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
Excessive neutrophil infiltration into the tumor microenvironment (TME) is an important factor that contributes to tumor overgrowth and limited immunotherapy efficacy. Neutrophils activate various receptors involved in tumor progression, while suppressing the infiltration and activity of cytotoxic T cells and creating optimal conditions for tumor growth. Therefore, the appropriate control of neutrophil infiltration is an effective strategy for tumor treatment. In the present study, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) inhibited tumor overgrowth by suppressing excessive neutrophil infiltration, resulting in >74.97â¯% reduction in tumor size in a Lewis lung carcinoma (LLC-1) mouse model. All subjects in the positive control group died during the 90-day survival period, whereas only four subjects in the PLAG treatment group survived. PLAG had a significantly higher tumor growth inhibitory effect and survival rate than other neutrophil infiltration-targeting inhibitors (e.g., Navarixin, lymphocyte antigen 6 complex locus G6D antibody [aLy6G]). The ability of PLAG to regulate neutrophil infiltration and inhibit tumor growth depends on thioredoxin-interacting protein (TXNIP). In tumors lacking TXNIP expression, PLAG failed to control neutrophil infiltration and infiltration-related factor release, and the inhibitory effect of PLAG on tumor growth was reduced. PLAG-mediated inhibition of neutrophil infiltration enhances the efficacy of immune checkpoint inhibitors (ICIs), increasing the antitumor efficacy and survival rate by 30â¯%. In conclusion, PLAG could be a novel alternative to anti-tumor drugs that effectively targets excessive neutrophil infiltration into cancer tissues.
Subject(s)
Carcinoma, Lewis Lung , Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Mice, Inbred C57BL , Neutrophil Infiltration , Tumor Microenvironment , Animals , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Lung Neoplasms/immunology , Carcinoma, Lewis Lung/drug therapy , Carcinoma, Lewis Lung/pathology , Carcinoma, Lewis Lung/immunology , Mice , Neutrophil Infiltration/drug effects , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/immunology , Tumor Microenvironment/drug effects , Diglycerides/pharmacology , Cell Line, Tumor , Neutrophils/drug effects , Neutrophils/immunology , Neutrophils/metabolism , Disease Models, Animal , Male , Antineoplastic Agents/pharmacology , GlyceridesABSTRACT
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
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: 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
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
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
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
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.
Subject(s)
CD8-Positive T-Lymphocytes , Carcinoma, Lewis Lung , Ferroptosis , Glycyrrhetinic Acid , Mice, Inbred C57BL , Animals , Carcinoma, Lewis Lung/drug therapy , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/pathology , Glycyrrhetinic Acid/pharmacology , Glycyrrhetinic Acid/analogs & derivatives , Glycyrrhetinic Acid/therapeutic use , Ferroptosis/drug effects , Mice , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Lung Neoplasms/drug therapy , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Cell Line, TumorABSTRACT
BACKGROUND: Radioimmunotherapy has become one of the most promising strategies for cancer treatment. Preclinical and clinical studies have demonstrated that antiangiogenic therapy can improve the efficacy of immunotherapy and sensitize radiotherapy through a variety of mechanisms. However, it is undefined whether angiogenesis inhibitors can enhance the effect of radioimmunotherapy. In this study, we aim to explore the role of anlotinib (AL3818) on the combination of radiotherapy and immune checkpoint inhibitors in Lewis lung carcinoma mouse. METHODS: C57BL/6 mouse subcutaneous tumor model was used to evaluate the ability of different treatment regimens in tumor growth control. Immune response and immunophenotyping including the quantification and activation were determined by flow cytometry, multiplex immunofluorescence, and multiplex immunoassay. RESULTS: Triple therapy (radiotherapy combined with anti-PD-L1 and anlotinib) increased tumor-infiltrating lymphocytes and reversed the immunosuppressive effect of radiation on the tumor microenvironment in mouse model. Compared with radioimmunotherapy, the addition of anlotinib also boosted the infiltration of CD8+ T cells and M1 cells and caused a decrease in the number of MDSCs and M2 cells in mice. The levels of IFN-gamma and IL-18 were the highest in the triple therapy group, while the levels of IL-23, IL-13, IL-1 beta, IL-2, IL-6, IL-10, and Arg-1 were significantly reduced. NF-κB, MAPK, and AKT pathways were downregulated in triple therapy compared with radioimmunotherapy. Thus, the tumor immune microenvironment was significantly improved. As a consequence, triple therapy displayed greater benefit in antitumor efficacy. CONCLUSION: Our findings indicate that anlotinib might be a potential synergistic treatment for radioimmunotherapy to achieve better antitumor efficacy in NSCLC patients by potentiating the tumor immune microenvironment.
