Rapid method for determining risk of cell malignancy

FIELD: medicine.

SUBSTANCE: to determine a risk of cell malignancy, a biopsy material taken from an individual is used to prepare a cell smear or a cell suspension in the hypoxic environment. The material is exposed to a pathogenic agent specified in UV light. The recurrent UV light exposure is followed by spectrofluorometry of the sample, and if the dark environment is characterised by an increase of NAD(P)H fluorescence intensity, the cells are diagnosed as malignant.

EFFECT: invention provides detecting the high-resistant cancer cells as negative cancer prediction measures in vivo for the cancer that are used to take the sample; a time of malignancy detection and cancer cell resistance makes a few minutes.

6 dwg, 1 ex

The invention relates to cancer medicine, in particular to testirovanie malignant process, highly-resistant cells which can be detected by physical analysis of biological materials that can be used to predict high multiple of the resistance of cancer cells in vivo when tested in vitro.

Despite the serious and comprehensive research in recent years in the field of Oncology are still many unsolved problems, which are associated primarily with the fact that radiation and chemotherapy remains largely ineffective. The main factor limiting the successful application of anticancer funds is the high resistance portion of tumor cells to various injuries, often emerging in hypoxia. Such resistance can be primary, and therefore the property of malignant cells before treatment; or secondary, which develops in response to exposure to radiation and/or drug administration.

Usually at the beginning of treatment there is a positive trend, but after a while it disappears. High variability of tumor cells and a permanent selection of cells with greater survival and malignancy leads to the fact that the original treatment could decrease the TB size of the tumor due to the death of tumor cells with low resistance. The remaining subpopulation of cells acquires an increased ability to survive in adverse conditions and immediately becomes resistant to radiation exposure and to a group of medicines that is developing the so-called multiple resistance, often accompanied by recurrence of the disease and the appearance of metastases. One of the areas of timely detection of such tumors is the search for new, specific indicators, typical of highly-resistant tumor cells, as well as the development of methods for their rapid determination of in vitro using as already known and new tumor markers.

Usually the tumor markers referred to as compounds (proteins, bioactive peptides, hormones, enzymes and metabolites), which are synthesized by cancer cells and not in normal cells. The majority of known tumor markers belong to oncofetal proteins that are found in embryonic tissues and blood during fetal development. They disappear completely or remain in trace quantities after birth. During tumour progression, they begin to be synthesized again and secreted into the blood. However, the specific Onco-fetal compounds are synthesized only in certain embryonic and tumor tissues. For example, alpha-fetoprotein is normalnym whey protein embryo and is synthesized in the liver of the fetus, and with the development of liver cancer. As Onco-fetal markers are often used proteins of the placenta (chorionic gonadotropin or placental alkaline phosphatase). Immunohistochemical detection of such proteins is a good oncological markers of cancer of the ovaries or testes that allows you to monitor the progress of their treatment. For prostate cancer as a sensitive marker used prostate-specific antigen PSA (from the English., prostate specific antigen). However, the large material obtained not only in experimental animals but also in humans, it was shown that the vast majority of Onco-fetal biomarkers is Dane-specific. Moreover, these tumor markers are often not detected at an early stage of the disease and can coil also in inflammatory diseases of the liver, pancreas and lungs. In addition, it is known that the Onco-fetal markers do not exist for all solid tumors.

Although in the process of malignant transformation violated some of the mechanisms controlling the growth and differentiation of tissues, but the number of tumors is still not completely escapes from under the regulatory influence of the body, keeping the receptors of hormones and neurotransmitters on the surface or inside the cells. These include, in particular, are tumors originating in hormone-dependent the x tissue: mammary gland, uterus, ovaries, pituitary, adrenal, thyroid and prostate glands. More than half of breast tumors, ovarian and endometrial contain receptors for estrogen and progesterone, which serve as tumor markers hormone-sensitive tumors. Tumors that do not contain receptors sensitive to hormonal therapy is, therefore, determine the level of receptors of estrogens and progestins are widely used for predicting the effectiveness of hormonal therapy for tumors of the breast, ovaries and uterus. However, these tumor markers are not allow to test the increased resistance of cancer cells to the damaging effects of chemotherapy or radiation.

