Solid tumor radiotherapy sensitization target and curative effect prediction biomarker

Abstract

The invention relates to a solid tumor radiotherapy sensitization target and a curative effect prediction biomarker. The biomarker is prostaglandin E2 (PGE2). Compared with the traditional treatment means, the biomarker disclosed by the invention has the advantages of wider indications, better specificity and simplicity in application; compared with traditional detection of gene mutation frequency, the method can be used for detection in serum of a patient, the detection means is more economical, simple and convenient, and the method has more opportunities to be applied clinically and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a solid tumor radiotherapy sensitization target and efficacy prediction biomarker. Background Art

[0002] Radiotherapy kills tumors and inhibits the progression of solid tumors by inducing DNA damage to cause tumor cell apoptosis or inhibiting mitosis. It is an indispensable adjuvant treatment for clinical tumor treatment. For example, adjuvant radiotherapy is included in the treatment plan for each subtype of breast cancer, which can not only significantly reduce the recurrence rate of breast cancer, but also improve the survival rate of early and some advanced breast cancer. Surgical resection of the tumor and adjuvant TMZ chemotherapy and adjuvant radiotherapy have always been the standard treatment for glioblastoma. However, various studies have shown that irradiation can induce global changes in the tumor microenvironment, such as changes in the immune system and tumor microvasculature, which can cause tumor recurrence and radiation resistance. The effectiveness of irradiation, the durability of the response, and the tolerance are the main challenges to achieving ideal irradiation results. However, there are currently no good drug targets in clinical practice to effectively improve the radiotherapy tolerance of tumor cells. A new sensitization target for solid tumor irradiation is urgently needed.

[0003] In addition, although radiotherapy has improved the mortality rate of most tumors, from an individual perspective, it is still unclear who can benefit from radiotherapy and who may be insensitive to radiotherapy or have toxic reactions. For example, there are currently very limited ways to determine whether breast cancer patients are suitable for radiation therapy and to predict the possible side effects of radiation therapy. Different biomarkers have become an indispensable method to assist breast cancer patients in early diagnosis, prognosis and possible toxic side effects. For example, the frequency of BRCA1 / BRCA2 gene mutations can be used to assess the genetic risk of breast cancer, and the overexpression of HER2 molecules is associated with a shortened recurrence time and a significantly reduced overall survival rate in breast cancer patients. However, these detection methods are relatively complex and can only cover part of the prediction needs. At present, there is an urgent need for a biomarker detection method that is more economical and simple to predict the efficacy of breast cancer radiotherapy.

[0004] PGE2 is an important growth regulator in cells and a highly active inflammatory mediator in inflammatory responses. It can affect other cells in the tumor microenvironment in an autocrine or paracrine manner. It stimulates tumor angiogenesis and cancer cell invasion by upregulating multiple cell signaling pathways, promoting tumor cell progression and survival. In addition, PGE2 secreted by tumor cells can also transform M1 macrophages with tumor suppressor effects into tumor M2 macrophages with tumor-promoting effects and stimulate myeloid cells to secrete tumor-promoting CXCL1, IL-6 and granulocyte colony-stimulating factor (G-CSF). However, there are no reports on the role of PGE2 in radiosensitization and efficacy prediction. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a solid tumor radiotherapy sensitization target and efficacy prediction biomarker, which has a wider indication, better specificity and simpler application than traditional treatment methods; compared with traditional detection of gene mutation frequency, it can be detected in patient serum, the detection method is more economical and simple, has a greater chance of clinical application, and has good application prospects.

[0006] The present invention provides a solid tumor radiotherapy sensitization target and efficacy prediction biomarker, wherein the biomarker is prostaglandin E2.

[0007] Preferably, the solid tumors include pancreatic cancer, breast cancer and glioma.

[0008] Preferably, the prostaglandin E2 is specifically and highly expressed in invasive breast cancer.

[0009] Preferably, the PGE2 concentration in the serum of breast cancer patients is significantly downregulated after irradiation.

[0010] The present invention also provides a biomarker detection product, which includes a prostaglandin E2 receptor non-selective inhibitor.

[0011] Preferably, the non-selective inhibitor of prostaglandin E2 receptor is AH6809.

