In recent years, targeted therapies using tumorigenic stem cells as vehicles carrying various therapeutic factors have become a hot topic of research in malignant glioma treatment strategies. Although neural stem cells (NSCs) have shown encouraging results in preclinical trials [[i]], their translation to the clinic is limited by technical difficulties in collection, isolation and in vitro expansion, potential immune rejection of allogeneic transplantation, and ethical and legal factors. Bone marrow-derived mesenchymal stem cells (BM-MSCs), which are relatively easy to obtain, have tumorigenic capacity similar to that of NSCs [[ii]], and are less technically difficult to collect and require relatively less demanding operating conditions. However, the number of BM-MSCs is relatively limited and their proliferative capacity decreases with age [[iii]]. In this regard, human adipose tissue derived mesenchymal stem cells (hAT-MSCs), which have similar tumorigenic effects [3], are available in larger numbers, free of ethical and legal concerns, easy to obtain and isolate, and can be autologously transplanted, are expected to be a more desirable gene targeting vehicle for glioma human adipose tissue derived mesenchymal stem cells (hAT-MSCs) are expected to become more ideal vectors for gene targeted therapy of glioma [[iv]]. Comparative studies on the tumorigenic effects of hAT-MSCs have revealed that hAT-MSCs have similar tumorigenic effects to BM-MSCs and NSCs in Transwell in vitro migration assays and in vivo migration assays to rat brainstem gliomas (F98) [3]. hAT-MSCs injected far from the tumor site were able to migrate toward and encircle the glioma; hAT-MSCs injected directly into the tumor interior were widely distributed in the tumor bed but did not invade normal brain tissue [[v]]. Gene transfection studies of hAT-MSCs showed that although the transfection efficiency of hAT-MSCs was significantly improved by adenoviral vectors modified with arginine-glycine-aspartate fibers, the tumor-tending ability of hAT-MSCs was reduced by 55% after adenoviral transfection in vitro, and the ability to track non-infiltrating glioma tumors in vivo was completely lost. The reason for this may be related to the fact that the viral proteins of adenovirus-transfected hAT-MSCs may have induced a local acute inflammatory response, causing hAT-MSCs to be cleared by immune cells and thus affecting their tumor homing ability. This suggests that first-generation adenoviruses are not suitable as vectors for presenting genes to hAT-MSCs, and that less immunogenic retroviruses, lentiviruses, adeno-associated viruses, or second-generation adenovirus vectors may be more desirable vector systems [6]. Subsequent studies have shown that hAT-MSCs carrying specific therapeutic factors by retrovirus [[vi], [vii]], mucosal tumor virus [[viii]] and nuclear transfection [5, [ix], [x]] are not significantly affected in their tumorigenic ability and can effectively exert anti-tumor efficacy in vitro and in vivo. 2. hAT-MSCs and enzyme/antitumor prodrugs Enzyme/antitumor prodrug combinations using MSCs as cell carriers are ideal therapeutic strategies for tumor gene targeting therapy. MSCs genetically modified to express specific enzymes can tend to migrate into and around tumors and form gap junctions with tumor cells [7], thus transforming relatively non-toxic antitumor prodrugs inside tumor cells into pharmacologically active cytotoxic drugs, effectively avoiding the toxicity of systemic drug administration while killing tumor cells. In addition, the prodrugs carried by MSCs can kill not only tumor cells but also the proliferating MSCs themselves [7], thus ensuring the safety of MSCs in treating tumors. Currently, the three most studied enzyme/antitumor prodrug combinations are the herpes simplex virus- thymidine kinase (HSV-tk)/ganciclovir (GCV) system, yeast cytosine deaminase::uracil phosphate ribosyltransferase (yeast cytosine deaminase::uracil phosphoribosyltransferase (CDy::UPRT)/5-fluorocytosine (5-FC) system and rabbit carboxylesterase (rCE)/irinotecan irinotecan-7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11) system. 