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Extracellular vesicles (EVs) are gaining ground as next-generation drug delivery modalities. Genetic fusion of the protein of interest to a scaffold protein with high EV-sorting ability represents a robust cargo loading strategy. To address the paucity of such scaffold proteins we conducted a large-scale comparative study involving 244 candidate proteins. Their EV-sorting potential was evaluated using a simple but reliable assay that can distinguish intravesicular cargo proteins from surface and non-vesicular proteins. Notably, 24 proteins with conserved EV-sorting abilities across five types of producer cells were identified. Most of these are first to be reported including TSPAN2 and TSPAN3, which emerged as lead candidates, outperforming the well-known CD63 scaffold. Importantly, these engineered EVs show promise as delivery vehicles as demonstrated byin vitroandin vivointernalization studies with luminal cargo proteins as well as surface display of functional domains. The discovery of these novel scaffolds provides a new platform for EV-based engineering.

Extracellular vesicles (EVs) are promising tools to transfer macromolecular therapeutic molecules to recipient cells, however, efficient functional intracellular protein delivery by EVs remains challenging. Here, we have developed novel and versatile systems that leverage selected molecular tools to engineer EVs for robust cytosolic protein delivery bothin vitroandin vivo. These systems, termed VSV-G plus EV-sorting Domain-Intein-Cargo (VEDIC) and VSV-G-Foldon-Intein-Cargo (VFIC), exploit an engineered mini-intein (intein) protein with self-cleavage activity to link cargo to an EV-sorting domain and release it from the EV membrane inside the EV lumen. In addition, we utilize the fusogenic protein VSV-G to facilitate endosomal escape and cargo release from the endosomal system to the cytosol of recipient cells. Importantly, we demonstrate that the combination of the self-cleavage intein, fusogenic protein and EV-sorting domain are indispensable for efficient functional intracellular delivery of cargo proteins by engineered EVs. As such, nearly 100% recombination and close to 80% genome editing efficiency in reporter cells were observed by EV-transferred Cre recombinase and Cas9/sgRNA RNPs, respectively. Moreover, EV-mediated Cre delivery by VEDIC or VFIC engineered EVs resulted in significantin vivorecombination in Cre-LoxP R26-LSL-tdTomato reporter mice following both local and systemic injections. Finally, we applied these systems for improved treatment of LPS-induced systemic inflammation by delivering a super-repressor of NF-ĸB activity. Altogether, this study describes a platform by which EVs can be utilized as a vehicle for the efficient intracellular delivery of macromolecular therapeutics for treatments of disease.Abstract FigureGraphic summary: Development of VEDIC and VFIC systems for high-efficiency intracellular protein delivery in vitro and in vivo.Intein in tripartite fusion protein (EV-sorting Domain-Intein-Cargo) performs C-terminal cleavage during the process of EV-biogenesis, resulting in enriched free cargo proteins inside of vesicles. Together with fusogenic protein, VSV-G, these engineered EVs achieve high-efficiency intracellular delivery of cargo protein (Cre and super repressor of NF-ĸB) or protein complex (Cas9/sgRNA RNPs) both in reporter cells and in mice models.

