WO2012045716A1 - Verfahren zur diagnose und/oder prognose von krebserkrankungen durch analyse der mechanischen eigenschaften von tumorzellen - Google Patents
Verfahren zur diagnose und/oder prognose von krebserkrankungen durch analyse der mechanischen eigenschaften von tumorzellen Download PDFInfo
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- WO2012045716A1 WO2012045716A1 PCT/EP2011/067271 EP2011067271W WO2012045716A1 WO 2012045716 A1 WO2012045716 A1 WO 2012045716A1 EP 2011067271 W EP2011067271 W EP 2011067271W WO 2012045716 A1 WO2012045716 A1 WO 2012045716A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
Definitions
- the invention relates to a method for the diagnosis and / or prognosis of cancers, for the diagnosis of the origin of tumor cells, for therapy optimization of cancer patients, and for the screening of drugs for oncology in which the mechanical properties of tumor cells and reference cells are analyzed from normal tissue and from Stretching of the cells that results from the entry of a directed mechanical stress, the diagnosis and / or prognosis of cancers is made.
- the invention finds application in research, medicine and pharmacy.
- cancer and cancer is a whole class of diseases that have in common that they form malignant tumors. To date, more than 200 different tumors have been identified. Characteristic of all malignant tumors is the uncontrolled proliferation of cells, the ability to displace healthy tissue (invasion of neighboring tissue) and to be able to form metastases in tissue of the whole body (distant metastases). These three processes are characteristic of the progression of cancer and are used as criteria to classify cancer into cancer stages I, II, III and IV (or A - D) or staging by TNM classification, and thus to testify the aggression of the disease. From stage III (C) it can be determined that the tumor grows beyond its area of origin and displaces the surrounding tissue. From stage IV (D) remote keys are detectable.
- Tumor markers are predominantly proteins or peptides that are detected in the blood or other body fluids of the patient or on the cell surface and their increased concentration indicates a tumor. Since malignant tumor cells develop from mutated cells of normal tissue, the tumor markers are also detectable on cells of normal tissue and are characterized in tumors only by their different frequencies. A variety of different tumor markers have been linked to various cancers brought. Due to their low specificity for tumor cells, these are for the most part unsuitable for diagnostic purposes. Classification in cancer stages based on tumor markers is currently not possible. For the final diagnosis, a pathological examination of tumor sections is currently being carried out. In order to analyze how far a tumor has already progressed, it is necessary with current research methods to search for the tumor metastasis directly, for example, to remove suspicious tissue (eg lymph nodes).
- suspicious tissue eg lymph nodes
- Metastases can only be detected at the time when they have formed sufficiently large cell aggregates so that they are visible in the respective procedure.
- incipient metastasis is difficult to detect.
- actin filaments of the cells which are present in the interphase, are regressed into the mitosis when the cell enters the cell, and actin is then present in a diffuse distribution in the cytoplasm of the cells [Sanger et al. 1975]. It is further assumed that actin is down-regulated in tumor cells [Rao & Cohen 1991]. Just actin in the form of filaments contributes to the stabilization of the cells, so that the higher deformability of cells can be used as a suitable marker for an uncontrolled proliferation of cells. This increased deformability of the tumor cells is observed under mechanical stress, which results in a linear deformation of the cell. In addition to actin, other cytoskeletal elements such as microtubules, intermediate filaments, and their associated cross-linking and motor proteins may also affect cell mechanics.
- US Pat. No. 6,067,859 discloses with the so-called optical stretcher a method by which the deformation of cells under load can be effected.
- a deforming mechanical stress is exerted on the cells to be analyzed by two counter-rotating laser beams, resulting in a viscoelastic deformation of the cell.
- the Tensile stress generated by the laser beams results at the time of loading (application of stress) in an extension of the cell along the major axis of the cell, which is oriented along the laser beams.
- the determined elongation ie the relative change in length of the cell along the stress direction, is greater for tumor cells than for normal tissue.
- the change in length of the cell is optically followed by a microscope, so that the strain can be determined.
- metastasis can currently only be determined at a relatively late stage, namely after the formation of the metastasis, using imaging techniques.
- the object of the invention is to provide a method with which a statement about the risk of tumor metastases and possibly the presence of invasive cells in a patient can be made by analyzing previously unknown mechanical properties of tumor cells.
- a further object of the invention is to provide a screening method which identifies potential drugs for cancer therapy which influence the biomechanical properties of the tumor cells.
- a further object of the invention is to enable therapy tailored to the needs of the patient by targeted drug screening. It is another object of the invention to make conclusions about the origin of a tumor by analyzing the biomechanical properties of tumor cells of a patient.
- the object is achieved according to the invention by a method for the diagnosis and / or prognosis of cancers, which comprises the analysis of the elongation of cells under mechanical stress, wherein the cells are obtainable from a sample of biological material of a patient (hereinafter patient sample).
- a) on each cell a deforming mechanical stress as a mechanical load so exerted that a linear deformation of the cell is to be expected.
- the strain of the cell along the stress direction is determined.
- the proportion of cells in the patient sample that exhibit a strain opposite to the stress direction under mechanical stress is compared with reference data. In this case, a higher proportion of cells which have a strain under mechanical stress which is opposite to the direction of stress indicates a higher risk of tumor metastases in the patient sample than in the reference data.
- the invention is based on the discovery that in the analysis of tumor cells in an optical Strecker entry of a low mechanical stress of 1-2 Pa, a small proportion of cells showed no positive strain in the direction of stress, ie a positive relative change in length, but on the contrary showed a contractile behavior (stretching opposite to the direction of tension).
- a reduction in cell diameter (“cell contraction") was noted, although the intrinsic stress would actually cause an increase in cell diameter, which is an elongation opposite to the direction of stress.
- the frequency of cells which under strain exerted against strained stress under directed mechanical stress was estimated to be about one cell per 100 cells in exemplary tissue tissue samples.
- tumor tissue which have a positive strain under mechanical stress in the optical straightener. These properties indicate that these cells are involved in metastasis.
