The concepts of mean and variance serve complementary roles in biological research. While the former captures the central tendencies of a trait in populations, the latter defines the degree of spread or dispersion of the data points relative to that central tendency. Most biological studies have focused on the differences in means. Here, we explore the relevance of variance in protein expression as a mechanism of therapeutic resistance for glioblastomas. Preclinical studies. Temozolomide treatment induced polyribonucleotide nucleotidyltransferase 1 (PNPT1) dependent degradation of miR-181d, a methyl-guanine methyl transferase (MGMT) regulating miRNA, resulting in de-repression of MGMT expression and acquired temozolomide resistance. In addition to MGMT, miR-181d additionally downregulates PNPT1. Degradation of miR-181d, therefore, creates a feed-forward cycle, expediting an increase in the steady-state MGMT level and acquired temozolomide resistance. The degradation requires the activation of the DNA damage response through Ataxia Telangiectasia and Rad3-related (ATR) kinase. The degradation of miR-181d in glioblastoma cells increased both the mean and the variance of MGMT expression in the cell population. Subclone reconstituted glioblastoma populations with similar populational mean MGMT levels but with differences in the variance of MGMT expression exhibited differential temozolomide sensitivity, with the higher MGMT variance population showing increased resistance. This variance is one defining feature of intra-tumoral heterogeneity, and the variance-mediated temozolomide resistance is suppressed by exogenous administration of miR-181d. Our study reveals a novel mechanism of temozolomide resistance in glioblastoma, wherein chemotherapy-induced miR-181d degradation increases MGMT variance. This finding offers a mechanistic basis for understanding the contribution of intra-tumoral heterogeneity to acquired temozolomide resistance and highlights the potential for miRNA delivery as a therapeutic strategy.

Intratumoral heterogeneity plays a pivotal role in cancer evolution, providing the substrate for adaptation to selective pressures, including chemotherapy treatment. Here, we demonstrate that miR-181d modulates variability in methyl-guanine methyl transferase (MGMT) expression, contributing to this heterogeneity in glioblastoma, the most common form of adult primary brain tumor. Treatment with standard-of-care temozolomide (TMZ) chemotherapy triggers a feedforward loop that accelerates polyribonucleotide nucleotidyltransferase 1 (PNPT1)-dependent miR-181d degradation. This degradation requires the activation of ataxia-telangiectasia and Rad3-related (ATR) kinase. The degradation of miR-181d in glioblastoma cells increases the variance of MGMT expression in the cell population, contributing to acquired TMZ resistance. This resistance is suppressed by exogenously transfected miR-181d. These findings suggest that microRNA regulates intratumoral heterogeneity by modulating the transcriptional variability of key DNA repair enzymes, providing a compelling rationale for miRNA delivery as a platform for glioblastoma therapy.

Intratumoral heterogeneity plays a pivotal role in cancer evolution, providing the substrate for adaptation to selective pressures, including treatment with chemotherapy. Here, we show that micro-RNA regulation of variance in the expression of the DNA repair protein methyl-guanine methyl transferase (MGMT) contributes to this heterogeneity and acquired therapeutic resistance. In cell lines derived from glioblastomas, the most common form of primary brain tumor, treatment with standard-of-care temozolomide chemotherapy triggers a feed-forward loop between polyribonucleotide nucleotidyltransferase 1 (PNPT1) and miR-181d, an MGMT regulating miRNA, expediting miR-181d degradation. This degradation requires the activation of Ataxia Telangiectasia and Rad3-related (ATR) kinase. The degradation of miR-181d in glioblastoma cells increased both the mean and the variance of MGMT expression in the cell population. Subclone reconstituted cell populations with similar populational mean MGMT levels but with differences in the variance of MGMT expression exhibited differential temozolomide sensitivity, with the higher MGMT variance population showing increased resistance. This resistance is suppressed by exogenously transfected miR-181d. These findings suggest a key role for miRNA in regulating intra-tumoral heterogeneity through modulation of key DNA repair enzymes and provide a compelling rationale for miRNA delivery as a platform for glioblastoma therapy. Significance Statement This study demonstrates a mechanistic link between a feed-forward loop mediating microRNA degradation and cell-to-cell variance in gene expression, and the contribution of this mechanism to intratumoral heterogeneity and therapeutic resistance. We show that when glioblastoma, the most common form of adult primary brain tumor, is treated with standard-of-care chemotherapy, temozolomide, a feed-forward loop between miR-181d and PNPT1 is initiated, causing rapid degradation of miR-181d. This degradation increases the cell-to-cell variability in methyl-guanine methyl transferase (MGMT) expression, expanding intra-tumoral heterogeneity and contributing to acquired temozolomide resistance. This process can be suppressed by therapeutic delivery of microRNA, providing compelling considerations for clinical translation. ### Competing Interest Statement The authors have declared no competing interest.