Messenger RNA (mRNA) has emerged as an attractive therapeutic molecule for a range of clinical applications. Forin vivofunctionality, mRNA therapeutics require encapsulation into effective, stable, and safe delivery systems to protect the cargo from degradation and reduce immunogenicity. Here, we developed a bioengineering platform for efficient mRNA loading and functional delivery using naturally-derived nanoparticles, Extracellular Vesicles (EVs). By expressing the highly specific PUFe RNA-binding domain fused to CD63 in EV producer cells stably expressing the target mRNA, our system exceeds mRNA loading efficiencies by up to 4.5-fold over previously reported EV-based approaches. By combining with an mRNA-stabilizing protein, PABPc, and a fusogenic endosomal escape moiety, VSVg, functional mRNA deliveryin vivois achieved at doses substantially lower than clinically used lipid nanoparticles. Our technology solves substantial drawbacks currently associated with EV-based nucleic acid delivery systems and could enable new applications for mRNA therapeutics.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers and ranks third inmortality. Mitochondria are the energy manufacturers of cells. Disruption of mitochondrial energy metabolism pathways is strongly correlated with the onset and progression of HCC. Aberrant genes in mitochondrial energy metabolism pathways may represent a unique diagnostic and therapeutic targets that act as indicators for HCC.Gene expression data from 374 HCC patients and 50 controls were acquired from TCGA database. A total of 188 mitochondrial energy metabolism-related genes (MMRGs) were obtained from KEGG PATHWAY database. A total of 368 patients with survival data were randomly split into training and validation groups in a 7: 3 ratio. Prognosis-related MMRGs were selected by univariate Cox and LASSO analyses. Kaplan-Meier and ROC curves were employed to analyze the model precision, whereas the validation set was used for model verification. Furthermore, clinical examinations, immune infiltration analysis, GSVA, and immunotherapy analysis were conducted in the high- and low-risk groups. Finally, the risk model was combined with the clinical variables of HCC patients to perform univariate and multivariate Cox regression analyses to obtain independent risk indicators and draw a nomogram. Therefore, we evaluated the accuracy of the predictions using calibration curves.A total of 6032 differentially expressed genes (DEGs) were detected in the HCC and control samples. After overlapping DEGs with 188 MMRGs, 42 mitochondrial energy metabolism-related DEGs (DEMMRGs) were identified. A 17 specific genes-based risk score model of HCC was created, which revealed effectiveness in each TCGA training and validation dataset. Moreover, patients categorized by risk scores exhibited distinct immune infiltration status, immunotherapy responsiveness, and functional properties. Finally, univariate and multivariate Cox regression analyses revealed that risk score and stage T were independent predictive variables. Based on the T stage and risk score, a nomogram for estimating the survival of HCC patients was created. The calibration curves demonstrated that the prediction model had a high level of accuracy.Our study constructed a mitochondrial energy metabolism-related risk model, that may be utilized to anticipate HCC prognosis and represent the immunological microenvironment of HCC.

Objective There are limited data from retrospective studies on whether therapeutic outcomes after regular pancreatectomy are superior to those after enucleation in patients with small, peripheral and well-differentiated non-functional pancreatic neuroendocrine tumors. This study aimed to compare the short- and long-term outcomes of regular pancreatectomy and enucleation in patients with non-functional pancreatic neuroendocrine tumors. Methods Between January 2007 and July 2020, 227 patients with non-functional pancreatic neuroendocrine tumors who underwent either enucleation (n = 89) or regular pancreatectomy (n = 138) were included. Perioperative complications, disease-free survival, and overall survival probabilities were compared. Propensity score matching was performed to balance the baseline differences between the two groups. Results The median follow-up period was 60.76 months in the enucleation group and 43.29 months in the regular pancreatectomy group. In total, 34 paired patients were identified after propensity score matching. The average operative duration in the enucleation group was significantly shorter than that in the regular pancreatectomy group (147.94 ± 42.39 min versus 217.94 ± 74.60 min, P Conclusion Enucleation in patients with non-functional pancreatic neuroendocrine tumors was associated with shorter operative time, lesser intraoperative bleeding, similar overall morbidity of postoperative complications, and comparable 5-year disease-free survival and overall survival when compared with regular pancreatectomy.

