Encapsulation of temozolomide in a tumor-targeting nanocomplex enhances anti-cancer efficacy and reduces toxicity in a mouse model of glioblastoma.
ABSTRACT: Although temozolomide (TMZ) is the current first-line chemotherapy for glioblastoma multiforme (GBM), most patients either do not respond or ultimately fail TMZ treatment. Both intrinsic tumor resistance and limited access of TMZ to brain tumors as a result of the blood-brain barrier (BBB) contribute to poor response and ultimately to poor prognosis for GBM patients. We have developed a "dual-targeting" nanomedicine that both actively crosses the BBB and actively targets cancer cells once in the brain parenchyma. This nanomedicine (termed scL-TMZ) is sized ~40?nm and comprised of a cationic liposome (DOTAP:DOPE) encapsulating TMZ. The surface of liposome is decorated with anti-transferrin receptor single-chain antibody fragments to facilitate the crossing of the BBB by the scL-TMZ in addition to targeting GBM in the brain. This novel formulation was found to be markedly more effective than standard TMZ in both TMZ-resistant and TMZ-sensitive GBM. Encapsulation of TMZ also markedly enhanced its efficacy in killing a variety of non-GBM tumor cells. The scL-TMZ nanocomplex was shown to target cancer stem cells, which have been linked to both drug resistance and recurrence in GBM. Most significantly, systemically administered scL-TMZ significantly prolonged survival in mice bearing intracranial GBM tumors. The improved efficacy of scL-TMZ compared to standard TMZ was accompanied by reduced toxicity, so we conclude that the scL-TMZ nanomedicine holds great promise as a more effective therapy for GBM and other tumor types.
Project description:Temozolomide (TMZ)-resistance in glioblastoma multiforme (GBM) has been linked to upregulation of O(6)-methylguanine-DNA methyltransferase (MGMT). Wild-type (wt) p53 was previously shown to down-modulate MGMT. However, p53 therapy for GBM is limited by lack of efficient delivery across the blood brain barrier (BBB). We have developed a systemic nanodelivery platform (scL) for tumor-specific targeting (primary and metastatic), which is currently in multiple clinical trials. This self-assembling nanocomplex is formed by simple mixing of the components in a defined order and a specific ratio. Here, we demonstrate that scL crosses the BBB and efficiently targets GBM, as well as cancer stem cells (CSCs), which have been implicated in recurrence and treatment resistance in many human cancers. Moreover, systemic delivery of scL-p53 down-modulates MGMT and induces apoptosis in intracranial GBM xenografts. The combination of scL-p53 and TMZ increased the antitumor efficacy of TMZ with enhanced survival benefit in a mouse model of highly TMZ-resistant GBM. scL-p53 also sensitized both CSCs and bulk tumor cells to TMZ, increasing apoptosis. These results suggest that combining scL-p53 with standard TMZ treatment could be a more effective therapy for GBM.
Project description:Intrinsic therapeutic resistance especially in cancer stem cells (CSCs) together with extensive tumor cell infiltration and restricted permeation of the blood-brain barrier (BBB) by drugs may all contribute to the treatment failure in patients with glioblastoma multiforme (GBM). Accumulating evidence suggests that long non-coding RNA (lncRNA), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays a role in tumor cell infiltration and therapeutic resistance of GBM. Using our tumor-targeted nanocomplex, we have modulated the expression of MALAT1 and investigated its impact on GBM cells. Importantly, our nanocomplex is able to target CSCs that are considered to be the prime culprits in therapeutic resistance and recurrence of GBM. Attenuation of MALAT1 by RNA interference significantly lowered the growth, motility and stemness of GBM cells. In addition, silencing of MALAT1 clearly improved the sensitivity of GBM cells to chemotherapeutic agents including the current first-line therapy of GBM [temozolomide (TMZ)]. In animal models of GBM, tumor involution with a modest but statistically significant survival benefit was achieved with concurrent treatment of TMZ and nanocomplex-mediated silencing of MALAT1. These results suggest that combining standard TMZ treatment with lncRNA-targeting therapies using our nanocomplex could substantially enhance the very poor prognosis for GBM patients.
Project description:The purpose of this study is to assess the preclinical therapeutic efficacy of magnetic resonance imaging (MRI)-monitored focused ultrasound (FUS)-induced blood-brain barrier (BBB) disruption to enhance Temozolomide (TMZ) delivery for improving Glioblastoma Multiforme (GBM) treatment. MRI-monitored FUS with microbubbles was used to transcranially disrupt the BBB in brains of Fisher rats implanted with 9L glioma cells. FUS-BBB opening was spectrophotometrically determined by leakage of dyes into the brain, and TMZ was quantitated in cerebrospinal fluid (CSF) and plasma by LC-MS\MS. The effects of treatment on tumor progression (by MRI), animal survival and brain tissue histology were investigated. Results demonstrated that FUS-BBB opening increased the local accumulation of dyes in brain parenchyma by 3.8-/2.1-fold in normal/tumor tissues. Compared to TMZ alone, combined FUS treatment increased the TMZ CSF/plasma ratio from 22.7% to 38.6%, reduced the 7-day tumor progression ratio from 24.03 to 5.06, and extended the median survival from 20 to 23 days. In conclusion, this study provided preclinical evidence that FUS BBB-opening increased the local concentration of TMZ to improve the control of tumor progression and animal survival, suggesting its clinical potential for improving current brain tumor treatment.
