Single-cell Molecular Analysis Research Articles

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues.

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.

Abstract 2860A: Evolution and characterization of carboplatin resistance at single-cell resolution

Abstract Acquired resistance to carboplatin is a major obstacle to the cure of ovarian cancer, but its molecular underpinnings are still poorly defined or inconsistently reported. Multiple cellular functions have been identified as being capable of modulating sensitivity to the platinum drugs; however, results from single-gene or transcriptome-wide studies have failed to disclose any one actionable gene or set of genes that are consistently altered across different cell lines or studies. We hypothesized that heterogeneity within the treated cell populations is responsible for this situation and that cloning and molecular analysis of single cells can capture the heterogeneity and dynamics of resistance acquisition, revealing cellular and molecular principles driving the phenotype. Following a rigorous experimental design, starting with a single ovarian cancer cell, we selected 8 subclones for resistance to carboplatin using repeated cycles of drug exposure. An additional 4 clones were left untreated for comparison. The resistant clones did not show significant mutations or gene copy number variations in common or reduced drug uptake, but displayed important phenotypic and gene expression heterogeneity. A gene set enrichment analysis revealed that each clone altered different molecular processes to become resistant. Suspecting that clonal heterogeneity was also present within each experimental replicate, we measured mRNA expression from 26,892 single cells from both sensitive and resistant clones, which resulted in the identification of 7 transcriptional states associated with resistance evolution and clone identity. Interestingly, the resistant states were not stable and continued to evolve after removal of the drug, without noticeable change in resistance level. Reduced proliferation and active interferon signaling were identified as two processes shared by most resistant states, becoming apparent early in the course of the resistance acquisition. Up to 12% of untreated cells were found to already be in a resistant state and this fraction diminished with each round of sub-cloning. Up to 18% of the cells from resistant clones showed extreme levels of key resistance genes and pathways, suggesting the importance of cellular heterogeneity in the development of resistance. Finally, six expression signatures derived from the in vitro resistant states distinguished primary from recurrent high-grade serous ovarian cancers and predicted both response to primary therapy and overall survival. The analysis of the biologic processes associated with each signature suggested functional differences between intrinsic and acquired resistance. This multidimensional, single-cell analysis decomposed a multigenic phenotype into tangible resistant states, offering new insights into the dynamics of the acquisition of resistance to carboplatin, a drug of major importance to the treatment of ovarian and other cancers. Citation Format: Devora Champa, Si Sun, Cheng-Yu Tsai, Stephen Howell, Olivier Harismendy. Evolution and characterization of carboplatin resistance at single-cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2860A.

Relapse pathway of glioblastoma revealed by single-cell molecular analysis.

Glioblastoma multiforme (GBM) remains an incurable brain tumor. The highly malignant behavior of GBM may, in part, be attributed to its intraclonal genetic and phenotypic diversity (subclonal evolution). Identifying the molecular pathways driving GBM relapse may provide novel, actionable targets for personalized diagnosis, characterization of prognosis and improvement of precision therapy. We screened single-cell transcriptomes, namely RNA-seq data of primary and relapsed GBM tumors from a patient, to define the molecular profile of relapse. Characterization of hundreds of individual tumor cells identified three mutated genes within single cells, involved in the RAS/GEF GTP-dependent signaling pathway. The identified molecular pathway was further verified by meta-analysis of RNA-seq data from more than 3000 patients. This study showed that single-cell molecular analysis overcomes the inherent heterogeneity of bulk tumors with respect to defining tumor subclonal evolution relevant to GBM relapse.

Molecular analysis of single circulating tumour cells following long‐term storage of clinical samples

The CellSearch® semiautomated CTC enrichment and staining system has been established as the ‘gold standard’ for CTC enumeration with CellSearch® CTC counts recognized by the FDA as prognostic for a number of cancers. We and others have gone on to show that molecular analysis of CellSearch® CTCs isolated shortly after CellSearch® enrichment provides another valuable layer of information that has potential clinical utility including predicting response to treatment. Although CellSearch® CTCs can be readily isolated after enrichment, the process of analysing a single CellSearch® patient sample, which may contain many CTCs, is both time‐consuming and costly. Here, we describe a simple process that will allow storage of all CellSearch®‐enriched cells in glycerol at −20 °C for up to 2 years without any measurable loss in the ability to retrieve single cells or in the genome integrity of the isolated cells. To establish the suitability of long‐term glycerol storage for single‐cell molecular analysis, we isolated individual CellSearch®‐enriched cells by DEPArray™ either shortly after CellSearch® enrichment or following storage of matched enriched cells in glycerol at −20 °C. All isolated cells were subjected to whole‐genome amplification (WGA), and the efficacy of single‐cell WGA was evaluated by multiplex PCR to generate a Genome Integrity Index (GII). The GII results from 409 single cells obtained from both ‘spike‐in’ controls and clinical samples showed no statistical difference between values obtained pre‐ and postglycerol storage and that there is no further loss in integrity when DEPArray™‐isolated cells are then stored at −80 °C for up to 2 years. In summary, we have established simple yet effective ‘stop‐off’ points along the CTC workflow enabling CTC banking and facilitating selection of suitable samples for intensive analysis once patient outcomes are known.