Abstract O6 methyl-guanine methyltransferase (MGMT) restores alkylated DNA to its undamaged form in a stochiometric manner to prevent the cytotoxic effects of temozolomide (TMZ), a DNA alkylating agent used as a standard-of-care treatment for glioblastomas. We previously demonstrated that he microRNA, miR-181d, post-transcriptionally repressed MGMT expression. Here, we show that treatment of glioblastoma cells with TMZ induced a feed-forward cascade resulting in an ataxia telangiectasia and Rad3 (ATR) and polyribonucleotide nucleotidyl transferase 1 (PNPT1)-dependent degradation of miR-181d. Single-cell analyses revealed such miR-181d degradation induced an: 1) increased mean level of MGMT expression and 2) widened the variance in MGMT expression in the cell population. While the former is known to confer increased population fitness to temozolomide, the importance of the latter is unknown. By mixing cultures derived from isogenic glioblastoma subclones with distinct MGMT expression levels, we generated glioblastoma cell populations with comparable mean MGMT expression levels while differing in the populational variance in MGMT expression (classified as “wide” or “narrow”). Despite comparable mean levels of MGMT expression, the glioblastoma population with wide variance exhibited increased temozolomide resistance relative to the population with the narrow variance, in vitro and in vivo. Transfection of miR-181d into the wide-variance population narrows the variance of MGMT expression and restores TMZ sensitivity to levels comparable to that observed in miR-181d transfected narrow-variance populations. These findings suggest that TMZ-induced miR-181d degradation enhanced TMZ resistance through modulation of both the mean and variance of MGMT expression, mechanisms which may be extrapolated to microRNA biology in general.

Background:In this study, we investigated the feasibility of quantitative ultrashort echo time (qUTE) magnetic resonance (MR) imaging techniques in the detection and quantification of iron oxide nanoparticle (IONP)-labeled stem cells.Methods:A stem cell phantom containing multiple layers of unlabeled or labeled stem cells with different densities was prepared. The phantom was imaged with quantitative UTE (qUTE) MR techniques [i.e., UTE-T1 mapping, UTE-T2* mapping, and UTE-based quantitative susceptibility mapping (UTE-QSM)] as well as with a clinical T2 mapping sequence on a 3T clinical MR system. For T1 mapping, a variable flip angle (VFA) method based on actual flip angle imaging (AFI) technique was utilized. For T2* mapping and UTE-QSM, multiple images with variable, interleaved echo times including UTE images and gradient recalled echo (GRE) images were used. For UTE-QSM, the phase information from the multi-echo images was utilized and processed using a QSM framework based on the morphology-enabled dipole inversion (MEDI) algorithm. The qUTE techniques were also evaluated in an ex vivo experiment with a mouse injected with IONP-labeled stem cells.Results:In the phantom experiment, the parameters estimated with qUTE techniques showed high linearity with respect to the density of IONP-labeled stem cells (R2>0.99), while the clinical T2 parameter showed impaired linearity (R2=0.87). In the ex vivo mouse experiment, UTE-T2* mapping and UTE-QSM showed feasibility in the detection of injected stem cells with high contrast, whereas UTE-T1 and UTE-T2* showed limited detection. Overall, UTE-QSM demonstrated the best contrast of all, with other methods being subjected more to a confounding factor due to different magnetic susceptibilities of various types of neighboring tissues, which creates inhomogeneous contrast that behaves similar to IONP.Conclusions:In this study, we evaluated the feasibility of a series of qUTE imaging techniques as well as conventional T2 mapping for the detection of IONP-labeled stem cells in vitro and ex vivo. UTE-QSM performed superior amongst other qUTE techniques as well as conventional T2 mapping in detecting stem cells with high contrast.

Non-invasive, clinically applicable tracking of therapeutic cells by magnetic resonance imaging (MRI) offers unparalleled insight into the safety and efficacy of cell-based therapies in the body. Here we used a series of 3D quantitative UTE techniques including UTE-QSM, UTE-T 1 , and UTE-T 2 * mapping to evaluate the MR characteristics of stem cells labeled with a proprietary nanoparticle formulation that simultaneously labels cells for both optical and MR imaging. In a phantom experiment, all quantitative UTE parameters showed strong correlation with concentrations of labeled stem cells. Interestingly, in ex vivo mouse imaging, only UTE-QSM and UTE-T 2 * mapping detected the injected, labeled stem cells.

