RU2508542C1 - Rapid method for determining risk of cell malignancy - Google Patents

Abstract

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.

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Description

The invention relates to oncological medicine, in particular to testing a malignant process, highly resistant cells of which can be detected using physical analysis of biological materials, which can be used to predict increased multiple resistance of cancer cells in vivo, when it is tested in vitro.

Despite serious and comprehensive studies of oncology in recent years, many unresolved problems remain, which are associated primarily with the fact that radiation and chemotherapy in many cases remains ineffective. The main factor limiting the success of the use of anticancer drugs is the high resistance of some tumor cells to various injuries, often formed under conditions of hypoxia. Such resistance can be: primary and, therefore, the property of malignant cells before treatment; or secondary, which develops in response to radiation exposure and / or drug administration.

Usually, positive dynamics are observed at the beginning of treatment, but after a while it disappears. The high variability of tumor cells and the constant selection of cells with greater survival and malignancy leads to the fact that initially with the help of treatment it is possible to reduce the 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 becomes resistant immediately to radiation exposure and to a whole group of drugs, that is, the so-called multiple resistance develops, often accompanied by a relapse of the disease and the appearance of metastases. One of the directions for the timely detection of such tumors is the search for new, specific indicators characteristic of highly resistant tumor cells, as well as the development of methods for their rapid determination in vitro using both already known and new tumor markers.

Typically, tumor markers are compounds (proteins, biologically active peptides, hormones, enzymes and metabolites) that are synthesized by cancer cells and are absent in normal cells. Most known tumor markers relate to oncofetal proteins that are found in human and blood embryonic tissues during fetal development. They disappear completely or remain in trace amounts after birth. During tumor progression, they begin to be synthesized again and secreted into the blood. However, specific oncofetal compounds are synthesized only in certain embryonic and tumor tissues. For example, alpha-fetoprotein is a normal fetal whey protein and is synthesized in the fetal liver, as well as in the development of liver cancer. Placental proteins (chorionic gonadotropin or placental alkaline phosphatase) are often used as oncofetal markers. Immunohistochemical detection of such proteins serves as good oncological markers of ovarian or testicular cancer, which allows monitoring the progress of their treatment. For prostate cancer, the prostate-specific antigen PSA (from the English, prostate specific antigen) is used as a sensitive tumor marker. However, on large material obtained not only on experimental animals, but also on humans, it has been shown that the vast majority of oncofetal biomarkers are tissue-specific. Moreover, these tumor markers are often not detected at an early stage of the disease and can also be induced in inflammatory diseases of the liver, pancreas and lungs. In addition, it is known that oncofetal markers do not exist for all solid tumors.

Although some mechanisms controlling the growth and differentiation of tissues are violated during the malignant transformation, a number of tumors still do not completely escape from the regulatory influence of the body, while retaining hormone and neurotransmitter receptors on the surface or inside the cells. These include, in particular, tumors originating from hormone-dependent tissues: mammary gland, uterus, ovaries, pituitary gland, adrenal glands, thyroid and prostate glands. More than half of breast, ovarian and endometrial tumors contain estrogen and progesterone receptors, which serve as tumor markers of hormone-sensitive tumors. Tumors that do not contain receptors are insensitive to hormone therapy, therefore, the determination of estrogen and progestin receptor levels is widely used to predict the effectiveness of hormone therapy in tumors of the breast, ovaries and uterus. However, these tumor markers do not allow testing the increased resistance of cancer cells to the damaging effects of chemotherapy or radiation.

