WO2012069095A1

WO2012069095A1 - Monitoring changes in supra-cellular structures

Info

Publication number: WO2012069095A1
Application number: PCT/EP2010/068347
Authority: WO
Inventors: Christoph RIETHMÜLLER
Original assignee: Serend-Ip Gmbh
Priority date: 2010-11-26
Filing date: 2010-11-26
Publication date: 2012-05-31

Abstract

The present disclosure relates to the use of a method based on atomic force microscopy on biological meshworks. A method is provided to detect the Local Deviational Volume (LDV) of defined supra-cellular structures irrespective of a biochemical characterisation, allowing the quantification of changes in the supra-cellular meshwork.

Description

MONITORING CHANGES IN SUPRA-CELLULAR STRUCTURES

Description

FIELD OF THE INVENTION

[0001] The present invention relates to the use of a method for quantifying changes in the supra-cellular meshwork (SCM).

BACKGROUND OF THE INVENTION

[0002] Fibroblast cells produce extracellular matrix (ECM) fibers. The ECM is a mesh of collagen fibers spun from proteoglycans that are released mainly by fibroblasts. In turn, the fibers are the mechanical scaffold for the cells to adhere via integrins and crawl along to recolonise the area of the former insult. Hormones and inflammatory peptide mediators govern the complex balance of the ECM. The ECM may be disrupted by mechanical stress. Wound healing is a process, which is necessary to restore proper cell-cell contacts so that a higher organism can survive. An overshoot of wound healing cascades leads to scar formation and facilitates cancer formation. [0003] Different methods are known from the state of the art for quantifying wound healing. In a biochemical approach the quantification is done via measuring the amount of extracellular matrix (ECM) proteins, i. e. staining of collagen with Sirius Red followed by a readout of the colour intensity, which indicates the amount of ECM-proteins (Pickering, J. G., Boughner, D. R. Am J Pathol. 1991 May; 138(5): 1225-1231.)

[0004] It is also possible to quantify wound healing via video microscopy (phase contrast microscopy). A confluent layer of living cells is scratched with sharp instrument and the recolonialisation of the area is monitored. Basic read-out of this method is the speed of closing the gap, modified by the homogeneity of the migration front (Tremel, A. et al. Chemical Engineering Science Volume 64, Issue 2, January 2009, Pages 247-25.)

[0005] As an alternative for monitoring the recolonialisation optically is monitoring the recolonialisation by measuring the electric impedance after scratching the cell layer by a high voltage pulse (Wegener, J., Keese, C. R. & Giaever, I. (2002) BioTechniques 33 , 348-357). [0006] Electron microscopy (EM) may also be employed according to the state of the art to image single fibers of the ECM (Ross and Benditi, 1964 JCB vol. 22 no. 2 365-398). The diameter of the fibers can be assessed via manual measuring in the micrographs.A disadvantage of EM is the complex sample preparation, which includes drying and reflective coating of the sample, because EM works only in a vacuum.

[0007] Atomic force microscopy (AFM) was invented two decades ago and became a versatile tool for biological studies on single biomolecules, aggregates, viruses, cells or tissue models. The method bridges the gap between the nm-resolution technique electron microscopy (EM) and the μιη-scale optical microscopy (OM). As such, it combines the advantages of high resolution and the ability to investigate cells under physiological buffer conditions. At the same time, no disadvantageous sample drying or coating as necessary for EM or chromophore labelling as in fluorescence microscopy (FM) are needed. Additionally, local mechanical properties of the sample can be obtained (Riethmuller C, Schaffer T.E., Kienberger F., Stracke W. and Oberleithner H.; 2007; Ultramicroscopy 107:895-901; Rotsch C. and Radmacher M.; 2000; Biophys. J. 78:520-535). Hence, investigation of biological specimen very close to physiological conditions is possible with only a minimum of procedure-derived artifacts. [0008] Recording the topography of cellular surfaces and multicellular ensembles -either living or fixed- has been the basis of biologically inspired AFM studies ever since (Braet F., de Zanger R. and Wisse E.; 1997; J. Microsc. 186 (Pt l):84-87.; Oberleithner H., Giebisch G. and Geibel, J.; 1993; Pflugers Arch. 425:506-510; Chang L., et al.; 1993; BiophysJ. 64: 1282- 1286). But distinct structures are difficult to identify, especially on whole cells, where only cytoskeletal structures are obviously recognisable. They almost exclusively consist of fibrillary actin, contributions of microtubuli can be neglected. However, since morphological features are difficult to classify, a reproducible quantification is hard to perform. Therefore, most studies use a qualitative, rather descriptive approach., so that often an overlay of AFM and fluorescence marker is desired to prove the claimed structure.

