Patent Application: US-201514678156-A

Abstract:
in a method for diagnosing and / or prognosis of cancers , diagnosing origin of tumor cells , optimizing cancer therapy , and screening active substances for oncology , the mechanical properties of tumor cells and reference cells are analyzed under mechanical load that causes linear or non - linear deformation of the loaded cell . the engineering strain of the cells caused by directed mechanical stress being introduced is used to determine metastasis risk and the presence of uncontrollably proliferating and / or invasive cells , or the origin of the tumor . the metastasis risk is determined based on the proportion of cells in the sample exhibiting engineering strain in a direction opposite to the stressing direction . the risk of the presence of uncontrollably proliferating cells for non - linear deformation of the cell is determined in the sample based on the mean value of the engineering strain in the direction of cell stressing .

Description:
analysis of the engineering strain of tumor cells at low loads shows the appearance of cells exhibiting engineering strain in a direction opposite to the direction of stressing . in an optical stretcher , the engineering strain of cells from malignant human breast tumors ( fig1 a , black bars respectively on the left ) were analyzed , which originate from tissue samples from patients with a t4 - classification ( distant metastases ). the cells were processed and individualized using conventional methods . subsequently , the cells were analyzed in an optical stretcher at a stress of 2 pa . at this mechanical stress , linear deformation of the cells in the optical stretcher is to be expected . as a reference sample , cells from human breast tissue samples were analyzed from breast reductions individualized in the optical stretcher at a stress of 2 pa ( fig1 a , white bars , reference example with cells from a healthy individual ). furthermore , cells were analyzed from malignant human breast tumors from tissue samples of patients with a t1b - classification . at this stage , there are no metastases ( fig1 a , black bars respectively on the right ). fig1 shows the distribution of engineering strain in the samples analyzed . only in human tissue samples from breast cancer patients with t4 - classification in which there are metastases was a proportion of cells detected exhibiting engineering strain in a direction opposite to the direction of stressing (“ contractile cells ”). in the direction of the mechanical tension stress introduced , the diameter of these cells decreases under load application ( negative engineering strain ). about one in 100 cells from the tissue samples of the t4 - classification exhibits this behavior . in normal tissue , the proportion of contractile cells was below the detection limit . this also applies to cells from human tissue samples from breast cancer patients with t1b - classification . the unexpected mechanical behavior of the tumor cells , that they exhibit engineering strain under a mechanical load being in a direction opposite to the direction of stressing , is detected only in tissue samples from patients with metastases . contrary to non - metastatic tumor cells , these ( metastatic ) cells are able to leave the cell structure of the tumor tissue and pass into tissues of other origin . it is believed that certain properties of the cytoskeleton of the metastatic cells is altered in a manner that they are able to overcome the surface stress effects which normally prevent tissue cells from leaving the original cell structure ( tissue ). apparently these changes in the cytoskeleton , which are present in metastatic in contrast to non - metastatic tumor cells , cause a change of the biomechanical properties to the extent , that these cells under introduced mechanical stress respond in a totally unexpected manner and show engineering strain that is not in the direction of stressing , but is directed oppositely . embodiment 2 was conducted as evidence for the fact that tumor cells , which under a mechanical load exhibit engineering strain in a direction opposite to the direction of stressing , exhibit biological properties than differ from other tumor cells ( which in an optical stretcher exhibit engineering strain in the direction of stressing ). it was examined on the basis of a cell culture experiment whether tumor cells , which in the optical stretcher under a mechanical load of 2 pa ( range of linear deformation ) exhibit contractile behavior , in the biological behavior differ from tumor cells , which under a mechanical load exhibit positive engineering strain . for this , the cells of the following different tissue samples were examined : cells from healthy tissue of the patient &# 39 ; s cervix were provided as normal tissue . they were in co - culture grown in droplet culture with either cells