WO2003058192A2

WO2003058192A2 - System and method for differentiating between cells with normal and pathologically altered cytoskeleton

Info

Publication number: WO2003058192A2
Application number: PCT/IL2003/000019
Authority: WO
Inventors: Shmuel Safran; Alexander D. Bershadsky; Elisha Moses; Roy Bar-Ziv; Tsvi Tlusty
Original assignee: Yeda Research And Development Co. Ltd.
Priority date: 2002-01-09
Filing date: 2003-01-07
Publication date: 2003-07-17
Also published as: AU2003209605A1; IL147544A0; WO2003058192A3

Abstract

Methods and systems for classifying a cell as having a normal or an abnormal phenotype related to its cytoskeleton are provided. One disclosed method includes contacting the cell with a substance capable of disrupting the cell cytoskeleton, monitoring a change in morphology of the cell; and classifying the cell phenotype according to a degree and/or kinetics of the change in the morphology. Further disclosed is a method including exposing the cell to multiple concentrations the substance and classifying the cell phenotype according to a concentration at which a morphological change in the cell occurs. Further disclosed is a system for classifying the cell including a substrate for supporting the cell, an image capture unit designed and configured for capturing image data the cell adhered to the substrate and a data processing unit capable of receiving the image data and determining a degree of pearling in the cell from the image data.

Description

SYSTEM AND METHOD FOR DIFFERENTIATING BETWEEN CELLS WITH

NORMAL AND PATHOLOGICALLY ALTERED CYTOSKELETON

FIELD AND BACKGROUND OF THE INVENTION The present invention relates to a system and method for differentiating between cells with normal and pathologically altered cytoskeleton and, more particularly, to a system and method for identifying cancerous cells in a sample.

Determining whether a small sample of single cells contains diseased or malignant cells is a major problem in modern medical pathology. A clinical cyto-pathologist is required to give, often within a matter of minutes, a decision as to whether a given sample of cells includes cancerous cells, and to identify their type and level of differentiation. This is far more difficult in samples containing individual cells than in whole tissue samples. Individual cells are often few in number, broken or malformed, and obscured by debris. Traditionally, cyto-pathologists use an optical microscope, relying on their experience in visually identifying cells that are deformed, slightly enlarged or bearing any other morphological irregularities. This is time consuming, painstaking and needs extensive training; further, the results are not 100 percent reliable.

Therefore, newer techniques incorporating fluorescent markers with specificity to various types of cancerous cells have been developed. These markers increase the accuracy of conventional pathological evaluation, however, currently available markers can select only for specific types of cancerous cells since suitable markers are not available for every malignant cell type.

As such, concurrent utilization of several types of markers is often undertaken, even though having the right marker in the mix cannot be assured.

One difference between cancerous (malignant) cells and normal ones is that cancerous cells are unable to build normal tissue and tend to detach from original tissue and invade into neighboring tissue. Malignant cells also tend to metastasize into other tissue regions. Malignant growth and metastasis occur because of an underlying difference in the structure of the actin cytoskeleton of cancerous cells.

It is widely accepted that cancerous cells have an actin cytoskeleton that is weaker than that of a healthy cell [Janmey and Chaponnier, Curr. Opin. Cell Biol. 7, 111 (1995); Pawlak and Helfinan, Curr. Opin. Genet. Dev. 1 1 , 41 (2001)]. This is usually understood in the literature as deriving from the weakness of the actin cortex - a thin shell of cytoskeletal filaments underlying the cell's membrane. Therefore, the

Young's modulus of the cortex may serve as an indication of malignancy; however, little use has been made of this fact to date. Earlier work published in the Proceedings of the National Academy of

Sciences (Bar-Ziv et al, PNAS 96,10140, (1999)), demonstrates that the effect of latrunculin A on the strength (elastic rigidity) of the cell is quantifiable and precise.

The rigidity of a cell is determined by its Young's modulus E. Specifically, Bar-Ziv et al. have shown that the Young's modulus decreases linearly with the concentration, φ, of latrunculin A added to the cell:

E = Eo - α φ. (Εq. 1) wherein Eo is the intrinsic Young's modulus of the cell in the absence of the drug. However, this published work is purely theoretical in nature and does not establish any practical use for the described properties. U.S. patent no. 6,067,859 issued to Kas et al. teaches a novel optical micromanipulation tool (optical stretcher), which may use a tunable laser to trap and deform cells between two counter-propagating beams generated by the laser. Kas teaches that it is possible to detect the deformation of cancer cells. However, the teachings of Kas contain neither a hint nor a suggestion that the morphological response of a cell to a drug or chemical can provide information concerning malignancy of the cell. Further, all teachings of Kas require the use of expensive laser equipment which is not typically found in a clinical pathology laboratory.

There is thus a widely recognized need for, and it would be highly advantageous to have, an improved system and method for differentiating between normal and abnormal cells devoid of the above limitations.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided a method of classifying a cell as having a normal or an abnormal phenotype. The method includes: (a) contacting the cell with at least one substance capable of disrupting at least one component of a cell cytoskeleton; (b) monitoring a change in a morphology of the cell; and (c) classifying the cell as having the normal or abnormal phenotype according to a degree and/or kinetics of the change in the morphology.

According to another aspect of the present invention there is provided a system for classifying a cell as having a normal or an abnormal phenotype. The system includes: (a) a substrate being for supporting a biological specimen; (b) an image capture unit designed and configured for capturing image data from a cell adhered to the substrate; and (c) a data processing unit being for receiving image data from the image capture unit, the data processing unit being for determining a degree of pearling in the cell from image data captured prior to and following exposure of the cell to at least one substance capable of disrupting at least one component of a cell cytoskeleton. The degree of pearling serves for classifying the cell as having a normal or an abnormal phenotype.

