Low-abundance Cells Research Articles

Efficient Strategy to Discover DNA Aptamers Against Low Abundance Cell Surface Proteins in Scarce Samples.

Molecular recognition probes targeting cell surface proteins such as aptamers play crucial roles in precise diagnostics and therapy. However, the selection of aptamers against low-abundance proteins in situ on the cell surface, especially in scarce samples, remains an unmet challenge. In this study, we present a single-round, single-cell aptamer selection method by employing a digital DNA sequencing strategy, termed DiDS selection, to address this dilemma. This approach incorporates a molecular identification card for each DNA template, thereby mitigating biases introduced by multiple PCR amplifications and ensuring the accurate identification of aptamer candidates. Through DiDS selection, we successfully obtained a series of high-quality aptamers against cell lines, clinical specimens, and neurons. Subsequent analyses for target identification revealed that aptamers derived from DiDS selection exhibit recognition capabilities for proteins with varying abundance levels. In contrast, multiple rounds of selection resulted in the enrichment of only one aptamer targeting a high-abundance target. Moreover, the comprehensive profiling of cell surfaces at the single-cell level, utilizing an enriched aptamer pool, revealed unique molecular patterns for each cell line. This streamlined approach holds promise for the rapid generation of specific recognition molecules targeting cell surface proteins across a broad range of expression levels and expands its applications in cell profiling, specific probe identification, biomarker discovery, etc.

The proteome of the blood–brain barrier in rat and mouse: highly specific identification of proteins on the luminal surface of brain microvessels by in vivo glycocapture

BackgroundThe active transport of molecules into the brain from blood is regulated by receptors, transporters, and other cell surface proteins that are present on the luminal surface of endothelial cells at the blood–brain barrier (BBB). However, proteomic profiling of proteins present on the luminal endothelial cell surface of the BBB has proven challenging due to difficulty in labelling these proteins in a way that allows efficient purification of these relatively low abundance cell surface proteins.MethodsHere we describe a novel perfusion-based labelling workflow: in vivo glycocapture. This workflow relies on the oxidation of glycans present on the luminal vessel surface via perfusion of a mild oxidizing agent, followed by subsequent isolation of glycoproteins by covalent linkage of their oxidized glycans to hydrazide beads. Mass spectrometry-based identification of the isolated proteins enables high-confidence identification of endothelial cell surface proteins in rats and mice.ResultsUsing the developed workflow, 347 proteins were identified from the BBB in rat and 224 proteins in mouse, for a total of 395 proteins in both species combined. These proteins included many proteins with transporter activity (73 proteins), cell adhesion proteins (47 proteins), and transmembrane signal receptors (31 proteins). To identify proteins that are enriched in vessels relative to the entire brain, we established a vessel-enrichment score and showed that proteins with a high vessel-enrichment score are involved in vascular development functions, binding to integrins, and cell adhesion. Using publicly-available single-cell RNAseq data, we show that the proteins identified by in vivo glycocapture were more likely to be detected by scRNAseq in endothelial cells than in any other cell type. Furthermore, nearly 50% of the genes encoding cell-surface proteins that were detected by scRNAseq in endothelial cells were also identified by in vivo glycocapture.ConclusionsThe proteins identified by in vivo glycocapture in this work represent the most complete and specific profiling of proteins on the luminal BBB surface to date. The identified proteins reflect possible targets for the development of antibodies to improve the crossing of therapeutic proteins into the brain and will contribute to our further understanding of BBB transport mechanisms.

ScPipe: an extended preprocessing pipeline for comprehensive single-cell ATAC-Seq data integration in R/Bioconductor.

scPipe is a flexible R/Bioconductor package originally developed to analyse platform-independent single-cell RNA-Seq data. To expand its preprocessing capability to accommodate new single-cell technologies, we further developed scPipe to handle single-cell ATAC-Seq and multi-modal (RNA-Seq and ATAC-Seq) data. After executing multiple data cleaning steps to remove duplicated reads, low abundance features and cells of poor quality, a SingleCellExperiment object is created that contains a sparse count matrix with features of interest in the rows and cells in the columns. Quality control information (e.g. counts per cell, features per cell, total number of fragments, fraction of fragments per peak) and any relevant feature annotations are stored as metadata. We demonstrate that scPipe can efficiently identify 'true' cells and provides flexibility for the user to fine-tune the quality control thresholds using various feature and cell-based metrics collected during data preprocessing. Researchers can then take advantage of various downstream single-cell tools available in Bioconductor for further analysis of scATAC-Seq data such as dimensionality reduction, clustering, motif enrichment, differential accessibility and cis-regulatory network analysis. The scPipe package enables a complete beginning-to-end pipeline for single-cell ATAC-Seq and RNA-Seq data analysis in R.

