Method for acquiring data and method for acquiring prediction data for determining the presence of cancer cells and / or resistance to anticancer drugs, and use of said determination marker and determination kit

Abstract

The present invention addresses the problem of providing a method for acquiring data for distinguishing the presence of cancer cells and / or distinguishing anticancer drug resistance, a method for acquiring prediction data, a use of a distinction marker in said method, and a distinguishing kit, and in particular addresses the problem of distinguishing anticancer drug resistance before an anticancer drug is administered to a patient. Polysulfides, which are markers for anticancer drug resistance, are distinguished in cancerous tissue of a cancer patient prior to administration of an anticancer drug, thereby making it possible to distinguish anticancer drug resistance to the cancerous tissue of the cancer patient.

Description

[Technical field]

[0001] The present invention relates to a method for acquiring data and a method for acquiring predictive data for determining the presence of cancer cells and / or resistance to anticancer drugs, as well as use of the determination marker and a determination kit. [Background technology]

[0002] Cancers generally produce various metabolites that are advantageous to their own survival due to genetic mutations, and exhibit metabolic characteristics that differ from those of surrounding non-cancerous tissues. It has also been revealed that they actively rob surrounding normal tissues of metabolites necessary for their own survival and proliferation. Among these metabolites, sulfur-containing metabolites such as glutathione, cysteine, and hydrogen sulfide have been suggested to be involved in the survival, proliferation, and drug resistance of cancer cells as powerful antioxidants, but the actual situation has not been clear. Therefore, the present inventors have conducted various analyses to obtain knowledge about the survival, proliferation and drug resistance of cancer cells, and as a result, have found through an analysis using a glioblastoma model mouse that polysulfides are abundantly contained in the glioblastoma part of this disease model (Non-Patent Document 1). However, no analysis of polysulfides has been conducted on humans, and no analysis has been conducted on cancers other than glioblastoma. In the case of ovarian cancer, the cancer is often already in an advanced stage by the time it is discovered. The standard treatment for such advanced ovarian cancer is to perform an ovarian removal surgery, followed by chemotherapy using anti-cancer drugs to kill any ovarian cancer cells remaining in the body. In general, anticancer drugs have strong side effects, such as nausea, general fatigue, hair loss, rashes, and anemia. They can also cause serious side effects such as acute renal failure, liver dysfunction, pancytopenia, and anaphylaxis, so anticancer drugs do not necessarily provide only useful effects to all cancer patients. Furthermore, even when treatment that can cause such various side effects is performed, there is a certain frequency of ovarian cancer recurrence, and some cancer patients have not achieved sufficient therapeutic effects. Under these circumstances, in order to improve the balance between the invasiveness of anticancer drug treatment for cancer patients and the therapeutic effects of that anticancer drug treatment, it has been desirable to be able to predict the therapeutic effects of anticancer drugs before they are administered to cancer patients, in order to select the appropriate anticancer drug for each patient. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Nature Communications, 2018, 9, Article number:1561 [Non-Patent Document 2] Analyt Chem, 2006, 78, 2631-2639 [Non-Patent Document 3] Inorg. Chem., 1976, 15, 1759-1763 [Non-Patent Document 4] Phys Chem Chem Phys., 2015, 17, 5155-5171 Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention aims to provide a method for obtaining data and a method for obtaining predictive data for determining the presence of cancer cells and / or resistance to anticancer drugs, as well as the use of such discrimination markers and a discrimination kit, in particular to determine resistance to anticancer drugs before administering the anticancer drug to a patient. [Means for solving the problem]

[0005] The present inventors have found that there are cancer tissues for which anticancer drugs are effective and cancer tissues for which anticancer drugs are ineffective, i.e., cancer tissues that are resistant to anticancer drugs. For example, when cisplatin is used as an anticancer drug, it has been shown that cisplatin is less effective against ovarian clear cell carcinoma (CCC) (i.e., resistant to anticancer drugs), but is more effective against ovarian serous carcinoma (SC) (i.e., not resistant to anticancer drugs). Then, when Raman spectroscopy was performed on anticancer drug-resistant cancer tissues, it was found that there is a specific peak in the spectrum of CCC. Furthermore, the specific peak seen in this anticancer drug-resistant cancer tissue is a peak at a Raman shift of 480 cm in Raman spectroscopy analysis. -1 (Kaiser) This specific peak was found to indicate that polysulfides were abundantly expressed in cancer tissue. That is, since anticancer drug resistant cancer tissues express a large amount of polysulfides that serve as anticancer drug resistance markers, by detecting these polysulfides that serve as anticancer drug resistance markers in a cancer patient, it is possible to determine whether the cancer tissues of the cancer patient have resistance to anticancer drugs. Even before the administration of anticancer drugs, it is possible to determine whether the cancer tissues of the cancer patient have resistance to anticancer drugs.

