Function and biomedical implications of exosomal microRNAs delivered by parenchymal and nonparenchymal cells in hepatocellular carcinoma.

Small extracellular vesicles (exosomes) are important components of the tumor microenvironment. They are small membrane-bound vesicles derived from almost all cell types and play an important role in intercellular communication. Exosomes transmit biological molecules obtained from parent cells, such as proteins, lipids, and nucleic acids, and are involved in cancer development. MicroRNAs (miRNAs), the most abundant contents in exosomes, are selectively packaged into exosomes to carry out their biological functions. Recent studies have revealed that exosome-delivered miRNAs play crucial roles in the tumorigenesis, progression, and drug resistance of hepatocellular carcinoma (HCC). In addition, exosomes have great industrial prospects in the diagnosis, treatment, and prognosis of patients with HCC. This review summarized the composition and function of exosomal miRNAs of different cell origins in HCC and highlighted the association between exosomal miRNAs from stromal cells and immune cells in the tumor microenvironment and the progression of HCC. Finally, we described the potential applicability of exosomal miRNAs derived from mesenchymal stem cells in the treatment of HCC.

Serpin Family A Member 1 Is Prognostic and Involved in Immunological Regulation in Human Cancers.

Serpin family A member 1 (SERPINA1) encodes a protease inhibitor participating in many human diseases, but its value in immunoregulation and prognosis of human cancers remains unclear. In this study, through comprehensive analysis of data from The Cancer Genome Atlas (TCGA) datasets, we found that SERPINA1 was dysregulated in many cancers compared with normal tissues. SERPINA1 expression was significantly associated with prognosis, immune subtype, molecular subtype, immune checkpoint (ICP) genes, tumor mutational burden (TMB), microsatellite instability (MSI), and the estimation of stromal and immune cells in malignant tumor tissues using expression data (ESTIMATE) score. There was a strong connection between SERPINA1 expression and tumor-infiltrating lymphocytes, and SERPINA1 showed significant relation to gene markers of immune cells in digestive tumors. Fluorescence-based multiplex immunohistochemistry confirmed that SERPINA1 protein expression was related to clinicopathologic features and immune infiltrates in hepatic cancer. This study suggests that SERPINA can potentially serve as a novel biomarker for cancer prognosis and immunotherapy.

High-Dosage NMN Promotes Ferroptosis to Suppress Lung Adenocarcinoma Growth through the NAM-Mediated SIRT1-AMPK-ACC Pathway.

BACKGROUND: Nicotinamide mononucleotide (NMN) is the physiological circulating NAD precursor thought to elevate the cellular level of NAD+ and to ameliorate various age-related diseases. An inseparable link exists between aging and tumorigenesis, especially involving aberrant energetic metabolism and cell fate regulation in cancer cells. However, few studies have directly investigated the effects of NMN on another major ageing-related disease: tumors. METHODS: We conducted a series of cell and mouse models to evaluate the anti-tumor effect of high-dose NMN. Transmission electron microscopy and a Mito-FerroGreen-labeled immunofluorescence assay (Fe2+) were utilized to demonstrate ferroptosis. The metabolites of NAM were detected via ELISA. The expression of the proteins involved in the SIRT1-AMPK-ACC signaling were detected using a Western blot assay. RESULTS: The results showed that high-dose NMN inhibits lung adenocarcinoma growth in vitro and in vivo. Excess NAM is produced through the metabolism of high-dose NMN, whereas the overexpression of NAMPT significantly decreases intracellular NAM content, which, in turn, boosts cell proliferation. Mechanistically, high-dose NMN promotes ferroptosis through NAM-mediated SIRT1-AMPK-ACC signaling. CONCLUSIONS: This study highlights the tumor influence of NMN at high doses in the manipulation of cancer cell metabolism, providing a new perspective on clinical therapy in patients with lung adenocarcinoma.

Combination of chemotherapy and immune checkpoint therapy by the immunoconjugates-based nanocomplexes synergistically improves therapeutic efficacy in SCLC.

