EP2010907A1

EP2010907A1 - Method for the detection of biological molecules in cells

Info

Publication number: EP2010907A1
Application number: EP07728160A
Authority: EP
Inventors: Roberta Carbone; Andrea Zanardi; Pier-Giuseppe Pelicci
Original assignee: TETHIS Srl
Current assignee: Tethis SpA
Priority date: 2006-04-20
Filing date: 2007-04-16
Publication date: 2009-01-07
Also published as: WO2007122133A1

Abstract

The present invention relates to a method for the detection of biological molecules in or on cells and a kit for carrying out the method. The method comprises the steps of (a) providing a solid support having at least one distinct area for receiving living cells; (b) treating at least the at least one area of the solid support with a reagent promoting the adhesion of the cells; (c) inoculating the at least one area with living cells; (d) incubating the solid support under conditions that allow proliferation of the cells; (e) spotting at least one reagent capable of interacting with a component of the cells onto predetermined multiple spatially distinct sites of the at least one area comprising the proliferated cells; and (f) detecting whether or not the at least one reagent has reacted with a component of the cells.

Description

Method for the detection of biological molecules in cells

The present invention relates to a method for the detection of biological molecules in or on cells and a kit for carrying out the method. The method comprises the steps of

(a) providing a solid support having at least one distinct area for receiving living cells;

(b) treating at least the at least one area of the solid support with a reagent promoting the adhesion of the cells; (c) inoculating the at least one area with living cells; (d) incubating the solid support under conditions that allow proliferation of the cells; (e) spotting at least one reagent capable of interacting with a component of the cells onto predetermined multiple spatially distinct sites of the at least one area comprising the proliferated cells; and (f) detecting whether or not the at least one reagent has reacted with a component of the cells.

The knowledge of signalling pathways in living cells triggered by physiological, pathological conditions or drugs is essential for the comprehension of biological events.

Recently, novel protein array platforms based on "reverse phase protein arrays" (Chan et al. (2004) Nature Med. 10 (12), 1390-1396) have proven to be very powerful in the study of different pathways, but still lack the possibility to detect events in their complexity in a "cellular" context, where dynamic phenomena need to be analyzed for the comprehension of biological responses.

The mis-regulation or hyperactivity of intracellular and extracellular signalling cascades may represent the molecular signature of human diseases: the capability of monitoring the complexity of all biochemical networks involved in specific pathologies is essential for the realisation of patient-tailored therapies and for the characterization of drug responses. Kinase activity, post-translational modification, protein overexpression- downreguiation or derealization, are only few of the putative mechanisms that can be relevant to human diseases (Megan et al. (2006) Nature Reviews Cancer 6,184- 192; Jonathan et al. (2006) Nature Reviews Cancer 6, 146-155; McLean et al. (2005) Nature Reviews Cancer 5, 505-515; Colombo et al. (2006) Cancer Res. 66 (6),3044- 50). Signalling pathways are complex (Hall et al. (2006) J. Pathol. 208, 1-6; Cadigan et al. (2005) J. Cell Sci. 119, 395-402) and they are regulated by specific spatio- temporal dynamics that occur in a cellular context. This complexity requires the utilization of different technologies that can describe "when and where" (Belkowski et al. (2005) Current Topics in Medicinal Chemistry, 5 (11), 1047-1051) a single event takes place.

New genomic and proteomic technologies have recently been developed (Carr et al. (2004) Hum Genomics. 1 (2), 134-40; Kingsmore (2006) Nature Reviews Drug Discovery 5, 310-321) allowing the simultaneous analysis of multiple events and the characterisation of complex samples.

Among them reverse microarrays (Nishizuka et al. (2003) Proc. Natl. Acad. Sci. USA 100 (24), 14229-14234) represent one approach to analyse tissue or cell lysates with a large number of antibodies or patient sera to allow the identification of new biomarkers, the analysis of protein expression profiles and drug candidates' efficacy and toxicity. In this context a multiplexed reverse phase protein microarray (RPP) has been developed by Chan and co-workers (2004, Nature Med., 10 (12), 1390-1396) and applied in the study of different molecular pathways.

However, since the reverse microarray technology detects molecules in cell lysates, it does not allow the detection of multiple events in a "cellular" context of compartments and organels, where phenomena need to be analyzed for the comprehension of the biological networks, either in normal or pathological conditions.

US Patent Application No. 2003/0189850 A1 discloses a method for carrying out continuous or simultaneous detection of a subject for assay in plural cells by chemiluminescence, bioluminescence or fluorescence comprising a device which comprises a substrate, and an array of spatially-separated plural sections formed on the substrate, each of the sections having an area enough to immobilise plural independent cells. In particular, the cells are immobilised in an array of small spots and are, without further proliferation, analysed, e.g. with respect to DNA ploidy, chromosomal aberration, or antibody expression.

Stephan et al. (2002, Am. J. Pathol. 161 , 787-797) describe the development of a frozen cell array for performing high-throughput cell-based analysis. The construction of the frozen cell array is carried out by a relatively complicated method involving several steps performed under dry ice conditions.

The technical problem underlying the present invention is the provision of an improved system for carrying out multiple analyses of biologically relevant molecules in a cellular context.

The solution to the above technical problem is characterised by the embodiments defined in the claims.