[0012] Preferably, the detection product includes a test kit, a test paper or a chip.

[0013] Beneficial Effects

[0014] Compared with traditional treatment methods, the biomarkers of the present invention have wider indications, better specificity and simpler application; compared with traditional detection of gene mutation frequency, they can be detected in patient serum, the detection method is more economical and simple, and has a greater chance of clinical application, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Serum samples from patients with breast cancer in situ (n=7) and invasive carcinoma (n=40) were tested using PGE2 ELISA kit.

[0016] Figure 2 A small animal in vivo imaging instrument was used to detect the changes in pancreatic cancer in situ tumor volume after the application of AH6809, a non-selective inhibitor of PGE2 receptor.

[0017] Figure 3 This is a statistical chart showing the changes in pancreatic cancer in situ tumor volume after the application of AH6809, a non-selective inhibitor of PGE2 receptor, detected by small animal in vivo imaging instrument.

[0018] Figure 4To detect the changes in the in situ tumor volume of gliomas after the application of AH6809, a non-selective inhibitor of PGE2 receptor, using magnetic resonance imaging. Figure 5 This is a statistical chart showing the changes in the in situ tumor volume of gliomas after the application of AH6809, a non-selective inhibitor of PGE2 receptor, detected by magnetic resonance imaging.

[0019] Figure 6 The PGE2 enzyme-linked immunosorbent assay kit was used to detect the changes in PGE2 concentration in serum samples of breast cancer patients before and after irradiation (n=31). DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0021] Example 1

[0022] 1. Experimental Methods

[0023] ① Pancreatic cancer:

[0024] 1) Construction of in situ animal models of solid tumors

[0025] 1. Culture the mouse pancreatic cancer cell line Panc02 with Luciferase in a TC-treated T225 culture flask to 80%-90% cell confluence;

[0026] 2. Pour out the culture medium and wash once with PBS;

[0027] 3. Add appropriate amount of trypsin to digest pancreatic cancer cells into single cells;

[0028] 4. Add an appropriate amount of complete culture medium to neutralize the trypsin and transfer to a 15 ml centrifuge tube. Confirm under a microscope that there are no residual cells at the bottom of the culture flask; 5. Centrifuge at 1000 rpm for 5 minutes;

[0029] 6. Discard the supernatant, add 10 ml of pure DMEM to resuspend the cells, and centrifuge at 1000 rpm for 5 minutes;

[0030] 7. Repeat step 6 4 times to remove the residual FBS on the cell surface;

[0031] 8. Discard the supernatant, add pure DMEM to a final volume of 10 ml and resuspend the cells, and count them using a cell counting plate;

[0032] 9. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add pure DMEM and respin to a final concentration of 5X 10^7 cells / ml. The injection volume for each C57BL / C mouse is calculated as 20 μl;

[0033] 10. Anesthetize C57BL / C mice with 0.9% sodium pentobarbital 0.01 ml / g intraperitoneally;

[0034] 11. After the mouse is completely paralyzed, evenly apply the depilatory cream on the lower edge of the left costal arch. After 5 minutes, remove the hair with a cotton swab, disinfect it in a circular pattern with an alcohol cotton ball 3 times, and cut the skin and subcutaneous fat along the longitudinal incision under the left costal arch. The incision is about 1 cm.

[0035] 12. Open the peritoneum at the approximate location of the spleen and make a longitudinal incision of about 1 cm;

[0036] 13. Bluntly separate the spleen and pancreas, making sure there is no tissue attached to the two organs;

[0037] 14. Pull the pancreas out through the incision to allow easier access to the tail;

[0038] 15. Grasp the pancreas with forceps in your non-dominant hand and carefully cut the ligaments that prevent the pancreas from being pulled out, making sure the organ is not folded;

[0039] 16. Expose the pancreatic tail and use your dominant hand to carefully insert the needle of the syringe through the pancreatic capsule and penetrate about 0.5 cm along the longitudinal axis of the pancreas (be careful not to puncture the blood vessels of the pancreas)

[0040] 17. Inject 20 μl of cell suspension into the pancreatic capsule. After successful injection, a transparent bubble will form at the tail of the pancreas. Keep the pancreas and the inserted syringe still and carefully pull it out after 1 minute.