Abstract INTRODUCTION Lactate dehydrogenase A (LDHA) encodes an enzyme that catalyzes the inter-conversion between pyruvate and lactate in glycolysis. Here, we demonstrate that LDHA mediates a novel role in DNA repair independent of this metabolic function. METHODS siRNA screen, The Cancer Genome Atlas (TCGA) survival analysis, ionizing radiation (IR), g-H2AX, and chromatin assays, site-directed mutagenesis. RESULTS In an orthogonal siRNA-informatic screen to identify genes 1) when silenced caused IR sensitivity in patient-derived glioblastoma lines and 2) lowered expression is associated with improved survival in TCGA, LDHA surfaced as the top candidate. The survival association was validated by LDHA immunohistochemical staining in an independent collection of glioblastoma samples. In vitro and in vivo, silencing of LDHA sensitized glioblastoma lines to IR and enhanced radiation-induced g-H2AX accumulation. Such sensitization was not observed after treatment with an LDHA inhibitor, suggesting the metabolic function of LDHA is distinct from its role in DNA repair. Supporting this hypothesis, truncation mutations that suppressed the LDHA glycolysis function minimally affected its role in DNA repair. Mechanistically, cytoplasmic LDHA translocates into the nucleus in response to IR. This translocation was associated with subsequent chromatin transition into an open conformation and enhanced homologous recombination. CONCLUSION The novel LDHA function in DNA repair suggests intricate crosstalks between glycolytic metabolism and DNA repair, offering a new platform for glioblastoma therapeutic development.

You have accessJournal of UrologyImaging/Radiology: Uroradiology II (MP22)1 Sep 2021MP22-16 DEVELOPMENT OF NON-INVASIVE CLINICALLY APPLICABLE IN VIVO TRACKING OF EXTRACELLULAR VESICLES USING MAGNETIC RESONANCE IMAGING (MRI) Johnny Akers, Paola Aguiari, Hasmik Soloyan, Seda Mkhitaryan, Gevorg Karaptyan, Roger De Filippo, Mya Thu, Laura Perin, and Sargis Sedrakyan Johnny AkersJohnny Akers More articles by this author , Paola AguiariPaola Aguiari More articles by this author , Hasmik SoloyanHasmik Soloyan More articles by this author , Seda MkhitaryanSeda Mkhitaryan More articles by this author , Gevorg KaraptyanGevorg Karaptyan More articles by this author , Roger De FilippoRoger De Filippo More articles by this author , Mya ThuMya Thu More articles by this author , Laura PerinLaura Perin More articles by this author , and Sargis SedrakyanSargis Sedrakyan More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002013.16AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSC) hold great potential for the treatment of chronic kidney diseases (CKD). We have already shown that AFSC-EVs are renoprotective in a mouse model of CKD, Alport syndrome. However, there is an important unmet need for real-time in vivo monitoring of these therapeutic EVs after they are injected into a subject to understand their safety, targeting, and effectiveness. While current optical imaging solutions like bioluminescence and fluorescence are useful for EV tracking studies in animal models, there is limited utility in clinical applications. Here we present a novel in vivo tracking solution for our therapeutic EVs in Alport mice, utilizing clinically applicable MRI technology. METHODS: To generate trackable EVs, AFSC were labeled with a novel magnetic agent (VSCM). EVs secreted by the labeled AFSC were isolated by ultracentrifugation. The viability and morphology of labeled-cells were evaluated, and the in vitro MR properties of EVs were analyzed by magnetometer. Purity, potency and identity of labeled EV was compared to non-labeled EVs. In vivo biodistribution of labeled EVs was evaluated in WT and Alport mice by MRI at 10 min and 3 hr post injection, and retro-orbital and intra-cardiac routes of delivery were compared. RESULTS: The magnetic label did not affect the physiological characteristics of the cells and did not change identity, purity and potency (therapeutic effect in vivo) of EVs. MRI phantom studies confirmed the in vitro/ex vivo detectability of labeled-EVs. Importantly, as expected MRI studies showed that EV homing to the kidney injected intra-cardiacally into Alport mice was more efficient vs the retro-orbital route, and Prussian blue staining of sections confirmed EV homing to the kidney. CONCLUSIONS: We have developed a clinically applicable novel magnetic nanoparticle agent that can be used to label and track the biodistribution of EVs in the kidney and other organs using non-invasive, safe, and effective MRI technology that’s widely available. This technology is highly adaptable and can be deployed in both preclinical and clinical settings. Source of Funding: The GOFARR Fund; Research Career Development Award, TSRI, CHLA © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e397-e398 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Johnny Akers More articles by this author Paola Aguiari More articles by this author Hasmik Soloyan More articles by this author Seda Mkhitaryan More articles by this author Gevorg Karaptyan More articles by this author Roger De Filippo More articles by this author Mya Thu More articles by this author Laura Perin More articles by this author Sargis Sedrakyan More articles by this author Expand All Advertisement PDF downloadLoading ...

