Blood-tumor Barrier Permeability Research Articles

FXR1 stabilizes SNORD63 to regulate blood-tumor barrier permeability through SNORD63 mediated 2’-O-methylation of POU6F1

How selectively increase blood-tumor barrier (BTB) permeability is crucial to enhance the delivery of chemotherapeutic agents to brain tumor tissues. In this study, we established in vitro models of the blood-brain barrier (BBB) and BTB using endothelial cells (ECs) co-cultured with human astrocytes (AECs) and glioma cells (GECs), respectively. The findings revealed high expressions of the RNA-binding protein FXR1 and SNORD63 in GECs, where FXR1 was found to bind and stabilize SNORD63. Knockdown of FXR1 resulted in decreased expression of tight-junction-related proteins and increased BTB permeability by down-regulating SNORD63. SNORD63 played a role in mediating the 2’-O-methylation modification of POU6F1 mRNA, leading to the downregulation of POU6F1 protein expression. POU6F1 showed low expression in GECs and acted as a transcription factor to regulate BTB permeability by binding to the promoter regions of ZO-1, occludin, and claudin-5 mRNAs and negatively regulating their expressions. Finally, the targeted regulation of FXR1, SNORD63, and POU6F1 expressions, individually or in combination, effectively enhanced doxorubicin passage through the BTB and induced apoptosis in glioma cells. This study aims to elucidate the underlying mechanism of the FXR1/SNORD63/POU6F1 axis in regulating BTB permeability, offering a novel strategy to improve the efficacy of glioma chemotherapy.

TMIC-55. TURNING ENEMY INTO ALLY: HIJACK BRAIN TUMOR CELLS TO BREAK DOWN THE BLOOD-TUMOR BARRIER

Abstract A major obstacle in brain cancer treatment is the blood-tumor barrier (BTB), which limits the delivery of a vast majority of therapeutic agents. Medulloblastoma (MB), the most common pediatric malignant brain tumor, consists of four subgroups (WNT, SHH, Group 3, Group 4). Increasing BTB permeability of non-WNT MB is essential for effectively treating patients by overcoming this drug delivery impediment. Since the BTB is exposed to biofluids such as blood and brain interstitial fluid, which present dynamic osmotic challenges, we hypothesized that cell volume regulation plays a crucial role in BTB integrity. Using a genetically engineered mouse model of SHH MB, we show that tumor-specific knockout of volume-regulated ion channel, Lrrc8a, significantly increases BTB permeability and enhances the therapeutic response of MB-bearing mice to vismodegib, an FDA-approved antagonist of the SHH pathway. Lrrc8a-deficient tumor cells develop ectopic contact with blood vessels, permitting tumor cell-derived Tgfβ2 to activate Tgfβ signaling in endothelial cells. Consequently, endothelial cells in Lrrc8a knockout MB lose endothelial characteristics and gain mesenchymal features, indicative of Tgfβ signaling-induced endothelial-mesenchymal transition (EndMT). Furthermore, tumor-specific LRRC8A knockdown enhances BTB permeability in a xenograft human Group 3 MB model. Collectively, our findings establish Lrrc8a/LRRC8A as a therapeutic target to increase the therapy response of MB and provide evidence that inducing EndMT could be a strategy for enhancing BTB permeability to treat brain cancer.

BSBM-11 COMBINING IMMUNOTHERAPY AND WHOLE-BRAIN RADIATION THERAPY IN A NOVEL SYNGENEIC LUNG CANCER BRAIN METASTASIS PRECLINICAL MODEL: DOES TIMING OF ADMINISTRATION MATTER?

Abstract Approximately 50% of all brain metastasis cases originate from primary lung tumors. Whole-brain radiation therapy (WBRT) is a common treatment for patients with multiple brain metastases, but patients rarely survive longer than one year after diagnosis. Recent studies demonstrate improved outcomes with combinatorial radiotherapy and immunotherapy, but the optimal timing of immunotherapy administration is unclear. Eliciting a robust immune response post-immunotherapy is vital for therapeutic efficacy. Our work shows that the BBB is disrupted and proinflammatory cytokines are increased in the brain 12 hours post-WBRT in immunocompetent but not immunocompromised mice. Additionally, efflux transporter activity is decreased at this time point. This study utilizes a novel Lewis Lung Carcinoma cell line, LLC-Br, which was developed in our laboratory to preferentially metastasize to the brain. Mice were treated with immune checkpoint inhibitors (ICIs) before or after WBRT then we evaluated tumor progression, survival, and blood-tumor barrier (BTB) permeability. We hypothesized administration of ICI following WBRT would increase survival, decrease tumor burden, and increase BTB permeability in our preclinical syngeneic lung cancer brain metastasis model.

