CN114292895A

CN114292895A - Application of substance for detecting PARP inhibitor tolerance in preparation of product for evaluating sensitivity of lung cancer patient to proton radiotherapy

Info

Publication number: CN114292895A
Application number: CN202210133317.7A
Authority: CN
Inventors: 杨庆源
Original assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2022-02-10
Filing date: 2022-02-10
Publication date: 2022-04-08

Abstract

The invention relates to the technical field of biological medicines, in particular to application of a substance for detecting PARP inhibitor tolerance in preparation of a product for evaluating the sensitivity of a lung cancer patient to proton radiotherapy. The invention discovers that NSCLC cells have half inhibitory concentration IC to Olaparib₅₀Value and RBE of proton radiotherapy_0.1The values are positively correlated, and the half inhibitory concentration IC₅₀The larger the value, the RBE_0.1The larger the value, i.e., the more resistant the NSCLC cell to Olaparib, indicates that the NSCLC cell is more sensitive to proton radiation therapy. In addition, cells with no significant change in the percentage of RAD51foci positive cells were found to be more prone to Olaparib resistance following Olaparib treatment. Therefore, the percentage of RAD51foci positive cells after Olaparib effect appears to be a combined predictive molecular marker for the sensitivity of proton radiotherapy in NSCLC patients.

Description

Application of substance for detecting PARP inhibitor tolerance in preparation of product for evaluating sensitivity of lung cancer patient to proton radiotherapy

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of a substance for detecting PARP inhibitor tolerance in preparation of a product for evaluating the sensitivity of a lung cancer patient to proton radiotherapy.

Background

According to the tumor epidemiological report of 2019, the incidence rate of lung cancer is about 13%, and the most common histopathological type is non-small cell lung cancer (NSCLCs). Unfortunately, the 5-year survival period of the NSCLC patients after diagnosis only accounts for 25 percent at present, so that the improvement of the early diagnosis of the NSCLC and/or the enrichment diagnosis of the targeted therapy population has wide clinical application prospect. Although NSCLC patients currently benefit from targeted chemotherapeutic drugs and immunotherapy, we can see that radiation therapy is the treatment of choice in patients with early stage and localized NSCLC according to the NCCN guidelines. The main advantage of proton therapy over photon therapy is that it allows SOBP (Spread-out bragg peak) to reach the tumor site directly with little or no radiation to surrounding normal tissues, and therefore, with little or no short-term and long-term side effects; the life quality of the cancer patient is improved; reducing the impact on developing pediatric patients; reducing the risk of secondary cancer. The optimal position (Spread-out bragg peak) of proton radiotherapy directly reaches the tumor, the energy of X-rays is highest on the body surface, and the energy is weakened after reaching the tumor, so that the irradiation intensity is not enough to cause great radiation to surrounding tissues, thereby increasing the side effect of a patient and the incidence rate of secondary tumor.

Is currently the most advanced and ideal tumor radiotherapy technology internationally recognized in the field of clinical radiotherapy. Because of high technical requirements, high infrastructure cost and long time consumption of proton radiotherapy, at present, only the proton heavy ion hospital in tumor hospitals and the Ruijin proton heavy ion therapy which is declared to be put into use in 2021 are put into real clinical application, and 9 proton centers in other China are still in the process of construction.

After the proton heavy ion hospital in Shanghai city is put into use from 2015, 31 cases of lung cancer (early lung cancer) in the stage I-II are treated by 12 months in 2018, and clinical data show that the 2-year local control rate of tumor of patients is up to 96%, the 2-year survival rate is 91%, the toxic and side effects are slight, and only 1 case of patients has grade III toxic and side effects which are far lower than the grade III toxic and side effects of conventional photon treatment (the incidence rate can be reduced by about 50%). Has no wound, high tumor control rate and slight toxic and side effects, and is a prominent advantage for treating early lung cancer by proton radiotherapy. Meanwhile, clinical data of proton heavy ion hospitals in Shanghai city show that the indications of proton therapy are greatly widened compared with ordinary photon radiotherapy due to high energy level and strong penetrability, and the proton therapy is mainly recommended to be used for central nervous system tumors (meningioma, pituitary tumor, acoustic neuroma and astrocytoma), skull base tumors (chordoma and chondrosarcoma), head and neck tumors (nasopharyngeal carcinoma, oral cancer, pharyngeal tumor, laryngeal tumor and adenoid cystic cancer), chest tumors (lung cancer, esophageal cancer and thymoma), abdominopelvic tumors (liver tumor, prostate cancer, pancreatic cancer and recurrent rectal cancer) and other tumors (breast cancer, bone and soft tissue sarcoma).

However, in clinical proton therapy, the radiation dose of a patient is mainly determined according to the radiobiological effect value (RBE), and a 'one-cutting' scheme is adopted at present, namely, the proton radiation dose (RBE) required by 90 percent of the tumor lethality of all tumors compared with photon therapy is adopted_0.1X-ray dose/Proton dose) to calculate the total radiation dose for the tumor patient. However, in actual clinical practice, researchers have found that some tumors are more sensitive to proton radiation therapy and some tumors are more resistant to proton radiation therapy, if still in accordance with RBE_0.11.1, toxic and side effects on some sensitive tumors still exist, or unnecessary economic burden is brought to sensitive tumor patients (according to the global tumor doctor network, the cost of proton treatment in China is 25-30 ten thousand RMB). Although preclinical data show that tumors deficient in homologous recombination injury repair (HR deficiency) are more sensitive to proton radiation therapy, cytological studies suggest RBE in HR-deficient cells_0.1Still less than 1.15 of the mean value of in vitro tumor cells, and the clinical radiation dose is reduced by only about 5 percent, and the clinical transformation potential is very limited.

The data of the currently developed proton therapy-related clinical research (clinicaltal. com) show that the main research aims at exploring the toxic and side effects of proton therapy, and no proton radiotherapy-related clinical experiments accompanied with diagnosis of molecular marker dependence exist, which indicates that the precise implementation of proton therapy currently faces the dilemma of effective molecular markers.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide the use of substances that are resistant to PARP inhibitors for the preparation of a product for assessing the susceptibility of lung cancer patients to proton radiation therapy.

The invention discovers that the cells have half inhibitory concentration IC to Olaparib by treating 15 NSCLC cells through Olaparib₅₀Value and RBE of proton radiotherapy_0.1The values are positively correlated, and it is also found by the GDSC database that the more resistant the NSCLC cell is to Olaparib, the more sensitive the proton treatment is; further, by establishing the availabilityVerifying the Olaparib drug-resistant cells, and finding that the acquired Olaparib drug-resistant cells are more sensitive to proton radiotherapy; furthermore, cells with no significant change in the percentage of RAD51foci positive cells after Olaparib treatment were more prone to Olaparib resistance. In conclusion, the combined detection of the RAD51foci positive cell percentage after the PARPi effect in the NSCLC cells is proposed to be capable of remarkably predicting the sensitive population of the tumor to proton treatment and provide reliable accompanying diagnostic molecular markers for precise proton treatment.

One of the objects of the present invention is to provide the use of a substance that is resistant to PARP inhibitors for the preparation of a product for assessing the susceptibility of lung cancer patients to proton radiation therapy.

In certain embodiments of the invention, the detecting a substance resistant to a PARP inhibitor comprises detecting a half maximal inhibitory concentration IC of the PARP inhibitor₅₀A value; and/or, detecting the percentage of RAD51foci positive cells.

It is a further object of the present invention to provide a target for resistance to PARP inhibitors, said target being RAD 51.

