Phosphoglycerate mutase 1 promotes breast cancer progression through inducing immunosuppressive M2 macrophages.

Immunosuppressive tumor microenvironment (TME) contributes to tumor progression and causes major obstacles for cancer therapy. Phosphoglycerate mutase 1 (PGAM1) is a key enzyme involved in cancer metabolism while its role in remodeling TME remains unclear. In this study, we reported that PGAM1 suppression in breast cancer (BC) cells led to a decrease in M2 polarization, migration, and interleukin-10 (IL-10) production of macrophages. PGAM1 regulation on CCL2 expression was essential to macrophage recruitment, which further mediated by activating JAK-STAT pathway. Additionally, the CCL2/CCR2 axis was observed to participate in PGAM1-mediated immunosuppression via regulating PD-1 expression in macrophages. Combined targeting of PGAM1 and the CCL2/CCR2 axis led to a reduction in tumor growth in vivo. Furthermore, clinical validation in BC tissues indicated a positive correlation between PGAM1, CCL2 and macrophage infiltration. Our study provides novel insights into the induction of immunosuppressive TME by PGAM1 and propose a new strategy for combination therapies targeting PGAM1 and macrophages in BC.

Anlotinib and anti-PD-1 mAbs perfected CIK cell therapy for lung adenocarcinoma in preclinical trials.

Murine cytokine-induced killer (CIK) cells are heterologous cells that kill various allogeneic and isogenic tumors and have functional and phenotypic characteristics of natural killer cells and T lymphocytes. However, the effect of CIK alone on solid tumor therapy is only limited. To enhance the therapeutic effect, it is vital to discover a mix of several therapy approaches. Immune cell function is inhibited by abnormal tumor vessels and the tumor microenvironment, which block lymphocyte entry into tumor tissue. To increase the effectiveness of CIK cells' anti-tumor activity, anti-vascular therapy and CIK cell therapy can be combined. Furthermore, anlotinib is a tiny drug with multi-target tyrosine kinase inhibitors (TKI) that can block cell migration, delay angiogenesis, and decrease blood vessel density. Compared with other anti-angiogenesis drugs, anlotinib stands out due to the wider target of action and lower effective dose. In this work, anlotinib and murine CIK cells were coupled to boost CD3+ T cell infiltration, CD3 + CD4+ T cell infiltration, and expression of granzyme B and IFN-γ from CD3 + CD8+ T cells, which increased the anti-tumor activity. Through the generation of cytotoxic cytokines by T lymphocytes, the therapeutic group using anti-PD-1 monoclonal antibodies (anti-PD-1 mAbs) in conjunction with anlotinib and CIK cells was more successful than the group receiving dual therapy. The preclinical study contributes to exploring the therapeutic alternatives for patients with lung adenocarcinoma, thus prolonging their lives.

Multiomics profiling reveals the benefits of gamma-delta (γδ) T lymphocytes for improving the tumor microenvironment, immunotherapy efficacy and prognosis in cervical cancer.

BACKGROUND: As an unconventional subpopulation of T lymphocytes, γδ T cells can recognize antigens independently of major histocompatibility complex restrictions. Recent studies have indicated that γδ T cells play contrasting roles in tumor microenvironments-promoting tumor progression in some cancers (eg, gallbladder and leukemia) while suppressing it in others (eg, lung and gastric). γδ T cells are mainly enriched in peripheral mucosal tissues. As the cervix is a mucosa-rich tissue, the role of γδ T cells in cervical cancer warrants further investigation. METHODS: We employed a multiomics strategy that integrated abundant data from single-cell and bulk transcriptome sequencing, whole exome sequencing, genotyping array, immunohistochemistry, and MRI. RESULTS: Heterogeneity was observed in the level of γδ T-cell infiltration in cervical cancer tissues, mainly associated with the tumor somatic mutational landscape. Definitely, γδ T cells play a beneficial role in the prognosis of patients with cervical cancer. First, γδ T cells exert direct cytotoxic effects in the tumor microenvironment of cervical cancer through the dynamic evolution of cellular states at both poles. Second, higher levels of γδ T-cell infiltration also shape the microenvironment of immune activation with cancer-suppressive properties. We found that these intricate features can be observed by MRI-based radiomics models to non-invasively assess γδ T-cell proportions in tumor tissues in patients. Importantly, patients with high infiltration levels of γδ T cells may be more amenable to immunotherapies including immune checkpoint inhibitors and autologous tumor-infiltrating lymphocyte therapies, than to chemoradiotherapy. CONCLUSIONS: γδ T cells play a beneficial role in antitumor immunity in cervical cancer. The abundance of γδ T cells in cervical cancerous tissue is associated with higher response rates to immunotherapy.