- a co-culture of cells from normal tissue with unsorted cells from tumor tissue (classification Tlb) showed that the tumor cells during normal culture isolate from the normal tissue and form clusters. The tumor cells thus show an affinity to like cells during growth and thus accumulate during culture. There is hardly any mixing with normal tissue. Especially during metastasis, however, a transition of the tumor cell into tissues of other origin takes place.
- a co-culture of cells from normal tissue with sorted contractile cells from tumor tissue showed that the sorted contractile tumor cells do not form clusters and the mixing with the cells of the normal tissue is maintained during culture.
- the sorted, contractile cells have thus lost their affinity for similar cells (FIG. 2).
- a higher proportion of cells in the sample, which have an elongation opposite to the stress direction under mechanical stress compared to reference data from normal tissue thus means that the analyzed sample contains more cells that can metastasize.
- cells from tumor tissue under mechanical stress in a region in which a linear deformation of the cell is to be expected contain a higher proportion of cells, which has an increased positive strain under tensile load (ie an elongation in the direction of tension ) compared to normal tissue cells and, on the other hand, that the range of elongation is greater than that of normal tissue cells. This was due to the dissolution of the actin filaments of the cytoskeleton of the cells after entry into mitosis.
- This higher extensibility of tumor cells under mechanical stress, which causes a linear deformation of the cell can additionally be used as a measure of the presence of uncontrollably proliferating cells.
- Metastatic cells have an even higher deformability compared to non-metastatic tumor cells.
- the bandwidth of the elongation is also higher (FIG. 3).
- a higher deformability of the cells of the patient sample compared to cells of normal tissue can be used both to determine an increased risk for the presence of uncontrolled proliferating cells and to determine the risk of metastasis. Since the analysis of the elongation of cells of a patient sample under mechanical stress is usually carried out with only one reference sample, the proportion of cells which have an elongation opposite to the stress direction under mechanical stress must be taken into account for a reliable statement about the metastatic risk.
- the method according to the invention preferably additionally comprises determining whether there is a risk of uncontrollably proliferating cells in the sample, wherein
- both a higher proportion of cells which have an elongation opposite to the stress direction under the load and a higher mean value of the elongation in the stress direction in each case in comparison to the reference data, the patient is at high risk for an aggressive tumor, as caused by uncontrolled proliferation and by the presence of tumor cells that can form metastases.
- the mean value of the elongation of the cells which have strain in the stress direction
- a narrower distribution is used when the standard deviation (for normal distribution) or the distance between 25% and 75% quantile (for non-normal distribution) is lower compared to reference data.
- the relaxation behavior of the cell is also analyzed.
- the relative relaxation is determined, which indicates how far the cell in its Initial state (before exerting a mechanical load) returns. The following parameters are determined for the determination of the relative relaxation:
- the relative relaxation R of the cell can be described with the following relationship:
- the relaxation behavior it is advantageously possible to make a statement about the risk of the presence of uncontrolled proliferating cells and the presence of invasive cells. Furthermore, the smaller the mean value of the relative relaxation R of the cells of the patient sample, the higher the risk for tumor metastases.
- cells are viscoelastic objects that can actively change their cell shape.
- deformation and relaxation are mirror-image processes, whereas in the case of purely viscous objects, no relaxation takes place at all.
- a viscoelastic object such as a cell
- cells that are stiffer that is to say have a lower elongation when mechanical stress is introduced
- a weaker relaxation than in cells of normal tissue would be expected.
- tumor cells have exactly the opposite behavior and relax even more than cells of normal tissue, here breast tissue (FIG. 6). It could be shown that Tumor cells relax faster than normal tissue cells, ie, their distribution function shows more negative values, and this effect increases as the cancer progresses (tumor staging).
- metastatic cells have a strain behavior directed against the stress direction (and thus an active deformation behavior of the metastatic cells under voltage entry) is confirmed by the observations of the relaxation behavior of the tumor cells. It could be shown that the progression of a cancer disease increasingly more cells occur, which show such s active deformation behavior. While this property of the cells is only detectable by the determination of the strain under stress, if the cells are already such that they can leave the cell composite and form metastases (tumors from stage 3), then the active deformation behavior of the cells is based on the relaxation already observed in non-metastatic cells (stages 1 and 2). This can be explained by the different force required by the cell for the respective deformation.
- the cell does not have to counteract any additionally introduced tension, as is the case when determining the elongation under stress entry. Therefore, less force is needed by the cell for the deformation process of relaxation than for deformation under stress. As disease progresses, the number of cells exhibiting active deformation behavior increases so that the mean value of relative relaxation continues to shift to negative levels.
- the invention also encompasses a method of diagnosing and / or prognosing cancers to determine relaxation behavior includes analyzing the elongation of cells from a sample of biological material of a patient under mechanical stress, and then stretching the cell in the absence of mechanical stress.
- the method for diagnosis and / or prognosis according to the invention is carried out as follows:
- the expansion L R of the cell after its relaxation is determined at a time t R.
- the relative relaxation R is determined by determining the elongation y (t s ) of the cell under voltage entry at time ts and determining the strain y (t R ) of the cell after relaxation of the cell at time t R and determining the difference of y (t R ) and y (ts) is formed.
- the strain is as defined above and indicates the relative deformation of the cell.
- the relative relaxation is calculated using formula (1).
- the relative relaxation of the cells in the patient sample is compared with reference data.
- An average lower relative relaxation in the patient sample compared to the reference data indicates a higher risk of the presence of uncontrolled proliferating cells.
- a lower number of cells in the patient sample having a relative relaxation greater than 0, as compared to the reference data indicate a greater risk of the presence of uncontrollably proliferating cells.
- a maximum of 1% of the cells of the patient sample having a relative relaxation greater than 0 indicates a higher risk for the presence of invasive cells.
- the mean of the relative relaxation of all cells analyzed is compared to the reference data, and a reduced mean in the patient sample indicates an increased risk for the presence of uncontrolled proliferating cells.
- a mean relative relaxation of all analyzed cells of the patient sample of ⁇ -0.01, preferably -0.05 to -0.01, indicates a higher risk for the presence of uncontrolled proliferating cells.
- the method according to the invention for the diagnosis and / or prognosis of cancers under analysis of the relaxation behavior of cancer cells after mechanical stress alone is sufficient to make a statement about the risk of the presence of a cancer.
