Fluidigm (United States)

This article addresses the healthcare disparities and potential impact on mortality among incarcerated patients with chronic kidney disease (CKD). Incarcerated individuals are more likely to suffer from acute and chronic health conditions and have inconsistent access to healthcare services. Demographic trends, behavioral health trends, and lack of insurance coverage are major contributing factors to healthcare disparities among incarcerated individuals. This article highlights the need for a new model of care, which includes clinical programs focusing on the transition period from incarceration to the community and linking individuals to post-incarceration healthcare utilizing community health workers and clinicians to establish rapport with individuals prior to their release. Addressing healthcare disparities and providing adequate healthcare to incarcerated individuals is crucial because a conviction should not deprive an individual of basic human rights, including the right to healthcare.

BackgroundImmune profiling studies using whole blood (WB) specimens are increasingly common in research and clinical research settings. It is crucial to standardize sample collection, shipping, and downstream processing of specimens for large studies. WB preservation reagents have been developed to address these logistics challenges.Using a lyophilized CyTOF® 30-marker immune profiling panel, this study aims to assess the effect of Streck Cyto-Chex®, BD PAXgene® Blood DNA Tube, Smart Tube Proteomic Stabilizer PROT1, and Cytodelics preservatives on data quality.CyTOF® flow cytometry allows for simultaneous detection of 50-plus parameters in a single tube without the need for single-stain controls. CyTOF uses typical antibody staining protocols and overcomes spillover and autofluorescence by using antibodies conjugated to heavy metal isotopes.MethodsWe compared 37+ immune populations in WB drawn from three healthy donors into BD heparin, Cyto-Chex, and PAXgene blood collection tubes. As a baseline, blood from the BD heparin donors was stained with the Maxpar® Direct® Immune Profiling Assay kit and T Cell Panel 3 with <24h post-blood draw. For Cytodelics stabilizer and Prot1, aliquots from BD heparin donor tubes was combined with stabilizer and frozen <24h post-blood draw. Remaining blood from the BD heparin, Cyto-Chex, and PAXgene tubes was stored at room temperature (RT). At 48h post-draw, blood stabilized in Cytodelics stabilizer and PROT1 was thawed and RBC lysed. All samples were stained following the Maxpar Direct Immune Profiling Assay protocol.Samples were acquired on the CyTOF XT™. Data was analyzed manually and by automated analysis using Maxpar Pathsetter™ software.ResultsCompared to the baseline at 24h, the frequencies of most populations remained similar across conditions however, the median staining intensity of most markers was reduced for all conditions. Major immune cell populations were identified when WB was stored at RT in Cyto-Chex and PAXgene tubes. Major populations were also preserved in PROT1 and Cytodelics, however, the expression of chemokine receptors was altered.ConclusionsThis study provides a comparison of WB preservation methods and their compatibility with a lyophilized CyTOF immune profiling panel. All stabilizers allowed major immune populations to be identified at 48h post thaw, although signal intensities of markers were reduced. An appropriate preservation method combined with lyophilized CyTOF antibody panels enables deep immune profiling when access to instruments, expertise and sample volume is limited in large/multi-site studies.Ethics ApprovalThe samples obtained for this study were sourced from an accredited commercial provider.