According to yet another aspect of the present invention there is provided a method of classifying a cell as having a normal or an abnormal phenotype. The method includes: (a) exposing the cell to multiple concentrations of at least one substance capable of disrupting a cell cytoskeleton; and (b) classifying the cell as having a normal or an abnormal phenotype according to a concentration from among the multiple concentrations of the at least one substance at which a morphological change in the cell.

According to further features in preferred embodiments of the invention described below, the at least one substance is capable of disrupting actin filaments of the cell cytoskeleton.

According to still further features in the described preferred embodiments the at least one substance includes at least one compound selected from the group consisting of Latrunculin A (Lat A), Latrunculin B, cytochalasin D, swinholide A, misaconilide A, tolytoxin; mycalolide B and aplyronine A.

According to still further features in the described preferred embodiments the change in the morphology of the cell is effected over a predetermined time period.

According to still further features in the described preferred embodiments classifying the cell as having a normal or an abnormal phenotype is effected by determining a degree of pearling of the cell.

According to still further features in the described preferred embodiments, the method further includes adhering the cell to a substrate prior to or following step (a). According to still further features in the described preferred embodiments classifying is relative to a second cell of a known phenotype contacted with the at least one substance capable of disrupting the at least one component of the cell cytoskeleton. According to still further features in the described preferred embodiments the second cell has a phenotype selected from the group consisting of a normal phenotype and an abnormal phenotype.

According to still further features in the described preferred embodiments the image capture unit includes at least one item selected from the group consisting of a still camera and a video camera.

According to still further features in the described preferred embodiments the substrate includes an adherent surface of a substance selected from the group consisting of glass and a plastic.

According to still further features in the described preferred embodiments the adherent surface is further coated with a substance selected from the group consisting of a peptide, a protein, a polysaccharide, a lipid, a lipo-polysacharide and a mucopolysacharide.

According to still further features in the described preferred embodiments the data processing unit is further for comparing the degree of pearling to a degree of pearling of a control cell, the control cell having a known phenotype.

According to still further features in the described preferred embodiments the data processing unit is designed and configured for performing at least one function selected from the group consisting of: (a) analyzing a single image of the cell, (b) comparing an image of the cell captured by the image capture unit prior to application of the at least one substance to the cell to an image of the cell captured after application of the at least one substance to the cell and (c) determining kinetics of the pearling over time.

According to still further features in the described preferred embodiments the kinetics are determined by at least one approach selected from the group consisting of: (i) comparing an image of the cell captured by the image capture unit prior to application of the at least one substance to the cell to each of a series of images of the cell captured at known times after application of the at least one substance to the cell; (ii) comparing each of a series of images of the cell captured at known times after application of the at least one substance to the cell to a first image in the series; and

(iii) comparing each image in a series of images of the cell captured at known times after application of the at least one substance to the cell to a previous image in the series.

According to still further features in the described preferred embodiments the at least one component of the cytoskeleton includes actin polymers.

According to still further features in the described preferred embodiments the at least one substance is capable of disrupting actin filaments of the cell cytoskeleton. According to still further features in the described preferred embodiments the to the concentration at which said morphological change in the cell occurs further facilitates differentiation between a malignant phenotype and a normal phenotype.

According to still further features in the described preferred embodiments monitoring the change in the morphology of the cell is effected over a predetermined time period.

According to still further features in the described preferred embodiments exposing the cell to each of the multiple concentrations refers to exposure for a predetermined time period.

According to yet another aspect of the present invention there is provided a method of determining the effect of a drug candidate on cells having a normal or an abnormal phenotype, the method comprising: (a) contacting a first cell culture with at least one substance capable of disrupting at least one component of a cell cytoskeleton; (b) determining a percentage of cells of the cell culture having an abnormal phenotype according to a response thereof to the at least one substance capable of disrupting at least one component of a cell cytoskeleton; (c) contacting a second cell culture with the drug candidate; (d) determining the percentage of cells of the second cell culture negatively affected by the drug candidate; and (e) comparing the percentage determined in step (b) and the percentage determined in step (d) to thereby determine the effect of the drug candidate on cells having the normal or the abnormal phenotype.

According to still further features in the described preferred embodiments the method further comprises the step of: (f) contacting the second cell culture with the at least one substance capable of disrupting at least one component of a cell cytoskeleton following step (d), thereby qualifying the phenotype of cells of the second culture unaffected by the drug candidate.

According to still further features in the described preferred embodiments the at least one substance is capable of disrupting actin filaments of the cell cytoskeleton.

According to still further features in the described preferred embodiments the at least one substance comprises at least one reagent selected from the group consisting of Latrunculin A (Lat A), Latrunculin B, cytochalasin D, swinholide A, misaconilide A, tolytoxin, mycalolide B and aplyronine A.

According to still further features in the described preferred embodiments step (b) is effected by monitoring a change in a morphology of cells of the first cell culture over a predetermined time period.

According to still further features in the described preferred embodiments the change monitored is a change in curvature of the cell leading to the "sun" morphology and/or the degree of cell pearling.

According to still further features in the described preferred embodiments the first cell culture and the second cell culture are similar.