Abstract 2713: Modulation of the blood-tumor barrier by 6-bromoindirubin acetoxime increases intra-tumoral drug accumulation and extends survival in murine glioblastoma models

Abstract Introduction: Glioblastoma (GBM) is the most frequent primary malignant adult brain tumor. Median survival is ~15 months after diagnosis. Therapies to effectively manage glioblastoma are hindered due to the presence of the blood-tumor barrier (BTB). Indirubins are 3,2’-bisindole compounds of the Indigo naturalis used in traditional Chinese medicine to treat proliferative diseases. Herein, we demonstrate that systemic delivery of 6-bromoindirubin-3’-acetoxime (BIA), an indirubin derivative, increases cisplatin delivery to GBM tumors through alteration of the BTB integrity and synergy of BIA with platinum therapeutics to potentiate anti-tumor killing. Methods: Pre-clinical studies involved G9-pCDH glioblastoma cell line implantation in naïve nude mice. Cisplatin uptake 24 hours after BIA tail-vein administration into healthy brain and GBM murine tumors was assessed via inductively-coupled plasma mass spectrometry (ICP-MS). DNA-damage was assessed by γH2AX immunofluorescence. BIA combination with cisplatin in vitro studies were performed via luminescent viability assays. Treatment of human cerebrovascular endothelial cells (HCMEC/D3) with BIA for BBB studies in vitro involved trans-endothelial electrical resistance (TEER) measurement, transcriptome analysis, VE-cadherin western blotting and immunofluorescence. Results: Combined addition of cisplatin and BIA showed enhanced anti-GBM killing effects than cisplatin alone in G9-pCDH (IC50 cisplatin=1, BIA+cisplatin=0.2), G34-pCDH (IC50 cisplatin=3, BIA+cisplatin=1.7) and G30-LRP (IC50 cisplatin=1, BIA+cisplatin=0.3) lines. Efficacy studies using the G9-pCDH xenograft murine model showed extended survival when using cisplatin in combination with BIA (median 27d, p=0.0016) when compared to cisplatin (18.5d), BIA (21.5d) and control (18d). Increased intra-tumoral accumulation of cisplatin was observed when used in combination with BIA (mean 100 pg/mg, p=0.012) rather than cisplatin alone (5 pg/mg). No increased platinum uptake was seen in healthy brain tissue under any conditions. γH2AX foci in tumor were more frequent in the BIA and cisplatin combination arm (p=0.0086) than each single agent and control groups. Evaluation of HCMEC/D3 cells treated with BIA showed a decrease in TEER levels (p<0.001) and transcriptomic dysregulation of members of the WNT pathway and downregulation of VE-cadherin (~4-fold). BIA exerted cytoskeleton changes in HCMEC/D3 cells and decreased VE-cadherin expression at the protein level and its distribution on cellular surface, suggesting loss of adherens junctions as a potential mechanism for BIA-induced permeability of the BTB. Conclusion: Overall, our data support the applicability of BIA for modulating the BTB permeability and increasing intra-tumoral drug accumulation for improving GBM therapeutic efficacy. Citation Format: Jorge L. Jimenez Macias. Modulation of the blood-tumor barrier by 6-bromoindirubin acetoxime increases intra-tumoral drug accumulation and extends survival in murine glioblastoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2713.

Transcription factor ZNF22 regulates blood-tumor barrier permeability by interacting with HDAC3 protein.

The primary goals of this study were to investigate the potential roles of ZNF22 and HDAC3 as a histone deacetylase in regulating an increases in blood-tumor barrier (BTB) permeability and some of the possible molecular mechanisms associated with this effect. The expression of ZNF22 and HDAC3 in glioma-exposed endothelial cells (GECs) of BTB were detected transcription real-time PCR or western blot. The interaction of ZNF22 and HDAC3 in GECs associated with transcript effect was analyzed by means of Co-Immunoprecipitation and luciferase reporter assay. In the present investigation, GECs expressed higher levels of ZNF22 as a zinc finger transcription factor and HDAC3 than endothelial cells. We then affirmed that silencing HDAC3 or ZNF22 led to a reduction in BTB permeability. By bioinformatics analysis, chromatin immunoprecipitation (ChIP) assays and luciferase assay, we found that ZNF22 had a target binding relationship with the promoter regions of ZO-1, Occludin, and Claudin-5 and negatively regulated the expression of ZO-1, Occludin, and Claudin-5. Furthermore, we revealed that HDAC3, as a co-transcript repressor with histone deacetylase activity, could interact with ZNF22 to hinder the expression of TJ-associated proteins, thereby further facilitating the permeability of BTB. ZNF22 acted as a transcription factor in conjunction with HDAC3 to modulate the expression of TJ-associated proteins, which was correlated with an increase in BTB permeability. These results may provide new strategies and targets for the chemotherapy of gliomas as well as intracranial infections.