Abstract While advancement in chimeric antigen receptors (CARs) has greatly improved the anti-tumor activity of immune cell-based therapies utilizing modified T lymphocytes (CAR-T) and natural killer cells (CAR-NK), there remains a critical unmet need for diagnostic technologies to help evaluate their biological performance effectively and efficiently after injecting into the body.The ability to better understand the biodistribution of injected cells and devise dosing strategies are key determinants to the success or failure of cell therapy. However, clinicians are limited by the available technology for real time in-vivo tracking of cell therapy products in clinical settings. Here we present a clinically applicable novel magnetic agent that can effectively label a variety of cell types used in cancer cell therapy development including T cells, NK cells, and neural stem cells (NSCs), and when combined with the 3D ultrashort echo time Cones (3D UTE-Cones) sequence, can enable quantitative assessment of cell therapy products using MRI. In this study, we optimized loading of our tracking agent into T cells, NK cells, and NSCs, evaluated the morphology and viability of the labeled cells, and confirmed internalization of the labeling agent by Prussian blue staining and TEM. The in vitro MRI properties of the labeled cells were quantitated by the 3D UTE-Cones sequence. Ex vivo MRI analysis was performed by injecting labeled cells into harvested mouse livers and brains to ascertain their MRI visibility in tissues using a clinical MRI scanner. We confirmed that our magnetic labeling agent efficiently labeled each cell line tested, and the morphology or viability of the labeled cells were unaffected. The MRI detectability of labeled cells were confirmed by in vitro MRI phantoms. Further, using quantitative susceptibility mapping (QSM), T1, and T2* mapping based on 3D UTE-Cones sequences, we found near perfect correlations between cell density and MRI properties. By applying the parameters established in the MRI phantoms, we were able to detect and quantity the number of labeled cells injected into mouse livers and brains ex vivo. In summary, we have developed a clinically applicable imaging-based platform that empowers cell therapy researchers to better understand the results of their biodistribution and dosing studies through real-time cell tracking and quantitative assessments using MRI. Citation Format: Johnny C. Akers, Zhao Wei, Hyungseok Jang, Eric Chang, Jiang Du, Mya S. Thu. Development of a non-invasive clinically applicable real time cell tracking platform for evaluating cell-based cancer therapy using magnetic resonance imaging (MRI) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-024.

Background: Technology platforms that afford biomarker discovery in patients suffering from traumatic brain injury (TBI) remain an unmet medical need. Here, we describe an observational pilot study to explore the utility of an alternating current electrokinetic (ACE) microchip device in this context. Methods: Blood samples were collected from participating subjects with and without minor TBI. Plasma levels of glial fibrillary acidic protein (GFAP), Tau, ubiquitin C-terminal hydrolase L1 (UCH-L1), and cell-free DNA (cfDNA) were determined in subjects with and without minor TBI using ACE microchip device followed by on-chip immunofluorescent analysis. Post-concussive symptoms were assessed using the Rivermead Post Concussion Symptoms Questionnaire (RPCSQ) at one-month follow-up. Results: Highest levels of GFAP, UCH-L1, and Tau were seen in two minor TBI subjects with abnormality on head computed tomography (CT). In patients without abnormal head CT, Tau and GFAP levels discriminated between plasma from minor-TBI and non-TBI patients, with sensitivity and specificity of 64-72 and 50%, respectively. Plasma GFAP, UCH-L1, and Tau strongly correlated with the cumulative RPCSQ score. Plasma UCH-L1 and GFAP exhibited highest correlation to sensitivity to noise and light (r = 0.96 and 0.91, respectively, p < 0.001). Plasma UCH-L1 and Tau showed highest correlation with headache (r = 0.74 and 0.78, respectively, p < 0.001), sleep disturbance (r = 0.69 and 0.84, respectively, p < 0.001), and cognitive symptoms, including forgetfulness (r = 0.76 and 0.74, respectively, p < 0.001), poor concentration (r = 0.68 and 0.76, respectively, p < 0.001), and time required for information processing (r = 0.77 and 0.81, respectively, p < 0.001). cfDNA exhibited a strong correlation with depression (r = 0.79, p < 0.01) and dizziness (r = 0.69, p < 0.01). While cfDNA demonstrated positive correlation with dizziness and depression (r = 0.69 and 0.79, respectively, p < 0.001), no significant correlation was observed between cumulative RPCSQ and cfDNA (r = 0.07, p = 0.81). Conclusion: We provide proof-of-principle results supporting the utility of ACE microchip for plasma biomarker analysis in patients with minor TBI.