There are various mechanisms for the emergence of multiple resistance of tumor cells, among which the multiple drug resistance is the most studied. Such resistance is usually tested by reducing the accumulation of anti-tumor drugs inside the cells or by the high level of expression of ATP-dependent transmembrane P-glycoprotein, which is able to efficiently pump out various anti-tumor drugs or dyes from resistant, tumor cells, thereby increasing their survival. It is important to note that P-glycoprotein is detected not only in tumor cells, but also in normal tissues, in particular in the kidneys, liver, and blood-brain barrier. The level of expression of P-glycoprotein may also increase with inflammation [But E., et al. Regulation of MDR by pro-inflammatory cytokines. "Curr. Cancer Drug Targets", 2006, 5 (4), 295-301]. Moreover, the performance of P-glycoprotein is closely dependent on the level of ATP production in 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]. Tumor mitochondria are characterized by a decrease in the level of ATP formation, especially in conditions of hypoxia, which limits the work of P-glycoprotein. However, under unfavorable conditions, such cells often maintain high survival rates, which is achieved by activating alternative metabolic pathways, in particular by activating aerobic glycolysis, as well as by the pentose phosphate pathway, which intensively produces reduced NAD (P) H [nicotinamidine nucleotide phosphate].

Recent studies have convincingly shown that resistant tumor cells, for their survival under conditions of hypoxia and oxidative stress, require the constant formation of NAD (P) H. [Tamada M., 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 activating the key antioxidant defense of tumor cells by activating enzymes that support glutathione synthesis [Gruning NM & Raiser M. Sacrifice for survival" Nature ", 2011, 480: 190-191; Hamanaka R & Chandel N. Warburg effect and redox balance. Science, 2011, 334: 1219-1220]. However, NAD (P) H cannot be classified as a classic tumor marker, so however, it can be synthesized in normal cells. However, in vitro NAD (P) H / NADH endogenous fluorescence indices can be used to predict in vivo metastasis [Xu HN, Nioka S., Clickon JD, Chance B., Li LZ Quantitative mitochondrial r edox imaging of breast cancer metastatic potential. "J Biomedical. Optics ", 2010, 15 (3): 136010].

The technical problem to which the invention is directed is to provide the ability to test the presence of malignant cells and the degree of their resistance to damage (degree of malignancy) by measuring live cells without staining.

The problem is solved in that an express method for determining the risk of cell malignancy is proposed, which consists in preparing a cell fingerprint or cell suspension from a biopsy sample obtained from a subject, in which hypoxia conditions are created, and subjected to repeated exposure to a damaging agent selected from UV -light, a spectro-fluorometric study of the sample is carried out after repeated exposure to UV light, and if in the dark conditions an increase in the fluorescence intensity of NAD (P) H is detected, the diagnosis Steer cells as malignant.

As a damaging agent, in particular, UV light or another agent, for example, chemical or pharmacological, can be used.

The ability to longer survival of malignant cells under conditions of hypoxia and repeated injuries (increased resistance) is assessed by repeatedly repeated repair of the NAD (P) H pool, which is characteristic only of tumor cells. The repair of the NAD (P) H pool under hypoxic conditions is assessed by the increase in the NAD (P) H fluorescence intensity after a short exposure of the test sample of cells in dark conditions, the presence of which follows repeated damage (in particular UV light), and the number of repair cycles NAD (P) H fluorescence and the magnitude of their growth in each cycle assess in vitro the potential resistance of various tumor cells. This allows us to identify highly resistant cancer cells as indicators of negative tumor prognosis in vivo for the tumor from which the test sample was taken.

The proposed method for the rapid determination of malignancy is non-specific and allows within a few minutes to identify malignant cells and establish the degree of their resistance for all types of cancer, both ascites and solid.

The proposed method for the rapid determination of the degree of malignancy and resistance of cancer cells consists in preparing a cell fingerprint or suspension of cells from a biopsy sample, which creates hypoxia conditions and repeated exposure to a damaging agent, such as UV light, and conducts spectro-fluorometric studies of these cells in the dynamics of the process of photodegradation of NAD (P) H fluorescence, and the increase in the fluorescence intensity of NAD (P) H in dark conditions, judge the presence of tumor cells and their potential sustainability.

Hypoxia conditions can be created by placing a cell suspension or cell imprint between two glasses, the edges of which are embedded in paraffin, for subsequent cytological studies. Spectrofluorometric studies can be carried out in the range of 440-470 nm, with an excitation wavelength of 365-370 nm.

