CN114099682A - Application of PI3K gamma inhibitor in treating radiofrequency ablation residual tumor - Google Patents
Application of PI3K gamma inhibitor in treating radiofrequency ablation residual tumor Download PDFInfo
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- CN114099682A CN114099682A CN202111484270.0A CN202111484270A CN114099682A CN 114099682 A CN114099682 A CN 114099682A CN 202111484270 A CN202111484270 A CN 202111484270A CN 114099682 A CN114099682 A CN 114099682A
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Abstract
The invention discloses application of a PI3K gamma inhibitor in preparing a medicament for treating radiofrequency ablation residual tumor. The PI3K gamma inhibitor targeting lymphocytes can reprogram macrophages infiltrated in residual tumors, and can promote anti-PD-1 mediated tumor regression, enhance the anti-PD-1 treatment sensitivity of radiofrequency ablation residual tumors and overcome the drug resistance of radiofrequency ablation residual tumors in the prior art besides the anti-tumor effect of the PI3K gamma inhibitor. The treatment strategy of the residual tumor after the combined treatment of IRFA by the PI3K gamma inhibitor and the anti-PD-1 is provided, and the combined use effect is more obvious compared with the single use. The PI3K gamma inhibitor and the treatment strategy of the PI3K gamma inhibitor and the PD-1 blocking agent are cooperated, so that more clinical treatment schemes and development directions of related medicines can be provided for the residual tumor after clinical IRFA, the recurrence of the residual tumor after IRFA can be inhibited, and the survival period of a patient can be prolonged.
Description
Technical Field
The invention relates to the field of immunotherapy, and more specifically relates to the use of a PI3K gamma inhibitor in the treatment of radiofrequency ablation residual tumors.
Background
Radiofrequency ablation (RFA) is a thermal ablation technique and is one of the major curative therapies for early hepatocellular carcinoma (HCC). RFA causes heating of the tumor tissue as well as surrounding liver tissue, resulting in tumor necrosis. RFA treatment is effective and minimally invasive with low incidence of complications. However, in some cases, patients receiving RFA treatment experience a phenomenon known as "RFA insufficiency" (IRFA), i.e., the tumor cannot be completely ablated because its size or location causes the ablation local temperature to fail to reach the target temperature. While the tumor cells remaining from radiofrequency ablation may be a major source of recurrence and metastasis. Recently, a rapid and aggressive recurrence of metastases has occurred in some patients with IRFA, and HCC development following IRFA has been reported in several studies. However, the detailed mechanism of onset of tumor invasion after IRFA has not been fully elucidated.
Non-malignant cells of the Tumor Microenvironment (TME) may account for more than 50% of the mass of the primary tumor and its metastases. They have also been shown to play a complex role in the progression of residual tumors. Multiple preclinical animal model studies indicate that RFA local tumor ablation can release tumor antigens and induce systemic T cell-mediated anti-tumor immunity. However, RFA-induced immune responses are insufficient to prevent tumor recurrence. Some studies have indicated that macrophages infiltrate the boundary region between residual tumor and the ablated zone, and that due to the presence of macrophages, IRFA can induce anti-PD-1 resistance. Based on the expression of IRFA residual tumor, the prior art lacks a more effective treatment means.
Therefore, there is a need in the art for a drug or therapeutic strategy that can effectively treat IRFA tumors to inhibit their recurrence, so as to overcome the deficiencies of the prior art in treatment of residual tumors by radiofrequency ablation.
Disclosure of Invention
The present invention is directed to overcoming at least one of the deficiencies of the prior art described above and providing the use of PI3K γ inhibitors in the treatment of radiofrequency ablation residual tumors. According to the invention, researches show that the PI3K gamma inhibitor is used for targeting radiofrequency ablation of macrophage cells in residual tumor, so that the tumor immune microenvironment can be remodeled, the cytotoxic T cell mediated tumor immunotherapy effect can be promoted, and the anti-tumor effect can be realized.
The invention aims to provide application of a PI3K gamma inhibitor in preparing a medicament for treating radiofrequency ablation residual tumor. The treatment of the radiofrequency ablation residual tumor comprises killing radiofrequency ablation tumor cells, inhibiting the progression and recurrence of the radiofrequency ablation tumor, and promoting or cooperating with other medicines to kill the tumor. In one embodiment of the invention, radiofrequency ablation residual tumors were found to be highly expressed as PD-L1, indicating that anti-PD-1 therapy may be an effective treatment for this type of tumor, however, past studies have revealed that it is resistant to PD-1. In this regard, the present inventors have studied on the drug resistance mechanism, and found that the inhibition of PI3K γ can reduce the M2 phenotype transformation caused by macrophages phagocytizing radiofrequency ablation residual tumor through LAP pathway and the immune suppression environment generated thereby, and can promote the progress of anti-tumor immunity to promote the killing of tumor cells. Therefore, PI3K γ inhibitors can be used in radiofrequency ablation of residual tumors and achieve corresponding therapeutic effects.
Further, the drugs include drugs that enhance the sensitivity of anti-PD-1 therapy for radiofrequency ablation of residual tumors. In one embodiment of the invention, after inhibition by PI3K γ inhibitor, the immunosuppressive environment in the rf ablated residual tumor is attenuated, which can increase T cell proliferation, and more importantly, can enhance the anti-PD-1 treatment sensitivity of the rf ablated residual tumor, reverse the drug resistance of the rf ablated residual tumor, especially reverse the drug resistance to PD-1 blocker.
