WO2005116648A2 - Method and reagent kit for evaluation of multidrug-resistance activity - Google Patents

Abstract

The invention provides an in vitro diagnostic method for detecting multi-drug resistance in a biological specimen comprising a heterogeneous population of discrete cells, said method comprising the steps of contacting a first portion of the biological specimen with MDR substrate derivative and a viability reagent; contacting a second portion of the biological specimen with a general inhibitor of transport protein-mediated cell efflux, the MDR substrate derivative, and the viability reagent; measuring the signal generated by the MDR substrate within the viable cells of said second portion; defining an MDR substrate signal intensity range based on data measured above; determining a first rate of MDR substrate accumulation within a population of cells of said first portion of the biological specimen; determining a second rate of MDR substrate accumulation within a population of cells of said second portion of the biological specimen; and calculating the difference in said first and second rates of MDR substrate accumulation, wherein data showing a reduced MDR substrate accumulation in said populations of cells between said first portion and said second portion is indicative of the presence of multi-drug resistance in said biological specimen.

Description

Description METHOD AND REAGENT KIT FOR EVALUATION OF MULTIDRUG-RESISTANCE ACTIVITY Technical Field

[1] The invention provides an in vitro diagnostic method for detecting multi-drug resistance in a biological specimen comprising a heterogeneous population of discrete cells, said method comprising the steps of contacting a first portion of the biological specimen with MDR substrate derivative and a viability reagent; contacting a second portion of the biological specimen with a general inhibitor of transport protein- mediated cell efflux, the MDR substrate derivative, and the viability reagent; measuring the signal generated by the MDR substrate within the viable cells of said second portion; defining an MDR substrate signal intensity range based on data measured above; determining a first rate of MDR substrate accumulation within a population of cells of said first portion of the biological specimen; determining a second rate of MDR substrate accumulation within a population of cells of said second portion of the biological specimen; and calculating the difference in said first and second rates of MDR substrate accumulation, wherein data showing a reduced MDR substrate accumulation in said populations of cells between said first portion and said second portion is indicative of the presence of multi-drug resistance in said biological specimen. Background Art

[2] The efficiency of chemotherapy of tumors is seriously affected or hindered by the onset of the so called multidrug-resistance: a clinical phenomenon originally described as acquired resistance of tumors subsequent to anticancer chemotherapy to a wide variety of structurally unrelated substances. One major mechanism discovered behind this phenomenon is the overexpression of certain membrane bound proteins. These extrude xenobiotic substances including cytotoxic drugs via an-energy dependent mechanism coming from ATP hydrolysis, a common feature with a large family of transporters coined the ATP-Binding-Cassette proteins. (ABC-transporters). Transporters involved in the mediation of clinical drug resistance, e.g. the MDR1 protein (alternatively termed P-glycoprotein), MRP1 protein and related proteins are also called multidrug-resistance transporters (or MDR- transporters). Substrates of the latter transporters include most clinically used cytostatic agents (e.g. Vinca alkaloids, anthracycline derivatives, etc.) that normally enter cells by simple diffusion through the plasma membrane.

[3] The transport activity can be detected by fluorescent substrates of MDR proteins. Hydrophobic cell-permeable ester derivatives of some fluorescent dyes are actively extruded from the cells by the MDR proteins present in cell membranes, before the ester derivatives can reach the cytosol. However, once the ester derivatives reach the cytosol, intracellular esterases cleave the esters from the fluorescent dyes, and the MDR proteins can not extrude the resulting free dye compounds [Homolya, L. et al, J. Biol. Chem., 268 21493 (1993)]. It has been demonstrated that the commercially available compound, calcein-AM (calcein-acetoxy-methylester), generally used in cell viability assays, is - unlike free calcein - an excellent activator of the MDR dependent ATPase (K < 1 μmol/1). It has also been shown that calcein accumulation in the cell following calcein-AM uptake is reduced by the presence of MDR activity.

[4] Calcein-AM is practically non-fluorescent and highly lipid soluble (hydrophobic) and rapidly penetrates through the cell membrane. Intracellular esterases rapidly cleave the ester bond present in the compound, producing a highly fluorescent water soluble (hydrophilic) compound. When living cells are contacted with calcein-AM, the molecules continuously penetrate the cells because of the established concentration gradient and cleavage products accumulate within the cell. The use of calcein-AM in cell viability assays is also based on this principle, since in damaged or dead cells entering calcein-AM molecules are not transformed to a fluorescent product [Handbook of fluorescent probes and research chemicals, pp. 172-173, ed. Haugland, R. P., Molecular Probes Inc., Eugene, Oreg. (1992-94)].

[5] It has been demonstrated that calcein-AM is useful for the qualitative functional analysis of the presence of multidrug-resistance in cells (Hollo, Zs. et al, Biochim. Biophys. Acta 1191, 384 (1994); application Ser. No. 08/928,528 by Sarkadi et al, filed Sep. 12, 1997, hereby incorporated by reference; International PCT Publication No. WO 96/06945). If any of the overexpressed proteins that exhibit MDR-type cell transport are present in the cell membrane, the cell extrudes the penetrating calcein- AM molecules via an active transport mechanism, and thus the rate of transformation of calcein-AM to fluorescent calcein (or other fluorescent calcein derivatives) and of accumulation of the fluorescent product(s) within the cells will be significantly reduced, relative to wild type cells. The resulting in vitro clinical diagnostic assay method makes possible, by applying a relatively simple and inexpensive measurement, the reliable pre-estimation of the measure of multidrug-resistance of different tissue types.

[6] Clinicians are especially interested in learning the drug resistance profile, and the substrate specificity and drug extrusion activity of the various multidrug-resistance proteins in a given tissue sample (including solid tissues and body fluids). The demonstration of the transport activity of various multidrug-resistance proteins (e.g. MDR1, MRP1, cMOAT) in the plasma membrane requires a sensitive and re- producible in vitro assay.

[7] Disintegration of solid tissues has attracted much interest in the past decades in an attempt to establish ex vivo test systems that enable high throughput study of cells in multiple parallel conditions, save animal life and cut costs. At the same time various methods have been established to select the cell types of interest ranging from morphological to highly sophisticated molecular biological methods. On the other hand, it became clear that cells surviving in the artificial environment change during culture conditions and are not necessarily representative of the phenotype of the cells in vivo.

[8] Although techniques have been established to grow ex vivo tumors and study their responsiveness to chemotherapy drugs, including successfully marketed products (see EDR - Extreme Drug Resistance - assay of OnchoTech Inc., CA., www.onchotech.com), many attempts have been directed towards establishment of methods by which freshly isolated cells of interest can be studied. However, these techniques do not allow to study functionally active cells with regard to their MDR protein function, and cellular markers at the same time to allow focused analysis of cell types in a heterogeneous cell suspension [Farrell, RJ. et al.: Gastroenterology, 118(2): 279-88 (2000)]. Depending on the markers of interest, targets differ significantly in terms of optimal conditions when observations are to be done.