Multiplex Protein Profiling by Low-Signal-Loss Single-Cell Western Blotting with Fluorescent-Quenching Aptamers.

Single-cell western blotting (scWB) is a prevalent technique for high-resolution protein analysis on low-abundance cell samples. However, the extensive signal loss during repeated antibody stripping precludes multiplex protein detection. Herein, we introduce Fluorescent-quenching Aptamer-based Single-cell Western Blotting (FAS-WB) for multiplex protein detection at single-cell resolution. The minimal size of aptamer probes allows rapid in-gel penetration, diffusion, and elution. Meanwhile, the fluorophore-tagged aptamers, coordinated with complementary quenching strands, avoid the massive signal loss conventionally caused by antibody stripping during repeated staining. Such a strategy also facilitates multiplex protein analysis with a limited number of fluorescent tags. We demonstrated FAS-WB for co-imaging four biomarker proteins (EpCAM, PTK7, HER2, CA125) at single-cell resolution with lower signal loss and enhanced signal-to-noise ratio compared to conventional antibody-based scWB. Being more time-saving (less than 25 min per cycle) and economical (1/1000 cost of conventional antibody probes), FAS-WB offers a highly efficient platform for profiling multiplex proteins at single-cell resolution.

CeDAR: incorporating cell type hierarchy improves cell type-specific differential analyses in bulk omics data

Bulk high-throughput omics data contain signals from a mixture of cell types. Recent developments of deconvolution methods facilitate cell type-specific inferences from bulk data. Our real data exploration suggests that differential expression or methylation status is often correlated among cell types. Based on this observation, we develop a novel statistical method named CeDAR to incorporate the cell type hierarchy in cell type-specific differential analyses of bulk data. Extensive simulation and real data analyses demonstrate that this approach significantly improves the accuracy and power in detecting cell type-specific differential signals compared with existing methods, especially in low-abundance cell types.

Low input capture Hi-C (liCHi-C) identifies promoter-enhancer interactions at high-resolution

Long-range interactions between regulatory elements and promoters are key in gene transcriptional control; however, their study requires large amounts of starting material, which is not compatible with clinical scenarios nor the study of rare cell populations. Here we introduce low input capture Hi-C (liCHi-C) as a cost-effective, flexible method to map and robustly compare promoter interactomes at high resolution. As proof of its broad applicability, we implement liCHi-C to study normal and malignant human hematopoietic hierarchy in clinical samples. We demonstrate that the dynamic promoter architecture identifies developmental trajectories and orchestrates transcriptional transitions during cell-state commitment. Moreover, liCHi-C enables the identification of disease-relevant cell types, genes and pathways potentially deregulated by non-coding alterations at distal regulatory elements. Finally, we show that liCHi-C can be harnessed to uncover genome-wide structural variants, resolve their breakpoints and infer their pathogenic effects. Collectively, our optimized liCHi-C method expands the study of 3D chromatin organization to unique, low-abundance cell populations, and offers an opportunity to uncover factors and regulatory networks involved in disease pathogenesis.