Further, PI3K gamma inhibitors include TG100-115, IPI-549, AZD3458, ZX-4081. In more than one embodiment of the invention, experiments such as treatment of radiofrequency ablation residual tumor, enhancement of curative effect of PD-1 blocking agent, adjustment of radiofrequency ablation residual tumor immune environment and the like are carried out by taking TG100-115 as PI3K gamma inhibitor representative, and the experiments are verified. Similarly, the PI3K gamma inhibitors of IPI-549, AZD3458 and ZX-4081 which are clinically used at present can also play the same effect.
Further, the drugs include drugs for enhancing the sensitivity of anti-PD-1 therapy for radiofrequency ablation of residual tumors, and the PI3K gamma inhibitors include TG100-115, IPI-549, AZD3458, and ZX-4081.
Further, the drugs include drugs that enhance the sensitivity of radiofrequency ablation residual tumors to treatment with PD-1 blockers, including InVivoMab anti-mouse PD-1, JS001, SHR-120, BGB-A317, IBI308, GLS-010, Nivolumab.
Still another object of the present invention is to provide the use of an LAP-IL-4-PI3K gamma pathway inhibitor for the preparation of a medicament for the treatment of radiofrequency ablation residual tumors. In one or more embodiments of the present invention, the present inventors found that, in the case of a radiofrequency ablation residual tumor, heat-treated dying cells of the radiofrequency ablation residual tumor cause macrophages to phagocytose via the LAP pathway, and the macrophages after LAP phagocytosis exhibit a tendency to shift from M1 type to M2 type, and form an immunosuppressive environment favorable for tumor progression, which also inhibits proliferation of T cells, and the like. And the interaction of radiofrequency ablation residual tumor cells and macrophages can also cause the drug resistance of the IRFA tumor to PD-1 blockers. In one or more embodiments of the invention, the main pathways leading to an immunosuppressive environment appear as: after macrophages undergo LAP, IL-4 mediated programming is enhanced, a PI3K gamma/AKT pathway is activated, cytokines including IL-10, CCL2 and CCL7 are secreted, immunosuppression is formed, and drug resistance of IRFA tumor cells to PD-1 blocking agents is promoted; and by applying the LAP inhibitor, the expression of IL-4 induced genes can be effectively reduced, so that the negative influence of LAP on T cell proliferation is eliminated, and the negative influence of tumor resistance brought by a downstream channel is reduced. The PI3K gamma inhibitor can reduce the negative effect of macrophages on phagocytosis of radiofrequency ablation residual tumor dying cells through LAP pathway in downstream, and the immune inhibitory tumor microenvironment can be remodeled.
Further, the drugs include drugs that enhance the sensitivity of anti-PD-1 therapy for radiofrequency ablation of residual tumors. By inhibiting on LAP-IL-4-PI3K gamma pathway, the drug resistance of tumor cells to PD-1 blocking agents can be reversed, and the treatment sensitivity to the PD-1 blocking agents is enhanced.
Further, LAP-IL-4-PI3K gamma pathway inhibitors include LAP inhibitors, PI3K gamma inhibitors.
Further, PI3K gamma inhibitors include TG100-115, IPI-549, AZD3458, ZX-4081, LAP inhibitors include Nox2 inhibitors, RUBCN siRNAs.
Further, the Nox2 inhibitor was diphenyleneiodonium chloride and the RUBCNsiRNA interfered with a target sequence of 5'-CCCACTCGGACACCAACAT-3'.
The invention further aims to provide a pharmaceutical composition which contains a PD-1 blocking agent and one or more of a PI3K gamma inhibitor and a LAP inhibitor.
Further, PD-1 blockers include: InVivoMab anti-mouse PD-1, JS001, SHR-120, BGB-A317, IBI308, GLS-010, Nivolumab, PI3K gamma inhibitors comprise TG100-115, IPI-549, AZD3458 and ZX-4081, LAP inhibitors comprise Nox2 inhibitor and RUBCN siRNA.
Compared with the prior art, the invention has the beneficial effects that: the invention discloses that macrophages are recruited after IRFA and phagocytosed by the LAP pathway, and that the LAP of macrophages promotes IL-4 mediated macrophage programming, activates the PI3K γ/AKT pathway, and expresses anti-inflammatory cytokines that induce immunosuppression. The macrophage infiltrated in the residual tumor can be reprogrammed by the selective inhibitor PI3K gamma of the target lymphocyte, and the inhibitor can promote the tumor regression mediated by anti-PD-1, enhance the treatment sensitivity of the radiofrequency ablation residual tumor to the PD-1 and overcome the drug resistance of the radiofrequency ablation residual tumor in the prior art besides the anti-tumor effect of the PI3K gamma inhibitor. Further, a treatment strategy for treating residual tumor after IRFA by combining a PI3K gamma inhibitor and anti-PD-1 is provided, the blocking of the PI3K gamma/AKT pathway can enhance the anti-tumor activity of the PD-1 blocking agent, inhibit malignant growth and improve the survival rate of a model after IRFA, and compared with the single application of the PD-1 blocking agent or the PI3K gamma inhibitor, the combined application effect is more obvious. The PI3K gamma inhibitor and the PD-1 blocker synergistic treatment strategy provided by the invention can provide more clinical treatment schemes and development directions of related medicaments for the residual tumor after clinical IRFA, so as to inhibit the recurrence of the residual tumor after IRFA and prolong the life cycle of a patient.
Drawings
Figure 1 increase in macrophage number in residual tumor after IRFA. (A, B) shows representative immunohistochemistry of CD68 and PDL1 in HCC sections of normal tumors and post-IRFA tumors in clinical patients. Scale bar 2000 μm and 500 μm (c) the proportion of F4/80+ and CD206+ cells in mouse tumor tissue after treatment was quantified by flow cytometry. (D) Representative immunofluorescence of F4/80 in mouse tumor sections. The area where DAPI is less frequent represents the ablation zone, where macrophages infiltrate. Scale bar 100 μm. (E) Representative immunohistochemistry for CD206 in mouse tumor sections. The section was divided into residual tumor, ablation zone and transition zone 3, where CD206+ cells were accumulated. Scale bar 500 μm.