In particular, according to a first aspect, the present invention provides a method for detecting biological molecules in/on cells comprising, in the following order, the steps of

(b) treating at least the at least one area of the solid support with a reagent promoting the adhesion of the cells; (c) inoculating the at least one area with living cells;

(d) incubating the solid support under conditions that allow proliferation of the cells;

(e) spotting at least one reagent capable of interacting with a component of the cells onto predetermined multiple spatially distinct sites of the at least one area comprising the proliferated cells; and

(f) detecting whether or not the at least one reagent has reacted with a component of the cells. According to the present invention any kind of culture cells can be used. Suitable cells can be obtained from American Type Culture Collection (ATCC), Manassas, VA, USA. Preferred cells are mammalian cells such as osteoblasts, melanocytes, keratinocytes, fibroblasts, haematopoietic cells, epithelial cells, endothelial, neuronal, macrophage, dendritic cells, embryonal/adult stem cells, hepatocytes etc. Especially preferred are cells capable of adherent proliferation.

The solid support according to step (a) of the present method may be any support suitable for receiving the cells and allowing proliferation thereof. Therefore, preferred materials of the solid support are biocompatible. If the support material itself is not biocompatible, it is desirable to cover the primary support or at least the area for receiving the cells with a biocompatible material. Preferred embodiments of the solid support are supports made of glass, ceramics, silicon, plastics etc. As mentioned before, the surface of the solid support but at least the at least one distinct area for receiving living adherent cells may be surface treated with suitable agents such as materials favouring the adherence of the cells.

According to a further embodiment, the one or more area(s) on the solid support or the complete upper surface of the support is/are coated with a nanostructured TiO₂ film. Preferred embodiments of such a nanostructured TiO₂ film are disclosed in European Patent Application No. 05 015 869.0 the respective contents of which is herewith enclosed in the present description by reference. Thus, a "nanostructured" TiO₂ film means an at least partial coating of the at least one area with nanoparticles of TiO₂ which are preferably deposited by supersonic cluster beam deposition (SCBD) as described in US 6,392,188 wherein the nanoparticles are produced by a pulsed microplasma cluster source (PMCS) and selected by aerodynamic separation effects. The deposited film is a complex mixture where amorphous material ("matrix") typically coexists, at the nanoscale, with anatase and rutile crystal phases. The nanocrystalline fraction of such a film is usually characterised by crystal sizes of between about 5 nm (or even less) to about 100 nm, preferably about 10 nm to about 20 nm. The spreading of the crystals in the amorphous matrix leads to a nanoporous structure having a large surface area. Such films show, at the nanoscale, a porosity and granularity comparable to typical extracellular matrix (ECM) structures. Furthermore, the solid support according to the present invention may have any suitable shape such as squares, triangles, circles and so on. The same holds true for the at least one area for receiving the living adherent cells.

The at least one area of the solid support is treated with a suitable reagent promoting the adhesion of the cells. Suitable reagents are for example gelatine or solutions thereof, polyornitine, polylysine, matrigel^® , aminosilane, fibronectin, vitronectin, laminin, collagen, vitronectin, biocompatible polymers that favour adhesion (see Anderson, D.G. (2004) Nature Biotechnology 22 (7), 863-866).

Thereafter, the at least one area containing the medium is inoculated with a suitable number of cells such as between about 4x10⁵ to about 6x10⁵ cells, preferably 5x10⁵ to about 6x10⁵ cells depending on the type of cells, later incubation conditions, growth rate, medium, and surface area to be finally covered. The person skilled in the art is readily able to determine a suitable number of cells by carrying out routine experiments.

Then, the solid support containing the inoculated cells is incubated under suitable conditions allowing the proliferation of the cells. The incubation is typically carried out in a suitable growth medium (e.g. DMEM, optionally supplemented with fetal calf serum (FCS), antibiotics, growth factors etc.) and, in particular for mammalian cells, under atmospheric conditions of 5 % CO₂ in a humidified atmosphere. The incubation time is chosen such that the cells on the at least one distinct area reach a suitable percentage of confluence (i.e. percentage of coverage of the surface area in terms of a cell monolayer) such as at least about 50%, more preferred at least 60%, even more preferred at least 70% and particularly preferred at least 80% or even 90 % depending on the cell type, conditions of the further method steps (reagents, application techniques for the reagents, detection means etc.). It is, however, as a rule, not desirable to proliferate the cells to such an extent that the at least one distinct area is more than covered with a monolayer of the cells. Suitable growth media, proliferation conditions etc. are described, e.g. in Bonifacino et al. (eds.) Current Protocols in Cell Biology, 1998-2005, John Wiley & Sons, New York. According to a further preferred embodiment of the present invention the cells are treated with at least one biochemically active compound or means during step (d), i.e. during the proliferation step. Suitable biochemically active compounds or means are any compounds or means that at least potentially exert an effect on the cells that is measurable by any biochemical, chemical or physical method. Thus, the biochemically active compound may be selected from the group consisting of pharmaceutical drugs or candidates thereof, nucleic acids, peptides, proteins, antibodies, chemokins, antibiotics, growth factors, X rays, UV, nanoparticles, vital stainings, free radicals releasing compounds, dendrimers, quantum dot-conjugated molecules.

Thus, the cells can be treated with suitable factors which at least are potentially capable of changing the biochemical status of the cells before the cells are further processed during the next steps. This represents a substantial benefit of the present invention in that the cells may be treated in any way that is imaginable in a cell biological/biochemical context. For example, the cells can be treated with pharmaceutical drugs or at least candidates thereof in order to later detect the effect of such drugs or drug candidates on specific cell components or signalling pathways. Other exemplary cell biological treatments are by influencing the expression profile of the cells or by starting or preventing the expression of endogenous or exogenous genes contained in the cells. Exogenous genes are genes present on exogenous DNA or RNA vehicles such as plasmids, viruses and so on. Preferred exogenous gene vehicles are recombinant constructs.

