Background art The study of the intracellular distribution of fluorescent probes and drugs by confocal fluorescence microscopy has opened new possibilities to the analysis of living cells. This sensitive methodology allows the fine detection of morphological and functional alterations in single living cells. There is increasing evidence that these alterations are related to functional changes in the cell physiology. For example it is possible to detect multidrug resistance (MDR) in human tumor cells through the analysis of their fluorescence imaging. In particular by means of the confocal microscope MRC 600 (Bio-Rad, Microscience Division) with Argon laser excitation (488 and 515 nm) and photon counting detection, the study of doxorubicin localization in human leukemia K562 and K562/DX has allowed us to discriminate between sensitive and multidrug resistant cells on the basis of their different doxorubicin intracellular distribution (EP 0568 126 B1 by the same applicants).

Distinctive features of resistant cells are: 1) lack of nuclear fluorescence; 2) intense membrane fluorescence; 3) a pattern of fluorescent organelles distributed in the cytoplasm (punctate pattern). These signals are not found in sensitive cells and therefore allow the diagnosis of resistance by a fast and non destructive method.

Similar results have been obtained also for human carcinoma adherent cells, with an even better resolution pattern than that observed in suspension cells. It has therefore become appealing to search for new probes with fluorescent yields higher than that of doxorubicin for the fine tuned detection of the state of a cell, in analogy to what has been done for the multidrug resistance with this methodology.

Brief description of the figures Figure 1. Ethidium Bromide and Rhodamine staining in sensitive and resistant MCF-7 cells: a) MCF-7 sensitive cells; b) MCF-7/DX resistant cells.

Figure 2. Intracellular distribution of doxorubicin in sensitive and resistant MCF-7 cetts : a) MCF-7 sensitive cells; b) MCF-7/DX resistant cells.

Figure 3. Ethidium Bromide and Rhodamine staining in sensitive MCF-7 cells.

Figure 4. Reversion of the resistant fluorescence MCF-7 pattern by Verapamil.

Figure 5. Single Rhodamine staining in sensitive and resistant fluorescence MCF- 7, a) MCF-7 sensitive cells, b) MCF-7/DX resistant cells.

Figure 6. Single Ethidium Bromide staining in sensitive and resistant fluorescence MCF-7 ; a) MCF-7 sensitive cells, b) MCF-7/DX resistant cells.

Disclosure of the invention By means of confocal fluorescence microscopy the applicants have studied, in sensitive and Multi-Drug-Resistant (MDR) tumor cells, the intracellular distribution of a number of fluorescent probes, such as rhodamine 123 (R123), ethidium bromide (EB), 5,5', 3,3'-tetra-ethyl-benzimidazol-carbocyanine iodide (JC-1), 2- (p-dimethylaminostiryl)-1-methylpyridinio iodide (DASPMI), all purchased from Molecular Probes. By the use of these particular probes, it has been found that the system of mitochondria in the two cell lines is different for morphology and localization. The mitochondria with the known S-shape and/or with ring-and globular-shape have been found in the cytoplasm of sensitive cells, whereas punctate mitochondria appeared in the subplasmalemamma region of resistant cells.

It has been surprisingly found that a rhodamine 123 and ethidium bromide double staining is extremely useful for the diagnosis of multidrug resistant cells on the basis of the described differences in their mitochondrial system. Indeed these two probes, which have high fluorescent yield and good spectral separation of their fluorescent emissions (respectively emission maxima in the green at 520 nm (R123) and in the red at 620 nm (EB)), stain with different efficiency the S-shaped and the punctate mitochondria. The red fluorescence of ethidium bromide is higher at the level of mitochondria in the periphery of the cells than on the central mitochondria that spread all through the cytoplasm with exception of the submembrane region. In figure 1a is shown the fluorescence pattern of MCF-7 cells treated with Ethidium Bromide followed by R123 and analysed by laser

scanning confocal microscopy. The green and red signals are distinct and separated in sensitive cells, where the red fluorescence, localized on peripheral mitochondria, is due to Ethidium Bromide (indicated as region 1 by an arrow in the picture), while the green fluorescence, localized on central mitochondria, is due to R123 (indicated as region 2 in the picture). In figure 1b is shown the different fluorescence pattern of resistant MCF-7/DX cells: in this case, the EB and R123 fluorescences overlap, resulting in a brown-yellow signal.

By this simple and non invasive technique the pattern of MDR sensitive and resistant cells has been easily discriminated, with powerful diagnostic implications.