from cervical tumors of t 1b - classification ( clinically apparent lesions limited to the cervix uteri ) or with contractile cells from cervical tumors of t3b - classification ( infestation of the lower third of the vagina and / or the pelvis wall ), and t4 ( distant metastases ). contractile cells were obtained from a sorting , in which the cells were deformed respectively using the optical stretcher and those cells were sorted which under mechanical stress of 2 pa exhibited negative engineering strain . these cells were used for co - culturing with cells of normal tissue . the cells used were individualized from the culture . for the co - culture , the same number of cells from normal tissue was respectively mixed with the respective cells from tumor tissue and grown in droplet culture in humec ready medium ( invitrogen ) for 24 h . for this , the cells were stained prior to cultivation using red and green fluorescent dyes of the celltracker ™ series from invitrogen and analyzed by fluorescence microscopy . fig2 a shows the state of the cells prior to the start of culturing . formation of a cell structure of the tumor cells ( at the center ) was observed between the tumor cells from patients with t1b - classification and the cells of normal tissue ( fig2 b ). the tumor cells cluster together with similar cells , namely , the other tumor cells , into one cell cluster . any mixing with cells of the normal tissue , even in the peripheral areas of the cell structure , occurs only to a small degree . the cells from the co - culture of the sorted contractile cells from cervical tumors with t3b - and t4 - classification and the normal tissue cells present a different image . after 24 hours of culturing under the same growing conditions , no formation of any cell clusters of tumor cells and cells of normal tissue was observed . the tumor cells , which under mechanical loads exhibit engineering strain behavior in a direction opposite to the direction of load application , also in the cell culture behave differently from the tumor cells , which under a mechanical load exhibit engineering strain in the direction of the stressing . the changes in the cytoskeleton are therefore associated with such a biological change of the cells , so that they lose the affinity to tissue of the same kind and are present in a homogeneous mix with other types of cells ( here , the cells of the normal tissue ). metastatic cells exhibit precisely 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 engineering strain under a mechanical load is directed in opposite to the load application were only observed in tissue samples from patients with the formation of metastases . it can therefore be inferred by the presence of cells with these biomechanical properties in a tissue sample , that there is an increased risk of tumor metastases . it is known from prior art that the deformability ( and thus the engineering strain under a mechanical load ) in tumor cells increases compared to cells from normal tissue . fig3 shows this by means of a histogram demonstrating the engineering strain of the cells analyzed in the investigation following the principle of the optical stretcher . three cell populations from cell lines were examined : cells of the cell line mcf10 were analyzed as the normal tissue , a cell line of human non - cancerous breast epithelial cells . the cell line was derived from the breast tissue of a 36 - year - old woman with mastopathy . the cell line mcf7 was used as a tumor cell line , an adenocarcinoma cell line , which was prepared from cells of a 69 - year old breast cancer patient . these cells are a model for non - metastatic and non - invasive cells . if cells of the cell line mcf7 were added phorbol ester 12 - o - tetradecanoylphorbol - 13 - acetate ( tpa ) ( addition of 100 nmol / l tpa for 18 h in culture ), then a significant increase in the invasive potential and metastasis - forming potential was observed in these cells ( johnson et al . 1999 ). mcf7 - cells with added tpa are here referred to as “ modmcf7 ”- cells and were used in the experiment as a model cell line for metastatic cells . the engineering strain was determined based on the principle of the optical stretcher at a stress of 5 pa ( fig3 ). mcf10 - cells on average exhibited the least engineering strain in the direction of stressing . mcf7 - cells , in comparison to mcf10 - cells , on average exhibited a higher engineering strain in the direction of stressing . furthermore , it was found that , within the sample , the engineering strain of the single cells fluctuates more , so that the standard deviation of the engineering strain , as compared to the cells in mcf10 - cells , is higher for mcf7 - cells . the analysis of the metastatic modmcf7 - cells shows that the engineering strain on average is higher than with mcf10 - as well as with mcf7 - cells . the standard deviation compared to unmodified non - metastatic mcf7 - cells is even higher [ guck et . al . 