FIG. 2 macrophages after IRFA represent immunosuppressive phenotypes. (A) T-SNE plots using scRNA-seq data from cells from normal tumors and tumor classification 7 days post IRFA. Cells from normal tumors were used as controls. Control and IRFA samples were pooled. (B) Expression of different immunoresponsive mRNA clusters in tumor cells before and after IRFA. (C) The violin plots show the probability distribution of gene expression for the immune response. (D) The volcano plots show differentially expressed chemokines (after/before IRFA) between the two lines. Genes with statistically significant differential expression (. gtoreq.1.2-fold, p <0.05) were located in the upper right and upper left quadrants. (E) Characteristic profiles of CCL2, CCL7 expression across cell clusters after IRFA identified in figure 2A. (F) Fluorescence microscopy images demonstrated CCL2, CCL7 (red) expression and DAPI nuclei (blue) and F4/80 (green) counterstaining in residual tumors. Scale bar 100 μm.
FIG. 3 macrophages engulf heat-treated tumor cells by LC 3-associated phagocytosis (LAP). (A) Representative flow cytometry plots illustrate macrophage phagocytosis of heat-treated GFP 1-6 cells in vitro and in vivo. (B)3D fluorescence microscopy images showing the location of GFP + tumor cells (green) with DAPI nuclei (blue) and F4/80 (red) counterstaining in residual tumors. (C) The hepa1-6 cells treated at 60 ℃ and 37 ℃ are stained by the membrane dye Wheat Germ Agglutinin (WGA), and the process of phagocytosis of tumor cells by macrophages is detected by a high content microscope. (D) Representative confocal images from mouse BMDM incubated with 37 ℃ and 60 ℃ GFP-transfected Hepa1-6 tumor cells and immunostained with LC3 antibody (red). Scale bar 50 μm; representative confocal images of Ethd-1 labeled Hepa1-6 tumor cells in mouse BMDM expressing GFP-LC3 (green). Scale bar 50 μm. (E) immunoblotting of lysates from murine BMDM with LC3 antibody. (F) BMDMs that phagocytose dead cells are stained by CM-H2 DCFDA. Flow cytometry was performed to assess global ROS production. BMDMs were treated with or without DPI (10 μ M) 1 hour prior to stimulation with dying cells. Data shown are the percentage of ROS + cells (left panel) and the Mean Fluorescence Intensity (MFI) of total cells (right panel).
FIG. 4 macrophages that underwent LAP exhibited the M2 phenotype. (A) CD206 expression on the surface of macrophages that phagocytose dying cells, measured by flow cytometry. (B) Fluorescence microscopy images showed F4/80 (green) and CD206 (red) in macrophages that engulfed dying cells compared to untreated macrophages. Scale bar 50 μm. (C) mRNA expression of M1 markers INOS, IL-6, IFN gamma, IL-12b, M2 markers Arg-1, IL-10 and chemokines CCL2, CCL 7. (D) IL-10, Arg-1, IL-12b and IFN gamma. (E) A cytokine expression array displaying cytokines from cell-free supernatants of untreated macrophages and macrophages that phagocytose dying cells; CCL7 ELISA results for cell culture conditioned media from group 3. (F) Chemotactic assays were used to assess the chemotactic capacity of monocytes isolated from bone marrow.
FIG. 5 LAP in macrophages enhances IL-4 sensitivity and regulates T cell proliferation. (A) M2 marked mRNA expression of Arg-1 and MRC 1. (B) Lysates of BMDM exposed to dying cells for 1 hour were analyzed by western blot to determine the protein expression levels of phosphorylated AKT Ser473 and total AKT. (C) Lysates from BMDM exposed to dying cells were immunoblotted with an antibody against LC 3. (D) mRNA expression of INOS, IL-10, IFN γ and IL-12b between groups 2. (E) Effect of TG100-115 on BMDM-mediated inhibition of splenic CD3+ T lymphocytes. T cell proliferation was quantified using carboxyfluorescein succinimidyl ester (CFSE).
FIG. 6 tumor progression of residual in situ tumors was inhibited by the combination of TG100-115 and anti-PD-1. (A) Representative ultrasound images of tumors on days 7, 11, 14, and 17. (B) Representative images of residual in situ tumors. (C) Growth curve of residual tumor. (D) representative flow cytometric analysis and quantification of CD8+ and CD4+ T cell populations. (E) Representative flow cytometry analysis and quantification of CD206+ cell populations. (F) Representative immunofluorescence and quantification of CD4, CD8, CD206 in tumor sections of different groups of mice. Scale bar 100 μm. (G) Fold change in mRNA expression in 4 groups of tumor tissues.
FIG. 7 schematic of macrophage induced LAP mechanism following IRFA: macrophages in the remaining tumor phagocytose the dead tumor cells, activating NOX2 and producing ROS for LAP formation. LAP enhances IL-4 mediated macrophage programming and activates the PI3K γ/AKT pathway. Cytokines including CCL2 and IL-10 are produced after LAP. At the same time, inhibition of LAP and blockade of the PI3K γ/AKT pathway can remodel the immunosuppressive state of macrophages.
FIG. 8(A) H & E staining to assess the establishment of IRFA model. (B) Tumor tissue was stained with ki67 antibody at a scale bar of 500 μm. (C) The ratio of mouse tumor tissue CD4 and Treg cells after treatment was quantified by flow cytometry.