The invention is here described in more details in the following non limiting examples.

METHODS Adherent MCF-7 cells have been used for all of these confocal microscopy studies. These cells can be taken as a model system for mitochondrial analysis, thanks to their high retention of mitochondrial probes (1). The apparatus used throughout these studies is the confocal microscope MRC-600 (Bio-Rad Microscience Division) coupled to a fluorescence microscope Nikon Optiphot II that has been employed under the following conditions: oil immersion objective 60X with N. A. 1.4; fluorescence excitation by the Argon ion laser at 488 nm (with power<0.1 mW), two channels double fluorescence detection (green for R123 and red for EB) by photon counting through band pass at 540nm (BW 30 nm) on the rhodamin channel and through a long pass filter above 600 nm on the ethidium channel.

Example 1. Double staining with Ethidium Bromide and Rhodamine in MDR sensitive and resistant cells and analysis of their confocal fluorescence pattern.

MCF-7 sensitive and MCF-7/DX resistant cells, grown as monolayer in RPMI (Hyclone) with fetal calf serum 10% (Hyclone) and glutamin (1%) (Gibco), were incubated at 37° C in culture medium containing ethidium bromide at a concentration of 1pM for 15'at 37°C, followed by incubation at 37°C for 10 minutes in a culture medium containing R123 at the concentration of 1pM. After the first and the second staining, cells were rinsed twice in PBS at 4° C.

As a comparison, a doxorubicin staining (1.7 M in culture medium for 1 hour) has been performed on the same human carcinoma cell line. Results are reported in Figure 1 where is shown the intracellular distribution of doxorubicin in human breast carcinoma MCF-7 sensitive (Figure 2 a) and resistant MCF-7/DX (Figure 2b) cells.

Figure 1 shows the fluorescent images of sensitive MCF-7 and resistant MCF- 7/DX cells, treated by the double R123 and EB staining described above. It is possible to appreciate that with EB and R123 staining in sensitive cells (figure 1a), the red EB fluorescence is localized on the peripheral mitochondria (region 1 in the picture), whereas the green R123 fluorescence (region 2 in the picture) is localized on the mitochondria that are distributed all through the cytoplasm with the exception of the subplasmalemma region. The two fluorescences are spatially separated in sensitive cells, leading to a two-coloured image in red and green.

In resistant cells (figure 1b) the EB fluorescence and the R123 fluorescence are both localized on the peripheral mitochondria of the cell. The overlapping of the two fluorescences-red and green-produces a brown-yellow image. In the experiments reported here merging of the two images, collecte respectively on the green fluorescence channel of R123 and on the red channel of EB, was performed. This is clearly shown in a further example shown in figure 3, where the R123 and EB double staining of MCF-7 cells is given and where the peripheral mitochondria of the cell are in red (red EB fluorescence indicated as region 1 in the picture; green R123 fluorescence indicated as region 2).

In the case of doxorubicin staining the differences in the fluorescence pattern of sensitive and resistant cells are instead mainly at the level of the nucleus (not fluorescent in resistant cells) and at the level of punctate cytoplasm (characteristic only of resistant cells) as shown in figure 2a and 2b.

Example 2. Treatment of resistant cells with Verapamil and double staining.

To confirm the hypothesis that the observed differences in the mitochondrial pattern are directly related to the multidrug resistance and, more generally, to cell conditions, the effect of Verapamil has been studied. Verapamil is a well known revertant of multidrug resistance (2,3). In Figure 4 is shown the double R123 and

EB fluorescence image of resistant cells MCF-7/DX in the presence of Verapamil, used at 100 M concentration. In this case the double staining with R123 and EB is performed in the continuous presence of Verapamil, used at 100 M concentration. It is evident from fig. 4 that Verapamil has re-established in resistant cells the fluorescence distinctive pattern of sensitive cells mitochondria. The green fluorescence (indicated as region 2 in the picture), due to R123, on peripheral mitochondria has disappeared and has reappeared instead on the central mitochondria, not fluorescent in resistant cells. The red fluorescence of EB (region 1 in the picture) has not changed, remaining mainly localized on peripheral mitochondria.

As a result, the two-coloured image characteristic of sensitive cells is restored in resistant cells by Verapamil, as expected on the basis of its known MDR revertant effect.

Example 3. Single dye staining with rhodamine 123 or ethidium bromide.

