The present invention relates to a drug screening method on cells cultured in a culturing environment according to the preamble of the independent claim 1.
Cell-based screening methods represent a crucial source of information in the decision-making process to evaluate mode of action, such as efficacy and toxicity, of new compounds, e.g. new anti-cancer drugs, in early phases of preclinical drug development (Michelini, Cevenini et. al., (2010) “Cell-based assays: fuelling drug discovery.” Anal Bioanal Chem 398(1): 227-238.; An and Tolliday (2010), “Cell-based assays for high-throughput screening.” Mol Biotechnol 45(2): 180-186; Sharma, Haber et al., (2010) “Cell line-based platforms to evaluate the therapeutic efficacy of candidate anticancer agents.” Nature Reviews Cancer 10: 241-253.).
Given their key role in drug development, a large number of novel approaches were investigated in the last decade towards the development of more physiologically relevant cell-based assays (Yamada and Cukierman (2007), “Modeling tissue morphogenesis and cancer in 3D.” Cell 130(4); 601-610; Pampaloni, Reynaud et al. (2007); “The third dimension bridges the gap between cell culture and live tissue.” Nat Rev Mol Cell Biol 8(10): 839-845). For instance, in cancer cell biology and anti-tumour drug development, a growing body of scientific evidence suggests that methods allowing cancer cells to organize and grow in three-dimensional structures (three-dimensional culture models), compared to conventional cultures on flat plastic surfaces (two-dimensional culture models), reflect more closely behaviours observed in vivo; in particular, with regard to cell growth, cell, signalling and gene expression (Abbott. (2003). “Cell culture: Biology's new dimension,” Nature 424(6951): 870-872, Kunz-Schughart, Freyer et al. (2004), “The use of 3-D cultures for high-throughput screening: the multicellular spheroid model.” J Biomol Screen 9(4): 273-283; Bissell. (2007), “Modelling molecular mechanisms of breast cancer and invasion: lessons from the normal gland.” Biochem Soc Trans 35(Pt 1): 18-22,; Friedrich, Seidel et al. (2009). “Spheroid-based drug screen: considerations and practical approach,” Nat Protoc 4(3): 309-324; Debnath and Brugge (2005), “Modelling glandular epithelial cancers in three-dimensional cultures,” Nat. Rev Cancer 5(9): 675-688).
Several techniques have been employed to produce these more physiological three-dimensional culture models to test anticancer drugs (Nyga 2011, Cheema et al, (2011), “3D tumour models: novel in vitro , approaches to; cancer studies” J. Cell Commun. Signal, 5, 239-248). The most common three-dimensional techniques use either (i) gel matrices from natural (Weaver, Petersen et al. (1997), “Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.” J Cell Biol 137(1): 231-245.; Fiebig, Maier et al. (2004), “Clonogenic assay with established human tumour xenografts: correlation of in vitro to in vivo activity as a basis for anticancer drug discovery.” Eur J Cancer 40(6): 802-820; Krausz, de Hoogt et al. (2013), “Translation of a tumor microenvironment mimicking 3D tumor growth co-culture assay platform to high-content screening.” J Biomol Screen 18 (1): 54-66) or synthetic (Loessner, Stok et al. (2010). “Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells.” Biomaterials 31(32): 8494-8506; Sieh, Taubenberger et al. (2012), “Phenotypic characterization, of prostate cancer LNCaP cells cultured within a bioengineered microenvironment. ” PLoS One 7(9): e40217.; Yang and Zhao (2011). “A 3D model of ovarian cancer cell lines on peptide nanofiber scaffold to explore the cell-scaffold interaction and chemotherapeutic resistance of anticancer drugs.” Int J Nanomedicine 6: 303-310) origin for cell encapsulation, (ii) rigid polymeric three-dimensional scaffolds (Fischbach, Chen, et al. (2007), “Engineering tumors with 3D scaffolds.” Nat Methods 4(10): 855-860; Knight, Murray et al. (2011). “Alvetex(R): polystyrene scaffold technology for routine three dimensional cell culture.” Methods Mol Biol 695: 323-340) for cell seeding, or (iii) matrix-free cell aggregation processes (WO 2010/031194 A1; (Friedrich, Seidel et al. (2009), “Spheroid-based drug screen: considerations and practical approach.” Nat Protoc 4(3): 309-324; Karlsson, Fryknas et al. (2012). “Loss of cancer drug activity in colon cancer HCT-116 cells during spheroid formation in a new 3-D spheroid cell culture system.” Exp Cell Res 318(13): 1577-1585; Tung, Hsiao et al. (2011). “High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array.” Analyst 136(3): 473-478; Lama, Zhang et al. (2013). “Development, validation and pilot screening of an in vitro multi-cellular three-dimensional cancer spheroid assay for anti-cancer drug testing.” Bioorg Med Chem 21(4): 922-931) to grow three-dimensional cancer cell structures (e.g. spheroids, tissue).