[0009] However, the fluorescence identification is hampered by mutually disturbing fixation protocols for AFM and FM. Unequivocally accepted structural characteristics are actin stress fibers (Haga H., Sasaki S., Kawabata K., Ito E., Ushiki T. and Sambongi T.; 2000; Ultramicroscopy 82:253-258), microvilli (Poole K., Meder D., Simons K. and Muller D.; 2004; FEBS Lett. 565:53-58) and cell junctions (RiethmuUer C, Oberleithner H., Wilhelmi M., Franz J., Schlatter E., Klokkers J. and Edemir B.; 2008; Biophys. J. 94:671-678). Recently, also subcellular structural elements could be quantified through their specific sectional area and cellular mechanics (Thoelking G, Reiss B, Wegener J, Oberleithner H, Pavenstaedt H, RiethmuUer C. Nanotechnology. 2010 Jul 2;21(26):265102.).

[0010] Beyond that, only very delicate setups can deliver more detailed information; in special cell models, where one type of receptor is abundantly expressed, some aggregates can be imaged marker-free (Hoogenboom, B.W., Suda K., Engel A. and Fotiadis D.; 2007; J. Mol. Biol. 370:246-255) or with the TREC procedure, where topography can be recorded simultaneously with localization of one specific target using antibody-modified tips (Kienberger F., Ebner A., Gruber H.J. and Hinterdorfer P.; 2006; Acc. Chem. Res. 39:29-36).

[0011] The known methods are not suitable for quantifying changes in the meshwork, which connects or surrounds cells or cell tissues. The US 6,357,285 Bl discloses a method for the quantitative and objective correlation of data from a local sensitive force detector. Data are acquired from two axes of a biological structure, wherein the axes are perpendicular. Data are quantified as a function of scan position along the third axis and the quantified data are plotted and displayed to generate a two dimensional representation of the biological structure's morphology. Basically, the disclosure classifies and types biological structures by converting the local sensitive force detector data into a two-dimensional graphical fingerprint, which may be compared to standard data generated from known biological structures. The disclosure of the US 6,357,285 Bl does not relate to the volume of biological structures and will not enable an artisan to determine the volume or changes in the volume of supra- or sub- cellular structures.

[0012] There is a need for quantifying processes of disrupting or restoring cell-cell contacts. The above-mentioned process of wound healing is one example for a naturally occurring processes changing the supra-cellular meshwork (SCM) surrounding cells or cell agglomerations. Another example, which results in the disruption or damaging of the SCM is the invasiveness of malignant tumour cells.

SUMMARY OF THE INVENTION [0013] Coming from this state of the art, it is an object of the present invention to provide the use of a method for quantifying changes in the supra-cellular meshwork.

[0014] Within the context of the present disclosure "wound healing" shall mean an in vivo model of meshwork production by cells, not disclosing tissue sections from animal models or patients, that can be used directly.

[0015] The term "metastasising" shall be understood as the potential of a cell to invade a standardised cell culture model, with or without a supra-cellular meshwork (SCM) or a tissue section taken from an animal model or a patient.

