Rare Population Research Articles

JAK/STAT signaling maintains an intermediate cell population during prostate basal cell fate determination.

Unipotent basal and luminal stem cells maintain prostate homeostasis, with an intermediate cell population emerging during prostate inflammation or cancer. However, the identities of basal stem cell and intermediate cell population remain unclear. Here we identified a rare intermediate cell population expressing luminal markers (termed Basal-B) with enhanced organoid formation capacity, and a larger basal population (termed Basal-A). Genetic lineage tracing revealed Basal-B cells represented a transient basal stem cell state during prostate homeostasis and androgen-mediated regeneration. Activated JAK/STAT signaling was identified in Basal-B cells, and its inhibition significantly reduced Basal-B markers expression. Inflammation increased Basal-B-to-luminal cell transdifferentiation, but JAK/STAT inhibition notably attenuated this effect. Pten gene deletion increased Nkx3.1-expressing Basal-B-like cell population and led to neoplasia. In humans, h-Basal-B cells were more prevalent in benign prostate hyperplasia. This study reveals the identities of intermediate Basal-B cells and underscores the role of JAK/STAT signaling in prostate cell fate determination.

EPCO-60. EXPRESSION OF EARLY PROGENITOR MARKERS WITHIN NF2-ASSOCIATED SPINAL EPENDYMOMA

Abstract Spinal ependymomas (SP-EPNs) are intramedullary spinal tumors commonly found in patients with NF2-related schwannomatosis. Recent work using bulkRNA sequencing has suggested that SP-EPNs express a molecular signature most similar to ependymal cells. However, this type of sequencing may miss rare stem cell populations within tumors. The aim of this study was to assess the spatial heterogeneity within NF2-associated SP-EPNs and explore whether a stem cell population exists within SP-EPNs. Spatial transcriptomics (ST) was performed on a SP-EPN sample previously resected from a patient with NF2-related schwannomatosis using the 10X Genomics Visium Cytassist Platform. Libraries were sequenced on the Illumina NovaSeq 6000. 28-bp reads including spatial barcode and UMI sequences and 50-bp probe reads were generated with Illumina NovaSeq 6000 at the Center for Medical Genomics at Indiana University School of Medicine. Space Ranger 2.1 was used to process the raw files after sequencing. Data analysis was performed with R package Seurat 4.9.9.9041 following preprocessing with spaceranger. Sctransform normalized the spatial transcriptome dataset. Clusters were identified using “FindClusters” and “FindNeighbors” functions. There was diffuse expression of astrocyte and ependymal cell markers throughout the SP-EPN sample with smaller areas expressing radial glia and stem cell markers, as well as overlap between these regions. Expression of vascular and connective tissue markers varied throughout the tumor. There was minimal to no expression of neural genes in the sample. ST on our SP-EPN sample revealed a tumor with marked cellular and molecular heterogeneity. Although the primarily expressed ependymal cell and astrocyte markers, there does appear to be small, stem cell-like populations. This suggests that NF2-associated SP-EPNs may have a developmental hierarchy with radial glia-like cells and more mature progenitors within the ependymal cell lineage. This proof-of-concept study offers important findings that provide justification for further investigation using single-cell sequencing.

Identification of CD141+vasculogenic precursor cells from human bone marrow and their endothelial engagement in the arteriogenesis by co-transplantation with mesenchymal stem cells