[9] A first class of techniques, i.e. receptor-based assays, e.g. when a single ligand interaction induces one receptor-specific response run in relatively slow cycles; therefore detection of receptor-ligand interaction predicts related responses. In other words, there is no need for the study of the 'function' of the cell; specific ligand-binding safely predicts related responses in non-viable cells or even in membranes, or specific cellular compartments.

[10] On the contrary, a second type of techniques takes advantage of the great numbers of proteins that regulate membrane conductance (e.g. ion channels, xenobiotic transport proteins, etc.). In these cases the resulting change of the intracellular concentrations of its substance exceeds the number of required transporters by numbers of magnitude. In addition, its functional activity can be modulated by internal or external factors (e.g. phosphorylation, mutation, etc.) that make correlation between 'presence' and 'activity' even more difficult. In these cases, sensitivity and specificity of assays targeting the function of these proteins exceed by far the efficacy of 'conventional' expression studies. Such approaches have been introduced in a wide range of applications from analysis of protein kinase function in isolated cellular compartments to intact-cell assays.

[11] However, no method exists so far to disintegrate solid-tissues into cell-suspensions containing cells that are at the same time both separated from each other and intact; both of these characteristics are crucial to enable the functional study of MDR proteins on distinguished cell-types.

[12] The major requirements concerning current methods are the following: a) high sensitivity for the detection of relatively low levels of various MDR proteins; b) standardization, which is suitable for inter-laboratory comparison; c) functional characterization of the actual transport activity and substrate specificity of a given tumor sample; and d) straightforward evaluation of potential inhibitors of MDR transport activity.

[13] The principles underlying the calcein assay method have permitted the formulation of highly advantageous assays that meet the above requirements. No method exists at present to meet these criteria using solid tissue samples or suspension samples containing a diversity of cells providing reliable, quantitative measurement of both the degree of multidrug-resistance being exhibited (to what extent MDR-type transport is occurring in the sample cells) as well as the nature of the multidrug-resistance being exhibited (what type of expressed proteins are responsible and transport pathways utilized) in a single assay.

[14] In addition, there is a continuous need for the evaluation of potential mediators and effectors of multidrug-resistance, such as drugs, multidrug-resistance inhibitors, or environmental toxins. These screening assays are well-suited for high-throughput methods in a clinical setting. The clinical relevance and usefulness of the calcein-AM based multidrug-assay has been demonstrated in acute myeloid leukemia [Karaszi, E et al., Br J Haematol. (2001)]. However, the assay in this form was only suitable to analyze cells in fluids (blood) without immunological identification of target populations.

[15] The aim of this invention is to provide the method and the reagents that enable functional assays particularly the calcein based MDR-activity assay, typically by flow cytometry on viable single cell-suspensions derived from either body fluids or solid tissue samples.

[16] It was surprisingly found that the combined effects of application of a special enzymatic digestion mixture (SEM), identification of a novel combination of fluorescent markers, and application of a suitable cell surface antigen, together with novel data analysis means, makes possible the development of a flow cytometric method which can accomplish all the above-mentioned goals of a quantitative diagnostic method on multidrug-resistance activity.

[17] The present invention is directed to methods of screening test substances for their effect on multidrug-resistance in cells, and of characterizing the type and degree of multidrug-resistance exhibited by cells. The present invention utilizes a quantitative in vitro diagnostic assay method for determining the activity and concentration of transport proteins that cause multidrug-resistance in tumors (MDR proteins). The invention further relates to reagent kits for performing said assay method.

[18] In a specific embodiment, the invention provides method and reagents to isolate functionally intact single cells from solid tissue samples suitable for MDR analysis. Further, the invention provides methods and reagents to combine MDR testing with the immunological identification of target cell populations. The methods and reagent kits of the present invention provide comprehensive solution for the functional determination of multidrug-resistance in large variety of solid mammalian tissues with the special emphasis on human carcinomas.

[19] In one embodiment, the invention provides an in vitro diagnostic method for detecting multi-drug resistance in a biological specimen comprising a heterogeneous population of discrete cells, said method comprising the steps of:

[20] (a) contacting a first portion of the biological specimen with • a cell permeable, non-detectable derivative of an MDR transporter substrate (MDR substrate derivative) which, after penetration, is modified in viable cells to become a non cell permeable, detectable MDR transporter substrate (detectable MDR substrate), and • a viability signaling reagent capable of distinguishing between viable and non- viable cells of the biological specimen by selectively attaching to non- viable cells (viability reagent);

[21] (b) contacting a second portion of the biological specimen with • an inhibitor of the MDR protein to be measured, advantageously, a general inhibitor of transport protein-mediated cell efflux, • the MDR substrate derivative, and • the viability reagent;

[22] (c) measuring the signal generated by the detectable MDR substrate within the viable cells of said second portion according to (b), wherein viability of said cells is determined by measuring the signal generated from the attached viability reagent;

[23] (d) defining an MDR substrate signal intensity range based on data measured in (c), wherein the lower limit of the range is set to be about one-tenth of the mean value of the measured signal intensities and the upper limit is set to be at least ten-times of the mean value, thereby excluding from the said signal intensity range most of the signal originating from non-cellular debris appearing viable because of non-specific non- attachment of the viability reagent;

[24] (e) determining a first rate of MDR substrate accumulation within a population of cells of said first portion of the biological specimen defined by generating MDR substrate signal within the signal intensity range defined in (d);

[25] (f) determining a second rate of MDR substrate accumulation within a population of cells of said second portion of the biological specimen defined by generating MDR substrate signal within the signal intensity range defined in (d);

[26] (g) calculating the difference in said first and second rates of MDR substrate accumulation, wherein data showing a reduced MDR substrate accumulation measured in said first population of cells defined in (e) compared to that measured in said second population of cells defined in (f) is indicative of the presence of multi-drug resistance in said biological specimen.

[27] In a further, preferred, embodiment, the invention relates to a method wherein the cells of the biological specimen are further exposed in steps (a) and (b) above to an immunological reagent suitable for immunological identification of a specific cell type, t he said populations of cells in steps (e) and (f) are further divided into subpopulations of cells according to their ability to bind the said immunological reagent, and the determinations in steps (e) and (f), as well as the calculation in step (g) are performed on the said subpopulations of cells, wherein data showing a reduced MDR substrate accumulation measured in said subpopulation of cells defined in (e) compared to that measured in said subpopulation of cells defined in (f) is indicative of the presence of multi-drug resistance in said immunologically characterized cell subpopulation of said biological specimen.

[28] In another embodiment of the method of the invention, the said MDR substrate derivative is a calcein compound, preferably an acetoxymethyl ester or acetate ester of calcein.

[29] In preferred embodiments the methods of the invention are performed using a flow cytometer, and the MDR substrate signal is measured in FL-2, and the said viability reagent is measured in FL-3.