CPEB2 m6A methylation regulates blood–tumor barrier permeability by regulating splicing factor SRSF5 stability

The blood–tumor barrier (BTB) contributes to poor therapeutic efficacy by limiting drug uptake; therefore, elevating BTB permeability is essential for glioma treatment. Here, we prepared astrocyte microvascular endothelial cells (ECs) and glioma microvascular ECs (GECs) as in vitro blood–brain barrier (BBB) and BTB models. Upregulation of METTL3 and IGF2BP3 in GECs increased the stability of CPEB2 mRNA through its m6A methylation. CPEB2 bound to and increased SRSF5 mRNA stability, which promoted the ETS1 exon inclusion. P51-ETS1 promoted the expression of ZO-1, occludin, and claudin-5 transcriptionally, thus regulating BTB permeability. Subsequent in vivo knockdown of these molecules in glioblastoma xenograft mice elevated BTB permeability, promoted doxorubicin penetration, and improved glioma-specific chemotherapeutic effects. These results provide a theoretical and experimental basis for epigenetic regulation of the BTB, as well as insight into comprehensive glioma treatment.

Abstract 3461: Focused ultrasound induced blood-tumor barrier permeability of combinatorial chemotherapy

Abstract Background: Incidence of patients having brain metastasis of breast cancer has increased due to enhanced treatment for peripheral disease. Breast cancer brain metastasis is often fatal and occurs in 15-20% of patients diagnosed with stage IV breast cancer. Traditional chemotherapy delivery to the brain is limited by the blood-brain barrier (BBB) and blood-tumor barrier (BTB). Low intensity focused ultrasound (LIFU) is a unique technique recently investigated to non-invasively disrupt these barriers and aid neuro-modulation. Herein, we demonstrate the use of a clinical LIFU device to modulate blood-tumor permeability and deliver combined chemotherapy in a novel preclinical model of brain metastases of breast cancer. Methods: Brain colonizing MDA-MB-231Br cells transfected with luciferase were injected into athymic Nu/Nu female mice by intracardiac injection into the left ventricle (per mouse 175,000 cells). Brain metastasis was allowed to develop, and tumor burden was monitored by bioluminescent imaging IVIS Spectrum CT (Perkin Elmer). Mice were randomized into sham or single treatment or combined treatment groups on Day 21 (Doxil nanoparticles 5.6 mg/kg or Paclitaxel 10mg/kg). Mice receiving LIFU were placed on a specially designed animal restraint with the skull partially immersed in degassed water. T1 weighted MRI was used to identify and localize sonication spots within the cortex, and mice were sonicated using 0.4uL/kg Definity© at 0.3 cavitation dose for 60sec. Treatment with LIFU was continued once weekly for three consecutive weeks. Survival and tumor burden was recorded until mice developed neurological symptoms. LIFU induced blood-brain barrier breakdown in healthy mice was also analyzed by in-situ brain perfusion with 14CSucrose, 3HGlucose, and 3HIvermectin to monitor BBB integrity and transporter function. Results: Mice receiving LIFU+paclitaxel+Doxil-NP showed significantly lower tumor burden (peak area 635.9) and higher median survival of 60 days as compared to vehicle (29d) and LIFU+vehicle (32d) groups. Analysis of BBB permeability in healthy mice demonstrated significantly higher passive permeability marker accumulation than non-sonicated contralateral hemisphere. Conclusion: Timing the combined chemotherapy with LIFU induced windows of maximal BBB opening resulted in slower tumor progression and increased survival in a preclinical model of breast cancer brain metastasis. Future directions aim to combine these results and evaluate underlying mechanisms of enhanced permeability post-LIFU. Citation Format: Tasneem A. Arsiwala, Kathryn E. Blethen, Samuel A. Sprowls, Ross A. Fladeland, Brooke Keilkowski, Morgan Glass, Trenton Pritt, Peng Wang, Ali Rezai, Paul R. Lockman. Focused ultrasound induced blood-tumor barrier permeability of combinatorial chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3461.

