A facile and scalable in production non-viral gene engineered mesenchymal stem cells for effective suppression of temozolomide-resistant (TMZR) glioblastoma growth

Abstract

Background Mesenchymal stem cells (MSCs) serve as an attractive vehicle for cell-directed enzyme prodrug therapy (CDEPT) due to their unique tumour-nesting ability. Such approach holds high therapeutic potential for treating solid tumours including glioblastoma multiforme (GBM), a devastating disease with limited effective treatment options. Currently, it is a common practice in research and clinical manufacturing to use viruses to deliver therapeutic genes into MSCs. However, this is limited by the inherent issues of safety, high cost and demanding manufacturing processes. The aim of this study is to identify a facile, scalable in production and highly efficient non-viral method to transiently engineer MSCs for prolonged and exceptionally high expression of a fused transgene: yeast cytosine deaminase::uracil phosphoribosyl-transferase::green fluorescent protein (CD::UPRT::GFP). Methods MSCs were transfected with linear polyethylenimine using a cpg-free plasmid encoding the transgene in the presence of a combination of fusogenic lipids and β tubulin deacetylase inhibitor (Enhancer). Process scalability was evaluated in various planar vessels and microcarrier-based bioreactor. The transfection efficiency was determined with flow cytometry, and the therapeutic efficacy of CD::UPRT::GFP expressing MSCs was evaluated in cocultures with temozolomide (TMZ)-sensitive or TMZ-resistant human glioblastoma cell lines. In the presence of 5-fluorocytosine (5FC), the 5-fluorouracil-mediated cytotoxicity was determined by performing colometric MTS assay. In vivo antitumor effects were examined by local injection into subcutaneous TMZ-resistant tumors implanted in the athymic nude mice. Results At > 90% transfection efficiency, the phenotype, differentiation potential and tumour tropism of MSCs were unaltered. High reproducibility was observed in all scales of transfection. The therapeutically modified MSCs displayed strong cytotoxicity towards both TMZ-sensitive and TMZ-resistant U251-MG and U87-MG cell lines only in the presence of 5FC. The effectiveness of this approach was further validated with other well-characterized and clinically annotated patient-derived GBM cells. Additionally, a long-term suppression (> 30 days) of the growth of a subcutaneous TMZ-resistant U-251MG tumour was demonstrated. Conclusions Collectively, this highly efficient non-viral workflow could potentially enable the scalable translation of therapeutically engineered MSC for the treatment of TMZ-resistant GBM and other applications beyond the scope of this study.

NPC2 as a Prognostic Biomarker for Glioblastoma Based on Integrated Bioinformatics Analysis and Cytological Experiments

“…In order to verify the function of NPC2 in GBM, we silenced NPC2 in GBM cells. U87-MG and U251 are cell lines often used in the study of GBM ( Gao et al, 2020 ; Tu et al, 2020 ; Xia et al, 2020 ; Zhang G. et al, 2020 ). CCK-8 assay has shown that cell proliferation was inhibited in U87-MG and U251 cells with Sh-NPC2 transfection compared with that in negative control (Sh-NC) ( Figure 6A ).…”

Mesenchymal stem cell‐glioblastoma interactions mediated via kinin receptors unveiled by cytometry

“…They are present in bone marrow, adipose tissue, dental pulp, blood, amniotic fluid, and umbilical cord [57–61]. Due to their accessibility, multilineage differentiation capacity as well as relatively easy and scalable manufacturing, native or engineered MSCs alone or in combination with biomaterials are being extensively tested for therapeutic applications worldwide [8, 62–65] including primary or adjuvant therapy for solid tumors and blood neoplasia (http://clinicaltrials.gov). Not only MSCs per se are applicable as cytotherapeutic agents, but also their secretomes, comprised of exosomes, microvesicles, cytokines, and other exocytic factors, are being increasingly investigated for acellular and regenerative therapies [66–68].…”

“…Thus, primed MSCs or MSCs loaded with anti‐tumor agents can be considered feasible for GBM therapy. For example, cytosine deaminase fused with uracil phosphoribosyltransferase (CDy::UPRT) expressing MSCs have shown anti‐tumor effects in several cancer types [6–8]. According to Altaner and colleagues, the infusion of CDy::UPRT‐transfected MSCs after GBM resection may present a valid strategy to prevent tumor relapse [6].…”

“…According to Altaner and colleagues, the infusion of CDy::UPRT‐transfected MSCs after GBM resection may present a valid strategy to prevent tumor relapse [6]. Suppression of U251 tumor growth has been demonstrated by injecting CDy::UPRT‐transfected MSCs with 5‐fluorocytosine (5‐FC), a widely used antifungal prodrug, directly into the subcutaneous tumor [7, 8]. MSCs can also be engineered to secrete higher amounts of exosomes containing antitumor miRNAs and proteins that inhibit tumor growth after systemic administration [76].…”

“…MSCs are endogenous, immunomodulatory and mobile tumor‐tropic cells that can be harvested from adipose tissue, oral cavity, umbilical cord and various other tissues with the least discomfort for the patient. They are easily propagated and engineered in vitro and have shown promising results as anti‐GBM agents [6–8]. Although, not only beneficial traits, but also tumorigenic properties were reported regarding MSCs in oncology studies [9–11].…”