Application of L-ASNase in preparation of medicine for treating bladder cancer and kit

Abstract

The invention belongs to the technical field of biological medicines, and relates to application of L-ASNase in preparation of a medicine for treating bladder cancer and a kit. After the ASNS molecule is knocked down by the shRNA technology, the subcutaneous tumor-bearing growth of the immune healthy mouse can be inhibited. The knock-down ASNS has no influence on the growth of subcutaneous tumor-bearing mice in nude mice with immunodeficiency. And asparagine can promote the growth of tumors. The influence of knocking down ASNS and feeding asparagine on subcutaneous tumor-bearing growth may be dependent on an immune system. Flow cytometry discovers that knock-down of ASNS promotes recruitment of CD8 + T cells in a tumor immune microenvironment, but the knock-down of ASNS has no influence on the number of CD4 + T cells, and knock-down of the influence of ASNS on subcutaneous tumor-bearing growth may be achieved by recruitment of more CD8 + T cells and promotion of activation of the CD8 + T cells, so that the capability of killing tumor cells is achieved. In conclusion, the asparagine can promote the growth of the bladder tumor, and the growth of the bladder tumor can be inhibited through the treatment of knocking down ASNS or L-ASNase.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an application of L-ASNase in preparing a drug for treating bladder cancer and a kit. Background Art

[0002] Bladder cancer is a malignant tumor originating from the urothelium of the bladder and is one of the most common tumors in the urinary system. Clinically, about 25% of patients are diagnosed with muscle-invasive bladder cancer at the first visit, that is, their bladder tumors have infiltrated the muscle layer of the bladder. At present, cisplatin-based combination chemotherapy is the preferred strategy for neoadjuvant and adjuvant treatment of muscle-invasive bladder cancer, but most patients gradually develop drug resistance after several cycles of chemotherapy, and the side effects are obvious. In recent years, the development of immune checkpoint inhibitors has revolutionized the first-line treatment of bladder cancer. Under normal circumstances, the human immune system has immune surveillance functions. When a malignant tumor appears, the body's T cells can recognize and kill the tumor to inhibit tumor growth. However, the binding of PD-1 on T cells to PD-L1 on tumor cells effectively inhibits T cell activation, and even leads to T cell apoptosis, reduced cytokine production, and induced antigen tolerance, thereby allowing the tumor to escape immune surveillance. PD-1 / PD-L1 inhibitors bind to PD-1 or PD-L1, respectively, preventing the interaction between PD-1 and PD-L1, restoring the recognition and killing effects of immune cells, and avoiding the immune escape of tumor cells. The first-line indications of the currently approved pembrolizumab (PD-1 inhibitor) and atezolizumab (PD-L1 inhibitor) are for patients with locally advanced or metastatic urothelial carcinoma who are intolerant to cisplatin, but only a few patients respond. Therefore, analyzing the mechanism by which bladder cancer cells evade immune surveillance and formulating new treatment strategies based on this are important ways to enhance anti-tumor effects and improve patient survival.

[0003] Although cisplatin-based chemotherapy is the first-line treatment for advanced and metastatic bladder cancer, the overall clinical effect of cisplatin is unsatisfactory due to the development of drug resistance. Patients often experience side effects, mainly bone marrow suppression, renal failure, neurotoxicity, gastrointestinal side effects and hearing loss. PD-1 and PD-L1 inhibitor therapy is a new tumor treatment strategy. As mentioned above, the immune checkpoint inhibitors currently approved for patients with locally advanced or metastatic urothelial carcinoma who are intolerant to cisplatin include pembrolizumab (PD-1 antibody) and atezolizumab (PD-L1 antibody). The basic principle of PD-1 and PD-L1 immunotherapy is to restore the suppressed immune system function by blocking the interaction between PD-1 molecules on the surface of CD8+T cells and PD-L1 molecules on the surface of tumor cells, thereby enhancing the body's immune response to tumors. Although PD-1 and PD-L1 immunotherapy have shown significant efficacy in some bladder cancer patients, not all patients can benefit from it. Some patients may experience immune non-response or immune-related adverse reactions, such as rash, abnormal liver function, etc. Studies have shown that the responsiveness of immunotherapy depends on the tumor infiltration of T lymphocytes, especially CD8+T cells that can recognize and kill tumor cells. Therefore, exploring the key mechanisms that mediate CD8+T cell infiltration and activation in the tumor immune microenvironment can provide important clues for effectively reversing tumor immunotherapy resistance and is a breakthrough point for developing new treatment strategies for bladder cancer.