[0006] That is, this application provides the following inventions. [1] A data acquisition method for determining the presence of cancer cells and / or anticancer drug resistance of cancer cells in a target tissue, comprising a step of measuring a level of polysulfide in the target tissue, The method, wherein the anti-cancer drug is a polysulfide-reactive anti-cancer drug. [2] The method according to [1], wherein the measurement is performed before administration of an anticancer drug. [3] The method according to [1] or [2], wherein the measuring step is a step of detecting a peak specific to polysulfides by Raman spectroscopy. [4] When the specific peak is detected by Raman spectroscopy in cancer tissue, the Raman shift is 480±10 cm -1 The method according to [3], wherein the peak appears at the position [5] The method according to any one of [1] to [4], wherein the anticancer drug is cisplatin or gemcitabine. [6] The method according to any one of [1] to [5], wherein the target tissue is an ovarian cancer tissue or a pancreatic cancer tissue. [7] The method according to [6], wherein the ovarian cancer tissue is ovarian clear cell cancer tissue. [8] A method for obtaining predictive data on the presence of cancer cells and / or the resistance of cancer cells to an anticancer drug, the method using a polysulfide level as an index, the method indicating the presence of cancer cells in the target tissue and / or the resistance of cancer cells to an anticancer drug when the polysulfide level in the target tissue is higher than the polysulfide level in a control tissue, comprising: The method, wherein the anti-cancer drug is a polysulfide-reactive anti-cancer drug. [9] Use of polysulfides as a marker for determining the presence of cancer cells and / or the resistance of cancer cells to anticancer drugs, comprising: The use, wherein the anti-cancer drug is an anti-cancer drug reactive with polysulfides.

[10] A kit for determining the presence of cancer cells and / or anticancer drug resistance of cancer cells, comprising a substrate for measuring a level of polysulfide by Raman spectroscopy, The kit, wherein the anticancer drug is an anticancer drug reactive with polysulfides. Effect of the Invention

[0007] The data acquisition method and prediction data acquisition method for determining the presence of cancer cells and / or anticancer drug resistance according to the present invention, as well as the determination marker and determination kit thereof, make it possible to determine anticancer drug resistance. Based on the results of determining anticancer drug resistance, a treatment plan can be made as to whether to administer an anticancer drug or to perform other treatment such as surgery. [Brief description of the drawings]

