Microwave ablation combined with PD-L1 blockade synergistically promotes Cxcl9-mediated antitumor immunity.

Although microwave ablation (MWA) is an important curative therapy in colorectal cancer liver metastasis, recurrence still occurs clinically. Our previous studies have shown that the expression of programmed cell death 1 ligand 1 (PD-L1) is upregulated following MWA, suggesting that MWA combined with anti-PD-L1 treatment can serve as a promising clinical therapeutic strategy against cancer. Using MWA-treated preclinical mice models, MWA combined with αPD-L1 treatment decreased tumor growth and prolonged overall survival (OS). Furthermore, through flow cytometry and single-cell RNA sequencing analysis, we determined that the MWA plus αPD-L1 therapy significantly suppressed CD8+ T cell exhaustion and enhanced their effector function. A significant increase in γ-interferon (IFN-γ) stimulated transcription factors, specifically Irf8, was observed. This enhancement facilitated the polarization of tumor-associated macrophages (TAM1s and TAM2s) through the nuclear factor-κB/JAK-STAT1 signaling pathway. Furthermore, the combination therapy stimulated the production of CXC motif chemokine ligand (CXCL9) by TAM1s and tumor cells, potentially increasing the chemotaxis of CD8 T cells and Th1 cells. Knocking out Cxcl9 in MC38 tumor cells or using CXCL9 blockade enhanced tumor growth of untreated tumors and shortened OS. Taken together, our study showed that blocking the IFN-γ-Cxcl9-CD8+ T axis promoted tumor progression and discovered a potential involvement of IRF8-regulated TAMs in preventing T cell exhaustion. Collectively, we identified that the combination of MWA with anti-PD-L1 treatment holds promise as a therapeutic strategy to rejuvenate the immune response against tumors. This merits further exploration in clinical studies.

Enhanced abscopal anti-tumor response via a triple combination of thermal ablation, IL-21, and PD-1 inhibition therapy.

Despite the success of immune checkpoint inhibitors (ICIs) in treating solid tumors, lots of patients remain unresponsive to this therapy. Microwave ablation (MWA) stimulates systemic adaptive immunity against tumor cells by releasing tumor antigens. Additionally, IL-21 has demonstrated importance in stimulating T-cell effector function. The combination of these three therapies-MWA, IL-21, and anti-PD-1 monoclonal antibodies (mAbs)-has yet to be explored in the context of cancer treatment.In this study, we explored the impact of thermal ablation on IL-21R expression in tumor-infiltrating lymphocytes (TILs). Subsequently, we assessed alterations in the tumor microenvironment (TME) and peripheral lymphoid organs. Additionally, we conducted a thorough examination of tumor-infiltrating CD45+ immune cells across various treatment groups using single-cell RNA sequencing (scRNA-seq). Moreover, we determined the potential anti-tumor effects of the triple combination involving MWA, IL-21, and anti-PD-1 mAbs.Our findings revealed that MWA upregulated the expression of IL-21R on various immune cells in the untreated tumors. The combination of MWA with IL-21 exhibited a robust abscopal anti-tumor effect, enhancing the effector function of CD8+ T cells and facilitating dendritic cells' maturation and antigen presentation in the untreated tumor. Notably, the observed abscopal anti-tumor effect resulting from the combination is contingent upon T-cell recirculation, indicating the reliance of systemic adaptive immunity for this treatment regimen. Additionally, the combination of MWA, IL-21, and PD-1 mAbs demonstrated profound abscopal anti-tumor efficacy. Our findings provide support for further clinical investigation into a triple combination therapy involving MWA, IL-21, and ICIs for the treatment of metastatic cancer.

Ferredoxin 1: a gatekeeper in halting lung adenocarcinoma progression through activation of the GPRIN2 signaling pathway.