Project description:Glioblastoma (GBM) is a primary malignant brain tumor with a dismal prognosis, partially due to our inability to completely remove and kill all GBM cells. Rapid tumor recurrence contributes to a median survival of only 15 months with the current standard of care which includes maximal surgical resection, radiation, and temozolomide (TMZ), a blood-brain barrier (BBB) penetrant chemotherapy. Radiation and TMZ cause sphingomyelinases (SMase) to hydrolyze sphingomyelins to generate ceramides, which induce apoptosis. However, cells can evade apoptosis by converting ceramides to sphingosine-1-phosphate (S1P). S1P has been implicated in a wide range of cancers including GBM. Upregulation of S1P has been linked to the proliferation and invasion of GBM and other cancers that display a propensity for brain metastasis. To mediate their biological effects, SMases and S1P modulate signaling via phospholipase C (PLC) and phospholipase D (PLD). In addition, both SMase and S1P may alter the integrity of the BBB leading to infiltration of tumor-promoting immune populations. SMase activity has been associated with tumor evasion of the immune system, while S1P creates a gradient for trafficking of innate and adaptive immune cells. This review will explore the role of sphingolipid metabolism and pharmacological interventions in GBM and metastatic brain tumors with a focus on SMase and S1P.
Project description:Introduction: To overcome the blood-brain barrier (BBB) which interferes with the effect of chemotherapeutic agents, we performed multiple disruptions of BBB (BBBD) with magnetic resonance-guided focused ultrasound on patients with glioblastoma (GBM) during standard adjuvant temozolomide (TMZ) chemotherapy [clinical trial registration no.NCT03712293 (clinicaltrials.gov)]. We report a 1-year follow-up result of BBBD with TMZ for GBM. Methods: From September 2018 to January 2019, six patients were enrolled (four men and two women, median age: 53 years, range: 50-67 years). Of the six patients, five underwent a total of six cycles of BBBD during standard TMZ adjuvant therapy. One patient underwent three cycles of BBBD but continued with TMZ chemotherapy. The 1-year follow-up results of these six patients were reviewed. Results: The mean follow-up duration was 15.17 ± 1.72 months. Two patients showed a recurrence of tumor at 11 and 16 months, respectively. One underwent surgery, and the other patient was restarted with TMZ chemotherapy due to the tumor location with a highly possibility of surgical complications. The survival rate up to 1 year was 100%, and the other four patients are on observation without recurrence. None of the six patients had immediate or delayed BBBD-related complications. Conclusion: Multiple BBBDs can be regarded as a safe procedure without long-term complications, and it seems to have some survival benefits. However, since TMZ partially crosses the BBB, a further extended study with large numbers would be needed to evaluate the benefits of BBBD resulting in an increase of TMZ concentration. This study opened a new therapeutic strategy for GBM by combining BBBD with a larger molecular agent.
Project description:Introduction:Glioblastoma (GBM) is the most common and lethal of the central nervous system (CNS) malignancies. The initiation, progression, and infiltration ability of GBMs are attributed in part to the dysregulation of microRNAs (miRNAs). Thus, targeting dysregulated miRNAs with RNA oligonucleotides (RNA interference, RNAi) has been proposed for GBM treatment. Despite promising results in the laboratory, RNA oligonucleotides have clinical limitations that include poor RNA stability and off-target effects. RNAi therapies against GBM confront an additional obstacle, as they need to cross the blood-brain barrier (BBB). Methods:Here, we developed gold-liposome nanoparticles conjugated with the brain targeting peptides apolipoprotein E (ApoE) and rabies virus glycoprotein (RVG). First, we functionalized gold nanoparticles with oligonucleotide miRNA inhibitors (OMIs), creating spherical nucleic acids (SNAs). Next, we encapsulated SNAs into ApoE, or RVG-conjugated liposomes, to obtain SNA-Liposome-ApoE and SNA-Liposome-RVG, respectively. We characterized each nanoparticle in terms of their size, charge, encapsulation efficiency, and delivery efficiency into U87 GBM cells in vitro. Then, they were administered intravenously (iv) in GBM syngeneic mice to evaluate their delivery efficiency to brain tumor tissue. Results:SNA-Liposomes of about 30-50 nm in diameter internalized U87 GBM cells and inhibited the expression of miRNA-92b, an aberrantly overexpressed miRNA in GBM cell lines and GBM tumors. Conjugating SNA-Liposomes with ApoE or RVG peptides increased their systemic delivery to the brain tumors of GBM syngeneic mice. SNA-Liposome-ApoE demonstrated to accumulate at higher extension in brain tumor tissues, when compared with non-treated controls, SNA-Liposomes, or SNA-Liposome-RVG. Discussion:SNA-Liposome-ApoE has the potential to advance the translation of miRNA-based therapies for GBM as well as other CNS disorders.
