miRNA Signature of Schwannomas: Possible Role(s) of “Tumor Suppressor” miRNAs in Benign Tumors

miRNAs have been recently implicated as drivers in several carcinogenic processes, where they can act either as oncogenes or as tumor suppressors. Schwannomas arise from Schwann cells, the myelinating cells of the peripheral nervous system. These benign tumors typically result from loss of the neurofibromatosis type 2 (NF2) tumor suppressor gene. We have recently carried out high-throughput miRNA expression profiling of human vestibular schwannomas using an array representing 407 known miRNAs in order to explore the role of miRNAs in the tumorigenesis of schwannomas. We found that miR-7 functions as a “tumor suppressor” by targeting proteins in three major oncogenic pathways - EGFR, Pak1, and Ack1. Interestingly, in this study, we also observed that several previously described potential tumor suppressor miRNAs that are down-regulated in malignant tumors were up-regulated in schwannomas. Here we discuss the possibility that “tumor suppressor” miRNAs may play a role in the transition stage(s) of cancer from benign to malignant forms

Circulating Tumor Biomarkers in Meningiomas Reveal a Signature of Equilibrium Between Tumor Growth and Immune Modulation

Meningiomas are primary central nervous system (CNS) tumors that originate from the arachnoid cells of the meninges. Recurrence occurs in higher grade meningiomas and a small subset of Grade I meningiomas with benign histology. Currently, there are no established circulating tumor markers which can be used for diagnostic and prognostic purposes in a non-invasive way for meningiomas. Here, we aimed to identify potential biomarkers of meningioma in patient sera. For this purpose, we collected preoperative (n = 30) serum samples from the meningioma patients classified as Grade I (n = 23), Grade II (n = 4), or Grade III (n = 3). We used a high-throughput, multiplex immunoassay cancer panel comprising of 92 cancer-related protein biomarkers to explore the serum protein profiles of meningioma patients. We detected 14 differentially expressed proteins in the sera of the Grade I meningioma patients in comparison to the age- and gender-matched control subjects (n = 12). Compared to the control group, Grade I meningioma patients showed increased serum levels of amphiregulin (AREG), CCL24, CD69, prolactin, EGF, HB-EGF, caspase-3, and decreased levels of VEGFD, TGF-α, E-Selectin, BAFF, IL-12, CCL9, and GH. For validation studies, we utilized an independent set of meningioma tumor tissue samples (Grade I, n = 20; Grade II, n = 10; Grade III, n = 6), and found that the expressions of amphiregulin and Caspase3 are significantly increased in all grades of meningiomas either at the transcriptional or protein level, respectively. In contrast, the gene expression of VEGF-D was significantly lower in Grade I meningioma tissue samples. Taken together, our study identifies a meningioma-specific protein signature in blood circulation of meningioma patients and highlights the importance of equilibrium between tumor-promoting factors and anti-tumor immunity.Peer reviewe

Genetically Engineered Microvesicles Carrying Suicide mRNA/Protein Inhibit Schwannoma Tumor Growth

Microvesicles (MVs) play an important role in intercellular communication by carrying mRNAs, microRNAs (miRNAs), non-coding RNAs, proteins, and DNA from cell to cell. To our knowledge, this is the first report of delivery of a therapeutic mRNA/protein via MVs for treatment of cancer. We first generated genetically engineered MVs by expressing high levels of the suicide gene mRNA and protein–cytosine deaminase (CD) fused to uracil phosphoribosyltransferase (UPRT) in MV donor cells. MVs were isolated from these cells and used to treat pre-established nerve sheath tumors (schwannomas) in an orthotopic mouse model. We demonstrated that MV-mediated delivery of CD-UPRT mRNA/protein by direct injection into schwannomas led to regression of these tumors upon systemic treatment with the prodrug (5-fluorocytosine (5-FC)), which is converted within tumor cells to 5-fluorouracil (5-FU)–an anticancer agent. Taken together, these studies suggest that MVs can serve as novel cell-derived “liposomes” to effectively deliver therapeutic mRNA/proteins to treatment of diseases

miQC : An adaptive probabilistic framework for quality control of single-cell RNA-sequencing data

Single-cell RNA-sequencing (scRNA-seq) has made it possible to profile gene expression in tissues at high resolution. An important preprocessing step prior to performing downstream analyses is to identify and remove cells with poor or degraded sample quality using quality control (QC) metrics. Two widely used QC metrics to identify a 'low-quality' cell are (i) if the cell includes a high proportion of reads that map to mitochondrial DNA (mtDNA) encoded genes and (ii) if a small number of genes are detected. Current best practices use these QC metrics independently with either arbitrary, uniform thresholds (e.g. 5%) or biological context-dependent (e.g. species) thresholds, and fail to jointly model these metrics in a data-driven manner. Current practices are often overly stringent and especially untenable on certain types of tissues, such as archived tumor tissues, or tissues associated with mitochondrial function, such as kidney tissue [1]. We propose a data-driven QC metric (miQC) that jointly models both the proportion of reads mapping to mtDNA genes and the number of detected genes with mixture models in a probabilistic framework to predict the low-quality cells in a given dataset. We demonstrate how our QC metric easily adapts to different types of single-cell datasets to remove low-quality cells while preserving high-quality cells that can be used for downstream analyses. Our software package is available at https://bioconductor.org/packages/miQC. Author summary We developed the miQC package to predict the low-quality cells in a given scRNA-seq dataset by jointly modeling both the proportion of reads mapping to mitochondrial DNA (mtDNA) genes and the number of detected genes using mixture models in a probabilistic framework. We demonstrate how our QC metric easily adapts to different types of single-cell datasets to remove low-quality cells while preserving high-quality cells that can be used for downstream analyses.Peer reviewe