Subject(s)
B7-H1 Antigen/antagonists & inhibitors , Carcinoma, Lewis Lung/drug therapy , Carcinoma, Lewis Lung/radiotherapy , Immune Checkpoint Inhibitors/administration & dosage , Indoles/administration & dosage , Lung Neoplasms/drug therapy , Lung Neoplasms/radiotherapy , Quinolines/administration & dosage , Radioimmunotherapy/methods , Tumor Microenvironment/immunology , Animals , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/radiation effects , Carcinoma, Lewis Lung/immunology , Cell Line, Tumor , Cytokines/metabolism , Disease Models, Animal , Female , Lung Neoplasms/immunology , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/radiation effects , Mice , Mice, Inbred C57BL , Radiotherapy Dosage , Signal Transduction/drug effects , Signal Transduction/immunology , Signal Transduction/radiation effects , Treatment Outcome , Tumor Microenvironment/drug effects , Tumor Microenvironment/radiation effectsABSTRACT
Tumor growth is associated with metabolic reprogramming of various organs including the liver. This metabolic reprogramming is responsible for the development of behavioral fatigue represented by decreased voluntary wheel running in a murine model of lung cancer. To determine whether interleukin (IL-)6 induced by the tumor is responsible for the metabolic reprogramming, mice injected with Lewis lung carcinoma cells in the flank were treated with an anti-mouse IL-6 monoclonal neutralizing antibody using a 2 × 2 factorial design (+/- tumor and +/- anti-IL-6 antibody). Endpoints were represented by behavioral, metabolic and immune phenotypes. Despite its ability to abrogate the increase in plasma levels of IL-6 that was apparent in tumor-bearing mice and decrease inflammatory signaling in the liver, immunoneutralization of IL-6 had no effect on voluntary wheel running and did not modify the tumor-induced alterations in hepatic gene expression of inflammatory cytokines and metabolic factors. These negative results indicate that IL-6 does not mediate the communication between tumor and host in mice implanted with Lewis lung carcinoma.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Carcinoma, Lewis Lung/immunology , Interleukin-6/immunology , Muscle Fatigue/physiology , Animals , Disease Models, Animal , Inflammation , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Motor Activity/physiologyABSTRACT
The combination of radiotherapy (RT) with immunotherapy represents a promising treatment modality for non-small cell lung cancer (NSCLC) patients. As only a minority of patients shows a persistent response today, a spacious optimization window remains to be explored. Previously we showed that fractionated RT can induce a local immunosuppressive profile. Based on the evolving concept of an immunomodulatory role for vagal nerve stimulation (VNS), we tested its therapeutic and immunological effects alone and in combination with fractionated RT in a preclinical-translational study. Lewis lung carcinoma-bearing C57Bl/6 mice were treated with VNS, fractionated RT or the combination while a patient cohort with locally advanced NSCLC receiving concurrent radiochemotherapy (ccRTCT) was enrolled in a clinical trial to receive either sham or effective VNS daily during their 6 weeks of ccRTCT treatment. Preclinically, VNS alone or with RT showed no therapeutic effect yet VNS alone significantly enhanced the activation profile of intratumoral CD8+ T cells by upregulating their IFN-γ and CD137 expression. In the periphery, VNS reduced the RT-mediated rise of splenic, but not blood-derived, regulatory T cells (Treg) and monocytes. In accordance, the serological levels of protumoral CXCL5 next to two Treg-attracting chemokines CCL1 and CCL22 were reduced upon VNS monotherapy. In line with our preclinical findings on the lack of immunological changes in blood circulating immune cells upon VNS, immune monitoring of the peripheral blood of VNS treated NSCLC patients (n=7) did not show any significant changes compared to ccRTCT alone. As our preclinical data do suggest that VNS intensifies the stimulatory profile of the tumor infiltrated CD8+ T cells, this favors further research into non-invasive VNS to optimize current response rates to RT-immunotherapy in lung cancer patients.