Figure 9(a) Kaplan-Meier survival analysis of patients with high (> 50%) and low expression stratification for CXCR4 in the abundant regions of liver cancer cells (time sequence p-value ═ 0.002). CCR2 mRNA expression of (B-E) TCGALIHC dataset and Pearson correlation coefficients of immune checkpoint markers, (B) CTLA4(r 0.6, p <0.0001), (C) PDCD1(r 0.35, p <0.0001), (D) LAG3(r 0.47, p <0.0001), and (E) TIMD4(r 39, p < 0.0001).
FIG. 10(A) Hepa1-6 cells treated at 60 ℃ and 37 ℃ were stained with FITC-labeled anti-Annexin V antibody and PI and analyzed by flow cytometry. (B) Hepa1-6 cells treated at 60 ℃ and 37 ℃ were stained with the membrane-permeable DNA marker EthD-I. Dying cells were labeled with the red fluorescence of EthD-1. Scale bar 200 μm. (C) immunoblotting the tumor cell lysate after different temperature treatments with caspase-7 antibody. (D) F4/80 expression of flow cytometric analysis results was used to check the purity of macrophages. (E) Lysates of BMDMs and tumor cells treated at different temperatures were immunoblotted with LC3 antibody. (F) Relative expression of mRNA for RUBCN in macrophages treated with siRNA. (G) Lysates of siRUBCN-transfected BMDMs and tumor cells treated at different temperatures were immunoblotted with LC3 antibody.
FIG. 11(A) mRNA expression of M1 markers INOS, IL-6, IFN γ, IL-12b, M2 markers Arg-1, IL-10 and chemotactic factors CCL2, CCL7 between two groups. (B) mRNA expression of different cytokines. (C) CCL7 ELISA detection of cell culture media from both groups. (D) Chemotactic assays were used to assess the chemotactic capacity of monocytes isolated from bone marrow. (D) Relative mRNA expression of AKT1 and AKT2 in siRNA-treated macrophages
FIG. 12 representative p-AKT immunohistochemistry images. (A) Mouse tumor sections and (B) patient sections of normal tumor and post-IRFA tumor.
FIG. 13(A) an anatomical view of a carcinoma in situ of the liver. (B) Anatomical mapping of carcinoma in situ of liver following RFA. H & E staining images show that the IRFA model of liver orthotopic tumor is successfully established.
FIG. 14(A) is a schematic diagram of liver cancer treatment. (B) Representative images of residual tumors. (C) Growth curve of residual tumor (n ═ 5). (D) residual tumor weight 12 days after IRFA. (E) Representative images of distal tumors. (F) Growth curve of distal tumor (n ═ 5). (G) weight of distal tumor 12 days after IRFA. (H) Kaplan-Meier survival curves of mice after IRFA.
Fig. 15(a) representative flow cytometric analysis and quantification of CD8+ and CD4+ cells. (B) Representative flow cytometric analysis and quantification of F4/80+ and CD206+ cells. (C) Representative immunofluorescence and quantification of CD4, CD8, CD206 in tumor sections of mice of each group. Scale bar 50 μm. (D) Fold change in mRNA expression in 4 groups of tumor tissues.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The present invention will now be further described with reference to specific examples, which are provided for the purpose of illustration only and are not to be construed as limiting the invention. The test samples and test procedures used in the following examples include the following (if the specific conditions of the experiment are not specified in the examples, generally according to the conventional conditions or according to the conditions recommended by the reagent company; reagents, consumables, etc. used in the following examples are commercially available without specific description).
1. Mouse and tumor transplantation
Wild type C57BL/6J mice, 3-4 weeks old, were purchased from the center of medical laboratory animals, Guangdong province. Cells from the mouse hepatoma cell line Hepa1-6 were inoculated to 75cm2The cell culture flask of (4), to which 15ml of Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) was added and maintained at 37 ℃ in a humidified atmosphere containing 5% carbon dioxide. The medium was changed every two days, and when the Hepa1-6 cells grew to-90% confluence, they were washed 3 times with PBS to remove debris and dead cells, and then trypsinized to obtain single cancer cells. The cells were then resuspended in a PBS and Matrigel (1:1) mixture on ice. Final cell count was 5 × 107Individual cells/ml, 0.1ml of cell suspension was injected subcutaneously into the flank of mice.
2. Establishment of RFA deficiency animal model
After the tumor xenograft model was established as described above, the tumor growth was observed every two days. By means of a cursorThe longest and shortest lengths are calipers calculated to determine the size of the tumor. Tumor size was determined by the following formula: w ═ V2xL/2, W and L refer to the width and length of the tumor, respectively.
(1) Establishing an IRFA pre-model and an IRFA post-model
When the tumor xenografts reached 10mm in length, mice were anesthetized with isoflurane (RWD Life Science Co, China), and the tumors on the left were removed and labeled as the 'pre-RFA' group. Another tumor (right) was treated with RFA and the mice were returned to cages. After 7 days, RFA-treated tumors were removed and labeled as "post-RFA" group. RFA was performed using a bipolar RFA device (radianics Inc, MA, USA) which is a miniature radiofrequency needle with an effective tip length of 10 mm. To simulate clinical IRFA, a radiofrequency needle was inserted into a non-central location of the tumor at 3 watts for 30 seconds (90 joules).
(2) Establishment of residual and distant IRFA subcutaneous tumors
For treatment, tumors were implanted bilaterally. One tumor was treated with IRFA and defined as the IRFA group, while the other tumor was defined as the untreated distant tumor.