[0041] 18. Reposition the pancreas and use a Pasteur tube to inject 1 ml of saline into the abdominal cavity to prevent organ adhesion;

[0042] 19.3-0 surgical suture is used to suture the abdominal skin;

[0043] 20. Observe the general condition of C57BL / C on the second day after surgery.

[0044] 2) Irradiation treatment of animal models: One week after model establishment, each group of C57BL / C received irradiation treatment according to experimental requirements.

[0045] 3) Injection of non-targeted PGE2 receptor inhibitor (AH6089): Starting from the second day after irradiation, AH6089 was dissolved in DMSO to prepare a working solution with a concentration of 0.67 mg / ml. Each C57BL / C mouse was intraperitoneally injected with AH6089 working solution according to body weight (7.5 ul / g). The injection was performed once a day for one week.

[0046] 4) In vivo imaging to detect pancreatic cancer tumorigenesis:

[0047] 1. One week after administration, C57BL / C were anesthetized with 0.9% sodium pentobarbital at a dose of 10 μl / g by intraperitoneal injection;

[0048] 2. Dissolve D-luciferin potassium salt in D-PBS to 15 mg / ml, and inject the corresponding D-luciferin potassium salt into the abdominal cavity according to the weight of the mouse (10 μl / g) after the C57BL / C mice are completely paralyzed;

[0049] 3. In vivo imaging of C57BL / C;

[0050] 4. C57BL / C were anesthetized with isoflurane, and the hearts were perfused once with normal saline and once with paraformaldehyde. The pancreas of the corresponding mice were then removed and the differences in tumor volume between the control group and the PGE2 non-selective receptor inhibitor group were analyzed and compared.

[0051] ②Glioma

[0052] 1) Construction of in situ animal models of solid tumors

[0053] 1. Human glioma cell line U87-MG was cultured in a TC-treated T225 culture flask until the cell confluence was 50%-60%; 2. The culture medium was poured out and washed once with PBS;

[0054] 3. Add 4 ml of trypsin to digest glioma cells into single cells;

[0055] 4. Add 8 ml of complete culture medium to neutralize the trypsin and transfer to a 15 ml centrifuge tube. Confirm under a microscope that there are no cells remaining at the bottom of the culture flask; 5. Centrifuge at 1000 rpm for 5 min;

[0056] 6. Discard the supernatant, add 10 ml of pure DMEM to resuspend the cells, and centrifuge at 1000 rpm for 5 minutes;

[0057] 7. Repeat step 6 4 times to remove the residual FBS on the cell surface;

[0058] 8. Discard the supernatant, add pure DMEM to a final volume of 10 ml and resuspend the cells, and count them using a cell counting plate. The specific calculation method varies depending on the cell counting plate;

[0059] 9. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add pure DMEM and respin to a final concentration of 1X 10^9 cells / ml. The injection volume for each nude mouse is calculated as 5ul;

[0060] 10. Anesthetize nude mice by intraperitoneal injection of 0.9% sodium pentobarbital at a dose of 10ul / g;

[0061] 11. Cut the scalp of the nude mouse longitudinally with scissors and apply 3% hydrogen peroxide solution on the surface of the nude mouse skull to expose the nude mouse bregma;

[0062] 12. Use the needle of a 1 ml syringe to make a hole 2 cm to the right and 1 cm above the nude mouse's anterior bregma (located in the basal ganglia area of ​​the nude mouse);

[0063] 13. Fix the nude mouse on the stereotaxic injection instrument;

[0064] 14. Take the prepared cells and slowly inject them at the opening. Each nude mouse is injected with 5ul of cells. The whole process lasts about 5min. Note that the injection speed should not be too fast, otherwise it will easily cause cell overflow.

[0065] 15. Remove the injected nude mouse from the stereotaxic apparatus and seal the opening with bone wax;

[0066] 16. Suture the scalp of the nude mouse with 3-0 sutures;

[0067] 17. Gently place the sutured nude mice on a 37°C heating blanket. After the nude mice wake up, put them back into the corresponding cages according to the groups and mark them.

[0068] 18. Observe the survival and movement of nude mice on the next day.