[30] Further preferred embodiments of the invention comprise the use of 7-amino-actinomycin-D (7-AAD) as the viability reagent.

[31] According to another aspect, the immunological reagent is targeted to a universal epithelial marker to identify carcinoma tissues, which in preferred embodiments is Ber- EP4, an antibody raised against epithelial antigen 17/1 A.

[32] In another preferred embodiment, the immunological reagent is labeled with a fluorescent compound measurable in FL-4. In further preferred embodiments this fluorescent label is allophycocyanin (APC).

[33] In another embodiment of the present invention the method is targeted to a solid biological specimen, and the solid biological specimen is disintegrated prior to carrying out the other steps by exposing the cells of the solid biological specimen to a special enzymatic digestion mixture having collagenase activity.

[34] In a preferred embodiment, the special enzymatic digestion mixture have sulfhydryl protease, clostripain, aminopeptidase and very low trypsin-like activity.

[35] In another preferred embodiment, the concentration of the enzyme mixture is about 1 mg/ml, preferably in the range of 0.1 to 4 mg/ml. [36] Preferred embodiments of the invention provide for an in vitro diagnostic method performed on biological samples isolated from a mammal. In some embodiments the mammal is a human. [37] In one embodiment, the invention provides a method for screening of cell type specific inhibitors of MDR transport comprising the steps of: [38] (a) contacting a first portion of a biological specimen comprising a population of specific discrete MDR positive cells with • a cell permeable, non-detectable derivative of an MDR transporter substrate (MDR substrate derivative) which, after penetration, is modified in viable cells to become a non cell permeable, detectable MDR transporter substrate (detectable MDR substrate), and • a viability signaling reagent capable of distinguishing between viable and non- viable cells of the biological specimen by selectively attaching to non- viable cells (viability reagent);

[39] (b) contacting a second portion of the biological specimen with • a test compound, suspected of being an inhibitor of transport protein-mediated cell efflux, • the MDR substrate derivative, and • the viability reagent;

[40] (c) measuring the signal generated by the detectable MDR substrate within the viable cells of said second portion according to (b), wherein viability of said cells is determined by measuring the signal generated from the attached viability reagent;

[41] (d) defining an MDR substrate signal intensity range based on data measured in (c), wherein the lower limit of the range is set to be about one-tenth of the mean value of the measured signal intensities and the upper limit is set to be at least ten-times of the mean value, thereby excluding from the said signal intensity range most of the signal originating from non-cellular debris appearing viable because of non-specific non- attachment of the viability reagent;

[42] (e) determining a first rate of MDR substrate accumulation within a population of cells of said first portion of the biological specimen defined by generating MDR substrate signal within the signal intensity range defined in (d);

[43] (f) determining a second rate of MDR substrate accumulation within a population of cells of said second portion of the biological specimen defined by generating MDR substrate signal within the signal intensity range defined in (d);

[44] (g) calculating the difference in said first and second rates of MDR substrate accumulation, wherein data showing an enhanced MDR substrate accumulation measured in said second population of cells defined in (f) compared to that measured in said first population of cells defined in (e) is indicative of the fact that the test compound is an inhibitor of MDR transport in the specific cell type provided in (a). [45] In a further embodiment a kit is also provided, said kit comprising instructions and reagent(s) for detecting multi-drug resistance in a biological specimen, wherein said instructions teach any of the methods of the invention. [46] In a preferred embodiment, the kit comprises at least one of the following: a MDR substrate derivative, a special enzymatic digestion mixture, a cell viability reagent, an immunological reagent and optionally an MDR transport inhibitor. [47] In a further preferred embodiment, the kit comprises at least one of the following: calcein-AM, a collagenase preparation, 7-amino-actinomycin-D (7-AAD), allophycocyanin (APC). [48] The invention features methods and reagents for functional and quantitative determination of multidrug-resistance in cells isolated from solid tissues and/or het- erogenous suspensions of cells in body-fluids. Disclosed herein are methods and reagents for: a) quantitative in vitro determination of multidrug-resistance in single cell suspensions; b) isolation of viable, functionally intact cells from solid tissue samples; c) identification of target cell populations for multidrug-resistance analysis in heterogeneous cell suspensions obtained from solid tissues. The method of the invention is based on the measurement of the accumulation rate of a substrate molecule of an MDR-transporter within the cells of the specimen (advantageously by fluorescence measurement), after in vitro exposure. One non-limiting example for such a substrate is Calcein-AM. In this specific case, the cell permeable (acethoxy-methylesther substituted) form of calcein is converted within the cell by intracellular enzymes to free calcein. Comparison of free calcein accumulation in the presence and absence of inhibitors of transport activity permits the rapid screening of such molecules. Additionally, evaluation of free calcein accumulation rates in the presence of inhibitors of all MDR transport in comparison with calcein accumulation rates in the presence of selective inhibitors of MDR transport permits the evaluation of the functional type of MDR being exhibited. [49] Selective measurement of MDR-activity in a selected cell population

[50] Identification of target cells by cell size

[51] In one method the analysis of MDR-activity of specific cell types in cell suspensions derived from solid tissue tissues is based on the size analysis of cells in FSC/SSC (forward and side scatter) analysis. In heterogeneous cell suspensions or cell suspensions obtained from solid tumor samples, two main populations of cells can be identified when instrument settings normally used for the analysis of peripheral white blood cell compartments are applied. There is a distinct population of small cells in the FSC/SSC dot plot characteristic for lymphocytes and a population of larger cells (Examples 3 and 6). Example 3 shows a sample of human carcinoma as solid tissue. In order to proximate the FSC/SSC pattern of epithelial carcinoma cells A431 human epidermoid carcinoma cell line with identical instrument settings was analyzed. It was observed that these cells had light scatter characteristics similar to the diffuse large cell population seen in the tumor sample. This indicated that the majority of tumor cells are among the cells represented by this upper larger cell population. However, normal granulocytes, monocytes or fibrocytes may have similar light scatter pattern. Therefore, we analyzed colon carcinoma samples by immunocytometry to distinguish epithelial carcinoma cells from non-epithelial other cell types. Cytokeratine is only expressed by cells of epithelial origin. Intracellular staining of cytokeratin after perme- bilisation revealed that the majority of the positive cells were in the upper right cell population as anticipated (Example 3). However, the percentage of positive cells within this cell population showed high variability among individual tumors ranging between 19% and 56%. It was not possible to identify a cell population more specific for tumor cells based on cell size within this upper right cell population probably due to the known heterogeneity of carcinomas in vivo. These results indicated the limited applicability of target cell population based exclusively on cell size.

[52] Exclusion of non-viable cells

[53] In one embodiment, the method comprises the addition of an additional reagent capable of distinguishing between viable and non- viable cells of the sample. Typically, the additional reagent is a cell membrane-impermeable fluorescent nucleic acid stain. In order to clearly differentiate the fluorescence of the additional reagent from that of calcein, it is preferred that the fluorescence of the additional reagent is detectably distinct from that of calcein.