It is a further object of the present invention to provide a product for assessing resistance to a PARP inhibitor, said product comprising a substance which detects resistance to a PARP inhibitor; and/or, a substance that measures the percentage of RAD51foci positive cells after exposure to a PARP inhibitor.

In certain embodiments of the invention, the product comprises a kit, a chip, a membrane strip.

The present invention also provides a method for assessing tolerance to a PARP inhibitor, comprising the steps of:

biological samples were treated with PARP inhibitors and the treated biological samples were then tested for percent positive cells for RAD51 foci.

The fourth purpose of the present invention is to provide a biomarker for evaluating the susceptibility of lung cancer patients to proton radiotherapy, wherein the biomarker is RAD 51.

The fifth purpose of the invention is to provide a method for evaluating the susceptibility of a lung cancer patient to proton radiotherapy, which comprises the following steps:

detecting biological samplesHalf inhibitory concentration of the present PARP inhibitor IC₅₀Values and/or percentage of positive cells for RAD51foci after PARP inhibitor exposure;

according to the half inhibitory concentration IC₅₀Values and/or percentage of positive cells for RAD51foci, lung cancer patients were assessed for sensitivity to proton radiation therapy.

In certain embodiments of the invention, at least one of the following is included:

1) susceptibility of lung cancer patients to proton radiation therapy and the median inhibitory concentration IC₅₀The values are positively correlated;

2) the sensitivity of lung cancer patients to proton radiation therapy correlates negatively with the percentage of RAD51foci positive cells.

In certain embodiments of the invention, 1) when said half inhibitory concentration IC₅₀If the value is more than or equal to 10 mu M, the lung cancer patient is sensitive to proton radiotherapy; when the half inhibitory concentration IC₅₀Value of<And when the concentration is 10 mu M, the lung cancer patient is not sensitive to proton radiotherapy.

When half inhibitory concentration IC₅₀The higher the value, the more resistant the lung cancer patient is to PARP inhibitors, indicating susceptibility to proton radiation therapy. Subsequently, proton radiotherapy is carried out.

When half inhibitory concentration IC₅₀Lower values indicate that the lung cancer patient is more sensitive to PARP inhibitors, indicating insensitivity to proton radiation therapy. Subsequent PARP inhibitor treatment.

In certain embodiments of the invention, the sensitivity of a lung cancer patient to proton radiation therapy is inversely related to the percentage of RAD51foci positive cells.

When the percentage of RAD51foci positive cells did not change significantly (P >0.05), it indicated that lung cancer patients were susceptible to proton radiation therapy. Subsequently, proton radiotherapy is carried out.

When the percentage of RAD51foci positive cells increased significantly (P <0.05), it indicated that lung cancer patients were not susceptible to proton radiation therapy. Followed by Olaparib treatment.

In certain embodiments of the present invention, the PARP inhibitor is selected from at least one of Olaparib, Rucaparib and Niraparib.

In certain embodiments of the present invention, the PARP inhibitor is Olaparib.

In certain embodiments of the invention, the lung cancer comprises non-small cell lung cancer and small cell lung cancer.

In certain embodiments of the invention, the lung cancer inhibitor is non-small cell lung cancer.

In certain more specific embodiments of the invention, the non-small cell lung cancer comprises squamous cell carcinoma, large cell carcinoma and adenocarcinoma of the lung.

Compared with the prior art, the invention has the following beneficial effects:

1) the present invention finds that the half inhibitory concentration IC of PARP inhibitor₅₀Value and proton radiotherapy RBE_0.1The values are positively correlated, and the half inhibitory concentration IC₅₀The larger the value, the RBE_0.1The larger the value, i.e., the more resistant the NSCLC cell to Olaparib, indicates that the NSCLC cell is more sensitive to proton radiation therapy.

2) According to the invention, cells with no significant change in the RAD51foci positive cell percentage after the action of Olaparib are more prone to Olaparib drug resistance, so that the RAD51foci positive cell percentage can be used as a combined predictive molecular marker for the proton radiation sensitivity of NSCLC patients. The percent of RAD51foci positive cells has no significant change after the Olaparib effect, so that NSCLC patients can be subjected to proton radiation therapy, and the corresponding radiation dose is more than that of 'one-knife cut' RBE_0.1The radiation dose calculated as 1.1 can be reduced by about 20% to 30%.

Drawings

FIG. 1 is a graph showing the survival rate of 15 NSCLC cells after 6 days of treatment with different concentration gradients of Olaparib solution in example 1 of the present invention.

FIG. 2 is a graph comparing the percentage of RAD51foci positive cells in 13 NSCLC cells after 8h of treatment with 10 μ M Olaparib solution in example 1 of the present invention. Wherein: p <0.05, p <0.01, p <0.001, p < 0.0001.

FIG. 3 is a graph showing the cell survival of H23 cells and H23PR cells after 6 days of treatment with different concentration gradients of Olaparib solution in example 1 of the present invention. Wherein Ctrl represents H23 cells, and PR represents H23PR cells.

Fig. 4 is a graph showing cell survival curves of a549 cells and a549PR cells after 6 days of treatment with varying concentration gradients of Olaparib solutions in example 1 of the present invention, respectively. Wherein Ctrl represents A549 cells, and PR represents A549PR cells.

FIG. 5 is a graph showing the percentage of RAD51foci positive cells among H23PR cells, A549PR cells and H460PR cells after 8 hours of treatment with 10. mu.M Olaparib solution in example 1 of the present invention.

FIG. 6 shows the half inhibitory concentration IC of Olaparib in 12 cells in example 2 of the present invention₅₀Value and RBE of proton radiotherapy_0.1Correlation plot of values.

FIG. 7 shows the half inhibitory concentration IC of Olaparib in 19 NSCLC cells extracted from GDSC database in example 2 of the present invention₅₀Value and RBE of proton radiotherapy_0.1Correlation plot of values.

Figure 8 shows a volcano plot of lung adenocarcinoma cell (LUAD) genetic mutations and Olaparib chemotherapy sensitivity and tolerance obtained from the COSMIC cell database in example 3 of the present invention. Wherein mut represents a mutation, the left arrow represents the more left-going, the more sensitive to Olaparib; the arrow on the right represents the more right-going, the more tolerant to Olaparib.

FIG. 9 shows the half inhibitory concentration IC of ARID1A truncated mutant NSCLC cells and ARID1A wild-type NSCLC cells against Olaparib obtained from GDSC cell database in example 3 of the present invention₅₀Bar graph of values. Wherein WT represents a wild type, and Mut represents a mutant type.

FIG. 10 is a graph showing RBE of proton radiation therapy of ARID1A truncated mutant NSCLC cells and ARID1A wild-type NSCLC cells in example 3 of the present invention_0.1Comparative plot of values. Wherein WT represents a wild type, and Mut represents a mutant type.

FIG. 11 is a immunoblot of ARID1A expression in ARID1A wild-type NSCLC cells and ARID1A truncated mutant NSCLC cells of example 4 of the present invention.

FIG. 12 is a graph showing the expression level of ARID1A in 8 NSCLC cells in example 4 and RBE in proton radiotherapy_0.1Pearson correlation analysis plot of values.

FIG. 13 shows RBE of proton radiation therapy on four subtypes of NSCLC cells in example 4 of the present invention_0.1A statistical map of values. Wherein Res WT represents orid 1A wild-type cells tolerant to Olaparib; res Mut represents ARID1A truncated mutant cells resistant to Olaparib; sen WT represents ARID1A wild-type cells sensitive to Olaparib; sen Mut represents ARID1A truncated mutant cells sensitive to Olaparib.