- this method is in combination with the determination of the proportion of cells having an elongation under mechanical stress, which is opposite to the direction of stress (fraction of contractile cells), determined.
- Higher levels of cells which under mechanical stress have an elongation opposite to the direction of stress show a higher risk of tumor metastases in the patient sample than in the reference data and on average a lower relative relaxation in the patient sample compared to the reference data at.
- the combination of the two methods advantageously makes a more reliable statement about the risk of tumor metastases possible.
- tumor cells under mechanical stress which is registered by a mechanical stress
- a statement can be made about the metastasis probability and proliferation of the cells as well as the presence of invasive cells.
- the inventors have found that tumor cells exhibit characteristic behavior even under mechanical stress resulting in non-linear deformation of the cells.
- tumor cells in the load area of non-linear deformation exhibit a higher degree of stiffening with increasing mechanical load than the cells of normal tissue. In this loading range (for non-linear deformation), the elongation of the tumor cells under load thus exhibits exactly the opposite tendency, as with loads that cause linear deformation.
- the elongation of the cell in the stress direction under mechanical stress in a range of non-linear deformation is lower than the elongation of the cells of normal tissue and the proportion of cells showing a low elongation under mechanical stress is higher than in the normal tissue. It has been shown that this higher rigidity of the tumor cells under mechanical stress, which results in a non-linear deformation of the cell, are characteristic of cells that are invasive. It is believed that the stiffening of the cells is caused by microtubules and intermediate filaments under mechanical stresses where nonlinear deformation is expected.
- Tumor cells that invasively spread into surrounding tissues unlike metastatic cells are usually not single cells that leave the cell cluster. Rather, the entire cell structure spreads into the surrounding tissue and displaces the surrounding tissue. This can only be done as long as the resistance caused by the cells of the surrounding tissue is lower than the force applied by the growth of the tumor. Since the tumor cells at higher loads have a higher stiffness than non-tumor cells, they can displace or dissolve the cells of the normal tissue, which are more elastic at these loads. In a demonstration experiment, in which tumor cells were cultured on a hydrogel with agarose, it was found that tumor cells in the cell assembly can withstand stresses of up to 10 kPa (FIG. 5).
- the diagnostic and / or prognosis method according to the invention By analyzing the elongation of cells from tissue samples under linear and non-linear deformation, it is thus possible to make a statement about uncontrolled proliferation, invasiveness and metastasis probability. It is advantageously possible by the diagnostic and / or prognosis method according to the invention to characterize tumor cells by analyzing their mechanical properties and not by the analysis of surface markers. This has the advantage that no change in the cell, for example by staining with antibodies or the like. which may affect their vitality and functional condition.
- the analysis of the mechanical properties of the cells in a diagnostic and / or prognosis method according to the invention advantageously makes it possible to classify the cancer into stages I, II, III and IV or into the staging of the TNM classification. It is even conceivable to make a more precise classification than the currently existing classification grid permits, since the risk of metastasis can be determined even before the actual formation of the metastasis from cells of the primary tumor (originating tumor).
- the cells which are analyzed in a diagnostic and / or prognosis method according to the invention, originate from a sample of biological material of a patient. Preference is given to tissue samples from tumors of the patient, which were taken invasive or minimally invasive. For minimally invasive removal, punch biopsy, fine needle biopsy or puncture are preferred. Particularly preferred are tissue samples from primary tumors of the patient, since it is advantageously possible in the analysis of cells from such samples to determine from cells of the primary tumor, if there is a risk of metastasis. For this purpose, cells from a thin slice from a surgically removed tumor of the patient are preferably analyzed in a diagnostic and / or prognosis method according to the invention.
- cells are also preferred in a diagnostic and / or prognostic procedure according to the invention, which have been taken from a patient noninvasively, for example by a swab sample.
- Cells from swab specimens have the advantage that they are already present in isolated form and therefore do not have to be separated with a complicated additional process step.
- Further preferred cells, which are analyzed in a diagnostic and / or prognosis method according to the invention originate from a sample of a body fluid of the patient, in particular a blood sample, a sample from a lumbar puncture or a sample from a thorax drainage. Very particularly preferred are cells from a blood sample.
- a diagnosis and / or a prognosis of cancers can be made.
- the analyzed mechanical properties apply to a variety of cancers. In that sense, the change is this Mechanical properties of tumor cells are not characteristic of a specific cancer, but may be transmitted to the diagnosis and / or prognosis of a variety of cancers, preferably cancers where solid tumors occur.
- a method according to the invention is used for the diagnosis and / or prognosis of breast cancer, oropharyngeal cancer, lung cancer, cervical cancer, skin cancer, stomach cancer, colon cancer or prostate cancer.
- leukemia cells show a higher proportion of cells compared to normal white blood cells, which have an elongation opposite to the direction of stress under mechanical load (linear deformation).
- the method according to the invention is preferably used for the diagnosis and / or prognosis of cancers by analyzing cells from a tumor tissue sample, in particular a primary tumor sample, of a patient and subsequently making a statement about the metastasis probability and possibly invasiveness and / or uncontrolled growth.
- a classification in the context of a diagnosis is advantageously carried out with methods of the invention from samples of tumor tissue. Particularly advantageous in comparison to methods which are known from the prior art, a statement on metastasis can be made from primary tumor samples.
- the diagnosis of cancers with subsequent classification by means of a method according to the invention is a preferred application.
- cells are preferably also analyzed from a non-invasive or minimally invasive sample from an individual.
- a non-invasive or minimally invasive sample from an individual.
- This can be, for example, a swab specimen, punch biopsy or fine needle biopsy or a tissue sample suspected tissue z.
- the analysis is preferably carried out on vital cells. If the sample contains cells that proliferate in an uncontrolled manner, the first sign of cancer is present. This is the case when, under mechanical loads where linear deformation is to be expected, there is a higher mean strain in the direction of the deforming stress of cells of the individual than in the reference data, and possibly differences in the distribution of strain in the Patient sample compared to the reference data.
- the post-stress relaxation behavior of the cell is determined, there is another indication of uncontrolled proliferating cells when there is a lower average of the relative relaxation of the cells in the patient sample compared to the reference data.
