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Chimeric antigen receptor macrophage (CAR-M) is a promising immunotherapy strategy of anti-tumor due to its high infiltration, direct phagocytosis of tumor cells, immunomodulation of tumor microenvironment (TME) and linkage of innate and adaptive immunity. Here a series of novelly designed CAR-Ms by targeting vascular endothelial growth factor receptor-2 (VEGFR2), which highly expressed in tumor cells and TME, were evaluated. Their activation signals were transduced by Tlr4 or Ifn-γ receptors either alone or in combination, which were designed to mediate M1 polarization of macrophages as the downstream of lipopolysaccharide or Ifn-γ that had been widely reported. Our results showed that VEGFR2-targeting CAR-Ms could be activated under the stimulation of VEGFR2-expressing cells. They exhibited higher expression of CD86, MHCII and TNF-α in vitro and enhanced tumor suppressive abilities in vivo. Implantation of these CAR-Ms into 4T1 breast cancer-bearing mice could obviously inhibit the progression of tumor without significant toxic side effects, especially the group of mmC in which constructed with Tlr4 as the intracellular domain of CAR. In conclusion, this research provides a promising design of CAR that induce macrophages activation by Tlr4 and/or Ifn-γ receptors, and these CAR-Ms could effectively inhibit tumor growth through targeting VEGFR2. [ABSTRACT FROM AUTHOR]

Head and neck cancers are a type of life-threatening cancers characterized by an immunosuppressive tumor microenvironment. Only less than 20% of the patients respond to immune checkpoint blockade therapy, indicating the need for a strategy to increase the efficacy of immunotherapy for this type of cancers. Previously, we identified a type B CpG-oligodeoxynucleotide (CpG-ODN) called CpG-2722, which has the universal activity of eliciting an immune response in grouper, mouse, and human cells. In this study, we further characterized and compared its cytokine-inducing profiles with different types of CpG-ODNs. The antitumor effect of CpG-2722 was further investigated alone and in combination with an immune checkpoint inhibitor in a newly developed syngeneic orthotopic head and neck cancer animal model. Along with other inflammatory cytokines, CpG-2722 induces the gene expressions of interleukin-12 and different types of interferons, which are critical for the antitumor response. Both CpG-2722 and anti-programmed death (PD)-1 alone suppressed tumor growth. Their tumor suppression efficacies were further enhanced when CpG-2722 and anti-PD-1 were used in combination. Mechanistically, CpG-2722 shaped a tumor microenvironment that is favorable for the action of anti-PD-1, which included promoting the expression of different cytokines such as IL-12, IFN-β, and IFN-γ, and increasing the presence of plasmacytoid dendritic cells, M1 macrophages, and CD8 positive T cells. Overall, CpG-2722 provided a priming effect for CD8 positive T cells by sharpening the tumor microenvironment, whereas anti-PD-1 released the brake for their tumor-killing effect, resulting in an enhanced efficacy of the combined CpG-2722 and anti-PD-1. [ABSTRACT FROM AUTHOR]

ICAM3 was reported to promote metastasis in tumors. However, the underlying mechanism remains elusive. Here, we disclosed that the expression of ICAM3 was closely correlated with the TNM stage of human breast and lung cancer, as well as the dominant overexpression in high aggressive tumor cell lines (231 and A549 cells). Moreover, the knockdown of ICAM3 inhibited tumor metastasis whereas the ectopic expression of ICAM3 promoted tumor metastasis both in vitro and in vivo . In addition, exploration of the underlying mechanism demonstrated that ICAM3 not only binds to LFA-1 with its extracellular domain and structure protein ERM but also to lamellipodia with its intracellular domain which causes a tension that pulls cells apart (metastasis). Furthermore, ICAM3 extracellular or intracellular mutants alternatively abolished ICAM3 mediated tumor metastasis in vitro and in vivo . As a therapy strategy, LFA-1 antibody or Lifitegrast restrained tumor metastasis via targeting ICAM3-LFA-1 interaction. In summary, the aforementioned findings suggest a model of ICAM3 in mediating tumor metastasis. This may provide a promising target or strategy for the prevention of tumor metastasis. [ABSTRACT FROM AUTHOR]