FIG. 14 is a diagram showing an immunoblot of H23 cells after 48H of interfering ARID1A expression by siRNA technique in example 5 of the present invention. Wherein siCtr1 is blank control siRNA, siARID1A represents siRNA targeting ARID1A knockout.

FIG. 15 is a graph showing the cell survival curves of H23 cells after 48H of interfering ARID1A expression by siRNA technology and then treated with different concentration gradients of Olaparib solution for 6 days in example 5 of the present invention. Wherein R represents tolerance.

FIG. 16 shows RBE of proton radiation therapy of siCtr1 group and siaRID1A group in example 5 of the present invention_0.1Comparative plot of values.

FIG. 17 is a immunoblot of ARID1A expression in CALU6 cells after 72h of treatment with 0. mu.M, 5. mu.M, 10. mu.M Olaparib solution in example 5 of the present invention.

FIG. 18 is a graph showing the cell survival of CALU6 cells, H23 cells, and A549 cells after 6 days of treatment with different concentration gradients of Olaparib solutions in example 5 of the present invention. Wherein R represents tolerance and S represents sensitivity.

FIG. 19 shows RBE of proton radiation therapy of CALU6 cells after 24h of Olaparib treatment and X-ray or proton radiation therapy in example 5 of the present invention_0.1A graph of values. Wherein, the Vehicle group represents a blank control group, and the cells are treated by DMSO for 24h and then receive X-ray or proton radiotherapy; the Olaparib group represents the experimental group, and the cells were treated with 2.5. mu.M Olaparib solution for 24h and then subjected to X-ray or proton radiation therapy.

FIG. 20 shows immunoblots of ARID1A from H23 cells and H23PR cells in example 6 of the present invention.

FIG. 21 is a graph showing statistical analysis of biological data of ARID1A expression levels in H23 cells and H23PR cells in example 6 of the present invention.

FIG. 22 shows immunoblots of A549 cells and A549PR cells ARID1A in example 6 of the present invention.

Fig. 23 shows a graph of statistical analysis of biological repeats expressed by ARID1A in a549 cells and a549PR cells in example 6 of the present invention.

FIG. 24 is a graph showing the survival rate of H23PR cells cloned after X-ray (X) and proton (P) irradiation, respectively, in example 6 of the present invention. Wherein X represents cells treated by X-ray radiotherapy, and P represents cells treated by proton-ray radiotherapy.

FIG. 25 is a graph showing RBE of proton radiation therapy of H23 cells and H23PR cells in example 6 of the present invention_0.1Comparative plot of values.

FIG. 26 is a graph showing the cell survival curves of H23 cells, H23PR cells, and H23PR ARID1A-V5 cells after 6 days of treatment with different concentration gradients of Olaparib solution in example 6 of the present invention. Wherein, H23PR ARID1A-V5 represents that ARID1A is overexpressed by H23PR cells.

FIG. 27 is a graph showing the survival rate of H23PR ARID1A-V5 cells cloned after X-ray (X) and proton (P) chemotherapy in example 6 of the present invention. Wherein, the graph A is a survival curve chart formed by cell cloning of H23PR ARID1A-V5 cells after chemotherapy by X rays (X) and proton rays (P); FIG. B is a graph showing the survival rate of H23PR cells after X-ray irradiation and proton ray irradiation (P) irradiation, and H23PR ARID1A-V5 cells after X-ray irradiation and proton ray irradiation (P) irradiation.

Fig. 28 is a diagram showing a549PR cells in example 6 of the present invention. Wherein: FIG. A is a graph showing the survival of A549PR cells cloned after X-ray and proton-ray chemotherapy, respectively; panel B shows RBE of proton radiation therapy of A549 cells and A549PR cells_0.1A comparison graph of values; FIG. C shows the efficiency of the immunoblotting to detect the interference of ARID1A after transfection of siCtrl or siARID1A 48h with A549PR cells; panel D shows A549 cells and A549PR cellsCell survival plots after Olaparib treatment after cell transfection with siCtrl or siARID 1A; FIG. E is a graph showing the survival rate of cell clones after transfection of siCtrl into A549PR cells and the respective actions of X-ray and proton ray (P); FIG. F is a graph showing the survival of cell clones after transfection of siARID1A with A549PR cells, respectively, by X-ray (X) or proton (P) radiation; panel G shows proton radiotherapy RBE after transfection of siCtrl or siARID1A with A549PR cells_0.1Comparative plot of values.

FIG. 29 is a technical scheme showing the sensitivity of NSCLC patients to proton radiation therapy.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

The invention discovers that the cells have half inhibitory concentration IC to Olaparib by treating 15 NSCLC cells through Olaparib₅₀Value and RBE of proton radiotherapy_0.1The values are positively correlated, and it is also found by the GDSC database that the more resistant the NSCLC cell is to Olaparib, the more sensitive the proton treatment is; furthermore, the acquired Olaparib drug-resistant cells are established for verification, and are found to be more sensitive to proton radiotherapy; furthermore, cells with no significant change in the percentage of RAD51foci positive cells after Olaparib treatment were more prone to Olaparib resistance. In summary, the following disclosure is providedThe combined detection of the RAD51foci positive cell percentage after the PARPi effect in the NSCLC cell can obviously predict the sensitive population of the tumor to the proton treatment, and provides a reliable concomitant diagnostic molecular marker for the precise treatment of the proton.

In a first aspect of the invention, there is provided the use of a substance that is resistant to a PARP inhibitor for the manufacture of a product for assessing the susceptibility of a patient with lung cancer to proton radiation therapy.

In the present invention, the detection of a substance resistant to a PARP inhibitor comprises detecting the median inhibitory concentration IC of the PARP inhibitor₅₀A value; and/or, detecting the percentage of RAD51foci positive cells.

In another aspect of the present invention, there is also provided a target for resistance to a PARP inhibitor, said target being RAD 51.

In another aspect of the present invention, there is also provided a product for assessing resistance to a PARP inhibitor, said product comprising a substance which detects resistance to a PARP inhibitor; and/or, a substance that measures the percentage of RAD51foci positive cells after exposure to a PARP inhibitor.

In the invention, the product comprises a kit, a chip and a membrane strip.

In another aspect of the present invention, there is also provided a method for assessing tolerance to a PARP inhibitor, comprising the steps of:

In another aspect of the present invention, there is also provided a biomarker for assessing susceptibility of a lung cancer patient to proton radiation therapy, wherein the biomarker is RAD 51.

In another aspect of the present invention, there is provided a method for assessing susceptibility to proton radiation therapy in a patient with lung cancer, comprising the steps of:

detection of half inhibitory concentration IC of biological samples against PARP inhibitors₅₀Values and/or percentage of positive cells for RAD51foci after PARP inhibitor exposure;

according to the half inhibitory concentration IC₅₀Value and/or percent positive cells for RAD51foci, evaluation of lung cancer patient tropismSusceptibility to radiotherapy.

In the present invention, at least one of the following is included:

In the present invention, 1) when the half inhibitory concentration IC₅₀If the value is more than or equal to 10 mu M, the lung cancer patient is sensitive to proton radiotherapy; when the half inhibitory concentration IC₅₀Value of<And when the concentration is 10 mu M, the lung cancer patient is not sensitive to proton radiotherapy.

The research of the invention finds that when the half inhibitory concentration IC₅₀The higher the value, the more resistant the lung cancer patient is to PARP inhibitors, indicating susceptibility to proton radiation therapy. Subsequently, proton radiotherapy is carried out. When half inhibitory concentration IC₅₀Lower values indicate that the lung cancer patient is more sensitive to PARP inhibitors, indicating insensitivity to proton radiation therapy. Subsequent PARP inhibitor treatment.