The failure to take into account the variation in disease progression may increase the probability to select false-positive drug candidates (drugs that will not work in humans) and to discard false-negative drug candidates (drugs that would have worked in human).
It is therefore an objective of the present invention to avoid disadvantages of the known drug screening methods, and specifically to provide data on drug activities tested on profiled cells displaying different key physiological in vivo disease states. This objective is solved with the drug screening method according to claim 1.
According to the present innovation the cell-based drug screening assay takes into account cells as well as their physiological changes and behaviour over time to understand the mode of action of drugs. It comprises determining at least two monitoring points in time in dependency of the cell type, the culturing environment, the physiological characteristics of the cells, the physiological characteristics of cell populations, the physiological characteristics of formed tissue, or the kind of drug substance, e.g. the targeted mechanism of action of a drug substance. The drug substances are applied to the cultured cells at least at one treatment point in time. The effect of a drug type on cell growth kinetics, behaviour and/or physiological characteristics are monitored at least at said two monitoring points in time. The points in time can be a specific moment but also a certain period of time.
The culturing environment includes physiological relevant microenvironments which take into account the specific physical, biological and biochemical features including cell-cell and cell-matrix interactions for specific cells and/or formed tissues.
Monitoring of cell proliferation at least at two points in time, for example at an “early-stage” and at a “late-stage”, may allow comparison of two stages during cell growth, and/or development of physiological features and/or effect of drug treatment. The cell-growth environment and physiological features may need to be adjusted for a given cell-type or drugs. The point in time of the monitoring is thus preferably chosen such as to provide information relevant in view of mode of action of a drug to e.g. early or late stages of cancer; a specific cancer may be considered as “early stage” preferably four to seven days after culturing in a physiologically relevant environment, and “late-stage” preferably fourteen to seventeen days after culturing in a physiologically relevant environment. “Late-stage” may be also defined by the absence or presence of a specific physiological, characteristic of feature, e.g. hypoxia or changes in cell growth kinetics or constitution of cells.
Monitoring of cells may continue one to seven or more days after termination of the drug application. The start and end of the drug-free time period has to be determined for a specific cell type and drug. As the cells are allowed to recover from the drug treatment this period is named “recovery phase”. Including points in time relevant for a recovery after a first and/or a second drug application may be essential for the identification of effective drugs or drugs that do not have an immediate effect on cells.
Preferred embodiments may refer e.g. to a process including treatment at an “early-stage” and monitoring at least at two related points in time with or without a “recovery phase”, or treatment at a “late-stage” and monitoring at least at two related points in time with or without a “recovery phase”, or treatments at an “early-stage” and a “late-stage” and monitoring at least two related points in time with or without a “recovery phase”.
The environment used for cell culturing may comprise a substrate matrix or medium that mimics the cellular microenvironment, preferably a three-dimensional matrix that consists of a structural compound and a linker compound to facilitate polymerization. The structural compound may be multi-branched polyethylene glycol comprising unsaturated end groups, preferably vinyl groups. The linker compound comprises a peptide with at least two cysteines or thiol groups enabling polymerization of the structural compound bridged by means of the linker compound. The three-dimensional matrix preferably comprises a matrix composition as described in the European Publication Number 2 561 005.
The environment used for cell culturing may promote three-dimensional growth (i) of a single cell to form a multicellular entity or (ii) of multiple cells to form a multicellular entity, when they are seeded into said environment. Preferentially, the cells grow to form a cell colony or a cell population. For example, colony arrangements of cancer cells form spheroids that mimic the three-dimensional arrangement of tumours where outer cells are easily accessible and at the same time shield cells at its core.
In order to produce a three-dimensional environment with a defined stiffness and a composition with appropriate physiological and biological characteristics, a recipient or a standard cell-culture device may be used, preferably a 384-well plate. The said environment and the cells may be dispensed into the device with cell culture media which may then be placed in a cell culture incubator. Monitoring of cell proliferation and activity may be measured using imaging and sensory equipment that is compatible with the recipient or the device, preferably equipment that is automated and maximize the reproducibility of the output monitoring data.
The cells used for culturing in the said environment may be any type of cell of any organism, cell or cell line cultured in vitro, differentiated or stem cell derived from an organism and cancer cell of an organism, preferentially colon, pancreatic and ovarian cancer cell, but also healthy cells of which the growth is promoted.
The drug substances applied to cultured cells may be any substances selected of the group of any chemical, natural or synthetic substance, any substance of known drug screens and substances recognized before as anti-cancer drugs. Drug substances may be applied in liquid form, wherein the liquid may be an organic solvent or an aqueous buffer comprising the drug substance, as powder, as pill or encapsulated in a carrier, preferably a nanoparticle.