The essence of the invention lies in the fact that experimentally established fundamental differences in the potential resistance of tumor and normal cells to survival under conditions of hypoxia and UV light. Moreover, cell survival was evaluated, in particular, by changing the intensity of NAD (P) H fluorescence and the ability inherent only in cancer cells to partially restore the pool of NAD (P) H in dark conditions. The invention is illustrated by graphic materials, where in FIG. Figure 1 shows a typical curve of the change in the total fluorescence intensity at λ = 460 nm (I ₄₆₀ ), which is characteristic of transplanted tumor cells of Ehrlich ascites carcinoma (AKE) under hypoxia under the influence of UV light (shaded areas) and in dark conditions ( after turning off the UV light; not shaded areas). The total intensity of the endogenous fluorescence of living cells (in the band 440-470 nm) is due to the fluorescence of two main fluorescent components: 1) fluorescent products of lipid peroxidation (FPPOL), which are highly sensitive to UV-induced photodestruction [after 0.5-1 min of UV light (λ-365 nm), fluorescence from FPPOL is not detected] and 2) a pool of reduced NAD (P) H, the fluorescence intensity of which reaches its maximum under hypoxia. The percentage of each of these components (in percent) in the total fluorescence band (with a maximum of I ₄₆₀ ), characteristic of most tumor AKE cells, is indicated on the ordinate scale.

A - kinetics of photodegradation of fluorescence (I ₄₆₀ ) of tumor AKE cells, with constant exposure to UV light (λ-365 nm)

B - NAD (P) H pool repair, measured by the increase in NAD (P) H fluorescence intensity after finding hypoxic tumor AKE cells in dark conditions [i.e. without the damaging effect of UV light (λ-365 nm)]. The increase in I ₄₆₀ (in percent) after each recovery cycle of NAD (P) H fluorescence is indicated by the magnitude of the up arrows.

Figure 2 shows the kinetic features of I ₄₆₀ photodestruction of lymphocytes isolated from the lymph node of healthy animals and placed in hypoxic conditions. For normal lymphocytes characteristic, permanent photodestruction fluorescence (with I ₄₆₀₎ was observed as when exposed to UV light (λ-365 nm) (shaded areas) and in darkness (when turning off the UV light; not shaded areas) . The value of I ₄₆₀ photodestruction is indicated by the size of the arrows pointing down, which indicates a low resistance of normal lymphoid cells to survival under hypoxia. The opposite effect is characteristic of tumor AKE cells (Fig. 1), which have increased potential resistance under similar conditions of hypoxia. The revealed differences between the behavior (namely, survival) of tumor and normal cells under conditions of damage can be used in vitro as an indicator of malignancy, with the help of which it is possible to identify single tumor cells and also to predict their survival in vivo in the hypoxic zones of the tumor.

In Fig. 3, similar studies are presented for another type of highly aggressive tumor cells - Seidel ascites hepatoma (AGD) [AGH cells in ascites fluid and when they metastasize to the spleen, as well as normal splenocytes surrounding metastasis, were studied (Fig. 4)]. Ascitic AGH cells, as well as AGH metastasis cells (Figure 3), were characterized by high potential resistance to the damaging effects of hypoxia and UV light, since they were capable of 3-5-fold restoration of the NAD (P) H pool, estimated by the increase in intensity I ₄₆₀ [the growth rate is indicated by numbers (in%) and marked by arrows pointing up]. The time of action of UV light on AGZ cells and their presence in dark conditions is indicated in seconds on the abscissa scale.

figure 4 - presents a similar spectro-fluorescence studies of cells from tissue imprint of the spleen of the tumor carrier (figure 3), carried out under conditions of hypoxia for splenocytes i.e. non-transformed spleen cells surrounding AGZ metastasis cells. Such splenocytes were characterized by low potential resistance to the damaging effect of UV light, since they were characterized by permanent photodegradation of NAD (P) H fluorescence at λ = 460 nm (I ₄₆₀ ), detected (in%) both under the influence of UV light, and in his absence.

