ABSTRACT
BACKGROUND: Neoantigen reactive T cell (NRT) has the ability to inhibit the growth of tumors expressing specific neoantigens. However, due to the difficult immune infiltration and the inhibition of tumor microenvironment, the therapeutic effect of NRT in solid tumors is limited. In this study, we designed NRT cells (7×19 NRT) that can express both interleukin-7 (IL-7) and chemokine C-C motif ligand 19 (CCL19) in mouse lung cancer cells, and evaluated the difference in anti-tumor effect between 7×19 NRT cells and conventional NRT cells. METHODS: We performed next-generation sequencing and neoantigen prediction for mouse Lewis lung carcinoma (LLC), prepared RNA vaccine, cultured NRT cells, constructed retroviral vectors encoding IL-7 and CCL19, transduced NRT cells and IL-7 and CCL19 were successfully expressed, and 7×19 NRT was successfully obtained. The anti-tumor effect was evaluated in vivo and in vitro in mice. RESULTS: The 7×19 NRT cells significantly enhanced the proliferation and invasion ability of T cells by secreting IL-7 and CCL19, achieved significant tumor inhibition in the mouse lung cancer and extended the survival period of mice. The T cell infiltration into tumor tissue and the necrosis of tumor tissue increased significantly after 7×19 NRT treatment. In addition, both 7×19 NRT treatment and conventional NRT treatment were safe. CONCLUSIONS: The anti-solid tumor ability of NRT cells is significantly enhanced by the arming of IL-7 and CCL19, which is a safe and effective genetic modification of NRT.
Subject(s)
Chemokine CCL19 , Interleukin-7 , Lung Neoplasms , Mice, Inbred C57BL , T-Lymphocytes , Animals , Mice , Interleukin-7/genetics , Interleukin-7/immunology , Chemokine CCL19/genetics , Chemokine CCL19/immunology , Lung Neoplasms/immunology , Lung Neoplasms/genetics , Lung Neoplasms/therapy , T-Lymphocytes/immunology , Cell Line, Tumor , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/genetics , Carcinoma, Lewis Lung/therapy , Antigens, Neoplasm/immunology , Antigens, Neoplasm/genetics , Female , Cell Proliferation , HumansABSTRACT
BACKGROUND: Phosphoribosyl pyrophosphate synthetase 2 (PRPS2) is known as an oncogene in many types of cancers, including lung cancer. However, its role in regulating tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) remains unclear. Our study aimed to explore the involvement of PRPS2 in TAM and MDSC regulation. METHODS: Stable Lewis lung cancer (LLC) cell lines were established using a lentivirus system. These LLC lines were then used to establish tumor model in mice. The levels of target genes were determined using qPCR, western blotting, and ELISA assays. The percentage of different immune cell types was analyzed using fluorescence-activated cell sorting. The chemotaxis ability of TAM and MDSC was evaluated using an in vitro transwell chemotaxis assay. RESULTS: Notably, PRPS2 was found to regulate the chemotaxis of TAM and MDSC in tumor cells, as evidenced by the positive correlation of PRPS2 expression levels and abundance of TAM and MDSC populations. In addition, the expression of CCL2, mediated by PRPS2, was identified as a key factor in the chemotaxis of TAM and MDSC, as evidenced by a significant reduction in macrophages and MDSC numbers in the presence of the CCL2 antibody. Furthermore, in vivo experiments confirmed the involvement of PRPS2 in mediating CCL2 expression. PRPS2 was also found to regulate immune cell infiltration into tumors, whereas knockdown of CCL2 reversed the phenotype induced by PRPS2 overexpression. In tumor tissues from mice implanted with LLC-PRPS2-shCCL2 cells, a notable increase in CD4+ and CD8+ T cell percentages, alongside a marked decrease in TAMs, M-MDSC, and PMN-MDSC, was observed. CONCLUSION: Taken together, PRPS2 plays a crucial role in modulating the antitumor immune response by reprogramming CCL2-mediated TAM and MDSC.
