Use of cutting-edge RNA-sequencing technology to identify biomarkers and potential therapeutic targets in canine and feline cancers and other diseases.

With the growing interest in companion animals and the rapidly expanding animal healthcare and pharmaceuticals market worldwide. With the advancements in RNA-sequencing (RNA-seq) technology, it has become a valuable tool for understanding biological processes in companion animals and has multiple applications in animal healthcare. Historically, veterinary diagnoses and treatments relied solely on clinical symptoms and drugs used in human diseases. However, RNA-seq has emerged as an effective technology for studying companion animals, providing insights into their genetic information. The sequencing technology has revealed that not only messenger RNAs (mRNAs) but also non-coding RNAs (ncRNAs) such as long ncRNAs and microRNAs can serve as biomarkers. Based on the examination of RNA-seq applications in veterinary medicine, particularly in dogs and cats, this review concludes that RNA-seq has significant potential as a diagnostic and research tool. It has enabled the identification of potential biomarkers for cancer and other diseases in companion animals. Further research and development are required to maximize the utilization of RNA-seq for improved disease diagnosis and therapeutic targeting in companion animals.

Genetically engineered neural stem cells expressing cytosine deaminase and interferon-beta enhanced T cell-mediated antitumor immunity against gastric cancer in a humanized mouse model.

AIMS: Gastric cancer (GC) is an invasive, fatal disease with a poor prognosis. Gene-directed enzyme prodrug therapy via genetically engineered neural stem cells (GENSTECs) has been widely studied in various malignancies, such as breast, ovarian, and renal cancer. In this study, the human neural stem cells expressing cytosine deaminase and interferon beta (HB1.F3.CD.IFN-ß) cells were applied to convert non-toxic 5-fluorocytosine to cytotoxic 5-fluorouracil and secrete IFN-ß. MATERIALS AND METHODS: Human lymphokine-activated killer cells (LAKs) were generated by stimulating human peripheral blood mononuclear cells (PBMCs) by interleukin-2, and we evaluated the cytotoxic activity and migratory ability of LAKs co-cultured with GNESTECs or their conditioned media in vitro. A GC-bearing human immune system (HIS) mouse model was generated by transplanting human PBMCs followed by subcutaneous engraftment of MKN45 cells in NSG-B2m mice to evaluate the involvement of T cell-mediated anti-cancer immune activity of GENSTECs. KEY FINDINGS: In vitro studies showed the presence of HB1.F3.CD.IFN-ß cells facilitated the migration ability of LAKs to MKN45 cells and activated their cytotoxic potential. In MKN45-xenografted HIS mice, treatment with HB1.F3.CD.IFN-ß cells resulted in increased cytotoxic T lymphocyte (CTL) infiltration throughout the tumor, including the central area. Moreover, the group treated to HB1.F3.CD.IFN-ß showed increased granzyme B expression in the tumor, eventually enhancing the tumor-killing potential of CTLs and significantly delaying tumor growth. SIGNIFICANCE: These results indicate that the HB1.F3.CD.IFN-ß cells exert anti-cancer effects on GC by facilitating the T cell-mediated immune response, and GENSTECs are a promising therapeutic strategy for GC.

Effects of CCN6 overexpression on the cell motility and activation of p38/bone morphogenetic protein signaling pathways in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancer types that is highly resistant to conventional treatments, such as chemotherapy and radiotherapy. As the demand for more effective therapeutics for PDAC treatment increases, various approaches have been studied to develop novel targets. The cellular communication network (CCN) family is a matricellular protein that modulates various cellular functions, including cell adhesion, proliferation, migration, and invasiveness. Despite this, little is known about the role of CCN6 in PDAC. The current study investigated the role of CCN6 in PDAC by generating CCN6-overexpressing PANC-1 cells (PANC-1-CCN6) by infecting lentivirus particles containing CCN6. PANC-1-CCN6 induces cell viability and tumorigenesis than PANC-1 cells with empty vector (control). The PANC-1-CCN6 formed more colonies, and the size of spheroids increased compared to the control. The upregulation of CCN6 enhances the expression of bone morphogenetic proteins (BMPs) genes and the upregulation of p38 mitogen-activated protein kinases (MAPKs). In PANC-1-CCN6 cells, the levels of N-cadherin, VEGF, and Snail expression were higher than the control, while E-cadherin expression was lower, which is associated with upregulation of epithelial-to-mesenchymal transition (EMT). Consistent with the changes in EMT-related proteins in PANC-1-CCN6, the migratory ability and invasiveness were enhanced in PANC-1-CCN6. Xenografted PANC-1-CCN6 in immunocompromised mice exhibited accelerated tumor growth than the control group. In immunohistochemistry (IHC), the PANC-1-CCN6 xenografted tumor showed an increased positive area of PCNA and Ki-67 than the control. These results suggest that CCN6 plays a tumorigenic role and induces the metastatic potential by the p38 MAPK and BMPs signaling pathways. Although the role of CCN6 has been introduced as an antitumor factor, there was evidence of CCN6 acting to cause tumorigenesis and invasion in PANC-1.