There are different mechanisms for the occurrence of multiple resistance of tumor cells, among which multi-drug resistance is the most studied. Such resistance is usually tested to reduce the accumulation of anti-tumor drugs inside cells or on the high level of expression of ATP-dependent transmembrane P-glycoprotein, are able to effectively pump out various anticancer drugs or dyes from sustainable, tumor cells, thus increasing their survival rate. It is important to note that P-glycoprotein is detected not only in the tumor, but also in normal tissues, particularly the kidneys, pécs is no, the blood-brain barrier. The level of expression of P-glycoprotein may also be increased by inflammation [But E., et al. Regulation of MDR by pro-inflammatory cytokines. "Curr. Cancer Drug Targets", 2006, 5(4), 295-301]. Moreover, the efficiency of P-glycoprotein closely depends on the education level of ATP in the mitochondria [Zhou Y., et al. Intracellular ATP levels are a pivotal determinant of chemoresistance in colon cancer cell. "Cancer Res.", 2012, 72(1), 304-314]. For tumor mitochondria are characterized by the decrease of the level of education Asia Pacific, especially in conditions of hypoxia, which limits the operation of P-glycoprotein. However, such cells under adverse conditions, often retain a high survival rate, which is achieved due to the activation of alternative pathways metabolism, in particular the activation of aerobic glycolysis, as well as work pentose-phosphate pathway, intensively producing restored NAD(P)H [nikotinamidadenindinukleotida].

Studies in recent years have clearly shown that resistant tumor cells for their survival in hypoxia and oxidative stress, need constant education NAD(P)H. [M. Tamada, et al. Modulation of glucose metabolism by CD44 contributes to antioxidant status and drug resistance in cancer cells. Cancer Res." 2012, 72(6), 1438-1448]. NAD(P)H is involved in the activation of key anti-oxidant protection of tumor cells, activating enzymes, supports the synthesis of glutathione [Gruning N-M & M. Raiser Sacrifice for survival "Nature", 2011, 480:190-191; Hamanaka R & Chandel N. Wrburg effect and redox balance. Science, 2011, 334: 1219-1220]. However, NAD(P)H cannot be regarded as a classic tumor markers, as it is synthesized in normal cells. However, the indicators of endogenous fluorescence of NAD(P)H/NADH in vitro can be used in forecasting processes metastasis in vivo [Xu H.N., Nioka S., Clickon J.D., Chance C., Li, L.Z. Quantitative mitochondrial redox imaging of breast cancer metastatic potential. "J Biomedical. Optics", 2010, 15 (3): 136010].

The technical task to be solved by the invention, is to provide opportunities to test the presence of malignant cells and the degree of their resistance to damage (degree of malignancy), conducting measurements on living cells without staining.

The problem is solved by the fact that offered a quick way to determine the risk of malignancy cells, namely, that from a biopsy sample obtained from the subject, preparing a cellular imprint or suspension cells, which create conditions of hypoxia, is subjected to repeated impacts damaging agent selected from UV light, hold Spectro-fluorometrically study sample after repeated exposure to UV light, and if in dark conditions detected increase in the fluorescence intensity of the NAD(P)H, diagnosed as malignant cells.

As damaging agent can be used in private the ti UV light, or another agent, such as chemical or pharmacological.

The ability to longer survival of malignant cells in hypoxia and repeated damage (increased resistance) estimated by repeated repair pool of NAD(P)H, which is characteristic for tumor cells. Repair a pool of NAD(P)H in hypoxia judged by the increase in the intensity of the NAD(P)H fluorescence after a short incubation, the sample cells in dark conditions, a finding which follows a recurring injuries (particularly UV light), and the number of repair cycles NAD(P)H fluorescence and the magnitude of the increase in each cycle assess in vitro the potential stability of various tumor cells. This allows you to identify highly-resistant cancer cells as negative indicators of tumor prognosis in vivo the tumor from which to take the sample.

The proposed method for the rapid determination of malignancy is non-specific and allows for several minutes to detect malignant cells and to establish the degree of their resistance to all types of cancer, as ascitic and solid.

