METHODS FOR IDENTIFYING AGENTS WHICH INDUCE (RE) DIFFERENTIATION IN UN- OR DEDIFFERENTIATED SOLID TUMOR CELLS

Abstract

The present invention comprises a test for the identification of an agent, which induces (re-)differentiation in a tumour cell based on the novel combined application of the two markers lactate as the end-product of the katabolic anaerobic glycolysis and neutral lipids as the end-product of the anabolic neutral lipid synthesis. The cell-based system is further characterized by a novel liquid handling procedure, which allows valid high throughput screening even over a longer time period of at least seven days, and a new viability test.

Claims

1. Method for the identification of compounds inducing (re-)differentiation in non- or dedifferentiated cells, comprising: a) provision of a cell culture sample consisting of dedifferentiated and/or undifferentiated tumour cells, b) bringing the compound of interest into contact with the cell culture sample, c) following the determination of the relative concentration of a first marker lactate in contrast to untreated cells, and d) following the determination of the relative concentration of a second marker neutral lipids in contrast to untreated cells, wherein steps c) and d) may be performed in reverse order.

2. Method according to claim 1, characterised by lipid-free or lipid-reduced cultivation of the cell culture sample

3. Method according to claim 1, characterised by cultivating the cell culture sample under aerobic conditions

4. Method according to claim 1, which further includes: c) determination of the amount of viable adherent cells via quantification of absorbance after centrifugation, and/or f) determination of complete induction of apoptosis in the cells among a complete signal reduction to background level

5. Method according to claim 4, for utilisation as viability test and/or for utilisation as test for apoptosis.

6. Method according to claim 1, characterised by cultivating the cell culture sample in the presence of insulin.

7. Method according to claim 1, characterised by cultivating (tumour) cell culture sample either negative for/or low expressing insulin receptor isotype B or possessing a higher expression ratio of insulin receptor isotype A/B than differentiated cells in the presence vs. absence of insulin.

8. Method according to claim 1, characterised by a change in one or both of the first and second marker(s) in a presence vs. absence of insulin towards the direction of an anabolic change in the cell's metabolism enabling the analysis of functional insulin receptor subtype B expression and ratio of subtype A to B towards a relative higher subtype B expression, respectively.

9. Method according to claim 1 further, characterised by cultivating a breast cell culture sample negative for or poorly expressing prolactin receptor in a presence vs. absence of prolactin.

10. Method according to claim 1, characterised by a change in one or both of the first and second marker(s) in a presence vs. absence of prolactin towards the direction of an anabolic change in the cell's metabolism enabling the analysis of functional prolactin receptor expression.

11. Method according to claim 1, characterised by the provision of the cell culture sample in a microtiter plate.

12. Method according to claim 1, characterised by a gas permeable foil sealing the microtiter plate.

13. Method according to claim 1, characterised by quantification of lactate concentration in bicarbonate buffered cell culture medium incubated in an appropriate CO.sub.2-atmosphere by measuring the pH-dependent change of optical phenol red absorption.

14. Method according to claim 1, characterised by single/multiple media and/or washing buffer removal from the cell culture sample via centrifugation prior to addition of new media for further culturing or measurement of marker in step 1d).

15. Method according to claim 1, characterised by performing single/multiple changes in media and/or washing buffer via partial device (A) and (B).

16. Method according to claim 1, characterised by the combined utilisation of the markers lactate and neutrals lipids for the identification of compounds inducing (re-)differentiation in un- or dedifferentiated cells, especially tumour cells.

17. Method according to claim 1, characterised by quantification of lactate concentration in bicarbonate buffered cell culture medium incubated in an appropriate constant CO.sub.2-atmosphere by measuring the pH-dependent change of optical phenol red absorption and by quantification of neutral lipids using a neutral lipid staining dye.

18. Use of the markers lactate and neutrals lipids for the identification of compounds inducing (re-)differentiation in un- or dedifferentiated cells, especially tumour cells, wherein the markers are used combined.