- the sample has a higher proportion of cells that are stretched opposite to the direction of stress under mechanical stress (compared to reference data), there is evidence of metastasis in cancer.
- there is a lower mean strain (in the tension direction) in the sample of the individual than in the reference sample in the case of mechanical stresses in which a non-linear deformation of the cells is to be expected there is an indication of the presence of invasive Cells given in case of cancer. These invasive cells allow the tumor to grow against the pressure of the surrounding tissue.
- a first sample of an individual can be used to test for signs of cancer and if cancer is present, how far it has progressed. It is not necessary to analyze cells from a tumor tissue sample, but it can also analyze cells from non-invasive or minimally invasive samples, such as blood samples, puncture samples, or patient swab specimens.
- the diagnostic and / or prognosis method according to the invention always requires a comparison of the elongation of the analyzed tumor cells or of the potential tumor cells with reference data.
- reference data the data of analysis of elongation under stress of cells of a suitable reference sample, which were analyzed in the same way as the cells of the patient sample.
- cells of the same tissue of one or more healthy individuals are preferably used for the diagnosis and / or prognosis.
- cells of healthy tissue of the same patient, which is not affected by the tumor are equally preferably also suitable. For this, especially after the resection of a tumor from a patient, tumor cells and non-tumor cells are separated and analyzed separately.
- tumor cells have differences in biomechanical properties compared to the original tissue from which they originated. Nevertheless, it is the case that the tumor cells in the range of their extensibility closely resemble the tissue of origin. Due to the fact that also cells of the original tissue differ in their extensibility (for example, cells of the lung tissue are more flexible than cells of the breast tissue), the magnitude of the strain (absolute value), which is determined under mechanical stress, can be traced back to the place of origin of the Tumors are taken.
- tumor cells from a sample of biological material of a patient under mechanical stress are analyzed and the mean value of the strain in the direction of stress is determined.
- This mean value is compared with the mean strain (in the tension direction) of reference data, in each case the elongation of cells of different human tissue was determined.
- the likelihood that the tumor cells are derived from a particular source tissue is highest in the dataset (tissue) of the reference data, where the absolute difference between the two means of elongation is lowest.
- the invention also encompasses a method for the diagnosis of the original tissue of tumor cells of a patient, which comprises the analysis of the elongation of tumor cells under mechanical stress, wherein the tumor cells are obtainable from a sample of biological material of a patient.
- the diagnosis of the original tissue of tumor cells is
- a deforming mechanical stress is exerted on one tumor cell as a mechanical load, so that a linear or non-linear deformation of the tumor cell is to be expected.
- the elongation of the tumor cell is determined at the time of mechanical stress.
- the mean strain in the stress direction of the tumor cells is determined and compared with different reference data sets, wherein in each case a reference data set contains the mean value of the elongation in the tension direction of cells of a particular human tissue.
- the tissue of origin of the reference data set in which the difference between the mean values of the elongation between the patient sample and the reference data set is the smallest is assigned as the tissue of origin of the tumor.
- the cells which are analyzed in a method according to the invention for the diagnosis of the tissue origin, are obtained from a sample of biological material of a patient and isolated by known methods and separated.
- the sample is a non-minimally or minimally invasive sample of the patient and not a tissue sample of the primary tumor.
- the tumor cells are preferably obtained from a sample of a body fluid of a patient, more preferably from a blood sample, a sample from a lumbar puncture or a sample from a thorax drainage.
- the tissue of origin of circulating tumor cells from a blood sample of a patient is determined with this method according to the invention.
- the reference data sets with which the expansion of the circulating tumor cells is adjusted are preferably identical to the reference data of a diagnosis and / or prognosis method according to the invention. Since the site of origin of the tumor cells from the patient sample must first be determined, different reference data sets are compared with the strain of the tumor cells from the patient sample determined in the method.
- a reference data set contains results of the strain analysis of a defined human tissue, particularly preferably of normal human tissue.
- data sets are used as reference data, each containing results of the strain analysis of defined human tumor tissue.
- data of at least Two reference patients are included and the values of elongation are averaged over the different reference patient samples.
- the invention also includes a method for screening substances as potential drugs for oncology (screening method), in which the influence of the substances on biomechanical properties of tumor cells is examined by analyzing the elongation of a plurality of tumor cells under mechanical stress.
- a deforming mechanical stress is exerted on the tumor cell as a mechanical load in such a way that the tumor cell is deformed linearly or non-linearly.
- the elongation of the tumor cell is determined at the time of exercise.
- the elongation of the tumor cells contacted with the substance is compared with reference data of the analysis of the elongation of similar untreated tumor cells.
- the substance is then classified as a potential agent for oncology, if in mechanical stress, in which a linear deformation of the cells is to be expected, the proportion of tumor cells that have an elongation under mechanical stress, which is opposite to the direction of stress, with the the substance contacted tumor cells compared to the untreated tumor cells is lower.
- the substance is classified as a potential agent for oncology, when, under mechanical loads where linear deformation of the cell is to be expected, the mean strain in the stress direction of the substance contacted tumor cells is lower compared to the untreated tumor cells and / or
- the mean of the elongation of the substance contacted tumor cells is higher compared to the untreated tumor cells.
- Substances that are analyzed in a screening method of the invention include molecules that are of natural or synthetic origin.
- Preferred substances which are analyzed in a screening method according to the invention are selected from natural molecules, in particular peptides, proteins, nucleic acids, in particular siRNA, synthetic organic molecules, in particular monomers, polymers, synthetic inorganic molecules and small molecules.
- tumor cells are examined, which either originate from a tissue sample of a patient or originate from a cell line.
- tumor cells from cell lines are used in a screening method according to the invention because of their ease of culturing and availability.
- the reference data used here is the analysis of the elongation of similar cells which were not contacted with the substance but were otherwise handled identically (herein also "untreated” cells) .
- untreated cells the analysis of the elongation of untreated tumor cells serves as a reference for the respective cell line
- tissue samples preferably tumor tissue samples
- untreated cells from the same tissue sample are analyzed as reference.
- the data are also compared with normal reference data.
- Non-tumor cells of the same type for which the strain is determined according to the same principle as for the tumor cells, serve to collect the data of the normal reference.