Extracellular Vesicles and Circulating Nucleic Acids is an open access journal, focusing on extracellular vesicles and circulating nucleic acids including DNA, RNA, and miRNA and their therapeutic use.

Background: Recurrence after radiation therapy is nearly universal for glioblastomas, the most common form of adult brain cancer. The study aims to define clinically pertinent mechanisms underlying this recurrence. Methods: microRNA (miRNA) profiling was performed using matched pre- and post-radiation treatment glioblastoma specimens from the same patients. All specimens harbored unmethylated O6-methylguanine-DNA methyltransferase promoters (umMGMT) and wild-type isocitrate dehydrogenase (wtIDH). The most altered miRNA, miR-603, was characterized. Findings: While nearly all miRNAs remained unchanged after treatment, decreased levels of few, select miRNAs in the post-treatment specimens were observed, the most notable of which involved miR-603. Unbiased profiling of miR-603 targets revealed insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF1R). Ionizing radiation (IR) induced cellular export of miR-603 through extracellular vesicle (EV) release, thereby de-repressing IGF1 and IGF1R. This de-repression, in turn, promoted cancer stem-cell (CSC) state and acquired radiation resistance in glioblastomas. Export of miR-603 additionally de-repressed MGMT, a DNA repair protein responsible for detoxifying DNA alkylating agents, to promote cross-resistance to these agents. Ectopic miR-603 expression overwhelmed cellular capacity for miR-603 export and synergized with the tumoricidal effects of IR and DNA alkylating agents. Interpretation: Profiling of matched pre- and post-treatment glioblastoma specimens revealed altered homeostasis of select miRNAs in response to radiation. Radiation-induced EV export of miR-603 simultaneously promoted the CSC state and up-regulated DNA repair to promote acquired resistance. These effects were abolished by exogenous miR-603 expression, suggesting potential for clinical translation. Funding: NIH 1R01NS097649-01, 9R44GM128223-02, 1R01CA240953-01, the Doris Duke Charitable Foundation Clinical Scientist Development Award, The Sontag Foundation Distinguished Scientist Award, the Kimmel Scholar Award, and BWF 1006774.01 (C.C.C).

Extracellular vesicles (EVs) are small, membrane-bound particles released by all cells that have emerged as an attractive biomarker platform. We study the utility of a dielectrophoretic (DEP) micro-chip device for isolation and characterization of EVs derived from plasma specimens from patients with brain tumors. EVs were isolated by DEP chip and subjected to on-chip immunofluorescence (IF) staining to determine the concentration of glial fibrillary acidic protein (GFAP) and Tau. EVs were analyzed from the plasma samples isolated from independent patient cohorts. Glioblastoma cell lines secrete EVs enriched for GFAP and Tau. These EVs can be efficiently isolated using the DEP platform. Application of DEP to clinical plasma samples afforded discrimination of plasma derived from brain tumor patients relative to those derived from patients without history of brain cancer. Sixty-five percent (11/17) of brain tumor patients showed higher EV-GFAP than the maximum observed in controls. Ninety-four percent (16/17) of tumor patients showed higher EV-Tau than the maximum observed in controls. These discrimination thresholds were applied to plasma isolated from a second, independent cohort of 15 glioblastoma patients and 8 controls. For EV-GFAP, we observed 93% sensitivity, 38% specificity, 74% PPV, 75% NPV, and AUC of 0.65; for EV-Tau, we found 67% sensitivity, 75% specificity 83% PPV, 55% NPV, and AUC of 0.71 for glioblastoma diagnosis. This proof-of-principle study provides support for DEP-IF of plasma EVs for diagnosis of glioblastoma.