Subject(s)
Chemokine CCL2 , Lung Neoplasms , Myeloid-Derived Suppressor Cells , Tumor-Associated Macrophages , Animals , Mice , Myeloid-Derived Suppressor Cells/metabolism , Myeloid-Derived Suppressor Cells/immunology , Chemokine CCL2/metabolism , Chemokine CCL2/genetics , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lung Neoplasms/genetics , Tumor-Associated Macrophages/immunology , Tumor-Associated Macrophages/metabolism , Humans , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/pathology , Carcinoma, Lewis Lung/genetics , Mice, Inbred C57BL , Cell Line, TumorABSTRACT
The durability of an antitumor immune response is mediated in part by the persistence of progenitor exhausted CD8+ T cells (Tpex). Tpex serve as a resource for replenishing effector T cells and preserve their quantity through self-renewal. However, it is unknown how T cell receptor (TCR) engagement affects the self-renewal capacity of Tpex in settings of continued antigen exposure. Here we use a Lewis lung carcinoma model that elicits either optimal or attenuated TCR signaling in CD8+ T cells to show that formation of Tpex in tumor-draining lymph nodes and their intratumoral persistence is dependent on optimal TCR engagement. Notably, attenuated TCR stimulation accelerates the terminal differentiation of optimally primed Tpex. This TCR-reinforced Tpex development and self-renewal is coupled to proximal positioning to dendritic cells and epigenetic imprinting involving increased chromatin accessibility at Egr2 and Tcf1 target loci. Collectively, this study highlights the critical function of TCR engagement in sustaining Tpex during tumor progression.
Subject(s)
CD8-Positive T-Lymphocytes , Carcinoma, Lewis Lung , Hepatocyte Nuclear Factor 1-alpha , Mice, Inbred C57BL , Receptors, Antigen, T-Cell , Animals , CD8-Positive T-Lymphocytes/immunology , Receptors, Antigen, T-Cell/metabolism , Receptors, Antigen, T-Cell/immunology , Mice , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/pathology , Carcinoma, Lewis Lung/metabolism , Hepatocyte Nuclear Factor 1-alpha/metabolism , Cell Differentiation/immunology , Dendritic Cells/immunology , Signal Transduction/immunology , Mice, Knockout , Lymphocyte Activation/immunology , Cell Self Renewal , Mice, Transgenic , Early Growth Response Protein 2ABSTRACT
BACKGROUND: It is amply demonstrated that cigarette smoke (CS) has a high impact on lung tumor progression worsening lung cancer patient prognosis and response to therapies. Alteration of immune cell types and functions in smokers' lungs have been strictly related with smoke detrimental effects. However, the role of CS in dictating an inflammatory or immunosuppressive lung microenvironment still needs to be elucidated. Here, we investigated the effect of in vitro exposure to cigarette smoke extract (CSE) focusing on macrophages. METHODS: Immortalized murine macrophages RAW 264.7 cells were cultured in the presence of CS extract and their polarization has been assessed by Real-time PCR and cytofluorimetric analysis, viability has been assessed by SRB assay and 3D-cultures and activation by exposure to Poly(I:C). Moreover, interaction with Lewis lung carcinoma (LLC1) murine cell models in the presence of CS extract were analyzed by confocal microscopy. RESULTS: Obtained results indicate that CS induces macrophages polarization towards the M2 phenotype and M2-phenotype macrophages are resistant to the CS toxic activity. Moreover, CS impairs TLR3-mediated M2-M1 phenotype shift thus contributing to the M2 enrichment in lung smokers. CONCLUSIONS: These findings indicate that, in lung cancer microenvironment of smokers, CS can contribute to the M2-phenotype macrophages prevalence by different mechanisms, ultimately, driving an anti-inflammatory, likely immunosuppressive, microenvironment in lung cancer smokers.
Subject(s)
Lung Neoplasms , Macrophages , Tumor Microenvironment , Animals , Mice , Lung Neoplasms/pathology , Macrophages/drug effects , Macrophages/metabolism , Macrophages/immunology , Tumor Microenvironment/drug effects , RAW 264.7 Cells , Cell Survival/drug effects , Macrophage Activation/drug effects , Smoke/adverse effects , Cell Polarity/drug effects , Humans , Carcinoma, Lewis Lung/pathology , Carcinoma, Lewis Lung/immunologyABSTRACT
Cryoablation is a therapeutic modality for lung adenocarcinoma that destroys target tumors using lethal levels of cold, resulting in the release of large amounts of specific antigens that activate immune responses. However, tumor immune checkpoint escape mechanisms prevent these released self-antigens from inducing effective anti-tumor immune responses. To overcome this challenge, we propose the use of immune checkpoint inhibitors to relieve T cell inhibition by immune checkpoints and enhance the anti-tumor immune response mediated by cryoablation. We used bilateral tumor-bearing mouse models and a specific cryoablation instrument to study the efficacy of cryoablation combined with PD-1 inhibitors in Lewis lung adenocarcinoma model mice. We found that cryoablation combined with PD-1 inhibitors significantly inhibited the growth of mouse lung adenocarcinoma, prolonged mouse survival, and enhanced the anti-tumor immune response. Moreover, this combined regimen could synergistically promote the activation and proliferation of T cells via the PI3K/AKT/mTOR pathway. The present study provides a strong theoretical basis for the clinical combination of cryoablation and PD-1 inhibitors.