Subject(s)
Carcinoma, Lewis Lung/radiotherapy , Carcinoma, Lewis Lung/therapy , Carcinoma, Non-Small-Cell Lung/radiotherapy , Carcinoma, Non-Small-Cell Lung/therapy , Lung Neoplasms/radiotherapy , Lung Neoplasms/therapy , Vagus Nerve Stimulation , Aged , Animals , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/pathology , Carcinoma, Non-Small-Cell Lung/immunology , Carcinoma, Non-Small-Cell Lung/pathology , Combined Modality Therapy , Female , Humans , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lymphocytes, Tumor-Infiltrating/immunology , Male , Mice, Inbred C57BL , Middle Aged , Tumor BurdenABSTRACT
Regulatory B cells (Breg) are IL-10 producing subsets of B cells that contribute to immunosuppression in the tumor microenvironment (TME). Breg are elevated in patients with lung cancer; however, the mechanisms underlying Breg development and their function in lung cancer have not been adequately elucidated. Herein, we report a novel role for Indoleamine 2, 3- dioxygenase (IDO), a metabolic enzyme that degrades tryptophan (Trp) and the Trp metabolite L-kynurenine (L-Kyn) in the regulation of Breg differentiation in the lung TME. Using a syngeneic mouse model of lung cancer, we report that Breg frequencies significantly increased during tumor progression in the lung TME and secondary lymphoid organs, while Breg were reduced in tumor-bearing IDO deficient mice (IDO-/-). Trp metabolite L-Kyn promoted Breg differentiation in-vitro in an aryl hydrocarbon receptor (AhR), toll-like receptor-4-myeloid differentiation primary response 88, (TLR4-MyD88) dependent manner. Importantly, using mouse models with conditional deletion of IDO in myeloid-lineage cells, we identified a significant role for immunosuppressive myeloid-derived suppressor cell (MDSC)-associated IDO in modulating in-vivo and ex-vivo differentiation of Breg. Our studies thus identify Trp metabolism as a therapeutic target to modulate regulatory B cell function during lung cancer progression.
Subject(s)
B-Lymphocytes, Regulatory/immunology , Carcinoma, Lewis Lung/immunology , Cell Differentiation/immunology , Indoleamine-Pyrrole 2,3,-Dioxygenase/immunology , Receptors, Aryl Hydrocarbon/immunology , Tumor Microenvironment/immunology , Animals , Carcinoma, Lewis Lung/metabolism , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Mice , Myeloid-Derived Suppressor Cells/immunology , Myeloid-Derived Suppressor Cells/metabolism , Receptors, Aryl Hydrocarbon/metabolism , Tryptophan/metabolismABSTRACT
Macrophages are often prominently present in the tumor microenvironment, where distinct macrophage populations can differentially affect tumor progression. Although metabolism influences macrophage function, studies on the metabolic characteristics of ex vivo tumor-associated macrophage (TAM) subsets are rather limited. Using transcriptomic and metabolic analyses, we now reveal that pro-inflammatory major histocompatibility complex (MHC)-IIhi TAMs display a hampered tricarboxylic acid (TCA) cycle, while reparative MHC-IIlo TAMs show higher oxidative and glycolytic metabolism. Although both TAM subsets rapidly exchange lactate in high-lactate conditions, only MHC-IIlo TAMs use lactate as an additional carbon source. Accordingly, lactate supports the oxidative metabolism in MHC-IIlo TAMs, while it decreases the metabolic activity of MHC-IIhi TAMs. Lactate subtly affects the transcriptome of MHC-IIlo TAMs, increases L-arginine metabolism, and enhances the T cell suppressive capacity of these TAMs. Overall, our data uncover the metabolic intricacies of distinct TAM subsets and identify lactate as a carbon source and metabolic and functional regulator of TAMs.