BACKGROUND: Lung adenocarcinoma (LUAD) is a highly lethal form of lung cancer. Despite advancements in treatments, managing LUAD is still challenging due to its aggressive behavior. Recent studies indicate that various molecular pathways, including the dysregulation of ferredoxin 1 (FDX1), play roles in LUAD progression. FDX1, a crucial protein in cellular redox reactions and energy metabolism, has been linked to several cancers. However, its exact role in the development of LUAD is not yet fully understood. METHODS: We investigated the role of ferredoxin 1 (FDX1) in LUAD progression through analysis of its expression in LUAD tissues and its impact on patient survival. Functional assays were performed to assess the effects of FDX1 overexpression on LUAD cell proliferation, migration, and invasion. A xenograft model was employed to evaluate the tumorigenesis potential of LUAD cells with FDX1 overexpression. Mechanistic insights into FDX1 regulation were gained through depletion experiments targeting the G protein-regulated inducer of neurite outgrowth 2 (GPRIN2)/PI3K signaling pathway. RESULTS: FDX1 expression was down-regulated in LUAD tissues, correlating with shorter patient survival. Overexpression of FDX1 suppressed LUAD cell proliferation, migration, and invasion in vitro, and inhibited tumorigenesis in vivo. Mechanistically, the GPRIN2/PI3K signaling pathway was implicated in FDX1 regulation, as depletion of GPRIN2 reversed the effects of FDX1 overexpression on cellular functions. CONCLUSIONS: Our findings highlight FDX1 as a potential tumor suppressor in LUAD, acting through modulation of the GPRIN2/PI3K signaling pathway. These results suggest FDX1 as a promising therapeutic target for LUAD treatment, warranting further investigation into its clinical relevance.

Decreased RIG-I expression is associated with poor prognosis and promotes cell invasion in human gastric cancer.

BACKGROUND: Retinoic acid-induced protein I (RIG-I), known as a cytoplastic pattern recognition receptor, can recognize exogenous viral RNAs, and then initiate immune response. Recently, numerous studies also showed that RIG-I play an important role in oncogenesis and cancer progression as well. As of now, the expression pattern and the role of RIG-I in gastric cancer still remain largely unexplored. In this study, we investigated the clinical associations of RIG-I expression in human gastric cancer tissues and further explore its important contribution in the regulation of malignant phenotype of gastric cancer cells. METHODS: Immunohistochemistry was performed to study the correlation between patients' clinical parameters and RIG-I expression in gastric cancer tissues. Knockdown of RIG-I was achieved by RNAi technology to examine the contribution of RIG-I in the regulation of biological functions in the cell lines of human gastric cancer. The Affymetrix GeneChip was performed to figure out the differential gene expression profile between RIG-I wild type and RIG-I knockdown cell lines of gastric cancer. RESULTS: Immunohistochemistry result demonstrated that the expression of RIG-I in gastric cancer tissues significantly correlated with pathological stage and patients' prognoses. Furthermore, decreased RIG-I expression in human gastric cancer cell lines could significantly increase the cell migration, cell viability, and the ratio of cells in G2/M phase. Our microarray analysis also revealed that the differentially expressed gene profiles were enriched in related signal pathways or biological processes in KEGG or GO analysis respectively. CONCLUSIONS: Our present findings showed that the decreased RIG-I expression significantly correlated with patients' prognoses, and such down-regulation could promote the cell invasion in this malignancy.

TIGIT Blockade Reshapes the Tumor Microenvironment Based on the Single-cell RNA-Sequencing Analysis.