PRISM : recovering cell-type-specific expression profiles from individual composite RNA-seq samples

Motivation: A major challenge in analyzing cancer patient transcriptomes is that the tumors are inherently heterogeneous and evolving. We analyzed 214 bulk RNA samples of a longitudinal, prospective ovarian cancer cohort and found that the sample composition changes systematically due to chemotherapy and between the anatomical sites, preventing direct comparison of treatment-naive and treated samples. Results: To overcome this, we developed PRISM, a latent statistical framework to simultaneously extract the sample composition and cell-type-specific whole-transcriptome profiles adapted to each individual sample. Our results indicate that the PRISM-derived composition-free transcriptomic profiles and signatures derived from them predict the patient response better than the composite raw bulk data. We validated our findings in independent ovarian cancer and melanoma cohorts, and verified that PRISM accurately estimates the composition and cell-type-specific expression through whole-genome sequencing and RNA in situ hybridization experiments.Peer reviewe

Longitudinal single-cell RNA-seq analysis reveals stress-promoted chemoresistance in metastatic ovarian cancer

Chemotherapy resistance is a critical contributor to cancer mortality and thus an urgent unmet challenge in oncology. To characterize chemotherapy resistance processes in high-grade serous ovarian cancer, we prospectively collected tissue samples before and after chemotherapy and analyzed their transcriptomic profiles at a single-cell resolution. After removing patient-specific signals by a novel analysis approach, PRIMUS, we found a consistent increase in stress-associated cell state during chemotherapy, which was validated by RNA in situ hybridization and bulk RNA sequencing. The stress-associated state exists before chemotherapy, is subclonally enriched during the treatment, and associates with poor progression-free survival. Co-occurrence with an inflammatory cancer-associated fibroblast subtype in tumors implies that chemotherapy is associated with stress response in both cancer cells and stroma, driving a paracrine feed-forward loop. In summary, we have found a resistant state that integrates stromal signaling and subclonal evolution and offers targets to overcome chemotherapy resistance.Peer reviewe

Locus-specific LINE-1 expression in clinical ovarian cancer specimens at the single-cell level

Long interspersed nuclear elements (LINE-1s/L1s) are a group of retrotransposons that can copy themselves within a genome. In humans, it is the most successful transposon in nucleotide content. L1 expression is generally mild in normal human tissues, but the activity has been shown to increase significantly in many cancers. Few studies have examined L1 expression at single-cell resolution, thus it is undetermined whether L1 reactivation occurs solely in malignant cells within tumors. One of the cancer types with frequent L1 activity is high-grade serous ovarian carcinoma (HGSOC). Here, we identified locus-specific L1 expression with 3′ single-cell RNA sequencing in pre- and post-chemotherapy HGSOC sample pairs from 11 patients, and in fallopian tube samples from five healthy women. Although L1 expression quantification with the chosen technique was challenging due to the repetitive nature of the element, we found evidence of L1 expression primarily in cancer cells, but also in other cell types, e.g. cancer-associated fibroblasts. The expression levels were similar in samples taken before and after neoadjuvant chemotherapy, indicating that L1 transcriptional activity was unaffected by clinical platinum-taxane treatment. Furthermore, L1 activity was negatively associated with the expression of MYC target genes, a finding that supports earlier literature of MYC being an L1 suppressor.Peer reviewe

PRISM: Recovering cell type specific expression profiles from individual composite RNA-seq samples

Motivation: A major challenge in analyzing cancer patient transcriptomes is that the tumors are inherently heterogeneous and evolving. We analyzed 214 bulk RNA samples of a longitudinal, prospective ovarian cancer cohort and found that the sample composition changes systematically due to chemotherapy and between the anatomical sites, preventing direct comparison of treatment-naive and treated samples.Results: To overcome this, we developed PRISM, a latent statistical framework to simultaneously extract the sample composition and cell type specific whole-transcriptome profiles adapted to each individual sample. Our results indicate that the PRISM-derived composition-free transcriptomic profiles and signatures derived from them predict the patient response better than the composite raw bulk data. We validated our findings in independent ovarian cancer and melanoma cohorts, and verified that PRISM accurately estimates the composition and cell type specific expression through whole-genome sequencing and RNA in situ hybridization experiments.</p

Longitudinal single-cell RNA-seq analysis reveals stress-promoted chemoresistance in metastatic ovarian cancer

Chemotherapy resistance is a critical contributor to cancer mortality and thus an urgent unmet challenge in oncology. To characterize chemotherapy resistance processes in high-grade serous ovarian cancer, we prospectively collected tissue samples before and after chemotherapy and analyzed their transcriptomic profiles at a single-cell resolution. After removing patient-specific signals by a novel analysis approach, PRIMUS, we found a consistent increase in stress-associated cell state during chemotherapy, which was validated by RNA in situ hybridization and bulk RNA sequencing. The stress-associated state exists before chemotherapy, is subclonally enriched during the treatment, and associates with poor progression-free survival. Co-occurrence with an inflammatory cancer-associated fibroblast subtype in tumors implies that chemotherapy is associated with stress response in both cancer cells and stroma, driving a paracrine feed-forward loop. In summary, we have found a resistant state that integrates stromal signaling and subclonal evolution and offers targets to overcome chemotherapy resistance