Subject(s)
Carcinoma, Lewis Lung , Cryosurgery , Immune Checkpoint Inhibitors , Lung Neoplasms , Phosphatidylinositol 3-Kinases , Programmed Cell Death 1 Receptor , Proto-Oncogene Proteins c-akt , Signal Transduction , TOR Serine-Threonine Kinases , Animals , Proto-Oncogene Proteins c-akt/metabolism , Cryosurgery/methods , Mice , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/pathology , Carcinoma, Lewis Lung/drug therapy , TOR Serine-Threonine Kinases/metabolism , TOR Serine-Threonine Kinases/antagonists & inhibitors , Phosphatidylinositol 3-Kinases/metabolism , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Signal Transduction/drug effects , Lung Neoplasms/immunology , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Mice, Inbred C57BL , Cell Line, Tumor , Combined Modality Therapy , T-Lymphocytes/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Female , Adenocarcinoma of Lung/immunology , Adenocarcinoma of Lung/drug therapy , Adenocarcinoma of Lung/pathology , Adenocarcinoma of Lung/metabolism , Adenocarcinoma of Lung/surgeryABSTRACT
BACKGROUND: Metastatic disease is a major and difficult-to-treat complication of lung cancer. Considering insufficient effectiveness of existing therapies and taking into account the current problem of lung cancer chemoresistance, it is necessary to continue the development of new treatments. METHODS: Previously, we have demonstrated the antitumor effects of reprogrammed CD8+ T-cells (rCD8+ T-cells) from the spleen in mice with orthotopic lung carcinoma. Reprogramming was conducted by inhibiting the MAPK/ERK signalling pathway through MEKi and the immune checkpoint PD-1/PD-L1. Concurrently, CD8+ T-cells were trained in Lewis lung carcinoma (LLC) cells. We suggested that rCD8+ T-cells isolated from the spleen might impede the development of metastatic disease. RESULTS: The present study has indicated that the reprogramming procedure enhances the survival and cytotoxicity of splenic CD8+ T-cells in LLC culture. In an LLC model of spontaneous metastasis, splenic rCD8 + T-cell therapy augmented the numbers of CD8+ T-cells and CD4+ T-cells in the lungs of mice. These changes can account for the partial reduction of tumors in the lungs and the mitigation of metastatic activity. CONCLUSIONS: Our proposed reprogramming method enhances the antitumor activity of CD8+ T-cells isolated from the spleen and could be valuable in formulating an approach to treating metastatic disease in patients with lung cancer.
Subject(s)
CD8-Positive T-Lymphocytes , Carcinoma, Lewis Lung , Spleen , Animals , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/pathology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Mice , Spleen/pathology , Spleen/immunology , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Mice, Inbred C57BL , Cellular Reprogramming , Cell Line, Tumor , Disease Models, AnimalABSTRACT
Combined medication has attracted increasing attention as an important treatment option for tumors due to the serious adverse effects of chemotherapy. In this study, as a new therapy strategy, a combination treatment of MDP (a polysaccharide from the rhizome of Menispermum dauricum DC.) with cyclophosphamide (CTX) was investigated. The results showed that combination treatment with MDP and CTX exerted a significantly synergistic anti-tumor effect in Lewis tumor-bearing mice, improved CTX-induced emaciation and hair loss, as well as increased the number of leukocytes, erythrocytes, hemoglobin, and platelets in the peripheral blood. In addition, compared with CTX alone, the thymus index and spleen index of the MDP + CTX group were increased, the number of CD3 + T cells, CD8 + T cells, white blood cells and B cells in spleen also increased significantly. MDP could also ameliorate the increase in liver and kidney index caused by CTX. In the Lewis lung cancer model, MDP showed a certain degree of anti-tumor effects, which may be related to its promotion of tumor-associated macrophages (TAMs) to M1 phenotype polarisation, enhancement of the number of T cells in tumor tissues and promotion of Th cells in tumor tissues to Th1 phenotype polarisation, thus alleviating the immunosuppressive microenvironment in tumor tissues. This study laid the foundation for the development of MDP as a polysaccharide drug for the treatment or adjuvant therapy of tumors and has important significance for the further clinical application of polysaccharides.