In the invention, in 2), when the percentage of RAD51foci positive cells has no significant change (P >0.05), the lung cancer patient is sensitive to proton radiotherapy, and then the proton radiotherapy is carried out; when the percentage of RAD51foci positive cells increased significantly (P <0.05), it indicated that lung cancer patients were not susceptible to proton radiation therapy, followed by Olaparib treatment. In particular, FIG. 29 is a technical scheme of susceptibility of NSCLC patients to proton radiation therapy.

In certain embodiments of the invention, the biological sample comprises a biological tissue or fluid. In some embodiments, the biological sample may be or comprise bone marrow; blood; blood cells; ascites fluid; tissue or fine needle biopsy samples; a body fluid containing cells; free floating nucleic acids; sputum; saliva; (ii) urine; cerebrospinal peritoneal fluid; pleural fluid; feces; lymph; a skin swab; orally administering the swab; a nasal swab; washings or lavages such as catheter lavages or bronchoalveolar lavages; (ii) an aspirate; scraping scraps; bone marrow specimen; a tissue biopsy specimen; a surgical specimen; feces, other body fluids, secretions and/or excretions; and/or cells therein, and the like. In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from a source of interest by any suitable means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of: biopsies (e.g., fine needle aspirates or tissue biopsies), surgical tissue, collection of bodily fluids (e.g., blood, lymph, stool, etc.), and the like.

In certain embodiments of the invention, the biological sample is NSCLC cells.

In certain embodiments of the invention, the level of the biomarker is compared to a reference control level. A "reference control level" or "reference control expression level" used interchangeably herein may be established using, for example, a reference control sample. Non-limiting examples of reference control samples include lung cancer patient specimens that are sensitive to proton radiation therapy.

In the following specific examples of the present application, the concentration gradient of the Olaparib solution with different concentration gradients is: 0. mu.M, 0.01. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M, 50. mu.M, 80. mu.M, 100. mu.M.

Example 1

In this example, the percentage change in RAD51foci positive cells in NSCLC cells after Olaparib treatment was examined and included as follows:

1. half inhibitory concentration IC of 15 NSCLC cells against Olaparib₅₀Value of

Experimental cells: 15 NSCLC cells, including H23 cell, H1792 cell, H1838 cell, H3122 cell, A549 cell, H460 cell, HCC44 cell, H1666 cell, CALU6 cell, H1703 cell, H1563 cell, JPC3 cell, H441 cell, COR-L105 cell, H1793 cell.

Reagent: olaparib (Olaparib) was dissolved in dimethyl sulfoxide and stored at-20 ℃ as a stock solution, and the drug Olaparib was diluted with a complete medium corresponding to cell culture to 12 Olaparib solutions of different concentration gradients of 0. mu.M, 0.01. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M, 50. mu.M, 80. mu.M, and 100. mu.M, respectively, before use.

15 experimental cells in logarithmic growth phase were seeded in 96-well plates at 8000 cells/mL, 100. mu.L per well. After 24h of cell culture and adherence, each cell was treated with Olaparib solution of different concentration gradient for 6 days, followed by 30. mu.l of CTG reagent

The operation of the kit instruction for detecting the cell viability by the luminescence method is used for detecting the cell viability. Percent cell survival was converted to a control of 0 μ M treatment, and half maximal Inhibitory Concentration (IC) of drug was calculated by plotting dose-response curves using Graph Prism 8 data processing software₅₀) Value, half inhibitory concentration IC of Olaparib in 15 NSCLCs cells₅₀The values are shown in Table 1.

TABLE 1

#	Cell Lines	Olaparib_AZD2281 IC₅₀(μM)
			1	H23	1.671
2	H1792	3.288
			3	H1838	5.566
4	H3122	6.146
			5	A549	6.438
6	H460	6.868
			7	HCC44	9.297
8	H1666	12.31
			9	CALU6	15.78
10	H1703	13.61
			11	H1563	19.53
12	JPC3	21.84
			13	H441	13.6
14	COR-L105	17.61
			15	H1793	25.32

As can be seen from Table 1, the half inhibitory concentration IC of Olaparib was observed after different cells were treated with Olaparib₅₀The values are different. According to half inhibitory concentration IC₅₀At a value of 10. mu.M, NSCLC cells were classified into endogenous Olaparib-resistant cells including H1666, CALU6, H1703, H1563, JPC3, H441, COR-L105, H1793, and endogenous Olaparib-sensitive cells including H23, H1792, H1838, H3122, A549, H460, HCC 44.

Using IC₅₀Two bases were used as threshold for Olaparib sensitivity and tolerance at 10 μ M: one is the clinical Olaparib blood concentration Cmax of 13.1 μ M, i.e. the highest Olaparib concentration in human blood that can be achieved is 13.1 μ M, and the references are: clin Cancer Res 2017Jul 15; 23(14) 3489-3498.doi 10.1158/1078-0432. CCR-16-3083; one is to define the clinical Olaparib drug-resistant system with IC₅₀>10 μ M is critical and references are: cancer Discov.2018 Nov; 8(11) 1404-1421.doi:10.1158/2159-8290. CD-18-0474.

FIG. 1 is a graph showing the cell survival rate of 15 NSCLC cells treated with different concentrations of Olaparib solution for 6 days in this example.

As can be seen from Table 1 and FIG. 1, the half inhibitory concentration IC of Olaparib in NSCLC cells₅₀The larger the value, the more tolerant the cell is to Olaprib.

2. Percentage of RAD51foci positive cells changed in 13 NSCLC cells after Olaparib treatment

Subject: 13 NSCLC cells, Olaparib sensitive cells including H23, H1792, H3122, a549, H460 and HCC 44; olaparib resistant cells include H1666, CALU6, H1703, H1563, JPC3, H441 and H1793.

After 13 NSCLC cells were treated with 10. mu.M Olaparib solution for 8h, the percentage of RAD51foci positive cells was determined by immunofluorescence and statistical analysis by t-test. Meanwhile, a control group is set up, and the reagent treated by the control group is DMSO and is marked as a Vehicle group.

The percentage of RAD51foci positive cells was: percentage of cells with RAD51foci number >10 out of 100 cells.

An immunofluorescence method:

4×10⁴cells were seeded in 8-well plates overnight, treated with 10 μ M Olaparib solution for 8h, washed with PBS, fixed with 4% PFA at room temperature for 15min, disrupted with 0.5% Triton X in PBS, and blocked with goat serum antibody (5% coat-serum in PBS) added dropwise for 45min at room temperature. Then primary antibody (3% GS in PBS) is added dropwise to incubate at 4 ℃ overnight, after the treatment is finished, the primary antibody is washed by PBS, then the secondary antibody is added dropwise to treat at 37 ℃ for 45min, DAPI is used for staining nuclei, and mounting agent is added dropwise to perform laser confocal observation.

FIG. 2 is a graph comparing the percentage of RAD51foci positive cells in 13 NSCLC cells treated with 10 μ M Olaparib solution for 8h in this example. Wherein: p <0.05, p <0.01, p <0.001, p < 0.0001.

As can be seen from fig. 2, the percentage of RAD51foci positive cells was significantly increased after treatment of endogenous Olaparib sensitive cells with Olaparib, while the percentage of RAD51foci positive cells was not significantly changed after treatment of endogenous Olaparib resistant cells with Olaparib.