Subject(s)
Carcinoma, Lewis Lung/pathology , Carcinoma, Non-Small-Cell Lung/pathology , Lactates/metabolism , Lung Neoplasms/pathology , T-Lymphocytes/immunology , Tumor Microenvironment , Tumor-Associated Macrophages/immunology , Animals , Carcinoma, Lewis Lung/genetics , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/immunology , Carcinoma, Non-Small-Cell Lung/metabolism , Female , Glycolysis , Humans , Lung Neoplasms/genetics , Lung Neoplasms/immunology , Lung Neoplasms/metabolism , Major Histocompatibility Complex , Metabolome , Mice , Mice, Inbred C57BL , TranscriptomeABSTRACT
Background: Malignant tumors accompanied with malignant pleural effusion (MPE) often indicate poor prognosis. The therapeutic effect and mechanism of intrapleural injection of anti-programmed cell death protein 1 (PD1) on MPE need to be explored. Methods: A preclinical MPE mouse model and a small clinical study were used to evaluate the effect of intrapleural injection of anti-PD1 antibody. The role of immune cells was observed via flow cytometry, RNA-sequencing, quantitative PCR, western blot, immunohistochemistry, and other experimental methods. Results: Intrathoracic injection of anti-PD1 monoclonal antibody (mAb) has significantly prolonged the survival time of mice (P = 0.0098) and reduced the amount of effusion (P = 0.003) and the number of cancer nodules (P = 0.0043). Local CD8+ T cells participated in intrapleural administration of anti-PD1 mAb. The proportion of CD69+, IFN-γ+, and granzyme B+ CD8+ T cells in the pleural cavity was increased, and the expression of TNF-α and IL-1ß in MPE also developed significantly after injection. Local injection promoted activation of the CCL20/CCR6 pathway in the tumor microenvironment and further elevated the expression of several molecules related to lymphocyte activation. Clinically, the control rate of intrathoracic injection of sintilimab (a human anti-PD1 mAb) for 10 weeks in NSCLC patients with MPE was 66.7%. Local injection improved the activity and function of patients' local cytotoxic T cells (CTLs). Conclusions: Intrapleural injection of anti-PD1 mAb could control malignant pleural effusion and the growth of cancer, which may be achieved by enhancing local CTL activity and cytotoxicity.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Carcinoma, Lewis Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Pleural Effusion, Malignant/drug therapy , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Animals , Carcinoma, Lewis Lung/immunology , Carcinoma, Non-Small-Cell Lung/immunology , Cell Line, Tumor , Humans , Injections , Lung Neoplasms/immunology , Male , Mice, Inbred C57BL , Pleural Cavity/immunology , Pleural Effusion, Malignant/immunology , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunology , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunologyABSTRACT
Lung cancer is the leading cause of cancer-related deaths. While the recent use of immune checkpoint inhibitors significantly improves patient outcomes, responsiveness remains restricted to a small proportion of patients. Conventional dendritic cells (DCs) play a major role in anticancer immunity. In mice, two subpopulations of DCs are found in the lung: DC2s (CD11b+Sirpα+) and DC1s (CD103+XCR1+), the latest specializing in the promotion of anticancer immune responses. However, the impact of lung cancer on DC populations and the consequent influence on the anticancer immune response remain poorly understood. To address this, DC populations were studied in murine models of Lewis Lung Carcinoma (LLC) and melanoma-induced lung metastasis (B16F10). We report that direct exposure to live or dead cancer cells impacts the capacity of DCs to differentiate into CD103+ DC1s, leading to profound alterations in CD103+ DC1 proportions in the lung. In addition, we observed the accumulation of CD103loCD11b+ DCs, which express DC2 markers IRF4 and Sirpα, high levels of T-cell inhibitory molecules PD-L1/2 and the regulatory molecule CD200. Finally, DC1s were injected in combination with an immune checkpoint inhibitor (anti-PD-1) in the B16F10 model of resistance to the anti-PD-1 immune checkpoint therapy; the co-injection restored sensitivity to immunotherapy. Thus, we demonstrate that lung tumor development leads to the accumulation of CD103loCD11b+ DCs with a regulatory potential combined with a reduced proportion of highly-specialized antitumor CD103+ DC1s, which could promote cancer growth. Additionally, promoting an anticancer DC signature could be an interesting therapeutic avenue to increase the efficacy of existing immune checkpoint inhibitors.