19. A vessel for the removal of liquids from cells via centrifugation (partial device (A)) characterized by comprising: 4 side walls (a.sub.1, a.sub.2, b.sub.1, b.sub.2), where the two opposing side walls have the same length (l.sub.a, l.sub.b), so that a rectangular shape is obtained, a flat bottom (c) being connected in such a way with each of the side walls over all of the connecting area in a liquid proof way, each of the side walls having a protrusion (d.sub.a, d.sub.b) directed towards the inside of the vessel, 2 of the 4 side walls being opposite to each other having recesses (c.sub.1, c.sub.2) positioned in the middle of the lengths l.sub.a of the side wall on the upper surface of the respective side wall.

20. A microtiter plate (partial device (B)) for culturing cells enabling addition of liquid via centrifugation, said plate comprising a surface and wells, said wells being tapered towards the bottom where an opening, is present.

21. Microtiter plate according to claim 20 wherein the diameter of the opening is proportional to the surface tension of a liquid inside each of the wells and adjusted in such way that liquid can escape only under influence of forces stronger than the gravitational force.

Description

[0058] The present invention thus refers in a further embodiment to a vessel for the removal of liquids from cells via centrifugation (partial device (A)) characterized by comprising: [0059] 4 side walls (a.sub.1, a.sub.2, b.sub.1, b.sub.2), where the two opposing side walls have the same length (l.sub.a, l.sub.b), so that a rectangular shape is obtained, [0060] a flat bottom (c) being connected in such a way with each of the side walls over all of the connecting area in a liquid proof way, [0061] each of the side walls having a protrusion (d.sub.a, d.sub.b) directed towards the inside of the vessel, [0062] 2 of the 4 side walls being opposite to each other having recesses (e.sub.1, e.sub.2) positioned in the middle of the lengths l.sub.a of the side wall on the upper surface of the respective side wall.

[0063] In a further embodiment, the present invention discloses a microtiter plate (partial device (B)) for culturing cells enabling addition of liquid via centrifugation, said plate comprising a surface (1) and wells (2), said wells being tapered towards the bottom (3) where an opening, especially a circular opening, is present. Said opening at the bottom (3) of the microtiter plate being adjusted in such way that the diameter of the opening is proportional to the surface tension of a liquid inside each of the wells and adjusted in such way that liquid can escape only under influence of forces stronger than the gravitational force.

[0064] Said partial devices (A) and (B) will be described in the following together with the inventive method.

[0065] Inventively, in order to determine markers mentioned in steps c) and d) the cell culture samples can be washed and/or one or more medium changes can be performed. For instance, this step can be performed using known pipetting methods. Inventively preferred, this step is made via centrifugation.

[0066] In contrast to common (multi-) pipetting systems aspirating the liquids, the invented process prefers to remove serum-containing media or low in-serum media or serum-free media from the single wells of a microtiter plate before the addition of compounds or fluorescent ligands via cost-effective, brief centrifugation.

[0067] Therefore, a vessel (partial device (A)) was developed for the use with commonly available centrifuge hangers. Microtiter plates are incorporated into the vessel with the opening of the wells pointing downwards into the vessel lumen whereby the microtiter plates are fixated by the container walls. As a result, the liquids from the single wells can be removed into the cavity provided by the container at the same time (FIG. 2-7; partial device (A)). This makes the efficient, gentle, sterile and complete removal of the liquids from the single wells possible in contrast to known pipetting methods using aspiration to remove liquids which often results in the loss of adherently growing cells and/or leads to liquid residues permanently remaining in the well. If needed partial device (A) can be installed together with various pipetting robots.

[0068] For assays using cell culture techniques based on adherently growing cells the media can be completely removed and collected after a few seconds of centrifugation (e.g. 100-300g) whereby no liquid residues (residual volume) remain in the microplate wells. Thus, the efficient reduction and/or prevention of washing steps is made possible further resulting in shorter test durations and less amounts of washing solutions. A reduction in viability of the cells/cell lines in following cell culture trials could not be observed.