3. Percentage change in RAD51foci positive cells in acquired Olaparib-resistant cells following Olaparib treatment

(1) Construction of acquired Olaparib drug-resistant cells

H23 cells, A549 cells and H460 cells are treated by a high-concentration pulse method (the Olaparib concentration is 100 mu M, the average time is 8 months) to construct acquired Olaparib resistant cells, and the acquired resistant cells are respectively marked as H23PR cells, A549PR cells and H460PR cells.

The references of the construction method of the drug-resistant cell are as follows: sharma SV, Lee DY, Li B, Quinlan MP, Takahashi F, mahanshan S, et al.a. chromium-mediated reversible drug-tolerant state in cancer cells subpartictions.cell.2010; 141(1):69-80.

Treating the H23PR cells and A549PR cells with Olaparib solution with different concentration gradients, and performing cell survival rate and IC₅₀And verifying whether the drug resistance model is constructed successfully.

FIG. 3 is a graph showing the cell survival of H23 cells and H23PR cells after 6 days of treatment with different concentration gradients of Olaparib solution in this example. Wherein Ctrl represents H23 cells, and PR represents H23PR cells.

As can be seen from FIG. 3, the half inhibitory concentration IC of H23 cells₅₀Value 2.584. mu.M, and half inhibitory concentration IC of H23PR cells₅₀The value is 19.43 mu M, which indicates that the H23 cell-acquired Olaparib drug-resistant model (H23PR) is successfully constructed.

FIG. 4 is a graph showing the cell survival of A549 cells and A549PR cells after treatment with different concentration gradients of Olaparib solutions for 6 days in this example. Wherein Ctrl represents A549 cells, and PR represents A549PR cells.

As can be seen from FIG. 4, the cell halves of A549Number inhibition concentration IC₅₀The value was 9.903. mu.M, whereas the median inhibitory concentration IC of A549PR cells₅₀The value was 27.39. mu.M, indicating that the acquired Olaparib resistance model (A549PR) was successfully constructed.

(2) Study of the percentage change in RAD51 foci-positive cells in H23PR cells, A549PR cells and H460PR cells after Olaparib treatment

The percentage of RAD51foci positive cells in each cell was determined after 8H of treatment with 10. mu.M Olaparib solution using H23PR cells, A549PR cells and H460PR cells as subjects. Meanwhile, a blank control group is set up, and the reagent treated by the blank control group is DMSO and is marked as a Vehicle group.

FIG. 5 is a graph showing the percentage of RAD51foci positive cells in H23PR cells, A549PR cells and H460PR cells after 8H of treatment with 10. mu.M Olaparib solution in this example.

As can be seen from fig. 5, the percentage of RAD51foci positive cells in the acquired Olaparib resistant cells was not significantly changed after treatment with Olaparib.

4. Relationship between Olaparib and percentage of cells RAD51foci positive

Subject: olaparib sensitive cells and Olaparib resistant cells; olaparib sensitive cells include H23, H1792, H3122, a549, H460, and HCC 44; olaparib resistant cells include H1666, CALU6, H1703, H1563, JPC3, H441, H1793.

The percentage of RAD51foci positive cells in the cells was obtained by treating the Olaparib sensitive cells and the Olaparib resistant cells with 10. mu.M of Olaparib solution for 8h, respectively, and then statistically analyzing the correlation between the change in the percentage of the Olaparib sensitive cells and the change in the percentage of the Olaparib resistant cells and the change in the RAD51foci positive cells, respectively, by using the Pearson chi's chi-square test. P >0.05 after t-test is NO, i.e. not significantly changed, p <0.05 is YES, i.e. significantly changed, and the results are shown in table 2.

TABLE 2

As can be seen from table 2, after 8h of Olaparib treatment, the percentage of RAD51foci positive cells in Olaparib sensitive cells significantly changed to 100%, while the percentage of RAD51foci positive cells in Olaparib resistant cells significantly changed to 43%, and Pearson chi-square statistical analysis found that p was 0.026, indicating that the two significantly different, i.e., the percentage of RAD51foci positive cells in Olaparib sensitive cells was more significantly changed.

From the results of this example 1, it can be seen that the percentage of RAD51foci positive cells in NSCLC cells without significant change after treatment with Olaparib is more resistant to Olaparib, while in the case of breast cancer cells with BRCA germline mutation causing defect in repair of homologous recombination injury (HR detoxification), the absence of RAD51foci after PARPi action suggests that the breast cancer patients are effective in PARP inhibitor treatment (ref: antibodies of Oncology 29:1203-1210,2018), therefore, RAD51foci predicts that Olaparib sensitivity is of completely opposite clinical value in breast cancer and NSCLC. In NSCLC cells, the percentage of RAD51foci positive cells can be used as a tolerance assessment molecular marker of Olaparib, namely, the percentage of RAD51foci positive cells after the cells are affected by the Olaparib is not changed remarkably, which indicates that NSCLC patients are tolerant to the Olaparib.

Example 2

In this example, half inhibitory concentration IC of NSCLC cells against Olaparib was investigated₅₀Value and proton radiotherapy RBE_0.1Correlations between values, including the following:

1. RBE of proton radiotherapy of 12 NSCLC cells_0.1Value of

Cell: 12 NSCLC cells including H23, H1792, H3122, a549, H460, HCC44, H1666, CALU6, H1703, H1563, JPC3, H1793.

RBE of proton radiotherapy of 12 NSCLC cells was tested by cell cloning experiments_0.1The value is obtained.

For cell cloning experiments see literature (1): (1) liu Q, Ghosh P, Magpayo N, Testa M, Tang S, Gheorghiu L, et al.Lung cancer cell line scan lines noise aspect/BRCA path defects to create a correlated biological effect of proton radiation. 91(5):1081-9.

Proton radiotherapy RBE_0.1Method of calculating values, reference (2): (2) a plasmid in medicine and biology.2014, a plasmid in molecular and biology transfer, a plasmid in biology, a plasmid in 2014; 59(22) R419-72.

RBE of proton radiotherapy of 12 NSCLC cells_0.1The values are shown in Table 3.

TABLE 3

2. Half inhibitory concentration IC of NSCLC cells against Olaparib₅₀Value and RBE of proton radiotherapy_0.1Correlation analysis of values

Half inhibitory concentration IC of Olaparib of 15 NSCLC cells obtained in step 1 of example 1₅₀RBE of proton radiation therapy of 12 NSCLC cells obtained in step 1 of example 2_0.1Values, Pearson correlation analysis was performed.

FIG. 6 shows the half inhibitory concentration IC of Olaparib in 12 cells of this example₅₀Value and RBE of proton radiotherapy_0.1Correlation plot of values.

As can be seen in FIG. 6, the half inhibitory concentration IC of the NSCLC cells against Olaparib₅₀Value and RBE of proton radiotherapy_0.1The values are positively correlated, r is 0.7274,^**p is 0.0073; indicating a half inhibitory concentration IC for Olaparib₅₀The larger the value, the RBE_0.1The larger the value, i.e., the more resistant the nsclc cell to Olaparib, the more sensitive the cell to proton radiation therapy.

3. Analysis of half inhibitory concentration IC of NSCLC cells against Olaparib based on GDSC database₅₀Value and RBE of proton radiotherapy_0.1Correlation of values

From cancer drug sensitive genomesIn the database of science (GDSC), the half inhibitory concentration IC of Olaparib of 19 NSCLC cells after Olaparib treatment for 72h is extracted₅₀Value and RBE of proton radiotherapy obtained in step 2_0.1Values were analyzed for Pearson correlation. 19 NSCLC cells include H23, H1792, H3122, A549, H460, HCC44, H1666, CALU6, H1703, H1563, JPC3, H1793, ABC-1, H1869, PC14, H299, H520, H1915, HCC 827.