[0004] As mentioned above, some bladder cancer patients are not sensitive to cisplatin-based chemotherapy and immune checkpoint inhibitors, and may even develop drug resistance and toxic side effects. It is urgent to find new targets to improve the efficacy of bladder cancer. The present invention uses L-ASNase (asparaginase) in mice to enhance the infiltration and activity of CD8+T cells in the tumor microenvironment, thereby inhibiting tumor growth, providing a new idea for the treatment of bladder cancer.

[0005] ASNS, or asparagine synthetase, converts aspartic acid and glutamine into asparagine and glutamate. Previous studies have reported that asparagine in the tumor microenvironment is closely related to the activation of CD8+T cells, thereby affecting anti-tumor function. However, there have been no reports on the regulation of immune cell function by asparagine inside tumor cells. Summary of the invention

[0006] The present invention specifically relates to an application of L-ASNase activated CD8+T cell-mediated tumor immunity in the treatment of bladder cancer.

[0007] The present invention firstly provides an application of L-ASNase in preparing a medicine for treating bladder cancer.

[0008] The present invention also provides an application of L-ASNase in preparing an in vitro medicine for treating bladder cancer.

[0009] In certain embodiments, said in vitro is in a cell.

[0010] The present invention also provides use of an agent for inhibiting or knocking down ASNS in preparing a drug for treating bladder cancer.

[0011] In certain embodiments, the agent comprises a shRNA that knocks down ASNS.

[0012] In certain embodiments, the agent comprises siRNA that knocks down ASNS.

[0013] Finally, the present invention provides a kit for diagnosing the prognosis of bladder cancer, wherein the kit comprises a reagent for detecting ASNS.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects:

[0015] We first investigated the effect of ASNS in bladder cancer cells on bladder tumor growth. Figure 1A-Figure 1C As shown in Figure 2, we found that knocking down ASNS molecules using shRNA technology could inhibit the growth of subcutaneous tumors in immune-competent mice (C57BL / 6). However, knocking down ASNS had no effect on the growth of subcutaneous tumors in immune-deficient nude mice (BALB / c-nu). Figure 1D-1F Similarly, we observed that asparagine promoted tumor growth in immune-competent subcutaneous tumor-bearing mice (e.g. Figure 1G-1H ). However, this phenomenon was not observed in immunodeficient nude mice (e.g. Figure 1I-1J ). Since BALB / c-nu mice have no thymus and lack T lymphocytes compared to C57BL / 6 mice, the effects of ASNS knockdown and asparagine feeding on subcutaneous tumor growth may be dependent on the immune system, that is, caused by T lymphocytes.

[0016] Subsequently, we performed flow cytometric analysis on subcutaneous tumor-bearing mice with ASNS knockdown and found that ASNS knockdown promoted the recruitment of CD8+ T cells in the tumor immune microenvironment, but had no effect on the number of CD4+ T cells (e.g. Figure 2A In addition, knocking down ASNS can also enhance the ability of CD8+ T cells to secrete the killer factors TNF-α and IFN-γ (such as Figure 2B-2C). It can be seen that the effect of ASNS knockdown on subcutaneous tumor growth may be through recruiting more CD8+T cells and promoting their activation, thereby playing a role in killing tumor cells. Next, we used mouse anti-CD8 antibody neutralization experiments, that is, intraperitoneally injecting anti-CD8 antibodies into C57BL / 6 mice to observe the effect of ASNS knockdown on subcutaneous tumor growth. Figure 2D-2F As shown, the growth of subcutaneous tumors in mice treated with anti-CD8 antibodies was not affected by knockdown of ASNS. In summary, knockdown of ASNS inhibited the growth of subcutaneous tumors in a CD8+ T cell-dependent manner.