[0069] The efficient removal of media and the prevention of washing steps significantly increase the robustness of cell based assays. Furthermore, the efficient removal of supernatant via the partial device (A) and centrifugation, in contrast to using various aspiration attachments, also leads to a reduction/prevention of washing steps, enables efficient liquid removal without residues and prevents artefacts from occurring in non-cell based assay systems (e.g. ELISA-based assays).

[0070] Conventionally, the simultaneous addition of media or liquid to all wells of a microtiter plate can be realised via multichannel robotic heads (e.g. 96 to 1536 channels). Alternatively, it could be shown for the first time that this can also be done using commonly available multi-/single channel pipettes or single-/multichannel robotic heads (e.g. 1-16 channels) in combination with a specially designed microtiter plate (FIG. 8-11; partial device (B)). [0071] In a first step the specially designed microtiter plate, consisting of wells slightly tapered towards the bottom where a (e.g. circular) opening has been built-in is used to add the liquids to be added. Every single well can be individually filled with liquids. Due to the surface tension the fluids will not flow through the e.g. circular opening at the bottom of the single wells slightly tapered towards the opening at the bottom. Here, the diameter of the drill hole must be adjusted to the surface tension of the liquid. Low surface tensions, e.g. due to the presence of detergents, require a smaller diameter. Liquids with higher surface tensions enable a bigger diameter. Alternatively, the filling of the microtiter plate consisting of wells slightly tapered towards the opening at the bottom can be done in one step via the full immersion of the microtiter plate into the liquid. [0072] In a second step the microtiter plate, consisting of wells slightly tapered towards the (e.g. circular) opening at the bottom and filled with the liquid that should be transferred into a commercially available microtiter plate, is appropriately set onto the microtiter plate whereby the wells slightly tapered towards the opening at the bottom do not get into contact with the wells of or the liquids in the wells of the commercially available microtiter plate. To transfer the liquid under sterile conditions a fitting cover lid is added to partial device (B). Now, the transfer of the liquids from the microtiter plate, consisting of wells slightly tapered towards the opening at the bottom to the commercially available microtiter via centrifugation plate can take place. In doing so the liquids will automatically and independently from the applied rotational speed and at the lowest possible pressure be pressed from the wells of the microtiter plate which are slightly tapered towards the opening at the bottom to the wells of the commercially available microtiter plate. The reason this process is independent from the applied rotational speed of the centrifuge is, as at the moment when the radial force becomes stronger than the surface tension retaining the liquid in the wells slightly tapered towards the opening at the bottom, the liquids will be transferred into the wells of the commercially available microtiter plate. A further increase in rotational speed may lead to total transfer of liquid residues from the wells of the microtiter plate which are slightly tapered towards the opening at the bottom which have been left due to adhesion. This allows the perforated microtiter plate to be used for further applications without any cleaning steps needed in between.

[0073] In sum it has been shown that through the usage of partial device (B) together with a commercially available cell culture centrifuge and an applied rotational speed increasing from zero to up to e.g. 300g single liquids can be transferred into the single wells of a microtiter plate in a targeted, gentle, simultaneously, even and complete as well as cost-efficient way.

[0074] Furthermore, it has been shown, that using centrifugation and the mentioned partial device (A) a new, cost-efficient viability test for adherently growing cells can be integrated in the field of cell culture using microtiter plates.

[0075] The preferred embodiment of the invented process further includes the following steps: [0076] e) Determination of cell viability of adherently growing cells via quantification of absorbance after centrifugation, and [0077] f) Determination of complete induction of apoptosis in the cells among a complete signal reduction to background level

[0078] In sum, the preferred embodiment of the invented process includes the following steps: [0079] a) provision of a cell culture sample consisting of de-/ or undifferentiated tumour cells, [0080] b) bringing the compound of interest into contact with the cell culture sample [0081] c) following the determination of the relative concentration of the first marker lactate in contrast to untreated cells, and [0082] d) following the determination of the relative concentration of the second marker neutral lipids in contrast to untreated cells,
wherein steps c) and d) may be performed in reverse order if necessary [0083] e) Determination of cell viability of adherently growing cells via quantification of absorbance after centrifugation, and [0084] f) Determination of complete induction of apoptosis in the cells among a complete signal reduction to background level.

