In vivo CAR T-cell generation in nonhuman primates using lentiviral vectors displaying a multidomain fusion ligand.

Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high costs and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVec platform, a lentiviral vector capable of generating CAR T cells in vivo.

Single Circulating-Tumor-Cell-Targeted Sequencing to Identify Somatic Variants in Liquid Biopsies in Non-Small-Cell Lung Cancer Patients.

Non-small-cell lung cancer (NSCLC) accounts for most cancer-related deaths worldwide. Liquid biopsy by a blood draw to detect circulating tumor cells (CTCs) is a tool for molecular profiling of cancer using single-cell and next-generation sequencing (NGS) technologies. The aim of the study was to identify somatic variants in single CTCs isolated from NSCLC patients by targeted NGS.

Beyond Circulating Tumor Cell Enumeration: Cell-Based Liquid Biopsy to Assess Protein Biomarkers and Cancer Genomics Using the RareCyte Platform.

The practice of medicine has steadily employed less invasive methods to obtain information derived from the tumor to guide clinical management of patients. Liquid biopsy-the sampling of blood-is a non-invasive method for generating information previously only available from tissue biopsies of the tumor mass. Analysis of fragmented circulating tumor DNA in the plasma is clinically used to identify actionable mutations and detect residual or recurrent disease.

Non-Invasive Profiling of Advanced Prostate Cancer via Multi-Parametric Liquid Biopsy and Radiomic Analysis.

Integrating liquid biopsies of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) with other minimally invasive measures may yield more comprehensive disease profiles. We evaluated the feasibility of concurrent cellular and molecular analysis of CTCs and cfDNA combined with radiomic analysis of CT scans from patients with metastatic castration-resistant PC (mCRPC). CTCs from 22 patients were enumerated, stained for PC-relevant markers, and clustered based on morphometric and immunofluorescent features using machine learning.

Identification and characterization of effusion tumor cells (ETCs) from remnant pleural effusion specimens.

Background: Cancer is a leading cause of death worldwide, and patients may have advanced disease when diagnosed. Targeted therapies guided by molecular subtyping of cancer can benefit patients significantly. Pleural effusions are frequently observed in patients with metastatic cancer and are routinely removed for therapeutic purposes; however, effusion specimens have not been recognized as typical substrates for clinical molecular testing because of frequent low tumor cellularity.

Multiple Molecular Mechanisms Rescue mtDNA Disease in C. elegans.

Genetic instability of the mitochondrial genome (mtDNA) plays an important role in human aging and disease. Thus far, it has proven difficult to develop successful treatment strategies for diseases that are caused by mtDNA instability. To address this issue, we developed a model of mtDNA disease in the nematode C.

Cyclophosphamide leads to persistent deficits in physical performance and in vivo mitochondria function in a mouse model of chemotherapy late effects.

Fatigue is the symptom most commonly reported by long-term cancer survivors and is increasingly recognized as related to skeletal muscle dysfunction. Traditional chemotherapeutic agents can cause acute toxicities including cardiac and skeletal myopathies. To investigate the mechanism by which chemotherapy may lead to persistent skeletal muscle dysfunction, mature adult mice were injected with a single cyclophosphamide dose and evaluated for 6 weeks.

Age-related accumulation of phosphorylated mitofusin 2 protein in retinal ganglion cells correlates with glaucoma progression.

Dysregulation of axonal bioenergetics is likely a key mechanism in the initiation and progression of age-related neurodegenerative diseases. Glaucoma is a quintessential neurodegenerative disorder characterized by progressive deterioration of the optic nerve (ON) and eventual death of retinal ganglion cells (RGCs). Age and elevation of intraocular pressure are key risk factors in glaucoma, but the common early hallmarks of decreased axonal transport and increased bioenergetic vulnerability likely underlie disease initiation.

Massively parallel digital transcriptional profiling of single cells.

Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3' mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency.

Mitochondrial DNA exhibits resistance to induced point and deletion mutations.

The accumulation of somatic mitochondrial DNA (mtDNA) mutations contributes to the pathogenesis of human disease. Currently, mitochondrial mutations are largely considered results of inaccurate processing of its heavily damaged genome. However, mainly from a lack of methods to monitor mtDNA mutations with sufficient sensitivity and accuracy, a link between mtDNA damage and mutation has not been established.

Cells Comprising the Prostate Cancer Microenvironment Lack Recurrent Clonal Somatic Genomic Aberrations.

Unlabelled: Prostate cancer-associated stroma (CAS) plays an active role in malignant transformation, tumor progression, and metastasis. Molecular analyses of CAS have demonstrated significant changes in gene expression; however, conflicting evidence exists on whether genomic alterations in benign cells comprising the tumor microenvironment (TME) underlie gene expression changes and oncogenic phenotypes. This study evaluates the nuclear and mitochondrial DNA integrity of prostate carcinoma cells, CAS, matched benign epithelium and benign epithelium-associated stroma by whole-genome copy-number analyses, targeted sequencing of TP53, and FISH.

Epithelial mesenchymal-like transition occurs in a subset of cells in castration resistant prostate cancer bone metastases.