FIG. 7 is the half inhibitory concentration IC of Olaparib in 19 NSCLC cells extracted from GDSC database in this example₅₀Value and RBE of proton radiotherapy_0.1Correlation plot of values.

As can be seen from FIG. 7, the half inhibitory concentration IC of Olaparib was observed after 72 hours of Olaparib treatment₅₀Value and RBE of proton radiotherapy_0.1The values are positively correlated, r is 0.6054,^**p is 0.006; illustrating half inhibitory concentration IC of NSCLC cells to Olaparib₅₀The larger the value, the RBE of proton radiotherapy_0.1The larger the value, i.e., the more resistant the NSCLC cell to Olaparib, indicates that the cell is more sensitive to proton radiation therapy.

Taken together, the results of example 2 indicate that NSCLC cells are more resistant to Olaparib and more susceptible to proton therapy.

Example 3

In this example, half inhibitory concentration IC of NSCLC cells against Olaparib was analyzed₅₀Correlation of values with the ARID1A truncation mutation, including the following:

somatic mutation profiles of lung adenocarcinoma cells (LUAD) sensitive to and tolerant to Olaparib were analyzed by the COSMIC database (https:// cancer. sanger. ac. uk/cell _ lines).

FIG. 8 is a volcano plot of lung adenocarcinoma cell (LUAD) genetic mutations versus Olaparib chemotherapy sensitivity and tolerance obtained from the COSMIC cell database in this example. Wherein mut represents a mutation, the left arrow represents the more left-going, the more sensitive to Olaparib; the arrow on the right represents the more right-going, the more tolerant to Olaparib.

As can be seen from fig. 8, the ARID1A truncated mutant lung adenocarcinoma cells were more resistant to Olaparib than the wild-type lung adenocarcinoma cells (p ═ 0.0344); compared with wild lung adenocarcinoma cells, KRAS mutant lung adenocarcinoma cells have no significant statistical difference in sensitivity to Olaparib (p > 0.05); MET mutant lung adenocarcinoma cells were not statistically significantly different in sensitivity to Olaparib compared to wild-type lung adenocarcinoma cells (p > 0.05).

FIG. 9 shows the half inhibitory concentration IC of ARID1A truncated mutant NSCLC cells and ARID1A wild-type NSCLC cells against Olaparib in this example₅₀Bar graph of values. Wherein WT represents a wild type, and Mut represents a mutant type.

As can be seen in FIG. 9, the half inhibitory concentration IC of Olaparib in ARID1A truncated mutant NSCLC cells was compared with ARID1A wild-type NSCLC cells₅₀Significantly higher values (═ p — 0.0364); indicating that ARID1A truncated mutant cells are more tolerant to Olaparib.

FIG. 10 is a RBE from proton radiation therapy of ARID1A truncated mutant NSCLC cells and ARID1A wild type NSCLC cells of this example_0.1Comparative plot of values. Wherein WT represents a wild type, and Mut represents a mutant type.

FIG. 10 shows that ARID1A truncates the RBE of proton radiation therapy in mutant NSCLC cells compared to ARID1A wild-type NSCLC cells_0.1The values were significantly higher (═ p ═ 0.0006), indicating that ARID1A truncated mutant cells were more sensitive to proton radiotherapy.

The results of example 3 taken together demonstrate that ARID1A truncated mutant NSCLC cells are resistant to Olaparib but are sensitive specifically to proton radiation therapy.

Example 4

In this example, ARID1A wild-type NSCLC cells and ARID1A cut-off mutant NSCLC cells were examined for ARID1A expression level, and ARID1A expression level and RBE of proton radiotherapy_0.1Correlation analysis of values, including the following:

subject: 8 NSCLC cells, including ARID1A wild-type NSCLC cells and ARID1A truncated mutant NSCLC cells. Wherein the wild-type NSCLC cells of ARID1A are: h23, H1792, a549, CALU6, and JPC 3; ARID1A truncated mutant NSCLC cells were: h460, H1563 and H1793.

Beta-actin is taken as an internal reference,the expression of ARID1A in ARID1A wild-type NSCLC cells and ARID1A cut-off mutant NSCLC cells is detected by immunoblotting, and the relative expression level of ARID1A is calculated by analyzing the gray value of immunoblotting protein bands by using Image J. Meanwhile, the RBE of proton radiotherapy of each cell is detected by adopting a cell clone experiment_0.1The value is obtained.

1. ARID1A expression level in NSCLC cells and RBE of proton radiotherapy_0.1Correlation analysis of values

FIG. 11 is an immunoblot of ARID1A expression in ARID1A wild-type NSCLC cells and ARID1A truncated mutant NSCLC cells of this example.

As can be seen in fig. 11, the ARID1A truncated mutant NSCLC cells hardly expressed ARID 1A.

FIG. 12 is a graph showing the expression level of ARID1A in 8 NSCLC cells of this example and RBE of proton radiation therapy_0.1Pearson correlation analysis plot of values.

As can be seen from FIG. 12, ARID1A expression level and RBE of proton radiation therapy_0.1The values are significantly negatively correlated (×) p ═ 0.0084, r ═ 0.8442.

2. ARID1A expression level in NSCLC cells and half inhibitory concentration IC of Olaparib in NSCLC cells₅₀Value and proton radiation dose RBE_0.1Correlation analysis between values

Drug-resistant cells NSCLC cells were classified into wild-type cells of ARID1A and truncated mutant cells of ARID1A based on the mutation information of ARID 1A. Wherein the wild type ARID1A cell marker is WT, and the truncated mutant ARID1A cell marker is Mut.

Combined median inhibitory concentration IC on Olaparib₅₀Value and mutation information of ARID1A, dividing NSCLC cells into four subtypes, Res WT, Res Mut, Sen WT and Sen Mut, performing ARID1A truncation mutation in combination with Olaparib tolerance and RBE of proton radiotherapy by a one-way ANOVA statistical analysis method_0.1And (4) analyzing the difference of the values.

FIG. 13 shows RBE of proton radiation therapy on four NSCLC subtypes in this example_0.1A statistical map of values. Wherein Res WT represents orid 1A wild-type cells tolerant to Olaparib; res Mut stands for tolerance to OlaparibARID1A truncated mutant cells; sen WT represents ARID1A wild-type cells sensitive to Olaparib; sen Mut represents ARID1A truncated mutant cells sensitive to Olaparib.

As can be seen from FIG. 13, ARID1A truncation was combined with Olaparib-resistant NSCLC cells, i.e., Res Mut subtype NSCLC cells, which were extremely sensitive to proton radiation therapy (RBE)_0.1＝1.4)。

The results of example 4 taken together demonstrate that ARID1A truncated mutant NSCLC cells hardly express ARID 1A; ARID1A expression level and RBE of proton radiotherapy_0.1The values are significantly inversely correlated; ARID1A truncated mutant and Olaparib-resistant NSCLC cells were extremely sensitive to proton radiation therapy.

Example 5

In this example, endogenous Olaparib-resistant cells and endogenous Olaparib-sensitive cells were used as examples, and interference techniques or drug knockdown were used to suppress the expression of ARID1A in the cells, and sensitivity studies with proton radiotherapy were performed, including the following:

1. taking endogenous Olaparib sensitive cells as objects, adopting siRNA technology to interfere ARID1A expression in the cells, then carrying out Olaparib treatment, detecting the knockdown efficiency and the cell survival rate of ARID1A, and comparing RBE of proton radiotherapy_0.1The value is obtained.