[0085] The particularly preferred process includes the following steps: [0086] a) provision of a cell culture sample consisting of de-/ or undifferentiated tumour cells, [0087] b) bringing the compound of interest into contact with the cell culture sample [0088] c) following the determination of the relative concentration of the first marker lactate in contrast to untreated cells, and [0089] d) following the determination of the relative concentration of the second marker neutral lipids in contrast to untreated cells,
wherein steps c) and d) may be performed in reverse order if necessary [0090] e) Determination of cell viability of adherently growing cells via quantification of absorbance after centrifugation, and [0091] f) Determination of complete induction of apoptosis in the cells among a complete signal reduction to background level
wherein
the addition of media and/or buffer components is made via partial device (B) and/or
the removal of media and/or buffer components is made via partial device (A).

[0092] Surprisingly, it has become apparent that using centrifugation and the mentioned partial device (A) a new, cost-efficient viability test for adherently growing cells can be integrated in the field of cell culture using microtiter plate.

[0093] Hereby, cell viability of adherently growing cells can be determined via the viability criteria cell adhesion measured by the non-invasive physical determination of absorption in commercially available microtiter plates (e.g. polystyrene-well bottom without any changes in the plastic surface). This allows a valid, rapid, non-invasive and label-free normalization of distinct measurements per microtiter plate well to the amount of viable cells.

[0094] The label-free quantification of vital cells can be performed due to the characteristics of cellular nucleotides and/or aromatic amino acids to physiologically absorb light at an absorption maximum from 260 nm to 280 nm (absorption maximum DNA/RNA: 260 nm; absorption maximum proteins: 280 nm). In case of measuring proteins this could be made possible for the first time, because the complete removal of media by centrifugation via partial device (A) leaves the wells of the microtiter plate free from media components such as proteins or buffer components which would otherwise affect the measurement. As an alternative to light absorption, light diffusion or cellular auto fluorescence can be quantified. Furthermore, a method using fluorophores or chromophores interacting with or incorporated by the cells prior to measurement can be used in which case the amount of remaining vital cells can be quantified in relation to background signal. As an alternative, the number of vital cells remaining can be determined via (high content) microscopy. The method can be performed as end-point measurement or can be implemented in defined kinetics by set medium changes and measurements prior addition/after removal of media. If cells are seeded at a density considerably lower than the density at which cell growth is reduced due to contact inhibition, kinetic trials for the determination of proliferation can also be performed.

[0095] As a lysis of cell is not implemented within this non-invasive, label-free method, viable cells are directly available for further studies. The described method can be used for toxicity studies as well as immunological toxicity assays. In the latter case, non-adherent immune cells are removed by centrifugation and therefore do not disturb the absorption measurement.

[0096] As, in contrast to necrotic cells, a loss of cell adherence is characteristic for apoptotic cells resulting in a reduced attachment to plastic surfaces (Kwon, H.-K., Lee, J.-H., Shin, H.-J., Kim, J.-H. & Choi, S. Structural and functional analysis of cell adhesion and nuclear envelope nano-topography in cell death. Sci. Rep. (2015). doi:10.1038/srep15623) a method was developed which correlates the reduction of optical absorption to background level after centrifugation with the complete induction of apoptosis in cells previously treated with the compound of interest. Thereby, non-/low-adherently cells are removed via the radial force generated by centrifugation which results in a lower optical absorption in the microplate wells to be examined. The complete detachment of originally adherent cells correlates with an induction of apoptosis in all cells at any time point measured due to the non-reversible detachment of the cells after the termination of cell adherence induced by apoptosis. The same applies to apoptotic vesicles, a later step in apoptosis occurring after cell detachment. This method provides a new, very efficient, non-invasive, label-free, valid and common apoptosis marker independent way to determine apoptosis which is, especially in high throughput applications, a due to complex kinetics difficult to measure variable.