The present invention successfully addresses the shortcomings of the presently known configurations by providing a system and methods for differentiating between healthy and diseased cells which may be implemented using standard cyto-pathology laboratory equipment. Implementation of the claimed methods and system for differentiating between healthy and diseased cells involves performing or completing selected tasks or steps manually, automatically, or a combination thereof. Moreover, according to actual instrumentation and equipment of preferred embodiments of the method and system of the present invention, several selected steps could be implemented by hardware or by software on any operating system of any firmware or a combination thereof. For example, as hardware, selected steps of the invention could be implemented as a chip or a circuit. As software, selected steps of the invention could be implemented as a plurality of software instructions being executed by a computer using any suitable operating system. In any case, selected steps of the method and system of the invention could be described as being performed by a data processor, such as a computing platform for executing a plurality of instructions.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the drawings:

FIGs. 1 A-B are photomicrographs of a cell prior to (Figure 1A) and following (Figure IB) exposure to latrunculin A. FIGs. 2A-H illustrate the pearling process which occurs when a cell is contacted with a cytoskeleton disrupting substance. Figures 2A through 2D illustrate the process in a cell with three points of attachment to a substrate. Figures 2E through 2H illustrate the process in a cell with seven points of attachment to a substrate. FIGs. 3A-C illustrate the difference in response between cancerous (black) and normal (white) cells when contacted with a substance according to a method of the present invention. Figure 3A shows the cells prior to contact. Figure 3B shows the cells following contact; Figure 3C is an enlargement of the region of Figure 3B indicated by a dashed rectangle. FIGs. 4A-D are photomicrographs illustrating the effect of transfection with

GFP-mDial (ΔN3) on CHOK1 cells. Figures 4A and 4B show fixed cells stained with Rhodamine-phalloidin to visualize filamentous actin. Figures 4C and 4D depict

GFP fluorescence of the same fields illustrated in 4 A and 4B respectively.

FIGs. 5A-B are images illustrating the ability of the present methodology to differentiate between abnormal and normal cells. Application of a cytoskeleton disrupting substance induces the "sun" morphology in Cells with an abnormal phenotype (arrows in Figure 5A) but only mildly affects the morphology of a cell having a normal phenotype (center of Figure 5A, GFP visualization of this cell in

Figure 5B).

FIG. 6 is a schematic illustration of a system according to the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of an improved system and methods which can be used to, for example, to identify cancerous cells in a sample. Specifically, the present invention can be used to differentiate between cells in a sample based upon the strength of their cytoskeletons.

The principles and operation of a system and methods for differentiating between healthy and diseased cells according to the present invention may be better understood with reference to the drawings and accompanying descriptions. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

Traditional prior art techniques for determining whether a small sample of single cells contains diseased or malignant cells typically include a clinical cyto-pathologist making a decision based upon visual evaluation using an optical microscope. The decision is most often based upon identification of cells that are deformed, slightly enlarged or display any other morphological irregularities. These techniques required the availability of a trained cytopathologist. In many instances, the reliability of the result was in proportion to the training and expertise of the cytopathologist. Because of the involvement of a high degree of subject evaluation, a significant percentage of misdiagnoses were bound to occur using these methods.

More recently, prior art techniques incorporating fluorescent markers with specificity to various types of cancerous cells have been developed. These markers increase the accuracy of diagnosis by eliminating the need for subjective evaluation to some degree. However, markers are currently available only for specific types of cancerous cells. Thus it is still necessary to rely upon traditional prior art techniques in many cases, even if concurrent utilization of several types of markers is undertaken. While it has been known that there is an underlying difference in the structure of the actin cytoskeleton of cancerous cells relative to normal cells, this fact had remained largely a scientific curiosity until the advent of the disclosed invention. That is because although U.S. pat. no. 6,067,859 issued to Kas et al. teaches an optical stretcher capable of trapping and deforming cells between two counter-propagating beams generated by the laser so that it is possible to selectively detect the deformation of cancer cells. However, the teachings of Kas require the use of expensive laser equipment which is not typically found in a clinical pathology laboratory and the willingness of medical centers to acquire, install and employ such equipment has not manifested itself since the publication of those teachings. Thus, despite improvements in the prior art, there remained a need for an objective measure of cellular phenotype, which would require neither highly skilled operators nor extra-ordinary laboratory equipment.

Reduction of the present invention to practice, involved exploitation of the fact that cells that are characterized by an abnormal phenotype, such as cancer cells, are moφhologically transformed when exposed to substances capable of disrupting cytoskeletal structures. The present invention relies upon quantitative differentiation of the transformation of abnormal cells relative to that of normal cells. Therefore, the present invention makes possible, for the first time, identification of cells of an abnormal phenotype, including but not limited to cancer cells, by means of differences in cytoskeletal strength without employing laser technology. This represents a significant breakthrough because it is applicable to cells for which no fluorescent markers are available. As such, it is likely to reveal a wide spectrum of cancerous cells, even those resulting from previously uncharacterized genotypes.

Thus, according to one aspect of the present invention there is provided a method of classifying a cell as having a normal or an abnormal phenotype. The method is effected by contacting the cell with at least one substance capable of disrupting at least one component of a cell cytoskeleton. The at least one substance may be any substance capable of disrupting cytoskeletal filaments such as, for example, actin filaments. Examples include but are not limited to Latrunculin A (Lat

A), Latrunculin B, cytochalasin D, swinholide A, misaconilide A, tolytoxin, mycalolide B and aplyronine A. According to preferred embodiments of the invention, Lat A* is employed. Combinations thereof may also be used according to alternate preferred embodiments of the invention.