Adenosine A2A Receptor Activation Enhances Blood-Tumor Barrier Permeability in a Rodent Glioma Model.

The blood-tumor barrier (BTB) limits the entry of effective chemotherapeutic agents into the brain for treatment of malignant tumors like glioblastoma. Poor drug entry across the BTB allows infiltrative glioma stem cells to evade therapy and develop treatment resistance. Regadenoson, an FDA-approved adenosine A2A receptor (A2AR) agonist, has been shown to increase drug delivery across the blood-brain barrier in non-tumor-bearing rodents without a defined mechanism of enhancing BTB permeability. Here, we characterize the time-dependent impact of regadenoson on brain endothelial cell interactions and paracellular transport, using mouse and rat brain endothelial cells and tumor models. In vitro, A2AR activation leads to disorganization of cytoskeletal actin filaments by 30 minutes, downregulation of junctional protein expression by 4 hours, and reestablishment of endothelial cell integrity by 8 hours. In rats bearing intracranial gliomas, regadenoson treatment results in increase of intratumoral temozolomide concentrations, yet no increased survival noted with combined temozolomide therapy. These findings demonstrate regadenoson's ability to induce brain endothelial structural changes among glioma to increase BTB permeability. The use of vasoactive mediators, like regadenoson, which transiently influences paracellular transport, should further be explored to evaluate their potential to enhance central nervous system treatment delivery to aggressive brain tumors. IMPLICATIONS: This study provides insight on the use of a vasoactive agent to increase exposure of the BTB to chemotherapy with intention to improve glioma treatment efficacy.

BIOL-01. THE RELATIONSHIP OF BRAIN ENDOTHELIAL WNT SIGNAL INHIBITION ON BLOOD-TUMOR BARRIER INTEGRITY

The blood-tumor barrier (BTB) is the primary site of nutrient and drug transport to tumor cells such as malignant gliomas. Yet, signaling pathways and factors influencing BTB permeability are poorly understood. Previous studies demonstrate the role of WNT/β-catenin signaling in establishing and fortifying blood-brain barrier integrity in a non-diseased state. Additionally, WNT proteins are highly expressed in gliomas and their surrounding vasculature. Thus, we propose inhibition of WNT/β-catenin signaling at the brain endothelium of malignant glioma can impair BTB integrity to enhance permeability for select cytotoxic agents. We used immortalized mouse brain endothelial cells (bEnd.3), akin to brain tumor endothelium, treated for 24 hours with WNT inhibitors (ICG-001, IWR-1, and LGK974). Inhibition of WNT/β-catenin signaling was confirmed by gene expression of transcription factors (Tcf4 and Birc5). Cell viability was confirmed by CellTiter Glo®. Brain endothelial cell-cell interaction was evaluated by cell impedance and resistance via the Agilent xCELLigence and ABP TEER24 systems. Using qPCR and flow cytometry, we observed changes in expression and function of Abcb1 and Abcg2 transporters. Using an in vitro BTB (bEnd.3 cells and mouse H3.3WT/K27 glioma cells) we evaluated the effect of WNT inhibition on permeability and glioma viability. We found that all the inhibitors downregulated Tcf4 and Birc5 in brain endothelium dose-dependently. Viability with inhibitors demonstrated an IC50 of 28μM for ICG-001, and 42μM for both IWR-1 and LGK974. Endothelial cell-cell interaction was transiently decreased by approximately 50% with all inhibitors at 30 minutes; increasing closer to baseline after 2-4hrs. All WNT inhibitors dose-dependently decreased Abcg2 transporter expression and function. While In vitro BTB studies are ongoing, preliminary findings demonstrate increasing permeability of BTB amongst H3.3K27 glioma cells. Our results demonstrate potential of WNT inhibitors to modulate BTB integrity and drug efflux function. More studies are warranted to explore WNT/β-catenin signaling inhibition on BTB in vivo.

Cerebral microcirculation in glioblastoma: A major determinant of diagnosis, resection, and drug delivery.