Methods and reagents for genetic engineering of genetic constructs are disclosed, e.g. in Ausubel et al. (eds.) Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2001-2005.

Transfection of cells to be analysed by the inventive method may be carried out by any method known by a person skilled in the art (see, e.g., Bonifacino et al., supra). Preferred methods of gene delivery into cells according to the present invention are based on viral infection. In this regard, it is especially preferred to avoid any toxic polycations (such as polybrene) commonly used for improvement of the infection efficiency. Therefore, a particularly preferred embodiment of the present invention makes use of a so-called "reverse infection" mechanism directly on the area(s) where cells proliferate.

In particular, according to preferred embodiments of the present invention, viral infection of the proliferated or proliferating cells is improved by the use of a nanostructured TΪO2 film, preferably deposited on the at least one distinct area by cluster supersonic cluster beam deposition (SCBD), e.g. using the apparatus disclosed in US 6,392,188 (see also European Patent Application No. 05 015 869.0). Thus, the method of the present invention (particularly during step (d)) preferably comprises the sub-step of contacting the cells with an infectious viral particle. Particularly preferred viruses are retroviruses. As mentioned above, especially preferred embodiments of the sub-steps for viral infection are achieved by a so-called "reverse infection" process which comprises contacting the ns-Tiθ2 coated area(s) of the support with the virus (usually the virus is present in form of a virus supernatant) such that the virus adheres to the area (preferably, the ns-Tiθ₂ coated area is coated by a suitable adhesion molecule such as streptavidin and the virus is derivatised by the corresponding counterpart of a binding pair such as biotin) whereafter the cells are administered to the area(s) and incubated under appropriate conditions. The reverse infection mechanism, especially in combination with ns-Tiθ₂ films, provides for very high gene transduction efficiencies and avoids the use of toxic polycations leading to apoptosis and DNA damage. A detailed description of "reverse infection" of cells on ns-Tiθ₂-coated substrates is disclosed in EP 05 015 869.0 the disclosure content of which is hereby incorporated into the present description by reference.

After the cells have been proliferated to the appropriate confluency, multiple predetermined spatially distinct sites of the at least one area comprising the proliferated cells are reacted with at least one reagent capable of interacting at least temporarily with one or more components of the cells. The term "reacted with" means that the at least one reagent (typically a suitable solution of a molecule capable of interacting at least temporarily with one or more components of the cells) is spotted onto the multiple predetermined spatially distinct sites such that said at least one reagent is restricted to said multiple predetermined spatially distinct sites. The spotting of the reagents according to the present invention is typically carried out by applying a small volume, generally not more than 200 nl, e.g. 5 to 200 nl, preferably 10 to 100 nl, of a solution containing the reagent(s). As a rule, the volume of the spotted solution correlates with the desired density of the spots. The interaction between the at least one reagent and the component of the cell can be of any type but will usually be sufficient such that the interaction can be detected by suitable means. Thus, the interaction may be by hydrogen bonding, an electrostatic interaction, a covalent bonding or through Van der Waals interactions.

Typical examples of such reagents capable of interacting with a component of the cells are antigen-specific binding molecules such as immunoglobulins or antigen- binding fragments thereof. Particular preferred reagents are antibodies which may be polyclonal, monoclonal or recombinant.

Antibodies for use in the present invention may be directed against any cellular component (examples of typical antigens are given in Tables 1 and 2 herein below). As mentioned before, the term "antibody" comprises polyclonal as well as monoclonal antibodies, chimeric antibodies, humanised antibodies. Furthermore, an "antibody" according to the present invention may be a fragment or derivative of the afore-mentioned species. Such antibodies or antibody fragments may also be present as recombinant molecules, e.g. as fusion proteins with other (proteinaceous) components. Antibody fragments are typically produced through enzymatic digestion, protein synthesis or by recombinant technologies known to a person skilled in the art. Therefore, antibodies for use in the present invention may be polyclonal, monoclonal, human or humanised or recombinant antibodies or fragments thereof as well as single chain antibodies, e.g. scFv-constructs, or synthetic antibodies.

Polyclonal antibodies are heterogeneous mixtures of antibody molecules being produced from sera of animals which have been immunised with the antigen. Subject of the present invention are also polyclonal monospecific antibodies which are obtained by purification of the antibody mixture (e.g. via chromatography over a column carrying peptides of the specific epitope. A monoclonal antibody represents a homogenous population of antibodies specific for a single epitope of the antigen. Monoclonal antibodies can be prepared according to methods described in the prior art (e.g. Kohler und Milstein, Nature, 256, 495-397, (1975); US-Patent 4,376,110; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring, Harbor Laboratory (1988); Ausubel et al., (eds), 1998, Current Protocols in Molecular Biology, John Wiley & Sons, New York). The disclosure of the mentioned documents is incorporated in total into the present description by reference.

Genetically engineered antibodies for use in the present invention may be produced according to methods as described in the afore-mentioned references. Briefly, antibody producing cells are cultured to a sufficient optical density, and total RNA is prepared by lysing the cells using guanidinium thiocyanate, acidification with sodium acetate, extraction with phenol, chloroform/isoamyl alcohol, precipitations with mit isopropanol and washing with ethanol. mRNA is typically isolated from the total RNA by chromatography over or batch absorption to oligo-dT-coupled resins (e.g. sepharose). The cDNA is prepared from the mRNA by reverse transcription. The thus obtained cDNA can be inserted into suitable vectors (derived from animals, fungi, bacteria or virus) directly or after genetic manipulation by "site directed mutagenesis" (leading to insertions, inversions, deletions or substitutions of one or more bases pairs) and expressed in a corresponding host organism. Suitable vectors and host organisms are well known to the person skilled in the art. Vectors derived from bacteria or yeast such as pBR322, pUC18/19, pACYC184, Lambda oder yeast mu vectors may be mentioned as preferred examples. Such vectors are successfully used for cloning the corresponding genes and their expression in bacteria such as E. coli or yeast such as Saccharomyces cerevisiae.