2005 ]. the examination of breast tissue cell lines by means of scanning force microscopy ( sfm ) confirms these biomechanical properties ( fig4 ). the data was determined by constantly increasing the sfm adjustment value in the “ real - time scan ” mode at a rate of 1 nn / s , and recording the corresponding cantilever deflection rate at a rate of 10 hz . mcf10a - cells ( normal tissue , atcc - lgc promochem , germany ) and mcf7 - cells ( non - metastatic , non - invasive tumor tissue , atcc - lgc promochem , germany ) were used . as metastatic cells , the cell line mda - mb - 231 ( atcc - lgc promochem , germany ) was analyzed , which originates from a metastatic tumor . fig4 shows that the cells during linear deformation exhibit different engineering strain properties . mcf10a - cells exhibit the lowest compression whereas the tumor cells in comparison thereto exhibit significantly higher values for compression . the metastatic mda - mb - 231 - cells , when compared with non - metastatic mcf7 - cells , exhibit still significantly higher compression . therefore , from a higher mean value of the engineering strain in the direction of stressing , a statement can be made as to whether uncontrollably proliferating cells are present . if there is a higher mean value of the engineering strain directed in the direction of stressing in an analyzed patient sample compared with cells from normal tissues and if the cells from the patients analyzed sample simultaneously exhibit a higher proportion of cells that under stress exhibit engineering strain in a direction opposite to the direction of stressing , then there is an increased risk of the occurrence of tumor metastases . it is assumed that the stiffening of the cells at high loads causing non - linear deformation ( low engineering strain of tumor cells directed along the stressing for non - linear deformation ) is responsible for tumor cells being able to grow against the surrounding normal tissue and thus can invade surrounding tissue . for detection , cells from the tumor cell line ( mcf7 ) as well as in a further experiment , primary tumor cells from breast cancer patients were cultivated as a spheroid in a hydrogel containing 1 % agarose ( cell culture medium containing 1 % agarose ) ( fig5 ). analysis of these cells following the principle of the optical stretcher showed that the individualized cells exhibited maximum mechanical resistance of less than 1000 pa for linear deformation . after culturing in hydrogel , it was found that the cell structure can in the hydrogels grow against significantly higher resistances . the tumor cell spheroids in 18 days of culture grew in the hydrogel , which due to its stiffness , exerts a pressure of 6000 to 10 , 000 pa ( fig5 d ). the stiffness of the hydrogel was determined in the rheometer . it could thus be shown that tumor cell spheroids can withstand significantly higher mechanical stresses , than was determined by linear deformation . relaxation behavior of tumor cells from primary breast tumors and cells from normal tissue in an optical stretcher , 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 of human breast tissue samples from breast reductions ). the individualized cells were analyzed at 800 mw in the optical stretcher , where stress was applied for 2 seconds and the cell was observed for a further 2 seconds . with the mechanical stress introduced , linear deformation of the cells was to be expected in the optical stretcher . n1 ( comparative example ): primary breast epithelial cells from breast reductions ( invitrogen , hmec cells ) n2 ( comparative example ): primary breast epithelial cells from breast reductions ( promozell , hmec cells ) bt1 : primary breast tumor stage i bt2 : primary breast tumor stage ii bt3 : primary breast tumor stage iii bt4 : primary breast tumor stage iii prior to introducing mechanical stress , the length of the cell ( l 0 ) two seconds after starting introduction of the mechanical stress ( corresponds to the time t s ), the length of the cell ( l s ), two seconds after terminating introduction of the stress ( corresponds to the time t r ), the length of the cell ( l r ), based on the parameters determined , the relative relaxation of the analyzed cells is determined ( r =( l r − l s )/ l 0 ). the results are exhibited in fig6 where the distribution of the relative relaxation in the analyzed samples is shown . it is seen that cells from primary breast tumors from stage ii on average exhibit a lower relative relaxation , therefore exhibit stronger contraction in the original state . furthermore , it is clear that cells of primary breast tumors from stage ii do not relax only to a very small proportion ( relative relaxation & gt ; 0 ). 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