BackgroundCritical limb ischemia (CLI) is a condition characterized by insufficient blood flow to the lower limbs, resulting in severe ischemia and potentially leading to amputation. This study aims to identify novel vasculogenic precursor cells (VPCs) in human bone marrow and evaluate their efficacy in combination with bone marrow-derived mesenchymal stem cells (BM-MSCs) for the treatment of CLI.MethodsEx vivo cultured VPCs and BM-MSCs from bone marrow were characterized and their effects on neovascularization and long-term tissue regeneration were tested in a mouse CLI model.ResultsVPCs, expressing high levels of hepatocyte growth factor and c-MET, were identified from human bone marrow aspirates. These cells exhibited strong vasculogenic capacity in vitro but possessed a cellular phenotype distinct from those of previously reported endothelial precursor cells in circulation or cord blood. They also expressed most surface markers of BM-MSCs and demonstrated multipotent differentiation ability. Screening of 376 surface markers revealed that VPCs uniquely display CD141 (thrombomodulin). CD141+VPCs are present in BM aspirates as a rare population and can be expanded ex vivo with a population doubling time of approximately 20 h, generating an elaborate vascular network even under angiogenic factor-deficient conditions and recruiting BM-MSCs to the network as pericyte-like cells. Intramuscular transplantation of a combination of human CD141+VPCs and BM-MSCs at a ratio of 2:1 resulted in limb salvage, blood flow recovery, and regeneration of large vessels in the femoral artery-removed CLI model, with an efficacy superior to that of singular transplantation. Importantly, large arteries and arterioles in dual cell transplantation expressed human CD31 in the intima and human α-smooth muscle actin in media layer at 4 and 12 weeks, likely indicating their lineage commitment to endothelial cells and vascular smooth muscle, respectively, in vivo.ConclusionDual-cell therapy using BM-derived CD141+ VPCs and BM-MSCs holds potential for further development in clinical trials to treat peripheral artery disease and diabetic ulcers.

Studying Chinese immigrants’ spatial distribution in the Raleigh–Durham area by linking survey and commercial data using romanized names

Abstract Many population surveys do not provide information on respondents’ residential addresses, instead offering coarse geographies like zip code or higher aggregations. However, fine resolution geography can be beneficial for characterizing neighbourhoods, especially for relatively rare populations such as immigrants. One way to obtain such information is to link survey records to records in auxiliary databases that include residential addresses by matching on variables common to both files. We present an approach based on probabilistic record linkage that enables matching survey participants in the Chinese Immigrants in Raleigh–Durham Study to records from InfoUSA, an information provider of residential records. The two files use different Chinese name romanization practices, which we address through a novel and generalizable strategy for constructing records’ pairwise comparison vectors for romanized names. Using a fully Bayesian record linkage model, we characterize the geospatial distribution of Chinese immigrants in the Raleigh–Durham area of North Carolina.

Overloading And unpacKing (OAK) - droplet-based combinatorial indexing for ultra-high throughput single-cell multiomic profiling

Multiomic profiling of single cells by sequencing is a powerful technique for investigating cellular diversity. Existing droplet-based microfluidic methods produce many cell-free droplets, underutilizing bead barcodes and reagents. Combinatorial indexing on microplates is more efficient for barcoding but labor-intensive. Here we present Overloading And unpacKing (OAK), which uses a droplet-based barcoding system for initial compartmentalization followed by a second aliquoting round to achieve combinatorial indexing. We demonstrate OAK’s versatility with single-cell RNA sequencing as well as paired single-nucleus RNA sequencing and accessible chromatin profiling. We further showcase OAK’s performance on complex samples, including differentiated bronchial epithelial cells and primary retinal tissue. Finally, we examine transcriptomic responses of over 400,000 melanoma cells to a RAF inhibitor, belvarafenib, discovering a rare resistant cell population (0.12%). OAK’s ultra-high throughput, broad compatibility, high sensitivity, and simplified procedures make it a powerful tool for large-scale molecular analysis, even for rare cells.