- the screening method according to the invention is carried out with tumor cell lines, cell lines which do not contain tumor cells are used as the normal reference.
- the method according to the invention is carried out with tumor cells from tissue samples, it is preferable to use cells of the same tissue as the normal reference, but these cells are not degenerated into tumor cells, or alternatively cells of a healthy individual from the same tissue (for example, the screening method is performed on breast cancer cells) as normal reference either non-tumor cells of the breast tissue of the same patient or cells from breast tissue of a healthy individual are used).
- the mechanical properties of the tumor cells by contacting with the Substance in the direction of the properties of the normal reference changed, so this is an indication that the substance is a potential drug for oncology.
- the screening method according to the invention is based on the effect that the elongation of cells with linear or non-linear deformation can be used as a criterion for the aggressiveness of tumor cells.
- the strain of the cell is determined under mechanical stress, wherein the stress is exerted on the cell by a deforming mechanical stress and thereby the cell is deformed.
- a mechanical stress is exerted on the tumor cells in such a way that a linear deformation of the cell is to be expected.
- a mechanical stress is exerted such that a non-linear deformation of the cell is to be expected.
- the tumor cells both the strain under mechanical stress, in which a linear deformation is to be expected and the strain under a mechanical load, in which a non-linear deformation is expected to be determined.
- the elongation under mechanical stress is determined by the same tumor cell.
- the strain of the untreated tumor cell under load is determined.
- a method must be used in which the tumor cell is vital even after the determination of the strain.
- an optical Strecker is suitable.
- the tumor cell is contacted with the substance and then determined the elongation of the contacted with the substance tumor cell.
- the influence of the substance on the mechanical properties of the tumor cells can be determined particularly well, since identical cells are analyzed before and after contact with the substance.
- a suitable active ingredient for the cancer therapy of a single patient personalized therapy.
- tumor cells from a sample of biological material of a patient are contacted with different active substances and the active substance selected for the therapy, which has the greatest influence on the biomechanical properties of the patient cells of all investigated active substances, and preferably modifies these in such a way that the progression of the disease is counteracted becomes.
- This can be done by preventing the ability to divide or migrate, by killing the cells, or by reconstituting the biomechanical properties of the cell to be similar to non-tumor cells.
- active substances are preferably suitable which cause the elongation of the cell (under load in the region of linear deformation) to be lowered so that cell division is no longer possible.
- active substances which selectively kill tumor cells having a higher mean strain in the direction of tension (as compared to similar non-tumor cells) or which selectively kill only the tumor cells exhibiting strain opposite to the stress direction (when loaded in the linear deformation region) or those of the stress prevent inward stretching and thus inhibit metastasis.
- the invention also encompasses a method for optimizing the therapy of a patient with a cancer, in which the influence of different active substances on the biomechanical properties of tumor cells of the patient is examined.
- a large number of tumor cells of the patient is analyzed under mechanical stress, wherein
- a mechanical stress is applied as a mechanical load so that the tumor cell is deformed linearly or non-linearly and the elongation of the tumor cell is determined at the time of exposure.
- That active ingredient for the therapy in which the sample with tumor cells after contacting with the drug compared to untreated tumor cells a) under mechanical stress, in which a linear deformation of the tumor cell is expected, the lowest proportion of tumor cells having an elongation opposite to the direction of stress, and / or
- b) has the lowest average of the expansion of the tumor cells in the stress direction under mechanical loads, in which a linear deformation of the tumor cell is to be expected, and / or
- c) has the highest average of the elongation of the tumor cells under mechanical stress, in which a non-linear deformation of the cell is to be expected.
- a suitable active ingredient selected is an active ingredient which meets at least one of the criteria a) to c), preferably at least two of the criteria a) to c), ideally all three criteria.
- Tumor cells of the patient which are used in a method according to the invention for optimizing therapy can be obtained in the same manner as the samples with Patient cells that are used in a method according to the invention for diagnosis and / or prognosis. They preferably originate from a tumor tissue sample of the patient, which was taken invasively or minimally invasively. Preferably, cells of the same sample are first analyzed by a diagnostic and / or prognosis method according to the invention and then used in a method according to the invention for optimizing therapy. In this way, firstly the disease can be classified and based on the patient cells of the same sample a targeted drug screening for the respective patient can be made.
- tumor cells are preferably used from the same patient sample, but in any case tumor cells of the same patient.
- the cells in one of the methods according to the invention are preferably provided individually, so that in each case a mechanical load is exerted on a cell and the elongation under load is determined.
- the mechanical properties of single cells are analyzed.
- a multiplicity of cells preferably at least 10, in particular at least 100 cells, are analyzed in one of the methods according to the invention and from this the mean value and, if applicable, the distribution of the strain in the direction of stress and the proportion of cells which have an elongation opposite to the direction of stress and, if appropriate, the proportion of cells having an elongation in the direction of stress is calculated.
- elongation at linear and non-linear strain is determined by applying mechanical stress to the cell at a time such that linear deformation of the cell is expected and at a different time a mechanical load is exerted, so that a non-linear deformation of the cell is to be expected.
- mechanical stress to the cell at a time such that linear deformation of the cell is expected and at a different time a mechanical load is exerted, so that a non-linear deformation of the cell is to be expected.
- the strain of the examined cell is determined at each applied voltage.
- the elongation is in mechanics an indication of the relative change in length, ie the extension or shortening of a body, in this case the cell under investigation, under load.
- the strain gives the ratio of the change in length under load to the original length (along the direction of the deforming stress), ie it is the quotient of the change in length and the original length of the cell.
- the strain is thus a dimensionless size.
- the strain in the direction of stress has a positive sign (positive stretching in the direction of stress, eg when the cell is stretched under tension)
- the strain in the stress direction has a negative sign and is thus opposite to the stress direction This can be referred to as negative strain in the stress direction or as positive strain opposite to the stress direction.
- the elongation of the cell is determined in the inventive method at the time of mechanical stress and in the direction of the load.
- the cell diameter is preferably determined under mechanical load in the direction of the load, compared with the cell diameter before loading and calculated from the elongation.