Clonal evolution in leukemia.

Human leukemias are liquid malignancies characterized by diffuse infiltration of the bone marrow by transformed hematopoietic progenitors. The accessibility of tumor cells obtained from peripheral blood or through bone marrow aspirates, together with recent advances in cancer genomics and single-cell molecular analysis, have facilitated the study of clonal populations and their genetic and epigenetic evolution over time with unprecedented detail. The results of these analyses challenge the classic view of leukemia as a clonal homogeneous diffuse tumor and introduce a more complex and dynamic scenario. In this review, we present current concepts on the role of clonal evolution in lymphoid and myeloid leukemia as a driver of tumor initiation, disease progression and relapse. We also discuss the implications of these concepts in our understanding of the evolutionary mechanisms involved in leukemia transformation and therapy resistance.

Single-cell genomic analysis of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) incidence or rates have increased dramatically recently with little improvement in patient outcomes. There is an unmet need in HNSCC to develop reliable molecular markers capable of evaluating patient risks and advising treatments. This review focuses on recent developments in single-cell molecular analysis of cancer, and its applications for HNSCC diagnosis and treatments. For proof of concept, we examined gene expression levels of 62 patients with HNSCC, and correlate the gene expression profiles to single-cell gene expression profiles obtained from a pilot single-cell study of CCR5-positive breast carcinoma cells. The single-cell molecular analyses complemented the lysate data and reveals heterogeneity of oncogenesis pathways with the cancer cell population. Our single-cell molecular analysis indicated that molecular heterogeneity exists in HNSCC and should be addressed in treatment strategy of HNSCC. Single-cell molecular technology can have significant impact on diagnosis, therapeutic decision making, and prognosis of HNSCC.

Single-cell molecular analysis defines therapy response and immunophenotype of stem cell subpopulations in CML

AbstractUnderstanding leukemia heterogeneity is critical for the development of curative treatments as the failure to eliminate therapy-persistent leukemic stem cells (LSCs) may result in disease relapse. Here we have combined high-throughput immunophenotypic screens with large-scale single-cell gene expression analysis to define the heterogeneity within the LSC population in chronic phase chronic myeloid leukemia (CML) patients at diagnosis and following conventional tyrosine kinase inhibitor (TKI) treatment. Our results reveal substantial heterogeneity within the putative LSC population in CML at diagnosis and demonstrate differences in response to subsequent TKI treatment between distinct subpopulations. Importantly, LSC subpopulations with myeloid and proliferative molecular signatures are proportionally reduced at a higher extent in response to TKI therapy compared with subfractions displaying primitive and quiescent signatures. Additionally, cell surface expression of the CML stem cell markers CD25, CD26, and IL1RAP is high in all subpopulations at diagnosis but downregulated and unevenly distributed across subpopulations in response to TKI treatment. The most TKI-insensitive cells of the LSC compartment can be captured within the CD45RA− fraction and further defined as positive for CD26 in combination with an aberrant lack of cKIT expression. Together, our results expose a considerable heterogeneity of the CML stem cell population and propose a Lin−CD34+CD38−/lowCD45RA−cKIT−CD26+ population as a potential therapeutic target for improved therapy response.

Catch and Release: rare cell analysis from a functionalised medical wire

Enumeration and especially molecular characterization of circulating tumour cells (CTCs) holds great promise for cancer management. We tested a modified type of an in vivo enrichment device (Catch&Release) for its ability to bind and detach cancer cells for the purpose of single-cell molecular downstream analysis in vitro. The evaluation showed that single–cell analysis using array comparative genome hybridization (array-CGH) and next generation sequencing (NGS) is feasible. We found array-CGH to be less noisy when whole genome amplification (WGA) was performed with Ampli1 as compared to GenomePlex (DLRS values 0.65 vs. 1.39). Moreover, Ampli1-processed cells allowed detection of smaller aberrations (median 14.0 vs. 49.9 Mb). Single-cell NGS data obtained from Ampli1-processed samples showed the expected non-synonymous mutations (deletion/SNP) according to bulk DNA. We conclude that clinical application of this refined in vivo enrichment device allows CTC enumeration and characterization, thus, representing a promising tool for personalized medicine.

