Composition for improving anti-tumor immunity of advanced non-small cell lung cancer

Abstract

The invention provides a composition for improving anti-tumor immunity of advanced non-small cell lung cancer. The composition comprises a mitochondrial extracting solution extracted in vitro and a platinum chemotherapeutic drug. Experiments prove that the composition can improve immune infiltration of T cells and NK cells of the advanced non-small cell lung cancer, promote infiltration of immune cells to tumor tissues, activate anti-tumor immune response and enhance the anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to a drug for immune infiltration in tumors, and in particular to a composition for enhancing anti-tumor immunity and improving immune infiltration in advanced non-small cell lung cancer. Background Art

[0002] Lung cancer is the most common malignant tumor. Its incidence rate ranks second after breast cancer, and its mortality rate ranks first in the world. Non-small cell lung cancer (NSCLC) accounts for 85% of all pathological types of lung cancer.

[0003] Current treatment options for advanced NSCLC include chemotherapy, radiotherapy, targeted therapy, and immunotherapy, but the effectiveness of each approach is suboptimal. Chemotherapy, the most commonly used first-line treatment for advanced NSCLC, is limited by chemoresistance, leading to drug resistance, recurrence, and distant metastasis in many patients. Furthermore, chemotherapy is often associated with numerous adverse reactions, including damage to myeloid cells and decreased immune cells.

[0004] In recent years, significant progress has been made in tumor immunotherapy. Tumor immunotherapy stimulates or mobilizes the body's immune system, enhancing anti-tumor immunity within the tumor microenvironment, thereby controlling and killing tumor cells (Lymphatic Control of the Tumor Immune Microenvironment[J]. Blood, 2019, 134.). Tumor immunotherapy approaches are diverse and have been integrated with modern biotechnologies to become the fourth treatment modality for cancer, following surgery, chemotherapy, and radiotherapy (Role of the tumor microenvironment in PD-L1 / PD-1-mediated tumor immune escape[J]. Molecular Cancer, 2019, 18.). Furthermore, the high adaptability afforded by the diversity of T cell receptors offers unique advantages for addressing chemotherapy resistance. Therefore, enhancing immune infiltration during chemotherapy for non-small cell lung cancer is a key issue.

[0005] Mitochondria are one of the most important organelles in eukaryotic cells, their primary function being to provide energy for cell survival. Mitochondrial abnormalities can cause cell and even organ damage, and a variety of diseases, including cancer, are associated with mitochondrial dysfunction. A key difference between malignant tumors and normal cells lies in the metabolic reprogramming of tumor cells. Tumor mitochondria are key organelles in this metabolic reprogramming. Mitochondria within malignant tumor cells often undergo changes in number, structure, and function to adapt to the rapid growth of tumors in acidic and hypoxic environments.

[0006] Replacing dysfunctional mitochondria within cells with healthy ones is called mitochondrial transplantation. Mitochondrial transplantation is an invasive treatment method that involves isolating mitochondria from healthy tissues or cells and injecting them into damaged or missing areas, thereby healing damaged cells and restoring normal organ function. As a new therapeutic strategy, this approach has emerged in basic research for several diseases, particularly in the field of acute myocardial ischemia-reperfusion injury, and has already progressed to clinical trials.

[0007] There is growing evidence that mitochondrial transplantation plays an important role in cancer treatment (Mitochondrial Involvement in Cisplatin Resistance[J]. International Journal of Molecular Sciences, 2019, 20(14).). After mitochondria enter hypoxic and acidic tumor tissues, they will produce a large number of oxygen free radicals, inducing tumor cell death. This environmentally responsive pharmacological property of mitochondria can be used to eliminate tumor cells and restore damaged tissue function (Dissecting the alternation landscape of mitochondrial metabolism-related genes in lung adenocarcinoma and their latent mechanisms[J]. Aging-Us, 2023, 15(12): 5482-96.).