[0008] [Figure 1] The average surface-enhanced Raman spectra of tissue sections derived from ovarian clear cell carcinoma (CCC) or serous carcinoma (SC) and the difference spectrum between these spectra (CCC-SC) are shown. The top and middle panels show the average surface-enhanced Raman spectra of tissue sections derived from CCC (n=12) and SC (n=12), respectively. All tissue sections were collected from the NCC Biobank. [Diagram 2] This shows a comparison of the SERS signal intensity at the height of the Raman shift of 480 cm-1 between tissue sections derived from CCC and SC. The unit of intensity is arbitrary unit (au). In the false discovery rate (FDR) analysis, a P value of less than 0.05 was considered to be statistically significant. SERS signals were treated as the same signal when the Raman shift was within ±10 cm-1 from the peak position. [Diagram 3] Gel shift assay confirmed that the inhibition of double-stranded DNA supercoiling by cisplatin (CDDP) was attenuated by Na2S, Na2S2, Na2S3 and Na2S4 in vitro (photo). [Figure 4] 1 shows a representative conventional resonance Raman spectroscopy spectrum of a CDDP solution at a concentration of 3 mM. [Diagram 5] This shows that the addition of CDDP reduces the signal intensity of the peak at the Raman shift of 471 cm-1 of Na2S4 in the conventional resonance Raman spectroscopy spectrum in a CDDP concentration-dependent manner. [Figure 6] 1 shows representative surface-enhanced Raman spectroscopy spectra of gemcitabine solutions at concentrations of 50 μM and 200 μM. [Figure 7] This shows that, by adding gemcitabine, the signal intensity of the peak at the Raman shift of 456 cm −1 of Na 2 S 3 in the surface-enhanced Raman spectroscopy spectrum decreases depending on the concentration of added gemcitabine. [Figure 8] This shows that, by adding gemcitabine, the signal intensity of the peak at the Raman shift of 456 cm −1 of Na 2 S 4 in the surface-enhanced Raman spectroscopy spectrum decreases depending on the concentration of added gemcitabine. [Figure 9] This shows visualization of polysulfides by SERS imaging of tissue sections derived from pancreatic cancer and chronic pancreatitis as a control. The upper left image is a photograph of a pancreatic cancer tissue section stained with HE, and the upper right image is a photograph of a pancreatic cancer tissue section stained with Raman shift 480 cm-1. The lower left image is a photograph of a chronic pancreatitis tissue section stained with HE, and the lower right image is a photograph of a chronic pancreatitis tissue section stained with Raman shift 480 cm-1. After SERS imaging, the sections were used for HE staining. In the HE staining, black annotations indicate cancerous areas, and white annotations indicate interstitial areas. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention is a data acquisition method for determining the presence of cancer cells in a target tissue and / or the resistance of cancer cells to an anticancer drug, comprising a step of measuring the level of polysulfide in the target tissue, wherein the anticancer drug is an anticancer drug reactive with polysulfide.

[0010] In the present invention, a polysulfide refers to a polymer or a salt thereof that contains one or more disulfide bonds, and RS x It is a compound represented by the chemical formula: -R. In the above chemical formula, although not particularly limited, R is, for example, H, Na, or an alkyl group which may be substituted with any substituent, and is preferably Na. Furthermore, x is any integer of 2 or more, and x is preferably an integer of 2 to 4. In the present invention, the polysulfide is not particularly limited, but when R is Na, it is preferably Na2S2, Na2S3 or Na2S4.

[0011] The method for measuring the polysulfide level is not particularly limited as long as it is a method capable of measuring the polysulfide level, and for example, it can be measured by Raman spectroscopy or an electrochemical measurement method as described in Non-Patent Document 2, but it is preferable to measure it by Raman spectroscopy. There are several methods of Raman spectroscopy known to those skilled in the art, and it is not particularly limited, and for example, normal resonance Raman spectroscopy or surface-enhanced Raman spectroscopy (SERS) can be used. Due to the nature of the measurement method, Raman spectroscopy has a specific peak in the Raman shift of several centimeters for each measurement. -1 The peak shift for each measurement is often ±10 cm. -1 Within ±10cm from the peak position -1 Raman shifts within 100 .mu.m are considered to represent the same signal. The specific peak representing polysulfide shifts depending on the measurement target and is not particularly limited. For example, when measuring cancer tissue as the measurement target, the Raman shift is 480±10 cm -1 When the solution is measured, the Raman shift is 460±10cm. -1 A peak appears at the position. Here, the level of polysulfide refers to the height of a specific peak representing polysulfide when Raman analysis is used, but when other measurement methods are used, it may refer to an actual measurement value of the amount of polysulfide in the cancer tissue, a corrected value obtained by correcting the actual measurement value with a control measurement value, or an index value.

[0012] The anticancer drug is not particularly limited as long as it is an anticancer drug reactive with polysulfide. When the anticancer drug reacts with polysulfide, the anticancer effect of the anticancer drug is eliminated, and the therapeutic effect of the anticancer drug is not obtained. Examples of the anticancer drug reactive with polysulfide include cisplatin and gemcitabine, and preferably cisplatin.

[0013] The target tissue is a tissue to be measured that is collected from a patient. When the control tissue is a cancer tissue, the cancer tissue is not particularly limited, but for example, the cancer tissue is an ovarian cancer tissue or a pancreatic cancer tissue. Preferably, the cancer tissue is an ovarian cancer tissue. Preferably, the cancer tissue is isolated.