Glioblastoma (GBM) is the most common primary brain tumor with a dismal prognosis. Current standard of treatment is safe maximal tumor resection followed by chemotherapy and radiation. Altered cerebral microcirculation and elevated blood-tumor barrier (BTB) permeability in tumor periphery due to glioma-induced vascular dysregulation allow T1 contrast-enhanced visualization of resectable tumor boundaries. Newer tracers that label the tumor and its vasculature are being increasingly used for intraoperative delineation of glioma boundaries for even more precise resection. Fluorescent 5-aminolevulinic acid (5-ALA) and indocyanine green (ICG) are examples of such intraoperative tracers. Recently, magnetic resonance imaging (MRI)-based MR thermometry is being employed for laser interstitial thermal therapy (LITT) for glioma debulking. However, aggressive, fatal recurrence always occurs. Postsurgical chemotherapy is hampered by the inability of most drugs to cross the blood-brain barrier (BBB). Understanding postsurgical changes in brain microcirculation and permeability is crucial to improve chemotherapy delivery. It is important to understand whether any microcirculatory indices can differentiate between true recurrence and radiation necrosis. LITT leads to peri-ablation BBB opening that persists for several weeks. Whether it can be a conduit for chemotherapy delivery is yet to be explored. This review will address the role of cerebral microcirculation in such emerging ideas in GBM diagnosis and therapy.

Long non-coding RNA MIAT regulates blood tumor barrier permeability by functioning as a competing endogenous RNA

Blood–tumor barrier (BTB) presents a major obstacle to brain drug delivery. Therefore, it is urgent to enhance BTB permeability for the treatment of glioma. In this study, we demonstrated that MIAT, ZAK, and phosphorylated NFκB-p65 (p-NFκB-p65) were upregulated, while miR-140-3p was downregulated in glioma-exposed endothelial cells (GECs) of BTB compared with those in endothelial cells cocultured with astrocytes (ECs) of blood–brain barrier (BBB). MIAT inhibited miR-140-3p expression, increased the expression of ZAK, enhanced the ratio of p-NFκB-p65:NFκB-p65, and promoted the endothelial leakage of BTB. Our current study revealed that miR-140-3p was complementary to the ZAK 3′untranslated regions (3′-UTR), and luciferase activity of ZAK was inhibited by miR-140-3p in 293T cells. MiR-140-3p silencing resulted in an increase in BTB permeability by targeting ZAK, while overexpression of miR-140-3p had the opposite results in GECs of BTB. Overexpression of ZAK induced an increase in BTB permeability, and this effect was related to ZAK’s ability to mediate phosphorylation of NFκB-p65. Conversely, ZAK silencing get opposite results in GECs of BTB. As a molecular sponge of miR-140-3p, MIAT attenuated its negative regulation of the target gene ZAK by adsorbing miR-140-3p. P-NFκB-p65 as a transcription factor negatively regulated the expression of TJ-associated proteins by means of chip assay and luciferase assay. Single or combined application of MIAT and miR-140-3p effectively promoted antitumor drug doxorubicin (Dox) across BTB to induce apoptosis of glioma cells. In summary, MIAT functioned as a miR-140-3p sponge to regulate the expression of its target gene ZAK, which contribution to phosphorylation of NFκB-p65 was associated with an increase in BTB permeability by down-regulating the expression of TJ associated proteins, thereby promoting Dox delivery across BTB. These results might provide a novel strategy and target for chemotherapy of glioma.

IGF2BP2 stabilized FBXL19-AS1 regulates the blood-tumour barrier permeability by negatively regulating ZNF765 by STAU1-mediated mRNA decay

ABSTRACT Blood-tumour barrier (BTB) has been known to significantly attenuate the efficacy of chemotherapy for glioma. In this report, we identified that insulin-like grown factor 2 mRNA-binding protein 2 (IGF2BP2) was over-expressed in glioma microvessel and glioma endothelial cells (GECs). Knockdown of IGF2BP2 decreased the expression of lncRNA FBXL19-AS1 and tight junction-related proteins, thereby promoting BTB permeability. FBXL19-AS1 was over-expressed and more enriched in the cytoplasm of GECs. In addition, FBXL19-AS1 could bind to 3ʹ-UTR of ZNF765 mRNA and down-regulate ZNF765 mRNA expression through STAU1-mediated mRNA decay (SMD). The low expression of ZNF765 was discovered in GECs and verified to increase BTB permeability by inhibiting the promoter activities of tight junction-related proteins. Meanwhile, ZNF765 also inhibited the transcriptional activity of IGF2BP2, thereby forming a feedback loop in regulating the BTB permeability. Single or combined application of silenced IGF2BP2 and FBXL19-AS1 improved the delivery and antitumor efficiency of doxorubicin (DOX). In general, our study revealed the regulation mechanism of IGF2BP2/FBXL19-AS1/ZNF765 axis on BTB permeability, which may provide valuable insight into treatment strategy for glioma.