[0008] In addition, studies have reported that tumor cells can "steal" immune cell mitochondria through tunneling nanotubes, thereby weakening the immune cells' immune capabilities. Inhibiting nanotube formation can prevent tumor cells from "stealing" immune cell mitochondria and enhance the immune system's ability to kill tumor cells (Intercellular nanotubes mediate mitochondrial trafficking between cancer and immune cells [J]. Nature Nanotechnology, 2022, 17(1): 98-+.). Summary of the Invention

[0009] The purpose of the present invention is to provide a composition for enhancing anti-tumor immunity of advanced non-small cell lung cancer, so as to improve immune infiltration during chemotherapy of advanced non-small cell lung cancer and enhance the anti-tumor effect.

[0010] Currently, no studies have proven that mitochondrial transplantation can increase immune infiltration. This study, for the first time, found that mitochondrial transplantation combined with cisplatin chemotherapy can significantly increase immune infiltration in advanced non-small cell lung cancer.

[0011] Based on the above research findings, the present invention first provides a composition for enhancing the immune infiltration of advanced non-small cell lung cancer tumors, comprising: a) mitochondrial extracts extracted in vitro; and b) Platinum-based chemotherapy drugs.

[0012] Furthermore, the composition of the present invention is more specifically used to enhance the immune infiltration of T cells and NK cells in advanced non-small cell lung cancer tumors.

[0013] Furthermore, the platinum chemotherapy drug described in the present invention is cisplatin.

[0014] Furthermore, the in vitro extracted mitochondrial extract described in the present invention is mitochondria extracted from in vitro tissues or cells using various conventional mitochondrial extraction kits.

[0015] Furthermore, the present invention also provides the use of the above composition in the preparation of a drug for enhancing anti-tumor immunity in advanced non-small cell lung cancer, wherein the composition comprises an in vitro extracted mitochondrial extract and a platinum chemotherapy drug.

[0016] Both flow cytometry and immunohistochemical staining analysis found that the composition of the present invention significantly increased the immune infiltration of T cells and NK cells in mouse tumors, while there was no significant change in the immune infiltration of T cells and NK cells in mouse tumors treated with cisplatin alone or undergoing mitochondrial transplantation alone, demonstrating that cisplatin chemotherapy alone and mitochondrial transplantation alone cannot improve the immune infiltration of mouse tumors, while the composition of the present invention significantly improved the immune infiltration of mouse tumors.

[0017] Transcriptomic sequencing results further revealed that the composition of the present invention can significantly enhance the function of tumor-infiltrating immune cells, promote the infiltration of immune cells into tumor tissues, and activate anti-tumor immune responses. Gene set enrichment analysis also showed that the composition of the present invention can promote T cell differentiation, T cell activation, antigen presentation and processing, exogenous antigen peptide antigen processing and presentation, natural killer cell-mediated cytotoxicity, natural killer cell activation, and enrichment of adaptive immune responses, significantly leading to enrichment of anti-tumor immune responses including T cells, NK cells, and antigen presentation, which is in contrast to the downregulation of T cell activation, natural killer cell-mediated cytotoxicity, innate immunity, and adaptive immune response enrichment caused by cisplatin chemotherapy alone.

[0018] Therefore, the present invention actually provides the use of mitochondria in the preparation of drugs for enhancing the anti-tumor immune effect of platinum-based chemotherapy drugs.

[0019] The composition of the present invention can significantly improve the immune infiltration of advanced non-small cell lung cancer, overcome the defects of myeloid cell damage and immune cell reduction caused by platinum chemotherapy drugs, and enhance its anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the experimental flow chart of the subcutaneous transplantation tumor model of non-small cell lung cancer LLC cells in C57BL / 6 mice.

[0021] Figure 2 Figure 3 is the growth curve of tumor tissue in each group of mice during the 18-day intervention.

[0022] Figure 3 Figures 2 and 3 show the tumor images and final tumor volumes of mice in each group 18 days after intervention (compared with the control group, * p < 0.05; **** p < 0.0001; compared with the DDP group, # p < 0.05).

[0023] Figure 4 Figure 3 shows the changes in body weight of mice in each group after 18 days of intervention.

[0024] Figure 5 It is the gene signature displayed by gene set enrichment analysis (GSEA).

[0025] Figure 6Flow cytometry was used to analyze the changes in immune infiltration in the tumors of the mice in each group (compared with the control group, * p < 0.05; ** p < 0.01; compared with the DDP group, # p < 0.05; ## p < 0.01).