[0016] A "supra-cellular meshwork" (SCM) designates within the context of the present disclosure a layer of fibers secreted by cells and spun around. Thus, the SCM is more or less the extracellular matrix (ECM) in statu nascendi, i. e. the not yet three-dimensional representation of a fiber network on a flat or monolayer cell culture Alternativly the SCM designates a meshwork surrounding cells or celluar agglomerations like cotton. Thus, the quantification according to the disclosure may be performed at two different spatial levels. The growing ECM or the meshwork surrounding cells wiht the direct forming structures. [0017] Changes in the SCM comprise on one hand the disruption of adhesive mechanisms that normally keep cells tethered to their proper neighbours and to the finished extracellular matrix. Said process can be observed for instance when cancer cells invade local tissues or vessels during metastasis. Thus, the present disclosure provides the use of a method to quantify invasiveness of cells, like circulating tumour cells (CTC).

[0018] On the other hand the wording "changes in the SCM" comprises the restoration of integrity of traumatised tissue or cell structures, which can be observed during wound healing or angiogenesis. During such a process, cells establish or restore connections or contacts to their proper neighbours.

[0019] The term "cancer" comprises cancerous diseases or a tumor being treated or prevented that is selected from the group comprising mammary carcinomas, melanoma, skin neoplasms, gastrointestinal tumors, including colon carcinomas, stomach carcinomas, pancreas carcinomas, colon cancer, small intestine cancer, ovarial carcinomas, cervical carcinomas, lung cancer, prostate cancer, kidney cell carcinomas and/or liver metastases.

[0020] The invention provides the use of a method for quantifying and monitoring changes in the supra-cellular meshwork, comprising the steps of a. Preparing in vitro a single cell, cellular monolayer or tissue section;

b. Determining the local deviational volume (LDV) of subcellular or supra-cellular

meshwork structures in a predefined mask in xy-plane;

c. Normalizing the positive or negative volume of the predefined area;

d. Quantifying the local deviational volume (LDV);

e. Analysing the data by comparing them with characteristic topographical elements of a calibrated sample;

f. Evaluating the quantified structural elements to obtain parameter sets.

[0021] It is intended that the analysed elements are larger than one cell, but constitutive parts of it may be smaller, wherein the cell is an isolated cell, a tissue section or a sample of the extracellular matrix from humans or animals, i. e. of eukaryotic or mammalian origin. The use enables the quantification of changes in the supra-cellular meshwork of endothelial or tumour cells, comprising circulating cells. Isolated cells may also be applied to cultured cells to quantify their potential to change the supra-cellular meshwork of a cell layer, i.e. to disrupt or restore supra-cellular structures. Using overlapping scan areas for the analysis enables overviewing a larger area by assembling them. [0022] The parameters sets that are produced during the use of the method of the disclosure may be used to produce an image. It is further intended that the image showing local deviational volumes can be combined with images using fluorescence or raman microscopy for example, or other marker. [0023] For the evaluation of the data obtained during determination of the LDV a neuronal network may be used, wherein the neuronal network comprises preferably at least three layers. For the skilled person in the art it is obvious that any other known algorithm is applicable for the evaluation of the data, even in the step of analysing the data. [0024] The determination of the LDV takes place in a predefined part of the supra-cellular meshwork or cellular surface, wherein the SCM element or cell surface comprises a length of preferably 10 to 500μιη. With respect to the determination of the LDV it is intended that the subdivided part of the SCM comprises a deviational volume in the range of 0.1 to 100 μιη in xy-axis and <500 nm in z-axis.

[0025] The predefined mask in xy-plane for the determination of the LDV may be selected by using optical methods, comprising phase contrast, fluorescence or raman microscopy. Thus, the area for applying the disclosed method is chosen in a further embodiment by optical microscopy before the determination and quantification of topographical elements of the biological surfaces takes place.

[0026] It is intended that the calibrated sample comprises data of biochemical marker, topographical or morphological structures. The data of the calibrated or standard sample are obtained preferably by the optical identification of biochemical marker or morphological structures. Specific patterns are used for the generation of a classification set or a classification matrix that is related to one or more diseases. It is also possible that the data of the calibrated sample are obtained by analysing the interaction of biochemical marker with topographical or morphological structures.