- all other parameters of the cell which can be used as a measure for a change in size, in particular the cross-sectional area of the cell, the eccentricity, the ratio of large to small main axis, the Taylor deformation parameter or the second moment are also suitable for determining the elongation and higher moments of cell shape.
- Elongation is determined for each individual cell at the time of a given mechanical stress. In the process, several cells are successively analyzed.
- the determination of the strain "at the time of loading” in the sense of the invention is to be understood as meaning that an elongation can also be determined after the end of the mechanical stress, since the cell does not relax immediately after the end of the stress to determine the strain at the actual time of stress.
- a mechanical stress is exerted in each of the methods according to the invention, which results in a deformation of the cell (deforming mechanical stress).
- This term is used herein analogously to its definition in mechanics and refers to the force acting per unit area in an imaginary sectional area on a body, in the present case on the cell.
- the mechanical stress exerted on the cell results in a deformation of the cell, preferably a compressive stress, in particular a tensile stress or a compressive stress is applied and / or a shear stress, in particular a shear stress.
- the deformation of the cell takes place in such a way that it acts in the direction of the tension. So if a tensile stress is applied, the cell expands in the direction of the tensile stress (positive strain), however, if a compressive stress is applied, the cell contracts in the direction of the compressive stress (negative strain).
- a tensile stress is preferably applied. If the cell increases along the tension (in this case: positive strain of the cell), the strain acts in Stress direction. When the cell is reduced (contraction of the cell, in this case: negative stretching of the cell), the strain is opposite to the direction of stress.
- Some cells of the tumor tissue do not expand in the direction of the mechanical stress exerted on the cell, but exhibit an elongation opposite to the stress direction. In such a manner, the cells contract or expand upon application of a tensile stress when compressive stress is applied to the cells. This characteristic is characteristic of tumor cells that are tissue-specific and can form metastases.
- the amplitude of the deformation of the cell depends on the applied load, i. H. the elongation of the cell depends on the mechanical stress applied to the cell.
- the voltage required for deformation, between different cell types and depending on the method used, with which the voltage is applied to the cells vary over many orders of magnitude from a few Pa to 100,000 Pa.
- Cells are complex biological and mechanical objects.
- the deformation of cells under mechanical stress is not purely elastic, but at least viscoelastic.
- a cell after completion of the applied mechanical stress, a cell only incompletely relaxes or requires at least a very long time for a complete relaxation (return to the original state).
- the strain is proportional to the stress.
- the area in which a linear deformation of cells is to be expected differs between cells of different origins.
- the stress that is suitable for cells of a particular tissue type to effect linear deformation of the cell can be readily determined by determining the elongation of the cells of the particular tissue type at a variety of different mechanical stresses, and linear range is read in a stress-strain diagram.
- a linear strain can be expected at a stress of preferably at most 5 Pa in the cells which are analyzed by methods according to the invention .
- a mechanical stress of 1 Pa to 5 Pa, preferably 1 Pa to 4 Pa, in particular 2 Pa to 3 Pa is exerted as a load.
- the strain of the cell is no longer proportional to the voltage. In this range, a non-linear deformation of the cell is detected as a function of the applied voltage.
- cells are analyzed in a diagnostic and / or prognosis method according to the invention in order to make a statement about the presence of invasive cells or in screening and screening methods according to the invention Therapy optimization method to identify a suitable substance or agent that influences this behavior. Again, it depends on the cell type, which mechanical stress must be exerted to effect a non-linear deformation of the cell. This range can also be read in the stress-strain diagram described above.
- a mechanical stress of 10 Pa to 50 kPa is applied to a cell.
- a mechanical stress in the range of more than 100 Pa to 50 kPa, more preferably between 5 kPa and 50 kPa is exercised.
- a mechanical stress is exerted on the cells, so that they experience a deformation.
- the mechanical stress is exerted on the cells by forces from electromagnetic radiation, which is preferably generated by the impingement of two counter-rotating laser beams on a single cell. This is preferably done according to the principle of the optical extender.
- the determination of the elongation is carried out by preferably optical methods with which a change in size of the cell can be detected.
- the determination of the elongation is preferably carried out with a microscope, the cell diameter being determined before and during the load (ie before and during the application of the mechanical stress) along the direction in which the deformation is applied and from this the elongation is calculated.
- the analysis of the mechanical properties of the cell is carried out with an optical stretcher.
- An analysis in the optical straightener or an input of mechanical stress by electromagnetic radiation, preferably laser radiation (which is not significantly absorbed by the cells) is particularly advantageous because no damage to the cell is caused and this can be further cultured after the determination of the strain.
- the cell can then be recovered after analysis and be available almost unaltered for further analysis or culture. This is not possible in this form by previous methods in which the surface properties of the cell is determined, for example, by staining with antibodies, since it is uncertain to what extent bound antibodies can influence the reactions of the cells.
- the method is determined by means of atomic force microscopy, dielectrophoretic forces, in particular by means of dielectrophoretic cages, microfluidic flows, optical tweezers, laser diode bars or ultrasound microscopy, of which atomic force microscopy is particularly preferred.
- the stress is applied to the cells by the respective devices, and the strain is determined by preferably optical methods. In principle, all methods which are suitable for exerting a mechanical stress on individual cells and for determining the elongation of the cell under load can be used to analyze the elongation in methods according to the invention.
- the invention also includes the use of devices that are suitable for a preferably isolated cell to exert a mechanical stress as a mechanical load and to determine the strain of the cell at the time of stress
- the devices are used in one of the methods of the invention.
- a particularly suitable and preferred device is the optical straightener.
- a further preferred device is an atomic force microscope.
- the following devices are preferred for use in the present invention: a dielectrophoretic field cage, a device for analyzing cell deformation in microfluidic flows, optical tweezers including diode laser bars, and / or an ultrasound microscope.
- the cell can be completely uncontaminated after the analysis of the mechanical properties for further analysis or for cultivation.
- the analysis of the mechanical properties of the cells has a considerable cost advantage over the use of molecular cell markers (surface markers, in particular tumor markers) or marker sets.