Although the etoposide and carboplatin (EP) combination strategy has been the first-line chemotherapy, patients with extensive-stage disease small-cell lung cancer (SCLC) still have poor survival outcomes. Our retrospective analysis revealed that 46 patients with SCLC only achieved medium overall survival (OS) of 11.6 months after treated by EP. Recently, it was demonstrated that combination therapy of PD1/PD-L1 immune checkpoint blocker and EP could significantly improve the OS of SCLC patients. However, the serious treatment-related toxicity leaded to a high rate of treatment-discontinuation or even death. In the present study, we have developed a novel TPP1-conjugated nanocomplex, abbreviated as TPP1NP-EP, which was co-loaded with carboplatin (CBP) and etoposide (VP16). The TPP1 was a PD-L1 targeting peptide and conjugated on the surface of nanocomplex by a matrix metalloproteinase (MMP-2/9)-cleavable peptide linker sequence PLGLAG. For dual-loading of CBP and VP16, the CBP was chemically conjugated with poly(ethylene glycol) (PEG)-poly(caprolactone) (PCL) by pH-sensitive hydrazone bond and the VP16 was physically encapsulated by emulsion-solvent evaporation method. In vitro and in vivo experiments demonstrated an excellent anti-tumor effect of TPP1NP-EP on SCLC and improved safety. In conclusion, the present study has provided a promising strategy for treatment of malignant SCLC.

Single-cell profiling of infiltrating B cells and tertiary lymphoid structures in the TME of gastric adenocarcinomas.

The occurrence and development of gastric adenocarcinoma (gADC) is closely related to the interaction between tumor cells and immune cells in the tumor microenvironment (TME). Our objective was to characterize the repertoire of immune cells in the TME of gADC. To analyze the transcriptomic, immune, and spatial information of TME in gADC, we constructed single-cell RNA sequencing, 10 × Genomics V(D)J analysis, multiple immunofluorescence techniques, and OSCmap analysis of 49,765 single cells in seven samples from four gADC patients. Our integrative analysis of B cells demonstrated that a large number of mucosal associated lymphoid tissue (MALT)-B cells were detected in the gADC tissues, which have mature tertiary lymphatic structures (mTLSs), and almost no MALT-B cells in peripheral blood sample. Moreover, MALT-B cells are a class of IgA+ plasma cells, which are characterized with high expression of complement pathway activation-related genes. Next, natural killer T (NKT) cells mainly exist in gADC tissues accompanied by mTLSs. This study also classified monocytes/macrophages and epithelial cells into benign and malignant types. Interestingly, CSOmap (q < .05) and multiple immunofluorescence (p < .05) results indicated more types of immune cells can be enriched in tissues with mTLSs than normal TLSs, and the density of mTLSs were higher than normal TLSs. Our findings provide novel insights for the signature of immune cells and tumor cells in the TME of gADC with TLSs and highlight the potential importance of IgA-mediated humoral immunity in gADC patients with TLSs.

Trop2 inhibition of P16 expression and the cell cycle promotes intracellular calcium release in OSCC.

Trop2 is an intracellular calcium signal transducer and a prognostic biomarker in many cancers. P16 is a cell cycle gene that negatively regulates cell proliferation and division in most human cancers. Oral squamous cell carcinoma (OSCC) is a common malignant tumor subgroup of head and neck squamous cell carcinoma worldwide. Both Ca2+-dependent and cell cycle signaling pathways play vital roles in OSCC, although the molecular mechanisms remain to be elucidated. We aimed to examine the function of Trop2 and P16 in regulating intracellular calcium ions and the cell cycle in OSCC cell lines. Furtherly, the mRNA and protein expression levels of Trop2 and P16 in OSCC tissue samples were assessed, and their function was evaluated as potential clinical prognostic biomarkers. Trop2 promoted intracellular calcium ion release in OSCC and induced S phase of the cell cycle. Moreover, Trop2-mediated Ca2+ inhibited P16 expression through the AMP-activated protein kinase pathway in OSCC. Interestingly, P16 overexpression could not reverse these phenomena in vitro. We also demonstrated that human OSCC tissues showed high Trop2 mRNA and protein expression, and Trop2+/P16- expression is an independent prognostic marker for OSCC patients. Our data suggest that Trop2+/P16- may be a valuable prognostic marker for OSCC and that Trop2 inhibits P16 expression and induces S phase by promoting intracellular calcium release in OSCC.

Trop2 Promotes Multidrug Resistance by Regulating Notch1 Signaling Pathway in Gastric Cancer Cells.