[0097] Measuring absorbance values at background level after centrifugation indicates, independently form the time point of measurement, that absolute induction of apoptosis has occurred in the cells of interest in the respective microplate well(s). As the ability of cells to undergo apoptosis represents a distinctive feature of (re-)differentiation (El-Metwally, T. H. & Pour, P. M. The retinoid induced pancreatic cancer redifferentiation-apoptosis sequence and the mitochondria: A suggested obligatory sequence of events. Journal of the Pancreas (2007)), the quantification of apoptosis especially with kinetics and/or serial dilutions can be very well combined with the previously described markers lactate and/or neutral lipids, e.g. for identifying differentiation inducing substances. Thus, an effective, time-equivalent, metabolic monitoring can be integrated into the trials.

[0098] In comparison to step e) the method can be performed as end-point measurement or can be implemented in defined kinetics by set medium changes and measurements prior addition/after removal of media. To reduce effects due to cell proliferation, cell density as well as cell culture techniques should be chosen individually for every cell line (see application example).

[0099] With another embodiment the underlying task of the present invention is solved by the utilization of the markers lactate and neutral lipids to identify compounds inducing (re-)differentiation in a tumour cell. With regards to the detection of the markers we refer to the invented process.

[0100] The method of the present invention is in a preferred embodiment therefore characterised by the combined utilisation of the markers lactate and neutrals lipids for the identification of compounds inducing (re-)differentiation in un- or dedifferentiated cells, especially tumour cells. Especially preferred it is characterised by quantification of lactate concentration (in bicarbonate buffered cell culture medium incubated in an appropriate constant CO.sub.2-atmosphere) by measuring the pH-dependent change of optical phenol red absorption and by quantification of neutral lipids using an appropriate neutral lipid staining (fluorescent) dye (e.g. BODIPY 493/503).

[0101] The following application example further describes the tasks underlying the present invention in a non-limiting manner:

EXAMPLE

[0102] To provide a homogenous, confluent cell layer, cells (cell lines: A549, MDA-MB-231 and PANC-1) were seeded with defined density and cultured for 24 h in a 384-well microtiter plate. The density of the cells is chosen in a way to reduce cell proliferation via growth arrest. In the following step, the medium is changed by centrifugation and the compound of interest is added. In case of MDA-MB-231 cells Na-Butyrate was added, in case of A549 cells Na-Butyrat and dexamethasone (DM) was added. The differentiation inducing effect of Na-Butyrate (including DM in case of A549) is known.

[0103] It might be necessary for some cell lines to change to serum-free media after the initial incubation of 24 h for a cell line dependent amount of time. Only afterwards, the addition of the compound of interest takes place. The compound's incubation time depends on the chosen tumour model and should be individually adjusted. If necessary, e.g. at incubation times from 24 h to 168 h, one/several medium changes with optional repeated additions of the compound of interest can be performed depending on the respective cell line.

[0104] Upon the expiry of the compound of interest's incubation time (from approx. 48 h to 168 h depending on the cell line) lactate concentration is determined in the supernatant. Therefore, changes in the pH-value of the samples are measured, using phenol red as indicator, and correlated to lactate concentration. For this purpose, the microtiter plate is analysed through the confluent cell layer.

[0105] Subsequently, a medium change via centrifugation was performed in order to remove the media completely. Any remaining media might have negatively affected the following measurement of the second marker neutral lipids.

[0106] After the complete exchange of the media, the fluorescent ligand BODIPY 493/503 was added and incubated for 1 h following the removal of the dye containing media via centrifugation. The concentration of neutral lipids was determined via fluorescence measurement.

[0107] Substances inducing (re-)differentiation lead to a decrease of the first marker lactate and an increase in the second marker neutral lipids. In order to analyse both markers in a positive way, the first marker is mathematically converted into positive values, meaning high values of marker 1 now represent a high reduction of marker 1. In comparison to the mean values of untreated cells various compounds can be examined, analysed and evaluated with regard to their (re-)differentiation inducing abilities.

[0108] Directly following the measurement of both markers the cell viability of adherent cells is determined via absorption measurement in a microplate-reader. Whereby, the execution of the measurement is independent from the previously conducted measurements and is not further affected by previously added fluorescent ligands.