TGFβ is a known driver of epithelial-mesenchymal transition (EMT) which is associated with tumor aggressiveness and metastasis. However, EMT has not been fully explored in clinical specimens of castration-resistant prostate cancer (CRPC) metastases. To assess EMT in CRPC, gene expression analysis was performed on 149 visceral and bone metastases from 62 CRPC patients and immunohistochemical analysis was performed on 185 CRPC bone and visceral metastases from 42 CRPC patients.

The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition.

For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs).

Targeted single molecule mutation detection with massively parallel sequencing.

Next-generation sequencing (NGS) technologies have transformed genomic research and have the potential to revolutionize clinical medicine. However, the background error rates of sequencing instruments and limitations in targeted read coverage have precluded the detection of rare DNA sequence variants by NGS. Here we describe a method, termed CypherSeq, which combines double-stranded barcoding error correction and rolling circle amplification (RCA)-based target enrichment to vastly improve NGS-based rare variant detection.

Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects.

Calorie restriction (CR) and rapamycin (RP) extend lifespan and improve health across model organisms. Both treatments inhibit mammalian target of rapamycin (mTOR) signaling, a conserved longevity pathway and a key regulator of protein homeostasis, yet their effects on proteome homeostasis are relatively unknown. To comprehensively study the effects of aging, CR, and RP on protein homeostasis, we performed the first simultaneous measurement of mRNA translation, protein turnover, and abundance in livers of young (3 month) and old (25 month) mice subjected to 10-week RP or 40% CR.

Skeletal muscle mitochondrial DNA deletions are not increased in CuZn-superoxide dismutase deficient mice.

Mitochondrial DNA (mtDNA) deletion mutations are proposed contributors to aging-related muscle fiber loss and atrophy, but evidence of a causal role for these mutations in muscle aging is lacking. Elucidating the etiology of in vivo mtDNA deletion mutations will help to better understand and test the possible roles of these mutations in aging. The implication of mtDNA mutations in aging is based on the susceptibility of mtDNA to oxidative damage by reactive oxygen species (ROS) due to residing in mitochondria, the primary source of endogenous ROS.

Digital genomic quantification of tumor-infiltrating lymphocytes.

Infiltrating T lymphocytes are frequently found in malignant tumors and are suggestive of a host cancer immune response. Multiple independent studies have documented that the presence and quantity of tumor-infiltrating lymphocytes (TILs) are strongly correlated with increased survival. However, because of methodological factors, the exact effect of TILs on prognosis has remained enigmatic, and inclusion of TILs in standard prognostic panels has been limited.

Targeted enrichment and high-resolution digital profiling of mitochondrial DNA deletions in human brain.

Due largely to the inability to accurately quantify and characterize de novo deletion events, the mechanisms underpinning the pathogenic expansion of mtDNA deletions in aging and neuromuscular disorders remain poorly understood. Here, we outline and validate a new tool termed 'Digital Deletion Detection' (3D) that allows for high-resolution analysis of rare deletions occurring at frequencies as low as 1 × 10(-8) . 3D is a three-step process that includes targeted enrichment for deletion-bearing molecules, single-molecule partitioning of genomes into thousands of droplets for direct quantification via droplet digital PCR, and breakpoint characterization using massively parallel sequencing.

Decreased mitochondrial DNA mutagenesis in human colorectal cancer.

Genome instability is regarded as a hallmark of cancer. Human tumors frequently carry clonally expanded mutations in their mitochondrial DNA (mtDNA), some of which may drive cancer progression and metastasis. The high prevalence of clonal mutations in tumor mtDNA has commonly led to the assumption that the mitochondrial genome in cancer is genetically unstable, yet this hypothesis has not been experimentally tested.

Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication.

DNA polymerase I (pol I) processes RNA primers during lagging-strand synthesis and fills small gaps during DNA repair reactions. However, it is unclear how pol I and pol III work together during replication and repair or how extensive pol I processing of Okazaki fragments is in vivo. Here, we address these questions by analyzing pol I mutations generated through error-prone replication of ColE1 plasmids.

Generation, function, and prognostic utility of somatic mitochondrial DNA mutations in cancer.

Exciting new studies are increasingly strengthening the link between mitochondrial mutagenesis and tumor progression. Here we provide a comprehensive review and meta-analysis of studies reporting on mitochondrial DNA mutations in common human cancers. We discuss possible mechanisms by which mitochondrial DNA mutations may influence carcinogenesis, outline important caveats for interpreting the detected mutations--particularly differentiating causality from association--and suggest how new mutational assays may help resolve fundamental controversies in the field and delineate the origin and expansion of neoplastic cell lineages.

Molecularly evolved thymidylate synthase inhibits 5-fluorodeoxyuridine toxicity in human hematopoietic cells.

Thymidylate synthase (TS) inhibitors, such as 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FUdR), are amongst the most frequently used chemotherapeutic drugs available, although their efficacy is often limited by myelotoxicity. An emerging strategy for overcoming bone marrow toxicity involves ex vivo genetic transfer of drug resistance to autologous hematopoietic progenitor cells, followed by reimplantation of the transfected cells before chemotherapy. Here we establish that expression of mutant TS genes, selected from millions of engineered variants, renders human hematopoietic cells resistant to 5-FUdR, and identify the most efficacious variant for gene therapeutic rescue of drug-induced myelosuppression.