OS10.4.A CXCL1 MEDIATES LEPTOMENINGEAL METASTASIS GROWTH

Abstract BACKGROUND Leptomeningeal metastasis (LM) is a fatal neurological complication of cancer characterized by the entry of metastatic cancer cells originated from solid or hematologic tumors into the cerebrospinal fluid (CSF)-filled leptomeningeal compartment encasing the brain and the spinal cord. LM occurs in 5-20% in patients with solid tumors, especially those with breast cancer, lung cancer, melanoma. Untreated LM patients succumb within 4-6 weeks after diagnosis, or 2-5 months when they are treated with the current available therapies. To date, the only efficacious life-prolonging intervention was the application of proton craniospinal irradiation (pCSI) as demonstrated in the recent clinical trial, NCT04343573, with an average overall survival of 9 months. While a cohort of pCSI-treated patients survived over 18 months (responders), another cohort did not respond to pCSI and succumbed to the disease within 3 months post-treatment (non-responders). In this project, we aimed to characterize the molecular basis of pCSI response to turn the non-responders into responders. MATERIAL AND METHODS We performed proteomic analyses on serially collected CSF from LM patients at baseline (before pCSI), and multiple time points post-treatment. We further established syngeneic mouse models of LM-CSI to define the molecular basis of pCSI resistance. RESULTS We found that pCSI resulted in a significant drop in the levels of the chemokine CXCL1, a CXC-motif chemokine with elevated baseline levels after the onset of LM. Patients with significantly higher CSF CXCL1 levels prior to pCSI had worse central nervous system (CNS) progression-free survival. Using our preclinical LM mouse models, we found that both the cancer and host cells, namely macrophages, choroid plexus epithelial cells, and leptomeningeal fibroblasts, were a source of CXCL1. Genetic manipulation of Cxcl1expression in cancer cells or host revealed that the CXCL1 produced by the cancer cells was an essential factor for their growth in the leptomeninges. Moreover, we found that a subset of cancer cells expressed a receptor for CXCL1 - CXCR2 - and transcriptomic profiling of this rare population revealed an enrichment in pathways associated with cell cycle progression. Finally, intrathecally delivered CXCR2 antagonist hampered the LM growth. CONCLUSION Our results indicate that CXCL1/CXCR2 signaling axis regulates LM growth and suggests interruption of this signaling axis as a possible actionable therapeutic intervention to halt LM progression.

The eROSITA Final Equatorial Depth Survey (eFEDS). The hard X-ray selected sample

During its calibration and performance verification phase, the eROSITA instrument aboard the Spectrum-RG satellite performed a uniform wide-area X-ray survey of approximately 140 deg2, known as the eROSITA Final Equatorial Depth Survey (eFEDS). The primary aim of eFEDS is to demonstrate the scientific performance to be expected at the end of the eight--pass eROSITA all--sky survey. This survey will provide the first focussed image of the whole sky in the hard X-ray ($>2$ keV) bandpass. The expected source population in this energy range is thus of great interest, particularly for AGN studies. We used a 2.3--5 keV selection to construct a sample of 246 point-like hard X-ray sources for further study and characterisation. These sources are classified as either extragalactic ($ 90$ <!PCT!>) or Galactic ($ 10$ <!PCT!>), with the former consisting overwhelmingly of AGN and the latter active stars. We concentrated our further analysis on the extragalactic AGN sample, describing their X-ray and multi-wavelength properties and comparing them to the eFEDS main AGN sample selected in the softer 0.2--2.3 keV band. The eROSITA hard band selects a subsample of sources that is a factor of more than ten brighter than the eFEDS main sample. The AGN within the hard population reach up to $z=3.2$ but on the whole, they are relatively nearby, with median $z$=0.34 compared to $z$=0.94 for the main sample. The hard survey probes typical luminosities in the range $ X = 43-46$. The X-ray spectral analysis shows significant intrinsic absorption (with $ H >21$) in $ 20$ <!PCT!> of the sources, with a hard X-ray power law continuum with mean $< which is typical of AGN, but slightly harder than the soft-selected eROSITA sample. Around $10$ <!PCT!> of the hard sample show a significant `soft excess' component. The sampled black hole mass distribution in the eFEDS broad-line AGN population is consistent with that of the deeper COSMOS survey that probes a higher redshift population. On the other hand, the Eddington ratios appear systematically lower, which is consistent with the idea that the decline in SMBH activity since $z 1$ is due to a reduction in the typical accretion rate, rather than a shift towards activity in lower-mass black holes. The eFEDS hard sample provides a preview of what can be expected from the eRASS final survey in terms of data quality. This pilot survey indicates the power of eROSITA to shed new light on the demographics and evolution of AGN, and the potential for discovery of new and rare populations.

On Combining Ratio and Product Type Estimators For Estimation of Finite Population Mean In Adaptive Cluster Sampling Design

This article introduces a novel class of estimators and several new novel member estimators, combining the ratio and product forms, within the framework of Adaptive Cluster Sampling (ACS) design for estimating finite population mean. Specifically designed for rare or hidden clustered populations, the new novel estimators developed from the proposed class offer enhanced efficiency in estimation. To study the proposed class comprehensively, we derive expressions for the bias and Mean Squared Error (MSE) up to the first order of approximation. Through comprehensive simulation studies, we demonstrate the superior efficiency of the new developed estimators over several existing alternatives considered in this study.

Cytokine-armed vaccinia virus promotes cytotoxicity toward pancreatic carcinoma cells via activation of human intermediary CD56dimCD16dim natural killer cells.

