AGS cell line xenograft tumor as a suitable gastric adenocarcinoma model: growth kinetic characterization and immunohistochemistry analysis

Objective(s): Gastric cancer is the third leading cause of cancer-related death worldwide. The overall survival rate of patients is poor because gastric cancers are usually diagnosed at the late stages. Therefore, further research is needed and appropriate research tools are required to develop novel therapeutic approaches.Materials and Methods: Eight female athymic nude mice with a C57BL/6 background were used in this study. AGS cells were inoculated into the flank. The tumor volumes were calculated and growth curves were drawn. When the volume of the tumors reached 1000 mm3, the animals were humanely euthanized with CO2 gas. After harvesting, tumors were analyzed with Hematoxylin and Eosin (H&E) and immunohistochemistry (IHC). A pathologist confirmed tumor entity through H&E staining. Tumors were evaluated for expression of HER-2, P53, Ki-67, CD34, cytokeratin 8 (CK8), vimentin, estrogen receptor (ER), and progesterone receptor (PR) utilizing immunohistochemistry.Results: The tumor take rate was 62.5%, mean doubling time was 40.984 d, and the latency period was 30.62 days. The H&E staining results showed highly malignant hyperchromatin epithelial cells. IHC assessment showed the mutation status of P53 gene. The expression score of the CK8 protein in the tumor cells was +3. Vimentin protein was not expressed and changes in mesenchymal phenotype were not observed. Ki-67 IHC indicated that the proliferation rate was >43% and angiogenesis was defined as high MVD.Conclusion: The respective AGS xenograft model provides an opportunity to understand the pattern of tumor growth as well as to evaluate new gastric cancer therapies in in vivo studies

CAR-T Regulatory (CAR-Treg) Cells: Engineering and Applications

Regulatory T cells are critical for maintaining immune tolerance. Recent studies have confirmed their therapeutic suppressive potential to modulate immune responses in organ transplant and autoimmune diseases. However, the unknown and nonspecific antigen recognition of polyclonal Tregs has impaired their therapeutic potency in initial clinical findings. To address this limitation, antigen specificity can be conferred to Tregs by engineering the expression of transgenic T-cell receptor (TCR) or chimeric antigen receptor (CAR). In contrast to TCR Tregs, CAR Tregs are major histocompatibility complex (MHC) independent and less dependent on interleukin-2 (IL-2). Furthermore, CAR Tregs maintain Treg phenotype and function, home to the target tissue and show enhanced suppressive efficacy compared to polyclonal Tregs. Additional development of engineered CAR Tregs is needed to increase Tregs’ suppressive function and stability, prevent CAR Treg exhaustion, and assess their safety profile. Further understanding of Tregs therapeutic potential will be necessary before moving to broader clinical applications. Here, we summarize recent studies utilizing CAR Tregs in modulating immune responses in autoimmune diseases, transplantation, and gene therapy and future clinical applications

Feasibility assessment of in vitro chemoresponse assay on stereotactic biopsies of glioblastoma multiforms: a step towards personalized medicine

Objective(s):P In vitro chemosensitivity and resistance assays (CSRAs) are a promising tool for personalized treatment of glioblastoma multiform (GBM). These assays require a minimum of 1 to 2 g of tumor specimen for testing, but this amount is not always accessible. We aimed to assess the feasibility and validity of utilizing stereotactic biopsies of GBM in CSRAs. Materials and Methods: Single cell suspension was prepared from 1 g weight explants of the established xenograft tumor of GBM. Also, primary culture was carried out on 35 mg weight specimens, as a surrogate for stereotactic biopsies. Then, chemoresponse profile of cells obtained by direct cell disaggregation and primary culture was determined using temozolomide and carmustine by clonogenic assay[AGA1] . Results: There was no statistically significant difference in the cytotoxicity of temozolomide and carmustine between cells obtained from both methods. Conclusion: This work supports the feasibility of using stereotactic biopsies of GBM in CSRAs

Novel Combinatorial MicroRNA-Binding Sites in AAV Vectors Synergistically Diminish Antigen Presentation and Transgene Immunity for Efficient and Stable Transduction

Recombinant adeno-associated virus (rAAV) platforms hold promise for in vivo gene therapy but are undermined by the undesirable transduction of antigen presenting cells (APCs), which in turn can trigger host immunity towards rAAV-expressed transgene products. In light of recent adverse events in patients receiving high systemic AAV vector doses that were speculated to be related to host immune responses, development of strategies to mute innate and adaptive immunity is imperative. The use of miRNA binding sites (miR-BSs) to confer endogenous miRNA-mediated regulation to detarget transgene expression from APCs has shown promise for reducing transgene immunity. Studies have shown that designing miR-142BSs into rAAV1 vectors were able to repress costimulatory signals in dendritic cells (DCs), blunt the cytotoxic T cell response, and attenuate clearance of transduced muscle cells in mice to allow sustained transgene expression in myofibers with negligible anti-transgene IgG production. In this study, we screened individual and combinatorial miR-BS designs against 26 miRNAs that are abundantly expressed in APCs, but not in skeletal muscle. The highly immunogenic ovalbumin (OVA) transgene was used as a proxy for foreign antigens. In vitro screening in myoblasts, mouse DCs, and macrophages revealed that the combination of miR-142BS and miR-652-5pBS strongly mutes transgene expression in APCs but maintains high myoblast and myocyte expression. Importantly, rAAV1 vectors carrying this novel miR-142/652-5pBS cassette achieve higher transgene levels following intramuscular injections in mice than previous detargeting designs. The cassette strongly inhibits cytotoxic CTL activation and suppresses the Th17 response in vivo. Our approach, thus, advances the efficiency of miRNA-mediated detargeting to achieve synergistic reduction of transgene-specific immune responses and the development of safe and efficient delivery vehicles for gene therapy