RareCyte® CTC Analysis Step 1: AccuCyte® Sample Preparation for the Comprehensive Recovery of Nucleated Cells from Whole Blood.

The RareCyte platform addresses important technology limitations of current circulating tumor cell (CTC) collection methods, and expands CTC interrogation to include advanced phenotypic characterization and single-cell molecular analysis. In this respect, it represents the "next generation" of cell-based liquid biopsy technologies. In order to identify and analyze CTCs, RareCyte has developed an integrated sample preparation, imaging and individual cell retrieval process. The first step in the process, AccuCyte®, allows the separation, collection, and transfer to a slide the nucleated cell fraction of the blood that contains CTCs. Separation and collection are based on cell density-rather than size or surface molecular expression-and are performed within a closed system, without wash or lysis steps, enabling high CTC recovery. Here, we describe our technique for nucleated cell collection from a blood sample, and the spreading of these nucleated cells onto glass slides permitting immunofluorescent staining, cell identification, and individual cell picking described in subsequent chapters. In addition to collection of rare cells such as CTCs, AccuCyte also collects cells of the circulating immune system onto archivable slides as well as plasma from the same sample.

Abstract 3160: Circulating tumor cell enrichment and dielectric manipulation for ultra-pure cell recovery in advanced bladder cancer

Abstract Background: Advanced transitional cell carcinoma (TCC) of the bladder is a lethal malignancy with few available biomarkers to guide therapy. Recent evidence suggests that circulating tumor cells (CTCs) are detectable in TCC and may have a prognostic role; however, most studies to date have focused on CTC enumeration. We leveraged novel enrichment and capture technologies to isolate and recover ultra-pure CTC (upCTC) that can be used for single cell molecular analysis. Methods: Patients with metastatic bladder cancer were enrolled under an institutional review board approved pilot study. Blood samples (7.5 ml each) were drawn into EDTA or Cell-Free DNA blood collection tubes (Streck), and preliminary CTC enrichment was performed using one of 3 microfluidic platforms: i) EpCAM-based LiquidBiopsy system (Cynvenio); ii) size/deformability-based ClearCell FX system (Clearbridge); or iii) size/deformability-based Parsortix system (Angle). Enriched CTCs were labeled with Cytokeratin (CK), EpCAM, and CD45 immunofluorescent antibodies and Hoechst nuclear stain. Identification and recovery of upCTCs was performed on a DEPArray v2 system (Silicon BioSystems). Additional experiments using cell lines were performed to gauge the recovery of mRNA from rare cancer cells collected in EDTA vs. preservative tubes. Results: To date, samples were collected from 8 patients of whom 5 (∼63%) had detectable CTCs. All 3 enrichment platforms successfully yielded CTC fractions from which Hoechst+/CK+/CD45- upCTCs were subsequently identified and recovered on the DEPArray v2 system. Additional cells that were Hoechst+/CK-/CD45- were identified and collected, and cells also were gated by integral Hoechst intensity and collected for correlation to aneuploidy. Comparison of mRNA recovery from 10 cancer cells collected into EDTA vs. fixative tubes (CellSave, Cynvenio, Streck Cell-free RNA) demonstrated that gene expression readings by qPCR from rare fixed cells was feasible but associated with variable results, likely due to variable proteinase K reversal of mRNA-protein cross-linkages. Conclusion: We leveraged new techniques to develop and optimize a workflow for identification and collection of upCTCs from bladder TCC patients, and we established that gene expression profiling is also feasible, albeit currently is still best achieved using EDTA collection and not any of the available preservative tubes. Single upCTC molecular profiles (DNA alterations, gene expression) from patient samples generated using these protocols may help to elucidate mechanisms of disease progression and ultimately may advance the management of advanced bladder cancer. Citation Format: Gareth Morrison, Cory Hugen, Tong Xu, Yucheng Xu, Dorff Tanya, David Quinn, Sarmad Sadeghi, Amir Goldkorn. Circulating tumor cell enrichment and dielectric manipulation for ultra-pure cell recovery in advanced bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3160.