Exosomes can mediate a dynamic method of communication between malignancies, including those sequestered in the central nervous system and the immune system. We sought to determine whether exosomes from glioblastoma (GBM)-derived stem cells (GSCs) can induce immunosuppression. We report that GSC-derived exosomes (GDEs) have a predilection for monocytes, the precursor to macrophages. The GDEs traverse the monocyte cytoplasm, cause a reorganization of the actin cytoskeleton, and skew monocytes toward the immune suppresive M2 phenotype, including programmed death-ligand 1 (PD-L1) expression. Mass spectrometry analysis demonstrated that the GDEs contain a variety of components, including members of the signal transducer and activator of transcription 3 (STAT3) pathway that functionally mediate this immune suppressive switch. Western blot analysis revealed that upregulation of PD-L1 in GSC exosome-treated monocytes and GBM-patient-infiltrating CD14+ cells predominantly correlates with increased phosphorylation of STAT3, and in some cases, with phosphorylated p70S6 kinase and Erk1/2. Cumulatively, these data indicate that GDEs are secreted GBM-released factors that are potent modulators of the GBM-associated immunosuppressive microenvironment.

When unrepaired, alkylated DNA can induce cell death or trigger mutagenesis. Cellular capacity for repair of these lesions by O 6 -methylguanine methyltransferase (MGMT) dictates the equilibrium between cell viability and genetic diversity. Treatment of glioblastoma cells with temozolomide (TMZ) induced ATM- and Rad3-related (ATR) kinase dependent polyribonucleotide nucleotidyltransferase 1 (PNPT1) degradation of miR-181d. miR-181d suppresses MGMT expression; its degradation increases the mean MGMT expression of the cell population. miR-181d degradation also magnifies the cell-to-cell variability in MGMT expression, expanding the genetic heterogeneity of the population. This expanded heterogeneity enhances the “fitness” of the population and constitutes a novel form of chemotherapeutic resistance. These effects can be suppressed by overexpression of miR-181d, suggesting miRNA delivery as a strategy for glioblastoma therapy. To characterize the mechanism of acquired resistance, we profiled the expression of 2400 miRNAs before and after TMZ treatment. In independent patient-derived neurosphere lines, the majority of miRNAs remained unchanged after treatment. However, miR-181d was consistently suppressed after TMZ treatment. Our previous work demonstrated miR-181d as the master regulator of MGMT. We confirmed TMZ-induced suppression of miR-181d using independent in vitro and in vivo models as well as matched pre- and post-TMZ treated clinical specimens. TMZ-induced miR-181d suppression persisted after transcriptional inhibition, suggesting degradation as the primary mechanism. We performed an siRNA screen and identified polyribonucleotide nucleotidyltransferase 1 (PNPT1) as the gene responsible for miR-181d degradation. CRISPR inactivation of PNPT1 eliminated TMZ-induced suppression of miR-181d; this was rescued by wild-type PNPT1 but not by PNPT1 harboring RNAse-inactivating mutations. TMZ-induced degradation of miR-181d requires ATR kinase. Silencing or inhibition of ATR eliminated binding of PNPT1 to miR-181d and prevented degradation of miR-181d. TMZ-sensitizing effects of ATR inhibition were reversed by anti-miR-181d, suggesting miR-181d is essential in this process. In addition to elevating the mean MGMT expression of the population, single-cell analysis revealed that miR-181d degradation broadened the cell-to-cell variability in MGMT expression in vitro. In matched clinical pre- and post-TMZ treated specimens, variability in MGMT expression was significantly elevated in post-TMZ samples. This was recapitulated using The Cancer Genome Atlas (TCGA) database. We propose that miR-181d degradation-mediated expansion of genetic heterogeneity enhances the “fitness” of the population, constituting a novel form of chemotherapeutic resistance. These effects are suppressed by miR-181d overexpression, suggesting miRNA delivery as a strategy for glioblastoma therapy. Citation Format: Valya Ramakrishnan, Johnny Akers, Thien Nguyen, Aaron Wang, Bandita Adhikari, Brian Hirshman, Jie Li, Jann Sarkaria, Wei Hua, Mao Ying, Masayuki Nitta, Tao Jiang, Bob Carter, Clark C. Chen. miR-181d degradation mediated genetic heterogeneity and acquired resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1956.