Zheyu Niu,* Faji Yang,* Hongguang Li,* Jianlu Wang, Qingqiang Ni, Chaoqun Ma, Huaqiang Zhu, Hong Chang, Xu Zhou, Jun Lu, Hengjun Gao Department of Hepatobiliary Surgery, Shandong Provincial Hospital, Shandong Provincial Hospital Affiliated to Shandong University, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jun Lu; Hengjun Gao, Department of Hepatobiliary Surgery, Shandong Provincial Hospital, Shandong Provincial Hospital affiliated to Shandong University, Shandong Provincial Hospital affiliated to Shandong First Medical University, Jinan, Shandong, People’s Republic of China, Email lujunsd@126.com; cghj_561@163.comPurpose: Monocarboxylate transporter 4 (MCT4) is an important component of cancer cell glycolytic metabolism. It has been confirmed that MCT4 is highly expressed in hepatocellular carcinoma (HCC) cells and tissues and is significantly associated with poor prognosis of HCC patients. However, research on its downstream molecules that affect HCC is still insufficient. The aim of current research was to investigate the MCT downstream molecule and its role of in HCC development.Patients and Methods: After MCT4 expression was knocked down by RNA interference, RNA sequencing and quantitative real-time PCR were used to screen for differentially expressed genes in an HCC cell line (HCCLM3). Immunohistochemistry in HCC tissue microarray was carried out to evaluate the Trafficking Protein Particle Complex Subunit 5 (TRAPPC5) expression. Cell proliferation, migration, and invasion were evaluated by CCK-8 assay, colony formation assay, transwell and wound-healing test, respectively. Xenograft experiment was employed to investigate the function of TRAPPC5 on tumor growth in vivo. Related signaling pathway proteins were evaluated by Western blot.Results: TRAPPC5 expression was significantly downregulated after knocking down of MCT4 in HCCLM3. TRAPPC5 was highly expressed in HCC tissues, and it could enhance the proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) process of HCC cells. In vivo experiment showed that TRAPPC5 could promote HCC tumorigenesis.Conclusion: In the process of MCT4 affecting the progression of HCC, TRAPPC5 is one of the most important related molecules. TRAPPC5 suppression could significantly reduce HCC cell proliferation, migration and invasion and could serve as a therapeutic target in HCC.Keywords: MCT4, hepatocellular carcinoma, TRAPPC5, RNA sequencing, tissue microarrays

Neuropilin-1 regulated by miR-320a participates in the progression of cholangiocarcinoma by serving as a co-receptor that activates multiple signaling pathways. The present study sought to investigate upstream lncRNAs that control the expression of miR-320a/neuropilin-1 axis and dissect some of the underlying mechanisms. Here we report lncRNA TTN-AS1 (titin-antisense RNA1) acts as a sponging ceRNA to downregulate miR-320a and is highly expressed in human cholangiocarcinoma tissues and cells. The expression of the above three molecules is correlated with the clinicopathologic parameters of cholangiocarcinoma patients. In this study, multiple bioinformatics tools and databases were employed to seek potential lncRNAs that have binding sites with miR-320a and TTN-AS1 was identified because it exhibited the largest folds of alteration between cholangiocarcinoma and normal bile duct epithelial cells. The regulatory role of TTN-AS1 on miR-320a was further evaluated by luciferase reporter and RNA pulldown assays, coupled with in situ hybridization and RNA immunoprecipitation analyses, which showed that TTN-AS1 bound to miR-320a through an argonaute2-dependent RNA interference pathway in the cytoplasm of cholangiocarcinoma cells. Knockdown and overexpression assays showed that the regulatory effect between TTN-AS1 and miR-320 was in a one-way manner. TTN-AS1 promoted the proliferation and migration of cholangiocarcinoma cells via the miR-320a/ neuropilin-1 axis. The function of TTN-AS1 on tumor growth and its interaction with miR-320a were confirmed in animal models. Further mechanistic studies revealed that TTA-AS1, through downregulating miR-320a, promoted cell cycle progression, epithelial–mesenchymal transition, and tumor angiogenesis by upregulating neuropilin-1, which co-interacted with the hepatocyte growth factor/c-Met and transforming growth factor (TGF)-β/TGF-β receptor I pathways. In conclusion, the present results demonstrate that lncRNA TTA-AS1 is a sponging ceRNA for miR-320a, which in turn downregulates neuropilin-1 in cholangiocarcinoma cells, indicating these three molecules represent potential biomarkers and therapeutic targets in the management of cholangiocarcinoma.