Abstract IA09: Single cell RNA-sequencing of circulating tumor cells

Abstract Isolation of circulating tumor cells (CTCs) in the blood allows noninvasive tumor sampling from patients with cancer. Molecular analysis of single CTCs may uncover heterogeneous cellular pathways that underlie disease progression and resistance to therapy. Using a microfluidic device, we isolated individual CTCs from patients with metastatic prostate cancer. Single candidate CTCs were micromanipulated after cell surface staining for epithelial (EpCAM) and mesenchymal (CDH11) markers, and then subjected to single cell RNA-sequencing. Digital gene expression profiles of lineage-confirmed CTCs were compared with each other, with primary prostate tumors, and with annotated markers of cellular signaling pathways. Single prostate CTCs displayed considerable heterogeneity in transcriptional profiles, but clustered according to patient of origin, indicating higher diversity in CTCs across different individuals (mean correlation 0.10 for CTCs within patients vs. 0.0014 for CTCs across patients, P=2.0x10E-11). Compared to primary tumors, CTCs were significantly enriched in 37 molecular pathways (FDR <0.1), with the majority implicated in growth factor, cell adhesion, and hormone signaling. Gene mutations and alternative splice variants of the Androgen Receptor (AR) gene were rare in primary prostate tumors and CTCs from untreated patients, but were prevalent in patients with castration-resistant prostate cancer. Distinct AR variants, including AR-V7, were present within different cells of individual patients, as well as within individual CTCs. Together, single cell molecular analysis of human CTCs points to multiple mechanisms of drug resistance in advanced prostate cancer, and suggests the role of heterogeneous signaling pathways that cooperate with co-existing abnormalities in AR in mediating disease progression. Citation Format: David T. Miyamoto, Yu Zheng, Ben S. Wittner, Richard J. Lee, Huili Zhu, Katherine T. Broderick, Rushil Desai, Brian W. Brannigan, Kshitij S. Arora, Douglas M. Dahl, Lecia V. Sequist, Matthew R. Smith, Ravi Kapur, Chin-Lee Wu, Toshi Shioda, Sridhar Ramaswamy, David T. Ting, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber. Single cell RNA-sequencing of circulating tumor cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr IA09.

Single-Cell Analysis of Circulating Tumor Cells as a Window into Tumor Heterogeneity.

Recent advances in microfluidic approaches have enabled the efficient isolation and detailed molecular characterization of circulating tumor cells (CTCs) in the peripheral blood of patients with cancer. Single-cell molecular analyses of CTCs reveal a tremendous degree of intracellular heterogeneity in CTC populations, reflective of heterogeneity across different patients as well as the underlying heterogeneity of tumors within each individual patient. These studies have enabled the identification of heterogeneous drug resistance mechanisms that can coexist in treatment refractory tumors. CTC analyses also enable serial noninvasive monitoring in patients and can capture the emergence of tumor heterogeneity over time, whether due to tumor evolution through genetic instability or through cellular plasticity. The presence and extent of intratumoral heterogeneity as revealed through the study of CTCs have important clinical implications for understanding and predicting the development of treatment resistance in a variety of solid tumors and for formulating appropriate therapeutic strategies in the effective treatment of cancer.

Abstract 224: Enrichment and isolation of uncontaminated breast cancer cells from human blood samples