The targeting sequence of the siRNA targeting ARID1A was derived from the literature: chang L, Azzolin L, Di Biagio D, Zanconato F, Battulana G, Lucon Xicaco R, et al, the SWI/SNF complex is a mechanified inhibitor of YAP and TAZ. Nature.2018; 563(7730):265-9..

Cell: h23 cells, endogenous Olaparib sensitive cells.

H23 cells were transfected with siRNA for 48H, and the knockdown efficiency of ARID1A was examined by immunoblotting, and the panel was labeled as the siARID1A panel. A blank control group was also established and labeled as siCtr1 group. Then, after the siARID1A group and the siCtr1 group were treated with different concentration gradients of Olaparib solution for 6 days, the cell viability was measured.

FIG. 14 is an immunoblot of H23 cells at 48H after interfering ARID1A expression with siRNA technology in this example. Wherein siCtr1 is blank control siRNA, siARID1A represents siRNA targeting ARID1A knockout.

As can be seen from fig. 14, ARID1A was successfully knocked down in H23 cells, i.e., siRNA was effective in reducing the protein level of ARID 1A.

FIG. 15 is a graph showing the cell survival curves of H23 cells after interfering ARID1A expression for 48H by siRNA technology and then treating with different concentration gradients of Olaparib solution for 6 days in this example. Wherein R represents tolerance, half inhibitory concentration IC for Olaparib₅₀Value of>10 mu M; s represents sensitive, half inhibitory concentration IC for Olaparib₅₀Value of<10μM。

As can be seen from FIG. 15, the half inhibitory concentration IC of the siCtr1 group against Olaparib₅₀The value was 3.61. mu.M; median inhibitory concentration IC of siaRID1A group for Olaparib₅₀The value was 1.89. mu.M, indicating that both the siARID1A and the siCtrl groups were Olaparib sensitive cells.

FIG. 16 shows RBEs used in proton radiotherapy of the siCtr1 group and the siARID1A group in this example_0.1Comparative plot of values.

As can be seen from FIG. 16, the siRNA interference technique was used to reduce ARID1A expression in H23 cells, and was insensitive to proton radiotherapy, and there was no significant difference between the sensitivity of the siCtr1 group and the sensitivity of the siARID1A group to proton radiotherapy (p > 0.05).

2. The endogenous Olaparib-resistant cells are taken as objects, ARID1A in the cells is knocked down by adopting drugs and then treated by the Olaparib, the knocking down efficiency and the cell survival rate of ARID1A are detected, and the RBE of proton radiotherapy is compared_0.1The value is obtained.

Cell: CALU6 cells, endogenous Olaparib resistant cells; h23 cells, endogenous Olaparib sensitive cells; a549 cells, which are endogenous Olaparib sensitive cells.

FIG. 17 is an immunoblot of ARID1A expression in CALU6 cells after 72h of treatment with 0. mu.M, 5. mu.M, 10. mu.M Olaparib solution in this example.

As can be seen from fig. 17, Olaparib significantly inhibited the expression of ARID1A in CALU6 cells.

FIG. 18 shows CALU6 cells, H23 cells and A549 cells after treatment with different concentrations of Olaparib solution for 6 days in this exampleSurvival plots. Wherein R represents tolerance, half inhibitory concentration IC for Olaparib₅₀Value of>10 mu M; s represents sensitive, half inhibitory concentration IC for Olaparib₅₀Value of<10μM。

As can be seen from FIG. 18, CALU6 cells were endogenous Olaparib-resistant cells (IC)₅₀Value of>10μM)。

Subsequently, CALU6 cells were treated with a 2.5. mu.M Olaparib solution for 24 hours, and then subjected to X-ray or proton radiation therapy to obtain an experimental group (labeled as Olaparib group) as RBE for proton radiation therapy of the Olaparib group_0.1The value is obtained. Meanwhile, CALU6 cells are taken as objects, treated with DMSO for 24h, and subjected to X-ray or proton radiotherapy to obtain RBE of proton radiotherapy in the Vehicle group as a blank control group (marked as Vehicle group)_0.1The value is obtained.

FIG. 19 is a graph showing RBE of CALU6 cells after Olaparib treatment for 24h and X-ray or proton radiation therapy_0.1A graph of values. Wherein, the Vehicle group represents a blank control group, and the cells are treated by DMSO for 24h and then receive X-ray or proton radiotherapy; the Olaparib group represents the experimental group, and the cells were treated with 2.5. mu.M Olaparib solution for 24h and then subjected to X-ray or proton radiation therapy.

Fig. 19 shows that CALU6 cells were significantly more sensitive to proton radiotherapy after treatment with Olaparib in combination with proton radiotherapy (p ═ 0.0003).

The combined results of this example 5 demonstrate that endogenous Olaparib-sensitive cells, after siRNA knockdown, inhibited the expression of ARID1A in the cells, but were still Olaparib-sensitive cells, which were not sensitive to proton radiation therapy. After the endogenous Olaparib drug-resistant cells are treated by the Olaparib, the expression of ARID1A in the cells to the drug-resistant cells can be inhibited; by treating endogenous Olaparib drug-resistant cells through the combination of Olaparib and proton radiotherapy, the sensitivity of the cells to the proton radiotherapy can be obviously improved. Therefore, ARID1A deficiency in endogenous Olaparib-resistant cells is more sensitive to proton radiation therapy.

Example 6

In this example, the expression of ARID1A in endogenous susceptible cells and acquired resistant cells was examined, interference techniques or drug knockdown were used to suppress the expression of ARID1A in acquired resistant cells, and sensitivity correlation studies with proton radiotherapy were performed, including the following:

1. expression of ARID1A in endogenous sensitive cells and in acquired-resistant cells

Subject: endogenous Olaparib-sensitive cells such as H23, a 549; the obtained Olaparib-resistant cells were targeted to the Olaparib-resistant cells constructed in example 1, such as H23PR and a549 PR.

Expression of ARID1A was examined in endogenous Olaparib-sensitive cells and in acquired Olaparib-resistant cells. After each cell was treated with Olaparib solution of different concentration gradient for 6 days, the cell survival rate was observed.

FIG. 20 is an immunoblot of ARID1A from H23 cells and H23PR cells in this example.

As is clear from FIG. 20, the expression level of ARID1A was reduced in the constructed H23PR cells.

FIG. 21 is a graph showing statistical analysis of biological data of ARID1A expression levels in H23 cells and H23PR cells in this example.

As can be seen from fig. 21, the expression level of ARID1A in H23PR cells was significantly lower than that in H23 cells.

FIG. 22 is an immunoblot of A549 cells and A549PR cells ARID1A in this example.

As can be seen from fig. 22, expression of ARID1A was not significantly changed in a549PR cells compared to a549 cells.

FIG. 23 is a graph showing statistical analysis of biological data for ARID1A expression in A549 cells and A549PR cells in this example.

As can be seen from fig. 23, there was no significant difference in expression of ARID1A in a549PR cells compared to a549 cells.

Although both models were significantly resistant, expression of ARID1A was completely inconsistent, with a significant loss of ARID1A expression in H23PR cells and no significant change in ARID1A expression in a549PR cells, indicating that expression of ARID1A was not associated with drug resistance of Olaparib.

2. RBE of proton radiotherapy detecting H23 cells and H23PR cells_0.1Value of

FIG. 24 is a graph showing the survival rate of H23PR cells cloned after X-ray (X) and proton (P) irradiation. Wherein X represents cells treated by X-ray radiotherapy, and P represents cells treated by proton-ray radiotherapy.