Novel strategies for cancer immunotherapy: counter-immunoediting therapy.

The advent of immunotherapy has made an indelible mark on the field of cancer therapy, especially the application of immune checkpoint inhibitors in clinical practice. Although immunotherapy has proven its efficacy and safety in some tumors, many patients still have innate or acquired resistance to immunotherapy. The emergence of this phenomenon is closely related to the highly heterogeneous immune microenvironment formed by tumor cells after undergoing cancer immunoediting. The process of cancer immunoediting refers to the cooperative interaction between tumor cells and the immune system that involves three phases: elimination, equilibrium, and escape. During these phases, conflicting interactions between the immune system and tumor cells result in the formation of a complex immune microenvironment, which contributes to the acquisition of different levels of immunotherapy resistance in tumor cells. In this review, we summarize the characteristics of different phases of cancer immunoediting and the corresponding therapeutic tools, and we propose normalized therapeutic strategies based on immunophenotyping. The process of cancer immunoediting is retrograded through targeted interventions in different phases of cancer immunoediting, making immunotherapy in the context of precision therapy the most promising therapy to cure cancer.

Immune-independent acquired resistance to PD-L1 antibody initiated by PD-L1 upregulation via PI3K/AKT signaling can be reversed by anlotinib.

Despite the benefit with cancer immunotherapies in clinical implication, immunotherapeutic resistance occurred in many patients and the mechanism remains unknown. Increasing evidence has revealed that cell-intrinsic programmed cell death ligand 1 (PD-L1) may play a non-negotiable part in immunotherapeutic resistance. Our present study aimed to elucidate the immune-independent acquired resistance mechanism to PD-L1 antibody. We found elevated PD-L1 expression induced by PD-L1 antibodies in cancer cell and vascular endothelial cells (VECs) with substantially acquired resistance to PD-L1 antibodies. Moreover, proliferation of resistant cells was accelerated and the apoptosis was reduced in the absence of immune compared with parental cells. Subsequently, we confirmed that the activation of the PI3K/AKT pathway is involved in the upregulation of PD-L1 expression. Finally, we found that low dose of anlotinib downregulated PD-L1 expression only in VECs via inhibiting the PI3K/AKT pathway; however, the same effect was not observed in cancer cells. To sum up, our findings revealed that upregulation of PD-L1 via activation of the PI3K/AKT signal pathway may promote acquired resistance to PD-L1 antibodies in an immune-independent manner. SIGNIFICANCE: These findings provide new mechanisms of immunotherapeutic resistance and effective evidence of anlotinib combined with immunotherapy.

Rational pemetrexed combined with CIK therapy plus anti-PD-1 mAbs administration sequence will effectively promote the efficacy of CIK therapy in non-small cell lung cancer.

Cytokine-induced killer (CIK) cells are heterogeneous cells composed mainly of CD3+CD56+ T cells. As an important treatment method of adoptive therapy, it has shown promising efficacy in many clinical trials, especially in combination with multidrug therapy. However, the maximal antitumor efficacy of CIK therapy in the combined administration of multidrug and CIK therapies and which administration scheme can maximize the antitumor efficacy of CIK therapy are still remain unclear. In this study, we observed that pemetrexed administration prior to the injection of CIK cells maximizes the efficacy of CIK therapy. Anti-PD-1 mAbs should be administered prior to CIK cell injection to maximize the efficacy of the therapy. However, administering anti-PD-1 mAbs after CIK cell injection significantly affects the binding rate of anti-PD-1 mAbs to the PD-1 receptor on CIK cells, affecting the efficacy of the antitumor therapy. In conclusion, our study observed that a rational administration sequence of pemetrexed combined with CIK therapy and anti-PD-1 mAbs significantly promotes the efficacy of CIK therapy, providing an experimental basis for the combination therapy mode and regimen of CIK therapy in clinical practice. We hope that this study can provide patients with lung adenocarcinoma with a prolonged survival time.

