Hepatocellular Carcinoma Is a Natural Target for Adeno-Associated Virus (AAV) 2 Vectors

Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current—or develop novel—strategies for treating HCC.

EBV+ tumors exploit tumor cell-intrinsic and -extrinsic mechanisms to produce regulatory T cell-recruiting chemokines CCL17 and CCL22

The Epstein-Barr Virus (EBV) is involved in the etiology of multiple hematologic and epithelial human cancers. EBV+ tumors employ multiple immune escape mechanisms, including the recruitment of immunosuppressive regulatory T cells (Treg). Here, we show some EBV+ tumor cells express high levels of the chemokines CCL17 and CCL22 both in vitro and in vivo and that this expression mirrors the expression levels of expression of the EBV LMP1 gene in vitro. Patient samples from lymphoblastic (Hodgkin lymphoma) and epithelial (nasopharyngeal carcinoma; NPC) EBV+ tumors revealed CCL17 and CCL22 expression of both tumor cell-intrinsic and -extrinsic origin, depending on tumor type. NPCs grown as mouse xenografts likewise showed both mechanisms of chemokine production. Single cell RNA-sequencing revealed in vivo tumor cell-intrinsic CCL17 and CCL22 expression combined with expression from infiltrating classical resident and migratory dendritic cells in a CT26 colon cancer mouse tumor engineered to express LMP1. These data suggest that EBV-driven tumors employ dual mechanisms for CCL17 and CCL22 production. Importantly, both in vitro and in vivo Treg migration was effectively blocked by a novel, small molecule antagonist of CCR4, CCR4-351. Antagonism of the CCR4 receptor may thus be an effective means of activating the immune response against a wide spectrum of EBV+ tumors.

CircRTN4 promotes pancreatic cancer progression through a novel CircRNA-miRNA-lncRNA pathway and stabilizing epithelial-mesenchymal transition protein

Background Circular RNAs (circRNAs) play important roles in many biological processes. However, the detailed mechanism underlying the critical roles of circRNAs in cancer remains largely unexplored. We aim to explore the molecular mechanisms of circRTN4 with critical roles in pancreatic ductal adenocarcinoma (PDAC). Methods CircRTN4 expression level was examined in PDAC primary tumors. The oncogenic roles of circRTN4 in PDAC tumor growth and metastasis were studied in mouse tumor models. Bioinformatics analysis, luciferase assay and miRNA pulldown assay were performed to study the novel circRTN4-miRNA-lncRNA pathway. To identify circRTN4-interacting proteins, we performed circRNA-pulldown and mass spectrometry in PDAC cells. Protein stability assay and 3-Dimensional structure modeling were performed to reveal the role of circRTN4 in stabilizing RAB11FIP1. Results CircRTN4 was significantly upregulated in primary tumors from PDAC patients. In vitro and in vivo functional studies revealed that circRTN4 promoted PDAC tumor growth and liver metastasis. Mechanistically, circRTN4 interacted with tumor suppressor miR-497-5p in PDAC cells. CircRTN4 knockdown upregulated miR-497-5p to inhibit the oncogenic lncRNA HOTTIP expression. Furthermore, we identified critical circRTN4-intercting proteins by circRNA-pulldown in PDAC cells. CircRTN4 interacted with important epithelial-mesenchymal transition (EMT)- driver RAB11FIP1 to block its ubiquitination site. We found that circRTN4 knockdown promoted the degradation of RAB11FIP1 by increasing its ubiquitination. Also, circRTN4 knockdown inhibited the expression of RAB11FIP1-regulating EMT-markers Slug, Snai1, Twist, Zeb1 and N-cadherin in PDAC. Conclusion The upregulated circRTN4 promotes tumor growth and liver metastasis in PDAC through the novel circRTN4-miR-497-5p-HOTTIP pathway. Also, circRTN4 stabilizes RAB11FIP1 to contribute EMT. Graphical abstract

Apoptin Mediates Endogenous Mitophagy and Apoptosis by Regulating the Level of ROS in Hepatocellular Carcinoma