Improvement and application of qPCR assay revealed new insight on early warning of Phaeocystis globosa bloom

Phaeocystis globosa bloom develops from its early solitary cells, providing clues for early warning of its bloom and timely responding to possible consequences. However, the early prediction requires quantification of the solitary cells for a thorough understanding of bloom formation. Therefore, we developed an accurate, sensitive, and specific qPCR assay for this need. Results show that the accuracy of qPCR was significantly enhanced by ameliorating DNA barcode design, improving genomic DNA extraction, and introducing a strategy of internal amplification control (IAC). This approach reached a quantification limit of 1 cell/reaction, making low-abundance cells (101−103 cells/L) detection possible, and we also observed a plunge in the abundance of the solitary cells before the bloom outbreak in two winters in 2019 and 2020 for the first time, which is quite unique from laboratory results showing an increase instead. The plunge in solitary-cell abundance might be associated with the attachment of solitary cells to solid matrices to form non-solitary attached aggregate, the precursor of colonies, which gains supports from other studies and needs more investigations in the future. Therefore, as the plunge in solitary-cell abundance is a sign of colony formation, it can be used as an early warning indicator to P. globosa bloom.

Metabolic engineering of oleaginous yeast Rhodotorula toruloides for overproduction of triacetic acid lactone

The plant‐sourced polyketide triacetic acid lactone (TAL) has been recognized as a promising platform chemical for the biorefinery industry. However, its practical application was rather limited due to low natural abundance and inefficient cell factories for biosynthesis. Here, we report the metabolic engineering of oleaginous yeast Rhodotorula toruloides for TAL overproduction. We first introduced a 2‐pyrone synthase gene from Gerbera hybrida (GhPS) into R. toruloides and investigated the effects of different carbon sources on TAL production. We then systematically employed a variety of metabolic engineering strategies to increase the flux of acetyl‐CoA by enhancing its biosynthetic pathways and disrupting its competing pathways. We found that overexpression of ATP‐citrate lyase (ACL1) improved TAL production by 45% compared to the GhPS overexpressing strain, and additional overexpression of acetyl‐CoA carboxylase (ACC1) further increased TAL production by 29%. Finally, we characterized the resulting strain I12‐ACL1‐ACC1 using fed‐batch bioreactor fermentation in glucose or oilcane juice medium with acetate supplementation and achieved a titer of 28 or 23 g/L TAL, respectively. This study demonstrates that R. toruloides is a promising host for the production of TAL and other acetyl‐CoA‐derived polyketides from low‐cost carbon sources.

Abstract 762: Practical guidelines for the design of single cell sequencing studies

Abstract Performance of Single Cell RNA sequencing (scRNA-seq) experiments depends on multiple factors including the number of cells loaded, cell recovery rates, and sequencing coverage. We optimized such factors in 10x Genomics gene expression profiling and compared targeted panels to whole transcriptome sequencing using Human donor PBMC samples. We expect that our findings will apply to scRNA-seq studies of other sample types as well. Optimizing cell recovery in scRNA-seq experiments is essential in order to ensure that changes in low-abundance cell populations (e.g. Treg) can be accurately quantified. Statistical analysis indicated that at least 100 cells are needed to assess cell-type specific state changes. We expect to recover 40-65% of cells loaded into the 10x chip. We found that loading 20k-30k cells/lane combined with cell hashtags for improved doublet detection improved cell recovery over the recommended loading of 10K-16K cells. We also surveyed internal single cell experiments and observed variable cell recovery across different sample types. The amount of ambient RNA in the library was correlated with lower performance in single cell studies, suggesting that cell death or damage was causing lower recovery. We found that using PBS for cell resuspension (which is less likely to cause cell rupture) performs as well as the nuclease-free water suggested by 10x. We also assessed the impact of sequencing depth and protocol on scRNA-seq data quality. Sequencing depth impacts both experiment cost and data quality due to dropouts. We performed computational simulations of lower depth sequencing by subsampling various number of reads from PBMC experiments to obtain coverages of 10K, 20K, 40K and 80K reads per cell. We observed that 40K reads per cell, yielding about 70% sequencing saturation, provided a good balance between cost and sequencing depth. Finally, we compared dropout rates and cell type annotation for two targeted panels, the Human Gene Signature and Human Immunology panels to whole transcriptome scRNAseq. On average we detected only 200 genes out of over 1000 represented in each panel. Dropout rates for targeted panels were only reduced at low coverage; at read depths higher than 40K reads per cell the whole transcriptome and targeted panels had similar dropout rates. Both methods detected major immune cell types, but targeted sequencing could not accurately identify some subtypes due to low number of detected genes. We conclude that for immune cell profiling whole-transcriptome analysis at coverage of 40K reads per cell or higher with inputs of 20k-30k cells and use of sample hashtag antibodies provides the best balance of experiment cost, cell recovery and transcriptome coverage. Although these guidelines were established for Human PBMCs we expect similar outcomes with other complex cell mixtures such as dissociated tissues. Citation Format: Amir Bayegan, Julien Tessier, Emma Wang, Adalis Maisonet, Shu Yan, Shannon McGrath, Donald G. Jackson, Jack Pollard. Practical guidelines for the design of single cell sequencing studies [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 762.