RBFOX1 Regulates the Permeability of the Blood-Tumor Barrier via the LINC00673/MAFF Pathway

The blood-tumor barrier limits the delivery of therapeutic drugs to brain tumor tissues. Selectively opening the blood-tumor barrier is considered crucial for effective chemotherapy of glioma. RNA-binding proteins have emerged as crucial regulators in various biologic processes. This study found that RNA-binding Fox-1 homolog 1 (RBFOX1) was downregulated in glioma vascular endothelial cells derived from glioma tissues, and in glioma endothelial cells obtained by co-culturing endothelial cells with glioma cells. Overexpression of RBFOX1 impaired the integrity of the blood-tumor barrier and increased its permeability. Additionally, RBFOX1 overexpression decreased the expression of tight junction proteins ZO-1, occludin, and claudin-5. Subsequent analysis of the mechanism indicated that the overexpression of RBFOX1 increased musculoaponeurotic fibrosarcoma protein basic leucine zipper [bZIP] transcription factor F (MAFF) expression by downregulating LINC00673, which stabilized MAFF messenger RNA (mRNA) through Staufen1-mediated mRNA decay. Moreover, MAFF could bind to the promoter region and inhibit the promoter activities of ZO-1, occludin, and claudin-5, which reduced its expression. The combination of RBFOX1 upregulation and LINC00673 downregulation promoted doxorubicin delivery across the blood-tumor barrier, resulting in apoptosis of glioma cells. In conclusion, this study indicated that overexpression of RBFOX1 increased blood-tumor barrier permeability through the LINC00673/MAFF pathway, which might provide a new useful target for future enhancement of blood-tumor barrier permeability.

Biosynthetic CircRNA_001160 induced by PTBP1 regulates the permeability of BTB via the CircRNA_001160/miR-195-5p/ETV1 axis

The presence of the blood-tumor barrier (BTB) severely impedes the transport of anti-neoplasm drugs to the central nervous system, affecting the therapeutic effects of glioma. Glioma endothelial cells (GECs) are the main structural basis of the BTB. Circular RNA is considered to be an important regulator of endothelial cell growth. In this study, we found that polypyrimidine tract binding protein 1 (PTBP1) and circRNA_001160 were remarkably upregulated in GECs. Knockdown of PTBP1 or circRNA_001160 significantly increased BTB permeability, respectively. As a molecular sponge of miR-195-5p, circRNA_001160 attenuated its negative regulation of the target gene ETV1 by adsorbing miR-195-5p. In addition, ETV1 was overexpression in GECs. ETV1 bounded to the promoter regions of tight junction-related proteins and increased the promoter activities, which significantly promoted the expression levels of tight junction-related proteins. The present study showed that the combined application of PTBP1, circRNA_001160, and miR-195-5p with the anti-tumor drug Dox effectively promoted Dox through BTB and extremely induced the apoptosis of glioma cells. Our results demonstrated that the PTBP1/circRNA_001160/miR-195-5p/ETV1 axis was critical in the regulation of BTB permeability and provided new targets for the treatment of glioma.

EXTH-35. LASER INTERSTITIAL THERMAL THERAPY INCREASES BLOOD-BRAIN BARRIER AND BLOOD-TUMOR BARRIER PERMEABILITY IN A MOUSE MODEL OF GLIOBLASTOMA

Abstract INTRODUCTION A central challenge in glioblastoma treatment is the presence of the blood-brain barrier (BBB) and blood-tumor barrier (BTB), which prevent access of drugs to the brain and tumor respectively. Recent evidence in patients suggests laser interstitial thermal therapy (LITT), used clinically for tumor ablation, locally disrupts BBB integrity, potentially creating a therapeutic window to deliver otherwise brain-impermeant agents. METHODS A mouse model for LITT, established using a Nd-YAG laser coupled to a 600 mm fiber optic and thermocouple probe, was inserted via burrhole to target the somatosensory cortex. Syngeneic GL261 tumor cells were stereotactically implanted prior to LITT. BBB and BTB permeability were assessed through measurement of fluorescein and doxorubicin after IV injection. Permeability of IV dextran (10 and 70 kDa) and human IgG was monitored by immunohistochemistry (IHC) analysis. Mechanisms of BBB breakdown in vivo were explored utilizing electron microscopy and IHC. RESULTS By fluorescein assay, LITT-induced BBB and BTB permeability began one day post-treatment and was sustained for at least 2 weeks. Additionally, both normal brain and brain tumors demonstrated an increase in Dextran 10 kDa, Dextran 70 kDa, and human IgG extravasation after IV injection in vivo. Mechanistically, we provide evidence that LITT triggers both a decrease in tight junction integrity and an increase in brain endothelial cell transcytosis. As proof-of-concept that LITT can enhance tumor delivery of systemic drugs, LITT increased IV doxorubicin permeability in brain in vivo. Moreover, LITT plus doxorubicin significantly increased survival in brain tumor-bearing mice compared to doxorubicin or LITT alone. CONCLUSIONS Our data suggest that LITT increases BBB and BTB permeability over a defined time window to large molecular weight agents, including antibodies, through multiple cellular mechanisms. Our preclinical results with LITT plus doxorubicin, which mirror a current clinical trial, indicate LITT can enhance the efficacy of systemically delivered drugs.