(3) Establishment of residual in situ tumors
When the tumor xenografts reached 10mm in diameter, the tumors were harvested and the non-necrotic tissue was cut into 1mm3The block was implanted into the left lobe of the liver of another tumor-free mouse. After 7 days, the cells were passed through a small animal ultrasound system (VisualSonics)
2100 systems, canada) to assess tumor volume and perform IRFA treatment. Tumor-bearing mice were then randomly divided into vehicle and different treatment groups, and tumor growth was monitored every three days.
3. 10X sample treatment and cDNA library preparation
The scRNA-Seq library was prepared from 10X Genomics chrome Controller instruments and chrome Single Cell 3' V2 Reagent Kits (10X Genomics, CA, USA). Cells were concentrated to 1000 cells/μ L, and approximately 17,000 cells were loaded into each channel to generate single cell gel bead emulsions (GEM). For each sample, 10,000 single cells were translated into mRNA barcodes. After the RT (real-time) step, GEM was destroyed, barcode-cDNA purified using a recovery reagent supplied at 10X, followed by silane Dyna Bead purification (Thermo Fisher, usa) and SPRI selection beads (Beckman, usa)), and then amplified. The amplified bar code cDNA is fragmented, A tail added with tail, connected with joint and index PCR amplified. The final cDNA library was quantified using a Qubit high sensitivity DNA assay (Thermo Fisher Scientific, USA) and the size distribution of the library was determined using a high sensitivity DNA chip on a Bioanalyzer 2200 (Agilent technologies, USA). All libraries were sequenced by HiSeqXten (Illumina, CA, USA) running at 150bp double ends.
4. Preparing bone marrow-derived macrophages: mononuclear cells were extracted from tibia and femur of C57BL/6 mice, and after removing erythrocytes with an erythrocyte lysis buffer, macrophage colony stimulating factor was added at a concentration of 20ng/ml to DMEM medium containing 10% fetal bovine serum and penicillin/streptomycin. After 7 days, bone marrow-derived macrophages were collected and their purity was verified by a cell sorter and flow cytometer.
5. T cell suppression assay: isolated spleen CD3+ cells were labeled with 1mM CFSE in pre-warmed PBS for 10 min at 37 ℃. CFSE-labeled CD8+ T cells were then placed in complete DMEM medium containing 1. mu.g/mL anti-CD 3 and 1. mu.g/mL anti-CD 28. BMDMs were cultured, matured, and treated with dying cells, and then co-cultured with CFSE-labeled CD8+ T cells. After 72 hours, cells were harvested and the CFSE signal was measured by flow cytometry.
6. Phagocytosis assay: macrophages were incubated with heat-treated GFP-transfected Hepa1-6 cells in a CO2 incubator at 37 ℃ for 4 hours, and free cells were washed away with PBS. Macrophages were labeled with anti-F4/80 antibody and characterized for phagocytosis by flow cytometry and confocal microscopy.
7. Study approval
Human clinical samples were obtained according to the protocol of human materials (approval No.: SYSEC-KY-KS-2020-212) approved by the ethical Committee of the commemorative Hospital of Sun-Yi of Zhongshan university. Animal experiments were conducted under guidelines approved by the institutional animal Care and use ethics committee of the university of Zhongshan (approval No.: SYSU-IACUC-2019-.
8. Statistical analysis
Statistical analysis of the partition maps was performed using GraphPad Prism v 8.0. The statistical tests used are listed in each chart title. Student's two-tailed t-test was used to compare different sets of quantitative data, and p <0.05 was considered statistically significant. P <0.001, p <0.01, p <0.05, NS ═ not significant (p > 0.05). Data are reported as mean ± Standard Error of Mean (SEM).
Example 1
Increased macrophage numbers in residual tumors after IRFA
(1) Liver cancer sample study
To investigate whether residual tumor recruitment macrophages after IRFA, a review study was performed on a subset of liver cancer patients at the university of zhongshan, grand asian, grand anecdotal memorial hospital, 21 patients who received RFA postoperative tumor resection due to recurrence were assigned to the RFA group, and 19 patients who received primary tumor resection without RFA were assigned to the non-RFA group, with no difference in clinical, biological and histological characteristics of the patients.
In the specimens, a greater number of tumor-infiltrating CD68 were observed in the RFA group than in the non-RFA group+Macrophages, and more infiltrated around the ablated zone (as shown in figure 1A). Furthermore, according to the prior studies, no high level of macrophage infiltration was shown in the simple recurrent tumor (non-RFA postoperative recurrent tumor), indicating that high level of macrophage infiltration is a characteristic expression of residual tumor after IRFA.
Meanwhile, the inventors also examined the expression of PD-L1, and found that the expression of PD-L1 in the RFA group is higher than that of PD-L1 in the non-RFA group (as shown in FIG. 1B), which indicates that anti-PD-1 treatment may be an effective treatment mode for IRFA-remaining tumors. However, in the present part of research, it is shown that macrophage infiltration leads to anti-PD-1 resistance of residual tumor, and therefore, how to provide effective anti-PD-1 therapy for treating residual tumor is still in need of research, and the following contents of the present invention are being studied.
(2) IRFA animal model study
To systematically understand the invasiveness and immunosuppressive status after IRFA, this example also established an IRFA animal model and developed the study as such. Specifically, subcutaneous tumors were inoculated on both sides of the mice, one tumor was excised and analyzed, and the cell status before IRFA was presented and defined as an untreated control group (NC). Another tumor was subjected to IRFA, excised 7 days later, and exhibited a cellular state after IRFA, defined as the IRFA group.
