Abstract
The poor outcome of patients with glioblastoma (GBM) is at least partly due to the inability to deliver therapeutic agents to the tumor. We have shown that exosomes, naturally occurring ...nano-size extracellular vesicles, are capable of delivering antiglioma microRNAs (MiRs) to brain tumors (Lang, FM et al. Neuro Oncol, 2018;20(3):380–390). However, our studies suggested that there is significant opportunity to increase packaging efficiency and delivery specificity of exosomes. To this end, we engineered exosomes to express specific viral proteins (called eExos) in order to enhance packaging and delivery capabilities of antiglioma genes. These eExos are created by transfecting HEK 293 cells with plasmids containing viral proteins and a plasmid of the therapeutic gene. After 72 hrs, differential ultracentrifugation was used to isolate the exosomes. To test the efficacy of these novel eExos, we transfected them with a plasmid containing Cre recombinase (as the therapeutic gene), and treated U87 cells harboring a dsRed/eGFP Cre recombinase/LoxP site (U87dsR/GFP). In in vitro studies, treatment of U87dsR/GFP with a single dose of eExos resulted in 82% conversion rate of cells from red to green, compared to control exosomes (< 18% green cells). In in vivo studies, a single intratumoral injection of eExos into mice harboring 7-day old intracranial U87dsR/GFP gliomas, resulted in significant increases in green cells compared to control exosomes when tumors were harvested at day 10. Mechanistic studies employing florescent microscopy demonstrate that in contrast to natural exosomes, eExos deliver their cargo to the nucleus rather than to lysosomes, avoiding degradation of the delivered agent and facilitating expression of the plasmid. We conclude that eExos, engineered to contain specific viral proteins, are capable of packaging and delivering antiglioma genes more effectively than natural exosomes and may overcome the current inability to deliver biological therapeutic agents to brain tumors.
Malignant gliomas are composed of heterogeneous cell populations and the clonal evolution of these cells is one of the key reasons for treatment resistance and tumor recurrence. A fundamental ...challenge in studying clonal evolution in these tumors is the difficulty in isolating the phenotype-associated (e.g. treatment resistant) sub-populations from the heterogeneous population. We developed an approach to individually barcode and isolate specific cell subpopulations using a CRISPR (clustered, regularly interspaced, short palindromic repeat)-Cas9D10A targetable, single-cell barcoding library with a complexity of 36 million unique barcodes. This approach enabled us to uniquely barcode > 1 million cells, identify enriching barcoded sub-populations following treatment and then isolate the resistant subpopulation using the subpopulation-specific barcode. Each barcode sequence carrying two guide RNAs so that CRISPR-Cas9D10A targeting can lead to “switching” (i.e. frameshift) of an EGFP (Enhanced Green Fluorescent Protein) reporter. Thus, targeted cells lose their EGFP signal and can be specifically isolated by cell sorting. Proof of principle studies showed that specific barcoding cells could be efficiently targeted to turn off EGFP and subpopulations isolated. Subsequent large-scale implementation of this approach has been performed to isolate resistant subpopulations following radiation and temozolomide. Our approach will lead to identification of the both pre-existing and acquired specific somatic genetic or epigenetic changes driving treatment resistance in patients with malignant gliomas.
Activation of central PPARgamma promotes food intake and body weight gain; however, the identity of the neurons that express PPARgamma and mediate the effect of this nuclear receptor on energy ...homeostasis is unknown. Here, we determined that selective ablation of PPARgamma in murine proopiomelanocortin (POMC) neurons decreases peroxisome density, elevates reactive oxygen species, and induces leptin sensitivity in these neurons. Furthermore, ablation of PPARgamma in POMC neurons preserved the interaction between mitochondria and the endoplasmic reticulum, which is dysregulated by HFD. Compared with control animals, mice lacking PPARgamma in POMC neurons had increased energy expenditure and locomotor activity; reduced body weight, fat mass, and food intake; and improved glucose metabolism when exposed to high-fat diet (HFD). Finally, peripheral administration of either a PPARgamma activator or inhibitor failed to affect food intake of mice with POMC-specific PPARgamma ablation. Taken together, our data indicate that PPARgamma mediates cellular, biological, and functional adaptations of POMC neurons to HFD, thereby regulating whole- body energy balance.