Pancreatic ductal adenocarcinoma (PDAC) remains a particularly aggressive disease with few effective treatments. The PDAC tumor immune microenvironment (TIME) is known to be immune suppressive. Oncolytic viruses can increase tumor immunogenicity via immunogenic cell death (ICD). We focused on tumor-selective (vvDD) and cytokine-armed Western-reserve vaccinia viruses (vvDD-IL2 and vvDD-IL15) and infected carcinoma cell lines as well as patient-derived primary PDAC cells. In co-culture experiments, we investigated the cytotoxic response and the activation of human natural killer (NK). Infection and virus replication were assessed by measuring virus encoded YFP. We then analyzed intracellular signaling processes and oncolysis via in-depth proteomic analysis, immunoblotting and TUNEL assay. Following the co-culture of mock or virus infected carcinoma cell lines with allogenic PBMCs or NK cell lines, CD56+ NK cells were analyzed with respect to their activation, cytotoxicity and effector function. Both, dose- and time-dependent release of danger signals following infection weremeasured. Viruses effectively entered PDAC cells, emitted YFP signals and resulted in concomitant oncolysis. The proteome showed reprogramming of normally active core signaling pathways in PDAC (e.g., MAPK-ERK signaling). Danger-associated molecular patterns were released upon infection and stimulated co-cultured NK cells for enhanced effector cytotoxicity. NK cell subtyping revealed enhanced numbers and activation of a rare CD56dimCD16dim population. Tumor cell killing was primarily triggered via Fas ligands rather than granule release, resulting in marked apoptosis. Overall, the cytokine-armed vaccinia viruses induced NK cell activation and enhanced cytotoxicity toward human PDAC cells in vitro. We could show that cytokine-armed virus targets the carcinoma cells and thus hasgreat potential to modulate the TIME in PDAC.

Regulatory network analysis of Dclk1 gene expression reveals a tuft cell-ILC2 axis that inhibits pancreatic tumor progression.

Dclk1 expression defines a rare population of cells in the normal pancreas whose frequency is increased at early stages of pancreatic tumorigenesis. The identity and the precise roles of Dclk1 expressing cells in pancreas have been matter of debate, although evidence suggests their involvement in a number of key functions, including regeneration and neoplasia. We employed a recently developed Dclk1 reporter mouse model and single cell RNAseq analysis to define Dclk1 expressing cells in normal pancreas and pancreatic neoplasia. In normal pancreas, Dclk1 epithelial expression identifies subsets of ductal, islet and acinar cells. In pancreatic neoplasia, Dclk1 expression identifies five epithelial cell populations, among which acinar-to-ductal metaplasia (ADM)-like cells and tuft-like cells are predominant. These two cell populations play opposing roles in pancreatic neoplasia, with Dclk1+ ADM-like cells sustaining tumor growth while Dclk1+ tuft-like cells restraining tumor progression. The differentiation of Kras mutant acinar cells into Dclk1+ tuft-like cells requires the activation of the transcription factor SPIB and is further supported by a cellular paracrine loop involving cancer group 2 innate lymphoid cells (ILC2) and cancer activated fibroblasts (CAFs) that provide IL13 and IL33, respectively. In turn, Dclk1+ tuft-like cells release angiotensinogen that plays protective roles against pancreatic neoplasia. Overall, our study provides novel insights on the biology of Dclk1+ cells in normal pancreas and unveils a protective axis against pancreatic neoplasia, involving CAFs, ILC2 and Dclk1+ tuft-like cells, which ultimately results in angiotensinogen release.

Single nucleus DNA sequencing of flow sorted archived frozen and formalin fixed paraffin embedded solid tumors.

