ suspension per well.

For aggregating the cells into 3-D spheroids, the loaded plate was placed into an incubator for 72 hours. Culture time information, where mentioned, will refer to the total time in culture of the cells, starting at day 0, the day of seeding the cells into the GravityPLUS™ plate.

For longtime cultivation and compound treatments, the microtissues were transferred three days after seeding from the GravityPLUS™ into the GravityTRAP™ plate. A medium exchange with microtissue maintenance medium was performed twice a week to prevent the microtumors from nutrient starvation. To observe the morphology of the microtissues over time, bright field (BF) images were taken on a regular basis.

Example 4: Drug testing

The compound treatment of the microtissues was initiated at the day of the transfer from the GravityPLUS™ into the GravityTRAP™ plate. Compounds were tested within a concentration range of 2.05 pM - 20 μΜ (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).

To track the microtissue size and morphology over time, the Dainippon SCREEN Cel iMager CC-5000 was used which allowed for scanning microtissues in the InSphero GravityTRAP™ 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.

To discriminate fibroblasts from cancer cells in the co-culture models, the fibroblasts were transfected with a reporter gene in order to secrete NanoLUC ^® luciferase. Within a screening run, 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 μΐ 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. As a biochemical endpoint of a screening run at treatment day 7, 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 μΐ 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.

For generation of dose-response curves and corresponding IC50 values, the data obtained from size measurements, NanoLUC assays and ATP assays were normalized to the vehicle control. The normalized data were processed using the GraphPad Prism ^® 6 Software. The program uses the subsequent formula for fitting a sigmoidal dose-response curve: _ (¾O )

1 + 10(Log iC _S0 -X)» HiilSlope

Ytop is the Y value at the top plateau; the Hill Slope variable, also called the Hill Slope coefficient, describes the steepness of the curve.

Example 5: Results

5.1 Tumor microtissue characterization Three heterotypic microtissue models were established: two ovarian co-culture systems with HEY-GFP and SKOV-3 as cancer cells, respectively, and NIH3T3 fibroblasts as stromal cells, referred to as HEY/NIH and SKOV/NIH microtissues, as well as one pancreatic co- culture systems with PANC-1 as cancer cells and NIH3T3 fibroblasts as stromal cells, referred to as PANC/NIH microtissues. In addition, 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.

The accumulation of the respective cells in hanging drops resulted in microtissue formation within 3 days. After microtissue formation, the spheroids were either harvested for histological analysis or transferred into a non-adhesive spheroid-specific 96-well plate for long-term culture (GravityTRAP™, InSphero). To investigate cell viability over time, the ATP content was measured regularly (CellTiter-Glo ^® , Promega) over a culture period of up to 13 days. To generate growth profiles, 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.

5.1.1 Growth and Viability Profiling of Microtissues Cultivation of HEY/NIH tumor spheroids over a culture period of 10 days resulted in a mean increase of microtissue size from 82,606 ± 6312 μιη ² to 373,532 ± 28,535 μιη ² . When subtracting the size of the non-proliferating fibroblast core in order to generate a baseline, the tumor cell growth corresponded approximately to a 1.85-fold increase in area per day. The growth rate of cancer cells cultivated in heterotypic microtissues was higher than in HEY- GFP homotypic spheroids.

In accordance with the growth profile, an increase in ATP contents from 54.0 ± 7.1 pmol ATP per microtissue at day 3 to 376.7 ± 17.1 pmol ATP per microtissue at day 13 could be observed.

To assess the cellular morphology, 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.

For SKOV/NIH and PANC/NIH heterotypic cancer spheroids, 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 μιη ² for SKOV/NIH microtissues and an area of 129,537 ± 11 ,797 μιη ² for PANC/NIH spheroids were measured. During the course of 7 days the area of these cultures increased to 262,963 ± 12,660 μιη ² , and to 287,348 ± 10,088 μιη ² , respectively. By subtracting the size of the non-proliferating fibroblast core in order to generate a baseline, approximately a 0.81-fold increase in area per day resulted for SKOV/NIH and a 0.66-fold increase in area per day for PANC/NIH spheroids.