The proposed method is rapid calculation of the degree of malignancy and resistance of cancer cells is that from a biopsy sample is otomat cellular imprint or cell suspension, which create conditions of hypoxia and repeated exposure to the damaging agent, such as UV light, and carried out Spectro-fluorometrically the study of these cells in the development of the process of photodegradation NAD(P)H fluorescence and the increase in the fluorescence intensity of the NAD(P)H in dark conditions, judged on the presence of tumor cells and their potential sustainability.

Conditions of hypoxia can be created by placing a suspension of cells or cellular imprint between two panes of glass, the edges of which are filled with paraffin for subsequent cytological studies. Spectrofluorometrically research can be conducted in the range of 440-470 nm, when the wavelength of excitation 365-370 nm.

The invention consists in the fact that experimentally have been established fundamental differences in the potential stability of normal and tumor cell survival under hypoxia and UV light. While the survival of cells was evaluated, in particular, by changing the intensity of the NAD(P)H fluorescence and abilities unique to cancer cells, in part to restore the pool of NAD(P)H in dark conditions. The invention is illustrated graphics, where Fig. 1 shows a typical curve of variation of total fluorescence intensity at λ=460 nm (I₄₆₀)characteristic of transplantable the x tumor cells in ascitic Ehrlich carcinoma (EAC), under conditions of hypoxia under the action of a UV-light (shaded areas) and in dark conditions (after turning off the UV light; not shaded sections). The total intensity of the endogenous fluorescence of living cells (lane 440-470 nm) due to the fluorescence of the two main fluorescent components: 1) fluorescent products of lipid peroxidation (FPOL), which are sensitive to UV-induced photodegradation [after 0.5-1 min UV light (λ 365 nm), the fluorescence from FPPA not detected] and 2) the pool restored NAD(P)H, the fluorescence intensity reaches a maximum in hypoxia. The share of each of these components (in percent) in the total band fluorescence (with a maximum of I₄₆₀)characteristic of most tumor EAC cells, indicated on the scale of the ordinate.

The photodegradation kinetics of fluorescence (I₄₆₀cancer EAC cells, with constant action of UV-light (λ 365 nm)

In the repair pool of NAD(P)H, as measured by the increase in the intensity of the NAD(P)H fluorescence after finding hypoxic tumor EAC cells in dark conditions [i.e. without the damaging effects of UV-light (λ 365 nm)]. The growth value of I₄₆₀(in percent) after each cycle recovery NAD(P)H fluorescence, indicated by the magnitude of the arrows upward.

Figure 2 p is the accumulated kinetic features of I ₄₆₀photodegradation of lymphocytes isolated from lymph nodes of healthy animals and placed in conditions of hypoxia. For normal lymphocytes characteristic, permanent photodestruction fluorescence (at I₄₆₀), which was observed as under the action of UV-light (λ 365 nm) (shaded areas)and dark conditions (when turning off the UV light; not shaded sections). The value of I₄₆₀photodegradation indicated by the magnitude of the arrows down, indicating low stability of normal lymphoid cells to survive under conditions of hypoxia. The opposite effect is characteristic for tumor EAC cells (figure 1), which has a higher potential resistance in similar conditions of hypoxia. The detected differences between the behavior (i.e. survival) of tumor and normal cells in terms of damage can be used in vitro as an indicator of malignancy, with which you can identify a single tumor cells, as well as to predict their survival in vivo in a hypoxic areas of the tumor.

Figure 3 a similar study presents for another type of highly aggressive tumor cells ascitic hepatoma Seidel Foundation (AHP) [examined AHP cells in ascitic fluid and their metastasis in the spleen, as well as normal splenocytes surrounding IU astas (figure 4)]. Ascitic AHP cells, as well as AHP cells metastasis (Figure 3) had a high potential resistance to the damaging effects of hypoxia and UV light, as they were able in a 3-5-fold recovery pool of NAD(P)H, as measured by the increase in the intensity of I₄₆₀[the amount of increase indicated by the digits (in %) and marked by arrows pointing upwards]. The duration of UV light at AHP cells and their presence in dark conditions indicated in seconds on the abscissa scale.

