Synergistic hyperactivation of both mTORC1 and mTORC2 underlies the neural abnormalities of PTEN-deficient human neurons and cortical organoids.

Mutations in the phosphatase and tensin homolog (PTEN) gene are associated with severe neurodevelopmental disorders. Loss of PTEN leads to hyperactivation of the mechanistic target of rapamycin (mTOR), which functions in two distinct protein complexes, mTORC1 and mTORC2. The downstream signaling mechanisms that contribute to PTEN mutant phenotypes are not well delineated. Here, we show that pluripotent stem cell-derived PTEN mutant human neurons, neural precursors, and cortical organoids recapitulate disease-relevant phenotypes, including hypertrophy, electrical hyperactivity, enhanced proliferation, and structural overgrowth. PTEN loss leads to simultaneous hyperactivation of mTORC1 and mTORC2. We dissect the contribution of mTORC1 and mTORC2 by generating double mutants of PTEN and RPTOR or RICTOR, respectively. Our results reveal that the synergistic hyperactivation of both mTORC1 and mTORC2 is essential for the PTEN mutant human neural phenotypes. Together, our findings provide insights into the molecular mechanisms that underlie PTEN-related neural disorders and highlight novel therapeutic targets.

Extracellular vesicles from biological fluids as potential markers in castration resistant prostate cancer.

PURPOSE: Extracellular vesicles (EV) secreted from cancer cells are present in various biological fluids, carrying distinctly different cellular components compared to normal cells, and have great potential to be used as markers for disease initiation, progression, and response to treatment. This under-utilised tool provides insights into a better understanding of prostate cancer. METHODS: EV from serum and urine of healthy men and castration-resistant prostate cancer (CRPC) patients were isolated and characterised by transmission electron microscopy, particle size analysis, and western blot. Proteomic and cholesterol liquid chromatography-mass spectrometry (LC-MS) analyses were conducted. RESULTS: There was a successful enrichment of small EV/exosomes isolated from serum and urine. EV derived from biological fluids of CRPC patients had significant differences in composition when compared with those from healthy controls. Analysis of matched serum and urine samples from six prostate cancer patients revealed specific EV proteins common in both types of biological fluid for each patient. CONCLUSION: Some of the EV proteins identified from our analyses have potential to be used as CRPC markers. These markers may depict a pattern in cancer progression through non-invasive sample collection.

Modeling Developmental Brain Diseases Using Human Pluripotent Stem Cells-Derived Brain Organoids - Progress and Perspective.

Developmental brain diseases encompass a group of conditions resulting from genetic or environmental perturbations during early development. Despite the increased research attention in recent years following recognition of the prevalence of these diseases, there is still a significant lack of knowledge of their etiology and treatment options. The genetic and clinical heterogeneity of these diseases, in addition to the limitations of experimental animal models, contribute to this difficulty. In this regard, the advent of brain organoid technology has provided a new means to study the cause and progression of developmental brain diseases in vitro. Derived from human pluripotent stem cells, brain organoids have been shown to recapitulate key developmental milestones of the early human brain. Combined with technological advancements in genome editing, tissue engineering, electrophysiology, and multi-omics analysis, brain organoids have expanded the frontiers of human neurobiology, providing valuable insight into the cellular and molecular mechanisms of normal and pathological brain development. This review will summarize the current progress of applying brain organoids to model human developmental brain diseases and discuss the challenges that need to be overcome to further advance their utility.

Modeling PTEN overexpression-induced microcephaly in human brain organoids.

The phosphatase and tensin homolog (PTEN) protein, encoded by the PTEN gene on chromosome 10, is a negative regulator of the phosphoinositide 3-kinase (PI3K) signaling pathway. Loss of PTEN has been linked to an array of human diseases, including neurodevelopmental disorders such as macrocephaly and autism. However, it remains unknown whether increased dosage of PTEN can lead to human disease. A recent human genetics study identifies chromosome 10 microduplication encompassing PTEN in patients with microcephaly. Here we generated a human brain organoid model of increased PTEN dosage. We showed that mild PTEN overexpression led to reduced neural precursor proliferation, premature neuronal differentiation, and the formation of significantly smaller brain organoids. PTEN overexpression resulted in decreased AKT activation, and treatment of wild-type organoids with an AKT inhibitor recapitulated the reduced brain organoid growth phenotypes. Together, our findings provide functional evidence that PTEN is a dosage-sensitive gene that regulates human neurodevelopment, and that increased PTEN dosage in brain organoids results in microcephaly-like phenotypes.

Duplex Proximity Sequencing (Pro-Seq): A method to improve DNA sequencing accuracy without the cost of molecular barcoding redundancy.