While Latrunculin A and the closely related substance, Latrunculin B are preferably employed in the practice of the present invention, several other macrolides are known to disrupt the sub-membranous cell cortex via specific and direct interactions with actin molecules. Therefore, these macrolides are all considered potentially useful in practice of the present invention. These macrolides include, for example, a family of cytochalasins, in particular, cytochalasin D; swinholide A, misaconilide A, tolytoxin, mycalolide B, aplyronine A, and related agents [reviewed in: Spector et al, Microscopy Research and Technique 47, 18 (1999)].

Latrunculin A and latrunculin B act by forming 1:1 complexes with actin monomers thereby preventing these monomers from re-polymerization into filaments. This feature makes these compounds preferable for the quantitative comparison of different types of cells as claimed herein. Swinholide A binds to two actin monomers in a configuration that prevents the actin subunits from participating in either actin-filament nucleation or elongation reaction. This mechanism of action has a result similar to that of the latrunculins, reduction of the amount of filamentous actin in a concentration-dependent fashion. Therefore alternate preferred embodiments of the present invention employ Swinholide A for probing the cell cortex.

In contrast, Cytochalasin D, which has typically been the compound of choice in previous cell biology studies, interacts with actin in a very complex manner. Cytochalasin D caps the barbed end of actin filaments, severs actin filaments, sequesters actin monomers, promotes nucleation, and stimulates the ATPase activity of G-actin. These multiple mechanisms of action serve to disorganize the actin cytoskeleton in different ways in different cell types. This makes the effect of Cytochalasin D more difficult to predict quantitatively and implementation of preferred embodiments which rely upon Cytochalasin D will be more difficult to achieve.

The exact molecular mechanisms of action of misaconilide A, tolytoxin, mycalolide B, aplyronine A, and related agents remain to be elucidated. Therefore, while latrunculin A, latrunculin B, and swinholide A are preferably employed in practice of the present invention, future elucidation of the exact molecular mechanisms of misaconilide A, tolytoxin, mycalolide B, aplyronine A, and related agents may lead to the development of additional preferred embodiments of the invention which rely upon those compounds. Following contacting the cell with the at least one substance described above, the method is further effected by (i) monitoring a change in morphology of the cell (further described hereinbelow with respect to Figure 6) and (ii) classifying the cell according to a degree and/or kinetics of the change monitored.

In order to effectively practice the present methodology, monitoring of the change in the moφhology of the cell is preferably effected over a predetermined time period. Determination of a suitable time period may be accomplished by calibration of the method with cells of known phenotypes. As an illustrative, non-limiting example, cells from axillary lymph nodes of a group of female patients with breast cancer might be analyzed with varying concentrations of Lat A according to the disclosed method. These analyses might reveal, for example, that at an applied concentration of 25 micro-molar Lat A, moφhologic changes occur in some samples at two minutes, in most samples at five minutes and that all samples which will undergo a moφhologic change at this concentration have done so by eleven minutes. Thus, in this example, a predetermined time of eleven minutes is clinically desirable, as this time period will serve to reveal cancerous cells in all patients. It will be appreciated that the larger the sample of patients screened initially, the greater the reliability of the time period selected. In some cases it may be feasible to shorten the predetermined time period by increasing the concentration of Lat A. However, this possibility has limits because, at a certain concentration of Lat A normal phenotype cells will also be moφhologically changed.

It will be appreciated that different predetermined exposure times and/or concentrations of the substance may be required for different clinical applications.

For example, one set of conditions might be employed for breast cancer patients, a different set of conditions might be employed for prostate cancer patients, and a third set of conditions might be employed for general screening to effect early diagnosis of a wide variety of cancers before any appreciable tumor growth is detected by conventional examination techniques. The disclosed invention is expected to find utility in screening for early detection, diagnosis of cancer type, monitoring of efficacy of treatment and follow-up examinations to detect possible remission.

According to the method of the present invention, classifying the cell as having a normal or an abnormal phenotype is preferably effected by determining a moφhological change. This moφhologic change is preferably arborization (Figures 2b, c, f ,g and 3b), more preferably cell pearling (Figures 2d, h and 3c) and most preferably a degree of pearling of the cell. This determination of moφhologic change may be effected by a skilled technician using a conventional microscope. Alternately, or additionally, this determination may be accomplished by a dedicated system as described hereinbelow and illustrated in Figure 6.

Determination of cellular phenotype can also be effected by exposing the cell tested to a series of concentrations of the cytoskeleton disrupting substance mentioned above and determining the concentration at which a moφhological change occurs. Thus, according to another aspect of the present invention there is provided a method of classifying a cell as having a normal or an abnormal phenotype which relies upon exposing the cell to multiple concentrations of at least one substance capable of disrupting a cell cytoskeleton and classifying the cell as having a normal or an abnormal phenotype according to a concentration at which a moφhological change in the cell is observed.

Preferably, the exposure time for each of the multiple concentrations is predetermined essentially as described hereinabove. According to this method, classifying the cell as having a normal or an abnormal phenotype is effected by determining the concentration at which a moφhological change in the cell occurs. Such classifying facilitates differentiation between, for example, a malignant phenotype and a normal phenotype. As described hereinabove, the moφhological change is preferably cell pearling, although other cytoskeleton related moφhologic changes are within the scope of the present invention. A high degree of cell pearling produces a moφhology commonly referred to a "sun" moφhology. The concentration at which a drastic moφhological change

(e.g. "sun" moφhology) occurs may be determined from direct measurements or can be extrapolated from initial measurements of changes in the cell shape prior to the appearance of the "sun" moφhology.