Antibodies for use in the present invention can belong to any one of the following classes of immunoglobulins: IgG, IgM, IgE, IgA, GILD and, where applicable, a subclass of the afore-mentioned classes, e.g. the sub-classes of the IgG class. IgG and ist sub-classes, such as IgGI , lgG2, lgG2a, lgG2b, lgG3 or IgGM, are preferred. IgG subtypes lgG1/k or lgG2b/k are especially preferred. A hybridoma clone which produces monoclonal antibodies for use in the present invention can be cultured in vitro, in situ oder in vivo. High titers of monoclonal antibodies are preferably produced in vivo or in situ.

Chimeric antibodies are species containing components of different origin (e.g. antibodies containing a variable region derived from a murine monoclonal antibody, and a constant region derived from a human immunoglobulin). Chimeric antibodies can be employed in order to improve the production yield. For example, in comparison to hybridoma cell lines, murine monoclonal antibodies give higher yields.. A further example is a monoclonal antibody in which the hypervariable complementarity defining regions (CDR) of a murine monoclonal antibody are combined with the further antibody regions of a human antibody. Such an antibody is called a humanised antibody. Chimeric antibodies and methods for their production are described in the prior art (see e.g. EP-A- 125023 and Harlow und Lane, Antibodies: A Laboratory Manual, supra). The disclosure content of the cited documents is incorporated in the present description by reference.

According to the present invention, the term ..antibody" comprises complete antibody molecules as well as fragments thereof being capable of binding to a cellular component. Antibody fragments comprise any deleted or derivatised antibody moieties having one or two binding site(s) for the antigen. Specific examples of such antibody framents are Fv, Fab or F(ab')₂ fragments or single strand fragments such as scFv. Double stranded fragments such as Fv, Fab or F(ab')2 are preferred. Fab und F(ab')₂ fragments have no Fc fragment contained in intact antibodies. Such fragments may be produced from intact antibodies by proteolytic digestion using proteases such as papain (for the production of Fab fragments) or pepsin (for the production of F(ab')2 fragments), or chemical oxidation.

Preferably, antibody fragments or antibody constructs are produced through genetic manipulation of the corresponding antibody genes. Recombinant antibody constructs usually comprise single-chain Fv molecules (scFvs, -3OkDa in size), in which the V_H and V_L domains are tethered together via a polypeptide linker to improve expression and folding efficiency. In order to increase functional affinity (avidity) and to increase the size and thereby reduce the blood clearance rates, the monomeric scFv fragments can be complexed into dimers, trimers or larger aggregates using adhesive protein domains or peptide linkers. An example of such a construct of a bivalent scFv dimer is a 60 kDa diabody in which a short, e.g. five-residue, linker between V_H- and VL-domains of each scFv prevents alignment of V-domains into a single Fv module and instead results in association of two scFv molecules. Diabodies have two functional antigen-binding sites. The linkers can also be reduced to less than three residues, which prevents the formation of a diabody and instead directs three scFv molecules to associate into a trimer (90 kDa triabody) with three functional antigen-binding sites. Association of four scFvs into a tetravalent tetrabody is also possible. Further antibody constructs for use in the present invention are dimers of scFv-CH3 fusion proteins (80 kDa; so-called "minibodies")

Further protocols for generating antibodies and their handling are described in, e.g. Coligan et al. (eds.) Current Protocols in Immunology, John Wiley & Sons, New York, 1999-2005.

Therefore, the method of the present invention is particularly useful for testing antibodies or antigen-binding fragments thereof, e.g. with respect to their antigen specificity in the cellular context.

Furthermore, it is possible to apply more than one reagent to the multiple predetermined spatially distinct (separated) sites in order to analyse more than one specific component of the cells at the same time. As an example, more than one antibody each specific for a particular cell component may be spotted onto the predetermined sites, whereafter the binding of these reagents can be detected, e.g. by indirect detection using differently labelled secondary antibodies (e.g. green, blue, red, far red such as appropriately chosen fluorescent markers).

Further reagents typically used for detecting cellular components such as organels, cytoskeletal molecules etc. are dyes, in particular fluorescent dyes, specifically binding or localising to a compartment or even single molecules. Suitable dyes for such purposes are commercially available and may be obtained from Invitrogen Moleculare Probes, Invitrogen Corp., Carlsbad, CA, USA. Furthermore, fluorescent ligands of cellular components (such as receptors, enzymes etc.) and quantum dot- conjugated reagents can be used as well.

Furthermore, the reagent capable of interacting with a component of the cell may be selected from nucleic acids for suitable hybridisation techniques. Therefore, the method according to the present invention can take the form of an in situ hybridisation protocol. The method according to the present invention provides a highly flexible system in that not only the multiple spatially distinct sites can be reacted with different reagents capable of binding to cellular components. Also, it is possible to contact the cells on the multiple spatially distinct sites with different amounts of the reagents. Furthermore, it is of course possible to form sub-groups of the multiple spatially distinct sites which may be reacted with different reagents or different amount of the reagent(s).

Moreover, since it is possible to provide multiple distinct areas on the solid support for receiving living cells, it is also possible to inoculate each distinct area with different cell types and/or different amounts of cells. Again, it is also possible to subgroup the multiple distinct areas such that different groups receive different cell types but within each group the same cell type is used.