Abstract Cancer cells in peripheral blood, known as circulating tumor cells (CTCs), play an important role in tumor dissemination. CTCs are shed from either the primary tumor or its metastases and circulate in the peripheral blood of patients; thus they can be regarded as “liquid biopsies” of metastasizing cells. Although the exact origin and physiology of CTCs is unknown, a fraction of these cells are thought to be viable metastatic precursors capable of initiating a clonal metastatic lesion. The molecular characterization of CTCs is important because it may provide insights into the molecular biology of metastasis, the association of their molecular profiles with treatment outcomes, and reveal the presence of potential therapeutic targets. The process of CTC enrichment represents a significant step toward the isolation of CTCs from whole blood. Given that there are approximately 5×109 erythrocytes and 107 leukocytes per ml of whole blood, purification must represent a reduction of many orders of magnitude. In this study, we show a method to isolate CTCs which combines a microfluidic cell separation device (Parsortix system) as an enrichment method followed by the DEPArray selection system. The Parsortix system is versatile in its ability to separate, capture and harvest CTCs in an enriched form compatible with the needs of downstream processes such as DEPArray. Here we demonstrate enrichment of CTCs via the Parsortix system using both breast cancer cell spiked healthy blood donor samples and blood from metastatic breast cancer patients. For these experiments, two milliliters of blood were processed. The cells were harvested and stained to identify CTCs in the enriched harvest product. Cells were stained with fluorescently labeled monoclonal antibodies specific for pan cytokeratin (CK-8/18/19-PE), leukocyte common antigen (CD45-APC), and nuclear stained with [4′,6-diamidino-2-phenylindole (DAPI)]. With spiked samples, tumor cells were isolated by DEPArray selection, which yielded a pure population of tumor cells for molecular characterization. Tumor cells were defined by presence of a clear DAPI-stained nucleus, CK-PE-positive cytoplasm and CD-45-APC negative. We demonstrated that by using a combination of enrichment and isolation/selection methods, we are able to isolate single, uncontaminated tumor cells to achieve single cell molecular analysis. The use of single cells is emerging as a powerful approach to molecular analysis in oncology, and this study demonstrates its potential application with circulating tumor cells. Citation Format: Sandra V. Fernandez, Christopher Wagner, Zahida Parveen, Lucy Aburto, Carmela Paolillo, George Hvichia, Zhaomei Mu, Laura Austin, Massimo Cristofanilli. Enrichment and isolation of uncontaminated breast cancer cells from human blood samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 224. doi:10.1158/1538-7445.AM2015-224

Live Cell Genomics: Cell-Specific Transcriptome Capture in Live Tissues and Cells.

The sensitivity of new transcriptomic techniques is rapidly improving to the point that single-cell molecular analysis is now becoming commonplace. However to obtain accurate transcriptome data, the initial experimental steps must strive to maintain the natural environment of cell and always get set in motion under in vivo conditions. Achieving these critical experimental parameters is technically challenging for investigators and currently the most frequently used molecular techniques experimentally commence with tissues or cells in artificial environments or under in vitro conditions. Here we review an innovative experimental approach that is called transcriptome in vivo analysis (TIVA) that was designed to overcome theses well-known limitations. The TIVA methods permit cell-specific transcriptome capture from viable intact heterogeneous tissues. Cell-penetrating peptides (CPPs) are used to deliver multifunctional transcriptome-capture tags (TIVA tags) to the cytoplasm of the cell under in vivo conditions. The TIVA capture tag enables investigators to target and isolate cell-specific transcriptomes in their natural microenvironments. The combination of maintaining in vivo conditions and selective cell-specific transcriptome capture provides investigators with the opportunity to yield the most biologically accurate and informative transcriptome data hitherto.

Interstitial Cells of Cajal and the Promise of Single Cell Molecular Analysis

Interstitial Cells of Cajal and the Promise of Single Cell Molecular Analysis

Molecular profiling of single circulating tumor cells with diagnostic intention.

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of > 90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance.

TP53 mutations detected in circulating tumor cells present in the blood of metastatic triple negative breast cancer patients

IntroductionCirculating tumor cells (CTCs) are tumor cells shed from either primary tumors or its metastases that circulate in the peripheral blood of patients with metastatic cancers. The molecular characterization of the CTCs is critical to identifying the key drivers of cancer metastasis and devising therapeutic approaches. However, the molecular characterization of CTCs is difficult to achieve because their isolation is a major technological challenge.MethodsCTCs from two triple negative breast cancer patients were enriched using CellSearch and single cells selected by DEPArray™. A TP53 R110 fs*13 mutation identified by next generation sequencing in the breast and chest skin biopsies of both patients was studied in single CTCs.ResultsFrom 6 single CTC isolated from one patient, 1 CTC had TP53 R110 delC, 1 CTC showed the TP53 R110 delG mutation, and the remaining 4 single CTCs showed the wild type p53 sequence; a pool of 14 CTCs isolated from the same patient also showed TP53 R110 delC mutation. In the tumor breast tissue of this patient, only the TP53 R110 delG mutation was detected. In the second patient a TP53 R110 delC mutation was detected in the chest wall skin biopsy; from the peripheral blood of this patient, 5 single CTC and 6 clusters of 2 to 6 CTCs were isolated; 3 of the 5 single CTCs showed the TP53 R110 delC mutation and 2 CTCs showed the wild type TP53 allele; from the clusters, 5 showed the TP53 R110 delC mutation, and 1 cluster the wild type TP53 allele. Single white blood cells isolated as controls from both patients only showed the wild type TP53 allele.ConclusionsWe are able to isolate uncontaminated CTCs and achieve single cell molecular analysis. Our studies showed the presence of different CTC sub-clones in patients with metastatic breast cancer. Some CTCs had the same TP53 mutation as their matching tumor samples although others showed either a different TP53 mutation or the wild type allele. Our results indicate that CTCs could represent a non-invasive source of cancer cells from which to determine genetic markers of the disease progression and potential therapeutic targets.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0445-3) contains supplementary material, which is available to authorized users.