The above described methodology of the present invention preferably also include a step of adhering the cell to a substrate prior to or following contacting the cell with the substance capable of disrupting the at least one component of the cell cytoskeleton. Although not imperative, such a step greatly enhances the ability to determine cellular moφhological changes and to track specific cells in a sample and an as such is preferably employed by the methods of the present invention.

In some cases it may be desirable to classify a tested cell relative to a second cell of a known phenotype, e.g., a normal phenotype or an abnormal phenotype (e.g. malignant).

As is mentioned hereinabove, the moφhological features suitable for use in classifying the cell according to the present invention include, but are not limited to, arborization and pearling or degrees thereof or kinetics thereof.

Preferably, the methods described above utilize cell pearling as a moφhological indicator for classifying the cell.

Figures 1A-B illustrate cell pearling as a result of an application of a cytoskeleton disrupting substance. Figure 1A depicts a cell prior to contact with a cytoskeleton disrupting substance while Figure IB depicts a cell following contact with a cytoskeleton disrupting substance illustrating the pearling effect induced by the cytoskeleton disrupting substance.

The phenomenon of cell pearling is induced by cytoskeleton disrupting substances since such substances influence cytoskeletal elasticity. Cytoskeletal elasticity along with cell surface tension, σ, determine the mechanical equilibrium of a cell adhered to a surface.

As a result, the shape of cells when adhered to a substrate at several distinct adhesion points is determined by the mechanical equilibrium, which is a function of σ and the cytoskeletal elasticity.

This means that cell shape is determined by an effective line tension, γ, whose magnitude is proportional to the product of:

(i) the elastic Young's modulus, E, of the cytoskeleton whose largest deformations occur at the cell-substrate boundary; and

(ii) the cross-sectional area over which this deformation occurs, related to the height of the cell.

As a result, the force balance predicts that the cell-substrate boundary has the shape of a circular section whose radius is given by the ratio of the effective line tension and the surface tension: R~ γ/ σ between two adjacent adhesion points. This relationship accounts for the arborization process depicted in Figures 2a-c and 2e-g.

The cytoskeletal elasticity, Young's modulus and effective line tension γ, are all reduced by the addition of the substance employed in the claimed methods. For small reductions the decrease is linear and is a function of the drug concentration, φ as presented in equation 1. Thus the observed radius of curvature between adhesion points is predicted to decrease as the modulus is weakened. When the radius becomes smaller than the distance between adhesion points, the cell shape shows a change to a star-like configuration with a central core and linear arms. As the radius further decreases, this core shrinks even further and the arms grow. The anticipated progression as the radius of curvature decreases for a cell with three adhesion points is represented schematically in Figures 2 A-D. The anticipated progression as the radius of curvature decreases for a cell with seven adhesion points is represented schematically in Figures 2 Ε-H.

The present invention relies upon the principle that if the surface tension is known, a measurement of the radius and thickness can be used to estimate the intrinsic Young's modulus, E₀. This intrinsic modulus is important because the modulus of cancerous cells is known to be smaller than that of healthy cells. However, an absolute determination of the elastic modulus from a measurement of the I radius requires good estimates of the height of the edge of the cell and the surface tension.

Therefore, a measure of the cytoskeletal modulus is obtained by measuring the boundary radius, R, as a function of the concentration of the applied substance.

Extrapolation to R— allows estimation of (see Eq. (1)) the critical concentration: φ_c~Eo /α. As used herein, the phrase "critical concentration" refers to any concentration which can be used to induce a visually discernable change in cell moφhology. Preferably, a critical concentration induces such a change in moφhology within a predetermined time period. Preferably this predetermined time period is approximately one hour, more preferably this predetermined time period is approximately 30 minutes, more preferably this predetermined time period is approximately ten minutes, most preferably this predetermined time period is between 1 minute and 5 minutes. Therefore, cells with a more rigid cytoskeleton will react to a higher critical concentration. This means that cells with an abnormally weak cytoskeleton will tend to exhibit a change in moφhology at a lower critical concentration than their normal counteφarts. The present invention is the first demonstration that one may determine a value of the critical concentration above which there is a high probability that the cell is healthy and below which there is a high probability that the cell is cancerous with a weakened cytoskeleton as depicted schematically in Figures 3A-C. Experimental support for this schematic representation is presented in Examples hereinbelow, most specifically in Figures 5A-B.

Apart from being effective in various diagnostic assays, the present methodology can also be utilized in drug screening assays directed at identifying agents effective against cancerous cells.

For example, the above described methodology can be utilized to verify that a drug candidate is selectively affective towards cancer cells only. As is mentioned hereinabove, cancer cells are characterized by a weak cytoskeleton which enables such cells to deform and move throughout the body. Such weakness can be controllably magnified using the cytoskeletal targeting substances described above (e.g., Latrunculin A). Since drug screening assays are preferably effected in cell cultures, the present methodology can be utilized to qualify the culture phenotype prior to and following drug candidate screening in order to determine the type of cells the drug candidate is most effective against. For an overview on screening procedures which are designed for identifying anti-cancer agents and as s such are suitable for use with the present invention, please see "Application of High-Throughput, Molecular-Targeted