Preferably, the cells are fixed and/or permeabilised between steps (d) and (e) of the inventive method. Reagents for fixation (such as paraformaldehyde or glutaraldehyde solutions or acetone/methanol or methanol) and permeabilisation (such as Tween^® or Triton-X 100, saponin) are well known in the art. Typically, such detergents and fixatives are used in phosphate buffered saline solutions such as Dulbecco's Phosphate Buffered Saline (DPBS).

The above mentioned step (f) of the protocol according to the present invention comprises a direct or indirect detection of whether or not the at least one reagent capable of binding to, i.e. interacting with, a component of the cell has factually reacted with this component.

Direct detection means that the reagent itself is typically labelled with a detectable marker which may be a radioactive label or dye, in particular a luminescent dye, more preferably a fluorophor.

An indirect detection means that the at least one reagent capable of interacting with the cellular component is detected by a second molecule that is specific for the reagent wherein this second molecule is either labelled or in itself detectable typically by physical means. In case the reagent is a proteinaceous molecule such as an antibody or at least an antigen-specific binding molecule or an antigen-binding fragment of such a molecule, the detection of whether or not the reagent has factually interacted or in other words reacted with a cellular component generally comprises the use of a (secondary) antibody directed against the reagent such as the antibody or fragment thereof.

Therefore, above step (f) comprises the sub-step of reacting one or more of the multiple spatially distinct sites with a molecule capable of interacting with the reagent(s).

In order to indirectly detect the reagent capable of interaction with a cellular component, the (second) molecule such as a secondary antibody is typically labelled with a detectable marker which may take the forms as described above. Again, fluorescent dyes are preferred for such purposes. Suitable fluorescent dyes and other components of detectable markers may be obtained from Invitrogen Molecular Probes, Invitrogen Corp., Carlsbad, CA, USA.

For carrying out detection step (f) according to the present invention it is finally necessary to detect the signal arising from the interaction of the reagent with the cellular component and/or the interaction of the secondary detection molecule with the initial reagent. Appropriate detecting means depend on the signal generated by the interaction or marker. In the case of fluorescent dyes fluorescence microscopes or spectrophotometers are suited for this purpose.

Furthermore, the method of the present invention typically comprises one or more washing steps between steps (d) and (e) and/or (e) and (f). Therefore, it is generally preferred to decrease the background of the final signal by washing away unbound (that is non-interacting) reagents (the first capable of interacting with the cellular component as well as optionally the secondary molecule in the case of indirect detection techniques) by using large volumes of suitable buffer solutions (e.g. PBS, DPBS, TBS, TBST etc.). Further washing steps are typically performed after fixation and/or permeabilisation steps. Typically, the surface of the at least one area is treated with a blocking solution before performing step (e) in order to block non-specific binding sites for the reagent(s) capable of interacting with a cellular component as well as of the secondary molecules in the case of indirect detection techniques. Suitable blocking solutions are phosphate or Tris buffered solutions of BSA or milk powder.

According to a further preferred embodiment, the method of the present invention comprises a further step (g) of detecting the localisation of the reagent within/on the cells.

Typically, microscope equipment is a suitable means for detecting the localisation of cellular components interacting with the reagents. Further preferred are automated fluorescence microscopes, optionally equipped with a CCD camera, and other well known high resolution techniques (confocal microscopes).

Step (e) of the present invention means that the at least one reagent capable of interacting with a cellular component is applied to the multiple predetermined spatially distinct sites of the at least one area comprising the proliferated cells in the form of multiple spots. Preferably, state of the art arraying techniques are used for the application of the reagent on a multitude of spatially distinct sites, e.g. by contact and/or non-contact spotting. Corresponding arrayers and accessory equipment are commercially available, e.g. from BioDot, Inc., Irvine, CA, USA.

"Spatially distinct" means that the predetermined multiple sides are spatially separated from one another. Preferred are regular arrangements, i.e. arrays of e.g. 8 x 8, 8 x 16 etc. spots, more preferred high density arrays wherein the multiple spatially distinct sides are separated by not more than about 2 mm, preferably not more than about 1 mm. "Separated" means that the centres of the multiple spatially distinct sites have a certain distance in a cartesian (x-y) system (also called "pitch") such that the spatially distinct sites do not overlap. Depending on the spotting technique it is possible to achieve much lower distances between the spots, e.g. about 500 μm or even less. In order to apply the reagent in small distances non- contact spotting using piezo-spotters is preferred. Especially preferred pitch ranges are between about 500 μm to about 2 mm. in the method according to the present invention, step (e) is carried out by spotting, preferably non-contact spotting using suitable and commercially available equipment. This technique is also preferably used in step (f) in the case of the application of suitable molecules for indirect detection (e.g. secondary antibodies) of the reagent capable of interacting with a cellular component.

As mentioned before, the solid support for carrying out the method of the present invention may be composed of different materials. However, preferred are glass slides, optionally coated with a nanostructured TΪO₂ film (or at least the distinct area(s) is/are coated with a film). Typically, such glass slides have dimensions such that they are compatible with usual high-throughput equipment for microarray purposes (typical surface dimensions of about 25 mm x 76 mm). Such microarray glass slides are commercially available, e.g. from TeleChem International, Inc., Arraylt^® Division, Sunnyvale, CA, USA.

The person skilled in the art readily recognises that a solid support can comprise more than one distinct area for receiving living adherent cells. The upper limit of the number of distinct areas is not critical for performing the invention and depends on the surface area of the solid support. A further parameter determining the upper limit of distinct areas for receiving living adherent cells are the type of the cells. Thus, the method of the present invention can be performed according to a multiplexed microarray format.