Breast cancer is one of the most malignant diseases in women worldwide. Serum microRNAs (miRNAs), with the characteristics of high sensitivity and specificity, have recently attracted more attentions to serve as potential biomarkers for tumor diseases. In this study, 194 breast cancer patients’ serum samples were collected before surgery and enrolled into different groups based on their diagnostic information. To search for breast cancer diagnostic biomarkers, serum miRNAs were screened by microarray in pooled samples of healthy volunteers and breast cancer patients in different clinical stages. The miRNAs were further verified in each individual patient’s serum samples in diagnostic and predictive sets. The serum level of miR-1915-3p was upregulated and miR-455-3p was downregulated significantly in breast cancer patients compared with healthy volunteers. Furthermore, the patients with infiltrating carcinoma or lymph node metastasis had a higher serum level of miR-1915-3p and lower serum level of miR-455-3p than patients with the carcinoma in situ or patients without lymph node metastasis. ROC analysis suggested that miR-1915-3p and miR-455-3p had the potential as a promising serum diagnostic and predictive biomarkers of breast cancer. miR-1915-3p was over-expressed in certain human breast cancer cells. Functional experiments in vitro showed that miR-1915-3p enhanced cell proliferative and migrational abilities. Overexpression of miR-1915-3p repressed target gene DUSP3 and activated ERK1/2. Collectively, this study provided a new insight that miR-1915-3p might play a role in the development of breast cancer and that serum miR-1915-3p and miR-455-3p could serve as diagnostic and predictive biomarkers for breast cancer. [ABSTRACT FROM AUTHOR]

The unfolded protein response (UPR) is widely activated in cancers. The mammalian UPR encompasses three signaling branches, namely inositol-requiring enzyme-1α (IRE1α), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6α (ATF6α). The functional significance of each branch in tumorigenesis is incompletely understood, especially in cancer stem cells (CSCs). Here, we report that inhibition and silencing of the three UPR sensors has differential effects on breast cancer growth and the CSC population. The levels of PERK and ATF6α strongly correlate with the expression of sex determining region Y (SRY)-box 2 (SOX2), a pluripotency regulator, in human breast cancer tissues. UPR activation is also elevated in the CSC-enriched mammospheres. Inhibition of the UPR sensors or excess ER stress markedly reduces the formation and maintenance of mammospheres, suggesting that an appropriate level of UPR activation is critical for the CSC survival. Mechanistically, transcription factors from UPR and pluripotency pathways interact and reciprocally influence each other. A transcription modulator, CCAAT-enhancer-binding protein delta (C/EBPδ), interacts with pluripotency regulator, SOX2, and UPR transcription factors, thus likely serving as a link to coordinate UPR and pluripotency maintenance in CSCs. Our findings demonstrate that UPR is critical for both cancer growth and pluripotency, and highlight the differential role and complexity of the three UPR branches in tumorigenesis. [ABSTRACT FROM AUTHOR]

In this study, we present a medium throughput siRNA screen platform to identify inflammation genes that regulate cancer cell stemness. We identified several novel candidates that decrease OCT4 expression and reduce the ALDH + subpopulation both of which are characteristic of stemness. Furthermore, one of the novel candidates ICAM3 up-regulates in the ALDH + subpopulation, the side population and the developed spheres. ICAM3 knockdown reduces the side population, sphere formation and chemo-resistance in MDA-MB-231 human breast cancer cells and A549 lung cancer cells. In addition, mice bearing MDA-MB-231-shICAM3 cells develop smaller tumors and fewer lung metastases versus control. Interestingly, ICAM3 recruits and binds to Src by the YLPL motif in its intracellular domain which further activates the PI3K-AKT phosphorylation cascades. The activated p-AKT enhances SOX2 and OCT4 activity and thereby maintains cancer cell stemness. Meanwhile, the p-AKT facilitated p50 nuclear translocation/activation enhances p50 feedback and thereby promotes ICAM3 expression by binding to the ICAM3 promoter region. On this basis, Src and PI3K inhibitors suppress ICAM3-mediated signaling pathways and reduce chemo-resistance which results in tumor growth suppression in vitro and in vivo . In summary, we identify a potential CSC regulator and suggest a novel mechanism by which ICAM3 governs cancer cell stemness and inflammation. [ABSTRACT FROM AUTHOR]