[0017] In summary, this study suggests that asparagine can promote the growth of bladder tumors, and knockdown of ASNS or L-ASNase can inhibit the growth of bladder cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A-1J Asparagine promotes tumor growth in an immune-dependent manner;

[0019] Figure 1A A gross photograph of the subcutaneous tumor of Example 1 is shown;

[0020] Figure 1B The volume of the subcutaneous tumor of Example 1 is shown;

[0021] Figure 1C The weight of the subcutaneous tumor of Example 1 is shown;

[0022] Figure 1D A gross photograph of the subcutaneous tumor of Example 2 is shown;

[0023] Figure 1E The volume of the subcutaneous tumor of Example 2 is shown;

[0024] Figure 1F The weight of the subcutaneous tumor of Example 2 is shown;

[0025] Figure 1G The gross photograph and volume of the subcutaneous tumor of Example 3 are shown;

[0026] Figure 1H The weight of the subcutaneous tumor of Example 3 is shown;

[0027] Fig. 1I The gross photograph and volume of the subcutaneous tumor of Example 4 are shown;

[0028] Figure 1J The weight of the subcutaneous tumor of Example 4 is shown.

[0029] Figure 2A-2F Inhibition of asparagine synthetase can promote CD8+T cell infiltration and activation;

[0030] Figure 2A The effect of ASNS knockdown on CD8+T and CD4+T cells is shown;

[0031] Figure 2B-2C The ability of ASNS knockdown to TNF-α and IFN-γ is shown;

[0032] Figure 2D A gross photograph of a subcutaneous tumor of Example 6 is shown;

[0033] Figure 2E The volume of the subcutaneous tumor of Example 6 is shown;

[0034] Figure 2F The weight of the subcutaneous tumor of Example 6 is shown.

[0035] Figure 3A-3E L-ASNase inhibits the growth of bladder cancer by promoting CD8+T cell infiltration;

[0036] Figure 3A A gross photograph of a subcutaneous tumor of Example 7 is shown;

[0037] Figure 3B The volume of the subcutaneous tumor of Example 7 is shown;

[0038] Figure 3C The weight of the subcutaneous tumor of Example 7 is shown;

[0039] Figure 3D The effect of L-ASNase on CD8+T is shown;

[0040] Figure 3E Shown is the effect of L-ASNase on the body weight of mice. DETAILED DESCRIPTION

[0041] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0042] Example 1: Effect of ASNS knockdown on subcutaneous tumors in C57BL / 6 mice

[0043] Animal experiment steps:

[0044] 1. Purchase 18 5-week-old female C57BL / 6 mice and randomly divide them into 3 groups, with 6 mice in each group.

[0045] 2. Expand the culture of MB49 control (scramble cells) and ASNS knockdown stable cell lines (shAsns#1 cells, shAsns#2 cells). Collect the cells, wash them with PBS, centrifuge and count the cells. Resuspend the cells with PBS so that 200ul of PBS contains 5 x 10 5 A tumor cell.

[0046] 3. Modeling: The mice were anesthetized with isoflurane, and the right axillary area was disinfected with iodine. 200ul of tumor cell suspension was injected subcutaneously into the right upper limb of the mouse using an insulin needle.

[0047] 4. Raising mice: After modeling, measure the length of the subcutaneous tumor with a vernier caliper every 3 days, and use this data to calculate the volume of the subcutaneous tumor.

[0048] 5. Sample collection: After 22 days, the mice were euthanized, the subcutaneous tumors were removed by dissection, photographed and weighed. Figure 1A The gross photo of the subcutaneous tumor is shown. Figure 1B The volume of the subcutaneous tumor shown, Figure 1C Weights of subcutaneous tumors are indicated.