According to a further aspect, the present invention provides a kit for performing the above-defined method, wherein the kit comprises a solid support having at least one distinct area for receiving living cells; a reagent providing a medium for the proliferation of cells; at least one strain of culture cells; - at least one reagent capable of binding to a biological molecule within and/or on the cells, which reagent is to be applied onto multiple predetermined spatially distinct sites of the at least one area; means for the detection of the at least one reagent; and an instruction manual containing information for carrying out the method as defined above.

With respect to preferred embodiments of the above components of the inventive kit it is referred to the respective sections with respect to the method constituting the first aspect of the present invention.

The figures show:

Fig. 1 shows immunocell-arrays of 8x8 immunostained spots of 100-150 cells each. Primary fibroblasts were fixed and permeabilized and stained in alternate spots with a monoclonal antibody against PML and BSA₁ as control for specific staining in localised spots. Panel A: image of a 2x2 array (enlarged magnification of the original image) where bright white staining indicates detection of PML, dark gray indicates detection of nuclei, not stained with the antibody. Panel B: image of an array of 8X8 immunostained spots of 100-150 cells each showing only PML staining in bright white; panel C: image enlargement of an area of panel B to exclude possible cross-contaminations among adjacent spots; inset: magnification of two cells of one spot. In panel B images were acquired at 2Ox magnification.

Fig. 2 Panel A shows an image of an 8x17 immunocell-array of NIH3T3 cells stained with 17 different antibodies; in panel B magnification of small areas of different spots are shown to evaluate specific protein localisation in detail (E3B1 , clathrin, vinculin, p-tyrosine, phalloidin, profilin, Wip, Vasp, AP1 -alpha, Eps8,

Eps15, tubulin). Panel C shows an image of an 8X16 immunocell-array of adult primary melanocytes with 15 different antibodies (BSA was spotted as control in the last lane); in panel D magnification of small areas of different spots are shown to evaluate specific protein localisation in detail (clathrin, tubulin, NPM, phalloidin, Eps8, Eps15). In panels A and C images were acquired at 2Ox magnification.

Fig. 3 shows images of an 8x24 immunocell-array of U2OS cells treated with bleomycin taken at different time points (control, 8h and 24h). Panel A: example of the 8x24 array. Panel B: small areas of different spots corresponding to the immunostained proteins in control and 24 h bleomycin treated slides. Differences in staining intensity or specific localisation of proteins can be observed.

Fig. 4 shows diagrams of statistical analyses of bleomycin response after image acquisition. Graph 1 shows an analysis of the mean fluorescence intensity of acetylases/deacetylases at 0-8-24h after treatment. Graph 2 shows an analysis of the mean fluorescence intensity of the other series of antigens involved in DNA damage response.

The present invention is further illustrated by the following non-limiting example.

EXAMPLE

Materials and Methods

Cell plating

Arraylt^® SuperClean2 glass slides are put into Vivascience^®Vivadish plates, microbiological grade. The glass slides are covered with a sterile solution of 0,2% Sigma^® type B gelatin from bovine skin in IxDPBS and incubated at room temperature for 30 min. After incubation the slides are washed once with IxDPBS. Then, growing cells (typically about 80% confluent) are collected from their growth dish and typically 4x10⁵ to 6x10⁵ cells are plated per slide in a volume of 4 ml of growth medium.

Treatments

In the bleomycin experiment (see below), after 48 h (when cells reached 80% confluence on the slides) the medium is changed (except for the control slide which is fixed without further treatment) to medium supplemented with 25 ug/ml bleomycin, and then fixed after 8 and 24 hours. Pre-Staininq

Cells covering slides are fixed with a solution of 4% paraformaldehyde in Pipes buffer (80 mM Pipes pH 6.8, 5 mM EGTA pH 8, 2 mM MgCI₂) for 15 min. After fixation slides are washed twice with 1 XDPBS and can be stored several days at 4° C. After washing, cells are permeabilized for 10 min with Triton 0.1% in a solution of IxDPBS

+ 0.2% BSA. The slides are then washed twice with IxDPBS, and blocked for 1 h with 2% BSA (diluted in IxDPBS). After blocking, the slides are washed twice with IxDPBS. Finally, the slides are dryed by keeping the slide holder tilted (about 45°) under the hood airflow.

Immunocell-arrav

A Biodot^® BioJet Plus spotter with Axsys software, aspirating antibodies from a 96 well plate and dispensing drops on a slide holder, was used. Usually, lines of 10 drops are spotted for each antibody with a pitch of 2 mm. For each drop 30 nl are generally dispensed. Usually, the humidifier is switched on 10' before spotting in order to reach constant and uniform 65% humidity in the spotting chamber. After drying, the slides are rapidly placed in the spotting chamber and left there for rehydration (in about 5 min slides lose their opacity which is indicative that the cells are not dry anymore). Then, primary antibodies are spotted which are incubated on the slide for 45 min at 4°C in a small chamber with a NaCI-saturated water solution, which keeps humidity at about 75% in the closed chamber; in addition a tiny film of DPBS is added on the bottom of the multiwell dish, were slides are placed, to ensure constant high humidity during antibody incubation. After incubation the slides are washed twice with IxDPBS + 0,1% Tween, and 4 times in IxDPBS. The slides are washed by dipping them using a slide rack in suitable boxes with large volumes of the solutions mentioned above. Slides are dryed as above, re-hydrated in the spotting chamber where after secondary antibodies are spotted. Secondary antibodies are incubated as above for 30 min. After washing twice in IxDPBS + 0.1% Tween, 4 times in IxDPBS, the slides are with DAPI for 5 min, for nuclei staining. Finally, the slides are washed once with IxDPBS, once with H₂O, and the cover glass is mounted with Mowiol.