Anlotinib Downregulates RGC32 Which Provoked by Bevacizumab.

Background: Bevacizumab is the representative drug in antiangiogenic therapy for lung cancer. However, it induced resistance in some neoplasm. Anlotinib, a novel multi-target tyrosine kinase inhibitor which has an inhibitory action on both angiogenesis and malignancy, is possible to reverse the resistance. Methods: Transwell migration and invasion experiments of bevacizumab with or without anlotinib were conducted to verify the activated/inhibited ability of lung adenocarcinoma cells. We sequenced A549 cells with enhanced migration and invasion abilities after bevacizumab treatment, screened out the differentially expressed gene and further confirmed by western blot and q-PCR assays. We also investigated immunohistochemical staining of tumor tissue in mice and human lung adenocarcinoma. Results: Bevacizumab facilitated migration and invasion of lung adenocarcinoma cells. Differentially expressed gene RGC32 was screened out. Bevacizumab upregulated the expression of RGC32, N-cadherin, and MMP2 through ERK-MAPK and PI3K-AKT pathways. Anlotinib downregulated their expression and reversed the effect of bevacizumab on A549 cells. In vivo experiments confirmed that higher-dose bevacizumab facilitated metastasis in tumor-bearing nude mice and upregulated the expression of RGC32, N-cadherin, and MMP2, whereas anlotinib abrogated its effect. Expression of both RGC32 and N-cadherin positively correlated with lymph node metastasis and stage in lung adenocarcinoma was found. Survival analysis revealed that higher expressions of RGC32 and N-cadherin were associated with poor progression-free survival and overall survival. Conclusions: Bevacizumab may promote invasion and metastasis of lung adenocarcinoma cells by upregulating RGC32 through ERK-MAPK and PI3K-AKT pathways to promote epithelial-mesenchymal transition, whereas anlotinib reverses the effect. RGC32 and N-cadherin are independent prognostic factors in lung adenocarcinoma.

CD4+ T cells are required to improve the efficacy of CIK therapy in non-small cell lung cancer.

As a widely studied adoptive treatment method, CIK (cytokine-induced killer cells) treatment has shown clinical benefits in many clinical trials on non-small cell lung cancer. As a heterogeneous cell population, however, CIK cells have a strong instability and individual differences in their efficacies, which are collaboratively regulated by the tumor microenvironment and CIK subpopulations. Among them, CD4+ T cells belong to a crucial subgroup of the CIK cell population, and their influence on CIK therapy is still unclear. Herein, we show how CD4+ T cells positively regulate the functions of CD3+CD56+ T and CD3+CD8+ T cells. During this process, we found that Th1/Th17 CD4+ subgroups can induce the phosphorylation of the AKT pathway by secreting IL-17A, and upregulate the expression of T-bet/Eomes transcription factors, thereby restoring the function of CD8+/CD3+CD56+ T cells and reversing the exhaustion of PD-1+Tim-3+ T cells. These findings will provide guidance for the clinical screening of suitable populations for CIK treatment and formulation of strategies for CIK therapy plus immune checkpoint treatment. Based on these findings, we are conducting an open-label phase II study (NCT04836728) is to evaluate the effects of autologous CIKs in combination with PD-1 inhibitor in the first-line treatment of IV NSCLC, and hope to observe patients' benefits in this clinical trial.

Molecular subtypes based on CNVs related gene signatures identify candidate prognostic biomarkers in lung adenocarcinoma.

The classical factors for predicting prognosis currently cannot meet the developing requirements of individualized and accurate prognostic evaluation in lung adenocarcinoma (LUAD). With the rapid development of high-throughput DNA sequencing technologies, genomic changes have been discovered. These sequencing data provide unprecedented opportunities for identifying cancer molecular subtypes. In this article, we classified LUAD into two distinct molecular subtypes (Cluster 1 and Cluster 2) based on Copy Number Variations (CNVs) and mRNA expression data from the Cancer Genome Atlas (TCGA) based on non-negative matrix factorization. Patients in Cluster 1 had worse outcomes than that in Cluster 2. Molecular features in subtypes were assessed to explain this phenomenon by analyzing differential expression genes expression pattern, which involved in cellular processes and environmental information processing. Analysis of immune cell populations suggested different distributions of CD4+ T cells, CD8+ T cells, and dendritic cells in the two subtypes. Subsequently, two novel genes, TROAP and RASGRF1, were discovered to be prognostic biomarkers in TCGA, which were confirmed in GSE31210 and Tianjin Medical University Cancer Institute and Hospital LUAD cohorts. We further proved their crucial roles in cancers by vitro experiments. TROAP mediates tumor cell proliferation, cycle, invasion, and migration, not apoptosis. RASGRF1 has a significant effect on tumor microenvironment. In conclusion, our study provides a novel insight into molecular classification based on CNVs related genes in LUAD, which may contribute to identify new molecular subtypes and target genes.