[0014] Resistance to anticancer drugs refers not only to cases where anticancer drugs are completely ineffective, but also to cases where the therapeutic effect of anticancer drugs is weak and insufficient, or where anticancer drugs are effective temporarily but then the cancer relapses soon after.

[0015] The data for determining resistance to an anticancer drug may be obtained either before or after administering the anticancer drug to the patient. However, in order to determine whether the cancer tissue of the patient has resistance to the anticancer drug before administering the anticancer drug to the patient, it is preferable to obtain the data before administering the anticancer drug to the patient.

[0016] Another aspect of the present invention is a method for obtaining predictive data on the presence of cancer cells and / or resistance of cancer cells to an anticancer drug, the method using polysulfide level as an indicator and indicating the presence of cancer cells in the target tissue and / or the resistance of cancer cells to an anticancer drug when the polysulfide level in the target tissue is higher than the polysulfide level in a control tissue, the method comprising the step of: (a) detecting a presence of cancer cells in the target tissue and / or indicating that the cancer cells have resistance to the anticancer drug; and (b) determining whether the anticancer drug is an anticancer drug reactive with polysulfides. With regard to the measurement method, anticancer agent, cancer tissue, etc. in this embodiment, those described above in the data acquisition method for determining the presence of cancer cells and / or the anticancer agent resistance of cancer cells can all be applied mutatis mutandis (the same applies to the other embodiments described below). If the cancer cells are determined to be resistant to anticancer drugs, the administration of the anticancer drug reactive with polysulfides can be discontinued, and the treatment can be switched to a step of administering an anticancer drug that is not reactive with polysulfides.

[0017] The control tissue refers to, for example, a normal tissue of a corresponding part in a healthy individual, or a cancer tissue of a corresponding part in another cancer patient having cancer in the corresponding tissue.

[0018] The expression "the polysulfide level in the cancer tissue is higher than the polysulfide level in the control tissue" preferably means that the polysulfide level in the cancer tissue is higher than the polysulfide level in the control tissue, preferably by 25% or more. A statistically significant difference is preferred, but does not necessarily have to be present.

[0019] Another aspect of the present invention is the use of polysulfides as markers for determining the presence of cancer cells and / or resistance of cancer cells to anticancer drugs, wherein the anticancer drug is an anticancer drug reactive with polysulfides.

[0020] The discriminative marker of the present invention may be used in combination with other discriminative markers.

[0021] Another aspect of the present invention is a kit for determining the presence of cancer cells and / or anticancer drug resistance of cancer cells, comprising a substrate for measuring polysulfide levels by Raman spectroscopy, The kit further comprises an anti-cancer drug that is reactive with polysulfides.

[0022] The kit may include, for example, a reagent or instrument for collecting cancer tissue, a substrate or instrument for measuring the level of polysulfides in cancer tissue by Raman spectroscopy, etc. The substrate is not particularly limited as long as it can measure the level of polysulfides by Raman spectroscopy, and an example of the substrate is a GNF (Gold-nanofeve) substrate. The GNF substrate is a substrate on which broad bean-shaped Au nanoparticles (Gold-nanofeve (GNF)) are randomly arranged. The method for producing the GNF substrate is not particularly limited, but can be carried out, for example, by the following method. First, aluminum (Al) is evaporated onto a glass plate. The evaporation method or the sputtering method, which are known to those skilled in the art, can be used for the evaporation, but the sputtering method is preferred from the viewpoint of increasing the adhesion of Al to the plate. For the sputtering method, for example, a reactive DC magnetron sputtering system (SPF-530H, ANELVA) can be used, and the evaporation rate is preferably 4 to 6 Å s -1 Al is evaporated onto the plate by vapor deposition. The thickness of evaporated Al is preferably 40 to 60 nm. Note that, before the evaporation of Al, the glass plate is preferably washed with a surfactant to prevent contamination with organic matter, rinsed with ultra-purified water in an ultrasonic bath, and dried. The plate on which Al has been deposited is then boiled in hot water (100°C) for preferably 10 to 20 minutes, thereby forming boehmite (AlO(OH)) on the plate, which may then be dried under nitrogen gas. Then, a GNF substrate can be produced by evaporating gold (Au) onto the plate by a deposition method known to those skilled in the art, preferably at a deposition angle of 75 to 85°. The deposition of Au can be carried out, for example, by an electron beam deposition system (EBX-8C, ULVAC). After placing the tissue slice on the substrate such as the GNF substrate prepared as described above, the substrate is irradiated with near-infrared laser light, and the Raman scattering light reflecting the atomic vibration of polysulfides is enhanced by using the near-field light generated by the irradiation. The enhanced Raman scattering light is detected by an inverted Raman microscope system (RAMANforce, Nanophoton Corporation, Suita, Osaka), and the polysulfide level can be measured. Additionally, it may include a package insert describing procedures and diagnostic criteria. EXAMPLES