Objective: The aim of this study was to investigate the role of matrix metallopeptidase 12 (MMP-12) in the development of hepatocellular carcinoma (HCC). Materials and Methods: A total of 343 HCC p...

Colorectal cancer (CRC) is the leading type of diagnosed cancer; globally, it resides in the fourth-leading origin of cancer-interrelated mortality in the globe. The treatment strategies were chemotherapy and potent radiotherapy. Although chemotherapy treatment can eliminate tumor cells, it remains with unnecessary toxic effects in cancer patients. Therefore, the identification of natural-based compounds, which have selectively inhibiting target proteins with limited toxicity that can facilitate the therapeutic approaches against CRC. In this existing approach, which highlights the binding efficacy of our anthraquinone compound, purpurin against phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) protein restrains the CRC cell growth by inhibiting phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT), cell proliferation, and inducing apoptosis signaling. Primarily, purpurin (36 μM) exposed to HCT-116 cells and incubated for 24 and 48 h could induce reactive oxygen species production, subsequently alter mitochondrion membrane, and increase the apoptotic cells in HCT-116. LHPP, a kind of histidine phosphatase protein, has been considered as a tumor suppressor in numerous carcinomas. However, purpurin-mediated LHPP proteins and its associated molecular events in CRC remain unclear. In our docking studies revealed that purpurin has been strongly interacts with LHPP via hydrophobic and hydrophilic binding interaction. Western blot results confirmed that purpurin enhances the expression of LHPP protein, thereby inhibits the expression of phosphorylated-PI3K/AKT, EGFR, cyclin-D1, PCNA in HCT-116 cells. Moreover, purpurin induces messenger RNA expression of apoptotic genes (Bax, CASP-9, and CASP-3) in HCT-116 cells. Thus, we conclude that purpurin could be a natural and useful compound, which inhibits the growth of CRC cells through the activation of LHPP proteins.

Determining an effective biomarker for predicting the prognosis of patients with hepatocellular carcinoma (HCC) may improve patient survival rates. The present study aimed to investigate the expression of glucose transporter 3 (GLUT-3) in HCC and to determine its predictive value for the survival of patients with HCC. Immunohistochemistry was used to detect GLUT-3 expression in HCC tissues of 275 and 140 patients with HCC from training and validation cohorts, respectively. The association between GLUT-3 expression and the clinicopathological characteristics of patients with HCC, and between GLUT-3 expression and patient survival rates were analyzed. The predictive value of GLUT-3 expression was confirmed using the validation cohort. The results demonstrated that the high GLUT-3 expression in HCC tissues was significantly associated with elevated α-fetoprotein level, large tumor size, poor histological differentiation and Tumor-Node-Metastasis stages III and IV (P

Interactions between pancreatic cancer cells and inflammatory cells play crucial roles in the biological behavior of pancreatic cancer. Abundant infiltration of immunoglobulin G4 (IgG4)-positive plasma cells in the pancreas is the most significant feature of autoimmune pancreatitis; however, the clinical significance of IgG4-positive plasma cell infiltration in pancreatic cancer has not previously been reported. Herein, we analyzed intratumoral and peritumoral infiltrations of IgG4-positive plasma cells in 95 pancreatic cancer cases after curative resection. The correlations between IgG4-positive plasma cell infiltration and the clinicopathologic traits and overall survival of pancreatic cancer were investigated. IgG4-positive plasma cells were found in 86 % of tumor tissue samples compared with 69 % of peritumoral tissue samples (P = 0.0063). The high-level infiltration of intratumoral IgG4-positive plasma cells was positively correlated with poor histological grade (P = 0.017). The high-level infiltration of intratumoral IgG4-positive plasma cells was significantly correlated with worse prognosis (P = 0.01) in multivariate analysis. We further found that intratumoral M2-polarized tumor-associated macrophages (TAMs) were positively, linearly correlated with IgG4-positive plasma cells. In conclusion, IgG4-positive plasma cell infiltration is correlated with the clinicopathologic traits and overall survival of pancreatic cancer. High-level intratumoral infiltration of IgG4-positive plasma cells is an independent predictor for poor overall survival in pancreatic cancer patients after curative resection. Intratumoral M2-polarized TAMs probably induce IgG4-positive plasma cells.