2.1 hAT-MSCs in combination with HSV-tk/GCV HSV-tk/GCV is the most widely used enzyme/antitumor prodrug combination [[xi]]. As purine nucleoside analogs, GCV is not toxic or has low toxicity when taken up by HSV-tk-negative cells, but can be phosphorylated by thymidine kinase to a cytotoxic phosphorylation product in HSV-tk-positive expressing cells, thereby inhibiting cellular DNA polymerase activity; or can be incorporated into synthetic DNA as a competitive inhibitor of deoxyguanosine triphosphate, blocking DNA strand lengthening, inhibiting cellular DNA synthesis and causing cell death [11]. Although the HSV-TK/GCV combination suicide gene therapy strategy was safe and effective in preclinical experiments, it was limited by the low transfection rate of target cells and was ineffective in clinical trials [[xii]]. For this reason, it has been proposed to improve the efficiency of tumor-selective targeting of HSV-tk/GCV by exploiting the tumor-tending properties of bone marrow-derived tumor-infiltrating cells and NSCs [[xiii], [xiv]]. Recently, studies of hAT-MSCs (hAT-MSCs-tk) with similar tumorigenic ability and expressing HSV-TK in combination with GCV for the treatment of human gliomas (8 MG-BA, 42 MG-BA and U-118 MG) have shown that the biological properties such as morphology, proliferation, immunophenotype and differentiation potential of hAT-MSCs-tk were unchanged after retroviral genetic modifications The GCV dose-dependent cytotoxic effect was observed in all three glioblastoma cell lines, especially in 8-MG-BA [7]. hAT-MSCs-tk can exert bystander killing effects on glioma cells through intercellular junctions, as well as on proliferating hAT-MSCs-tk itself through GCV transformation-mediated suicide effects. thus effectively killing tumor cells while avoiding the potential tumor growth-promoting effect of MSCs proliferation. In addition, the bystander effect induced by hAT-MSCs-tk also overcomes the titration of HSV-tk-mediated suicide effect by U-118 MG, suggesting that tumor cells resistant to the suicide effect can also be targeted by cellular vectors capable of producing and releasing toxic intermediates [7]. Although systemic injection of hAT-MSCs-tk during the exponential growth phase of the tumor resulted in in vivo tumor shrinkage, hAT-MSCs-tk injected via the tail vein during the tumor growth arrest phase or a later period failed to inhibit tumor growth, which may be related to the failure of hAT-MSCs-tk to integrate with tumor cells or insufficient number of integrated cells. hAT-MSCs-tk was also confirmed by the undetectable thymidine kinase gene in xenograft tumors after injection and before GCV treatment [7]. This suggests that the low proportion of MSCs effectively targeting gliomas after systemic injection may be one of the mechanisms for its failure to effectively inhibit tumor growth. 2.2 hAT-MSCs combined with CDy::UPRT/5-FC 5-Fluorouracil (5-FU) is a chemotherapeutic agent with a broad anti-tumor spectrum, but its wide application is limited by factors such as severe side effects and the need for high drug concentrations to work effectively [[xv]]. Since yeast cytosine deaminase (CDy) is capable of converting the relatively nontoxic 5-FC to the cytotoxic 5-FU with 15 times the efficiency of bacterial CD, the severe systemic toxicity of 5-FU can be overcome by genetically targeting the enzyme/prodrug Cdy/5-FC [[xvi]]. Furthermore, cells expressing the CDy::UPRT dual-energy fusion gene are 10,000-fold more susceptible to 5-FC [[xvii]], thereby significantly increasing the direct killing effect and bystander effect on tumor cells. Thus, the CDy::UPRT/5-FC system becomes ideal for effective 5-FU antitumor activity and avoiding systemic toxic reactions. Studies of hAT-MSCs expressing the suicide gene CDy::UPRT after retroviral transfection (hAT-MSCs-CDy) for the treatment of glioma (C6) showed that hAT-MSCs-CDy in the absence of 5-FC presence did not exert any cytotoxic effect on C6 glioma cells, but hAT-MSCs-CDy in combination with 5-FC could kill C6 glioma cells and even subpopulations of C6 cells that are resistant to 5-FU. After injection into the contralateral hemisphere of the tumor, superparamagnetic iron oxide nanoparticle-labeled hAT-MSCs-CDy could migrate and infiltrate into the interior of the tumor, effectively inhibiting tumor growth. In some of these rats, the tumors disappeared, survived for more than 90 days and showed weight gain, suggesting that hAT-MSCs-Cdy/5-FC could cure gliomas. Further studies showed that the progression-free survival of rats under the condition of daily intraperitoneal injection of 500 mg/kg 5-FC had a dose-dependent effect with the hAT-MSCs-CDy used; the survival time was further prolonged by continuous intracerebroventricular infusion of 5-FC and repeated injections of hAT-MSCs-CDy. hAT-MSCs-CDy-targeted treatment of glioma by a mechanism This may be related to the pericyte-like properties of hAT-MSCs: hAT-MSCs target both tumor vascular endothelial cells and peritumor cells, exerting a synergistic inhibitory effect on tumor angiogenesis and tumor growth, and also killing glioblastoma stem cells located in perivascular tumor nests [[xviii]]. Thus, hAT-MSCs-targeted tumor prodrug suicide gene therapy is a promising approach for the application in the treatment of glioblastoma. 