Early detection of brain cancer remains a challenging proposition. We applied a novel alternating current electrokinetic (ACE) chip device that relies on the dielectrophoretic (DEP) force to isolate and analyze the proteomic content of extracellular vesicles (EV) derived from undiluted patient plasma to identify biomarkers for early detection of brain tumors. EVs derived from cultured cells or plasma samples were isolated using the ACE chip. The concentration of intra-vesicular glial fibrillary acidic protein (GFAP) and Tau was determined using immunofluorescence staining. Improvement in model prognostication was quantified using net reclassification improvement (NRI>0) and integrated discrimination improvement (IDI). EVs secreted by cultured brain tumor cells (brain metastasis, meningioma, and glioma) harbored high levels of GFAP and Tau. We isolated EVs from plasma collected from brain tumor patients (5 meningiomas, 5 metastases, 6 gliomas) and 17 non-cancer controls. Compared to controls, plasma EVs from brain tumor patients exhibited greater fluorescence for GFAP (1.94 ± 0.139 vs. 1.28 ± 0.042, p<0.0001) and Tau (4.92 ± 0.43 vs. 1.79 ± 0.11, p<0.0001). Immunofluorescence did not differ between tumor types. Elevated EV GFAP was associated with a sensitivity of 88%, a specificity of 92%, and a AUC of 0.931 (95% CI 0.84–1.022) for brain tumor detection. Similarly, elevated EV Tau was associated with sensitivity of 94%, specificity of 94%, and AUC of 0.948 (95% CI 0.846–1.05) for brain tumor detection. The combination of EV GFAP and Tau improved test discrimination relative to GFAP alone (NRI>0 1.66, 95% CI 1.19–2.13, p<0.001; IDI 0.26, 95% CI 0.11–0.41, p<0.001) or Tau alone (NRI>0 1.54, 95% CI 1.03–2.05, p<0.001; IDI 0.18 0.038-0.32, p=0.013). We have provided proof-of-principle studies to demonstrated the utility of a novel DEP-based technology for minimally invasive brain cancer detection using undiluted patient plasma.

Temozolomide (TMZ), the standard-of-care chemotherapy for glioblastoma, is a DNA alkylating drug. At cytotoxic doses, TMZ Induces high levels of alkylated DNA, including O6 methyl-guanine (O6MG), ultimately triggering cell death. In sub-lethal levels, O6MGs undergo non-Watson-Crick pairing, facilitating mutagenesis and promote acquired resistance. Whether the cytotoxic or mutagenic effects of TMZ predominate is largely determined by the DNA repair enzyme, Methyl-guanine-methyl transferase (MGMT). MGMT restores TMZ-alkylated DNA into its native form. To date, studies of MGMT have largely focused on its expression in large populations of cells. Variation of MGMT expression at a single cell level and pertinence of this variation to acquired TMZ resistance remains poorly understood. In single cells derived from the same patient-derived glioblastoma line, we found that the cell-to-cell variability in MGMT mRNA expression differed by as much as 10-fold. The distribution of single cell MGMT expression approximated that of a normal distribution. Upon treatment with TMZ, the mean expression of MGMT increased by two-fold. Additionally, TMZ treatment increased the standard deviation of the MGMT expression distribution by ten-fold, causing significant “widening” of the range the MGMT expression. Both of these effects were caused by degradation of miR-181d, an MGMT suppressing miRNA. TMZ treatment triggered the activation of ATM- and Rad3- related (ATR) kinase, which in turn activated Polyribonucleotide nucleotidyltransferase 1 (PNPT1), an RNAse that ultimately degradation of miR-181d. Our results suggest that RNA degradation enhanced stochastic variation in MGMT expression, allowing a wider range of MGMT expression from which the optimally fit clone can emerge. This novel form of TMZ resistance can be suppressed by over-expression of miR-181d, suggesting the potential of microRNA delivery as a therapeutic strategy. Accordingly, miR-181d delivery combined with TMZ treatment effectively cured intracranial murine models of patient derived glioblastoma xenografts (PDX) that expressed high levels of MGMT.

The underlying mechanisms related to the prion-like propagation of tau is poorly understood. The interest in exosomes has grown dramatically over the past few years, specifically with their role in cell-to-cell communication and as carriers of Alzheimer's disease (AD)-related proteins. Here, we determined if exosomes mediate the transfer of human tau to wild-type mouse neurons, and induce AD-like pathology in vivo. Exosomes secreted into the cell culture media were isolated from neuronal cultures derived from non-demented controls and neuronally – differentiated, human induced pluripotent stem cells that express the tau P301L and V337M mutations (NiPSCEs – tau(LM)). Exosomes were characterized by size and Western Blot (WB). NiPSCEs – tau(LM) suspensions were injected into normal mice CNS and pathological changes were characterized by immunostaining at one and two months’ post injection. Following NiPSCEs – tau(LM) injection and animal sacrifice, mouse plasma exosomes were extracted, precipitated, and exosome protein cargo were quantified by ELISA. Size distribution for neuronal exosome preparations fluctuated significantly while NiPSCE – tau(LM) were uniform and smaller in size as demonstrated by Nanosight. KJ9A, a pan-human tau antibody recognized endogenous human tau only in NiPSCEs-tau(LM) samples as demonstrated by WB. Full length and pathological forms of tau, as measured by KJ9A, Alz50, MC1 and PHF1, and oligomeric amyloid beta (oAb) as measured by 6E10, were visualized in the hippocampus, entorhinal cortex, and subcortical thalamic regions at one and two months post – injection. Moreover, laminar disorganization and microtubule disruption, as measured by MAP2, SMI312, were observed in the brains of injected mice at both time points. Lastly, plasmas exosomes isolated from injected mice contained human tau species as measured by ELISA, suggesting exosome trafficking from the CNS to the periphery. Here, we are the first to demonstrate that secreted exosomes isolated from hiPSCs can propagate human tau and oAb species to multiple brain regions and traffic into the periphery. Our model system provides a means to study transcellular propagation of exosome cargo, and related neurodegenerative mechanisms. Together, our data suggests that exosomes are mediators of AD pathogenesis and can serve as potential targets for AD therapeutics.