[54] Typically, free calcein exhibits high intensity green fluorescence, while the additional reagent exhibits red fluorescence, both within the visible spectrum. In a preferred embodiment of the present invention, the fluorescent detection of intracellular calcein and the viability dye are performed using a flow cytometer. The use of flow cytometry in conjunction with the additional reagent above permits the exclusion of non- viable cells from the analysis of MDR activity, and permits evaluation of MDR function on a cell-by-cell basis.

[55] According to the prior art, typically propidium iodide (PI) or ethidium iodide was used in combination with calcein to exclude non- viable cells. This combination was well suited for the analysis of uniform (blast) cells from clinical samples in cases of acute (myeloid) leukemia, where vast majority of target cells can be identified based on size and granularity. The same applies for in vitro cultured tumor cell lines, which are derived from a single clone, making distinction among different cell types unnecessary. Utilization of these dyes presented a technical problem when used in samples with heterogeneous cell populations typically obtained after enzymatic tissue disintegration. Spectral emission of large cells with an intense calcein fluorescent signal reaches the red region of the spectrum which is almost impossible to distinguish from the signal of PL

[56] In flow cytometry fluorescent detectors (FLs) for specified light wavelength intervals are used. Typically, FL-1 detects in green (530 ± 30 nm), FL-2 detects in orange (585 + 42 nm) and FL-3 in far red (>670 nm). The maximum of the emission spectrum of calcein (515 nm) gives the maximum signal intensity in FL-1. However, due to the wide emission spectrum of calcein, 50% of the FL-1 signal can be detected also in FL-2 and even a few percent in FL-3. PI emission spectrum emits few percent of its signal in FL-1, maximum in FL-2 and 50% in FL-3. Since calcein emits a very intensive fluorescent signal, the electronic compensation between FL-1 (calcein) and FL-2 (PI) is very difficult. The solution used in prior art was the detection of calcein in FL-1 and PI in FL-3; this method was used in the calcein assay for MDR and also for calcein/PI for viability assays. However, this method leaves the remaining overlap between the two fluorescent dyes unsolved. The small overlap of calcein signal that can be detected in the FL-3 together with the PI signal is not a major problem when uniform cell populations are investigated. However, cells with variable sizes make distinction between bright green calcein fluorescence and PI positivity almost impossible: large cells with a strong calcein signal are detected as false positive (dead) cells in the FL-3 channel similar to smaller PI positive dead cells. Thus, they are either excluded from the analysis, or a variable proportion of dead cells will be included. This problem cannot be electronically adjusted in standard flow cytometers because compensation between FL-1 and FL-3 detectors is not possible.

[57] The solution for this problem was based on the observation that the high signal intensity of calcein can be detected on FL-2 without limiting the sensitivity or specificity of the assay (Example 6). Therefore, a carefully selected viability dye with an emission spectrum in FL-3 would solve these above-mentioned problems by eliminating the high background emission of calcein in the next FL-channel and would provide added benefits from electronic compensation.

[58] In a preferred embodiment of the present invention, the additional reagent is 7-amino-actinomycin-D (7-AAD). The emission spectrum of 7-amino-actinomycin-D (7-AAD) is more in the far red than the spectrum of PI, which allows the elimination of its signal with electronic compensation from FL-2 while detected in FL-3 (Examples 4 and 6). Taken together, the combination of detection of calcein signal in FL-2 and dead cell exclusion in FL-3 by 7-AAD instead of PI makes compensation between the two signals possible and the exclusion of dead cells objective.

[59] It will be now obvious for those experienced in the art to replace 7-AAD with another dye that bears similar characteristics to 7-AAD. Therefore, the invention provides the use of a fluorescent compound taken up by non- viable cells which has an emission spectrum in the far red detected in FL-3, detection of the calcein signal in FL- 2 and the electronic compensation between the two signals.

[60] Novel gating strategy to discriminate large cellular debris from viable cells.

[61] Cell suspensions from solid tissues presented another unexpected difficulty. These samples contain high amount of cellular debris. The size of these particles is in the range of the target cells. In addition, viability dyes (propidium iodide or 7-AAD) do not label them since they do not contain DNA. As a result, the traditional flow cytometric analysis methods, which gate the target population based on size (FSC/SSC) and lack of staining with the viability dyes, do not exclude large part of non-cellular debris from the analysis. This is not obvious to people with standard knowledge and experience in flow cytometry since the standard method is gating in the FL-3 (i.e., propidium iodide, 7-AAD)/FSC (size). However, these particles do not accumulate calcein or other cell permeable fluorescent dyes. These particles could be considered wrongly to be viable cells with extreme high MDR activity. However, when this debris is not excluded from the analysis, the MDR resistant phenotype of the sample is obscured since this low calcein signal is not influenced by the presence or absence of MDR inhibitors. To overcome this problem a novel gating strategy was developed. It was observed that the diffuse, homogenous population of flow cytometric events analyzed on a FSC/SSC dot plot forms three well distinguished population on the FL-2 /FL-3 dot plot (i.e., in a specific example, calcein/7-AAD dot plot). One population is negative for both dyes which corresponds to the large cellular debris, a population of high calcein (FL-2) signal without 7-AAD signal which corresponds to the viable cells and a population with high 7-AAD (FL-3) signal and decreased calcein signal (FL-2) which represent the non- viable cells. In the presented exemplary analysis the calcein is exploited as a positive viability dye. However, any other fluorescent dye, which labels viable cells, could be used. For example, doxorubicin enters viable cells and binds the DNA. We have previously described a method to quantify specific MDR activity [Karaszi, E et al. Br J Haematol. (2001)]. Briefly, MDR activity (designated as MAF value, multidrug resistance activity factor) is calculated based on the increase in mean calcein fluorescence in the presence of inhibitors of MDR activity for example verapamil. The set up of gates is performed in the control sample treated with the inhibitors. High MDR activity will decrease the calcein or doxorubicin fluorescent signal. However, a 10-fold decrease in fluorescent signal, which corresponds to a MAF value of 90 will decrease the mean signal to the channel 10 from channel 10 while the debris has lower than channel 10¹ fluorescence in FL-2. In previous results only genetically manipulated cells to overexpress MDR1 protein had MAF value of 90 but not higher (for example: p388MDR). The highest MAF values from clinical samples were around 50. Therefore, a gate set from the channel 10² in FL-2 and 10¹ in FL-3 will exclude both debris and non- viable cells but will not exclude multidrug resistant cells (see Example 6).

[62] Immunological identification of target cells

[63] In another embodiment of the invention, methods and reagents for immunological identification of tumor cells by flow cytometry are integrated into the MDR-activity measurement.