[0069] 2) Irradiation treatment of animal models: One week after modeling, each group of nude mice was irradiated according to experimental requirements.

[0070] 3) Injection of non-targeted PGE2 receptor inhibitor (AH6809): Starting from the second day after irradiation, AH6809 was dissolved in DMSO to prepare a working solution with a concentration of 0.67 mg / ml. Each nude mouse was intraperitoneally injected with AH6809 working solution according to body weight (7.5 ul / g). The injection was performed once a day for one week.

[0071] 4) MRI detection of tumor formation in nude mice:

[0072] 1. One week after administration, nude mice were anesthetized by intraperitoneal injection of 0.9% sodium pentobarbital at a dose of 10 μl / g;

[0073] 2. After the nude mice were completely paralyzed, gadolinium was injected intraperitoneally at a dose of 5 μl / g;

[0074] 3. MRI was used to take magnetic resonance images of nude mice, and the volume of intracranial tumors in nude mice was estimated according to the formula. The difference in tumor volume between the control group and the PGE2 non-selective receptor inhibitor group was analyzed and compared.

[0075] ③ Detection of serum PGE2 concentration in patients

[0076] The specific steps are as follows:

[0077] 1) Preparation of serum samples before and after irradiation

[0078] 1. Before irradiation and after the start of the irradiation treatment, collect a few milliliters of venous blood from the patient in the early morning when fasting and place it in a 5 ml EP tube. Let it stand at room temperature for 1 hour to allow the blood to coagulate and stratify.

[0079] 2. Centrifuge at 3000rpm at room temperature for 20min. Carefully remove the centrifuged blood sample from the centrifuge and place it on a test tube rack. The blood sample can be seen in two distinct layers with the naked eye. The upper layer is light yellow, which is the required serum, and the lower layer is the blood cell component. Use a pipette to divide the upper layer of serum evenly into two EP tubes and store them in a -80℃ refrigerator.

[0080] 2) Determination of PGE2 concentration in patient serum

[0081] 1. Take the serum sample out of the -80℃ freezer and thaw it at 4℃.

[0082] 2. Take out the PGE2 enzyme-linked immunosorbent assay kit (Elabscience, E-EL-0034) 20 minutes in advance and equilibrate it to room temperature, draw up the concentrated washing solution (25X), and dilute it with double distilled water at a ratio of 1:24.

[0083] 3. Standard preparation: Centrifuge the standard at 10000×g for 1 minute, add 1ml of standard & sample diluent and invert several times, and prepare 2000pg / mL standard working solution after it is fully dissolved. At the same time, take 7 autoclaved 1.5ml EP tubes, and pre-add 500μL of standard & sample diluent to each tube. Pipette 500μL from the 2000pg / mL standard working solution into the first EP tube and mix well to prepare 1000pg / mL standard working solution, and then pipette and mix well in the following tubes. The last tube is used as a blank tube and liquid is not drawn from the second to last tube. The following concentrations are prepared: 2000, 1000, 500, 250, 125, 62.5, 31.25, 0pg / ml.

[0084] 4. Preparation of biotinylated antibody working solution: 15 minutes before the experiment, centrifuge the concentrated biotinylated antibody (100X) at 800Xg for 1 minute, and dilute the concentrated biotinylated antibody to 1X working concentration with biotinylated antibody diluent according to the number of samples to be measured.

[0085] 5. Preparation of HRP enzyme conjugate working solution: HRP enzyme conjugate can bind to avidin. 15 minutes before the experiment, centrifuge the concentrated HRP enzyme conjugate (100x) at 800Xg for 1 minute, and dilute the concentrated HRP enzyme conjugate to 1X working concentration with enzyme conjugate diluent.

[0086] 6. Add standard / sample and biotinylated antibody: Set standard wells and sample wells respectively, add 50ul of 8 tubes of diluted standard in the standard wells in descending order, and add 50ul of the prepared sample to be tested in the sample wells (set up 2 duplicate wells for both standard wells and sample wells). When adding samples, add the samples to the bottom of the ELISA plate, try not to touch the well wall to avoid bubbles. Immediately add 50ul of the prepared biotinylated antibody working solution to each well, cover the ELISA plate with a sealing film to prevent the liquid from evaporating, gently shake to mix, and incubate at 37°C for 45min.