Archived samples, including frozen and formalin fixed paraffin embedded (FFPE) tissues, are a vast resource of clinically annotated materials for the application of high-definition genomics to improve patient management and provide a molecular basis for the delivery of personalized cancer therapeutics. Notably, FFPE tissues are stable, provide repeat sampling of tissues of interest, and can be stored indefinitely at ambient temperature. The development of single cell DNA sequencing (scDNA-seq) technologies provides an unparalleled opportunity for the study of tumor heterogeneity and the identification of often rare subclonal cell populations that drive tumor evolution and progression to advanced therapy resistant disease. However, major limitations to the use of archived tissues for scDNA-seq include the low yields of intact cells in the presence of high levels of subcellular debris in biopsies, and the highly variable quantity and quality of the DNA extracted from samples of interest. The latter is of high significance for the use of FFPE tissues due to the presence of DNA-protein crosslinks. In addition, many samples, notably tumors arising in solid tissues, contain admixtures of reactive stroma, inflammatory cells, and necrosis in immediate contact with tumor cells. To expand their use for translational studies, we optimized flow sorting and sequencing of single nuclei from archived fresh frozen (FF) and FFPE tumor tissues. Our methods, which include isolation of intact nuclei suitable for library preparations, quality control (QC) metrics for each step, and a single cell sequencing bioinformatic processing and analysis pipeline, were validated with flow sorted nuclei from matching FF and FFPE ovarian cancer surgical samples and a sequencing panel of 553 amplicons targeting single nucleotide and copy number variants in genes of interest. Our flow sorting based protocol provides intact nuclei suitable for snDNA-seq from archival FF and FFPE tissues. Furthermore, we have developed QC steps that optimize the preparation and selection of samples for deep single cell clonal profiling. Our data processing pipeline captures rare subclones in tumors with highly variable genomes based on variants in genes of interest.

Applying Deep Generative Neural Networks to Data Augmentation for Consumer Survey Data with a Small Sample Size

Questionnaire consumer survey research is primarily used for marketing research. To obtain credible results, collecting responses from numerous participants is necessary. However, two crucial challenges prevent marketers from conducting large-sample size surveys. The first is cost, as organizations with limited marketing budgets struggle to gather sufficient data. The second involves rare population groups, where it is difficult to obtain representative samples. Furthermore, the increasing awareness of privacy and security concerns has made it challenging to ask sensitive and personal questions, further complicating respondent recruitment. To address these challenges, we augmented small-sized datawith synthesized data generated using deep generative neural networks (DGNNs). The synthesized data from three types of DGNNs (CTGAN, TVAE, and CopulaGAN) were based on seed data. For validation, 11 datasets were prepared: real data (original and seed), synthesized data (CTGAN, TVAE, and CopulaGAN), and augmented data (original + CTGAN, original + TVAE, original + CopulaGAN, seed + CTGAN, seed + TVAE, and seed + CopulaGAN). The large-sample-sized data, termed “original data”, served as the benchmark, whereas the small-sample-sized data acted as the foundation for synthesizing additional data. These datasets were evaluated using machine learning algorithms, particularly focusing on classification tasks. Conclusively, augmenting and synthesizing consumer survey data have shown potential in enhancing predictive performance, irrespective of the dataset’s size. Nonetheless, the challenge remains to minimize discrepancies between the original data and other datasets concerning the values and orders of feature importance. Although the efficacy of all three approaches should be improved in future work, CopulaGAN more accurately grasps the dependencies between the variables in table data compared with the other two DGNNs. The results provide cues for augmenting data with dependencies between variables in various fields.

12484 Safety And Pharmacokinetics Of Anti-ACTH Antibody Lu AG13909 In Patients With Classic Congenital Adrenal Hyperplasia: Phase 1 Open-label, Multiple-Ascending-Dose Trial Protocol