Role of linc00174/miR-138-5p (miR-150-5p)/FOSL2 Feedback Loop on Regulating the Blood-Tumor Barrier Permeability

The blood-tumor barrier (BTB) limits the transport of chemotherapeutic drugs to brain tumor tissues and impacts the treatment of glioma. Long non-coding RNAs play critical roles in various biological processes of tumors; however, the function of these in BTB permeability is still unclear. In this study, we have identified that long intergenic non-protein coding RNA 174 (linc00174) was upregulated in glioma endothelial cells (GECs) from glioma tissues. Additionally, linc00174 was also upregulated in GECs from the BTB model in vitro. Knock down of linc00174 increased BTB permeability and reduced the expression of the tight junction-related proteins ZO-1, occludin, and claudin-5. Both bioinformatics data and results of luciferase reporter assays demonstrated that linc00174 regulated BTB permeability by binding to miR-138-5p and miR-150-5p. Furthermore, knock down of linc00174 inhibited FOSL2 expression via upregulating miR-138-5p and miR-150-5p. FOSL2 interacted with the promoter regions and upregulated the promoter activity of ZO-1, occludin, claudin-5, and linc00174 in GECs. In conclusion, the present study demonstrated that the linc00174/miR-138-5p (miR-150-5p)/FOSL2 feedback loop played an essential role in regulating BTB permeability.

Circular RNA USP1 regulates the permeability of blood-tumour barrier via miR-194-5p/FLI1 axis.

Recent studies indicate circular RNAs are related to dysregulation of vascular endothelial cell function, yet the underlying mechanisms have remained elusive. Here, we characterized the functional role of circular RNA USP1 (circ‐USP1) in the regulation of the blood‐tumour barrier (BTB) permeability and the potential mechanisms. In the current study, the circ‐USP1 expressing level was up‐regulated in glioma cerebral microvascular endothelial cells (GECs) of the BTB model in vitro. Knockdown of circ‐USP1 disrupted the barrier integrity, increased its permeability as well as reduced tight junction‐related protein claudin‐5, occludin and ZO‐1 expressions in GECs. Bioinformatic prediction and luciferase assay indicated that circ‐USP1 bound to miR‐194‐5p and suppressed its activity. MiR‐194‐5p contributed to circ‐USP1 knockdown‐induced increase of BTB permeability via targeting and down‐regulating transcription factor FLI1. Furthermore, FLI1 regulated the expressions of claudin‐5, occludin and ZO‐1 in GECs through binding to their promoter regions. Single or combined treatment of circ‐USP1 and miR‐194‐5p effectively promoted anti‐tumour drug doxorubicin across BTB to induce apoptosis of glioma cells. Overall, this present study identified the crucial regulation of circ‐USP1 on BTB permeability via miR‐194‐5p/FLI1 axis‐mediated regulation of tight junction proteins, which might facilitate the development of therapeutics against human gliomas.

Laser Therapy Enhances Blood-Brain Barrier and Blood-Tumor Barrier Permeability in a Mouse Model of Glioblastoma