SUMMARY: Immune checkpoint blockade therapy is a pivotal approach in treating malignant tumors. TIGIT has emerged as a focal point of interest among the diverse targets for tumor immunotherapy. Nonetheless, there is still a lack of comprehensive understanding regarding the immune microenvironment alterations following TIGIT blockade treatment. To bridge this knowledge gap, we performed single-cell sequencing on mice both before and after the administration of anti-TIGIT therapy. Our analysis revealed that TIGIT was predominantly expressed on T cells and natural killer (NK) cells. The blockade of TIGIT exhibited inhibitory effects on Treg cells by downregulating the expression of Foxp3 and reducing the secretion of immunosuppressive cytokines. In addition, TIGIT blockade facilitated the activation of NK cells, leading to an increase in cell numbers, and promoted cDC1 maturation through the secretion of XCL1 and Flt3L. This activation, in turn, stimulated the TCR signaling of CD8 + T cells, thereby enhancing their antitumor effect. Consequently, anti-TIGIT therapy demonstrated substantial potential for cancer immunotherapy. Our research provided novel insights into future therapeutic strategies targeting TIGIT for patients with cancer.

Single-cell RNA sequencing indicates cordycepin remodels the tumor immune microenvironment to enhance TIGIT blockade's anti-tumor effect in colon cancer.

Both preclinical and clinical studies have extensively proven the effectiveness of TIGIT inhibitors in tumor immunotherapy. However, it has been discovered that the presence of CD226 on tumor-infiltrating lymphocytes is crucial for the effectiveness of both anti-TIGIT therapy alone and when combined with anti-PD-1 therapy for tumors. In our investigation, we observed that cordycepin therapy significantly augmented the expression of the Cd226 gene. As a result, it was hypothesized that cordycepin therapy could enhance the effectiveness of anti-TIGIT therapy. By employing single-cell RNA sequencing analysis of immune cells in the MC38 tumor model, we discovered that cordycepin combined with anti-TIGIT therapy led to a significant increase in the proportion of NK cells within the tumor immune microenvironment. This increased NK cell activity and decreased the expression of inhibitory receptors and exhaustion marker genes. In the combination therapy group, CD8+ T cells had lower exhaustion state scores and increased cytotoxicity, indicating a better immune response. The combination therapy group increased DCs in the tumor immune microenvironment and promoted cellular interaction with CD4+ T cell and CD8+ T cell populations while decreasing Treg cell interactions. In conclusion, cordycepin with anti-TIGIT therapy in colon cancer could reshape the tumor immune microenvironment and have notable anticancer effects.

Precursor exhausted CD8+T cells in colorectal cancer tissues associated with patient's survival and immunotherapy responsiveness.

Exhausted CD8+T cells represent a distinct cellular lineage that emerges during both chronic infections and cancers. Recent studies have shown that persistent antigen exposure can drive the differentiation of precursor exhausted CD8+T cells, termed Tpex cells, which are characterized as TCF-1+PD-1+CD8+T cells. Elevated Tpex cell frequencies in the tumor microenvironment (TME) are associated with improved overall survival (OS) in cancer patients and heightened responsiveness to anti-PD-1 therapy. In our present study, we utilized multi-color immunohistochemistry (mIHC) to determine the localization and clinical implications of tumor-infiltrating Tpex cells within the TME of human colorectal cancer (CRC) tissues. We also conducted a multi-omics integrative analysis using single-cell RNA sequencing (scRNA-seq) data derived from both the murine MC38 tumor model and human CRC tissues. This analysis helped delineate the transcriptional and functional attributes of Tpex cells within the CRC TME. Furthermore, we employed spatial transcriptome sequencing data from CRC patients to investigate the interactions between Tpex cells and other immune cell subsets within the TME. In conclusion, our study not only established a method for Tpex cell detection using mIHC technology but also confirmed that assessing Tpex cells within the CRC TME could be indicative of patients' survival. We further uncovered the transcriptional and functional characteristics of Tpex cells in the TME and ascertained their pivotal role in the efficacy of immunotherapy against CRC.

The Role of RNA m6A Modification in Cancer Glycolytic Reprogramming.