Set up of an 'immunocell-array' protocol on slide in high-throughput format by spotting primary and secondary antibodies on fixed cells.

Primary human fibroblasts were plated on microarray grade glass slides coated with 0.2% gelatin, grown at the desired confluency and fixed by 4% paraformaldheyde for 10 min. Cells were subsequently permeabilized, blocked by BSA incubation, and further processed for primary and FITC conjugated secondary antibody spotting in a 8X8 array, by using a BioDot non-contact spotter. To ensure the specificity of staining in localised areas we deposited by automated spotting a specific monoclonal antibody against PML, a nuclear protein localised in nuclear dots (PML bodies) and BSA in alternate spots to easily check for antibody contamination in BSA adjacent spots. Slides were processed for DAPI staining to identify nuclei and mounted with Mowiol. As it is shown in Fig. 1 , it was possible to specifically identify, by analysis using automated time-lapse microscopy, PML stained spots with no contamination of staining in BSA spots (see panel C at 10X and panel D at 2OX magnification). The inset on panel D shows the PML localisation in nuclear bodies.

Adult primary melanocytes and NIH3T3 are suitable cellular model systems for immunocell-array by targeting cytoplasmic and nuclear proteins.

To explore the potential of this technology immunocell-array experiments were performed on different cellular types with different cellular target (nuclear, cytoplasmic and membrane proteins).

Adult primary melanocytes and NIH3T3 cells were tested: primary melanocytes represent important tools for biological studies related to diseases like pigmentation disorders, melanoma or others genetic abnormalities; the possibility to perform high throughput signaling studies on patient derived cells, where the availability is one of the major constraint, should represent a valuable resource. As shown in Fig. 2 panel B a 8X16 array of melanocytes was stained with a panel of Abs specific for different antigens (see Table 1): in panel B high resolution images show detailed staining of some of the tested Abs. In particular, NPM nucleolar staining (Colombo et al. (2006) Cancer Res. 66 (6), 3044-50), actin filaments by phalloidin staining, and clathrin containing vesicles (Edeling et al. (2006) Nat. Rev. MoI. Cell Biol. 7 (1), 32-44) was detected.

NIH3T3 cells represent a suitable model for cytoskeleton-associated signalling pathways. Therefore, an 8x17 immunocell-array with 12 different cytoskeleton related antibodies (see Table 1) was performed. As shown in Fig. 2 panel B specific staining is obtained.

Table 1: Antigens of primary antibodies

Bleomycin treated U2OS cells show deacetylation of chromatin and modulation of acetylase-deacetylase enzymes.

To evaluate the present immunocell-array system in dissecting molecular pathways the DNA damage response through bleomycin treatment of U2OS cells was studied by analysing a panel of selected antibodies that target different nuclear and cytoplasmic proteins (see Table 2). Table 2: Antigens of primary antibodies

As shown in Fig. 3 (in the upper panel an example of a complete array and in the lower panel enlarged images of control and 24h treated cells for each of the antibodies tested) U2OS cells were treated for 8 and 24 hours, processed for staining with antibodies against 21 different antigens (see Table 2) and analysed by time lapse microscopy. After image analysis a series of graphs representing the response in terms of Cy3 intensity/DAPI staining of each antibody were obtained (see Table 3 and Fig. 4).

Table 3: Image analysis of bleomycin treated cells reacted with antibodies against different antigens

As shown in Fig. 4, statistical imaging analysis reveals, as expected, an up-regulation of ATM and p53, induction of H2AX staining and no pH3 staining upon treatment. Interestingly, anti-acetylated T52 and T25 antibodies revealed down-regulation of respective targets (anti-acetylated lysines and anti-acetylated Histone H4) suggesting that DNA damage is inducing down-regulation of chromatin acetylation. In the same context CBP appeared to decrease after treatment, while HDAC was not altered. Moreover Ezh2, a putative target of p53, is also showing a decrease in staining. Further biochemical evaluations are required to confirm these observations, nevertheless the present data unravel the existence of a pathway of regulation of chromatin acetylation upon DNA damage in mammalian cells (Tamburini et al. (2005) MoI. Cell. Biology 25 (12), 4903-4913; Hassa et al. (2005) Biochem. Cell. Biol. 83 (3), 270-85).

Previous work done in yeast (Tamburini et al. (2005), supra) underlined a phenomenon of histone acetylation and deacetylation triggered by the double strand DNA repair pathway. The present observation in mammalian cells by using the method of the present invention further strengthens the relevance of this signalling pathway and the role of chromatin state in DNA repair and genomic stability.

Conclusions

The present invention provides a novel cell array-based method which can be conveniently used for immunofluorescence analysis of target proteins in different cellular models.

The present technology provides many advantages over existing immunofluorescence methods: it decreases the cost of the assay in terms of antibodies and cells, provides a flexible high content approach "on chip" for multiple targets and different kinetics, data can be analyzed through high resolution confocal microscopy, and finally the present method ensures a high degree of reproducibility and no well to well variability.

This technology can be used inter alia for the molecular dissection of signalling pathways (kinase response, post-translational modifications, protein localization and protein profiling) upon drug response in normal and disease states, for the characterization of "cancer" signatures (protein overexpression, mis-localization) on patient cells samples and, ultimately, for the screening of monoclonal and polyclonal antibodies for immunofluorescence applications. In particular, by using the method according to the present invention a pathway of deacetylation in mammalian cells upon DNA damage could be identified, which probably involves specific chromatin modifying enzymes. Detailed biochemical analysis at sites of DNA double strand breaks will help in elucidating specific molecular changes in chromatin state.