Screening to Anticancer Drug Discovery" by Robert H. Shoemaker which is available online at http://www.bentham.org/sample-issues/ctmc2-3/shoemaker/shoemaker-ms.htm. Such a screening assay is effected as follows. A cytoskeletal targeting substance such as Latrunculin A is applied to a cell culture at a predetermined concentration, and the percentage of cells exhibiting a marked change in moφhology is determined. This step defines the percentage (on average) of abnormal (cancerous) cells in the assay sample. Following such determination, an identical (untreated) cell culture is exposed to the drug candidate and the percentage of cells killed by the drug is determined. The percentage of cells that die indicate if the drug candidate has an unwanted effect of killing healthy cells. Latrunculin A can then be used (at a similar concentration) on the drug treated culture in order to determine the phenotype of the cells which were unaffected by the drug treatment. Alternatively, a drug candidate found effective against cancerous cells can be tested against normal cells in the presence of an otherwise ineffective dose of a cytoskeletal targeting substance such as Latrunculin A (e.g., a dose incapable of eliciting observed moφhological changes), in order to determine if the drug candidate has an otherwise undetectable and potentially deleterious effect on normal cells. The use of a cytoskeletal weakening substance in a drug screening assay in the manner described above significantly shortens the time it takes to validate a drug candidate since it enables to determine the effectiveness and possible toxicity of the drug candidate in a single assay.

In addition, since the above described approach determines the effect of a drug candidate on both healthy and abnormal cells, it can also be used to deteπnine the concentration of a drug which is effective against cancerous cells yet not toxic to healthy cells. The methods of the present invention are preferably effected using a dedicated cell classification system, which is referred to hereinunder as system 20.

As is illustrated in Figure 6, system 20 includes a substrate 26 for supporting a biological specimen. The substrate preferably includes an adherent surface of a substance such as, for example, glass or a plastic. Plastics suitable for use as substrates for cell growth are well known and are commercially available from many sources including Costar (Corning Inc., Corning, NY, USA). One of ordinary skill in the art of cell biology will be capable of choosing a suitable substrate for use as part of system 20. Preferably, the adherent surface is further coated with a substance such as a peptide, a protein, a polysaccharide, a lipid, a lipo-polysacharide a mucopolysacharide, or combinations thereof. Such coatings may be employed to regulate the degree of adherence of a cell, or of a certain type of cells. Many such coatings are known and one ordinarily skilled in the art of pathology or cell biology will be capable of selecting an available coating for use in conjunction with the present invention. Substrate 26 may be divided, according to some embodiments, into a series of discrete and addressable locations, as indicated by the grid pattern. For example, substrate 26 may be a multi-well microtiter plate. In such a case, repeated images of a single cell residing in a defined location might be easily collected over time. Many machines for reading discrete and addressable locations are commercially available and any of these could easily be incoφorated into system 20.

System 20 further includes an image capture unit 30 designed and configured for capturing image data from a cell adhered to substrate 26. The image capture unit may include, for example, a still camera or a video camera (e.g., a CCD camera such as the Micro-MAX-1300YHX CCD camera commercially available from Princeton Instruments and Roper Scientific).

System 20 further includes a data processing unit 40 capable of receiving image data (arrow) from image capture unit 30. If image capture unit 30 employs film or videotape, additional hardware and software for transformation of captured images to digital format will be further incoφorated into system 20. Such hardware and software is commercially available and will be readily amenable to use in conjunction with the present invention. Data processing unit 40 may be, for example, a computer platform executing a software application designed and configured for determining a degree of cell pearling. A conventional desktop or laptop computer is suited for use as data processing unit 40. Many software applications (e.g. those of

Clemex, Quebec, Canada and Visus Image Analysis, Rancho Cucamonga, CA) are commercially available. One of ordinary skill in the art of image analysis will readily be able to configure/modify such software applications for use in determining, for example, a degree of cell pearling.

Data processing unit 40 determines a degree of pearling in the cell from image data captured prior to and following exposure of the cell to the at least one substance capable of disrupting at least one component of a cell cytoskeleton. Image data is stored in memory 44 of data processor 40. The degree of pearling is used to classify the cell as having a normal (i.e. healthy) or an abnormal (i.e. diseased or malignant) phenotype. In some cases image data may be visible to an operator on display 42. Alternately, or additionally, an operator may use input device 46 (e.g. a keyboard) to instruct image capture unit 30 to capture images from specific locations on substrate 26. Preferably, data processing unit 40 is further for comparing the degree of pearling to a degree of pearling of a control cell, the control cell having a known phenotype. Preferably control cells reside in known locations on substrate 26.

The data processing unit may execute one or more analysis approaches. According to one approach, data processing unit 40 analyzes a single image of the cell. This image may be collected, for example, at a predetermined time after contacting a cell with a substance as described hereinabove. According to an additional/alternative approach, the data processing unit compares an image of the cell captured by the image capture unit prior to application of the at least one substance to the cell, to an image of the cell captured after application of the at least one substance to the cell. In addition, the data processing unit may also determine kinetics of the pearling of a cell over time. The kinetics of pearling over time may be determined by a variety of approaches. For example, one approach is to compare an image of the cell captured by image capture unit 30 prior to application of the substance to the cell to each of a series of images of the cell captured at known times after the application. An alternate approach is to compare each of a series of images of the cell captured at known times after application of the at least one substance to the cell to a first image in the series. Another alternate approach is to compare each image in a series of images of the cell captured at known times after application of the at least one substance to the cell to a previous image in the series.

One ordinarily skilled in the art of mathematical modeling will be capable of adapting these, and many other, approaches for determining kinetics of pearling for use in conjunction with the present invention.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non-limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H.

Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos.

3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;

5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);

"Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985);

"Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984);

"Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B.,

(1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:

A Guide To Methods And Applications", Academic Press, San Diego, CA (1990);

Marshak et al., "Strategies for Protein Purification and Characterization - A

Laboratory Course Manual" CSHL Press (1996); all of which are incoφorated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incoφorated herein by reference.