anlotinib alters tumor immune microenvironment by downregulating PD-L1 expression on vascular endothelial cells.

Aberrant vascular network is a hallmark of cancer. However, the role of vascular endothelial cells (VECs)-expressing PD-L1 in tumor immune microenvironment and antiangiogenic therapy remains unclear. In this study, we used the specimens of cancer patients for immunohistochemical staining to observe the number of PD-L1+ CD34+ VECs and infiltrated immune cells inside tumor specimens. Immunofluorescence staining and flow cytometry were performed to observe the infiltration of CD8+ T cells and FoxP3+ T cells in tumor tissues. Here, we found that PD-L1 expression on VECs determined CD8+ T cells', FoxP3+ T cells' infiltration, and the prognosis of patients with lung adenocarcinoma. Anlotinib downregulated PD-L1 expression on VECs through the inactivation of AKT pathway, thereby improving the ratio of CD8/FoxP3 inside tumor and remolding the immune microenvironment. In conclusion, our results demonstrate that PD-L1 high expression on VECs inhibits the infiltration of CD8+ T cells, whereas promotes the aggregation of FoxP3+ T cells into tumor tissues, thus becoming an "immunosuppressive barrier". Anlotinib can ameliorate the immuno-microenvironment by downregulating PD-L1 expression on VECs to inhibit tumor growth.

Clinical importance of VEGFC and PD-L1 co-expression in lung adenocarcinoma patients.

BACKGROUND: Vascular endothelial growth factor C (VEGFC), an activator of lymphangiogenesis, is newly identified as an immunomodulator which can regulate the immune system so that tumor cells more easily escape immune surveillance. Evidence has shown programmed cell death-ligand 1 (PD-L1) can also suppress the immune response. Nevertheless, the clinical significance of co-expression of VEGFC and PD-L1 for predicting outcomes in patients with lung adenocarcinoma has not yet been determined. METHODS: A total of 114 patients with lung adenocarcinoma who underwent surgeries at Tianjin Medical University Cancer Institute and Hospital between December 2011 and September 2016 were retrospectively reviewed. Tissue specimens were collected for immunohistochemistry of VEGFC and PD-L1 which were analyzed with an H-score system. RESULTS: In this study, 57 (50.0%) and 47 (41.2%) patients were classified as VEGFC high expression and PD-L1 high expression. Co-expression was observed in 33 (28.9%) patients. In addition, a positive correlation was found between VEGFC and PD-L1 (P = 0.0398, r = 0.1937). In a univariate analysis, both progression-free survival (PFS) and overall survival (OS) were significantly worse in the VEGFC high expression group and the PD-L1 high expression group, respectively. Furthermore, VEGFC/PD-L1 co-expression showed a worse OS (P = 0.03) and PFS survival (P = 0.01) than the other groups. CONCLUSIONS: Taken together, these results indicate that VEGFC/PD-L1 co-expression can forecast both poor OS and PFS in patients with resected lung adenocarcinoma. Co-expression of VEGFC and PD-L1 may serve as a significant prognostic factor for patients with lung adenocarcinoma. KEY POINTS: VEGFC/PD-L1 co-expression forecasts poor survival in patients with resected lung adenocarcinoma. VEGFC/PD-L1 co-expression may be used as a prognostic indicator and provide the theoretical possibility to screen the optimal population with a combination of anti-VEGFC and anti-PD-L1 therapy in the clinical treatment.

Anlotinib suppresses lymphangiogenesis and lymphatic metastasis in lung adenocarcinoma through a process potentially involving VEGFR-3 signaling.