[0026] Figure 7 Flow cytometry was used to analyze the changes in immune infiltration in tumors of mice treated with mitochondrial-independent intervention.

[0027] Figure 8 The immune infiltration of the tumors of mice in each group was analyzed by immunohistochemistry.

[0028] Figure 9 It is a heat map display of the pathways related to transcriptome changes, where red indicates activation and blue indicates repression. Implementation Method

[0029] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention so that those skilled in the art can better understand and utilize the present invention, but are not intended to limit the scope of protection of the present invention.

[0030] Unless otherwise specified, the production processes, experimental methods or detection methods involved in the embodiments of the present invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field and are very clear and unambiguous in the relevant fields of use. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment to implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0031] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in their sources and are all conventional products that can be purchased through regular commercial channels or prepared according to conventional methods well known to those skilled in the art. Example

[0032] Example 1

[0033] To study the effect of mitochondrial transplantation on cisplatin (DDP) chemotherapy in advanced non-small cell lung cancer, the present invention conducted a C57BL / 6 mouse non-small cell lung cancer LLC subcutaneous transplant tumor model experiment.

[0034] Thirty six-week-old male C57BL / 6 mice were housed in an SPF environment. After one week of adaptation, non-small cell lung cancer LLC cells were subcutaneously implanted on the right side of the mice. When the tumor volume reached 50-80 mm, the mice were placed in a 40-well plate. 3The mice were randomly divided into 6 experimental groups, including control group, DDP group, DDP+mitotail-1 group, DDP+mitotail-2 group, DDP+mitotail+ju-1 group and DDP+mitotail+ju-2 group, with 5 mice in each group.

[0035] The tumor-bearing mice in the control group were not treated; the tumor-bearing mice in the DDP group were treated with intraperitoneal injection of cisplatin (DDP) for two weeks, 5 mg / kg each time, twice a week; the tumor-bearing mice in the DDP+mito tail-1 group were first treated with intraperitoneal injection of DDP for one week, and then received tail vein injection of mitochondria once, 1×10 7 The tumor-bearing mice in the DDP+mito tail-2 group were first injected with DDP intraperitoneally for one week, and then injected with mitochondria twice through the tail vein, each time with 1×10 7 The tumor-bearing mice in the DDP+mitotail+local group were first given DDP intraperitoneal injection for one week, and then mitochondria were injected into the tail vein and the tumor once, with 1×10 7 The tumor-bearing mice in the DDP+mitotail+local group were first given DDP intraperitoneal injection for one week, and then mitochondria were injected into the tail vein and tumor twice, each time with 1×10 7 mitochondria, the experimental process is as follows Figure 1 shown.

[0036] The tumor size and body weight of the mice were measured every other day, recorded and counted. After 18 days of intervention, the mice were killed and samples were taken for photography.

[0037] The changes of tumor volume in each group of mice during the experiment are as follows Figure 2 Compared with the control group, the tumor growth rate of mice in each intervention group was significantly reduced after 18 days of intervention. Compared with the tumors in the DDP group, the tumor growth rate of mice treated with mitochondrial transplantation combined with DDP chemotherapy was also reduced. In particular, the tumor growth rate of mice treated with two injections of mitochondrial transplantation combined with DDP chemotherapy was significantly lower than that of the DDP group, and the difference was statistically significant.

[0038] Figure 3Figure 18 shows the tumor volume of mice after 18 days of intervention. (A) shows a tumor image, and (B) shows the final tumor volume. Compared to the control group, tumors in each intervention group were significantly smaller. The inhibition rates compared to the control group were: DDP group, 43.8%; DDP + mitotail-1 group, 62%; DDP + mitotail-2 group, 68%; DDP + mitotail + zodiac-1 group, 70%; and DDP + mitotail + zodiac-2 group, 74.5%. Furthermore, mice treated with two tail vein injections of mitochondrial transplantation combined with DDP chemotherapy exhibited significantly smaller tumors than those in the DDP group (the inhibition rate in the DDP + mitotail + zodiac-2 group was 54.5%, compared to the DDP group).