Assay readout modality to monitor cell growth kinetics and activity may be carried out by preferably any (i) of the selected group of metabolic activity, preferably Alamar Blue®, flow cytometry, Deoxyribonucleic acid (DNA) content, protein quantification, cell counting, image-based analysis, gene expression levels, mass spectrometry, label-based measurements (such as reporter gene technology, resonance energy transfer, protein-fragment- or split-complementation assays), or label-free based measurements (such as electrical biosensor/impedance systems, optical, biosensor systems, surface plasmon resonance systems, acoustic biosensor systems, and systems that exploit the calorimetry, change in electrical activity or by (ii) any combination of afore mentioned monitoring assays. Monitoring of cell proliferation gives direct information on the drug-induced effect on cellular shape and constitution.
Assay readout modality to determine other physiological and biological characteristics (e.g. hypoxic and/or angiogenic markers) may be carried out preferably by any (i) of she selected group of Ribonucleic acid (RNA) levels, protein expression levels, mass spectrometry, immunohistochemistry, image-based analysis, proteomics methods or by (ii) any combination of aforementioned monitoring assays.
The maximally tolerated dose or concentration of an applied drug substance may be determined for a given cell and cell type. Determination of the accepted drug dose of a cell, cell type or tissue allows adjustment of the applied drug doses when targeting diseased cells, preferably cancer cells.
The lowest effective dose of an applied drug substance may be determined for a given cell and cell type. Determination of the lowest effective dose of a cell, cell type or tissue allows adjustment of the applied drug doses, when targeting diseased cells, preferably cancer cells.
Another objective of the present invention is to provide use of the method to monitor (i) curing or killing diseased cells, supporting healthy cells and inducing healthy cells to enter a state of disease by drug treatment or (ii) reprogramming and/or differentiation of cells.
Reprogramming or differentiated cells into stem cells or progenitor cells with respect to an applied drug substance may be assayed by any (i) of the selected group of metabolic activity, preferably Alamar Blue®, flow cytometry, Deoxyribonucleic acid (DNA) content, protein quantification, cell counting, image-based analysis, gene expression levels, mass spectrometry, label-based measurements (such as reporter gene technology, resonance energy transfer, protein-fragment- or split-complementation assays), or label-free based measurements (such as electrical biosensor/impedance systems, optical biosensor systems, surface plasmon resonance systems, acoustic biosensor systems, and systems that exploit the calorimetry, change in electrical activity or by (ii) any combination of afore mentioned monitoring assays. The differentiated cell may be any cell, cell type, cell culture-derived cell or cell derived from a specific tissue. Here, the applied drug substance may trigger the reprogramming process.
The method may be used for analyzing the differentiation of stem cells into a differentiated cell with respect to an applied drug substance according to cell cell growth kinetics and activity including any (i) of the selected group of metabolic activity using Alamar Blue®, flow cytometric, hypoxic and angiogenic markers, Ribonucleic acid (RNA) levels, protein expression levels, mass spectrometry, DNA content, protein quantification, cell counting, image-based analysis, and immunohistochemistry or by (ii) any combination of afore mentioned monitoring assays. Here, the applied drug substance may trigger the differentiation process.
Further aspects and details of the present invention will become apparent from the figures and examples given in the following, which show:
Profiling of cancer cells is a key step in the present invention, as it permits to identify and investigate physiological characteristics (e.g. hypoxia) of cancer cell spheroids at different time points of growth. Importantly, these characteristics strongly depend on the origin of tumour tissue (as shown in
The “early-stage” comprises two time points for readout. The first point of time at day seven is at the end of the drug treatment, whereas the second point in time at day eleven follows a drug-free recovery phase. The “late-stage” comprises two time points for readout. The first point in time at day seventeen is at the end of a second (independent from “early-stage”) drug treatment phase. The second point in time at day twenty-one follows a drug-free recovery phase. Measurements at additional points in time can be performed before the start of treatment and/or after different drug-free recovery phases.
The “early-stage”, day four to seven, shows small spheroids with a diameter of 35 μm, highly proliferative cells (shown in
For reference two-dimensional assays, cells are normally tested with drugs within the logarithmic proliferations phase between day one to four (
In
In
Cancer cells normally grow as two-dimensional monolayer on tissue culture plastic as described by cell suppliers and scientific publications.
In
In
In
In
Number | Date | Country | Kind |
---|---|---|---|
13165419.6 | Apr 2013 | EP | regional |
This application is a National Stage completion of PCT/EP2014/058152 filed Apr. 22, 2014, which claims priority from European patent application serial no. 13165419.6 filed Apr. 25, 2013.
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/EP2014/058152 | 4/22/2014 | WO | 00 |