[0027] According to the present disclosure the surface marker comprise biochemical or topographical structures like fibers, knots, secreted extracellular structures or other morphological structures or combinations thereof or specific patterns of such structures. [0028] It is intended that the method according to the disclosure is used to produce a map of topographical elements or for mapping and quantifying of topographical or morphological structures of supra-cellular meshworks being either taken ex vivo as tissue sections or produced in vitro and isolated before applying the method. [0029] The use of the method according to the disclosure is intended for the detection of specific topographical or morphological structures related to diseases, wherein the diseases are chosen from the group of wound healing, tumour, cardiovascular, nephritic, fibrotic, inflammatory, arteriosclerotic or auto-immune diseases. It is obvious for a person skilled in the art that the present invention is not limited to the listed diseases but also applicable to any disease that is accompanied with topographical or morphological changes of the supra- cellular meshwork.

[0030] Within the context of tumour diseases or cancer the invasiveness of a tumour cell can be quantified by the use of a method according to the disclosure. The quantification of the invasive potential of a homogenous cell sample may contribute to predictions about the malignancy of a tumour or the diagnosis of metastasis. The test systems known from the state of the art allow only a yes-or-no result, while the present disclosure provides a test system or an assay to get reliable information about the invasive capacity.

[0031] The use is also suitable for quantifying and monitoring the chemo- sensitivity of cells, tissues or extracellular matrices in terms of quantifying the disruptive or restoring capacity of a substance onto cell-cell contacts or the surpa-cellular meshwork surrounding cells or cellular agglomerations. The use according to the disclosure is further intended for quantifying the barrier function against invasion of extracellular matrices or tissues.

[0032] The method according to the invention is further intended for determining cell surface marker, topographical or morphological structures as diagnostic marker or quantifying and monitoring changes within the supra-cellular meshwork, topographical or morphological structures in the prophylaxis, diagnosis, therapy, follow-up and/or aftercare of a therapy in any of the diseases mentioned above. Besides, the method is suitable and intended for determining mechanical or contractile forces, of supra-cellular meshwork.

[0033] The method according to the invention may be used in the production or screening of a drug for the treatment of any of the diseases mentioned above comprising pharmaceutical compositions, antibodies, proteins, peptides, nucleic acids or chemicals, but is not limited to this substances.

[0034] The method according to the invention is also intended for a cell-culture based classification system in diagnosis. Specific patterns of topographical or morphological structures will be related to disease induced changes of the supra-cellular meshwork or cell surface, so that changes of the SCM can be used for the identification of specific disease patterns. Additionally it is possible to determine the local extension of a disease, if cell or tissue samples from different parts of the body are used as template for the method according to the invention.

[0035] The present invention provides a method to determine the Local Deviational Volume (LDV) of supra-cellular structures irrespective of their biochemical characterisation while disregarding the lack of knowledge about their exact function. The LDV shall define a nanoscale excursion in z-direction (height) over an expected mask in the xy-plane, no matter whether they are linear or not. They use a fuzzy definition of patterning elements including meshwork topolgy. Then, the local protruding or depressed volume as compared to the mean surface level is evaluated. The new method bases on the observation that the meshwork topology changes within a nanometer range in z (height), when cells and their SCM are growing, developing, differentiating or are being stressed or undergo a transformation. Moreover, their physiological function sometimes correlates to the LDV in some respect. [0036] Different stimuli to distinct alterations in SCM. Some examples are: a. Signalling molecules -like for instance TGFbeta- lead to both alteration of cell surface topography and production of extracellular matrix proteins like collagen, fibronectin etc. This influences the assembly of the supra-cellular meshwork (SCM) as a precursor of the final ECM.

b. Virtually all endothelial cells form stress fibers, when challenged, resulting in a markedly structured cytoskeleton, whose force traces exceed single cells at cellular junctions. The quantitation of this intracellular fibers, which transduce forces in supra-cellular patterns would give a stress factor.

c. Tumor cell invasion is coupled to an intensive remodelling of the extracellular matrix via secretion of proteolytic enzymes. The refibrosation of a migratory path/channel leaves the meshwork altered in terms of topology,

d. Endothelial cells are key to the control of leukocyte invasion into an inflamed tissue.