Parathyroid carcinoma is a rare tumor associated with poor prognosis, thus early preoperative recognition and surgical resection are critical. However, because of a lack of definitive diagnostic markers and typical clinical features, it is difficult to diagnose this disease in its early stages and it is often misdiagnosed as parathyroid adenoma. Additionally, little is known about the molecular pathogenesis of parathyroid carcinoma because of its rarity. To better understand the molecular genetics of parathyroid carcinoma in the Chinese population, we undertook gene expression profiling of eight parathyroid tumors (five parathyroid adenomas and three parathyroid carcinomas) and five normal parathyroid samples. Differential gene sets between the groups were identified, which may serve as diagnostic biomarkers. Using both gene and protein expression, we demonstrated that CD24, HMOX1, VCAM1 and KCNA3 are useful markers for parathyroid carcinoma. Our findings provide insights into the molecular mechanisms of parathyroid tumorigenesis and may help to improve the diagnosis and treatment of parathyroid carcinoma.

Background Interactions of inflammatory cells with pancreatic cancer cells play crucial roles in pancreatic cancer, however the dynamic changes of inflammatory cell populations in pancreatic cancerogensis and after chemotherapy have not been well eclucidated. The combinational use of aspirin and atrovastatin (Lipitor) have been widely prescribled for cardio-cerebral vascular diseases mainly by regulation of inflammations, and they have been also reported to have plausible anti-tumor effects, however their potential roles in pancreatic cancerogenesis and chemotherapeutic effects have been seldom investigated. We scanned the dynamic changes of pan-inflammatory cell populations in pancreatic cancerogensis and after chemotherapy and found the potential target cell populations. Then we tested the roles of aspirin and Lipitor to regulate these inflammatory cell populations and their effects on pancreatic cancerogenesis and chemotherapeutic effects. Methods Cancerogen, dimethylbenzanthracene (DMBA), was used to induce pancreatic cancerogenesis and subcatunous implantation of syngenic murine Panc02 pancreatic cancer cells was adopted as well. Gemcitabine was used for chemotherapy. The peripheral blood, pancreatic lesions and tumor samples were harvested and analyzed to search for the potential target cell populations. The roles of aspirin and Lipitor to regulate these cell populations and their potential effects on pancreatic cancerogenesis and chemotherapeutic efficacy were investigated both in vitro and in vivo. Results We found progressive accumulations of myeloid-derived suppressor cells (MDSC) and M2-polarzied tumor associated macrophages(M2) in pancreatic lesions accompanied with dynamic reducations of cytotoxic T cells(CTL) and helper T cells(Th) in the progression of pancreatic cancerogenesis. After gemcitabine treatment, the MDSC significantly reduced, however M2 soared up unexpectedly. Aspirin could significantly inhibit the MDSC and M2 to prevent pancreatic cancerogenesis and improve chemotherapeutic effects of gemcitabine, however Lipitor did not significantly affect MDSC, instead it could promote M2 to attenuate the postive effects of aspirin and gemcitabine. Conclusions MDSC and M2 accumulate in progression of pancreatic cancerogenesis and gemcitabine can induce M2. Aspirin could prevent pancreatic cancerogenesis and improve efficacy of gemcitabine partially by inhibiting MDSC and M2, however when used in combination, Lipitor could weaken the efficacy of aspirin and gemcitabine partially by promoting M2. Electronic supplementary material The online version of this article (doi:10.1186/s13046-016-0304-4) contains supplementary material, which is available to authorized users.