Abstract INTRODUCTION A main challenge in the treatment of glioblastoma is the blood-brain barrier (BBB). In this study, we established a laser interstitial thermotherapy (LITT) platform in mice to test the hypothesis that LITT can increase BBB and blood-tumor barrier (BTB) permeability, which could potentially be exploited to enhance delivery of antitumor agents. METHODS We established a preclinical mouse model for LITT using an Nd-YAG laser coupled to a 600 μm fiber optic and thermocouple probe in C57BL/6 mice. Both the fiber optic and temperature probes were inserted through a small burrhole to target somatosensory cortex. In other experiments, mice were stereotactically implanted with syngeneic GL261 tumor cells prior to LITT. Post-LITT, brain tissue was assessed for BBB and BTB permeability through quantitative measurement of brain fluorescein (FL) after intraperitoneal injection and immunohistochemistry (IHC) after intravenously injected dextran (MWs 10 and 70 kDa) and human IgG (MW 150 kDa) to define spatial characteristics. Electron microscopy and IHC was used to delineate the mechanisms of BBB break down. RESULTS Using BBB permeability tracers, noninjected mice and mice orthotopically implanted with GL261 tumor cells were used to characterize the effect of laser therapy on the BBB and BTB, respectively. We found LITT substantially disrupts the BBB in the first day post-treatment and is sustained for 4 wk. Additionally, tumor-implanted mice demonstrated an increase in Dextran 10 and 70 kDa and human IgG extravasation after IV injection of these tracers. Mechanistically, we provide evidence, by IHC and electron microscopy, that LITT triggers both a decrease in tight junction integrity and an increase in brain endothelial cell transcytosis to increase overall BBB permeability. CONCLUSION Our data suggest that LITT increases BBB and BTB permeability over a defined time window in a mouse model and that large molecular size agents, including human IgG, can infiltrate brain parenchyma and brain tumors after systemic administration. Future experiments will focus on testing delivery of antitumor agents following LITT to enhance survival in brain tumor-bearing mice.

Overexpression of RBFOX1 Increases Blood-Tumor Barrier Permeability via linc00673/MAFF Pathway

Background: RNA binding protein have recently emerged as critical regulators of various biologic processes and therapeutic targets. RNA-binding Fox 1(RBFOX1) involved in in non-small cell lung cancer progress. However, there is no research about the functional role of RBFOX1 in glioma endothelial cells (GECs). Methods: The in vitro blood-tumor barrier (BTB) model and RBFOX1 overexpressed or silenced GECs were established. Transendothelial electric resistance (TEER) measurements and HRP flux assays to detect the integrity and permeability of BTB. Immunofluorescence and Chromatin Immunoprecipitation (ChIP) assays were used to detect the potential mechanism. Findings: RBFOX1 was downregulation in GECs. Overexpression of RBFOX1 resulted in an impaired integrity and increased permeability of BTB and decreased the tight-related protein zonula occludin (ZO)-1, occludin, claudin-5 expression. Long non-coding RNA linc00673, was targets of RBFOX1, knockdown increased BTB permeability and destabilized MAFF mRNA through Staufen 1-mediated messenger RNA decay (SMD). MAFF bound to the promoter regions and inhibited the promoter activity of ZO-1, occludin, and claudin-5 in GECs. Interpretation: Those results indicated the potential role of RBFOX1/linc00673/MAFF pathway in regulation of BTB permeability, thereby may provide new targets of glioma therapy. Funding Statement This work is supported by grants from the Natural Science Foundation of China (81573010, 81672511 and 81602726), Liaoning Science and Technology Plan Project (No. 2017225020, 2015225007), Project of Key Laboratory of Neuro-oncology in Liaoning Province (112-2400017005), special developmental project guided by central government of Liaoning Province (No. 2017011553-301). Declaration of Interests: The authors declared that they have no conflict of interest. Ethics Approval Statement: All the participants signed and provided the informed consent, and this study was approved by the Institutional Review Board of Shengjing Hospital of China Medical University.

KHDRBS3 regulates the permeability of blood\u2013tumor barrier via cDENND4C/miR-577 axis

The existence of blood–tumor barrier (BTB) severely restricts the efficient delivery of antitumor drugs to cranial glioma tissues. Various strategies have been explored to increase BTB permeability. RNA-binding proteins and circular RNAs have recently emerged as potential regulators of endothelial cells functions. In this study, RNA-binding protein KH RNA-binding domain containing, signal transduction associated 3 (KHDRBS3) and circular RNA DENND4C (cDENND4C) were enriched in GECs. KHDRBS3 bound to cDENND4C and increased its stability. The knockdown of cDENND4C increased the permeability of BTB via downregulating the expressions of tight junction-related proteins. The miR-577 was lower expressed in GECs. The overexpressed miR-577 increased the permeability of BTB by reducing the tight junction-related protein expressions, and vice versa. Furthermore, cDENND4C acted as a molecular sponge of miR-577, which bound to miR-577 and inhibited its negative regulation of target genes ZO-1, occludin and claudin-1 to regulate BTB permeability. Single or combined treatment of KHDRBS3, cDENND4C, and miR-577 effectively promoted antitumor drug doxorubicin (DOX) across BTB to induce apoptosis of glioma cells. Collectively, the present study indicated that KHDRBS3 could regulate BTB permeability through the cDENND4C/miR-577 axis, which enhanced doxorubicin delivery across BTB. These findings may provide a novel strategy for chemotherapy of brain tumors.