[64] Example 6 shows epithelial cells in the cell suspension of human carcinoma samples targeted for MDR-activity analysis. This is a potentially important application of the invention since carcinomas (tumors of epithelial origin) are responsible for the majority of tumor related human deaths. Since all epithelial cells in a carcinoma tissue are tumor cells and non-epithelial cells are non-tumor cells, a universal epithelial marker would be suitable for the analysis of carcinomas of diverse origin or localization. Cytokeratin-labeling described above requires membrane permeabilization which makes it unsuitable for functional analysis of MDR activity with the calcein assay which requires viable cells with intact cell membrane. Therefore, a cell surface marker specific for all but only the epithelial cells which can be detected by an antibody without damaging the functionality of the cell membrane had to be found. The third special characteristic of a suitable immunological identification was the stability of antigen-antibody interaction after enzymatic disintegration of the solid tumor cell surface antigens. Further, in the prior art, there have been no routine diagnostic applications to detect epithelial cells by flow cytometry. For example, a monoclonal antibody raised against the most known cell surface epithelial antigen, epithelial membrane antigen (or EMA) has proven un-suitable for flow cytometry in our experiments. However, we found that Ber-EP4, an antibody raised against another epithelial antigen, 17/1 A, did label 100% of intact, viable A431 (and other epithelial cell lines) but not fibroblasts or lymphocytes separated from peripheral blood, two of the major component cell-types in neoplastic tissues other than cancer cells. Incubation of epithelial cells with the special enzymatic mixture used for tissue disintegration did not alter the detection of this cell surface antigen (Example 5).

[65] Introduction of immunofluorescent labeling enables extension of the assay to non- epithelial tumor types (lymphomas, sarcomas, neuronal tumors) by replacing anti- BerEP4 with the corresponding antibody suitable for flow cytometry.

[66] It is obvious for the skilled person in the art that specific markers exist for many different cell types or populations. Therefore, the present method can be equally applied for targeting different cell types by selecting different markers on their surface, and therefore the present method can be equally applied to assess and quantitatively determine multidrug-resistance in those cells. This determination then can be used in the assessment of different conditions where MDR testing is useful. A few non- limiting example of these conditions are inflammatory diseases (Crohn' disease, Ulcerative Colitis, Rheumatoid arthritis), as well as infectious diseases like HIV, where infected target cells expressing MDR proteins can escape drugs.

[67] Fluorescent labeling of target cells

[68] In a preferred embodiment of the present invention, immunological identification of target cells is simultaneous to the analysis of the fluorescent calcein signal and the detection of the fluorescent viability dye such as 7-AAD. It is necessary that the emission spectrum of the fluorescent label of the antibody used for the immunological detection can be distinguished electronically from calcein and the viability dye. Calcein signal is very strong in the FL-1 and FL-2 channels. FL-3 is used to detect typical viability dyes including the preferred 7-AAD. However, it is known that the red diode laser excites neither calcein nor 7-AAD significantly. This observation makes possible the utilization of fluorescent dyes excited by the second red laser found in many flow cytometers used in combination with calcein and 7-AAD. It was found that allophycocyanin (APC) is suitable for this specific purpose and can be combined with calcein and 7-AAD. Emission of APC is in the far red region of the spectrum (660 nm), resulting in minimal compensation between APC and other fluorochromes. The APC fluorochrome is maximally excited at 650 nm and can be used on instruments with a helium-neon, dye, or red-diode laser. FACSCalibur™ (with FL-4 option), FACSVantage™ SE, and FACStarPLUS™ cytometers from BD Biosciences are designed to detect APC fluorescence. It will be obvious for those experienced in the art to replace APC with another dye which bears similar characteristics to APC. Therefore, the invention provides the use of a fluorescent compound with excitation and emission spectrum in the far red detected in FL-4, in combination with Calcein and a viability dye. Examples of such dyes include, without limitation, APC, Texas Red, Cy5, etc. [69] Enzymatic dissociation of solid tissues for functional assays

[70] In further embodiments of the present invention, the source of the heterogeneous cell population to be analyzed is a solid tissue, particularly a solid tumor tissue. The significance of being capable of analyzing MDR activity in solid tumor tissues is enormous. Up till now, no MDR diagnostic methods have been accepted for clinical use. Potential siginificance of a specific and sensitive assay can only be predicted by rough estimations. In a Bottom-up approach the test could add valuable information in three clinical settings:

[71] 1. Patients who need personalized therapy: For the 11 mentioned indications, this represents about 4 million patients in the USA alone (see Table 1). IBD counts for 500 000 and the various solid tumors for almost 400,000. 2. New treatment indications for marketed chemotherapies / development of new personalized chemotherapies: For the 11 mentioned indications, about 120 Phase III clinical trials are conducted in the US for chemotherapies alone this year (see www.clinicaltrials.gov). In the presence of MDR proteins, patients assigned in the treatment arm may show resistance to therapy for a reason unrelated to the pathomechanism and therefore potentially hazard statistical evaluation in clinical development. 3. Advanced/late stage oncology patients: More than 250,000 patients die in the US of the seven mentioned cancer indications yearly (see Table 1). Unnecessary heroism in 'palliatve' chemotherapy seriously affects the quality of life of these patients, whereas potential responsive cases are not treated currently because of the lack of a predictive assay.

[72] Table 1. Epidemiology of selected diseases in the US

[73] * 1998-2000; Source for cancers: WHO Globocan 2000; US population estimated at 280 million (CIA World Factbook 2002) [74] 2 www.niams.nih.gov/ne/press/1998/05_05.htm

[75] ³ http://129.170.61.64/Powerpoints HIVl 102slides.pdf - Dartmouth-Hichtcock Medical Center [76] ⁴Datamonitor estimates that CLL represent 30% of all leukemia cases

[77] ⁵ www.niddk.nih.gov/health/digest/pubs/ddstats/ddstats.htm andwww.aafp.org/afp/980101ap/botoman.html - result is average of these two estimates [78] Datamonitor estimates colon cancer patients for 150,000 p.a. η

[79] www.ustransplant.org

[80]

[81] The basic concepts of enzymatic cell isolation techniques have been described in the past decades for several purposes, including establishment of primary cultures, preparation of cellular membranes, and subcellular fractions [United States Patent 5,989,888; Journal of Bone and Medical Research, vol. 2, No. 6, 1987 (pp. 505-516); McShane, et al., Diabetes,. vol. 38, Suppl. Jan. 1, 1989 pp. 126-128; Wolters, et al., Di- abetologia 35, 1992, pp. 735-742; Bond, et al., Biochemistry, vol. 23, No. 13, 1984, pp. 3077-3085; Bond, et al., vol. 23, 1984, pp. 3085-3091]. In these cases, either cell viability (in the case of primary cultures) or preservation of cell-surface antigens (in case of membrane-preparation) or unaffected functional properties are required during the separation process. Exemplary methodologies in each direction have been described ('Worthington Enzyme Manual: Enzymes and Related Biochemicals,' Worthington Biochemical Corporation, 1988, pp. 93-101), but no reliable protocol optimized to meet these requirements in one assay at the same time exists that is mandatory for a functional assay of MDR-transporters in viable cells combining im- munofluorescent separation of cells by flow cytometry.