As one of the main characteristics of neoplasia, metabolic reprogramming provides nutrition and energy to enhance cell proliferation and maintain environment homeostasis. Glycolysis is one of the most important components of cancer metabolism and the Warburg effect contributes to the competitive advantages of cancer cells in the threatened microenvironment. Studies show strong links between N6-methyladenosine (m6A) modification and metabolic recombination of cancer cells. As the most abundant modification in eukaryotic RNA, m6A methylation plays important roles in regulating RNA processing, including splicing, stability, transportation, translation and degradation. The aberration of m6A modification can be observed in a variety of diseases such as diabetes, neurological diseases and cancers. This review describes the mechanisms of m6A on cancer glycolysis and their applications in cancer therapy and prognosis evaluation, aiming to emphasize the importance of targeting m6A in modulating cancer metabolism.

Single-cell RNA sequencing reveals the effects of anti-PD-L1 therapy on 3LL lung cancer model and its tumor microenvironment.

PD-L1 is expressed on antigen-presenting cells and tumor cells, thus allows tumor cells to escape immune surveillance. Moreover, targeting PD-L1 was also recommended and selected as important immune checkpoint inhibitors (ICIs) strategy in the treatment of advanced cancers due to the safety and activity. However, the detailed alteration of tumor microenvironment (TME) upon anti-PD-L1 therapy in lung cancer tumor model still needs to be resolved. In our present study, first, we characterized PD-L1 expression in human lung adenocarcinoma tissues by using public data, then we established the subcutaneous tumor-bearing model by using murine lung cancer cell line 3LL to perform the anti-PD-L1 therapy and the single-cell RNA sequencing (scRNA-seq) to reveal the remodeling of TME. We confirmed that PD-L1 blockade significantly inhibited tumor progression in 3LL mouse lung cancer model. The scRNA-seq depicted the detailed TME landscape of 3LL tumor model upon anti-PD-L1 treatment. Five major populations according to the marker genes were identified, including tumor cells, stromal cells, myeloid cells, T cells, and NK cells. In addition, we found that anti-PD-L1 treatment enhanced tumor immunogenicity and promoted inflammation in TME and promoted cancer-associated fibroblasts (CAFs)-mediated T-cell migration and infiltration. We also found that anti-PD-L1 treatment can increase dendritic cells (DCs) population and enhance the antigen-presenting ability to CD8+T cells and promote the transition of monocytes to macrophages and tumor-associated macrophages 2 (TAM2) to TAM1. We also revealed that Nfatc1 was up-regulated in the anti-PD-L1 treatment group, the frequencies of effector CD8+T cells, exhausted CD8+T cells, cycling T cells, and NKT were increased, and the frequencies of conventional CD4+T cells, Treg, IFN-induced T cells, and γδT cells were decreased. Therefore, our scRNA-seq data of the lung cancer tumor model upon anti-PD-L1 treatment made a comprehensive presentation and description about the remodeling of TME and will benefit us to understand the underlying mechanisms and to design combinational therapeutic strategies based on anti-PD-L1 therapy against lung cancer.

Cordycepin remodels the tumor microenvironment of colorectal cancer by down-regulating the expression of PD-L1.

PURPOSE: Colorectal cancer, as a common malignant tumor, poses a serious threat to human life. Cordycepin, derived from Cordyceps militaris extract, which was established as a capable inhibitor of tumor growth. Nevertheless, the precise antitumor mechanism of cordycepin in colorectal cancer cells remains elusive. METHODS: Herein, our initial focus was to explore the tumor-suppressive impact of cordycepin through its influence on various biological functions in murine colorectal cancer cells, conducted by an in vitro setting. First, we investigated the tumor-suppressive effect of cordycepin on the regulation of biological functions in murine colorectal cancer cells in vitro. Furthermore, we evaluated the in vivo antitumor potential of cordycepin using a mouse preclinical tumor model, and further explored the antitumor mechanism. RESULTS: Our findings revealed that cordycepin effectively inhibit the proliferation, invasion, and migration of murine colon cancer cells. Moreover, there is a substantial reduction in the expression of PD-L1 observed in tumor cells, in response to cordycepin treatment. Collectively, these results demonstrate the significant tumor-suppressive attributes of cordycepin against colorectal cancer. Consequently, our study lays a solid foundation for the potential clinical utilization of cordycepin in cancer therapy. CONCLUSION: Cordycepin inhibits the biological functions of colorectal cancer cells and suppresses tumor growth by reducing the expression of PD-L1.