Abstract Disclosure: U. Srirangalingam: Advisory Board Member; Self; Lundbeck. Consulting Fee; Self; Diurnal. Research Investigator; Self; Neurocrine. A. Velusamy: Consulting Fee; Self; Novo Nordisk. M. Binderup: Employee; Self; H. Lundbeck A/S. B. Baker: Employee; Self; H. Lundbeck A/S. M. Folden Flensburg: Employee; Self; H. Lundbeck A/S. L. Pickering Boserup: Employee; Self; H. Lundbeck A/S. J. Luthman: Employee; Self; H. Lundbeck A/S. H. Benecke: Employee; Self; H. Lundbeck A/S. R.J. Auchus: Consulting Fee; Self; Neurocrine Biosciences, Diurnal, Corcept Therapeutics, Recordati Rare Diseases, Crinetics Pharmaceuticals, Quest Diagnostics, Xeris Pharmaceuticals, Novo Nordisk, H. Lundbeck A/S, Sparrow Pharmaceuticals. Research Investigator; Self; Adrenas Therapeutics, Spruce Biosciences, Neurocrine Biosciences, Diurnal, Corcept Therapeutics, Recordati Rare Diseases, Crinetics Pharmaceuticals. Introduction: Classic congenital adrenal hyperplasia (CAH) is a rare, autosomal recessive disorder characterized by cortisol and aldosterone deficiency, elevated adrenocorticotropic hormone (ACTH) and subsequent hyperandrogenemia, most commonly due to 21-hydroxylase deficiency. Balancing physiological glucocorticoid replacement and control of hyperandrogenism remains a challenge with the risk of long-term consequences of glucocorticoid overtreatment. Lu AG13909 is a novel, high-affinity, anti-ACTH monoclonal antibody that neutralizes ACTH-induced signaling. In preclinical models, Lu AG13909 dose-dependently and persistently reduced plasma corticosterone and cortisol concentrations. No adverse effects were observed after 6 months of intravenous dosing every 2 weeks in cynomolgus monkeys. Here, we describe the ongoing first-in-human trial of Lu AG13909, investigating the safety, tolerability, pharmacokinetic (PK) properties, and pharmacodynamic (PD) effects of Lu AG13909 in adults with classic CAH. Methods: This is a Phase 1, open-label, multiple-ascending-dose trial with intra-participant dose escalation (EUCT #2023-503711-15-00). The trial enrolls men and women with classic CAH aged between ≥18 and ≤70 years under stable glucocorticoid and mineralocorticoid dosing, with morning 17-hydroxyprogesterone >4 times the upper limit of normal. Each participant receives up to 6 doses of Lu AG13909 every 28-35 days at increasing dose levels. The trial consists of 2 parts: Part A has a 3+3 design, in which 3 participants are given a certain dose, and escalation to the next dose is based on a 28-day safety observation period. Part B explores the pharmacologically relevant Lu AG13909 doses identified in Part A in a larger number of participants. The aim is for a total of 12 participants to receive each of the dose levels identified as relevant. Outcomes The primary safety and tolerability endpoints include adverse events and clinical safety tests. PK endpoints include maximum observed exposure and area under the Lu AG13909 concentration-time curve in a dosing interval. The PD endpoints are the relative reductions in morning 17-hydroxyprogesterone, androstenedione, and unbound ACTH, from baseline to the day after each Lu AG13909 dose. Conclusions This innovative, multi-dose, intra-participant dose escalation trial design was developed specifically for the rare classic CAH population, who have a high unmet medical need due to the challenges with current glucocorticoid treatment. The aim of the trial is to gather safety, PK, and PD data while minimizing participant risk and burden. Data from this trial will reinforce the development of Lu AG13909 as a novel treatment strategy in conditions characterized by increased levels of ACTH, such as classic CAH and Cushing disease. Presentation: 6/3/2024

Exploring the Origin of Cold Gas and Star Formation in a Rare Population of Strongly Bulge-dominated Early-type Galaxies

We analyze the properties of a rare population, the strongly bulge-dominated early-type galaxies (sBDEs) with significant H i gas, using the databases from the FAST All Sky H i survey (FASHI) and the Arecibo Legacy Fast ALFA (ALFALFA) survey. We select the sBDEs from the Sloan Digital Sky Survey and crossmatch with the FASHI-ALFALFA combined H i sample, resulting in 104 H i-rich sBDEs. These sBDEs tend to have extremely high H i reservoirs, which is rare in previous studies such as ATLAS3D. A total of 70% of the selected sBDEs are classified as quiescent galaxies, even though they have a large H i reservoir. We study the properties of these sBDEs from four main aspects: stellar population, gas-phase metallicity, stacked H i spectra, and environment. The majority of H i-rich sBDEs appear to show lower gas-phase metallicity and are located in significantly lower-density environments, suggesting an external origin for their H i gas. We find that star-forming sBDEs exhibit statistically higher star formation efficiency and slightly older stellar populations compared to normal star-forming galaxies, suggesting a recent star formation on the Gyr timescale. They also show narrower and more concentrated H i profiles compared to control star-forming galaxies, which may explain their higher star formation efficiency.

Essential Fluidics for a Flow Cytometer.