[0049] See also Figure 1A-Figure 1C : In immunocompetent C57BL / 6 mice, ASNS knockdown was found to inhibit the growth of subcutaneous tumors.

[0050] Example 2: Effect of ASNS knockdown on subcutaneous tumors in nude mice

[0051] Animal experiment steps:

[0052] 1. Purchase 18 5-week-old male BLAB / c-nu mice and randomly divide them into 3 groups, with 6 mice in each group.

[0053] 2. Expand the culture of MB49 control (scramble cells) and ASNS knockdown stable cell lines (shAsns#1 cells, shAsns#2 cells). Collect the cells, wash them with PBS, centrifuge and count the cells. Resuspend the cells with PBS so that 200ul of PBS contains 5 x 10 5 A tumor cell.

[0054] 3. Modeling: The mice were anesthetized with isoflurane, and the right axillary area was disinfected with iodine. 200ul of tumor cell suspension was injected subcutaneously into the right upper limb of the mouse using an insulin needle.

[0055] 4. Raising mice: After modeling, measure the length of the subcutaneous tumor with a vernier caliper every 2-3 days, and use this data to calculate the volume of the subcutaneous tumor.

[0056] 5. Sample collection: After 10 days, the mice were euthanized and the subcutaneous tumors were removed, photographed and weighed. Figure 1D The gross photo of the subcutaneous tumor is shown. Figure 1E The volume of the subcutaneous tumor shown, Figure 1F Weights of subcutaneous tumors are indicated.

[0057] See also Figure 1D-1F MB49 cells were subcutaneously implanted in nude mice, and it was found that knockdown of ASNS had no effect on tumor growth.

[0058] Example 3: Effect of Asparagine on Subcutaneous Tumors in C57BL / 6 Mice

[0059] Animal experiment steps:

[0060] 1. Purchase 12 5-week-old female C57BL / 6 mice and randomly divide them into 2 groups, with 6 mice in each group.

[0061] 2. Expand the MB49 cell line. Collect the cells, wash them with PBS, centrifuge and count the cells. Resuspend the cells with PBS so that 200ul of PBS contains 5 x 10 5 A tumor cell.

[0062] 3. Modeling: The mice were anesthetized with isoflurane, and the right axillary area was disinfected with iodine. 200ul of tumor cell suspension was injected subcutaneously into the right upper limb of the mouse using an insulin needle.

[0063] 4. Feeding mice: After modeling, asparagine was added to the drinking water of mice on the 4th day. Asparagine was fed to the mice every day at a concentration of 5 mmol / L. The drinking water with asparagine was changed every three days until the end of the experiment. The length of the subcutaneous tumor was measured with a vernier caliper every 4 days. The subcutaneous tumor volume was calculated based on this data.

[0064] 5. Sample collection: After 20 days, the mice were euthanized, the subcutaneous tumors were removed by dissection, photographed and weighed. Figure 1G The gross photo of the subcutaneous tumor and the volume of the subcutaneous tumor are shown. Figure 1H Weights of subcutaneous tumors are indicated.

[0065] See also Figure 1G-1H : In immunocompetent C57BL / 6 mice, asparagine was found to promote the growth of subcutaneous tumors.

[0066] Example 4: Effect of Asparagine on Tumors in Nude Mice

[0067] Animal experiment steps:

[0068] 1. Purchase 12 5-week-old male BLAB / c-nu mice and randomly divide them into 2 groups, with 6 mice in each group.

[0069] 2. Expand the MB49 cell line. Collect the cells, wash them with PBS, centrifuge and count the cells. Resuspend the cells with PBS so that 200ul of PBS contains 5 x 10 5 A tumor cell.

[0070] 3. Modeling: The mice were anesthetized with isoflurane, and the right axillary area was disinfected with iodine. 200ul of tumor cell suspension was injected subcutaneously into the right upper limb of the mouse using an insulin needle.

[0071] 4. Feeding mice: After modeling, feed mice with asparagine at a concentration of 5 mmol / L on the 4th day until the end of the experiment. Measure the length of the subcutaneous tumor with a vernier caliper every 2-3 days. Use this data to calculate the volume of the subcutaneous tumor.