H & E staining was performed and successful construction of the IRFA model was confirmed (fig. 8A). IHC staining of tumor sections with Ki67 showed a higher proportion of circulating tumor cells after IRFA compared to cells before IRFA and that circulating tumor cells after IRFA were mainly located in the Transition Zone (TZ) between the ablation zone and the residual tumor, (fig. 8B), indicating the invasive status of the tumor cells.
Further analysis, by multi-label flow cytometry, the inventors detected a higher proportion of macrophages, in particular M2 macrophages, after IRFA in the mouse model (as shown in fig. 1C).
Further Immunofluorescence (IF) showed that macrophages were predominantly expressed in the Transition Zone (TZ) (fig. 1D, E), indicating that there may be some interactions between dying cells and macrophages.
Furthermore, a higher proportion of tregs was detected in the mouse model, indicating an immunosuppressive state following IRFA (fig. 8C).
Example 2
Identification of immunosuppressive function of IRFA-post-macrophage by single-cell transcriptome
Tumor cells from the NC and IRFA groups were collected using an IRFA animal model. Two groups of freshly isolated tumors were rapidly isolated and subjected to Fluorescence Activated Cell Sorter (FACS) to obtain viable single cells. Then, cDNA is prepared from the single cells, a single-cell RNA-seq library is constructed, and next-generation sequencing (NGS) is performed. The NC group analyzed 10690 cells in total, and the IRFA group analyzed 9672 cells. Among them, 99.85% of cells in the NC group and 99.7% of cells in the IRFA group pass quality control. The average number of mapped reads per cell in the NC group was about 8000 and the IRFA group was 7000. For the NC and IRFA groups, the median gene per cell was 726 and 1038, respectively. For non-malignant cells, these clusters were annotated as T cells, macrophages, Dendritic Cells (DCs), granulocytes, B cells and fibroblasts according to the specific profile of genes previously established to define such cells (fig. 2A).
M2-type macrophages are known to be a hallmark of various tumor immunotherapies. The inventors studied the cytokine profile of macrophages after IRFA and found that Mrc-1, IL-10 (gene associated with M2 marker) increased after IRFA, while Gbp3, Gbp5, Fcgr4 (gene associated with innate immunity), and Nod1 (gene associated with antigen presentation) decreased after IRFA (fig. 2B, C).
Furthermore, the expression of CCL2 (shown to be associated with residual tumor progression), CCL7, CXCL1, CXCL2, CXCL16 and CCL24 was higher in post-IRFA macrophages compared to normal tumors (fig. 2D). Furthermore CCL2 and CCL7, which are known as immunosuppressive-associated chemokines, were predominantly expressed in macrophages after IRFA, as evidenced by the IF results (fig. 2E, F).
Meanwhile, the inventors searched for chemokines indicating progression-free survival of HCC in TCGA gene expression, and found that the expression of CCL2 and CCL7 receptors CCR2 was correlated with disease-free survival (DFS), and that CCR2 expression was strongly and positively correlated with immunosuppressive markers such as CTLA4, PDCD1, TIM-4, and LAG3 (fig. 9).
These results indicate that macrophages recruit monocytes to the residual tumor after IRFA, and that monocytes switch to an immunosuppressive state, which may block the immune response after IRFA.
Example 3
Phagocytosis of heat-treated tumor cells by macrophages
As shown in example 1, macrophages aggregated around the Transition Zone (TZ) following IRFA (fig. 1D). In order to obtain the relationship between heat-treated tumor cells and macrophages, the inventors first established a heat-treated tumor cell model based on previous studies. Hepa1-6 cells were exposed for 10 min at 60 ℃ and then stained with PI/Annexin V. Tumor cells after heat treatment showed a significantly higher proportion of Annexin V and PI positive cells compared to tumor cells at 37 ℃ (fig. 10A). The heat-treated cells were further evaluated by cell membrane impermeable nuclear fluorescent dye, Ethidium Homodimer 1(EthD-1) and cleaned-Caspase-7 (see FIG. 10B, C).
We then extracted bone marrow-derived macrophages (BMDMs) and induced them to mature, and the purity of the macrophage cells was determined using flow cytometry (fig. 10D).
Further experiments were performed using the hpa 1-6 cell line stably expressing fluorescent GFP (hpa 1-6 cells expressing GFP). Transfection of tumor cells with heat-treated GFP stimulated BMDMs, which were detected by flow cytometry, and higher amounts of BMDMs phagocytosed GFP + heat-treated tumor cells (25.87% versus 10.46%) compared to untreated cells (fig. 3A).
In addition, a GFP + Hepa1-6 tumor-bearing mouse model was also established in this example. Mice received IRFA and were euthanized after 24 hours. By flow cytometry, it was found that more F4/80+ macrophages engulfed GFP + tumor cells (20.49% versus 5.77%) in mice receiving IRFA treatment compared to untreated tumor-bearing mice (fig. 3A). In tumor sections, GFP + tumor cells overlapped with F4/80+ macrophages under 3D confocal microscopy (FIG. 3B). The process of phagocytosis of heat-treated cells by macrophages was also observed by high content microscopy. Macrophages hardly phagocytosed normal tumor cells, but engulfed heat-treated cells within 3 hours (fig. 3C). These results indicate that macrophages engulf dying tumor cells after IRFA.
Second, macrophages engulf dying tumor cells through LC 3-related phagocytosis
Macrophages are reported to phagocytose apoptotic, necrotic and RIPK3 dependent necrotic cells by LC 3-associated phagocytosis (LAP). The inventors investigated whether IRFA induced LAP, and by confocal microscopy, the inventors found that macrophages stimulated by heat-treated tumor cells expressed higher levels of LC3 protein, while normal tumor cells did not induce high expression of LC3 and were hardly phagocytosed by macrophages (fig. 3D).