Flow cytometry is an inherently fluidic process that flows particles on a one-by-one basis through a sensing region to discretely measure their optical and physical properties. It can be used to analyze particles ranging in size from nanoparticles to whole organisms (e.g., zebrafish). It has particular value for blood analysis, and thus most instruments are fluidically optimized for particles that are comparable in size to a typical blood cell. The principles of fluid dynamics allow for particles of such size to be precisely positioned in flow as they pass through sensing regions that are tens of microns in length at linear velocities of meters per second. Such fluidic systems enable discrete analysis of cell-sized particles at rates approaching 100 kHz. For larger particles, the principles of fluidics greatly reduce the achievable rates, but such high rates of data acquisition for cell-sized particles allow rapid collection of information on many thousands to millions of cells and provides for research and clinical measurements of both rare and common cell populations with a high degree of statistical confidence. Additionally, flow cytometers can accurately count particles via the use of volumetric sample delivery and can be coupled with high-throughput sampling technologies to greatly increase the rate at which independent samples can be delivered to the system. Due to the combination of high analysis rates, sensitive multiparameter measurements, high-throughput sampling, and accurate counting, flow cytometry analysis is the gold standard for many critical applications in clinical, research, pharmaceutical, and environmental areas. Beyond the power of flow cytometry as an analytical technique, the fluidic pathway can be coupled with a sorting mechanism to collect particles based on desired properties. We present an overview of fluidic systems that enable flow cytometry-based analysis and sorting. We introduce historical approaches, explanations of commonly implemented fluidics, and brief discussions of potential future fluidics where appropriate. © 2024 Wiley Periodicals LLC.

Systemic Therapy Options for Peritoneal Mesothelioma.

Peritoneal mesothelioma is an uncommon malignancy affecting approximately 300 new patients annually in the United States. Due to its low incidence, prospective clinical trials specific to this disease are scant. Recommendations regarding systemic therapy are mostly extrapolated from clinical trials conducted among patients with pleural mesothelioma. At present, the recommended first-line systemic treatment options may include immunotherapy with nivolumab plus ipilimumab or chemotherapy with pemetrexed plus either cisplatin or carboplatin. For second-line treatment, the other previously unchosen first-line option can be used. Off-label bevacizumab may be considered in combination with chemotherapy among carefully selected patients. The benefit of third-line treatment or beyond is less clear. Nonetheless, a number of single-agent regimens show modest activity. Anecdotal reports of children or young adults with peritoneal mesothelioma harboring ALK rearrangement have suggested the efficacy of ALK inhibitors for this rare population. In summary, there is a growing number of systemic therapy options for peritoneal mesothelioma. To gain a better insight into this disease, future prospective trials in mesothelioma should include more patients with peritoneal mesothelioma.

Evidence for Intrinsic X-Ray Weakness among Red Quasars at Cosmic Noon

Quasar feedback is a key ingredient in shaping galaxy evolution. A rare population of extremely red quasars (ERQs) at z = 2−3 are often associated with high-velocity [O iii]λ5008 outflows and may represent sites of strong feedback. In this paper, we present an X-ray study of 50 ERQs to investigate the link between the X-ray and outflow properties of these intriguing objects. Using hardness ratio analysis, we confirm that the ERQs are heavily obscured systems with gas column density reaching N H = 1023−24 cm−2. We identify 20 X-ray-nondetected ERQs at high mid-infrared (MIR) luminosities of ν L ν,6 μm ≳ 3 × 1046 erg s−1. By stacking the X-ray observations, we find that the nondetected ERQs are on average underluminous in X-rays by a factor of ∼10 for their MIR luminosities. We consider such X-ray weakness to be due to both heavy gas absorption and intrinsic factors. Moreover, we find that the X-ray-weak sources also display higher-velocity outflows. One option to explain this trend is that weaker X-rays facilitate more vigorous line-driven winds, which then accelerate the [O iii]-emitting gas to kiloparsec scales. Alternatively, super-Eddington accretion could also lead to intrinsic X-ray weakness and more powerful continuum-driven outflow.