In this study, we present a medium throughput siRNA screen platform to identify inflammation genes that regulate cancer cell stemness. We identified several novel candidates that decrease OCT4 expression and reduce the ALDH + subpopulation both of which are characteristic of stemness. Furthermore, one of the novel candidates ICAM3 up-regulates in the ALDH + subpopulation, the side population and the developed spheres. ICAM3 knockdown reduces the side population, sphere formation and chemo-resistance in MDA-MB-231 human breast cancer cells and A549 lung cancer cells. In addition, mice bearing MDA-MB-231-shICAM3 cells develop smaller tumors and fewer lung metastases versus control. Interestingly, ICAM3 recruits and binds to Src by the YLPL motif in its intracellular domain which further activates the PI3K-AKT phosphorylation cascades. The activated p-AKT enhances SOX2 and OCT4 activity and thereby maintains cancer cell stemness. Meanwhile, the p-AKT facilitated p50 nuclear translocation/activation enhances p50 feedback and thereby promotes ICAM3 expression by binding to the ICAM3 promoter region. On this basis, Src and PI3K inhibitors suppress ICAM3-mediated signaling pathways and reduce chemo-resistance which results in tumor growth suppression in vitro and in vivo. In summary, we identify a potential CSC regulator and suggest a novel mechanism by which ICAM3 governs cancer cell stemness and inflammation. [ABSTRACT FROM AUTHOR]

Treatment of mice with lipopolysaccharide (LPS) and the liver-specific transcriptional inhibitor D-(+)-galactosamine (GalN) induces fatal hepatitis, which is mediated by tumor necrosis factor α (TNF-α) and characterized by massive hepatic apoptosis. Previous studies suggest that GalN increases the sensitivity to LPS/TNF-α, probably by blocking the transcription of protective factors, but the identity of most of these factors is still unclear. Here, we report that Ifit1 protects against LPS/GalN-induced fatal hepatitis. Forced expression of Ifit1 in hepatocytes significantly diminished TNF-α-mediated apoptosis. Moreover, targeted expression of Ifit1 in the liver by recombinant adeno-associated virus serotype 8 protected mice from LPS/GalN-induced lethal hepatitis, which was associated with the inhibition of TNF-α-mediated activation of the c-Jun N-terminal kinase (JNK)-Bim cascade. Furthermore, Ifit1 bound to a scaffolding protein Axin and inhibited its function to mediate JNK activation. Together, our data demonstrate that Ifit1 is a novel protective factor that inhibits LPS/GalN-induced (TNF-α-mediated) fatal hepatitis, suggesting that Ifit1 is a potential therapeutic target for treatment of inflammatory liver diseases. [ABSTRACT FROM AUTHOR]

Although Oct4 is known as a critical transcription factor involved in maintaining “stemness”, its role in tumor metastasis is still controversial. Herein, we overexpressed and silenced Oct4 expression in two breast cancer cell lines, MDA-MB-231 and 4T1, separately. Our data showed that ectopic overexpression of Oct4 suppressed cell migration and invasion in vitro and the formation of metastatic lung nodules in vivo . Conversely, Oct4 downregulation increased the metastatic potential of breast cancer cells both in vitro and in vivo . Furthermore, we identified Rnd1 as the downstream target of Oct4 by ribonucleic acid sequencing (RNA-seq) analysis, which was significantly downregulated upon Oct4 overexpression. Chromatin immunoprecipitation assays revealed the binding of Oct4 to the promoter region of Rnd1 by ectopic overexpression of Oct4. Dual luciferase assays indicated that Oct4 overexpression suppressed transcriptional activity of the Rnd1 promoter. Moreover, overexpression of Rnd1 partially rescued the inhibitory effects of Oct4 on the migration and invasion of breast cancer cells. Overexpression of Rnd1 counteracted the influence of Oct4 on the formation of cell adhesion and lamellipodia, which implied a potential underlying mechanism involving Rnd1. In addition, we also found that overexpression of Oct4 led to an elevation of E-cadherin expression, even in 4T1 cells that possess a relatively high basal level of E-cadherin. Rnd1 overexpression impaired the promoting effects of Oct4 on E-cadherin expression in MDA-MB-231 cells. These results suggest that Oct4 affects the metastatic potential of breast cancer cells through Rnd1-mediated effects that influence cell motility and E-cadherin expression. [ABSTRACT FROM AUTHOR]