The illustration of the procedures of DMBA-induced pancreatic cancerogenesis model. Table S1. The list of antibodies. Table S2. The list of the primers of the real time RT-PCR. (DOCX 2437 kb)

Glucose-regulated protein 78 (GRP78) is a member of the heat-shock protein 70 family. We evaluated the expression of GRP78 using tissue microarray-based immunohistochemistry in tumor tissues and adjacent nontumor tissues from 180 pancreatic ductal adenocarcinoma (PDAC) patients. The associations between the expression levels of GRP78, clinicopathological factors and overall survival were evaluated. The results showed that the expression of GRP78 was significantly higher in PDAC cells than in normal pancreatic duct cells within adjacent nontumor tissues (p in vitro study, the regulation of GRP78 in the PDAC cell lines affected the proliferation, migration and invasion of PDAC cells through the regulation of CyclinD1, cyclin-dependent kinase (CDK) 4, CDK6, phospho-signal transducer, activator of transcription 3 (p-STAT3), janus kinase 2 (JAK2), ras homolog gene family member A (RhoA), Rho-associated kinase 1 (ROCK1) and sterile alpha motif domain containing protein 4 (Smad4). The present data suggest that GRP78 plays a crucial role in the proliferation, migration and invasion of pancreatic cancer cells and may be a suitable prognostic marker in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most highly malignant tumors with a very poor prognosis. In addition to the cancer cells, the stroma of tumor can expand by 50% and influence cancer cell growth. Cancer-associated fibroblasts (CAFs) are important components of tumor stroma. Cancer cells, normal fibroblasts, normal epithelial cells as well as bone marrow-derived myofibroblasts contribute to the emergence of CAFs through various cytokines (e.g., TGF-β, SHH, PDGF) and epithelial-to-mesenchymal transition. CAFs affect cancer growth, survival, metastasis, angiogenesis and immunosurveillance through the secretion of various cytokines, such as CXCL12 and secreted protein acidic and rich in cystein. Also, CAFs correlate to the prognosis and chemoresistance of PDAC patients. As novel therapeutic targets, CAFs, and their relative factors, represent an important role in PDAC therapy.

AIM: To investigate the prognostic significance of pretreatment standardized maximum uptake value (SUVmax) and serum carbohydrate antigen (CA)19-9 in pancreatic cancer. METHODS: From January 2007 to October 2011, 80 consecutive patients with pancreatic cancer who received positron emission/computed tomography before any treatment were enrolled in this study. The pretreatment SUVmax and CA19-9 level of the primary pancreatic tumor were obtained and compared with clinicopathological and prognostic factors. Student’s t test for unpaired data was used to analyze the differences between two groups. Univariate analysis and Cox proportional hazards regression were used to examine the independent effects of each significant variable. Survival was analyzed by the Kaplan-Meier method. RESULTS: There was a significant correlation between both the SUVmax and serum CA19-9 of pancreatic cancer and R0 surgical resection (P = 0.043 and P = 0.007). Lymph node metastasis was associated with SUVmax (P = 0.017), but not serum CA19-9 (P = 0.172). On the contrary, the tumor stage was significantly related to serum CA19-9 (P = 0.035), but not SUVmax (P = 0.110). The univariate analysis showed that survival time was significantly related to tumor stage (P < 0.001), lymph node metastasis (P = 0.043), R0 surgical resection (P < 0.001), serum CA19-9 (P = 0.001), SUVmax (P < 0.001) and SUVmax plus CA19-9 (P = 0.002). Multivariate analysis clearly showed that only tumor stage (hazard ratio = 0.452; P = 0.020) was an independent prognostic factor for overall survival in pancreatic cancer. Higher SUVmax or CA19-9 showed worse prognosis. We found that high serum CA19-9 plus SUVmax was the most significant variable. CONCLUSION: Higher pretreatment SUVmax and serum CA19-9 indicates poor prognosis. SUVmax plus serum CA19-9 is the most significant variable in predicting survival.