For GFP-LC3 transfected macrophages, heat-treated tumor cells were detected to be localized to LC3+In the compartment (fig. 3D). And at the cells which have been heat-treatedAfter treatment, a significant increase in LC3-II to LC3-I was observed by WB (fig. 3E).
LAP requires NADPH oxidase Nox2 to produce Reactive Oxygen Species (ROS), and the inventors found that heat-treated tumor cells induced macrophage ROS production (fig. 3F). In response to this manifestation, the inventors performed a related experiment using the Nox2 inhibitor diphenylene iodine chloride (DPI) one hour prior to treatment of macrophages with heat-treated tumor cells, with the result that DPI reverses the production of ROS in macrophages that phagocytose the heat-treated cells (fig. 3F).
In addition, inhibition of LAP process using rubicon (rubcn) siRNA (whose interfering target sequence is 5'-CCCACTCGGACACCAACAT-3') showed knockdown efficiency (fig. 10E). Silencing of RUBCN decreased the ratio of LC3-II to LC3-I, indicating that macrophages engulfed dying cells by LAP (FIG. 3E).
Example 4
Macrophages that undergo LC 3-associated phagocytosis exhibit the M2 phenotype
In this example, the function of macrophages that phagocytose dying tumor cells was studied. Flow cytometry analysis and immunofluorescence of the cell surface phenotypic marker CD206 confirmed modulation of the M2 phenotypic marker compared to untreated BMDM (figure 4A, B), qPCR results against M1(INOS, IFN γ, IL-12b) and M2(Arg-1, IL-10) phenotypic markers showed: in BMDM phagocytosing dying cells, the M2 phenotypic marker was upregulated (fig. 4C, fig. 11A).
Inhibition of LAP with RUBCN siRNA reduced IL-10 and Arg-1 levels and increased IFN γ and IL-12b levels (FIG. 4D).
The inventors further investigated chemokines after phagocytosis of dying cells by macrophages, in which the expression of CCL2, CCL7, CXCL1, CXCL2, CXCL16, CCL24 was increased (fig. 11B). The cytokine array map showed significant upregulation of CCL2, CXCL1, CXCL2 and CXCL16 (FIG. 4E). ELISA showed a significant upregulation of CCL7 (FIG. 4E). CCL2 and CCL7 were shown to be major chemokines for the recruitment of monocytes, and further, the inventors conducted a co-culture system of BMDM and bone marrow cells. Bone marrow cells were seeded in the upper compartment of the transwell insert and BMDM in the lower compartment. The results showed that macrophages treated with dying cells recruited more bone marrow-derived Ly6C + monocytes within one hour (fig. 4F).
In addition, macrophages showed higher expression levels of M1 gene (INOS, IL-6, TNF α, IL-12b), reduced expression levels of M2 gene (IL-10, Arg-1) and chemokines (CCL2, CCL7) (FIG. 4C) by DPI treatment. ELISA showed lower expression of macrophages secreting CCL7 (fig. 4E). Using the co-culture system described, DPI reduced the number of cells that underwent BMDM recruitment of LAP (fig. 4F).
Example 5
One, LC 3-associated phagocytosis enhances IL-4 mediated macrophage polarization
In the tumor microenvironment, macrophages have shown the M2 phenotype driven by IL-4 or IL-13. To examine the effect of LAP on macrophage polarization, we treated BMDMs with IL-4 in the presence or absence of dying cells. Macrophages that underwent LAP showed increased expression of IL-4-induced genes Arg1 and Mrc1, and silencing of RUBCN reduced IL-4-induced gene expression (fig. 5A). These results show that LAP enhances IL-4 mediated macrophage polarization.
Second, macrophages that undergo LAP inhibit T cell infiltration and function through PI3K γ/AKT pathway
This example detected higher AKT phosphorylation in animal models and patients (fig. 12A, B). Western blot analysis showed that levels of phosphorylated AKT increased after phagocytosis of dead cells, and silencing of RUBCN decreased levels of phosphorylated AKT (fig. 5B).
Inhibition of PI3K phosphorylation with PI3K γ inhibitor (TG100-115) decreased AKT phosphorylation and the ratio of LC3-II to LC3-I (fig. 5C). In addition, PCR results showed that TG100-115 increased the expression of IFN γ, IL-12b and INOS and decreased the expression of IL-10 (FIG. 5D), suggesting that PI3K activity is crucial for LAP-enhanced programming.
The inventors subsequently tested the inhibitory function of LAP-receiving bone marrow cells on the proliferation of naive CD 3T cells. In co-culture systems of BMDMs and T cells, dying tumor cells stimulated T cell proliferation for untreated BMDMs culture systems, whereas this stimulation was eliminated in LAP-bearing BMDMs and, after addition of TG100-115, reversed the inhibitory effect on T cell proliferation in the LAP group (fig. 5E).
Example 6
PI3K gamma inhibitor enhances anti-tumor immunity after IRFA
As with the previous examples, it has been demonstrated that: after IRFA, macrophages penetrate more into TZ, activate the PI3K γ/AKT pathway, and mediate immunosuppression.
The inventors evaluated whether targeting macrophages with PI3K γ inhibitor (TG100-115) could inhibit progression of residual tumors. Residual tumors are reported to be resistant to anti-PD-1 therapy, and macrophages play an important role in anti-PD-1 resistance. In this regard, further studies were conducted in the present application to establish subcutaneous residual tumors, distant tumors and liver orthotopic tumors in C57BL/6J mice and to evaluate the establishment of an orthotopic tumor IRFA model with IRFA, H & E staining (fig. 13). And whether TG100-115 could overcome anti-PD-1 resistance in residual tumors was investigated (fig. 14A).