[0087] 7. Wash the plate: Tear off the sealing film and shake off the liquid in the plate, and pat the ELISA plate dry on absorbent paper. Add 300ul of washing solution to each well, shake for 1 minute, shake off the liquid in the plate, and pat dry on absorbent paper. Repeat 3 times.

[0088] 8. Add HRP enzyme conjugate: Add 100ul of the prepared HRP enzyme conjugate working solution to each well, cover the ELISA plate with a sealing film, shake gently to mix, and incubate in a 37°C constant temperature incubator for 30 minutes.

[0089] 9. Wash the plate: Tear off the sealing film and shake off the liquid in the plate, and pat the ELISA plate dry on absorbent paper. Add 300ul of washing solution to each well, shake for 1 minute, shake off the liquid in the plate, and pat dry on absorbent paper. Repeat 5 times.

[0090] 10. Color development: Add 90ul of substrate solution to each well in the dark, cover the ELISA plate with a sealing film, shake gently to mix, and incubate in a 37℃ incubator in the dark for 15min. At the same time, turn on the ELISA reader and preheat to 37℃.

[0091] 11. Stop: Add 50ul of stop solution to each well in the same order as the substrate solution.

[0092] 12. Measurement: Immediately measure the OD value of each well at 450 nm and 37°C using an ELISA reader.

[0093] Analysis: Calculate the average OD value of the standard and duplicate wells, use concentration as the horizontal axis and OD value as the vertical axis, draw the corresponding scatter plot in excel and fit the corresponding standard curve. Substitute the OD value measured in the sample well into the function of the standard curve to obtain the serum PGE2 concentration (pg / ml) corresponding to the patient sample.

[0094] 2. Experimental Results

[0095] like Figure 1 As shown, the PGE2 enzyme-linked immunosorbent assay kit detected differences in baseline PGE2 concentrations in serum samples from patients with breast cancer in situ (n=7) and invasive cancer (n=40);

[0096] like Figure 2 , 3As shown in Figure 4, after the C57BL / C cells were treated with the non-selective inhibitor of PGE2 receptor AH6809, the irradiation effect on the growth of pancreatic cancer in situ tumors was more significant than that of the group without treatment;

[0097] like Figure 5 , 6 As shown, after nude mice were treated with the non-selective inhibitor of PGE2 receptor AH6809, the irradiation effect of glioma in situ tumors was more significant than that of the group without treatment;

[0098] like Figure 6 As shown, the PGE2 enzyme-linked immunosorbent assay kit was used to detect the changes in PGE2 concentration in serum samples of breast cancer patients before and after irradiation (n=31);

[0099] All the results were compared among the groups and statistically analyzed using GraphPad software.

[0100] 3. Experimental Conclusion

[0101] Conclusion 1: Compared with breast carcinoma in situ, PGE2 is specifically overexpressed in invasive breast cancer.

[0102] Conclusion 2: The use of PGE2 receptor non-selective inhibitor AH6809 in orthotopic tumor models of pancreatic cancer and glioma can significantly sensitize the efficacy of radiation therapy.

[0103] Conclusion 3: Compared with before irradiation, the serum PGE2 concentration of most breast cancer patients was significantly decreased after irradiation.

Claims

1. A solid tumor radiotherapy sensitization target and efficacy prediction biomarker, characterized in that: The biomarker is prostaglandin E2.

2. The biomarker according to claim 1, characterized in that: The solid tumors include breast cancer, pancreatic cancer and glioma.

3. The biomarker according to claim 1, characterized in that: The prostaglandin E2 is specifically and highly expressed in invasive breast cancer.

4. The biomarker according to claim 1, characterized in that: The serum PGE2 concentration of breast cancer patients was significantly downregulated after irradiation.

5. A detection product comprising the biomarker according to claim 1, characterized in that: The test products include non-selective inhibitors of prostaglandin E2 receptors.

6. The biomarker according to claim 5, characterized in that: The non-selective inhibitor of prostaglandin E2 receptor is AH6809.

7. The detection product according to claim 3, characterized in that: The detection product includes a test kit, a test paper or a chip.

Images