[0023] The examples are set forth for purposes of disclosure and are not intended to limit the scope of the invention.

[0024] Materials and Methods Samples used The experimental data using cancer tissues described in the Examples were obtained with approval from the Internal Review Boards of the National Defense Medical College (NDMC, Tokorozawa) and the National Cancer Center (NCC, Tokyo). The ovarian cancer tissues from patients used were obtained from the NCC Biobank.

[0025] Gel shift assay To quantify the degree of DNA intercalation by cisplatin (CDDP), which inhibits DNA supercoiling, 1 μg of plasmid (pcDNA3.1 empty vector, #V79020, Invitrogen) was mixed with a given concentration of Na2Sn (n = integer 1 to 4) and preincubated in 10 mM phosphate-buffered saline (PBS, pH 6.0) at room temperature for 5 minutes. Then, CDDP solution was added to the solution to a final concentration of 60 μM. The mixture was then incubated at 37 °C for 60 minutes. After incubation, the reaction products in the mixture were separated by electrophoresis using a 1% agarose gel to confirm the DNA secondary structure.

[0026] Conventional Resonance Raman Spectroscopy Analysis The conventional resonance Raman spectroscopy analysis was carried out by a method known to those skilled in the art. Specifically, for example, it can be carried out by the method described in Non-Patent Document 3.

[0027] Surface-enhanced Raman spectroscopy (SERS) analysis <Creating the board> 24×24×0.5mm 3 The glass plate was washed with a surfactant (W304 / LB93, ADEKA Corp.) to prevent organic contamination, and rinsed with ultra-purified water in an ultrasonic bath. After drying the plate with a spin dryer, aluminum (Al) was reactively sputtered using a DC magnetron sputtering system (SPF-530H, ANELVA) for 5 Å s. -1The Al film was evaporated on the plate to a thickness of 50 nm using a deposition rate of 1000 nm. The Al film was then boiled in hot water (100 °C) for 15 min to form boehmite (AlO(OH)), which was then dried with nitrogen gas. Gold (Au) was then evaporated at a deposition angle of 80° using an electron beam evaporation system (EBX-8C, ULVAC). Using this method, a random array of broad bean-shaped Au nanoparticles, named Gold-nanofeve (GNF), was constructed on the substrate. Using this substrate, electromagnetic hot spots as an excitation source can be generated in surface-enhanced Raman spectroscopy (SERS) analysis, allowing the molecular vibrations of metabolites in tissue sections to be visualized over a wide area with sufficient sensitivity and uniformity. <Raman spectrum measurement> The Raman spectra were measured using an inverted Raman microscope system (RAMANforce, Nanophoton Corporation, Suita, Osaka, Japan) equipped with a 50x (NA = 0.65) objective lens. The microscope system was equipped with an xy-scan stage and a 24 mW 785 nm diode laser for a line-scan laser confocal system. The sensitivity and frequency of the microscope system were adjusted to the Raman shift of the silicon phonon mode of 520 cm before the SERS measurements. -1 was calibrated.

[0028] <GNF-SERSイメージング> Metabolites in air-dried tissue sections prepared from frozen blocks of pancreatic cancer were visualized by mounting 5-μm-thick tissue sections on GNF substrates and placing them in a vacuum drying chamber. Prior to the SERS imaging experiment, three micro-scratches were made in the outer area of ​​the tissue slice on the surface of the SERS substrate using a microneedle. The micro-scratches could be identified by optical microscopy and SERS imaging, allowing the orientation of SERS imaging and HE staining to match. To achieve SERS imaging with a higher signal-to-noise ratio, the micro-scratches were aligned at ±10 cm. -1 The SERS signal was accumulated at the central peak wavenumber of .