Background: Apoptin, as a tumor-specific pro-apoptotic protein, apoptin plays an important role in the field of anti-tumor, but its autophagy activation mechanism and the interaction between autophagy and apoptosis have not been accurately elucidated. Here, we studied the mechanism of apoptosis and autophagy induced by apoptin and the interaction between autophagy and apoptosis. Methods: Through crystal violet staining and CCK-8 assay, we analyzed the effect of apoptin in inhibiting liver cancer in vitro, and also analyzed the effect of inhibiting liver cancer in vivo by establishing a nude mouse tumor model. Flow cytometry and fluorescence staining were used to analyze the main types of apoptosis and autophagy induced by apoptin. Subsequently, the relationship between apoptosis and autophagy induced by apoptin was analyzed. Then, flow cytometry was used to analyze the effect of ROS on apoptosis and autophagy mediated by apoptin. Then, the affect of ROS on apoptosis and autophagy mediated by apoptin was analyzed. Finally, the key genes leading to autophagy were analyzed by silencing different genes.Results: The results showed that apoptin can significantly increase the apoptosis and autophagy of liver cancer cells, and apoptin can cause mitophagy through the increase of NIX protein. Apoptin can also significantly reduce the level of cellular ROS, which is related to the autophagy and apoptosis of liver cancer cells caused by apoptin. The change of ROS may be a key factor causing apoptosis and autophagy. Conclusion: The above results indicate that the increase of ROS level after apoptin treatment of liver cancer cells leads to the loss of mitochondrial transmembrane potential, which leads to endogenous apoptosis and mitophagy while recruiting NIX. Therefore, ROS may be a key factor connecting endogenous apoptosis and mitophagy induced by apoptin in liver cancer cells.

Nanopore Long-Read Transcriptomics Profiling Reveals Gene Expression Signatures of Mouse Tumor Endothelial Cells 2H-11

Background:Tumor endothelial cells (TECs) play an indispensable role in tumor growth and metastasis. Compared with normal endothelial cells (NECs), TECs exhibit unique phenotypic and functional heterogeneity in terms of metabolism, genetics, and transcriptomics. It is not only the key to coordinate tumor angiogenesis, but also an important factor of immune regulation in the tumor microenvironment. In recent years, the role of TECs in tumor metabolism and invasion has been continuously reported. However, the research on the mechanism behind the complex functions of TECs is still at the basic stage. We use Oxford Nanopore Technology (ONT) three-generation full-length transcriptome sequencing to detect all genetic structural changes in the transcriptome of mouse TECs 2H-11 and mouse NECs SVEC4-10.Results: In Tumor endothelial cells 2H-11,1847genes are up-regulated and 1202 genes are down-regulated. According to the Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs), we found that different functional trends related to metabolic processes, developmental processes, localization, immune system processes, and locomotion are the main reasons for the differences. DEGs are mainly enriched in signal pathways related to cancer, immunity and metabolism, involving Pathways in cancer,Antigen processing and presentation , Proteoglycans in cancer, Focal adhesion, MAPK signaling pathway ,Protein digestion and absorption，ECM-receptor interaction，PI3K-Akt signaling pathway and Glutathione metabolism. We also obtained the structural variation of transcripts such as alternative splicing, gene fusion, and alternative polyadenylation and accurately quantified the expression of the transcript. Some of our results have been confirmed in other documents. But other data have not been reported yet, which is the focus of our future exploration.Conclusion: We try to use transcriptomics and bioinformatics methods to characterize tumor endothelial cell-related genes and signaling pathways.It could help better understand the molecular mechanisms of tumor endothelial cells involved in tumorigenesis and development. DEGs in key pathways may be potential diagnostic markers or therapeutic targets of TECs. Our data also provide useful genetic resources for improving the genome and transcriptome annotations of TECs and NECs.

Two Laser Treatments Can Improve Tumor Ablation Efficiency of Chemophototherapy

Chemophototherapy is an emerging tumor ablation modality that can improve local delivery of chemotherapeutic agents. Long circulating doxorubicin (Dox) in porphyrin-phospholipid (PoP) liposomes (LC-Dox-PoP) has previously been developed as an effective chemophototherapy agent. In the present study, we observed that in mice, LC-Dox-PoP showed enhanced accumulation in human pancreatic tumor xenografts even with suboptimal light doses, as assessed by fluorometric analysis of tissue homogenates and microscopic imaging of Dox and PoP in tumor slices. A second laser treatment, at a time point in which tumors had greater drug accumulation as a result of the first laser treatment, induced potent tumor ablation. Efficacy studies were carried out in two human pancreatic cancer subcutaneous mouse tumor models; MIA PaCa-2 or low-passage patient derived pancreatic cancer xenografts. A single treatment of 3 mg/kg LC-Dox-PoP and an initial 150 J/cm2 laser treatment 1 h after drug administration, followed by second laser treatment of 50 J/cm2 8 h after drug administration, was more effective than a single laser treatment of 200 J/cm2 at either of those time points. Thus, this study presents proof-of-principle and rationale for using two discrete laser treatments to enhance the efficacy of chemophototherapy.