- the cell markers are very different for each type of cancer, so that for all different diseases, a separate marker set is used that determines a specific "signature" on molecular markers.However, it is not certain that with the selected set of molecular markers also every tumor detected However, because of the diversity that results from the mutations in the tumor cells, there may also be deviations in this signature, but the mechanical properties of the cells used in a method of prognosis or diagnosis according to the invention are for various cancers reproducible.
- the change in strain of the tumor cell which is analyzed in a method according to the invention, is based on the determination of changes in the cytoskeleton of the cell. Such changes can be detected with known devices with high sensitivity.
- a particular advantage of the method according to the invention for the prognosis and / or diagnosis of cancer is that the risk for the occurrence of tumor metastases can already be determined at a comparatively early point in time, for example from cells of the primary tumor.
- the risk for the occurrence of tumor metastases can be determined here before the formation of metastases or before the appearance of new tumors in distant tissue. Accordingly, the treatment of the patient can be adjusted by this information.
- the therapy can already be aligned with knowledge of the risk for metastases, without the metastases are already detectable in an imaging process or were detected.
- a decisive advantage of the diagnostic and / or prognosis method according to the invention and the method for determining the origin of the tumor is that it is already possible to make a statement about whether a sample of a body fluid, in particular a blood sample, of a patient Cancer is present and in which tissue the original tumor is located. In this way it is possible by a relatively simple and inexpensive test, for. For example, by analyzing a blood sample to obtain significantly more information about a cancer than is possible with current blood tests. For the detection of the origin of the tumor imaging methods are necessary according to the current state of the art. With a method according to the invention for therapy optimization of a patient, it is advantageously possible to individually select a suitable active substance for each individual patient.
- a screening method according to the invention is suitable for this purpose.
- Fig. 1 Expansion of cells from primary breast tumors (black bars) and healthy cells from breast reduction (white bars). The analysis was carried out with the optical straightener at a mechanical tension of 2 Pa. The black bars on the left are from a tumor with the classification T4. The black bars on the right are from a tumor with the classification Tlb. The double arrow shows the border between cells with negative and positive elongation. All cells were measured in HuMEC Ready Medium (Invitrogen).
- Fig. 2 Fluorescence microscopic analysis of sorted contractile cells from tumor tissue of cervical cancer in droplet culture. Contractile cells do not form their own cell structure after co-culture with cells from non-germ cells. Cultured cervix cells (T lb classification) and cervical cells a) at culture start and b) after 24 h culture (comparative example) c) co-culture of cervical contractor cervical tumor cells (classification T3b and T4) with cervical cells after 24 h culture. The staining was done with Invitrogen's CellTracker TM Red and Green Fluorescent Dye.
- Fig. 3 Elongation of cells of the cell line MCF10 (non-cancerous mammary epithelial cells), MCF7 (non-metastatic tumor cells) and modMCF7 (metastatic tumor cells) determined in the optical straightener at a mechanical tension of 5 Pa (linear deformation of the cells). Tumor cells have a higher elongation than cells from normal tissue. A further increase in stretch is noted in metastatic cells.
- Fig. 4 Compression of tumor cells determined by scanning force microscopy (SFM).
- SFM scanning force microscopy
- MCF 10A non-cancerous mammary epithelial cells, comparative example
- MCF7 non-metastatic cancer cells
- MDA-MB-231 metalastatic cancer cells.
- tumor cells MCF7 and MDA-MB-231 have a higher compression.
- the metastatic MDA-MB-231 show the highest compressibility.
- Fig. 5 Growth of tumor cells (MCF7) as a spheroid in a hydrogel of cell culture medium containing 1% agarose with a stiffness of 1 - 2 kPa in culture after a) 2 days, b) 4 days, c) 14 days and d) 18 days ,
- the spheroids displace the hydrogel and grow to a stiffness of 6 - 10 kPa.
- Fig. 6 Relative relaxation of tumor cells from primary breast tumors (BT 1 to 4) and cells from normal tissue (Nl, N2).
- the analyzed tumor cells originate from the following cancer stages: BT1 ... stage I, BT2 ... stage II, BT3 and BT4 ... stage III.
- Example 1 Analysis of the elongation of tumor cells at low loads shows the appearance of cells having an elongation opposite to the direction of stress
- FIG. 1a The elongation of cells from malignant human breast tumors (FIG. 1a, black bars, left), which originate from tissue samples from patients with a classification T4 (distant metastases), was analyzed in an optical stretcher.
- the cells were processed by conventional methods and isolated. Subsequently, the cells were analyzed in an optical straightener at a voltage of 2 Pa. With this mechanical stress, a linear deformation of the cells is to be expected in the optical straightener.
- Figure la white bars, comparative example with cells of a healthy individual.
- cells from malignant human breast tumors were analyzed from tissue samples of patients with a classification T lb. There are no metastases at this stage (Fig. La, black bars, each on the right).
- Fig. 1 shows the distribution of elongation in the analyzed samples. Only in human tissue specimens from breast cancer patients classified T4, in which metastases are present, a proportion of cells was found, which had an elongation opposite to the direction of tension ("contractile cells") In the direction of the registered mechanical tension, the diameter of these cells decreases under load (Negative Elongation) About one in 100 cells of the tissue samples of classification T4 shows this behavior.
- exemplary embodiment 2 was carried out.
- a cell culture experiment was used to investigate whether tumor cells exhibiting contractile behavior in the optical straightener under mechanical stress of 2 Pa (range of linear deformation) differ in the biological behavior of tumor cells which exhibit a positive strain under mechanical stress.
- the cells from the following different tissue samples were examined: as normal tissue cells of healthy cervical tissue of the patient were provided. These were co-cultured with either cervical tumor cells classified as Tlb (clinically identifiable lesions confined to the uterine cervix) or with contractile cells derived from cervical T3b (infiltrating the lower third of the vagina and / or pelvic wall) and T4 ( Distant metastases) cultivated in droplet culture.
- the contractile cells were obtained from a sorting process in which the cells were each deformed using the optical extensor and the cells were sorted, which showed a negative strain under mechanical stress of 2 Pa. These cells were used for co-culture with normal tissue cells.
- the cells used were separated before culture. For co-culture, the same number of cells from normal tissue were mixed with the respective cells from tumor tissue and cultured in droplet culture in HuMEC Ready Medium (Invitrogen) for 24 h.