Abstract Introduction We developed a unique panel of 54 target genes dedicated to hematological malignancies (HematoPanel) and screened 1,108 clinical samples. We also performed transcriptomic analysis and evaluated its relevance for patients with acute lymphoblastic leukemia (ALL). Methods DNA and RNA were extracted from cell pellets with the Maxwell® RSC (Promega®). Libraries for targeted high-throughput DNA sequencing (HTS) were generated in duplicate from 100 ng with the Advanta™ NGS Library Prep Assay kit using the LP 48.48 IFC (integrated fluidic circuit) on a Juno™ and sequenced on the NextSeq™ 550 (Illumina®) to a median depth of coverage of approximately 1,200X per sample. Transcriptomic libraries were generated from 50 ng RNA with the Advanta RNA-Seq XT NGS Library Prep Kit using the LP 48.Atlas™ IFC on Juno and sequenced on the NextSeq 550. A bioinformatics pipeline was developed for fusion transcript detection. Differential gene expression was analyzed with the DESeq2 package after STAR alignment. Results In 2020 we sequenced 1,108 consecutive samples in routine practice with HematoPanel. The main indications were the suspicion of myeloproliferative neoplasms (32%), the evaluation of chronic lymphocytic leukemia (23%), and the diagnostic workup of cytopenias (15%). We detected 1,306 mutations in 654 patients (59%) (1 to 8 mutations per sample). Eight genes (TET2, SF3B1, TP53, DNMT3A, ASXL1, JAK2, SRSF2 and MYD88) were reported as mutated for more than 50 patients. We sequenced 22 samples including the MOLT-4 cell line, healthy donor samples, and 16 ALL samples for transcriptomic evaluation. From ALL samples, we detected 5 fusion transcripts previously identified by specific PCR (1 BCR-ABL, 2 ETV6-RUNX1, 1 KMT2A-AFF1, 1 TCF3-PBX1). Moreover, we detected 4 other fusion transcripts for 3 patients (EBF1-PDGFRB, FGFR1-MYO18A, P2RY8-CRLF2, and PAX5-NOL4L). Interestingly, these patients clustered with the BCR-ABL positive sample based on their expression profile. It confirms the diagnosis of Phi-like ALL and thus offers a therapeutic target for these patients. Conclusion Microfluidics-based library prep using Juno offers a cost-effective strategy and straightforward workflows for both targeted HTS and RNA sequencing. We validated its use for hematological malignancy evaluation in routine practice. Citation Format: Cédric Pastoret, Amyra Aliouat, Marie-Laure Boulland, Florent Denoual, Anne Desmares, Marie De Tayrac, Thierry Fest, Celeste Letellier. Microfluidics-based library preparation strategies for targeted high-throughput sequencing and RNA sequencing in hematological malignancies in clinical samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5791.

Abstract Colorectal cancer (CRC) is the second-leading cause of cancer-related death worldwide, and treatment failure in patients with metastatic CRC portends a poor prognosis. Early detection of chemoresistance would help reduce the usage of ineffective and possibly toxic therapies and facilitate alternative treatment strategies. We present a new technique combining phasor-fluorescence lifetime imaging microscopy (phasor-FLIM) and a machine learning algorithm that quantitatively identifies chemoresistant single cells, which may also be potentially applicable for evaluating heterogeneous circulating tumor cells (CTCs) obtained by liquid biopsy. We used parental HCT116 CRC cells sensitive to 5-fluorouracil (5-FU), a common chemotherapeutic agent used in CRC, to generate a cell line resistant to 5-FU; we confirmed resistance by a cell proliferation assay (MTS). We then analyzed phasor-FLIM patterns in sensitive and resistant cells before and after treatment with 5-FU of two endogenously fluorescent biomarkers, NADH and NADPH, which are key co-enzymes involved in energy metabolism. This was performed at 21% and 1% oxygen, which simulates both standard cell culture and tumor hypoxic conditions. Differences in phasor-FLIM signatures were found in resistant cells that suggested upregulation of the electron transport chain under normoxia (21% oxygen) and reprogrammed glucose metabolism under hypoxia (1% oxygen). Such changes were confirmed using NADH, lactate, and pyruvate production colorimetric tests, which are readouts of energy metabolism. To improve the quantification of changes in phasor-FLIM patterns, a new drug resistance indexing algorithm was developed to separate sensitive and resistant cells based on fluorescence lifetime multiparametric analysis of the intrinsic fluorescent biomarkers at the single-cell level. We then used single-cell total RNA sequencing (scRNA-Seq) to explore gene expression differences between sensitive and resistant HCT116 cells. Principal component analysis (PCA) and Uniform Manifold Approximation and Projection (UMAP) clustering showed a clear separation of 5-FU-sensitive and -resistant cells. There was increased expression of proliferation markers in resistant cells and upregulation of genes involved in electron transport from NADPH to ferredoxin, consistent with our single-cell phasor-FLIM findings. We demonstrate proof of concept that the phasor-FLIM method combined with our new drug resistance indexing algorithm provides a marker-free technology for quantitatively assaying drug response and discriminating drug resistance at single-cell resolution. Citation Format: Ning Ma, Hui Ren, Naveen Ramalingam, David King, Banafshé Larijani, Stefanie S. Jeffrey. Label-free single-cell drug response determined by fluorescence lifetime imaging microscopy (FLIM) and RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2337.

Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are quiescent, insensitive to BCR-ABL1 tyrosine kinase inhibitors (TKIs) and responsible for CML relapse. Therefore, eradicating quiescent CML LSCs is a major goal in CML therapy. Here, using a G0 marker (G0M), we narrow down CML LSCs as G0M- and CD27- double positive cells among the conventional CML LSCs. Whole transcriptome analysis reveals NF-κB activation via inflammatory signals in imatinib-insensitive quiescent CML LSCs. Blocking NF-κB signals by inhibitors of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4 inhibitors) together with imatinib eliminates mouse and human CML LSCs. Intriguingly, IRAK1/4 inhibitors attenuate PD-L1 expression on CML LSCs, and blocking PD-L1 together with imatinib also effectively eliminates CML LSCs in the presence of T cell immunity. Thus, IRAK1/4 inhibitors can eliminate CML LSCs through inhibiting NF-κB activity and reducing PD-L1 expression. Collectively, the combination of TKIs and IRAK1/4 inhibitors is an attractive strategy to achieve a radical cure of CML.

Recent studies have provided insights into the pathology and immune response to coronavirus disease 2019 (COVID-19). However thorough interrogation of the interplay between infected cells and the immune system at sites of infection is lacking. We use high parameter imaging mass cytometry targeting the expression of 36 proteins, to investigate at single cell resolution, the cellular composition and spatial architecture of human acute lung injury including SARS-CoV-2. This spatially resolved, single-cell data unravels the disordered structure of the infected and injured lung alongside the distribution of extensive immune infiltration. Neutrophil and macrophage infiltration are hallmarks of bacterial pneumonia and COVID-19, respectively. We provide evidence that SARS-CoV-2 infects predominantly alveolar epithelial cells and induces a localized hyper-inflammatory cell state associated with lung damage. By leveraging the temporal range of COVID-19 severe fatal disease in relation to the time of symptom onset, we observe increased macrophage extravasation, mesenchymal cells, and fibroblasts abundance concomitant with increased proximity between these cell types as the disease progresses, possibly as an attempt to repair the damaged lung tissue. This spatially resolved single-cell data allowed us to develop a biologically interpretable landscape of lung pathology from a structural, immunological and clinical standpoint. This spatial single-cell landscape enabled the pathophysiological characterization of the human lung from its macroscopic presentation to the single-cell, providing an important basis for the understanding of COVID-19, and lung pathology in general.

Recent studies have provided insights into the pathology and immune response to coronavirus disease 2019 (COVID-19). However thorough interrogation of the interplay between infected cells and the immune system at sites of infection is lacking. We use high parameter imaging mass cytometry9 targeting the expression of 36 proteins, to investigate at single cell resolution, the cellular composition and spatial architecture of human acute lung injury including SARS-CoV-2. This spatially resolved, single-cell data unravels the disordered structure of the infected and injured lung alongside the distribution of extensive immune infiltration. Neutrophil and macrophage infiltration are hallmarks of bacterial pneumonia and COVID-19, respectively. We provide evidence that SARS-CoV-2 infects predominantly alveolar epithelial cells and induces a localized hyper-inflammatory cell state associated with lung damage. By leveraging the temporal range of COVID-19 severe fatal disease in relation to the time of symptom onset, we observe increased macrophage extravasation, mesenchymal cells, and fibroblasts abundance concomitant with increased proximity between these cell types as the disease progresses, possibly as an attempt to repair the damaged lung tissue. This spatially resolved single-cell data allowed us to develop a biologically interpretable landscape of lung pathology from a structural, immunological and clinical standpoint. This spatial single-cell landscape enabled the pathophysiological characterization of the human lung from its macroscopic presentation to the single-cell, providing an important basis for the understanding of COVID-19, and lung pathology in general.