A magnetic nanoparticle assisted microfluidic system for low abundance cell sorting with high recovery

Low abundance cell sorting has fundamental applications in cell research and therapy, such as cancer diagnosis, cell transplantation, and immunology. However, various methods for isolating low abundance cells are challenging due to the low cell recovery rate and throughput. In this paper, we propose a magnetic nanoparticle assisted microfluidic system (MNPAMS) for selecting low abundance cells with high cell recovery rate. This system integrates an active magnetic nanoparticles (MNPs)-based sorting mechanism and a passive size-based capture mechanism into one microfluidic chip. Polydimethylsiloxane (PDMS) was used to fabricate a bilayer microfluidic chip. The magnetic and fluid field models of the MNPAMS were analyzed using finite element method (FEM). Furthermore, the sorting performance was validated by mixed HeLa cell samples of three different concentrations. The experimental results show the target cells were concentrated in the arrays of structures 2 and structures 3. For different concentrations, the target cell recovery rate is relatively stable up to maximum 99.5%. Besides, the recovery efficiency of low abundance cells sorting can reach 88.6% for a 1/100 of the original sample concentration. Based on these results, the MNPAMS has the potential to facilitate liquid biopsy and fundamental biomedical studies with high target cells recovery. • A Magnetic Nanoparticle Assist Microfluidic System was proposed for cell sorting. • The system combined magnetic labeling with microstructures capturing mechanism. • The highest recovery efficiency could reach 88.6% for low abundance cell sorting.

Enrichment-Free Single-Cell Detection and Morphogenomic Profiling of Myeloma Patient Samples to Delineate Circulating Rare Plasma Cell Clones.

Multiple myeloma is an incurable malignancy that initiates from a bone marrow resident clonal plasma cell and acquires successive mutational changes and genomic alterations, eventually resulting in tumor burden accumulation and end-organ damage. It has been recently recognized that myeloma secondary genomic events result in extensive sub-clonal heterogeneity both in localized bone marrow areas and circulating peripheral blood plasma cells. Rare genomic subclones, including myeloma initiating cells, could be the drivers of disease progression and recurrence. Additionally, evaluation of rare myeloma cells in blood for disease monitoring has numerous advantages over invasive bone marrow biopsies. To this end, an unbiased method for detecting rare cells and delineating their genomic makeup enables disease detection and monitoring in conditions with low abundant cancer cells. In this study, we applied an enrichment-free four-plex (CD138, CD56, CD45, DAPI) immunofluorescence assay and single-cell DNA sequencing for morphogenomic characterization of plasma cells to detect and delineate common and rare plasma cells and discriminate between normal and malignant plasma cells in paired blood and bone marrow aspirates from five patients with newly diagnosed myeloma (N = 4) and monoclonal gammopathy of undetermined significance (n = 1). Morphological analysis confirms CD138+CD56+ cells in the peripheral blood carry genomic alterations that are clonally identical to those in the bone marrow. A subset of altered CD138+CD56- cells are also found in the peripheral blood consistent with the known variability in CD56 expression as a marker of plasma cell malignancy. Bone marrow tumor clinical cytogenetics is highly correlated with the single-cell copy number alterations of the liquid biopsy rare cells. A subset of rare cells harbors genetic alterations not detected by standard clinical diagnostic methods of random localized bone marrow biopsies. This enrichment-free morphogenomic approach detects and characterizes rare cell populations derived from the liquid biopsies that are consistent with clinical diagnosis and have the potential to extend our understanding of subclonality at the single-cell level in this disease. Assay validation in larger patient cohorts has the potential to offer liquid biopsy for disease monitoring with similar or improved disease detection as traditional blind bone marrow biopsies.