[0029] <Pathological annotation of cancer and interstitial cancer> Immediately after SERS imaging of the tissue sections, HE staining was performed on the same tissue sections. Microscopic images of HE-stained tissue sections mounted on optically transparent GNF substrates were imported as digital photo files using NanoZoomer v.2.0-HT (Hamamatsu Photonics). Cancer cells were annotated into cancerous and cancer stroma by a pathology expert using NDP view2 software (Hamamatsu Photonics). When cancer cells were contained in the candidate stroma area within the cancer tissue, the area was not annotated.

[0030] <Example 1: Identification of proteins expressed in CCC (ovarian clear cell carcinoma)> The present inventors have found that, for example, CCC (ovarian clear cell carcinoma) is an example of a cancer for which anticancer drugs are ineffective, while SC (ovarian serous carcinoma) is an example of a cancer for which anticancer drugs are effective. To investigate whether different proteins are expressed between CCC and SC, surface-enhanced Raman spectroscopy (SERS) analysis was performed. To investigate the number of peaks in the SERS spectrum specific to the cancerous region of CCC, spectral measurements were performed on 12 CCC patients and 12 SC patients as controls, and the respective spectra were averaged (Figure 1). Furthermore, a spectral diagram showing the difference between the spectra was created by subtracting the average spectrum of SC from the average spectrum of CCC. As a result, the peak significant to CCC was found to be at a Raman shift of 295 to 296 cm. -1 and 478~480cm -1 The peak significant for SC is shown at 720-722 cm -1 It was shown that these peaks appear at positions of ±10 cm. -1 The range was analyzed as one signal. In addition, based on the results of the inventors' research to date, the 298 cm -1 , 480cm -1 and 978 cm -1The peaks at 480 cm confirmed by the SERS spectrum showing the difference between CCC and SC are known to indicate the presence of active sulfur species such as reduced glutathione (GSH), polysulfides, and hypotaurine (Non-Patent Document 1). -1 It was confirmed that the peak at the position was polysulfide. Furthermore, when we confirmed whether the difference between CCC and SC was statistically significant, we found that the 480 cm peak suggested the presence of polysulfides. -1 Regarding the peak at 298 cm, a significant difference was confirmed between CCC and SC (Fig. 2). -1 and 978 cm -1 Regarding peak, there was no statistically significant difference between CCC and SC.

[0031] <Example 2: Inhibition of supercoiling of double-stranded DNA by cisplatin (CDDP) and the elimination of the supercoiling inhibition by polysulfide> It has been shown that cisplatin is inserted between the bases of the double-stranded structure of DNA, causing a structural change, which inhibits DNA supercoiling (Non-Patent Document 4). Here, we confirmed in vitro that the bulk molecular volume of cisplatin-induced supercoiled DNA changes, and that the bulk molecular volume is restored to its original state by the addition of polysulfide (Figure 3). First, cisplatin increased the bulk molecular volume of double-stranded DNA, suggesting that it inhibits DNA supercoiling. By gradually increasing the concentration of polysulfides, we showed that the increase in cisplatin-induced DNA molecular volume was suppressed in a concentration-dependent manner (Figure 3). It is noteworthy that the inhibitory effect on the cisplatin-induced increase in DNA molecular volume was strengthened as the number of sulfur atoms in each polysulfide increased. This is presumably because the reducing power of polysulfides increases with the number of sulfur atoms.

[0032] <Example 3: Confirmation of disappearance of conventional resonance Raman spectroscopy signals derived from polysulfides by cisplatin> To confirm whether cisplatin interacts directly with polysulfides such as Na2S4, we examined the non-SERS conventional resonance Raman spectroscopy (Figures 4 and 5). As can be seen from these figures, in conventional resonance Raman spectroscopy, cisplatin exhibits four main peaks (316, 327, 504, and 522 cm -1 ) (Figure 4). Furthermore, when 5 mM Na2S4 was added to cisplatin, the four peaks derived from cisplatin disappeared, and the peak derived from Na2S4 (471 cm -1 ) was also confirmed to disappear (Figure 5). These results demonstrated that the DNA intercalation activity of CDDP was eliminated by binding to polysulfides. Based on the above, we performed Raman spectroscopy analysis of cancer tissue and found that the 480 cm -1 It was suggested that a high peak, i.e., a high expression level of polysulfides in cancer tissue, can be used as an indicator to determine whether the cancer is resistant to anticancer drugs.