[0039] The results showed that in C57BL / 6 mice with LLC subcutaneous non-small cell lung cancer tumors, a full four-dose DDP chemotherapy regimen, or two DDP chemotherapy regimens combined with tail vein or tail vein plus local intratumoral injection of mitochondrial transplantation (one or two times) significantly inhibited LLC tumor growth and reduced tumor volume. Even if the number of DDP chemotherapy cycles was halved, the combination of mitochondrial transplantation and DDP chemotherapy could achieve the same effect as a full four-dose DDP chemotherapy regimen. The tumor growth rate and size of mice treated with tail vein plus two local intratumoral injections of mitochondrial transplantation combined with DDP chemotherapy were significantly lower than those of mice in the DDP group, significantly superior to the effect of a full four-dose DDP chemotherapy regimen.

[0040] To further evaluate the safety of DDP chemotherapy and mitochondrial transplantation, the body weight of mice was monitored during the experiment, e.g. Figure 4 As shown, compared to the control group, mice in the DDP chemotherapy group experienced weight loss, while the mice in the mitochondrial transplantation combined with DDP chemotherapy group gradually regained weight after stopping DDP chemotherapy and undergoing mitochondrial transplantation. These weight results indicate that mitochondrial transplantation does not cause weight loss in mice and is relatively safe.

[0041] Example 2

[0042] To investigate the effects of mitochondrial transplantation combined with cisplatin (DDP) chemotherapy on the anti-tumor immune-related functions of advanced non-small cell lung cancer, this example performed transcriptomic analysis on tumors in all groups of mice and performed functional enrichment analysis on differentially expressed genes between groups.

[0043] Chemotherapy is often accompanied by adverse reactions such as immune cell reduction. Figure 5The results were consistent with those in the previous study. Gene set enrichment analysis (GSEA) showed that cisplatin chemotherapy caused downregulation of T cell activation, natural killer cell-mediated cytotoxicity, innate immunity, and adaptive immune response enrichment. However, mitochondrial extract combined with cisplatin treatment promoted T cell differentiation, T cell activation, antigen presentation and processing, exogenous antigen peptide antigen processing and presentation, natural killer cell-mediated cytotoxicity, natural killer cell activation, and adaptive immune response enrichment. The specific results are as follows: Figure 5 shown.

[0044] This proves that mitochondrial extract combined with cisplatin chemotherapy has the effect of enhancing the anti-tumor immune infiltration of advanced non-small cell lung cancer, can significantly enhance the function of tumor-infiltrating immune cells, and activate anti-tumor immune response.

[0045] Example 3

[0046] To further investigate the effects of mitochondrial extract and cisplatin (DDP) chemotherapy on anti-tumor immune infiltration in advanced non-small cell lung cancer, flow cytometry was performed on tumors in all groups of mice in this example.

[0047] like Figure 6 As shown in the flow cytometry plots (A) and statistical graphs (B), compared with the control group, T cell immune infiltration in the tumors of mice treated with DDP+mitotail+ju-1 and DDP+mitotail+ju-2 groups was significantly increased, while T cell immune infiltration in the tumors of mice treated with DDP did not change significantly. Compared with the DDP group, T cell immune infiltration in the tumors of mice treated with DDP+mitotail-2, DDP+mitotail+ju-1, and DDP+mitotail+ju-2 groups was significantly increased. Compared with the control group, NK cell immune infiltration in the tumors of mice treated with DDP+mitotail-2 and DDP+mitotail+ju-2 groups was significantly increased, while NK cell immune infiltration in the tumors of mice treated with DDP did not change significantly. Compared with the DDP group, NK cell immune infiltration in the tumors of mice treated with DDP+mitotail-2 and DDP+mitotail+ju-2 groups was significantly increased.

[0048] At the same time, this example also specially set up a group of tumor-bearing mice with separate mitochondrial intervention: tail + local-2 group. The mice in this group were not given DDP intraperitoneal injection intervention, but were directly given mitochondria tail vein + local injection into the tumor twice. Flow cytometry analysis was performed and compared with the control group and DDP group. The results are as follows: Figure 7 As shown in the flow cytometry graph (A) and the statistical graph (B), the proportion of T cells and NK cells in the tumors of mice in the Tail+Ju-2 group was not significantly different from that in the control group.