Acral melanoma (AM) is a rare, aggressive type of cutaneous melanoma (CM) with a distinct genetic profile. We aimed to identify a methylome signature distinguishing primary acral lentiginous melanoma ...(PALM) from primary non-lentiginous AM (NALM), metastatic ALM (MALM), primary non-acral CM (PCM), and acral nevus (AN). A total of 22 PALM, nine NALM, 10 MALM, nine PCM, and three AN were subjected to genome-wide methylation analysis using the Illumina Infinium Methylation EPIC array interrogating 866,562 CpG sites. A prominent finding was that the methylation profiles of PALM and NALM were distinct. Four of the genes most differentially methylated between PALM and NALM or MALM were
,
,
, and
. However, when primary AMs (PALM + NALM) were compared with MALM,
and
were the most differentially methylated, highlighting their pivotal role in the metastatic potential of AMs. Patients with NALM had significantly worse disease-specific survival (DSS) than patients with PALM. Aberrant methylation was significantly associated with aggressive clinicopathologic parameters and worse DSS. Our study emphasizes the importance of distinguishing the two epigenetically distinct subtypes of AM. We also identified novel epigenetic prognostic biomarkers that may serve to risk-stratify patients with AM and may be leveraged for the development of targeted therapies.
Thrombocytosis is triggered by and promotes tumor growth. The relationship between the change in circulating platelets after chemoradiation therapy (CRT) or adjuvant temozolomide (TMZ) and survival ...in glioblastoma remains unclear. We hypothesized that an increase in platelets after these treatments would be predictive of a shorter survival.
We retrospectively reviewed data on 122 patients with newly diagnosed, pathologically proven glioblastoma who had been treated with surgery, followed by CRT and adjuvant TMZ, from 2007 to 2016. The association between the changes in blood count levels and survival was analyzed by the log-rank test. To adjust for confounding, we performed a multivariate analysis using known prognostic co-variates.
Patients were dichotomized on the basis of the relative change in platelets after CRT from the baseline: ≤30% increase, low (
= 101)
>30% increase, high (
= 12). The median survival for high
. low platelets were 11
28 months (
= 0.0062). No significant survival differences were observed on the basis of platelet changes during adjuvant TMZ. Similarly, changes in lymphocyte counts were not significantly prognostic. On multivariate analysis, MGMT, performance status, and an increase in platelets after CRT were significantly associated with survival (HR for platelets, 4.5; 95% confidence interval, 1.6-12.6).
Increased platelet counts after CRT are predictive of poor survival in glioblastoma. The effect is platelet specific and does not reflect bone marrow changes, as lymphocyte changes were not significantly prognostic. These results suggest an interaction between platelets and tumor aggressiveness. Thus, platelets serve as a novel, minimally invasive liquid biopsy for predicting outcome.
Glioma sphere-forming cells (GSCs) are important in glioblastoma (GBM) initiation, maintenance, and treatment resistance. We performed whole exome and transcriptome sequencing, DNA methylation ...profiling, DNA copy number determination, and functional characterization of 43 GSCs and matching tumors. Comparative analyses revealed that GSCs recapitulate the molecular landscape of GBM and provide a unique means for discovering the inter- and intra-tumor heterogeneity of GBM. We performed clonogenic assays to explore the relationship between methylation status and radiation response in twelve IDH wild type GSCs irradiated with 2-, 4-, and 6-Gy ionizing radiation. The survival fraction at 4Gy and 6Gy (SF4 and SF6, respectively) were used to dichotomize GSCs as either radiation-sensitive or resistant. DNA CpG methylomes of the GSCs were profiled using Infinium 450K methylation beadchip arrays. We observed that 304,458 out of 465,844 methylation probes (65.4%) showed increased methylation in radiation-resistant relative to radiation-sensitive GSCs (Fisher’s Exact Test, p < 1e-15). Using GSEA, we observed that fifteen of sixteen oxidative stress genes were methylated in radiation-resistant GSCs (p-value=0.019), suggesting an association between radiation-resistance and reactive oxygen species metabolism (ROS). To validate our finding, we derived a methylation signature differentiating the two GSC radiation response groups and used this to classify TCGA cases that received radiotherapy into a responder and non-responder group. We found that survival was significantly different between the two groups (median survival 84 vs. 61 weeks; HR 1.64 adjusting for patient age, p-value<0.008), suggesting that the methylation signature predicts clinical response to radiation treatment. This study identified a novel predictor of radiation response and confirms that the genomic landscape of GSCs can be used to determine clinical and functional properties of GBM.
Background Cesarean scar pregnancy （CSP） is a very rare but life-threatening entity and there is no optimal management strategy.Here we report a successfully conservative treatment of CSP.Methods We ...retrospectively analyzed the clinical data of 54 women with CSP,who underwent uterine artery embolization between January 2007 and September 2012 at the Peking University People＇s Hospital.We evaluated the clinical outcomes,the technique and the complications of uterine artery embolization.Results Of the 54 patients,2 patients with hemorrhage after induced abortion received bilateral uterine artery embolization treatment alone,and 52 patients underwent suction curettage after bilateral uterine artery embolization.All 54 women were successfully cured,without any severe complications,and uterine function was restored.During the follow-up,one patient had accidental normal interuterine pregnancy and received induced abortion during the first trimester.Conclution Uterine artery embolization combined with suction curettage is an effective and safe conservative treatment for cesarean scar pregnancy.
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