The process of transmigration is not completely understood, but the endothelial role has been underestimated. An important barrier is the basal lamina, produced from endothelial-secreted proteoglycans, e. g. laminins. They form an extracellular meshwork barrier for leukocytes. [0037] When quantitated, the above listed alterations can be used for determination of a cell's or a tissues' status in various kinds of disease models up to the development of diagnostic assays. One advantage of this method is its applicability to cells and supra-cellular arrangements on biomaterials, which are not suited for optical microscopy like metals, minerals or microporous membranes. Successful linking of Bio-AFM to diagnostic procedures has not been reported so far.

[0038] It has to be noted that tumour cells for instance disrupt supra-cellular structures, whereas leukocytes "travel" through cell agglomerations or tissues without damaging the supra- or extra-cellular matrix. So there is a fundamental difference in determining the invasiveness of a tumour cell in comparison to monitor changes caused by a leukocyte in inflammatory processes.

[0039] It is possible to apply the method of the disclosure on known xy-axis data in order to record data for the z-axis or to obtain the local deviation volume (LDV) of a cellular or supra- cellular meshwork.

[0040] The data of the standard sample are obtained preferably by the optical identification of biochemical marker or morphological structures. Specific patterns are used for the generation of a classification set or a classification matrix that is related to one or more diseases.

[0041] The use according to the present disclosure determines changes in structures surrounding the cells as well as the structures forming direct cellular contacts. Thus, the claimed use of the method relates to a layer surrounding the structures forming the direct cellular contacts. Said layer surrounds a cell or an agglomeration of cells like cotton. Consequently, this supra-cellular layer or meshwork will be changed in terms of disruption or restoration before the structures forming the direct cellular contacts will be affected. The ability to determine changes within the supra-cellular meshwork provide a much more sensitive assay and allows the quantification of different states of changes in a wider range. Methods relating to the extra-cellular matrix are not appropriate for such a sensitivity and do only allow a yes-or-no result.

[0042] The disclosure of the US 6,357,285 Bl does not relate to the volume of biological structures and will not enable an artisan to determine the volume of supra- or sub-cellular structures. It was technically simply not possible to determine a local sub- or supra-cellular volume at the time when the US 6,357,285 B l was granted. Consequently the method disclosed in the US document is not suitable to quantify sub- or supra-cellular structures in such a manner that the status of a disease could be predicted or determined, i. e. the determination of the invasive capacity of a tumour cell or the status of wound healing.

[0043] We here open up a possibility to classify and quantify the three-dimensionsional nanoarchitecture of cells as a holistic approach to evaluating a cell's or tissue's biological status.

DETAILED DESCRIPTION OF THE INVENTION

[0044] The invention will be further described by figures and examples without being limited to the described embodiments:

[0045] The invention is based on the experimental results, that the meshwork texture changes on the nanoscale, when cells are developing, are being stressed or undergo a transformation. Using the method according to the disclosure it was for the first time possible to quantify the effect of known stimulating distinct alterations in target cell models or supra- cellular meshwork topography.

[0046] Samples and AFM was performed as follows:

Sample preparation

[0047] Living cells and tissues were subjected to AFM-imaging either directly in growth medium or in physiological HEPES buffer (in contact mode using gold coated standard AFM tips) without further preparation.