2.3 hAT-MSCs in combination with rCE / CPT-11 CPT-11 is a non-biologically active antitumor prodrug that can be converted into a biologically active and potent topoisomerase I inhibitor SN-38 (7-ethyl-10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin) in the presence of CE, which has a with significant cytotoxicity [[xix]]. Injection of nuclear transfected hAT-MSCs expressing rCE (hAT-MSCs-rCE) into gliomas (F98), which could be distributed in the tumor bed and at the interface between tumor and normal brain parenchyma, converted intravenous CPT-11 into SN-38, effectively inhibited glioma growth and significantly prolonged median survival in rats with tumors, but had no significant effect on normal brain tissue. Although the prolongation of survival time was statistically significant, the actual prolongation time (only 5 days) was lower than expected. This may be related to the fact that the CPT-11 used was not the optimal concentration or that the single drug application was less effective in brainstem gliomas [10]. 3. hAT-MSCs and lysing viruses In recent years, lysing viruses have become a new research hotspot in the treatment of malignant brain tumors. Myxoma virus (vMyx) is a novel lysing virus with a broad antitumor spectrum, which has a relatively selective killing effect on tumor cells and avoids damage to normal non-transformed cells. vMyx is a rabbit-specific virus that is not pathogenic to vertebrates, including humans, who lack acquired immunity to such viruses. vMyx has a narrow host range and cannot effectively infect normal non-rabbit cells. It cannot effectively infect normal non-rabbit cells (e.g., untransformed brain cells), but is able to infect and kill human glioblastomas. However, its efficacy is limited to intratumoral injection sites and does not infect and kill glioma cells that infiltrate distantly [[xx], [xxi]]. Recent studies have found that vMyx can effectively infect and replicate within hAT-MSCs, with no significant effect on the activity and tumorigenic capacity of hAT-MSCs. hAT-MSCs expressing vMyx (hAT-MSCs-vMyx) were able to release toxicity into malignant glioma cells, resulting in nuclear sequestration (cell death), inhibition of tumor growth, and prolonged survival in tumor-bearing mice. In addition, multiple injections of hAT-MSCs-vMyx further improved the efficacy and prolonged the survival of tumor-bearing mice, with approximately 20% of mice achieving disease-free survival [8]. Therefore, hAT-MSCs can be used as an effective vehicle for vMyx treatment of brain tumors. 4. hAT-MSCs and tumor necrosis factor- related apoptosis-inducing ligand (TRAIL) TRAIL is a member of the tumor necrosis factor protein family, which can selectively induce apoptosis in tumor cells without causing normal cell TRAIL activates an extrinsic p53-independent pathway by binding to two cell surface death receptors (DR), DR4 (TRAIL-R1) and DR5 (TRAIL-R2/KILLER) [[xxii]]. The TRAIL secretion level of hAT-MSCs after nuclear transfection was cell number-dependent and could be widely distributed in the tumor bed and its interface with normal brain tissue after inoculation into tumors, resulting in a 56.3% reduction in tumor volume, a 3.03-fold increase in the number of apoptotic tumor cells, and a more than 3-fold increase in the survival of hormonal mice without any damage to normal brain parenchyma. In long-term surviving hAT-MSCs, some hAT-MSCs began to express markers of neural differentiation but not mesenchymal cell markers [5], suggesting that non-viral vector-transfected hAT-MSCs are safe and effective for the treatment of brainstem gliomas. 5. Summary hAT-MSCs have tumorigenic effects similar to those of NSCs and BM-MSCs, and have the advantages of being easy to obtain and isolate, high content, easy genetic modification, and autologous transplantation. Although the tumorigenic ability of hAT-MSCs was significantly reduced or even lost after adenovirus transfection, the tumorigenic ability of hAT-MSCs did not change significantly after retrovirus, mucosal tumor virus and nuclear transfection. The untransfected hAT-MSCs had no significant effect on the growth of gliomas and the median survival of tumor-bearing mice, suggesting that hAT-MSCs have a good safety profile as cellular vectors for oncogene therapy. The modified hAT-MSCs carrying HSV-tk, CDy::UPRT and rCE genes combined with GCV, 5-FC and CPT-11 antitumor prodrugs could exert effective anti-glioma effects, and hAT-MSCs carrying lysozyme virus and TRAIL could also exert good anti-tumor effects. Thus, hAT-MSCs are promising for gene targeting therapy of glioma, and are expected to be a new strategy to completely eliminate glioma cells and effectively solve the problem of glioma recurrence.