Moreover, with the increased availability of highly specific and selective antibodies to cancer relevant targets, the present technology could accelerate the drug development process and offer new tools to patient monitoring during disease therapy.

Thus, according to the present invention there is provided a methodology based on the investigation of living cells "on chip" where, typically upon fixation, it is possible to detect simultaneously, e.g. by using specific antibodies, the localisation and state of a multitude such as hundreds or more of cell components, in particular proteins, involved in different biochemical (e.g. signalling, metabolic etc.) pathways. Furthermore, at the same time, reagents and cell consumption are minimised and high quality detection signals, e.g. high resolution images by using corresponding microscopic equipment, are routinely obtainable. As an example, by using the method of the present invention a previously unidentified signalling pathway of deacetylation of chromatin upon DNA damage in mammalian cells was identified. The present invention thus permits the detailed analysis of dynamical events such as protein profiling, protein post-translational modification, protein migration and transportation inside cells, even at the single molecule level.

Claims

1. A method for detecting biological molecules in/on cells comprising the steps of

(b) treating at least the at least one area of the solid support with a reagent promoting the adhesion of the cells;

(c) inoculating the at least one area with living cells;

(T) detecting whether or not the at least one reagent has reacted with a component of the cells.

2. The method of claim 1 wherein the cells are fixed and/or permeabilised between steps (d) and (e).

3. The method of claim 1 or 2 comprising one or more washing steps between steps (d) and (e) and/or (e) and (f).

4. The method according to any one of the preceding claims further comprising the step of

(g) detecting the localisation of the reagent within/on the cells.

5. The method according to any one of the preceding claims wherein each of the multiple spatially distinct sites or groups thereof is/are reacted with a different reagent.

6. The method according to any one of the preceding claims wherein each of the multiple spatially distinct sites or groups thereof is/are reacted with different amounts of the reagent(s).

7. The method according to any one of the preceding claims wherein the reagent(s) is/are selected from the group of antigen-specific binding molecules, nucleic acids, fluorescent ligands, organel-specific dyes and quantum dots-conjugated reagents.

8. The method of claim 7 wherein the antigen-specific binding molecule is an immunoglobulin or antigen-binding fragment thereof.

9. The method of claim 8 wherein the immunoglobulin is a polyclonal, monoclonal or recombinant antibody or an antigen-binding fragment thereof.

10. The method according to any one of the preceding claims wherein step (c) is carried out for a time and under conditions such that the cells are at least 50 % confluent within the at least one area before performing step (e).

11. The method according to any one of the preceding claims wherein the cells are treated with at least one biochemically active compound or means during step (d).

12. The method of claim 11 wherein the biochemically active compound or means is selected from the group consisting of pharmaceutical drugs or candidates thereof, recombinant nucleic acids, X rays, UV, nanoparticles, vital stainings, free radicals releasing compounds, dendrimers, quantum dot-conjugated molecules and polymers.

13. The method according to any one of the preceding claims wherein the solid support is provided with multiple spatially distinctive areas each capable of receiving living adherent cells.

14. The method of claim 13 wherein each area is inoculated with different cells.

15. The method according to any one of the preceding claims wherein the area(s) is/are each inoculated with about 4 x 10^s to about 6 x 10^s cells.

S 16. The method according to any one of the preceding claims wherein step (f) comprising the sub-step of reacting one or more of the multiple spatially distinct sites with a molecule capable of interacting with the reagent(s).

17. The method of claim 16 wherein the molecule capable of interacting with the 0 reagent(s) is an antibody or antigen-binding fragment thereof.

18. The method of claim 17 wherein the reagent(s) is/are an antigen-binding molecule as defined in claim 8 or 9 and the molecule capable of interacting therewith is a secondary antibody directed against the antigen-binding 5 molecule.

19. The method according to any one of the preceding claims wherein the reagent(s) and/or the molecule capable of interacting therewith is/are labelled with a detectable marker. 0

20. The method of claim 19 wherein the marker is a luminescent, preferably fluorescent marker.

21. The method according to any one of the preceding claims wherein the solid 5 support comprises glass, plastic, quartz, silicon and/or ceramics.

22. The method of claim 21 wherein the area(s) is/are coated with a nanostructured TiO₂ film.

0 23. The method according to any one of the preceding claims wherein the centres of the multiple spatially distinct sites are separated by between about 500 μm to about 2 mm.

RECTIFIED SHEET (RULE 91) ISA/EP

24. The method according to any one of the preceding claims wherein step (e) is carried out by non-contact spotting.

25. The method according to any one of the preceding claims wherein step (b) S comprises coating of the area(s) with a gelatine solution, polyornitine, polylysine, matrigel^®, aminosilane, fibronectin, vitronectin, laminin or collagen.

26. The method according to any one of the preceding claims wherein step (f) comprises an inspection of the area(s) with an automated fluorescence 0 microscope, optionally being equipped with a CCD camera.

27. The method according to any one of the preceding claims wherein the cells are adherent cells.

5 28. A kit for performing the method according to any one of the preceding claims comprising a solid support having at least one distinct area for receiving living cells; at least one strain of culture cells; at least one reagent capable of interacting with a component of the 0 cells, which reagent is to be applied on predetermined multiple spatially distinct sites of the at least one area; means for the detection of the at least one reagent; and an instruction manual comprising information for carrying out the method according to any one of the preceding claims. 5

RECTIFIED SHEET (RULE 91) ISA/EP