Multiparametric profiling of HER2-enriched extracellular vesicles in breast cancer using Single Extracellular VEsicle Nanoscopy

BackgroundPatients with HER2-positive breast cancer can significantly benefit from HER2-directed therapy – such as the monoclonal antibody trastuzumab. However, some patients can develop therapy resistance or change HER2 status. Thus, we urgently need new, noninvasive strategies to monitor patients frequently. Extracellular vesicles (EVs) secreted from tumor cells are emerging as potential biomarker candidates. These membrane-delimited nanoparticles harbor molecular signatures of their origin cells; report rapidly on changes to cellular status; and can be frequently sampled from accessible biofluids.ResultsUsing Single Extracellular VEsicle Nanoscopy (SEVEN) platform that combines affinity isolation of EVs with super-resolution microscopy, here we provide multiparametric characterization of EVs with ~ 8 nm precision and molecular sensitivity. We first interrogated cell culture EVs affinity-enriched in tetraspanins CD9, CD63, and CD81; these transmembrane proteins are commonly found on EV membranes. SEVEN robustly provided critical parameters of individual, tetraspanin-enriched EVs: concentration, size, shape, molecular cargo content, and heterogeneity. Trastuzumab-resistant cells (vs. trastuzumab-sensitive) secreted more EVs. Additionally, EVs from trastuzumab-resistant cells had lower tetraspanin density and higher HER2 density. We also evaluated EVs affinity-enriched in HER2; we found that these EVs (vs. tetraspanin-enriched) were larger and more elongated. We further optimized analytical sample processing to assess a rare population of HER2-enriched EVs from patient plasma. In breast cancer patients with elevated HER2 protein expression (vs. controls), HER2-enriched EVs had distinct characteristics including typically increased number of tetraspanin molecules and larger size. Importantly, these EVs were on average 25-fold more abundant compared to no cancer controls.ConclusionsSEVEN revealed unique characteristics of HER2-enriched EVs in cultured cells and complex biological fluid. In combination with current clinical approaches, this method is well poised to support precise therapeutic decisions.Graphical abstract

Hereditary and clinical insights into paraganglioma and pheochromocytoma.

Approximately 30-40% of paragangliomas (PGLs) and pheochromocytomas (PCCs) harbor an underlying hereditary cause. Early identification of at-risk individuals is imperative given the early onset, aggressiveness of tumors, and other tumor/cancer risks associated with hereditary PGLs/PCCs. This study analyzes the clinical presentations and genetic histories of patients with PGL/PCC and/or hereditary risk to contribute to the expanding knowledge in this rare population. A retrospective chart review identified two cohorts of patients seen in cancer genetics clinics at an academic medical center and a safety-net hospital between August 2016 and December 2022. Cohort 1 consisted of patients with likely pathogenic variants (LPVs)/pathogenic variants (PVs) in hereditary PGL/PCC predisposition genes. Cohort 2 consisted of patients with a personal history of a PGL/PCC. Demographics, personal/family history, and genetic testing outcomes were analyzed. A total of 560 patients met the study criteria (Cohort 1, n = 364; Cohort 2, n = 269). In Cohort 1, 77 (21.1%) patients had an incidental LPV/PV in a PGL/PCC gene. Nearly half (n = 36, 46.8%) were in SDHx genes, with a majority in SDHA (n = 21). In Cohort 2, 86 patients tested positive for 87 LPV/PV in a hereditary cancer predisposition gene. The SDHx genes were most likely to have an LPV/PV identified (SDHB n = 24, SDHD n = 23). Multigene panels identify patients at risk for hereditary PGL/PCC, many of whom are incidentally found. While SDHA LPV/PVs were the most frequent incidental finding, they were less common in patients with PGL/PCC, indicating the need for longitudinal studies to better understand the prevalence and penetrance of these tumors.

Cell cycle expression heterogeneity predicts degree of differentiation.

Methods that predict fate potential or degree of differentiation from transcriptomic data have identified rare progenitor populations and uncovered developmental regulatory mechanisms. However, some state-of-the-art methods are too computationally burdensome for emerging large-scale data and all methods make inaccurate predictions in certain biological systems. We developed a method in R (stemFinder) that predicts single cell differentiation time based on heterogeneity in cell cycle gene expression. Our method is computationally tractable and is as good as or superior to competitors. As part of our benchmarking, we implemented four different performance metrics to assist potential users in selecting the tool that is most apt for their application. Finally, we explore the relationship between differentiation time and cell fate potential by analyzing a lineage tracing dataset with clonally labelled hematopoietic cells, revealing that metrics of differentiation time are correlated with the number of downstream lineages.