[82] Single cells obtained after the enzymatic dissociation must preserve: a) viability; b) functional activity of multidrug-resistance transporter proteins, c) cell surface antigen integrity for immunological identification.

[83] No cell dissociation protocols have been previously optimized to fulfill the requirements to provide high proportion of functionally inert cells. The basis of the presented assay technology was optimiziation of a proteolytic enzyme mixture, that avoid the problems suffered by the other preparation which interfered with functional integrity of cell surface transmembrane proteins.

[84] Description of special enzymatic digestion mixture (SEM) [85] This is a collagenase preparation from Clostridium histolyticum which contains not only several collagenases but also a sulfhydryl protease, clostripain, aminopeptidase and very low trypsin-like activity. This latter is crucial for the preservation of cell surface membrane receptor and transporter activity. A non-limiting example of such a preparation is the type 4 collagenase (CLS-4) distributed commercially (Worthington Biochemical Corporation, 730 VassarAve, Lakewood, NJ, 08701) The optimal concentration of the SEM is about 1 mg/ml (0.1%) for most tissue-applications for most of the previously described purposes. Therefore, the concentration of SEM can be preferably in the range of 0.1 to 4 mg/ml. Higher concentration maybe needed in some instances, which do not alter the multidrug-resistance activity (Examples 1 and 2). This allows the user to increase the concentration if the characteristics of new target tissue- types require. The major requirement for the cell dissociation buffer is to contain Ca⁺⁺ which is activator of the crucial collagenase enzymes in the SEM. Such buffers are the Hanks (0.14 g/1 CaCl ), Dulbecco's PBS (0.4 g/1 CaCl ) and media such DMEM (0.2 g/ 1 CaCl ).

[86] Solid tissue kit

[87] In preferred embodiments, the specimen or sample comprises solid tissue. Typically, the source of tissues is preferably animal cells, more preferably mammalian cells. In a preferred embodiment, the sample is solid tumor tissue, typically biopsy or surgical material of human tumors and/or inflammatory tissues.

[88] It is another aspect of the present invention to provide reagent kits, further comprising instructions for carrying out the methods of the invention.

[89] The reagent kits according to the invention typically comprise a calcein derivative and/or an inhibitor or a good substrate of a transport protein. Typically the inhibitor is verapamil. In another embodiment, the kit further comprises a selective inhibitor of either MDRl-Pgp or MRP1, preferably MK 571. In another embodiment, the kit further comprises an additional reagent capable of distinguishing between viable and non-viable sample cells, preferably 7-amino-actinomycin-D. In another embodiment, the invention comprises an additional reagent capable to identify target cell population, preferably an antibody that recognizes a specific cell surface antigen. In a preferred embodiment this antibody is a monoclonal antibody (Ber-EP4), which recognizes a partially formol resistant epitope on the protein moiety of two 34 kilodalton and 39 kilodalton glycopolypeptides on human epithelial cells. This antibody is labeled indirectly - using a labeled secondary antibody - or directly with a fluorescent compound which has an emission spectrum distinguishable from the emission spectrum of calcein and 7-AAD. In a preferred embodiment this fluorescent dye is al- lophycocyanin (APC). Description of Drawings [90] Figure 1 shows the influence of incubation with Worthington Type 4 collagenase on intracellular calcein-AM degradation by P388-MDR1 cells.

[91] Figure 2 shows the influence of Worthington Type 4 collagenase incubation on the MDR protein function in P388 MDR cells

[92] Figure 3 shows the analysis of freshly isolated cell suspension from colon carcinoma biopsy. Figure 3. A shows an FSC/SSC dot plot analysis. Gray dots represent cytokeratin positive (epithelial cells) back gated (Rl) from Figure 3.B. Figure 3.B represent cells gated on Figure 3.A (R2) incubated either with isotype control mouse IgG antibody or the anti-cytokeratin antibody.

[93] Figure 4 shows that 7-AAD can replace propidium ioide as viability dye without alteration in MDR activity.

[94] Figure 5 shows the fluorescence intensity of epithelial marker staining-efficacy on a colon carcinoma biopsy sample.

[95] Figure 6 shows the determination of elevated MRP1 activity in a colon carcinoma sample. Figure 6.A shows the gating strategy where viable cells are identified based on calcein (FL-2) and 7-AAD (FL-3) signal. Figure 6.B shows the positivity for the Ber- EP4 antibody in the carcinoma sample against isotype control in a histogram. Figure 6.C shows shows the mean fluorescence values determined in each sample.

[96] Figures 7. A, B and C show the determination of elevated MDR 1 -activity in another colon carcinoma sample.

[97] Figure 8 shows the determination of MDR-activity of lymphocytes in a biopsy sample of an IBD patient. Figure 8.A shows the gating strategy of viable CD8-positive lymphocytes in the biopsy sample. Figure 8.B shows the mean fluorescence activity of the CD8 positive lymphocytes in the presence or absence of MDR inhibitors. Example 1. Influence of 1-hour incubation with increasing doses of an enzymatic digestion cocktail on intracellular calcein-AM degradation by P388-MDR1 cells.

[98] P388 human lyrnphoma cells were plated and treated at a density of 4x10 cells/well in 96-well plates for 1 hr with increasing concentrations (0.015-2 mg/ml) of enzymatic digestion solution in 0.1 ml HPMI (Sigma) buffer at 37°C. Subsequently, 0.1 ml 0.52x10^" M Calcein-AM was added to the samples. Fluorescence signal of the free calcein was determined by fluorescent plate reader periodically at increasing incubation times. Data on Figure 1 represent the mean fluorescence values +/- standard deviation of multiple parallel samples. Example 2. Influence of 1-hour incubation with increasing doses of the enzymatic digestion cocktail on the corresponding MAF values of P388-MDR1 cells.

[99] P388-MDR1 human lyrnphoma cells (5x10 cells/well on 6-well plates) were incubated in the presence of increasing concentration (0.015-2 mg/ml) of enzymatic digestion solution at 37°C for 1 hr. The MDR activity (MAF value) was determined by standard procedure of the calcein assay. Calcein signal was determined by fluorescent plate reader. Data on Figure 2 represent the mean MAF values +/- standard deviation of multiple parallel samples. Example 3. FSC/SSC analysis is not sufficient to identify the target epithelial cell population.

