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Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with poor prognosis and limited treatment options. While 5-fluorouracil (5-FU) has not been widely employed in GBM therapy, emerging research indicates its potential for effectiveness when combined with advanced drug delivery systems to improve its transport to brain tumors. This study aims to investigate the role of THOC2 expression in 5-FU resistance in GBM cell lines. We evaluated diverse GBM cell lines and primary glioma cells for 5-FU sensitivity, cell doubling times, and gene expression. We observed a significant correlation between THOC2 expression and 5-FU resistance. To further investigate this correlation, we selected five GBM cell lines and developed 5-FU resistant GBM cells, including T98FR cells, through long-term 5-FU treatment. In 5-FU challenged cells, THOC2 expression was upregulated, with the highest increase in T98FR cells. THOC2 knockdown in T98FR cells reduced 5-FU IC50 values, confirming its role in 5-FU resistance. In a mouse xenograft model, THOC2 knockdown attenuated tumor growth and extended survival duration after 5-FU treatment. RNA sequencing identified differentially expressed genes and alternative splicing variants in T98FR/shTHOC2 cells. THOC2 knockdown altered Bcl-x splicing, increasing pro-apoptotic Bcl-xS expression, and impaired cell adhesion and migration by reducing L1CAM expression. These results suggest that THOC2 plays a crucial role in 5-FU resistance in GBM and that targeting THOC2 expression could be a potential therapeutic strategy for improving the efficacy of 5-FU-based combination therapies in GBM patients., Competing Interests: J.J.H., D.Y.C., N.B., and H.S.K. are employees of and stock and/or option holders in Cell&Brain Co., Ltd. The other authors have no potential conflicts of interest to disclose., (AJCR Copyright © 2023.)

Cancer immunotherapy has emerged as a promising approach for treating various malignancies. In this study, we investigated the combined therapeutic effects of mesenchymal stem cells expressing cytosine deaminase (MSC/CD) and 5-fluorocytosine (5-FC) with α-galactosylceramide (α-GalCer) in a colon cancer model. Our findings demonstrated that the combination of MSC/CD, 5-FC, and α-GalCer resulted in enhanced antitumor activity compared to the individual treatments. This was evidenced by increased infiltration of immune cells, such as natural killer T (NKT) cells, antigen-presenting cells (APCs), T cells, and natural killer (NK) cells, in the tumor microenvironment, as well as elevated expression of proinflammatory cytokines and chemokines. Furthermore, we observed no significant hepatotoxicity following the combined treatment. Our study highlights the potential therapeutic benefits of combining MSC/CD, 5-FC, and α-GalCer for colon cancer treatment and contributes valuable insights to the field of cancer immunotherapy. Future research should focus on elucidating the underlying mechanisms and exploring the applicability of these findings to other cancer types and immunotherapy strategies., Competing Interests: J.J.H., D.Y.C., N.B., and H.S.K. are employees of and stock and/or option holders in Cell&Brain Co., Ltd. The other authors have no potential conflicts of interest to disclose., (AJCR Copyright © 2023.)

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor, and current standard therapy provides modest improvements in progression-free and overall survival of patients. Innate tumor resistance and presence of the blood-brain barrier (BBB) require the development of multi-modal therapeutic regimens. Previously, cytosine deaminase (CD)-expressing mesenchymal stem cells (MSC/CD) were found to exhibit anticancer activity with a wide therapeutic index by converting 5-fluorocytosine (5-FC), a nontoxic prodrug into 5-fluorouracil (5-FU), a potent anticancer drug. In this study, we evaluated the efficacy of MSC/CD in a multi-modal combination regimen with temozolomide (TMZ). Cell viability test, cell cycle, and normalized isobologram analyses were performed. In vivo anticancer effects were tested in a mouse orthotopic glioma model. TMZ and MSC/CD with 5-FC synergistically interacted and suppressed U87 glioma cell line growth in vitro . Combined treatment with TMZ and 5-FU increased cell cycle arrest and DNA breakage. In an orthotopic xenograft mouse model, treatment with TMZ alone suppressed tumor growth; however, this effect was more intense with MSC/CD transplantation followed by the sequential treatment with 5-FC and TMZ. Therefore, we propose that sequential treatment with 5-FC and MSC/CD can be used in patients with GBM during the immediate postoperative period to sensitize tumors to subsequent adjuvant chemo- and radiotherapy., Competing Interests: None., (AJCR Copyright © 2020.)

Stem cells carrying a suicide gene have emerged as therapeutic candidates for their cytotoxic bystander effects on neighboring cancers, while being non-toxic to other parts of the body. However, traditional cytotoxicity assays are unable to adequately assess the therapeutic effects of bystander cells. Here, we report a method to assess bystander effects of therapeutic stem cells against 3-dimensionally grown glioma cells in real time. U87 glioma cells were stably transduced to express a green fluorescence protein and co-cultivated with mesenchymal stem cells engineered to carry a bacterial cytosine deaminase gene (MSC/CD). Following addition of a 5-fluorocytine (5-FC) prodrug to the co-culture, fluorescence from U87 cells was obtained and analyzed in real time. Notably, the IC50 of 5-FC was higher when U87 cells were grown 3-dimensionally in soft agar medium for 3 weeks, as compared to those grown for one week in two-dimensional monolayer cultures. Additionally, more MSC/CD cells were required to maintain a similar level of efficacy. Since three-dimensional growth of glioma cells under our co-culture condition mimics the long-term expansion of cancer cells in vivo, our method can extend to an in vitro assay system to assess stem cell-mediated anti-cancer effects before advancing into preclinical animal studies.