Although microtissue formation was performed using the double number of initial cancer cells, the growth rates of SKOV and PANC cells cultivated in heterotypic spheroids were significantly lower as compared to HEY/NIH spheroids. The respective intra-tissue ATP contents correlated to size measurements and resulted in 38.2 ± 9.1 pmol ATP per SKOV/NIH microtissue and 45.6 ± 9.8 pmol ATP per PANC/NIH microtissue at day 3 with an increase to 189.2 ± 10.7 pmol ATP per SKOV/NIH microtissue and 236.4 ± 45.6 pmol ATP per PANC/NIH microtissue at day 10.

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.

5.1.2 Histological Characterization of Microtissues

The 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.

5.1.3 NanoLUC ^® luciferase reporter expression

To enable a direct discrimination between non-proliferative stromal cells and the proliferating cancer cells within the heterotypic tumor models, a NanoLUC ^® luciferase reporter gene was introduced into the fibroblasts. Prior to spheroid formation, stromal fibroblasts were transiently transfected with CMV-driven constructs encoding secreted NanoLUC ^® luciferase by using the Nucleofector™ technology. Transfected cells were referred to as 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. For this purpose, 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. Tissue sizes of the spheroids were directlyproportional to the respective NanoLUC ^® secretion at different time points, indicating a linear relationship between spheroid size and NanoLUC ^® signal. 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.

To assess the stability and NanoLUC ^® expression pattern of transfected fibroblasts in the heterotypic microtissue model systems, 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 GravityPLUS™ into the GravityTRAP™ 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. Compounds were tested within a concentration range of 2.05 pM - 20 μΜ (11 doses, 1 :5 dilution series). For analysis of the data, the values from treated microtissues were normalized to the vehicle control group which consisted of microtissues treated with 1 % DMSO in microtissue maintenance medium (Figures 4, 5, 6).

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). 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. Within the pancreatic models, 13 out of 20 compounds tested were more potent in 3D than in 2D. Interestingly, 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.

Unspecific cytotoxic effects on the incorporated NIH3T3 fibroblasts were evaluated by quantifying a secreted reporter over time. The assay system allowed to discriminate between acute cytotoxic effects (3 -day exposure) and sub-chronic toxicity (7-day exposure). Whereas only a very limited number of compounds had acute cytotoxic effects, there were considerable more compounds with sub-chronic cytotoxicity. Based on the single endpoint classifications, an mTOR inhibitor, Torin-2, was chosen to further assess molecular mechanism of drug action for all three tumor microtissue models used in this study, applying reverse phase protein array (RPPA) and transcriptome analysis for signaling pathway profiling. First results of RPPA analysis revealed a clear down-regulation in the cell cycle and PI3K/Akt/mTOR signaling pathway, especially for S6 ribosomal protein phosphorylated at Ser 235, 236, 240 and 244, indicating a strong inhibitory effect on translation and cell growth control. Table 3 : Therapeutic indexes of different compounds corresponding to HEY/NIH

microtissues treated continuously with the respective compounds. The therapeutic index is shown as the ratio adverse effect / therapeutic effect, for acute and subchronictoxicity of the compounds. Therapeutic index Therapeutic index

Compound name [CTD ₅₀ anoLUC/ED ₅₀ ATP] [CTD ₅₀ NanoLUC/ED ₅₀ ATP] acute cytotoxicity subchronic cytotoxicity

Toiin-2 9.63 2.22 SR-3306 21.77 2.138

Obatoclax 71.20 5.62

Ponatinib 106.22 29,71 Cvr bewmide j; ^» 5.:-.'i 51.86

Carfilzomib 577.76 12.85

H SLL0084 'infinite 1.39

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.

Figure 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.

Figure 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.

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.

Figure 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). Poorly vascularized areas in solid tumors are characterized by irregular tissue architecture and proliferation as well as oxygen/nutrient gradients. In multicellular tumor spheroids large proliferating cancer cells are typically arranged in a concentric manner in the periphery and smaller non-proliferating cells in deeper regions. Depending on the microtissue size, the cancer cell type, and the culture condition, a necrotic core emerges (adapted from Mueller-Klieser, 2000). Figure 8: Vector map of pNL1.3CMV[secNluc/CMV]. 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.

Figure 9: Flow chart of the determination of In-Vitro Therapeutic Index; TI = therapeutic index; ED = effective dose; TD = toxic dose.

Figure 10: Flow chart of the determination of In-Vitro Metabolic Index; MI = metabolic index; TD = toxic dose. REFERENCES

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