Differences in the system of mitochondria of sensitive and resistant cells described above can be observed-in a more subtle way-also with rhodamine staining alone. The intracellular distribution of rhodamine in sensitive cells (in absence of EB) is presented in figure 5a, where MCF-7 and MCF-7/DX cells have been treated with Rhodamine 1M in culture medium for 10'at 37°C. Their fluorescent pattern has been analyzed by laser scanning confocal microscopy.

In this case, central mitochondria are fluorescent as in the case of the double R123 and EB staining (see figure 1a). In resistant cells, instead, rhodamine stains only peripheral punctate mitochondria in the sub-plasmalemma region as shown in figure 5b.

Taking into account that rhodamine 123 is a probe of mitochondrial potential (4, 5), the observed results suggest that the peripheral punctate mitochondria are fluorescent and therefore active-with high membrane potential-only in resistant cells, whereas only the central S-shaped mithocondria are active in sensitive cells.

This latter mitochondria system is activated in resistant cells only in the presence of Verapamil, as shown in fig 4.

In the case of staining with EB alone MCF-7 and MCF-7/DX cells have been

treated with Ethidium Bromide 1 uM in culture medium for 15'at 37°C and their fluorescence pattern analyzed by laser scanning confocal microscopy (figure 6).

Mitochondria of sensitive MCF-7 and resistant MCF-7/DX cells are stained in a similar way as in the case of the double staining (figure 1 a and figure 1 b). In figure 6a and 6b is possible to note that the red fluorescence of EB is particularly intense on peripheral mitochondria that cannot be seen by R123 in sensitive cells. Indeed, as newly shown here, the EB staining allows the observation of peripheral mitochondria (not active in sensitive cells) which are not easily detectable by the mitochondrial probes in use up to now. This new method of mitochondrial staining might then be extremely useful to the analysis mitochondrial alterations in other cellular systems (6,7).

Multidrug resistance therefore shows itself on the system of mitochondria of the cell. Differences in the morphology and localization of mitochondria in sensitive and resistant cells, are easily observable by confocal microscopy after fluorescent probes staining of these organelles, and therefore offer a possible standard for the diagnosis of antitumor drug resistance, with important clinical applications. It has also been found that this methodology is able to show the peripheral mitochondria of the cell and has been successfully applied to the study and the diagnosis of all the pathologies associated with mitochondrial dysfunctions, such as cardiac failure, myopathies and neuropathies, autoimmune as well as degenerative diseases such as Alzheimer's and Parkinson's diseases.

In addition to the laser scanning confocal microscopy herein described, performed with double or single probes, other techniques such as the new optical fluorescence microscopies have been found to be useful for the study of mitochondrial pattern with satisfactory results. Of particular importance is the fact that, when coupled with highly sensitive detection systems these techniques allow a low energy sample irradiation, preserving cell viability during measurements.

References 1) Davis S, Weiss MJ, Wong JR, Lampidis TJ, Chen LB"Mitochondriai and plasma memebrane potentials cause unusual accumulation and retention of rhodamine 123 by human breast adenocarcinoma-derived MCF-7 cells" (1985), J Biol Chem 260,13844-13850 2) Tsuruo, T., lida, H., Tsukagoshi, S., Sakurai, Y."Overcoming of Vincristine Resistance in P388 Leukemia in Vivo and in Vitro through Enhanced Cytotoxicity of Vincristine and Vinblasine by Verapamil" (1981) Cancer Res. 41,1967-1972 3) Tsuruo, T., lida, Nojiri, M., Tsukagoshi, S., Sakurai, Y."Circumvention of Vincristine and Adriamycin Resistance in Vitro and in Vivo by Calcium Influx Blockers" (1983) Cancer Res. 43,2905-2910 4) Johnson, L. J., Walsh, M. L., Chen, L. B."Localization of Mitochondria in Living Cells with Rhodamine 123" (1980) Proc. Natl. Acad. Sci. USA 77,990-994 5) Johnson, L. J., Walsh, M. L., Bockus, B. J., Chen, L. B."Monitoring of Relative Mitochondrial Membrane Potential in Living Cells by Fluorescence Microscopy" (1981) J. Cell Biol. 88,526-535 6) Current Topics in Bioenergetics."Molecular Basis of Mitochondrial Pathology", vol 17, Ed: C. P. Lee, Academic Press (1994) 7)"Mitochondrial Diseases and Aging"Sect. III in"Mitochondrial Biogenesis and Genetics"Eds GM Attardi and A Chomyn, Methods in Enzymology Part B, Vol 264, Academic Press (1996)