figure 5 presents the results of spectro-fluorescence studies of cells [from a biopsy of the axillary lymph node of the patient (46 years old)], which were diagnosed as cancerous. Under hypoxic conditions, these cells showed the ability to restore NAD (P) H fluorescence in dark conditions [when turning off UV light (not shaded areas)], which was measured by the increase in I ₄₆₀ (in%). The increase in I _{460 is} indicated by an arrow pointing up. The number of NAD (P) H fluorescence cycles in such cells was small (only 2 cycles), however, the increase in the NAD (P) H fluorescence intensity in the first cycle was very significant, indicating an increased resistance of cells of this metastasis to survival in adverse conditions in vivo .

figure 6 presents the results of spectro-fluorescence studies of the same cell suspension (from the biopsy material of the patient), which were diagnosed as not transformed. These cells were characterized by permanent UV-induced photodestruction I ₄₆₀ , both under the action of UV light (shaded areas) and in the absence of UV light (non-shaded areas). An increase in the content of FPPOL (in%) and a decrease in the proportion of basal NAD (P) H fluorescence (I ₄₆₀ , ordinate scale) were also typical for these cells.

The invention is also illustrated by an example of a specific implementation.

EXAMPLE.

Patient K. (46 years old), with suspicion of the development of metastasis of breast cancer in a nearby lymph node, was taken biopsy material (in the form of a cell suspension) for cytological and histological examination. Part of the seized biopsy specimen was placed in a moist, cooled chamber, in order to preserve the cells during their transportation to the laboratory. The detection of malignancy and cell resistance was carried out 1.5 hours after the removal of cellular material. For this purpose, the studied cell suspension was placed between the slide and cover glasses, the edges of which were tightly sealed with liquid paraffin to create hypoxic conditions in the studied cells. Further, on the fluorescent microscope MLD-2 using a mercury lamp DRSh-250-2 ultrahigh pressure as a source of UV light (1-365 nm), carried spektrofluorestsentnye photodegradation kinetics studies ₄₆₀ I in the analyzed cells are in conditions of hypoxia when exposed to UV light and when it is turned off (dark conditions). A search was made for cells that were capable of repairing the intensity of NAD (P) H fluorescence (I ₄₆₀ ) under dark conditions. Such cells were diagnosed as malignant (cancerous) [Fig. 5, the effect is indicated by an upward arrow]. Other cells, which were characterized by permanent UV-induced photodestruction, both under the influence of UV light and in the absence of UV exposure, were diagnosed as not transformed (Fig.6). The results are presented in FIG. 5 and FIG. 6. As can be seen from FIG. 5 and FIG. 6., under conditions of persistent hypoxia, among the analyzed cells were detected cells (FIG. 5) capable of increasing the intensity of NAD (P) H fluorescence (I ₄₆₀ ) in dark conditions, which allowed to identify them as malignant cells. These cells showed single NAD (P) H repair cycles of fluorescence under dark conditions (single up arrow), which allowed them to be identified as cancer cells, although they did not have as high potential resistance to hypoxia as AGZ cells (Figure 3). The entire testing process from the moment of creating hypoxia conditions for the studied cells to the analysis results was no more than 5 minutes.

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

The diagnosis was then confirmed histologically.

Thus, the above data confirm the possibility of rapid testing of samples (fingerprints or cell suspensions) for malignancy and potential resistance of the tested tumors to damaging effects.

Claims (1)

A method for determining the risk of cell malignancy, consisting in the fact that a cell fingerprint or a suspension of cells is prepared from a biopsy sample obtained from a subject, in which hypoxia conditions are created, subjected to repeated exposure to a damaging agent selected from UV light, and a spectrofluorometric study of the sample is performed after repeated exposure to UV light, and if in the dark conditions an increase in the fluorescence intensity of NAD (P) H is detected, the cells are diagnosed as malignant.

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