Objective: Lymphatic metastasis is one of the leading causes of malignancy dispersion in various types of cancer. However, few anti-lymphangiogenic drugs have been approved for clinical use to date. Therefore, new therapies to block lymphangiogenesis are urgently required. Methods: Immunohistochemistry, immunofluorescence, Western blot, migration assays, and lymphangiogenesis and lymphatic metastasis assays were used. Results: Anlotinib, a receptor tyrosine kinase inhibitor, suppressed the rate of new metastatic lesions (31.82% in the placebo arm and 18.18% in the anlotinib arm) in patients with advanced lung adenocarcinoma who were enrolled in our ALTER-0303 study. D2-40+-lymphatic vessel density was strongly correlated with disease stage, metastasis, and poor prognosis in 144 Chinese patients with lung adenocarcinoma. In mice bearing A549EGFP tumors, tumor lymphatic vessel density, tumor cell migration to lymph nodes, and the number of distant metastatic lesions were lower in the anlotinib group than in the controls. Anlotinib inhibited the growth and migration of human lymphatic endothelial cells (hLECs) and lymphangiogenesis in vitro and in vivo. Treatment of hLECs with anlotinib downregulated phosphorylated vascular endothelial growth factor receptor 3 (VEGFR-3). Conclusions: Anlotinib inhibits lymphangiogenesis and lymphatic metastasis, probably through inactivating VEGFR-3 phosphorylation. The results indicate that anlotinib may be beneficial for treatment in avoiding lymphangiogenesis and distant lymphatic metastasis in lung adenocarcinoma. (Trial registration: ALTER0303; NCT02388919; March 17, 2015.).

PD-L1 Expression Is Associated With VEGFA and LADC Patients' Survival.

Objectives: To elucidate the relationship between VEGFA and PD-L1 expression in lung adenocarcinoma (LADC). Methods: PD-L1 and VEGFA expression were determined by immunohistochemistry with H-score on formalin-fixed paraffin-embedded resected LADC specimens of 129 cases. Results: High PD-L1 expression in 53 (41.1%) patients, high VEGFA expression in 65 (50.4%), and co-expression in 18 (14.0%) were observed. Inverse correlation between expression of PD-L1 and VEGFA was found (P = 0.002, r = -0.274). VEGFA and PD-L1 expression were not significantly associated with the clinicopathological features. High PD-L1 expression was significantly association with all patients' poor progression-free survival and overall survival in a univariate analysis, but there was no significantly association with high VEGFA expression and prognosis. Co-expression of PD-L1 and VEGFA exhibited a worst overall survival compared to negative groups (P = 0.005). Conclusions: These findings indicate that high PD-L1 expression could impact both poor overall survival and progression-free survival in patients with resected LADC. Co-expression of PD-L1 and VEGFA may be considered as an important prognostic factor for patients with resected lung adenocarcinoma.

Effect of bevacizumab on the tight junction proteins of vascular endothelial cells.

The combination of anti-angiogenesis and chemotherapy can significantly prolong the survival period of patients with non-squamous non-small cell lung cancer (NSCLC). But drug resistance will inevitably occur, thereby causing increased tumor invasion and metastasis. Claudin-5 (CLDN5) is a protein member of tight junction (TJ) structures expressed in endothelial and epithelial cells and confirmed to be involved in the proliferation and leakage of endothelial cells (ECs) and malignant metastases. This study aimed to investigate how bevacizumab, a vascular endothelial growth factor A (VEGFA) neutralizing antibody applied in clinic, affects the tight junction protein CLDN5 and subsequently influences tumor cell invasion and potential metastasis. Western-blot, quantitative real-time polymerase chain reaction (q-PCR), immunofluorescence and immunohistochemistry revealed that low-dose bevacizumab up-regulated CLDN5, whereas high-dose bevacizumab down-regulated CLDN5. Cell migration, invasion and permeation assay demonstrated that high-dose bevacizumab enhanced the migration, invasion and permeation abilities of human umbilical vein endothelial cells (HUVECs). The migration and permeation abilities of HUVECs were also enhanced by silencing the CLDN5 expression. CLDN5 was regulated by JNK, PI3K and transforming growth factor-ß-1 (TGFß1), and these findings were confirmed by the inhibitor or siRNA of JNK, PI3K and TGFß1. Our data indicated that high-dose bevacizumab likely increased tumor invasion and potential metastatic abilities by down-regulating CLDN5, which was down regulated by TGFß1. Low-dose bevacizumab increased CLDN5 expression by up-regulating PI3K and JNK expression.