figure 4 - presents similar Spectro-fluorescence analysis of cells from tissue imprint spleen of ofwholesale (figure 3), held in hypoxia for splenocytes i.e. untransformed cells of the spleen, the surrounding cells AHP metastasis. These splenocytes were characterized by low potential resistance to the damaging effects of UV light, as for them, a typical permanent photodegradation fluorescence of NAD(P)H at λ=460 nm (I₄₆₀)detected (%) as under the action of UV light, and in him no.

figure 5 presents the results of Spectro-fluorescent studies of cells [biopsy axillary lymph node of the patient (age 46)], which were diagnosed as cancer. Under hypoxic conditions, these cells showed the ability to regenerate NAD(P)H fluorescence in dark conditions [when is iklyuchenie UV light (not shaded sections)], which was measured by the increase of I₄₆₀(in %). The growth value of I₄₆₀the arrow pointing up. The number of cycles NAD(P)H fluorescence in these cells was small (only 2 cycles), but increase the intensity of the NAD(P)H fluorescence in the first cycle was highly significant, indicating a higher stability of the cells of this metastasis to survive in adverse conditions in vivo.

figure 6 presents the results of Spectro-fluorescence study of the same cell suspension (from biopsy material of patients), who were diagnosed as not transformed. For these cells was characterized by permanent UV-induced photo-destruction of I₄₆₀as under the action of UV light (shaded areas), and in the absence of UV light (not shaded sections). For these cells was also typically increase the content FPOL (%) and a reduction of basal NAD(P)H fluorescence (I₄₆₀the scale of the ordinate).

The invention is illustrated well by the example of a specific implementation.

EXAMPLE.

The patient K. (46 years)with suspicion on the development process of metastasis breast cancer in nearby lymph node, biopsy was taken of the material (in the form of cell suspension) for cytological and histological studies. Of the seized biopsy specimen was placed in the most important, cooled camera, with the aim of preserving the cells during their transport to the laboratory. The detection of malignancy and resistance of the cells was carried out in 1.5 hours after removal of cellular material. To this end the investigated cell suspension was placed between the objective and the cover glass, the edges are tightly sealed with liquid paraffin to create conditions of hypoxia in the studied cells. Next on fluorescent microscope BN-2, using a mercury lamp, ultrahigh pressure DRS-250-2 as the source of UV light (1-365 nm), conducted spectrofluorimetry studies of the kinetics of photodegradation I₄₆₀in the analyzed cells under conditions of hypoxia under the action of a UV light and when it is turned off (dark conditions). Conducted a search of the cells that had the ability to repair the intensity of the NAD(P)H fluorescence (I₄₆₀in dark conditions. These cells were diagnosed as malignant (cancerous) [figure 5, the effect indicated by the arrow pointing up]. Other cells, characterized by a permanent UV-induced photo-destruction, as under the action of UV light and in the absence of UV irradiation was diagnosed as not transformed (Fig.6). The obtained results are shown in figure 5 and Fig. 6. As can be seen from figure 5 and 6., in the context of persistent hypoxia, is a single of the analyzed cells were detected cells (figure 5), able to increase the intensity of the NAD(P)H fluorescence (I₄₆₀in dark conditions, allowing to identify them as malignant cells. These cells were detected individual cycles of reparation NAD(P)H fluorescence in dark conditions (single arrow), which allowed them to identify how cancer cells, although they did not have such a high potential resistance to hypoxia as AHP cells (Figure 3). The entire testing process from the inception of hypoxia for the studied cells until the results of the analysis amounted to no more than 5 minutes.

Most of the analyzed cells in the sample did not show the ability to repair NAD(P)H fluorescence in dark conditions (6), which allowed them to refer to non-transformed cells.

The diagnosis was then confirmed histologically.

Thus, the above data confirm the possibility of the rapid testing of samples (prints or cell suspensions) for malignancy and the potential stability of the tested tumors to damage.

The way to determine the risk of malignancy cells, namely, that from a biopsy sample obtained from the subject, preparing a cellular imprint or suspension cells, which create conditions of hypoxia, is subjected to repeated impacts of damages is promoting agent, selected from UV light, hold spectrofluorometrically study sample after repeated exposure to UV light, and if in dark conditions detected increase in the fluorescence intensity of the NAD(P)H, diagnosed as malignant cells.