Subject(s)
Carcinoma, Lewis Lung , Dendritic Cells , Immune Checkpoint Inhibitors , Lung Neoplasms , Melanoma, Experimental , Neoplasm Proteins/immunology , Programmed Cell Death 1 Receptor/immunology , Animals , Carcinoma, Lewis Lung/drug therapy , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/pathology , Dendritic Cells/immunology , Dendritic Cells/pathology , Female , Lung Neoplasms/drug therapy , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Male , Melanoma, Experimental/drug therapy , Melanoma, Experimental/immunology , Melanoma, Experimental/pathology , Mice , Neoplasm MetastasisABSTRACT
Natural polysaccharides have shown promising effects on the regulation of immunity in animals. In this study, we examined the immune stimulatory effect of intranasally administered Codium fragile polysaccharides (CFPs) in mice. Intranasal administration of CFPs in C57BL/6 mice induced the upregulation of surface activation marker expression in macrophages and dendritic cells (DCs) in the mediastinal lymph node (mLN) and the production of interleukin-6 (IL-6), IL-12p70, and tumor necrosis factor-α in bronchoalveolar lavage fluid. Moreover, the number of conventional DCs (cDCs) was increased in the mLNs by the upregulation of C-C motif chemokine receptor 7 expression, and subsets of cDCs were also activated following the intranasal administration of CFP. In addition, the intranasal administration of CFPs promoted the activation of natural killer (NK) and T cells in the mLNs, which produce pro-inflammatory cytokines and cytotoxic mediators. Finally, daily administration of CFPs inhibited the infiltration of Lewis lung carcinoma cells into the lungs, and the preventive effect of CFPs on tumor growth required NK and CD8 T cells. Furthermore, CFPs combined with anti-programmed cell death-ligand 1 (PD-L1) antibody (Ab) improved the therapeutic effect of anti-PD-L1 Ab against lung cancer. Therefore, these data demonstrated that the intranasal administration of CFP induced mucosal immunity against lung cancer.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Antineoplastic Agents/administration & dosage , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/therapy , Chlorophyta/chemistry , Immunity, Mucosal , Immunotherapy/methods , Lung Neoplasms/immunology , Lung Neoplasms/therapy , Phytotherapy/methods , Plant Extracts/administration & dosage , Polysaccharides/administration & dosage , Administration, Intranasal/methods , Animals , CD8-Positive T-Lymphocytes/immunology , Carcinoma, Lewis Lung/pathology , Cell Line, Tumor , Dendritic Cells/immunology , Disease Models, Animal , Female , Killer Cells, Natural/immunology , Lung Neoplasms/pathology , Lymphocyte Activation/drug effects , Mice , Mice, Inbred C57BLABSTRACT
Ganoderma formosanum (GF) is a medicinal mushroom endemic to Taiwan. Previous research established the optimal culture conditions to produce exopolysaccharide rich in ß-glucan (GF-EPS) from submerged fermentation of GF. The present study investigated the antitumor effects of GF-EPS in a Lewis lung carcinoma cell (LLC1) tumor-bearing mice model. In the preventive model, GF-EPS was orally administered to mice before LLC1 injection. In the therapeutic model, GF-EPS oral administration was initiated five days after tumor cell injection. The tumor size and body weight of the mice were recorded. After sacrifice, the lymphocyte subpopulation was analyzed using flow cytometry. Spleen tissues were used to analyze cytokine mRNA expression. The results showed that GF-EPS (80 mg/kg) effectively suppressed LLC1 tumor growth in both the preventive and therapeutic models. GF-EPS administration increased the proportion of natural killer cells in the spleen and activated gene expression of several cytokines. Our results provide evidence that GF-EPS promotes tumor inhibition through immunomodulation in tumor-bearing mice.