Fixed samples

[0048] After time intervals determined by the experimental model and specific question, the cell samples or tissue- sections were fixed with glutardialdehyde (0.05 % to 5 % final concentration for 1-100 min in growth medium under physiological conditions (37°C, 19% 0₂, 5%C0₂) or in buffer at room temperature. If applicable, filter membranes were cut out and subjected to AFM contact imaging in HEPES buffered solution at room temperature (20°C). Images were taken with a Bioscope (Nanoscope Ilia Controller, Digital Instruments, CA, Santa Barbara, USA) using gold-coated MLCT-AUNM tips (spring constant 0,01 N/m) in contact mode. AFM

[0049] To obtain maximal resolution in z-height, the AFM was mounted on a specially designed construction for minimising the ambient mechanical noise. To isolate the setup well from vibration, it was put on an air cushioned table, which in turn bears a platform being suspended on rubber strings. Moreover, the whole construction was shielded by a foam- coated acoustic hood. Additionally, careful grounding of metal parts was performed to reduce electrical noise. Parameters in the software were always optimised for lowest noise and least artefact generation. The noise of the instrumentation using conditions as stated below was measured on atomically flat mica to yield < 0.5 nm of mean roughness. The force exerted on the sample was kept below 5 nN, the scan rates were 0.5-10 Hz/line and digital resolution usually was from 128 2 to 10242 pixels.

[0050] Images were processed using the Nanoscope software, version 5.12b48 which is supplied by the manufacturer (Digital Instruments). Image analysis and presentation was performed with the software SPIP (Scanning probe image processor V 3.3.9, Image Metrology, Lyngby, Denmark).

[0051] Force- volume imaging: To obtain the Young's Modulus (YM) quantifying the stiffness of the samples, 64*64 arrays of force-distance curves were recorded in "force volume" mode which records the deflection of the cantilever (in nm) as a function of piezo elongation (z-distance in nm). Piezo z travel speed was kept below 10 μιη/s. In order to reconstruct the respective maps for height and Young's Modulus (YM), raw data were processed with a routine written for the software "Igor Pro" based on the Hertz' model of elasticity as described in previous studies by M. Radmacher et al. (Science; 1992; 257: 1900).

[0052] The following examples were performed by using the method according to the disclosure:

1. Determination of a "stress factor" [0053] Virtually all cells react to stress when challenged, resulting in a markedly structured cytoskeleton. The actin stress fibers exceed cell borders in transmitting mechanical forces causing supra-cellular organisation of force transmission. The quantitation of the supra- cellular meshwork topology of intracellular fibers would give a supra-cellular "stress factor" in a two-dimensional cell culture. or tissue section

Biopsy isolation and cell cultivation:

[0054] Biopsies are isolated following international medical standards. Cells are cultivated along standard biological protocols applicable for growth of the specific cells. Livings cells are extremely soft - especially at 37 °C when the cellular surface basically is a fluid, as well as the cytosol. Hence, upon minute mechanical loads, they readily deform until a harder structure becomes detectable, which represent polymeric actin bundles or extracellular matrix components like collagen etc. These fibers are quickly reorganised by cells upon physiological or noxious stimuli. The mediators of this pro-fibrotic signaling may be among cytokines, interleukins, growth factors, (peptide) hormones etc. Due to the altered mechanical characteristics, physiological function of the cells or supra-cellular meshwork may be inhibited, eventually indicating a pathophysiological state. Results

[0055] Through morphometrical analysis by the method according to the disclosure, the amount of intra- and extracellular fiber-formation can be quantified. Both types of fibrous structures usually, but not necessarily exceed the single cell borders. Moreover, stiffness measurements (mechanical quantification via determination of Young's Modulus) can give the sum of local effects.

[0056] Cellular and extracellular fibers have been investigated so far by fluorescence or electron microscopy, which usually requiresample preparation and do not yield quantitative results.. Additionally, optically transparent media are needed to grow the cells on.

Determination of wound healing

[0057] Fibroblasts secrete collagen and other ECM -proteins. ALso in viro these proteins can build up supra-cellular fiber meshworks (SCM), a kind of two dimensional representation of what is termed extracellular matrix (ECM) in 3D. AFM is able to quantify the LDV and topology of the fibers, which can be taken as an indirect measure for viability of the connectice tissue. Using this kind of readout, cellular production of ECM can be quantified and classified In a standardised assay, to give an estimate of wound healing capacity of a certain setting.