Background Understanding the genetic heterogeneity of circulating immune and tumors cells at single-cell resolution will enable one to decipher the interplay between cancer and immune cells, and ultimately lead to better design of immunotherapy assays. Marker-free methods for isolation of cells are attractive because they provide an opportunity to analyze a larger set of these cells that may otherwise be missed due to variable or no expression of protein (marker) markers. Integration of the Biolidics ClearCell® FX System and the Fluidigm® Polaris™ system enabled us not only to develop a marker-free workflow to isolate immune and circulating tumor cells (CTCs) but also to seamlessly prepare amplified cDNA from immune and CTCs for full-length mRNA-seq analysis.Method and Results The ClearCell FX System processes blood samples and isolates CTCs in a marker-free manner. During the CTC isolation process the immune cells are separated from CTCs on the microfluidic device. To differentiate larger blood cells from CTCs, we stained the enriched cells with Alexa Fluor® 647-conjugated CD45 and CD31 to identify leukocytes and endothelial cells, respectively. Calcein AM (live cell marker) and CellTracker™ Orange (universal cell marker) were added to identify live cells. Different enrichment strategies on Polaris allowed us to either specifically select for CTCs (live+, universal cell marker+, CD45-, CD31-) or immune cells (live+, universal cell marker+, CD45+). The enriched cells were then lysed, mRNA were reverse-transcribed, and cDNA were preamplified on a Polaris integrated fluidic circuit (IFC). Sequencing libraries were generated (off-IFC) using the Nextera® XT DNA library prep protocol and sequenced on Illumina® MiSeq™ systems. We successfully profiled full-length mRNA of 81 CTCs from stage III and IV subjects. Based on hormone receptor status, these six subjects were categorized as three types: (1) ER-/PR-/HER2- (triple-negative breast cancer; n=1), (2) ER+/PR+/HER2- (n=3), and (3) ER+/PR+/HER2- (n=2). For immune cell comparative analysis, we included CD45+ (FACS-sorted) from healthy donor. Unsupervised hierarchal clustering of gene expression data showed clustering by subject with a unique set of genes expressed by triple-negative breast cancer only. Heterogeneity was noted in immune cells from subjects at baseline (pretreatment), under treatment with drugs and a healthy subject without cancer. Using TraCeR computational method, we successfully reconstructed full-length, paired T cell receptor (TCR) sequences from CD45+ single-cell RNA sequence data from breast cancer subjects.Conclusion We present the feasibility of integrating two microfluidics platforms to isolate circulating immune and tumor cells for transcriptome and functional study. Our data suggests that the heterogeneity of immune and cancer cells can be elucidated from single-cell mRNA sequencing data and full-length, paired T cell receptor sequences can be reconstructed from immune cells of breast cancer subjects.Citation Format: Naveen Ramalingam, Yifang Lee, Lukasz Szpankowski, Anne Leyrat, Brian Fowler, Ninez Delos Angeles, Chad Sanada, Andrew Wu, Yoon Sim Yap, Jay West, Ali Asgar Bhagat, Kyle Hukari, Mark Lynch, David King. Full-Length mRNA transcriptome analysis of matched circulating tumor and immune cells from breast cancer subjects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-326.

Single-cell transcriptomic profiling is a powerful tool to explore cellular heterogeneity. However, most of these methods focus on the 3′-end of polyadenylated transcripts and provide only a partial view of the transcriptome. We introduce C1 CAGE, a method for the detection of transcript 5′-ends with an original sample multiplexing strategy in the C1TM microfluidic system. We first quantifiy the performance of C1 CAGE and find it as accurate and sensitive as other methods in the C1 system. We then use it to profile promoter and enhancer activities in the cellular response to TGF-β of lung cancer cells and discover subpopulations of cells differing in their response. We also describe enhancer RNA dynamics revealing transcriptional bursts in subsets of cells with transcripts arising from either strand in a mutually exclusive manner, validated using single molecule fluorescence in situ hybridization.

Direct comparisons of human and non-human primate brain tissue have the potential to reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is largely inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee. First, we systematically evaluated the fidelity of organoid models to primary human and macaque cortex, finding organoid models preserve gene regulatory networks related to cell types and developmental processes but exhibit increased metabolic stress. Second, we identified 261 genes differentially expressed in human compared to chimpanzee organoids and macaque cortex. Many of these genes overlap with human-specific segmental duplications and a subset suggest increased PI3K/AKT/mTOR activation in human outer radial glia. Together, our findings establish a platform for systematic analysis of molecular changes contributing to human brain development and evolution.