Cordycepin synergizes with CTLA-4 blockade to remodel the tumor microenvironment for enhanced cancer immunotherapy.

The strategy of using immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment, leading to remarkable clinical outcomes. However, certain cancer types and patient demographics continue to face unique challenges. As a result, it is vital to investigate combination therapies that involve ICIs to boost therapeutic efficacy. Cordycepin, an adenosine derivative composed of adenine and pentose, holds immense promise for treating inflammation and cancer. Our recent research has demonstrated that the combined treatment of cordycepin and the anti-CD47 antibody significantly curtails tumor growth and extends the lifespan of tumor-bearing mice. In the current study, we showed that the combination of cordycepin and CTLA-4 blockade had a profound impact on suppressing tumor growth. We utilized the MC38 and CT26 tumor models to evaluate the therapeutic effect of cordycepin, CTLA-4 blockade, and their combined approach. Flow cytometry results unveiled that cordycepin, when combined with CTLA-4 blockade, considerably augmented the presence of tumor-infiltrating CD8+T cells and diminished the population of Foxp3+Tregs within the tumor microenvironment (TME). Additionally, we employed single-cell analysis to examine the TME's reconfiguration upon the combined treatment of anti-CTLA-4 and cordycepin. We observed a significant impact on inhibiting tumor growth and substantially extended survival in tumor-bearing mice. Our data also demonstrated an increased proportion of effector CD8+T cells in the combined treatment group compared to all other groups, while exhausted CD8+T cells diminished in the combined group compared to the anti-CTLA-4 treatment alone. In conclusion, our findings supported the idea that combining cordycepin and CTLA-4 blockade could modify the effector and exhaustion status of CD8+T cells, thereby bolstering CD8+T-cell-mediated anti-tumor immunity in the TME. Collectively, our current study successfully established a combination therapeutic strategy utilizing cordycepin and CTLA-4 blockade. This strategy demonstrated a significant synergistic effect against cancer, highlighting its importance in cancer treatment.

CD226 identifies functional CD8+T cells in the tumor microenvironment and predicts a better outcome for human gastric cancer.

It is well-known that CD226 serves as a critical activating receptor on various immune cells, such as lymphocytes and monocytes, and it is suggested to promote anti-tumor immunity in the tumor microenvironment (TME). Herein, we showed a crucial regulatory role of CD226 in CD8+T cell-mediated anti-tumor response in TME of human gastric cancer (GC). Specifically, the increased CD226 expression in cancer tissues was significantly associated with better clinical outcomes in GC patients. Moreover, the increased infiltrating CD226+CD8+T cells and the increased ratio of infiltrating CD226+CD8+T cells in CD8+T subpopulation within cancer tissues could also be valuable prognostic predictors for GC patients. Mechanically, the assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis revealed that the chromatin accessibility of CD226 in CD4+ and CD8+TILs was significantly higher than that in CD8+T cells in normal tissues. Further analysis showed that CD8+TILs highly expressed immune checkpoint molecules, such as TIGIT, LAG3, and HAVCR2, which means CD8+TILs are more exhausted. In addition, our multi-color immunohistochemical staining (mIHC) revealed that GC patients with higher frequency of IFN-γ+CD226+CD8+TILs showed poorer prognosis. Combined with the single-cell transcriptome sequencing (scRNA-seq) data analysis, we found that the expressions of IFN-γ and TIGIT in CD8+TILs were significantly and positively correlated. The expression of TIGIT in IFN-γ+CD226+CD8+TILs was higher, while that in IFN-γ-CD226+CD8+TILs was significantly lower. The correlation analysis showed that the expression of CD226 was positively correlated with the score of effector T cells but negatively correlated with that of immunosuppressive factors, such as Tregs and tumor-associated macrophages (TAMs). Collectively, we showed that the frequency of CD226+CD8+TILs was an excellent prognostic predictor for GC patients. Our findings provided insights into the interaction pattern between co-stimulatory receptor CD226 and tumor cells as well as the infiltrating immune cells in the TME in GC.