Identification and Characterization of Key Differentially Expressed Genes Associated With Metronomic Dosing of Topotecan in Human Prostate Cancer

Repetitive, low-dose (metronomic; METRO) drug administration of some anticancer agents can overcome drug resistance and increase drug efficacy in many cancers, but the mechanisms are not understood fully. Previously, we showed that METRO dosing of topotecan (TOPO) is more effective than conventional (CONV) dosing in aggressive human prostate cancer (PCa) cell lines and in mouse tumor xenograft models. To gain mechanistic insights into METRO-TOPO activity, in this study we determined the effect of METRO- and CONV-TOPO treatment in a panel of human PCa cell lines representing castration-sensitive/resistant, androgen receptor (+/−), and those of different ethnicity on cell growth and gene expression. Differentially expressed genes (DEGs) were identified for METRO-TOPO therapy and compared to a PCa patient cohort and The Cancer Genome Atlas (TCGA) database. The top five DEGs were SERPINB5, CDKN1A, TNF, FOS, and ANGPT1. Ingenuity Pathway Analysis predicted several upstream regulators and identified top molecular networks associated with METRO dosing, including tumor suppression, anti-proliferation, angiogenesis, invasion, metastasis, and inflammation. Further, the top DEGs were associated with increase survival of PCa patients (TCGA database), as well as ethnic differences in gene expression patterns in patients and cell lines representing African Americans (AA) and European Americans (EA). Thus, we have identified candidate pharmacogenomic biomarkers and novel pathways associated with METRO-TOPO therapy that will serve as a foundation for further investigation and validation of METRO-TOPO as a novel treatment option for prostate cancers.

Heterologous Prime-Boost Vaccination with a Peptide-Based Vaccine and Viral Vector Reshapes Dendritic Cell, CD4+ and CD8+ T Cell Phenotypes to Improve the Antitumor Therapeutic Effect

Heterologous prime-boost settings with a protein vaccine and the viral vector vesicular stomatitis virus, both expressing tumor-associated antigens (KISIMA-TAA and VSV-GP-TAA), have been previously shown to generate potent antitumor immunity. In the cold TC-1 model (HPV antigen) and the immune-infiltrate MC-38 model (Adpgk, Reps1 and Rpl18 neo-antigens), we further investigated pivotal immune cells that educate CD8+ T cells. Heterologous prime-boost vaccination induced a superior antitumor response characterized by the increase in number and functionality of antigen-specific CD8+ T cells, recruitment of cross-presenting dendritic cells, and polarization of CD4+ T cells towards an antitumor Th1 phenotype within the tumor and tumor-draining lymph nodes, turning the cold TC-1 tumor into a hot, inflamed tumor. In the inflamed MC-38 tumor model, treatment combination markedly prolonged the overall survival of mice. Treatment with multi-epitope vaccines also induced high frequencies of multiple antigen specificities in the periphery and in the tumor. Prime-boost treatment reduced tumor-infiltrating regulatory CD4+ T cells whilst increasing cross-presenting dendritic cells in tumor-draining lymph nodes. In conclusion, heterologous prime-boost vaccination possesses the ability to induce a potent anti-tumor response in both immune-excluded and immune-infiltrated mouse tumor models. Additionally, this study highlights the design of a multi-epitope vaccine for cancer immunotherapy.

Computational modeling of schedule-specific chemotherapy outcomes in mouse tumor model

Despite the rapid development of new innovative strategies in cancer treatment like immunotherapy, chemotherapy still remains a common choice in many cases. Standard protocols of chemotherapeutic administration rely on a maximal tolerated dose paradigm, however there is growing evidence that this approach is not always optimal. Alternative scheduling, like metronomic - low dose continuous drug administration - were recently proved their efficacy. The space of available variants of drug administration protocols is prohibitively large to be explored empirically, and there is an urgent need of predictive mathematical models for rational chemotherapeutic scheduling design. In this work we tested the ability of the minimal pharmacokinetic-pharmacodynamics model to describe schedule-specific tumor volume time evolution in different mouse tumor models.