As a result: inhibition of PI3K γ slowed tumor growth, and when synergistically acted with anti-PD-1 (this example used a PD-1 blocker, InVivoMab anti-mouse PD-1 from BioXcell), inhibited tumor growth, particularly in residual and distant tumors (fig. 14B-G) and in situ tumors (fig. 6A-C). TG100-115+ anti-PD-1 therapy also prolonged the survival of mice compared to TG100-115 and anti-PD-1 (FIG. 14H). Analysis of Tumor Infiltrating Lymphocytes (TILs) in dual treatments showed that TG100-115 decreased immunosuppression by increasing the M1/M2 ratio and improved the number of CD8+ T cells in residual and distant subcutaneous tumors as well as in situ tumors (fig. 15A-C, fig. 6D-F). Further, this example also investigated RNA expression of M1 and M2 markers in residual tumors after TG100-115, and the results showed that M1 markers (IFN γ and IL-12b) were more highly expressed in TG100-115 treated tumors (fig. 15D, fig. 6G).
With respect to the above examples, the present inventors first found that macrophages infiltrate around TZ in residual tumors of clinical patients and animal models and exhibit the M2 phenotype in animal models, and found that macrophages are one of the major components in creating an immunosuppressive microenvironment. This suggests that macrophages may be potential targets for residual tumor treatment after IRFA. Further, the experiments showed that macrophages respond to dying cells in residual tumors, undergo LAP (in this study, the inventors found that immunosuppression after IRFA is associated with LAP in macrophages. only heat-treated tumor cells induced LAP in macrophages, not just live tumor cells. reduction of ROS or silencing rubicon in macrophages both reduced Arg-1 and IL-10 and increased IL-12b and IFN γ), enhanced IL-4 mediated programming, activated the PI3K γ/AKT pathway, and secreted cytokines including IL-10, CCL2, and CCL 7. Inhibition experiments were then performed with PI3K γ inhibitors, and the results show that blockade of the PI3K γ/AKT pathway was demonstrated to remodel the immunosuppressive tumor microenvironment in vivo and in vitro (fig. 7). in one or more embodiments of the present invention, it was found that the PI3K γ/AKT pathway activates macrophages in response to LAP, and that blockade of PI3K γ reduces the levels of immunosuppressive cytokines (including Arg-1 and IL-10), increases T cell proliferation, and overcomes resistance to anti-PD-1 therapy in vivo. The invention applies three animal models: residual and distant subcutaneous tumors, as well as residual in situ tumors, were studied for their mechanism and effectiveness in three models of PI3K γ inhibitors, and the results showed that in all three models, PI3K γ inhibitors were effective and PI3K γ inhibitors were able to overcome the resistance exhibited by residual tumors against PD-1 therapy. After the PI3K gamma inhibitor and the anti-PD-1 are combined to treat IRFA, clinical patients have clinical treatment application prospect.
In general, the inventors have discovered a novel therapeutic strategy to target tumor-infiltrating macrophages in residual tumors via the PI3K γ/AKT pathway. The PI3K gamma inhibitor TG100-115 remodels the tumor immune microenvironment and promotes cytotoxic T cell-mediated tumor immunotherapy effects by targeting macrophages in residual tumors. The PI3K gamma inhibitor overcomes the drug resistance of the anti-PD-1 therapy to treat the residual tumor after IRFA, and provides a new combination treatment strategy for treating the residual tumor after IRFA.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.
Claims (10)
- Use of PI3K gamma inhibitor in preparing medicine for diagnosing, preventing and treating residual tumor caused by RF ablation.
- 2. The use of claim 1, wherein the medicament comprises a medicament that enhances the sensitivity of anti-PD-1 therapy for radiofrequency ablation of residual tumors.
- 3. Use according to claim 1 or 2, wherein the PI3K γ inhibitor comprises TG100-115, IPI-549, AZD3458, ZX-4081.
- 4. The use of claim 1, wherein the medicament comprises a medicament for enhancing the sensitivity of radiofrequency ablation residual tumors to treatment with PD-1 blockers, and the PD-1 blockers comprise InVivoMab anti-mouse PD-1, JS001, SHR-120, BGB-a317, IBI308, GLS-010, Nivolumab.
- Use of LAP-IL-4-PI3K gamma pathway inhibitor in preparation of medicine for diagnosing, preventing and treating radiofrequency ablation residual tumor.
- 6. The use of claim 5, wherein the medicament comprises a medicament that enhances the sensitivity of anti-PD-1 therapy for radiofrequency ablation of residual tumors.
- 7. The use according to claim 5 or 6, characterized in that the LAP-IL-4-PI3K gamma pathway inhibitor comprises LAP inhibitors, PI3K gamma inhibitors, LAP inhibitors comprise Nox2 inhibitors, rubbcnsina, PI3K gamma inhibitors comprise TG100-115, IPI-549, AZD3458, ZX-4081.
- 8. The use of claim 7, wherein the Nox2 inhibitor is diiphenylene iodochloride and the rub bcnsina interference target sequence is 5'-CCCACTCGGACACCAACAT-3'.
- 9. A pharmaceutical composition comprising a PD-1 blocker and one or more of a PI3K gamma inhibitor and a LAP inhibitor.
- 10. The pharmaceutical composition of claim 9, wherein the PD-1 blocker comprises: InVivoMab anti-mouse PD-1, JS001, SHR-120, BGB-A317, IBI308, GLS-010, Nivolumab, PI3K gamma inhibitors comprise TG100-115, IPI-549, AZD3458 and ZX-4081, LAP inhibitors comprise Nox2 inhibitor and RUBCNsiRNA.
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