Synergism Between IL21 and Anti-PD-1 Combination Therapy is Underpinned by the Coordinated Reprogramming of the Immune Cellular Network in the Tumor Microenvironment.

T cell-stimulating cytokines and immune checkpoint inhibitors (ICI) are an ideal combination for increasing response rates of cancer immunotherapy. However, the results of clinical trials have not been satisfying. It is important to understand the mechanism of synergy between these two therapeutic modalities. Here, through integrated analysis of multiple single-cell RNA sequencing (scRNA-seq) datasets of human tumor-infiltrating immune cells, we demonstrate that IL21 is produced by tumor-associated T follicular helper cells and hyperactivated/exhausted CXCL13+CD4+ T cells in the human tumor microenvironment (TME). In the mouse model, the hyperactivated/exhausted CD4+ T cell-derived IL21 enhances the helper function of CD4+ T cells that boost CD8+ T cell-mediated immune responses during PD-1 blockade immunotherapy. In addition, we demonstrated that IL21's antitumor activity did not require T-cell trafficking. Using scRNA-seq analysis of the whole tumor-infiltrating immune cells, we demonstrated that IL21 treatment in combination with anti-PD-1 blockade synergistically drives tumor antigen-specific CD8+ T cells to undergo clonal expansion and differentiate toward the hyperactive/exhausted functional state in the TME. In addition, IL21 treatment and anti-PD-1 blockade synergistically promote dendritic cell (DC) activation and maturation to mature DC as well as monocyte to type 1 macrophage (M1) differentiation in the TME. Furthermore, the combined treatment reprograms the immune cellular network by reshaping cell-cell communication in the TME. Our study establishes unique mechanisms of synergy between IL21 and PD-1-based ICI in the TME through the coordinated promotion of type 1 immune responses. Significance: This study reveals how cytokine and checkpoint inhibitor therapy can be combined to increase the efficacy of cancer immunotherapy.

Mobilizing phospholipids on tumor plasma membrane implicates phosphatidylserine externalization blockade for cancer immunotherapy.

In "healthy" tumor cells, phosphatidylserine (PS) is predominately localized in the inner plasma membrane leaflet. During apoptosis, PS relocates to the outer leaflet. Herein, we established PSout tumor models with tumor cells lacking PS flippase component CDC50A, constantly exposing PS but alive. PSout tumors developed bigger than wild-type (WT) tumors, featuring M2 polarized tumor-associated macrophages (TAMs) and fewer tumor-antigen-specific T cells. The PS receptor TIM-3 is responsible for PS recognition. Employing an opposite tumor model, PSin, with tumor cells lacking the PS scramblase Xkr8 and unable to expose PS during otherwise normal apoptosis, we find that the accumulated apoptotic tumor cells produce and release cyclic GAMP (cGAMP) to immune cells to activate the STING pathway, leading to TAM M1 polarization, suppressed interleukin (IL)-10 secretion, and natural killer (NK) cell cytotoxicity. Silencing Xkr8 in vivo by either short hairpin RNA (shRNA) or small interfering RNA (siRNA) to achieve a PS externalization blockade provides robust therapeutic anti-tumor efficiency.

The Half-Life-Extended IL21 can Be Combined With Multiple Checkpoint Inhibitors for Tumor Immunotherapy.