[100] Freshly isolated cell suspension (10 ) from colon carcinoma biopsy was washed in PBS and subsequently fixed and permeabilized in 0.05 ml Dako Intrastain buffer 'A' (Dako). Following washing with PBS, epithel specific antigen, the cytokeratin was detected with 5 microL fluorescein conjugated anti-cytokeratin monoclonal antibody (Dako) in 0.05 ml at room temperature. Fluorescence of the fluorescein dye and the size of the cells [the forward (FSC) and side scatter (SSC) of the laser beam] was detected and analyzed on a Becton Dickinson FacsCalibur flow cytometer. Figure 3. A exhibits the FSC/SSC dot plot analysis. Gray dots represent cytokeratin positive (epithelial cells) back gated (Rl) from Figure 3.B. Figure 3.B represent cells gated on Figure 3. A (R2) incubated either with isotype control mouse IgG antibody or the anti- cytokeratin antibody. Shift in fluoescent intensity identify a cell population (35%) positive for this immunmarker. Data demonstrate that based on FSC/SSC gating the cell population is very heterogenous. Example 4. 7-AAD can replace the propidium ioide as viability dye without alteration in MDR activity.

[101] P388-MDR cells (50xl0⁴) were incubated in the presence of calcein (50 nM) for 10 minutes in 600 μl at 37°C in presence or absence of verapamil (250 μM). Subsequently, non- viable cells were labeled either with propidium ioide (10 μg/ml) for 10 minutes on ice or Actinomycin D (2 μg/ml) for 30 minutes at room temperature. Fluorescence was detected as described in the standard calcein assay. Data on Figure 4 represent the mean MAF values +/- standard deviation of multiple parallel samples. MAF values calculated by these fluorescent values did not differ significantly (ρ=0.095). Example 5. BerEP4 antibody is suitable to identify epithelial cells in contrast to anti-EMA (EMA: epithelial membran antigen).

[102] Cell suspensions obtained from a colon carcinoma biopsy sample by enzymatic digestion solution were labeled by the berEP4 antibody (Dako) (the upper three histograms: first IgG isotype control, second and third BerEP4 staining of the cells) or anti-epithelial membran antigen (EMA) (Dako) (the lower three histograms: fourth isotype IgG control, fifth and sixth EMA staining of the cells) by standard procedure. Briefly, 0.5x10 cells were washed in PBS and subsequently incubated in 100 μl PBS containing 1 μg of each antibody and their isotype control (IgGl and IgG2a respectively) and 1 μg anti-mouse secondary antibody labeled with APC fluorescent dye on ice for 30 min. Fluorescent signal was analyzed by a FacsCalibur (Becton Dickinson) flow cytometer in channel 4 and compared to isotype control sample. Only BerEP4 identified a distinct cell population while anti-EMA failed to react with the epithelial cells in the sample. Figure 5 shows the fluorescence intensity epithelial marker staining-efficacy with the two different epithelial markers in histograms. The background instensity was similar with both IgG type isotype controls. However, BerEP4 antibody identified 45.79 and 47.01 percent epithelial cells in the parallel samples while the antibody specific for the EMA antigene positively labeled only 17.06 and 14.56 percent of the cells. Example 6. Determination of elevated MRP1 activity in a colon carcinoma sample. [103] During surgery an 3x3x3 mm large biopsy sample was removed from a colon carcinoma. The sample was cut into small pieces by a surgical blade, washed in DMEM buffer then incubated in 1 ml of 4 mg/ml SEM for 10 min at 37°C. Enzyme reaction was stopped by adding 200 μl foetal bovine serum. Subsequently, the sample was suspended in pipette several time then filtered through 40 micron mesh. Samples were washed in HBSS once and 600 μl of the cell suspension was aliquoted into seven 5-ml Falcon tubes and processed for calcein-assay with several modification. 200 μl of 250 μM Verapamil in HBSS was added into three samples, 200 μl of 250 μM MK571 in HBSS was added into another two samples and only HBSS buffer to another two samples. Subsequently, 200 μl calcein-AM (50 nM) was added to the samples and incubated for 10 minutes at 37 C. Samples were chilled on ice for 5 minutes spined down at (2000 g, 1 min) and resuspended in 200 μl HBSS containning 2 μg/ml 7-AAD. 1 μg isotype control mouse IgG was added to one verapamil treated sample and 1 μg berEP4 antibody to the other samples in 200 μl HBSS. Subsequently, 200 μl HBSS containing 1 μg secondary Cy5 conjugated anti-mouse IgG anitbody was added to the samples and incubated at room temperature for 30 min. Samples were spinned down (1000 g, 5 min) and resuspended in 500 μl HBSS containing 2 μg/ml 7-AAD. Flow cytometric analysis was performed on Becton Dickinson flow cytometer. Figure 6.A presents the gating strategy where viable cells are identified based on calcein (FL-2) and 7-AAD (FL-3) signal. Subsequently, BerEP4 positive cell detected in FL4 channel were identified. Gates Rl and R2 were combined and applied on hystograms of FL-2. Figure 6.B shows the positivity for the Ber-EP4 antibody in the carcinoma sample against isotype control in a histogram. Figure 6.C shows the mean fluorescence values deteremined in each sample. The shift can be seen both with the total MDR inhibitor and with MRP1 inhibitor indicating an elevated MRP1 activity. Example 7. Determination of elevated MDRl-activity in a colon carcinoma sample.

[104] The modified calcein assay was performed as in Example 6 on another sample taken from a patient with colorectal cancer. There were enough viable epithelial cells for analysis in the sample (Figure 7.A and 7.B). Analysis of the mean fluorescent activity in FL-2 in the presence or absence of MDR1/MRP1 inhibitors revealed a fluorescent intensity shift only in the presence of the general MDR inhibitor, but not in the presence of the specific MRP1 -inhibitor compared to the control samples (Figure 7.C). Results indicate an elevated MDR1 activity in the tumor sample. Example 8. Determination of MDR-activity of lymphocytes in a biopsy sample of an IBD patient.

[105] The modified calcein assay was performed as described in Example 6 except this time the source was a biopsy from a patient with inflammatory bowel disease (IBD). Further, instead of the epithelial cells, the CD8 positive cytotoxic lymphocytes were the target population of the calcein assay. To identify CD8 positive cells, anti-CD8 (Dako) monoclonal antibody was used. Figure 8. A shows the gating strategy of viable CD8-positive lymphocytes in the biopsy sample. Figure 8.B shows the mean fluorescence activity of the CD 8 positive lymphocytes in the presence or absence of MDR inhibitors. The shift in FL-2 fluorescence intensity indicates the presence of MDR1 activity in the target cell population. REFERENCES