EXAMPLE 1

Establishment of a tumor cell model

In order to demonstrate that similar cells with different content of filamentous actin react differently and exhibit different moφhological changes in response to latrunculin A, cells of identical origin but with different levels of filamentous actin were produced. To produce such cells unique features of a recently discovered regulatory protein (mDial; Watanabe et al, EMBO J. 16, 3044 (1997); Watanabe et al, Nat. Cell Biol. 1, 136 (1999); SwissProt name "DIAl_MOUSE, accession "O08808", gi :6014968) which is a potent stimulator of the level of filamentous actin in the cell. The protein mDial is a primary target of the signaling protein RhoA, which is a molecular switch responsible for the formation of actin containing stress fibers and focal adhesions. The mDial protein is regulated by RhoA by the binding of RhoA to mDial which converts mDial from a "closed" to an "open" configuration. In the "open" configuration some of the domains of mDial (most probably FH1 and

FH2) bind further, as yet unidentified, targets, which in turn activate processes of actin polymerization. This does not occur in the closed configuration. Deletion mutants of mDial lacking the RhoA binding site were shown to be always in the "open" configuration and therefore these deletion mutants are constitutively active.

A GFP (Genbank accession number AAB02572) derivative of the deletion mutant of mDial (GFP-mDial (ΔN3) described by ( Watanabe et al, Nat. Cell Biol.

1, 136 (1999) and demonstrating constitutive activity was used to transfect CHOK1 cells (ATCC number CCL-61). CHOK1 is a stable cell line, maintained in culture for many years, with a tumor origin. It is derived from the ovary tumor of a Chinese Hamster. Therefore CHOK1 cells have moderate levels of filamentous actin that can nevertheless form thin actin stress fibers when the cells are attached to solid substrates such as tissue culture dishes. Upon transfection with GFP-mDial (ΔN3) the level of filamentous actin in these cells increases significantly. This increase in the level filamentous actin in the cytoskeleton following transfection with GFP-mDial (ΔN3) is shown in Figures 4A-4D, where two examples of the effect of transfection are shown. Figures 4A and 4B show fixed cells stained with Rhodamine-phalloidin, a compound that specifically marks and visualizes filamentous actin. Fluorescence microscopy is used to identify the red Rhodamine marker of actin filament distribution, and also permits identification of transfected cells on the basis of the green GFP fluorescence (Figures 4C and 4D). Figures 4A and 4B show the stain with Rhodamine-phalloidin while Figures 4C and 4D depict GFP fluorescence of the same fields. Comparison of the left and right columns shows that cells containing GFP-mDial (ΔN3) have much higher levels of Rhodamine-phalloidin staining, indicating that these cells have more filamentous actin than neighboring, non-transfected CHOK1 cells. Thus, the GFP-mDial (ΔN3) transfected cells serve as an example of CHOK1 cells with a non-malignant phenotype. Transfection with GFP-mDial (ΔN3) can therefore be used to generate a mixed population of malignant/nonmalignant cells from a culture of CHOK1 cells. Correlation of GFP and Rhodamine-phalloidin fluorescence allows identification of the phenotype of each cell within the mixed population. This model system was employed to evaluate the present invention as explained hereinbelow in Example 2. EXAMPLE 2

Assay of a method according to the present invention in a tumor cell model

A mixed culture containing both CHOK1 cells transfected with GFP-mDial (ΔN3) and non-transfected CHOK1 cells was used in order to ascertain whether contacting cells with at least one substance capable of disrupting at least one component of a cell cytoskeleton could induce a change in moφhology which could be used to ascertain the phenotype of single cells. In this experiment latrunculin A was employed as the at least one substance capable of disrupting at least one component of a cell cytoskeleton, although other substances and combinations thereof are within the scope of the present invention.

Specifically, the culture was treated with 2.5μM of latrunculin A for 60 minutes. Under these conditions all non-transfected cells (i.e. malignant phenotype) acquire the typical "sun" moφhology described above and depicted in Figures 1-3, with almost all the cell contracted around its nucleus, and thin processes that can demonstrate "pearling" extending outwards (Indicated by arrows in Figure 5A). In contrast, cells that have been transfected with GFP-mDial (ΔN3) (i.e. induced normal phenotype), as visualized by GFP fluorescence, demonstrate a strikingly different moφhology. These cells, induced to normal phenotype, remain spread on the substrate, demonstrating only the curved invaginations that are typical of the reaction of malignant cells to much lower concentrations of latrunculin A.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incoφorated in their entirety by reference into the specification, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

Claims

WHAT IS CLAIMED IS:

1. A method of classifying a cell as having a normal or an abnormal phenotype, the method comprising:

(a) contacting the cell with at least one substance capable of disrupting at least one component of a cell cytoskeleton;

(b) monitoring a change in a moφhology of the cell; and

(c) classifying the cell as having the normal or abnormal phenotype according to a degree and/or kinetics of said change in said moφhology.

2. The method of claim 1, wherein said at least one substance is capable of disrupting actin filaments of said cell cytoskeleton.

3. The method of claim 1, wherein said at least one substance comprises at least one reagent selected from the group consisting of Latrunculin A (Lat A), Latrunculin B, cytochalasin D, swinholide A, misaconilide A, tolytoxin; mycalolide B and aplyronine A.

4. The method of claim 1, wherein said monitoring said change in said moφhology of the cell is effected over a predetermined time period.

5. The method of claim 1, wherein said classifying the cell as having a normal or an abnormal phenotype is effected by determining a degree of pearling of the cell.