In the era of immune checkpoint blockade cancer therapy, cytokines have become an attractive immune therapeutics to increase response rates. Interleukin 21 (IL21) as a single agent has been evaluated for cancer treatment with good clinical efficacy. However, the clinical application of IL21 is limited by a short half-life and concern about potential immune suppressive effect on dendritic cells. Here, we examined the antitumor function of a half-life extended IL21 alone and in combination with PD-1 blockade using preclinical mouse tumor models. We also determined the immune mechanisms of combination therapy. We found that combination therapy additively inhibited the growth of mouse tumors by increasing the effector function of type 1 lymphocytes. Combination therapy also increased the fraction of type 1 dendritic cells (DC1s) and M1 macrophages in the tumor microenvironment (TME). However, combination therapy also induced immune regulatory mechanisms, including the checkpoint molecules Tim-3, Lag-3, and CD39, as well as myeloid derived suppressor cells (MDSC). This study reveals the mechanisms of IL21/PD-1 cooperation and shed light on rational design of novel combination cancer immunotherapy.

Immune suppressed tumor microenvironment by exosomes derived from gastric cancer cells via modulating immune functions.

Gastric cancer is one of the leading causes of cancer-related death due to late diagnosis with high metastatic frequency. In this study, the impact of tumor secreted exosomes on immune function in the tumor environment was investigated using exosomes isolated from gastric cancer cell lines MKN-28, MKN-45, and SGC-7901. Results show that exosomes derived from all of these cell lines changed the gene expression and cytokine secretion levels of CD8+ T cells. They also block cell cycle progression, induced apoptosis in CD8+ T cells. Image analysis of fluorescent labeled exosomes derived from three cell lines injected systemically into C57BL/6 mice revealed these exosomes primarily localize to the lungs. We further showed exosomes were mainly taken up by natural killer cells and macrophages in the lung. After long-term exposure to inject exosomes from MKN-45 cells, mice developed an immunosuppressive tumor microenvironment in the lung with increased frequency of effector memory CD4+ T and MDSC, decreased CD8+ T cell and NK frequency. This immune suppressive environment promotes gastric cancer lung metastasis. Lung metastasis sites developed after mice were exposed to exosomes isolated from all three gastric cancer cell lines when the mice were injected with MFC cells. Results suggest that exosomes derived from gastric cancer cells (especially MKN-45 and MKN-28) changed CD8+ T cell gene expression and cytokine secretion patterns to create an immunosuppressive condition for metastatic niche formation in the lung. Overall, this study provides new insights into how gastric cancer derived exosomes modulate the immune response to promote lung tumor metastasis.

Checkpoint molecules coordinately restrain hyperactivated effector T cells in the tumor microenvironment.

The immune checkpoint blockade (ICB) immunotherapy has prolonged overall survival for cancer patients but the response rates are low. The resistance to ICB is likely due to compensatory upregulation of additional immune inhibitory molecules. In this study, we first systematically examined Tim-3 expression in immune cells in mouse tumors and found that Tim-3 was specifically up-regulated in a large number of Treg, conventional CD4+, CD8+ T cells, dendritic cell 1 (DC1), and macrophage 1 (M1) in the tumor microenvironment (TME). Interestingly, Tim-3+ T cells in the TME were phenotypically effector but not "exhausted" T cells because Tim-3+ PD-1+ CD8+ T cells had a higher number of mitochondria, greater levels of glycolysis, and higher tumor-specific cytolytic activities compared to Tim-3- PD-1- CD8+ T cells. The combination treatment with Tim-3 and PD-1 mAbs resulted in a synergistic antitumor activity but also increased the expression of Lag-3 and GITR in TIL, demonstrating cross-regulation between multiple checkpoint molecules. Furthermore, we found that the antitumor efficacy with triple combination of Tim-3, PD-1, and Lag3 mAbs was much greater than any two antibodies. Mechanistically, we demonstrated that simultaneous targeting of Tim-3, PD-1, and Lag-3 cooperatively increased the levels of granzyme B and tumor-specific cytolytic activities of CD8+ TIL. Our data indicate that multiple checkpoint molecules are coordinately upregulated to inhibit the function of hyperactivated T cells in the TME and requirement for the simultaneous blockade of PD-1, Tim-3 and Lag3 for cancer treatment.