A Magnetic Nanoparticle Assisted Microfluidic System for Low Abundance Cell

A Magnetic Nanoparticle Assisted Microfluidic System for Low Abundance Cell

Sheathless acoustic based flow cell sorter for enrichment of rare cells.

Cell enrichment is a powerful tool in many kinds of cell research, especially in applications with low abundance cell types. In this work, we developed a microfluidic fluorescence activated cell sorting device that was able to perform on-demand, low loss cell detection, and sorting. The chip utilizes three-dimensional acoustic standing waves to position all cells in the same fluid velocity regime without sheath. When the cells pass through a laser interrogation region, the scattering and fluorescent signals are detected, translated and transported to software. The target cells are then identified by gating on the plots. Short bursts of standing acoustic waves are triggered by order from PC to sort target cells within predefined gating region. For very low abundance and rare labeled lymphocytes mixed with high concentration unlabeled white blood cells (WBCs), (1-100 labeled lymphocytes are diluted in 106 WBCs in 1 ml volume fluid), the device is able to remove more than 98% WBCs and recover labeled lymphocytes with efficiency of 80%. We further demonstrated that this device worked with real clinical samples by successfully isolating fetal nucleated red blood cells (FNRBCs) in the blood samples from pregnant women with male fetus. The obtained cells were sequenced and the expressions of (sex determining region Y) SRY genes were tested to determine fetal cell proportion. In genetic analysis, the proportion of fetal cells in the final picked sample is up to 40.64%. With this ability, the device proposed could be valuable for biomedical applications involving fetal cells, circulating tumor cells, and stem cells.

TMOD-29. STANDARDIZED GENERATION OF TUMOR-ORGANOIDS AS NOVEL DRUG SCREENING MODEL IN MENINGIOMA

Abstract Tumor-organoids (TOs) are novel, complex three-dimensional ex vivo tissue cultures that under optimal conditions accurately reflect genotype and phenotype of the original tissue with preserved cellular heterogeneity and morphology. They may serve as a new and exciting model for studying cancer biology and directing personalized therapies. The aim of our study was to establish TOs from meningioma (MGM) and to test their usability for large-scale drug screenings. We were capable of forming several hundred TO equal in size by controlled reaggregation of freshly prepared single cell suspension of MGM tissue samples. In total, standardized TOs from 60 patients were formed, including eight grade II and three grade III MGMs. TOs reaggregated within 3 days resulting in a reducted diameter by 50%. Thereafter, TO size remained stable throughout a 14 days observation period. TOs consisted of largely viable cells, whereas dead cells were predominantly found outside of the organoid. H&E stainings confirmed the successful establishment of dense tissue-like structures. Next, we assessed the suitability and reliability of TOs for a robust large-scale drug testing by employing nine highly potent compounds, derived from a drug screening performed on several MGM cell lines. First, we tested if drug responses depend on TO size. Interestingly, drug responses to these drugs remained identical independent of their sizes. Based on a sufficient representation of low abundance cell types such as T-cells and macrophages an overall number of 25.000 cells/TO was selected for further experiments revealing FDA-approved HDAC inhibitors as highly effective drugs in most of the TOs with a mean z-AUC score of -1.33. Taken together, we developed a protocol to generate standardized TO from MGM containing low abundant cell types of the tumor microenvironment in a representative manner. Robust and reliable drug responses suggest patient-derived TOs as a novel drug testing model in meningioma research.

Asymmetrically coating Pt nanoparticles on magnetic silica nanospheres for target cell capture and therapy.