[0072] 5. Sample collection: After 10 days, the mice were euthanized and the subcutaneous tumors were removed, photographed and weighed. Fig. 1I The gross photo of the subcutaneous tumor and the volume of the subcutaneous tumor are shown. Figure 1J Weights of subcutaneous tumors are indicated.

[0073] See also Figure 1I-1J : MB49 cells were subcutaneously injected into nude mice and it was found that asparagine had no effect on tumor growth.

[0074] Example 5: Effect of asparagine on CD8+ T cells in the tumor immune microenvironment

[0075] Animal experiment steps:

[0076] 1. Wash the subcutaneous tumor of C57BL6 mice bearing MB49 cells (scramble cells, shAsns#1 cells, shAsns#2 cells) with PBS several times and then cut it into pieces with scissors.

[0077] 2. Prepare tissue digestion solution: 320ug / ml type IV collagenase + 5ug / ml DNase, i.e. 32mg type IV collagenase + 0.5mg DNase + 100ml complete culture medium.

[0078] 3. Place the chopped tissue into a 15 ml centrifuge tube, add 5 ml of tissue lysis buffer, and place it in a 37° constant temperature shaker for 1 hour.

[0079] 4. After the sample is fully lysed, filter the sample with a cell filter. The filtrate is the cell suspension. Centrifuge at 1500rpm for 6 minutes to collect the cell precipitate.

[0080] 5. Dilute the flow cytometry antibodies (CD45, CD4, CD8, TNF-α and IFN-γ) with PBS in proportion. Add to the cell pellet and mix the cells with a vortex oscillator. Incubate at room temperature for 30 minutes.

[0081] 6. After incubation, add 5 ml PBS to the cell suspension containing the antibody to wash it once, and centrifuge at 1500 rpm for 6 minutes. Fix the cell pellet with flow cytometry fixative. Then perform flow cytometry on the machine. Figure 2A As shown in Figure 2, knockdown of ASNS increased the number of CD8+ T cells in the tumor immune microenvironment, but had no effect on the number of CD4+ T cells. Figure 2B-2C As shown, knockdown of ASNS enhanced the ability of CD8+ T cells to secrete TNF-α and IFN-γ.

[0082] See also Figure 2A-2C : Flow cytometry analysis of subcutaneous tumors in C57BL / 6 mice bearing MB49 cells revealed that knocking down ASNS could increase the number of CD8+T cells in the tumor immune microenvironment and enhance the ability of CD8+T to secrete cytotoxic factors.

[0083] Example 6: Effect of knocking down ASNS on subcutaneous tumors after neutralizing CD8+ T cells

[0084] Animal experiment steps:

[0085] 1. Purchase 30 5-week-old female C57BL / 6 mice and randomly divide them into 6 groups, with 5 mice in each group.

[0086] 2. Expand the culture of MB49 control (scramble cells) and ASNS knockdown stable cell lines (shAsns#1 cells, shAsns#2 cells). Collect the cells, wash them with PBS, centrifuge and count the cells. Resuspend the cells with PBS so that 200ul of PBS contains 5 x 10 5 A tumor cell.

[0087] 3. Administration of anti-CD8 antibodies: Anti-CD8 antibodies were intraperitoneally injected 3 days before modeling, on the day of modeling, and on the 3rd, 8th, and 11th days after modeling, 200ug / mouse.

[0088] 4. Modeling: The mice were anesthetized with isoflurane, and the right axillary area was disinfected with iodine. 200ul of tumor cell suspension was injected subcutaneously into the right upper limb of the mouse using an insulin needle.

[0089] 5. Raising mice: After modeling, measure the length of the subcutaneous tumor with a vernier caliper every 4 days. Use this data to calculate the volume of the subcutaneous tumor. After the 24th day, euthanize the mice. Take the subcutaneous tumor, take a picture and weigh it. Figure 2DThe gross photo of the subcutaneous tumor is shown. Figure 2E The volume of the subcutaneous tumor shown, Figure 2F Weights of subcutaneous tumors are indicated.