[0033] <Example 4: Confirmation of disappearance of SERS signal derived from polysulfide by gemcitabine> To confirm whether gemcitabine interacts directly with the polysulfides of Na2S3 and Na2S4, the SERS spectra were examined (Figures 6-8). As can be seen in these figures, gemcitabine exhibits one major peak (435 cm -1 ) (Figure 6). Furthermore, when 100 μM of Na2S3 or Na2S4 was added to gemcitabine, one peak derived from gemcitabine disappeared, and a peak derived from Na2S3 or Na2S4 (456 cm) was observed. -1 ) was also eliminated (Figures 7 and 8). These results demonstrate that the binding of gemcitabine to polysulfides eliminates the anticancer drug activity of gemcitabine.

[0034] Example 5: Detection of polysulfides in pancreatic cancer tissue by SERS imaging The present inventors performed SERS imaging by the above-mentioned method and HE staining by a method known to those skilled in the art using pancreatic cancer tissue. In SERS imaging, the Raman shift was 480±10 cm -1 The range of 100 to 200 was considered to be the same signal. As a result of SERS imaging, in pancreatic cancer, the ion concentration was 480±10cm throughout the entire cancer tissue, including not only the cancerous area (the area annotated in black in the HE staining image) but also the surrounding area (the interstitial area, annotated in white in the HE staining image). -1 It was revealed that a strong SERS signal was observed in the chronic pancreatitis group (upper left and upper right figures). As a control, the results of SERS imaging of chronic pancreatitis are also shown, but only a weak signal was confirmed in the chronic pancreatitis group (lower left and lower right figures). Considering the above results, it was suggested that pancreatic cancer can also be determined to have resistance to anticancer drugs that are reactive with polysulfides because the level of polysulfides in the cancer tissue is high. In addition, pancreatic cancer testing usually involves inserting a needle through the stomach wall while checking with endoscopic ultrasound to obtain pancreatic tissue for testing, but in many cases surrounding CAF (cancer associated fibroblast) is obtained, making it difficult to determine whether the cancer is present. However, this invention makes it possible to clearly diagnose cancer.

Claims

1. A data acquisition method for determining anticancer drug resistance of cancer cells in a target tissue, comprising a step of measuring a level of polysulfide in the target tissue, the anticancer drug is a polysulfide-reactive anticancer drug; and The method, wherein the anti-cancer drug reactive with said polysulfides is cisplatin or gemcitabine.

2. The method according to claim 1 , wherein the measurement is performed before administration of an anticancer drug.

3. 3. The method according to claim 1 or 2, wherein the measuring step is by detecting peaks specific to polysulfides by Raman spectroscopy.

4. When the specific peak is subjected to Raman spectroscopy in cancer tissue, the Raman shift is 480±10 cm －１ The method according to claim 3, wherein the peak appears at the position

5. The method according to any one of claims 1 to 4, wherein the target tissue is ovarian cancer tissue or pancreatic cancer tissue.

6. The method according to claim 5 , wherein the ovarian cancer tissue is ovarian clear cell cancer tissue.

7. A method for obtaining predictive data on resistance of cancer cells to an anticancer drug, the method comprising the steps of: using a polysulfide level as an index; and, when the polysulfide level in a target tissue is higher than the polysulfide level in a control tissue, indicating that the cancer cells in the target tissue have resistance to the anticancer drug, comprising: The method, wherein the anti-cancer drug is an anti-cancer drug reactive with polysulfides, and the anti-cancer drug reactive with polysulfides is cisplatin or gemcitabine.

8. Use of polysulfides as a marker for determining anticancer drug resistance in cancer cells, comprising: The anti-cancer drug is an anti-cancer drug reactive with polysulfides, and the anti-cancer drug reactive with polysulfides is cisplatin or gemcitabine.

9. A kit for determining anticancer drug resistance of cancer cells, comprising a substrate for measuring a polysulfide level by Raman spectroscopy, The kit, wherein the anti-cancer drug is an anti-cancer drug reactive with polysulfides, and the anti-cancer drug reactive with polysulfides is cisplatin or gemcitabine.

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