A method for detecting protein expression in single cells using the C1™ Single-Cell Auto Prep System (TECH2P.874)

Abstract Recent improvements in microfluidics and biochemistry have enabled single-cell molecular analysis, providing new insight into the heterogeneity of cell populations. The C1 Single-Cell Auto Prep System is an automated platform that streamlines the isolation and processing of 96 individual, live cells for RNA and DNA analysis. Single-cell protein profiling is a direct complement to genomic analysis as it provides additional insights into key molecular mechanisms and system biology. To enable this, we adapted a highly multiplexed protein detection system (Proseek Multiplex Oncology, Olink Bioscience) for use on the C1 System. We analyzed 192 individual cells from two human promyelocytic leukemia cell lines (HL60 and K562) and compared these result against 16 FACS-sorted cells in tube format. These results indicated that four key proteins (i.e. CASP3, CSTB, CD69 and MPO) are differentially expressed between the two cell lines and vary from cell-to-cell. For example, the human protein MPO, produced by neutrophils for their microbicidal activity, was differentially expression between individual HL60 and K562 cells, suggesting varying levels of neutrophil activation within the cell populations. This work demonstrates highly-multiplexed protein detection at the single-cell level and shows promise to have sufficient sensitivity, reproducibility and scale to effectively interrogate a statistically significant numbers of individual cells.

Molecular analysis of circulating tumour cells—biology and biomarkers

Growing evidence for intratumour heterogeneity informs us that single-site biopsies fall short of revealing the complete genomic landscape of a tumour. With an expanding repertoire of targeted agents entering the clinic, screening tumours for genomic aberrations is increasingly important, as is interrogating the tumours for resistance mechanisms upon disease progression. Multiple biopsies separated spatially and temporally are impractical, uncomfortable for the patient and not without risk. Here, we describe how circulating tumour cells (CTCs), captured from a minimally invasive blood test-and readily amenable to serial sampling-have the potential to inform intratumour heterogeneity and tumour evolution, although it remains to be determined how useful this will be in the clinic. Technologies for detecting and isolating CTCs include the validated CellSearch(®) system, but other technologies are gaining prominence. We also discuss how recent CTC discoveries map to mechanisms of haematological spread, previously described in preclinical models, including evidence for epithelial-mesenchymal transition, collective cell migration and cells with tumour-initiating capacity within the circulation. Advances in single-cell molecular analysis are enhancing our ability to explore mechanisms of metastasis, and the combination of CTC and cell-free DNA assays are anticipated to provide invaluable blood-borne biomarkers for real-time patient monitoring and treatment stratification.

Odorant receptor gene choice and axonal wiring in mice with deletion mutations in the odorant receptor gene SR1

In the mouse, a mature olfactory sensory neuron (OSN) of the main olfactory epithelium (MOE) expresses one allele of one of the 1200 odorant receptor (OR) genes in the genome. The mechanisms that underlie the one receptor-one neuron rule remain poorly understood. A popular experimental paradigm for OR gene choice is to delete an OR coding region by gene targeting or in a transgene. Here we have applied this ∆OR paradigm to SR1, also known as MOR256-3 or Olfr124. This gene is expressed in OSNs of the MOE, and in ~50% of the OSNs of the septal organ. In heterozygous ∆SR1 mice, we observe an unprecedented biallelic expression rate of 30% at the SR1 locus. In homozygous ∆SR1 mice, we find a significant increase in the number of septal organ OSNs that undergo apoptosis. As a population, ∆SR1 OSNs project their axons to 81-85 glomeruli in each half of the OB, and coexpress at least 77 OR genes as evaluated by single-cell molecular analysis. There are no obvious or simple rules for the set of OR genes that are coexpressed with the ∆SR1 allele. The frequencies of coexpression are different for ∆SR1 OSNs in the septal organ compared to those in the MOE. We propose that there are as many as five scenarios for the fate of individual ∆SR1 OSNs.