BACKGROUND Chemotherapy is widely used in gastric cancer treatment, but multidrug resistance remains a leading cause of chemotherapy failure. Trop2 is highly expressed in gastric tumor tissues and greatly influences cancer progression. However, little is known about the relationship between Trop2 and drug resistance in gastric cancer. MATERIAL AND METHODS In the present study, Trop2 was knocked down in BGC823 cells and overexpressed in HGC27. CCK-8 assay was performed to explore the relationship of Trop2 expression and cell proliferation treated with anticancer drugs. Flow cytometry was performed to assess the relationship between Trop2 and cell apoptosis after chemotherapy. Subcutaneous xenograft models were generated to explore the curative effect of DDP to GC in vivo. MRP1 and Notch1 expressions were assessed by Western blot. RESULTS Trop2 decreased cell proliferation inhibition and apoptosis after chemotherapeutic treatments. DDP showed stronger therapeutic effects on Trop2-knockdown tumor than control in vivo. MRP1 and Notch1 signaling pathway were confirmed to participate in Trop2-induced drug resistance. CONCLUSIONS Our findings suggest that Trop2 promotes the resistance of gastric cancer to chemotherapy by activating the Notch1 pathway.

TROP2 increases growth and metastasis of human oral squamous cell carcinoma through activation of the PI3K/Akt signaling pathway.

Most malignant neoplasms of the oral cavity are oral squamous cell carcinoma (OSCC), which is a type of highly malignant tumor with a propensity for forming distant metastases. Trophoblast cell surface antigen 2 (TROP2) is a transmembrane protein that is overexpressed in several types of tumor cells, although its role and regulatory mechanism in OSCC have not been determined. The aim of the present study was to examine the effects of TROP2 in human OSCC cell lines. The present study demonstrated that TROP2 protein expression was upregulated in OSCC cell lines. Transfection of short hairpin RNA (shRNA) targeting TROP2 (sh­TROP2) reduced cell proliferation, migration and invasion of OSCC cell lines, whereas overexpression of TROP2 increased proliferation, migration and invasion. sh­TROP2 transfection in OSCC cell lines inhibited tumor growth in OSCC mouse models. Furthermore, TROP2 expression activated the phosphoinositide 3­kinase (PI3K)/Akt signaling pathway in human OSCC cells. These results suggest that TROP2 induces cell growth, migration and invasion through activation of the PI3K/Akt signaling pathway in OSCC cells.

High-Affinity Human Anti-c-Met IgG Conjugated to Oxaliplatin as Targeted Chemotherapy for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most mortality-causing solid cancers globally and the second largest cause of death among malignancies. Oxaliplatin, a platinum-based drug, has been widely utilized in the treatment of malignancies such as colorectal cancer and hepatocellular carcinoma, yet its usage is limited because of severe side effects of cytotoxicity to normal tissues. c-Met, a receptor tyrosine kinase, is expressed aberrantly on the surface of HCC. The purpose of this study was to synthesise a humanized antibody against c-Met (anti-c-Met IgG) and conjugate it to oxaliplatin to develop a novel antibody-drug conjugate (ADC). Anti-c-Met IgG was detected to be loaded with ~4.35 moles oxaliplatin per mole of antibody. ELISA and FCM confirmed that ADC retained a high and selective binding affinity for c-Met protein and c-Met-positive HepG2 cells. In vitro, the cytotoxicity tests and biological function assay indicated that ADC showed much higher cytotoxicity and functioning in c-Met-positive HepG2 cells, compared with shMet-HepG2 cells expressing lower levels of c-Met. Furthermore, compared with free oxaliplatin, ADC significantly improved cytotoxicity to c-Met-positive tumours and avoided off-target cell toxicity in vivo. In conclusion, by targeting c-Met-expressing hepatoma cells, ADC can provide a platform to reduce drug toxicity and improve drug efficacy in vitro and in vivo.

The role and molecular mechanism of Trop2 induced epithelial-mesenchymal transition through mediated ß-catenin in gastric cancer.

The present study elucidates the potential role of Trop2 in tumor invasion and the promotion of epithelial-mesenchymal transition (EMT) when binding ß-catenin in GC. The role of Trop2 in promoting EMT in GC cells was examined by a variety of experimental assays. Moreover, the underlying molecular mechanism of Trop2 in promoting EMT was studied by in vivo and in vitro assays. The Trop2 expression in relation to tumor metastasis status was detected by IHC in 248 cases of GC tissues and 86 cases of matched adjacent tissues. Trop2 promoted the metastasis and induces EMT in GC. Meanwhile, the elevated protein levels of Trop2 and mesenchymal markers were also found in the TGF-ß1-induced EMT model in GC cells. Importantly, Trop2 physically bound and activated ß-catenin to promote EMT; moreover, Trop2 increased the accumulation of ß-catenin in the nucleus to accelerate metastasis in GC cells. Inhibition of Trop2 expression in GC cells prevented the migration and invasion of GC cells in vivo. Trop2+/vimentin+ expression was higher in GC tissues than that in matched adjacent tissues, and Trop2+/vimentin+ expression in GC was associated with the differentiation, TNM stage, and distant metastases. These sets of data reveal a novel regulatory network of Trop2 in EMT and GC metastasis, suggesting Trop2 as a useful marker for inducing EMT and metastasis of GC, which may help to lead a better understanding of the pathogenesis of the GC.