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Claims

Claims

[1] In vitro diagnostic method for detecting multi-drug resistance in a biological specimen comprising a heterogeneous population of discrete cells, said method comprising the steps of: (a) contacting a first portion of the biological specimen with - a cell permeable, non-detectable derivative of an MDR transporter substrate (MDR substrate derivative) which, after penetration, is modified in viable cells to become a non cell permeable, detectable MDR transporter substrate (detectable MDR substrate), and - a viability signaling reagent capable of distinguishing between viable and non- viable cells of the biological specimen by selectively attaching to non- viable cells (viability reagent); (b) contacting a second portion of the biological specimen with - an inhibitor of the MDR protein to be measured, advantageously, a general inhibitor of transport protein-mediated cell efflux, - the MDR substrate derivative, and - the viability reagent; (c) measuring the signal generated by the detectable MDR substrate within the viable cells of said second portion according to (b), wherein viability of said cells is determined by measuring the signal generated from the attached viability reagent; (d) defining an MDR substrate signal intensity range based on data measured in (c), wherein the lower limit of the range is set to be about one-tenth of the mean value of the measured signal intensities and the upper limit is set to be at least ten-times of the mean value, thereby excluding from the said signal intensity range most of the signal originating from non-cellular debris appearing viable because of non-specific non-attachment of the viability reagent; (e) determining a first rate of MDR substrate accumulation within a population of cells of said first portion of the biological specimen defined by generating MDR substrate signal within the signal intensity range defined in (d); (f) determining a second rate of MDR substrate accumulation within a population of cells of said second portion of the biological specimen defined by generating MDR substrate signal within the signal intensity range defined in (d); (g) calculating the difference in said first and second rates of MDR substrate accumulation, wherein data showing a reduced MDR substrate accumulation measured in said first population of cells defined in (e) compared to that measured in said second population of cells defined in (f) is indicative of the presence of multi-drug resistance in said biological specimen.

[2] The method according to claim 1, wherein - cells of the biological specimen are further exposed in (a) and (b) to an immunological reagent suitable for immunological identification of a specific cell type, - the said populations of cells in (e) and (f) are further divided into sub- populations of cells according to their ability to bind the said immunological reagent, and - the determinations of (e) and (f), as well as the calculation of (g) are performed on the said subpopulations of cells, wherein data showing a reduced MDR substrate accumulation measured in said subpopulation of cells defined in (e) compared to that measured in said subpopulation of cells defined in (f) is indicative of the presence of multi-drug resistance in said immunologically characterized cell subpopulation of said biological specimen.

[3] The method according to claim 1 or 2, wherein the said MDR substrate derivative is a calcein compound, preferably an acetoxymethyl ester or acetate ester of calcein.

[4] The method according to any one of claims 1 to 3, wherein the method is performed using a flow cytometer.

[5] The method according to claim 4, wherein the MDR substrate signal is measured in FL-2.

[6] The method according to claims 4 or 5, wherein the said viability reagent is measured in FL-3.

[7] The method according to any one of claims 1 to 6, wherein the viability reagent is 7-amino-actinomycin-D (7-AAD).

[8] The method according to any one of claims 2 to 7, wherein the immunological reagent is targeted to a universal epithelial marker to identify carcinoma tissues.

[9] The method according to any one of claims 2 to 8, wherein an antibody raised against epithelial antigen 17/1 A is used as an immunological reagent.

[10] The method according to any one of claims 4 to 9, wherein the immunological reagent is labeled with a fluorescent compound measurable in FL-4.

[11] The method according to claim 10, wherein the fluorescent label is allo- phycocyanin (APC).

[12] The method according to any one of claims 1 to 11, wherein the specimen is a solid biological specimen and the solid biological specimen is disintegrated prior to carrying out the other steps by exposing the cells of the solid biological specimen to a special enzymatic digestion mixture having collagenase activity.

[13] The method according to claim 12, wherein the special enzymatic digestion mixture have sulfhydryl protease, clostripain, aminopeptidase and very low trypsin-like activity.

[14] The method according to claim 12 or 13, wherein the concentration of the enzyme mixture is about 1 mg/ml, preferably in the range of 0.1 to 4 mg/ml.

[15] The method according to any one of claims 1 to 14, wherein said biological sample is isolated from a mammal.

[16] The method according to claim 15, wherein said mammal is a human.

[17] A method for screening of cell type specific inhibitors of MDR transport comprising the steps of: (a) contacting a first portion of a biological specimen comprising a population of specific discrete MDR positive cells with - a cell permeable, non-detectable derivative of an MDR transporter substrate (MDR substrate derivative) which, after penetration, is modified in viable cells to become a non cell permeable, detectable MDR transporter substrate (detectable MDR substrate), and - a viability signaling reagent capable of distinguishing between viable and non- viable cells of the biological specimen by selectively attaching to non- viable cells (viability reagent); (b) contacting a second portion of the biological specimen with - a test compound, suspected of being an inhibitor of transport protein- mediated cell efflux, - the MDR substrate derivative, and - the viability reagent; (c) measuring the signal generated by the detectable MDR substrate within the viable cells of said second portion according to (b), wherein viability of said cells is determined by measuring the signal generated from the attached viability reagent; (d) defining an MDR substrate signal intensity range based on data measured in (c), wherein the lower limit of the range is set to be about one-tenth of the mean value of the measured signal intensities and the upper limit is set to be at least ten-times of the mean value, thereby excluding from the said signal intensity range most of the signal originating from non-cellular debris appearing viable because of non-specific non-attachment of the viability reagent; (e) determining a first rate of MDR substrate accumulation within a population of cells of said first portion of the biological specimen defined by generating MDR substrate signal within the signal intensity range defined in (d); (f) determining a second rate of MDR substrate accumulation within a population of cells of said second portion of the biological specimen defined by generating MDR substrate signal within the signal intensity range defined in (d); (g) calculating the difference in said first and second rates of MDR substrate accumulation, wherein data showing an enhanced MDR substrate accumulation measured in said second population of cells defined in (f) compared to that measured in said first population of cells defined in (e) is indicative of the fact that the test compound is an inhibitor of MDR transport in the specific cell type provided in (a).

[18] The method according to claim 17, wherein - cells of the biological specimen are further exposed in (a) and (b) to an immunological reagent suitable for immunological identification of a specific cell type, - the said populations of cells in (e) and (f) are further divided into sub- populations of cells according to their ability to bind the said immunological reagent, and - the determinations of (e) and (f), as well as the calculation of (g) are performed on the said subpopulations of cells, wherein data showing an enhanced MDR substrate accumulation measured in said subpopulation of cells defined in (f) compared to that measured in said subpopulation of cells defined in (e) is indicative of the fact that the test compound is an inhibitor of MDR transport in the specific cell type to which the said immunological reagent binds.

[19] A kit comprising instructions and reagent(s) for detecting multi-drug resistance in a biological specimen, wherein said instructions teach the method according to any one of claims 1 to 18.

[20] The kit according to claim 19, wherein said kit further comprises at least one of the following: - a MDR substrate derivative, - a special enzymatic digestion mixture, - a cell viability reagent, - an immunological reagent and optionally - an MDR transport inhibitor.

[21] The kit according to claim 20, wherein said kit comprises at least one of the following: - calcein-AM, - a collagenase preparation, - 7-amino-actinomycin-D (7-AAD), - allophycocyanin (APC).