Cell isolation and cultivation

[0058] The epithelial cell line NRK-52E (being cloned from a mixture of normal rat kidney cells) was received from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany). Cells were propagated and cultured in Dulbecco's Minimum Essential Medium (DMEM) containing 4.5 g/1 D-glucose and 3.7 g/1 NaHC0₃ (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (PAA, Linz, Austria), 2mM L-glutamin (Biochrom, Berlin, Germany) as well as 100 g/ml Penicillin and 100 μg/ml Streptomycin (Biochrom). Cells were routinely passaged once a week (1: 10). All cell cultures were maintained at 37° C in a humidified atmosphere of 5% C0₂ - 95% air. Cells were seeded into 6- and 12-well plates containing 15 mm or 24 mm coverslips which were partially gold-covered (50 nm) for AFM experiments. Cells were grown to confluence on all substrates used and the cell culture medium was exchanged 24 h prior to any experiment.

Results

[0059] Control cells appear like typical epithelial cells do; they form a cobblestone-like layer with high nuclear regions, decorated by microvilli and separated by furrow-type cell borders. After stimulation with cytokine for at least 30h, they not only develop stress fibers and elongate in shape, but they also form punctuate cell borders. These borders eventually protrude above cytosolic level up to 300 nm. This effect is accompanied by an increase in overall cell stiffness of 70%. The protrusions obviously are a counter-regulatory response of the cells to elevated intracellular tension. A quantification of the LDV gives a measure for the degree of transdifferentiation from an epithelial to a mesenchymal state. This measure has been proven sensitive to force-inhibiting agents and hence can report on the tensional status of a cell culture and its physiological barrier function.

Determination of invasive capacity of a cancer cell

[0060] Endothelial cells are key to the control of cancer cells invading a tissue. The process of invasion is not completely understood, but the endothelial role has been underestimated. [0061] The inventors were able to demonstrate that the endothelial cytoskeleton retracts to let pass the cancer cell (Fig. 1, unpublished data). The step is initiated by the secretion of proteases which lead to digestion of SCM fibers. Quantification of LDV of the resulting channels or holes in a standardised assay setup will give an estimate of the invasive capacity or aggressive behaviour of a given cancer cell. This model can be transferred (by a person skilled in the art) to a standardised cell-free substrate model that mimics interstitial tissue (ECM). The invasive capacity of a cancer cell sample can be quantified using the LDV which is cut out of the fibrous ECM. Cell isolation and cultivation

[0062] Human umbilical cords were obtained from normal births. Endothelial cells (HUVEC) were prepared as described. Briefly, veins were treated with collagenase and grown on gelatine coated culture flasks in a humidified chamber at 37°C, 5% C0₂ in M199 Medium (Gibco, purchased through Invitrogen, Karlsruhe, Germany) containing penicilline/streptomycine, heparin and 10% freshly isolated human serum. After reaching confluence, MV3 melanoma cells were seeded on top, left for lh and the sample was fixed. After nanosurgery procedure, the remaining hole or channel digged by the cancer cell into the underlying endothelium was quantified via its local deviational volume.

Results

[0063] After having developed a special kind of AFM-manipulation method ("nanosurgery") to specifically remove adhering cells (Riethmuller 2008, Isac et al. 2010), the method was transferred to cancer cells invading an endothelial layer. Albeit the cellular processes being different, a similar LDV is measurable after removal of the cancer cell quantitation of supra-cellular morphological structures could give at hand a readout parameter for the invasive capacity (permissivity) of an endothelial cell culture. This could help to estimate the pro- or anti-invasive or metastatic potential of pharmaceutical compounds in standardised cell based assays. Analogously, the reduction of fibrous components in a standardised ECM-could give at hand a chemosensitivity test for individual patient samples.

[0064] To test cancer cell invasion MV3 melanoma cells were seeded onto a confluent layer of endothelial cells (HUVEC). After 1 h of coincubation, the samples were fixed. MV3-cells were removed by„nano surgery" and the local deviational volume of the engraved hole or channel was quantified using the method described above.