Project description:Glioblastoma (GBM) is amongst the most aggressive brain tumors with a dismal prognosis. Despite significant advances in the current multimodality therapy including surgery, postoperative radiotherapy (RT) and temozolomide (TMZ)-based concomitant and adjuvant chemotherapy (CT), tumor recurrence is nearly universal with poor patient outcomes. These limitations are in part due to poor drug penetration through the blood-brain barrier (BBB) and resistance to CT and RT by a small population of cancer cells recognized as tumor-initiating cells or cancer stem cells (CSCs). Though CT and RT kill the bulk of the tumor cells, they fail to affect CSCs, resulting in their enrichment and their development into more refractory tumors. Therefore, identifying the mechanisms of resistance and developing therapies that specifically target CSCs can improve response, prevent the development of refractory tumors and increase overall survival of GBM patients. Small molecule inhibitors that can breach the BBB and selectively target CSCs are emerging. In this review, we have summarized the recent advancements in understanding the GBM CSC-specific signaling pathways, the CSC-tumor microenvironment niche that contributes to CT and RT resistance and the use of novel combination therapies of small molecule inhibitors that may be used in conjunction with TMZ-based chemoradiation for effective management of GBM.
Project description:BACKGROUND:Temozolomide (TMZ) is the most commonly used chemotherapeutic agent used to treat glioblastoma (GBM), which causes significant DNA damage to highly proliferative cells. Our observations have added to accumulating evidence that TMZ induces stress-responsive cellular programs known to promote cell survival, including autophagy. As such, targeting these survival pathways may represent new vulnerabilities of GBM after treatment with TMZ. METHODS:Using the T98G human glioma cell line, we assessed the molecular signaling associated with TMZ treatment, the cellular consequences of using the pan-PI3K inhibitor PX-866, and performed clonogenic assays to determine the effect sequential treatment of TMZ and PX-866 had on colony formation. Additionally, we also use subcutaneous GBM patient derived xenograft (PDX) tumors to show relative LC3 protein expression and correlations between survival pathways and molecular markers which dictate clinical responsiveness to TMZ. RESULTS:Here, we report that TMZ can induce autophagic flux in T98G glioma cells. GBM patient-derived xenograft (PDX) tumors treated with TMZ also display an increase in the autophagosome marker LC3 II. Additionally, O6-methylguanine-DNA-methyltransferase (MGMT) expression correlates with PI3K/AKT activity, suggesting that patients with inherent resistance to TMZ (MGMT-high) would benefit from PI3K/AKT inhibitors in addition to TMZ. Accordingly, we have identified that the blood-brain barrier (BBB) penetrant pan-PI3K inhibitor, PX-866, is an early-stage inhibitor of autophagic flux, while maintaining its ability to inhibit PI3K/AKT signaling in glioma cells. Lastly, due to the induction of autophagic flux by TMZ, we provide evidence for sequential treatment of TMZ followed by PX-866, rather than combined co-treatment, as a means to shut down autophagy-induced survival in GBM cells and to enhance apoptosis. CONCLUSIONS:The understanding of how TMZ induces survival pathways, such as autophagy, may offer new therapeutic vulnerabilities and opportunities to use sequential inhibition of alternate pro-survival pathways that regulate autophagy. As such, identification of additional ways to inhibit TMZ-induced autophagy could enhance the efficacy of TMZ.
Project description:Glioblastoma multiforme (GBM) is the most aggressive and lethal type of brain tumor. Both therapeutic resistance and restricted permeation of drugs across the blood-brain barrier (BBB) play a major role in the poor prognosis of GBM patients. Accumulated evidence suggests that in many human cancers, including GBM, therapeutic resistance can be attributed to a small fraction of cancer cells known as cancer stem cells (CSCs). CSCs have been shown to have stem cell-like properties that enable them to evade traditional cytotoxic therapies, and so new CSC-directed anti-cancer therapies are needed. Nanoparticles have been designed to selectively deliver payloads to relevant target cells in the body, and there is considerable interest in the use of nanoparticles for CSC-directed anti-cancer therapies. Recent advances in the field of nanomedicine offer new possibilities for overcoming CSC-mediated therapeutic resistance and thus significantly improving management of GBM. In this review, we will examine the current nanomedicine approaches for targeting CSCs and their therapeutic implications. The inhibitory effect of various nanoparticle-based drug delivery system towards CSCs in GBM tumors is the primary focus of this review.
Project description: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. <i>In vivo</i> 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 <i>in vitro</i>. 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.