Toll-like receptors 7 (TLR7) and 8 (TLR8) recognize viral single-stranded RNA and small molecular weight agonists to activate anti-viral immune responses. TLR8s from different species have distinct ligand recognitions. For example, human TLR8 is responsive to ligand stimulation, but mouse and rat TLR8 are activated by small molecular weight agonists only in the presence of polyT-oligodeoxynucleotides. TLR7 and TLR8 have been reported to be absent and pseudogenized, respectively, in rabbit ( Oryctolagus cuniculus ). In this study, we detected the expression of rabbit (rab)TLR8 in immune-cell-associated tissues. Cell proliferation and cytokine expressions in rabbit splenocytes were induced by the TLR7/8 ligand but not by the TLR7 ligands, suggesting that rabTLR8 is functional but rabTLR7 is not. In rabbits, CL075, a TLR7/8 ligand, activated an antigen-specific antibody response, although one not as potent as aluminum salt or Freund's adjuvant. Nevertheless, CL075, alone or in combination with aluminum salt, generates fewer adverse effects than Freund's adjuvant at the injection sites. To further investigate the activation of rabTLR8, we cloned its cDNA. In cell-based assay, this rabTLR8 is activated by TLR7/8 ligand but not activated by TLR7 ligand. Upon stimulation the rabTLR8 had a lower activation compared to the activation of TLR8 from other species, except the mouse and rat TLR8s. Using different deletion and human-rabbit chimeric TLR8 expressing constructs, we showed that an extra peptide in the undefined region results in reduced activity of rabTLR8. These results provide a molecular basis for the mild activities of TLR7/8 ligands in rabbits, and suggest TLR7/8 agonists may provide safer immune stimuli in rabbits than in other non-rodent species. [ABSTRACT FROM AUTHOR]

Vaccines are powerful tools for controlling microbial infections and preventing epidemic diseases. Efficient inactive, subunit, or viral-like particle vaccines usually rely on a safe and potent adjuvant to boost the immune response to the antigen. After a slow start, over the last decade there has been increased developments on adjuvants for human vaccines. The development of adjuvants has paralleled our increased understanding of the molecular mechanisms for the pattern recognition receptor (PRR)-mediated activation of immune responses. Toll-like receptors (TLRs) are a group of PRRs that recognize microbial pathogens to initiate a host's response to infection. Activation of TLRs triggers potent and immediate innate immune responses, which leads to subsequent adaptive immune responses. Therefore, these TLRs are ideal targets for the development of effective adjuvants. To date, TLR agonists such as monophosphoryl lipid A (MPL) and CpG-1018 have been formulated in licensed vaccines for their adjuvant activity, and other TLR agonists are being developed for this purpose. The COVID-19 pandemic has also accelerated clinical research of vaccines containing TLR agonist-based adjuvants. In this paper, we reviewed the agonists for TLR activation and the molecular mechanisms associated with the adjuvants' effects on TLR activation, emphasizing recent advances in the development of TLR agonist-based vaccine adjuvants for infectious diseases. [ABSTRACT FROM AUTHOR]

Lymph node metastasis is of major prognostic significance for breast cancer. Lymph node metastasis arises at a very early stage in some patients. Using the data downloaded from the TCGA database, we studied the differences between primary tumors with and without lymph node metastasis at the multi-omics level using bioinformatics approaches. Our study found that low mutation and neoantigen burdens correlated with lymph node metastazation of breast cancer. All three conserved domains in TP53 were mutated in lymph node-negative breast cancers, whereas only one domain was mutated in lymph node-positive samples. Mutations in microtubule-related proteins appear to help immune cells recognize tumors and inhibit their lymph node metastasis. Destroying microtubule-related proteins is a potential therapeutic strategy to inhibit lymph node metastasis of breast cancer. As the neoantigens specifically present in lymph node-positive breast cancers, MAPK10, BC9L, TRIM65, CD93, KITLG, CNPPD1, CPED1, CCDC146, TMEM185A, INO80D, and PSMD11 are potential targets for vaccine design. In the tumor microenvironment, reduced numbers of effector immune cells, especially activated memory CD4+ T cells and activated mast cells, facilitate breast cancer metastasis to the lymph nodes. According to transcriptome data, lymph node metastasis was mostly driven by gene mutation rather than by gene expression. Although differential gene expression analysis was based on lymph node metastasis status, many genes were shown to be differentially expressed based on estrogen receptor status. [ABSTRACT FROM AUTHOR]