Engineered adult stem cells: a promising tool for anti-cancer therapy.

Cancers are one of the most dreaded diseases in human history and have been targeted by numerous trials including surgery, chemotherapy, radiation therapy, and anti-cancer drugs. Adult stem cells (ASCs), which can regenerate tissues and repair damage, have emerged as leading therapeutic candidates due to their homing ability toward tumor foci. Stem cells can precisely target malicious tumors, thereby minimizing the toxicity of normal cells and unfavorable side effects. ASCs, such as mesenchymal stem cells (MSCs), neural stem cells (NSCs), and hematopoietic stem cells (HSCs), are powerful tools for delivering therapeutic agents to various primary and metastatic cancers. Engineered ASCs act as a bridge between the tumor sites and tumoricidal reagents, producing therapeutic substances such as exosomes, viruses, and anti-cancer proteins encoded by several suicide genes. This review focuses on various anti-cancer therapies implemented via ASCs and summarizes the recent treatment progress and shortcomings. [BMB Reports 2023; 56(2): 71-77].

Overexpression of KiSS1 Induces the Proliferation of Hepatocarcinoma and Increases Metastatic Potential by Increasing Migratory Ability and Angiogenic Capacity.

Liver cancer has a high prevalence, with majority of the cases presenting as hepatocellular carcinoma (HCC). The prognosis of metastatic HCC has hardly improved over the past decade, highlighting the necessity for liver cancer research. Studies have reported the ability of the KiSS1 gene to inhibit the growth or metastasis of liver cancer, but contradictory research results are also emerging. We, therefore, sought to investigate the effects of KiSS1 on growth and migration in human HCC cells. HepG2 human HCC cells were infected with lentivirus particles containing KiSS1. The overexpression of KiSS1 resulted in an increased proliferation rate of HCC cells. Quantitative polymerase chain reaction and immunoblotting revealed increased Akt activity, and downregulation of the G1/S phase cell cycle inhibitors. A significant increase in tumor spheroid formation with upregulation of ß-catenin and CD133 was also observed. KiSS1 overexpression promoted the migratory, invasive ability, and metastatic capacity of the hepatocarcinoma cell line, and these effects were associated with changes in the expressions of epithelial mesenchymal transition (EMT)-related genes such as E-cadherin, N-cadherin, and slug. KiSS1 overexpression also resulted in dramatically increased tumor growth in the xenograft mouse model, and upregulation of proliferating cell nuclear antigen (PCNA) and Ki-67 in the HCC tumors. Furthermore, KiSS1 increased the angiogenic capacity by upregulation of the vascular endothelial growth factor A (VEGF-A) and CD31. Based on these observations, we infer that KiSS1 not only induces HCC proliferation, but also increases the metastatic potential by increasing the migratory ability and angiogenic capacity.

Next-Generation Antisense Oligonucleotide of TGF-ß2 Enhances T Cell-Mediated Anticancer Efficacy of Anti-PD-1 Therapy in a Humanized Mouse Model of Immune-Excluded Melanoma.

Anti-programmed death-1 (PD-1) immunotherapy is one of the most promising therapeutic interventions for treating various tumors, including lung cancer, bladder cancer, and melanoma. However, only a subset of patients responds to anti-PD-1 therapy due to complicated immune regulation in tumors and the evolution of resistance. In the current study, we investigate the potential of a novel transforming growth factor-beta2 (TGF-ß2) antisense oligonucleotide (ngTASO), as a combination therapy with an anti-PD-1 antibody in melanoma. This study was conducted in a melanoma-bearing human immune system mouse model that recapitulates immune-excluded phenotypes. We observed that the TGF-ß2 blockade by ngTASO in combination with PD-1 inhibition downregulated the tumor intrinsic ß-catenin, facilitated the infiltration of CD8+ cytotoxic lymphocytes (CTLs) in the tumor, and finally, enhanced the antitumor immune potentials and tumor growth delays. Blockade of TGF-ß2 combined with PD-1 inhibition also resulted in downregulating the ratio of regulatory T cells to CTLs in the peripheral blood and tumor, resulting in increased granzyme B expression. In addition, co-treatment of ngTASO and anti-PD-1 augmented the PD-L1 expression in tumors, which is associated with an improved response to anti-PD-1 immunotherapy. These results indicate that the combination of ngTASO and anti-PD-1 exerts an enhanced T cell-mediated antitumor immune potential. Hence, co-inhibition of TGF-ß2 and PD-1 is a potentially promising immunotherapeutic strategy for immune-excluded melanoma.