Human monoclonal anti-protective antigen antibody for the low-dose post-exposure prophylaxis and treatment of Anthrax.

BACKGROUND: Disease caused by Bacillus anthracis is often accompanied by high mortality primarily due to toxin-mediated injury. In the early disease course, anthrax toxins are secreted; thus, antibiotic use is limited to the early stage. In this regard, antibodies against the toxin component, protective antigen (PA), play an important role in protecting against anthrax. Therefore, we developed PA21, a fully human anti-PA immunoglobulin G monoclonal antibody. METHODS: Combining human Fab was screened from a phage library with human heavy constant regions. Enzyme-linked immune sorbent assay, Western blot analysis and immunoprecipitation test evaluated the binding ability of PA21. Moreover, the affinity and neutralizing activity of the antibody was detected in vitro while the protective effectiveness in 60 rats was also examined in vivo. RESULTS: The Fischer 344 rats challenged with the lethal toxin can be protected by PA21 at a concentration of 0.067 mg/kg. All six rats remained alive although PA21 was injected 24 h before the toxin challenge. PA21 did not influence the binding of PA to cell receptors and that of a lethal factor to PA. CONCLUSION: The PA21 monoclonal antibody against PA can be used for emergency prophylaxis and anthrax treatment.

High expression of TROP2 is correlated with poor prognosis of oral squamous cell carcinoma.

Human trophoblastic cell-surface antigen 2 (TROP2) is a cell surface glycoprotein that exhibits high expression in various carcinomas but low or no expression in normal tissues. High TROP2 expression plays an important role in promoting tumor development and aggressiveness, which is correlated with reduced patient survival. However, there are few studies regarding TROP2 in relation to both oral squamous cell carcinoma (OSCC) and oral potentially malignant lesions. The expression of TROP2 protein and mRNA was investigated in OSCC tissues, oral potentially malignant lesion tissues, and normal oral tissues using immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). The association between TROP2 expression and clinicopathological characteristics of OSCC was also analyzed, and the prognostic value of TROP2 was evaluated. The expression of TROP2 protein and mRNA were both higher in OSCC tissues than in oral potentially malignant lesion tissues or normal oral tissues. Positive TROP2 expression was related to differentiation, lymph node metastases, TNM stage, perineural infiltration, and vascular invasion. Poor overall survival was associated with high TROP2 expression and other factors associated with poor overall survival including poor differentiation, lymph node metastasis, TNM stage, vascular invasion, and perineural invasion in univariate analyses. TROP2 expression as well as TNM stage and vascular invasion were independent prognostic factors associated with the overall survival of OSCC patients in multivariate analyses. In summary, High TROP2 expression is associated with poor overall survival and serves as an independent prognostic factor in OSCC. The results suggest that TROP2 expression could be an effective prognostic biomarker for OSCC.

Trop2 is a potential biomarker for the promotion of EMT in human breast cancer.

Trop2 is considered to have an important function in tumor metastasis and the promotion of epithelial­mesenchymal transition (EMT). E­cadherin is a crucial factor in intercellular adhesion and EMT transformation. In the present study, we detected the expression of Trop2 and E­cadherin in breast cancer (BC) to better define their prognostic value. The mRNA expression levels of these two genes in 20 cases of fresh BC tissues were detected by quantitative real­time polymerase chain reaction (qRT­PCR). We also detected the expression levels of these two genes by immunohistochemistry (IHC) in 312 BC tissues, and the correlations between the expression of these two genes and the clinicopathological characteristics in BC patients were analyzed. The mRNA and protein expression levels of the two genes in BC cell lines were studied by qRT­PCR and western blotting. The results indicated that Trop2+/E­cadherin­ was expressed in BC tissues more than that in the matched adjacent tissues. The protein expression results obtained via IHC were similar to the mRNA expression results. Trop2+/E­cadherin­ that was expressed in BC was associated with lymph node status, metastasis, tumor­node­metastasis (TNM) stage, and ER­/PR­/HER2­ expression. BC patients that expressed Trop2+/E­cadherin­ had poor overall survival rates. The results of Trop2 and E­cadherin expression levels obtained in the BC cell lines were the same as those obtained in the BC tissues. Overall, Trop2 has a potential role in the promotion of EMT in BC and it could be considered as a therapeutic target in the future.