As can be seen from FIG. 24, the survival rate of H23PR cells deficient in ARID1A expression was decreased after proton radiation therapy, indicating that they were more sensitive to proton radiation therapy. H23PR cells were more sensitive to proton radiation therapy than H23 cells.

FIG. 25 is a graph showing RBE of proton radiation therapy of H23 cells and H23PR cells in this example_0.1Comparative plot of values.

As can be seen from FIG. 25, RBE of proton radiation therapy with H23 cells_0.1Values comparison, RBE of proton radiotherapy in H23 PR-resistant cells_0.1The value was significantly higher.

Construction of H23PR ARID1A-V5 cells: mu.L of Lipo3000 diluted in 125. mu.LOpti-MEM constituted mixture 1, and then 125. mu.L of Opti-MEM diluted 5. mu. L P3000 and 2.5. mu.g of pcDNA6-ARID1A-V5(ARID1A-V5, addge #39311) constituted mixture 2, and

mixtures

1 and 2 were mixed homogeneously and incubated at room temperature for 12 min. Then 250 μ L of the mixture was dropped into the culture medium, and after 6 hours, the medium was changed to normal medium, 5-10 μ g/ml of Blasticidin (InvivoGen, # ant-bl-05) was added to each well 48 hours after transfection, the culture medium containing Blasticidin cells was periodically changed, and after 20 days, transfected clonal cells were selected, expanded and maintained, and verified by immunoblotting, thereby constructing H23PR ARID1A-V5 cells, i.e., ARID1A overexpressing H23PR cells.

After H23 cells, H23PR cells, and H23PR ARID1A-V5 cells were treated with Olaparib solutions having different concentration gradients for 6 days, the cell viability of each cell was observed.

FIG. 26 is a graph showing the cell survival of H23 cells, H23PR cells, and H23PR ARID1A-V5 cells after treatment with different concentration gradients of Olaparib solution for 6 days in this example. Wherein, H23PR ARID1A-V5 represents that ARID1A is overexpressed by H23PR cells.

As can be seen from FIG. 26, H23PR ARID1A-V5 cells were not significantly changed from H23PR cells and remained Olaparib resistant cells.

FIG. 27 is a graph showing the survival of H23PR ARID1A-V5 cells cloned after X-ray (X) and proton (P) chemotherapy in this example. Wherein, A is a survival curve chart formed by cell cloning of H23PR ARID1A-V5 cells after chemotherapy by X-ray (X) and proton ray (P); the B picture is the survival curve chart formed by the cell clone of H23PR cell after being irradiated by X ray and proton ray (P) and H23PR ARID1A-V5 cell after being irradiated by X ray and proton ray (P).

As can be seen in fig. 27, overexpression of ARID1A in ARID 1A-deficient H23PR cells rescued the expression of ARID1A, which showed no difference in the therapeutic response to photons (X-ray) and protons (Proton).

Fig. 28 is a map of a549PR cells. Wherein: FIG. A is a graph showing the survival of A549PR cells cloned after X-ray and proton-ray chemotherapy, respectively; panel B shows RBE of proton radiation therapy of A549 cells and A549PR cells_0.1A comparison graph of values; FIG. C shows the efficiency of the immunoblotting to detect the interference of ARID1A after transfection of siCtrl or siARID1A 48h with A549PR cells; FIG. D is a graph of cell survival after Olaparib treatment after transfection of siCtrl or siARID1A with A549 cells and A549PR cells; FIG. E is a graph showing the survival rate of cell clones after transfection of siCtrl into A549PR cells and the respective actions of X-ray and proton ray (P); FIG. F is a graph showing the survival of cell clones after transfection of siARID1A with A549PR cells, respectively, by X-ray (X) or proton (P) radiation; panel G shows proton radiotherapy RBE after transfection of siCtrl or siARID1A with A549PR cells_0.1Comparative plot of values.

As seen in FIG. 28, it was found that the expression level of ARID1A in A549PR cells was interfered with siRNA, and RBE was obtained by proton radiation therapy in cells of control group_0.1In comparison, RBE of proton radiotherapy of A549PR cells after knocking down ARID1A_0.1The value is remarkably increased, and the ARID1A is expressed and the expression of ARID1A can be inhibited by an Olaparib drug-resistant cell through a gene knockdown method or a drug knockdown method, so that the sensitivity of the NSCLC cell to proton radiotherapy is improved.

The results of comprehensive example 6 indicate that ARID1A is more sensitive to proton radiation therapy following a defect in acquired Olaparib-tolerant NSCLC cells.

The present invention finds that the half inhibitory concentration IC of PARP inhibitor₅₀Value and RBE of proton radiotherapy_0.1The values are positively correlated, and the half inhibitory concentration IC₅₀The larger the value, the RBE_0.1The larger the value, i.e., the more resistant the nsclc cell to Olaparib, indicates that the nsclc cell is more sensitive to proton radiation therapy. Furthermore, cells with no significant change in RAD51foci in NSCLC cells after Olaparib treatment were more prone to being resistant to Olaparib. Therefore, after the treatment of the Olaparib, the foci expression of the RAD51 can be used as a prediction molecular marker of the proton radiotherapy sensitivity of the NSCLC patient, after the Olaparib acts on the NSCLC, the RAD51foci has no significant change, so the NSCLC patient can carry out proton radiotherapy, and the corresponding radiotherapy dose of the NSCLC patient is higher than that of the 'one-knife-cut' RBE_0.1The radiation dose calculated as 1.1 can be reduced by about 20% to 30%.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Claims

1. Use of a substance that is resistant to a PARP inhibitor for the preparation of a product for assessing the susceptibility of a patient with lung cancer to proton radiation therapy.

2. The use of claim 1, wherein said detecting a substance resistant to a PARP inhibitor comprises detecting a median inhibitory concentration IC of the PARP inhibitor₅₀A value; and/or, detecting the percentage of RAD51foci positive cells.

3. A target for tolerance to a PARP inhibitor, wherein said target is RAD 51.

4. A product for assessing resistance to a PARP inhibitor, said product comprising a substance that detects resistance to a PARP inhibitor; and/or, a substance that measures the percentage of RAD51foci positive cells after exposure to a PARP inhibitor.

5. The product of claim 4, wherein the product comprises a kit, chip, membrane strip.

6. A biomarker for assessing susceptibility of a lung cancer patient to proton radiation therapy, wherein the biomarker is RAD 51.

7. A method of assessing susceptibility to proton radiation therapy in a patient having lung cancer, comprising the steps of:

8. The method of claim 6, comprising at least one of the following technical features:

9. The method of claim 8, wherein 1) when said half inhibitory concentration IC is reached₅₀If the value is more than or equal to 10 mu M, the lung cancer patient is sensitive to proton radiotherapy; when the half inhibitory concentration IC₅₀Value of<And when the concentration is 10 mu M, the lung cancer patient is not sensitive to proton radiotherapy.

10. The use according to claim 1 or 2 or the target according to claim 3 or the product according to claim 4 or 5 or the method according to claim 7 or 8, wherein said PARP inhibitor is selected from at least one of Olaparib, Rucaparib and Niraparib; preferably, the PARP inhibitor is Olaparib.

11. The use of claim 1 or 2 or the biomarker of claim 6 or the method of claim 7 or 8, wherein the lung cancer comprises non-small cell lung cancer and small cell lung cancer; preferably, the lung cancer is non-small cell lung cancer.