BACKGROUND:RNAs within extracellular vesicles (EVs) have potential as diagnostic biomarkers for patients with cancer and are identified in a variety of biofluids. Glioblastomas (GBMs) release EVs containing RNA into cerebrospinal fluid (CSF). Here we describe a multi-institutional study of RNA extracted from CSF-derived EVs of GBM patients to detect the presence of tumor-associated amplifications and mutations in epidermal growth factor receptor (EGFR).METHODS:CSF and matching tumor tissue were obtained from patients undergoing resection of GBMs. We determined wild-type (wt)EGFR DNA copy number amplification, as well as wtEGFR and EGFR variant (v)III RNA expression in tumor samples. We also characterized wtEGFR and EGFRvIII RNA expression in CSF-derived EVs.RESULTS:EGFRvIII-positive tumors had significantly greater wtEGFR DNA amplification (P = 0.02) and RNA expression (P = 0.03), and EGFRvIII-positive CSF-derived EVs had significantly more wtEGFR RNA expression (P = 0.004). EGFRvIII was detected in CSF-derived EVs for 14 of the 23 EGFRvIII tissue-positive GBM patients. Conversely, only one of the 48 EGFRvIII tissue-negative patients had the EGFRvIII mutation detected in their CSF-derived EVs. These results yield a sensitivity of 61% and a specificity of 98% for the utility of CSF-derived EVs to detect an EGFRvIII-positive GBM.CONCLUSION:Our results demonstrate CSF-derived EVs contain RNA signatures reflective of the underlying molecular genetic status of GBMs in terms of wtEGFR expression and EGFRvIII status. The high specificity of the CSF-derived EV diagnostic test gives us an accurate determination of positive EGFRvIII tumor status and is essentially a less invasive "liquid biopsy" that might direct mutation-specific therapies for GBMs.

Tauopathies are a class of neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia and progressive supranuclear palsy, which are associated with the pathological aggregation of tau protein into neurofibrillary tangles (NFT). Studies have characterized tau as a "prion-like" protein given its ability to form distinct, stable amyloid conformations capable of transcellular and multigenerational propagation in clonal fashion. It has been proposed that progression of tauopathy could be due to the prion-like propagation of tau, suggesting the possibility that end-stage pathologies, like NFT formation, may require an instigating event such as tau seeding. To investigate this, we applied a novel human induced pluripotent stem cell (hiPSC) system we have developed to serve as a human neuronal model. We introduced the tau repeat domain (tau-RD) with P301L and V337M (tau-RD-LM) mutations into hiPSC-derived neurons and observed expression of tau-RD at levels similar to total tau in postmortem AD brains. Tau aggregation occurred without the addition of recombinant tau fibrils. The conditioned media from tau-RD cultures contained tau-RD seeds, which were capable of inducing aggregate formation in homotypic mode in non-transduced recipient neuronal cultures. The resultant NFTs were thioflavin-positive, silver stain-positive, and assumed fibrillary appearance on transmission electron microscopy (TEM) with immunogold, which revealed paired helical filament 1 (PHF1)-positive NFTs, representing possible recruitment of endogenous tau in the aggregates. Functionally, expression of tau-RD caused neurotoxicity that manifested as axon retraction, synaptic density reduction, and enlargement of lysosomes. The results of our hiPSC study were reinforced by the observation that Tau-RD-LM is excreted in exosomes, which mediated the transfer of human tau to wild-type mouse neurons in vivo. Our hiPSC human neuronal system provides a model for further studies of tau aggregation and pathology as well as a means to study transcellular propagation and related neurodegenerative mechanisms.