6. The method of claim 1, further comprising adhering the cell to a substrate prior to or following step (a).

7. The method of claim l, wherein said classifying is relative to a second cell of a known phenotype contacted with said at least one substance capable of disrupting said at least one component of said cell cytoskeleton.

8. The method of claim 7, wherein said second cell has a phenotype selected from the group consisting of a normal phenotype and an abnormal phenotype.

9. A system for classifying a cell as having a normal or an abnormal phenotype, the system comprising:

(a) a substrate being for supporting a biological specimen;

(b) an image capture unit designed and configured for capturing image data from a cell adhered to said substrate; and

(c) a data processing unit being for receiving image data from said image capture unit, said data processing unit being for determining a degree of pearling in said cell from image data captured prior to and following exposure of said cell to at least one substance capable of disrupting at least one component of a cell cytoskeleton, said degree of pearling being for classifying the cell as having a normal or an abnormal phenotype.

10. The system of claim 9, wherein said image capture unit includes at least one item selected from the group consisting of a still camera and a video camera.

11. The system of claim 9, wherein said substrate includes an adherent surface of a substance selected from the group consisting of glass and a plastic.

12. The system of claim 11, wherein said adherent surface is further coated with a substance selected from the group consisting of a peptide, a protein, a polysaccharide, a lipid, a lipo-polysacharide and a mucopolysacharide.

13. The system of claim 9, wherein said data processing unit is further for comparing said degree of pearling to a degree of pearling of a control cell, said control cell having a known phenotype.

14. The system of claim 8, wherein said at least one substance comprises at least one reagent selected from the group consisting of Latrunculin A (Lat A), Latrunculin B, cytochalasin D, swinholide A, misaconilide A, tolytoxin; mycalolide B and aplyronine A.

15. The system of claim 9, wherein said data processing unit is designed and configured for performing at least one function selected from the group consisting of:

(a) analyzing a single image of the cell;

(b) comparing an image of the cell captured by said image capture unit prior to application of said at least one substance to the cell to an image of the cell captured after application of said at least one substance to the cell; and

(c) determining kinetics of said pearling over time.

16. The system of claim 15, wherein said kinetics are determined by at least one approach selected from the group consisting of:

(i) comparing an image of the cell captured by said image capture unit prior to application of said at least one substance to the cell to each of a series of images of the cell captured at known times after application of said at least one substance to the cell; (ii) comparing each of a series of images of the cell captured at known times after application of said at least one substance to the cell to a first image in said series; and (iii) comparing each image in a series of images of the cell captured at known times after application of said at least one substance to the cell to a previous image in said series.

17. The system of claim 9, wherein said at least one component of the cytoskeleton includes actin polymers.

18. A method of classifying a cell as having a normal or an abnormal phenotype, the method comprising:

(a) exposing the cell to multiple concentrations of at least one substance capable of disrupting a cell cytoskeleton; and (b) classifying the cell as having a normal or an abnormal phenotype according to a concentration from among said multiple concentrations of said at least one substance at which a moφhological change in the cell occurs.

19. The method of claim 18, wherein said at least one substance is capable of disrupting actin filaments of said cell cytoskeleton.

20. The method of claim 18, wherein said at least one substance comprises at least one reagent selected from the group consisting of Latrunculin A (Lat A), Latrunculin B, cytochalasin D, swinholide A, misaconilide A, tolytoxin; mycalolide B and aplyronine A.

21. The method of claim 18, wherein said classifying the cell according to said concentration at which said moφhological change in the cell occurs further facilitates differentiation between a malignant phenotype and a normal phenotype.

22. The method of claim 18, wherein said exposing the cell to each of said multiple concentrations refers to exposure for a predetermined time period.

23. The method of claim 18, wherein said moφhological change in the cell is cell pearling.

24. The method of claim 18, further comprising adhering the cell to a substrate prior to or following step (a).

25. The method of claim 18, wherein said classifying is relative to a second cell of a known phenotype.

26. The method of claim 25, wherein said second cell has a phenotype selected from the group consisting of a normal phenotype and an abnormal phenotype.

27. A method of determining the effect of a drug candidate on cells having a normal or an abnormal phenotype, the method comprising:

(a) contacting a first cell culture with at least one substance capable of disrupting at least one component of a cell cytoskeleton;

(b) determining a percentage of cells of said cell culture having an abnormal phenotype according to a response thereof to said at least one substance capable of disrupting at least one component of a cell cytoskeleton;

(c) contacting a second cell culture with the drug candidate;

(d) determining the percentage of cells of said second cell culture negatively affected by the drag candidate; and

(e) comparing said percentage determined in step (b) and said percentage determined in step (d) to thereby determine the effect of the drug candidate on cells having the normal or the abnormal phenotype.

28. The method of claim 27, further comprising the step of:

(f) contacting said second cell culture with said at least one substance capable of disrupting at least one component of a cell cytoskeleton following step (d), thereby qualifying the phenotype of cells of said second culture unaffected by the drug candidate.

29. The method of claim 27, wherein said at least one substance is capable of disrupting actin filaments of said cell cytoskeleton.

30. The method of claim 27, wherein said at least one substance comprises at least one reagent selected from the group consisting of Latranculin A (Lat A), Latrunculin B, cytochalasin D, swinholide A, misaconilide A, tolytoxin, mycalolide B and aplyronine A.

31. The method of claim 27, wherein step (b) is effected by monitoring a change in a moφhology of cells of said first cell culture over a predetermined time period.

32. The method of claim 31, wherein said change is a degree of cell pearling.

33. The method of claim 27, wherein said first cell culture and said second cell culture are similar.