[0090] Figure 2D-2F : Anti-CD8 antibodies were used to neutralize CD8+T cells in mice, and it was found that the inhibitory effect of ASNS knockdown on subcutaneous tumor growth in C57BL / 6 mice was dependent on CD8+T cells.

[0091] Example 7: Asparaginase ASNase can inhibit bladder cancer

[0092] 1. Purchase 10 5-week-old female C57BL / 6 mice and randomly divide them into 2 groups, with 5 mice in each group.

[0093] 2. Expand the MB49 cell line. Collect the cells, wash them with PBS, centrifuge and count the cells. Resuspend the cells with PBS so that 200ul of PBS contains 5 x 10 5 A tumor cell.

[0094] 3. Modeling: The mice were anesthetized with isoflurane, and the right axillary area was disinfected with iodine. 200ul of tumor cell suspension was injected subcutaneously into the right upper limb of the mouse using an insulin needle.

[0095] 4. Asparaginase administration regimen: L-ASNase was intraperitoneally injected on days 4, 6, 8, 11, 13, 15, 18, 20, and 22 after modeling, 2 U per mouse.

[0096] 5. Raising mice: After modeling, measure the length of the subcutaneous tumor with a vernier caliper every 4 days. Use this data to calculate the volume of the subcutaneous tumor. After the 24th day, euthanize the mice. Take the subcutaneous tumor, take a picture and weigh it. Figure 3A The gross photo of the subcutaneous tumor is shown. Figure 3B The volume of the subcutaneous tumor shown, Figure 3C Weights of subcutaneous tumors are indicated.

[0097] 6. Wash the subcutaneous tumor of MB49 cell-bearing C57BL / 6 mice with PBS several times and then cut it into pieces with scissors.

[0098] 7. Prepare tissue digestion solution: 320ug / ml type IV collagenase + 5ug / ml DNase, i.e. 32mg type IV collagenase + 0.5mg DNase + 100ml complete culture medium.

[0099] 8. Place the chopped tissue into a 15 ml centrifuge tube, add 5 ml of tissue lysis buffer, and place it in a 37° constant temperature shaker for 1 hour.

[0100] 9. After the sample is fully lysed, filter the sample with a cell filter. The filtrate is the cell suspension. Centrifuge at 1500rpm for 6 minutes to collect the cell precipitate.

[0101] 10. Dilute the flow cytometry antibodies (CD45 and CD8) with PBS in proportion. Add to the cell pellet and mix the cells using a vortex oscillator. Incubate at room temperature for 30 minutes.

[0102] 11. After the incubation, add 5 ml of PBS to the cell suspension containing the antibody to wash it once, and centrifuge at 1500 rpm for 6 minutes. Fix the cell pellet with flow cytometry fixative. Then perform flow cytometry on the machine. Figure 3D As shown, L-ASNase can increase the number of CD8+T cells in the tumor immune microenvironment.

[0103] 12. After modeling is completed, weigh the mice every 6 days. Figure 3E As shown, L-ASNase had no effect on the body weight of mice during the entire animal experiment.

[0104] See also Figure 3A-3E :A subcutaneous tumor-bearing model of C57BL / 6 mice was established using MB49 cells, and it was found that asparaginase ASNase can inhibit the growth of bladder cancer.

[0105] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of L-ASNase in the preparation of a drug for treating bladder cancer.

2. An application of L-ASNase in the preparation of in vitro drugs for the treatment of bladder cancer.

3. The use according to claim 2, wherein the in vitro is in cells.

4. Use of an agent for inhibiting or knocking down ASNS in the preparation of a drug for treating bladder cancer.

5. The use according to claim 4, characterized in that: The reagent comprises shRNA that knocks down ASNS.

6. The use according to claim 4, characterized in that: The reagent comprises siRNA that knocks down ASNS.

7. A kit for diagnosing the prognosis of bladder cancer, characterized in that: The kit comprises reagents for detecting ASNS.

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