Subject(s)
Cyclophosphamide , Polysaccharides , Rhizome , Tumor Microenvironment , Animals , Polysaccharides/pharmacology , Polysaccharides/chemistry , Polysaccharides/therapeutic use , Tumor Microenvironment/drug effects , Mice , Rhizome/chemistry , Cyclophosphamide/adverse effects , Cyclophosphamide/pharmacology , Male , Carcinoma, Lewis Lung/drug therapy , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/pathology , Antineoplastic Agents/pharmacology , Mice, Inbred C57BL , Spleen/drug effects , Spleen/immunologyABSTRACT
The remarkable efficacy of cancer immunotherapy has been established in several tumor types. Of the various immunotherapies, PD-1/PD-L1 inhibitors are most extensively used in the treatment of many cancers in clinics. These inhibitors restore the suppressed antitumor immune response and inhibit tumor progression by blocking the PD-1/PD-L1 signaling. However, the low response rate is a major limitation in the clinical application of PD-1/PD-L1 inhibitors. Therefore, combination strategies that enhance the response rate are the need of the hour. In this investigation, PT-100 (also referred to as Talabostat, Val-boroPro, and BXCL701), an orally administered and nonselective dipeptidyl peptidase inhibitor, not only augmented the effectiveness of anti-PD-1 therapy but also significantly improved T immune cell infiltration and reversed the immunosuppressive tumor microenvironment. The combination of PT-100 and anti-PD-1 antibody increased the number of CD4+ and CD8+ T cells. Moreover, the mRNA expression of T cell-associated molecules was elevated in the tumor microenvironment. The results further suggested that PT-100 dramatically reduced the ratio of tumor-associated macrophages. These findings provide a promising combination strategy for immunotherapy in lung cancer.
Subject(s)
Carcinoma, Lewis Lung , Programmed Cell Death 1 Receptor , Tumor Microenvironment , Animals , Mice , Carcinoma, Lewis Lung/drug therapy , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/pathology , Carcinoma, Lewis Lung/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , Cell Line, Tumor , Dipeptidyl Peptidase 4/metabolism , Dipeptidyl-Peptidase IV Inhibitors/pharmacology , Dipeptidyl-Peptidase IV Inhibitors/therapeutic use , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Immunotherapy/methods , Mice, Inbred C57BL , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/metabolism , Tumor Microenvironment/drug effectsABSTRACT
OBJECTIVE: Up-regulating programmed cell death ligand-1(PD-L1) expressed on tumor cells and tumor-infiltrating myeloid cells interacting with up-regulated programmed cell death-1 (PD-1) expressed on tumor-infiltrating lymphoid cells greatly hinder their tumor-inhibiting effect. It is necessary to explore the deep mechanism of this negative effect, so as to find the potential methods to improve the immunotherapy efficiency. METHODS AND RESULTS: In this study, we found that the PD-1 expression in lung cancer-infiltrating type II innate lymphoid cells (ILC2s) was highly up-regulated, which greatly restrained the activation and function of ILC2s. Furthermore, anti-PD-1 could restore the inhibition and effective cytokine secretion of ILC2s when co-cultured with tumor cells. In vivo studies proved that anti-PD-1 treatment promoted the activation of tumor-infiltrating ILC2s and inhibited the tumor growth of LLC-bearing nude mice. DISCUSSION: Our studies demonstrate a new PD-1/PD-L1 axis regulating mechanism on innate immune cells, which provide a useful direction to ILC2s-based immunotherapy to cancer diseases.
Subject(s)
Immunity, Innate , Lymphocytes , Mice, Nude , Programmed Cell Death 1 Receptor , Up-Regulation , Animals , Programmed Cell Death 1 Receptor/immunology , Programmed Cell Death 1 Receptor/metabolism , Mice , Up-Regulation/drug effects , Immunity, Innate/immunology , Lymphocytes/immunology , Lymphocytes/metabolism , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Cell Line, Tumor , Humans , B7-H1 Antigen/immunology , B7-H1 Antigen/metabolism , Mice, Inbred C57BL , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/drug effects , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/pathology , Carcinoma, Lewis Lung/metabolismABSTRACT
PURPOSE: 18ß-glycyrrhetinic acid (GA), the main metabolite of glycyrrhizic acid extracted from the root of licorice, has been reported to possess anti-cancer and immunomodulatory activity, but the mechanisms are not well understood. Recent studies have shown that ferroptosis of immune cells is involved in tumor-associated immune suppression. The purpose of this study was to investigate whether the enhanced immune response via inhibiting immune cell ferroptosis contributed to the anticancer effect of 18ß-GA. METHODS: Lewis Lung carcinoma mouse model and Murine CD8 + T cell culture model were used to examine the changes of immune response and ferroptosis of immune cells. RESULTS: We found that 18ß-GA was effective against lung cancer accompanied by enhanced activation of tumor-infiltrating CD8+ T cells in Lewis Lung carcinoma mouse model. Furthermore, we demonstrated that the boosted immune response by GA was attributed to its ability to inhibit arachidonic acid (AA)-mediated CD8+ T ferroptosis via suppressing CD36 expression. CONCLUSION: The findings of the present study unraveled a novel mechanism underlying the anti-cancer and immunomodulatory activity of 18ß-GA and support that 18ß-GA holds potential to be used as an immune enhancer for lung cancer prevention or treatment.