A challenge in the clinical adoption of cell-free DNA (cfDNA) liquid biopsies for cancer care is their high cost compared to potential reimbursement. The most common approach used in liquid biopsies to achieve high specificity detection of circulating tumor DNA (ctDNA) among a large background of normal cfDNA is to attach molecular barcodes to each DNA template, amplify it, and then sequence it many times to reach a low-error consensus. In applications where the highest possible specificity is required, error rate can be lowered further by independently detecting the sequences of both strands of the starting cfDNA. While effective in error reduction, the additional sequencing redundancy required by such barcoding methods can increase the cost of sequencing up to 100-fold over standard next-generation sequencing (NGS) of equivalent depth. We present a novel library construction and analysis method for NGS that achieves comparable performance to the best barcoding methods, but without the increase in sequencing and subsequent sequencing cost. Named Proximity-Sequencing (Pro-Seq), the method merges multiple copies of each template into a single sequencing read by physically linking the molecular copies so they seed a single sequencing cluster. Since multiple DNA copies of the same template are compared for consensus within the same cluster, sequencing accuracy is improved without the use of redundant reads. Additionally, it is possible to represent both senses of the starting duplex in a single cluster. The resulting workflow is simple, and can be completed by a single technician in a work day with minimal hands on time. Using both cfDNA and cell line DNA, we report the average per-mutation detection threshold and per-base analytical specificity to be 0.003% and >99.9997% respectively, demonstrating that Pro-Seq is among the highest performing liquid biopsy technologies in terms of both sensitivity and specificity, but with greatly reduced sequencing costs compared to existing methods of comparable accuracy.

Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers.

Targeted Next Generation Sequencing (NGS) is being adopted increasingly broadly in many research, commercial and clinical settings. Currently used target capture methods, however, typically require complex and lengthy (sometimes multi-day) workflows that complicates their use in certain applications. In addition, small panels for high sequencing depth applications such as liquid biopsy typically have low on-target rates, resulting in unnecessarily high sequencing cost. We have developed a novel targeted sequencing library preparation method, named Linked Target Capture (LTC), which replaces typical multi-day target capture workflows with a single-day, combined 'target-capture-PCR' workflow. This approach uses physically linked capture probes and PCR primers and is expected to work with panel sizes from 100 bp to >10 Mbp. It reduces the time and complexity of the capture workflow, eliminates long hybridization and wash steps and enables rapid library construction and target capture. High on-target read fractions are achievable due to repeated sequence selection in the target-capture-PCR step, thus lowering sequencing cost. We have demonstrated this technology on sample types including cell-free DNA (cfDNA) and formalin-fixed, paraffin-embedded (FFPE) derived DNA, capturing a 35-gene pan-cancer panel, and therein detecting single nucleotide variants, copy number variants, insertions, deletions and gene fusions. With the integration of unique molecular identifiers (UMIs), variants as low as 0.25% abundance were detected, limited by input mass and sequencing depth. Additionally, sequencing libraries were prepared in less than eight hours from extracted DNA to loaded sequencer, demonstrating that LTC holds promise as a broadly applicable tool for rapid, cost-effective and high performance targeted sequencing.

Exosomes confer pro-survival signals to alter the phenotype of prostate cells in their surrounding environment.

Prostate cancer (PCa) is the most frequently diagnosed cancer in men. Current research on tumour-related extracellular vesicles (EVs) suggests that exosomes play a significant role in paracrine signaling pathways, thus potentially influencing cancer progression via multiple mechanisms. In fact, during the last decade numerous studies have revealed the role of EVs in the progression of various pathological conditions including cancer. Moreover, differences in the proteomic, lipidomic, and cholesterol content of exosomes derived from PCa cell lines versus benign prostate cell lines confirm that exosomes could be excellent biomarker candidates. As such, as part of an extensive proteomic analysis using LCMS we previously described a potential role of exosomes as biomarkers for PCa. Current evidence suggests that uptake of EV's into the local tumour microenvironment encouraging us to further examine the role of these vesicles in distinct mechanisms involved in the progression of PCa and castration resistant PCa. For the purpose of this study, we hypothesized that exosomes play a pivotal role in cell-cell communication in the local tumour microenvironment, conferring activation of numerous survival mechanisms during PCa progression and development of therapeutic resistance. Our in vitro results demonstrate that PCa derived exosomes significantly reduce apoptosis, increase cancer cell proliferation and induce cell migration in LNCaP and RWPE-1 cells. In conjunction with our in vitro findings, we have also demonstrated that exosomes increased tumor volume and serum PSA levels in vivo when xenograft bearing mice were administered DU145 cell derived exosomes intravenously. This research suggests that, regardless of androgen receptor phenotype, exosomes derived from PCa cells significantly enhance multiple mechanisms that contribute to PCa progression.