- Fig. 2a shows the state of the cells before the start of cultivation.
- the tumor cells assemble with similar cells, namely the other tumor cells, into a cell cluster. A mixture with cells of the normal tissue takes place even in the margins around the cell structure only to a small extent.
- the cells from the co-culture of the sorted contractile cells from cervix tumors of the classification T3b and T4 with cells of the normal tissue show a different picture. After 24 hours of culture under identical culture conditions, no formation of cell aggregates from tumor cells and normal tissue cells was observed.
- the tumor cells which show a stress-directed strain behavior under mechanical stress, also behave differently in cell culture from the tumor cells, which show an elongation in the direction of stress under mechanical stress.
- the changes in the cytoskeleton are thus associated with such a biological change of the cells, so that they lose the affinity for similar tissue and in homogeneous mixing with other types of cells (here the cells of the normal tissue) are present.
- Metastatic cells show exactly this property of being able to leave the cell structure of the tumor and infiltrate other types of tissue. According to Embodiment 1, cells whose elongation under mechanical stress is directed opposite to the load are detected only in tissue samples from patients with metastasis. Thus, the presence of cells having these biomechanical properties in a tissue sample may indicate that there is an increased risk of tumor metastasis.
- Example 3 The deformability of tumor cells is associated with their aggressiveness.
- FIG. 3 shows this on the basis of a histogram which shows the elongation of the analyzed cells when examined according to the principle of the optical extender.
- the cell line MCF 10 Three cell populations from cell lines were examined: as normal tissue cells of the cell line MCF 10 were analyzed, a cell line of human non-cancerous mammary epithelial cells.
- the cell line was obtained from the breast tissue of a 36-year-old woman with mastopathy.
- the tumor cell line used was the cell line MCF7, an adenocarcinoma cell line that was produced from cells of a 69-year-old breast cancer patient. These cells are a model for non-metastatic and non-invasive cells.
- TPA-added MCF7 cells are referred to herein as "modMCF7" cells and have served as a model cell line for metastatic cells.
- the elongation was determined on the principle of the optical extender at a tension of 5 Pa (FIG. 3).
- MCF10 cells show on average the lowest strain in the stress direction.
- MCF7 cells show on average a higher strain in the direction of stress compared to MCF 10 cells.
- the elongation of the single cells fluctuates more, so that the standard deviation of elongation is higher in MCF7 cells compared to the MCF10 cells.
- Analysis of metastatic modMCF7 cells shows that elongation is higher on average compared to both MCFIO and MCF7 cells. The standard deviation is even higher compared to unmodified, non-metastatic MCF7 cells [Guck et al. 2005].
- MCF 10A cells normal tissue, ATCC-LGC-Promochem, Germany
- MCF7 cells non-metastatic, non-invasive tumor tissue, ATCC-LGC-Promochem, Germany
- metastatic cells the cell line MDA-MB-23 1 (ATCC-LGC-Promochem, Germany) derived from a metastatic tumor was analyzed.
- Fig. 4 shows that the cells exhibit different elongation properties when linearly deformed.
- MCF10A cells show the lowest compression whereas the tumor cells show significantly higher compression values.
- the metastatic MDA-MB-231 cells show a significantly higher compression compared to non-metastatic MCF7 cells.
- Example 5 Relaxation behavior of tumor cells from primary breast tumors and cells from normal tissue
- the relaxation behavior of cells from malignant human breast tumors was analyzed and compared with the relaxation behavior of cells from a reference sample (cells from human breast tissue samples from breast reduction).
- the cells were analyzed occasionally at 800 mW in the optical Strecker, wherein a voltage was applied for 2 seconds and the cell was observed for a further 2 seconds.
- a linear deformation of the cells is to be expected in the optical straightener.
- Nl primary breast epithelial cells from breast reduction (Invitrogen, HMEC cells)
- N2 primary breast epithelial cells from breast reduction (promo cell, HMEpC cells)
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CN105738254A (zh) * | 2016-02-03 | 2016-07-06 | 苏州大学 | 一种力学生物学耦合测试系统及方法 |
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DE102012211735B4 (de) | 2012-07-05 | 2015-04-09 | Universität Leipzig | Vorrichtung und Verfahren zur Charakterisierung von Zellen und deren Verwendung |
EP3201635B1 (de) * | 2014-10-03 | 2024-02-07 | Universität Basel | Verfahren, um das fortschreiten des krebs durch nanomechanische profilierung vorherzusehen |
AU2017219901B2 (en) * | 2016-02-19 | 2022-07-14 | Nant Holdings Ip, Llc | Methods of immunogenic modulation |
PL237087B1 (pl) | 2017-10-25 | 2021-03-08 | Univ Medyczny Im Piastow Slaskich We Wroclawiu | Sposób diagnozowania nowotworów układu chłonnego |
US20210247386A1 (en) * | 2018-04-26 | 2021-08-12 | Technion Research & Development Foundation Limited | A device and method for determining cell indentation activity |
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CN104758058A (zh) * | 2015-03-11 | 2015-07-08 | 苏州大学 | 一种血细胞机械应力形变脉冲激光同步显微成像观测装置 |
CN105738254A (zh) * | 2016-02-03 | 2016-07-06 | 苏州大学 | 一种力学生物学耦合测试系统及方法 |
CN105738254B (zh) * | 2016-02-03 | 2019-07-12 | 苏州大学 | 一种力学生物学耦合测试系统及方法 |
DE102017201252A1 (de) | 2017-01-26 | 2018-07-26 | Universität Ulm | Verfahren und Vorrichtung zur Untersuchung von Zellen |
WO2018138279A1 (de) | 2017-01-26 | 2018-08-02 | Universität Ulm | Verfahren und vorrichtung zur untersuchung von zellen |
US11788950B2 (en) | 2017-01-26 | 2023-10-17 | Universität Ulm | Method and device for analysing cells |
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JP2013541338A (ja) | 2013-11-14 |
DE102010041912B3 (de) | 2012-04-12 |
CA2813653A1 (en) | 2012-04-12 |
US20130316390A1 (en) | 2013-11-28 |
EP2624742A1 (de) | 2013-08-14 |
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