[0065] Figure 1 A shows a channel or hole in the endothelial cell layer as a leftover after removal of a adherent cancer cell. A representative height profile of a hole is shown below. In another example (Fig. 1 B), a typical channel area of approx. 2 μιη diameter is marked in black (Fig. 1 C) indicating the basis for calculation of the LDV.

[0066] Figure 1 D depicts the quantification of the local deviational volume (LDV) of a digged channel in the case of an non-invasive (A7) versus an invasive (MV3) cancer cell line on primary human endothelium. In the first examples, the channel LDV yielded a mean of 15000 cubic nanometers for MV3 cells as compared to 2800 nm in controls.

[0067] To visualize a supra-cellular meshwork during wound healing, primary mouse fibroblasts were isolated and cultivated for 2-6 days with or without the addition of purified decorin. The local deviational volume (LDV) of the extracellular fibers spun across the cell culture was quantified using the method described above.

[0068] Figure 2 A shows an image of SCM fibers. Primary fibroblasts isolated from mice were cultivated for two days and fixed. The supra-cellular meshwork assembly of mostly collagen fibers are shown. It is possible to quantify the number and diameter of the supra- cellular meshwork fibers.

[0069] Pharmacological intervention as quantified via Specific LDV. In a cellular model of inflammation, the LDV values are quantitated and divided by cell border length to yield the Specific LDV independent of cell size. Inhibition of the cytokine-induced signalling reduced the Specific LDV almost down to control values.

Claims

The use of a method for quantifying and monitoring changes in the supra-cellular meshwork, comprising the steps of

a. Preparing in vitro single cell, cellular monolayer or tissue section;

b. Determining the local deviational volume (LDV) of cellular or

pericelluar structures in a predefined mask in xy-plane; c. Normalizing the positive or negative volume of the predefined area; d. Quantifying the local deviational volume (LDV);

f. Evaluating the quantified structural elements to obtain parameter sets.

The use according to claim 1, wherein the analysis is performed with isolated cells, a tissue sections or a sample of the extracellular matrix from humans or animals.

The use according to claim 1 to 2, wherein the analysed area is larger than one cell, but the structural element in one dimension is much smaller than one cell, wherein the cell is an eukaryotic or a mammalian cell.

The use according to any of the preceding claims, wherein the cells are endothelial or tumour cells, comprising circulating tumour cells.

The use according to any of the preceding claims, wherein a neuronal network is used for the evaluation of the data, wherein the neural network comprises at least three layers.

The use according to any of the preceding claims4, wherein the subdivided part of the cell comprises a length of preferably 10 to 500μιη.

The use according to any of the preceding claims, wherein the supra-cellular topology is parallel organised by knots, comprising a deviational volume in the range of 10 to 500 μιη in xy-axis and <1 μιη in z-axis. The use according to any of the preceding claims, wherein the calibrated sample comprises data of biochemical marker, topographical or morphological structures.

The use according to any of the preceding claims, wherein the cell surface marker, topographical or morphological structures comprise protrusions, depressions, secreted extracellular structures or other morphological structures or combinations thereof or specific patterns of such structures.

The use according to any of the preceding claims, wherein the parameter sets are used to produce a map of topographical elements.

The use according to any of the preceding claims, for quantifying and monitoring wound healing.

The use according to any of the preceding claims for quantifying and monitoring the invasive capacity of a single cell or a cell ensemble or a heterogeneous cell population.

The use according to any of the preceding claims for quantifying and monitoring chemo- sensitivity of cells or tissues.

The use according to any of the preceding claims for quantifying the barrier function of supra-celluar meshwork against invasion of extracellular matrices or tissues.

The use according to any of the preceding claims in the production or for the identification of a drug stimulating or supporting the disruption or restoration of cell- cell contacts or supra-cellular meshworkcomprising pharmaceutical compositions, antibodies, proteins, peptides, nucleic acids or chemicals.

The use according to any of the preceding claims, wherein the predefined mask in xy- plane for the determination of the LDV is determined by optical methods, comprising phase contrast, fluorescence or raman microscopy.