[0049] The above flow cytometry results of mouse tumors indicate that DDP chemotherapy alone or mitochondrial extract intervention alone cannot improve the immune infiltration of mouse tumors, but mitochondrial transplantation combined with cisplatin chemotherapy can significantly improve the immune infiltration of mouse tumors.

[0050] Likewise, in Figure 8 Immunohistochemical staining analysis of tumors in all groups of mice found that compared with the control group, CD3 staining in the tumors of mice in the DDP+mitotail-1, DDP+mitotail-2, DDP+mitotail+ju-1, and DDP+mitotail+ju-2 groups was significantly darker, indicating a significant increase in T cell immune infiltration, while there was no significant change in T cell immune infiltration in the tumors of mice in the DDP group. Compared with the DDP group, CD3 staining in the tumors of mice in the DDP+mitotail-1, DDP+mitotail-2, DDP+mitotail+ju-1, and DDP+mitotail+ju-2 groups was significantly darker, indicating a significant increase in T cell immune infiltration. Compared with the control group, NKP46 staining in the tumors of mice in the DDP+mitotail-1, DDP+mitotail-2, DDP+mitotail+ju-1, and DDP+mitotail+ju-2 groups was significantly darker, indicating that NK cell immune infiltration in the tumors of mice was significantly increased, while there was no significant change in NK cell immune infiltration in the tumors of mice in the DDP group. Compared with the DDP group, NKP46 staining in the tumors of mice in the DDP+mitotail-1, DDP+mitotail-2, DDP+mitotail+ju-1, and DDP+mitotail+ju-2 groups was significantly darker, indicating that NK cell immune infiltration in the tumors of mice was significantly increased.

[0051] The above immunohistochemical staining results of mouse tumors also show that DDP chemotherapy alone cannot improve the immune infiltration of mouse tumors, while mitochondrial transplantation combined with cisplatin chemotherapy can significantly improve the immune infiltration of mouse tumors.

[0052] Example 4

[0053] In order to study the effects of mitochondrial extract and cisplatin (DDP) chemotherapy on anti-tumor immune activation in advanced non-small cell lung cancer, this example further analyzed the transcriptomic sequencing results and found that Figure 9 As shown in the results, mitochondrial extract and cisplatin (DDP) chemotherapy intervention can significantly induce the enrichment of anti-tumor immune responses including T cells, NK cells and antigen presentation, which is consistent with the GSEA results, indicating that mitochondrial extract and cisplatin (DDP) chemotherapy significantly activate anti-tumor immunity.

[0054] In addition, pathways related to tumor cell apoptosis (MAPK, programmed cell death) were significantly upregulated in mitochondrial extracts and cisplatin (DDP) chemotherapy, while pathways related to tumor malignancy (Wnt) were significantly inhibited.

[0055] The above experimental results show that mitochondrial extract combined with cisplatin treatment can not only significantly enhance the function of tumor-infiltrating immune cells, but also significantly promote the infiltration of immune cells into tumor tissues and significantly activate anti-tumor immune responses.

[0056] The above embodiments of the present invention do not describe all details in detail, nor do they limit the present invention to the above embodiments. Various changes, modifications, substitutions, and variations made by those skilled in the art without departing from the principles and purpose of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. A composition for enhancing anti-tumor immunity in advanced non-small cell lung cancer, comprising: a) mitochondrial extract extracted in vitro; and b) Platinum-based chemotherapy drugs.

2. The composition according to claim 1 is used to enhance the immune infiltration of T cells and NK cells in advanced non-small cell lung cancer tumors.

3. The composition according to claim 1, wherein the platinum chemotherapy drug is cisplatin.

4. Use of a composition in the preparation of a drug for enhancing anti-tumor immunity in advanced non-small cell lung cancer, the composition comprising an in vitro extracted mitochondrial extract and a platinum chemotherapy drug.

5. The application of mitochondria in the preparation of drugs for enhancing the anti-tumor immune effect of platinum-based chemotherapy drugs.

6. The use according to claim 5, wherein the anti-tumor immune effect includes overcoming the damage of myeloid cells and the decrease of immune cells caused by chemotherapy.

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