AJanus cargo has been developedvia the combination of magnetic mesoporous silica (MMS) with asymmetric decoration of Pt nanoparticles (PtNPs). Mesoporous morphology of MMS provides plenty of space for loading photosensitizers and targeting agents; the magnetic feature endows the as-formed nanospheres with satisfactory isolation function in removal of low abundant target cells. The excellent catalytic ability of PtNPs can effectively alleviate the hypoxia condition of tumor microenvironment via the decomposition of hydrogen peroxide (H2O2), as well as an O2-drived nanomotor for highly efficient drug release. Using CCRF-CEM as the model target cell, the Janus cargo is demonstrated to possess significantly improved performance in cell capture and photodynamic therapy. Specially, owing to the patchy Pt decoration, the loaded photosensitizers exhibit a more efficient release behavior. More importantly, asymmetric O2-emission from one side of the nanocargo acts as a driving force, which could effectively accelerate the motion ability of cargo in cell media, thus leading to an enhanced therapeutic effect compared withthe traditionally symmetric nanocargo. This Janus cargo would offer a new paradigm to design highly efficient drug carrier for gaining an improved photodynamic therapy in hypoxic cancer cells.

Engineering of a Small Protein Scaffold To Recognize Sulfotyrosine with High Specificity.

Protein tyrosine O-sulfation is an essential post-translational modification required for effective biological processes such as hemostasis, inflammatory response, and visual phototransduction. Because of its unstable nature under mass spectrometry conditions and residing on low-abundance cell surface proteins, sulfated tyrosine (sulfotyrosine) residues are difficult to detect or analyze. Enrichment of sulfotyrosine-containing proteins (sulfoproteins) from complex biological samples are typically required before analysis. In this work, we seek to engineer the phosphotyrosine binding pocket of a Src Homology 2 (SH2) domain to act as an antisulfotyrosine antibody mimic. Using tailored selection schemes, several SH2 mutants are identified with high affinity and specificity to sulfotyrosine. Further molecular docking simulations highlight potential mechanisms supporting observed characteristics of these SH2 mutants. Utilities of the evolved SH2 mutants were demonstrated by the detection and enrichment of sulfoproteins.

Profiling Active Enzymes for Polysorbate Degradation in Biotherapeutics by Activity-Based Protein Profiling.

Polysorbate is widely used to maintain stability of biotherapeutic proteins in pharmaceutical formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual host cell enzymes such as lipases and esterases plays a major role for polysorbate degradation. Their high activity, often at very low concentration, constitutes a major analytical challenge in the biopharmaceutical industry. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using an optimized chemical probe, we established the first global profile of active serine hydrolases in harvested cell culture fluid (HCCF) for monoclonal antibodies (mAbs) production from two Chinese hamster ovary (CHO) cell lines. A total of eight known lipases were identified by ABPP with enzyme activity information, while only five lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study with downstream samples, phospholipase A2 group VII (PLA2G7) was only identified by ABPP and confirmed to contribute to polysorbate-80 degradation for the first time. The established ABBP approach is approved to be able to identify low-abundance host cell enzymes and fills the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.

Fluorescent Droplet Cytometry for On-Cell Phenotype Tracking.

Profiling the heterogeneous phenotypes of live cancer cells is a key capability that requires single-cell analysis. However, acquiring information at the single-cell level for live cancer cells is challenging when small collections of cells are being targeted. Here, we report single-cell analysis for low abundance cells enabled by fluorescent droplet cytometry (FDC), an approach that uses a biomarker-specific enzymatic fluorescent assay carried out using a droplet microfluidic platform. FDC utilizes DNA-functionalized antibodies in droplets to achieve specific on-cell target detection and enables characterization and profiling of live cancer cells with single-cell resolution based on their surface phenotype. Using this approach, we achieve live-cell phenotypic profiling of multiple surface markers acquired with small (<40 cells) collections of cells.

Single-Cell On-Probe Derivatization–Noncontact Nanocarbon Fiber Ionization: Unraveling Cellular Heterogeneity of Fatty Alcohol and Sterol Metabolites

Currently in single-cell mass spectrometry, the analysis of low-abundance cell metabolites such as fatty alcohols and sterols remains a challenge. In most research studies, single-cell samples are analyzed directly after sampling. However, this workflow may exclude many effective sample pretreatment methods such as derivatization for the improvement of detection sensitivity for specific cell metabolites in a single-cell sample. Metabolites in low abundance in a cell may not be detected. Herein on-probe derivatization coupled with noncontact nanocarbon fiber ionization is proposed for sensitive fatty alcohol and sterol metabolite analysis at the single-cell level. Fatty alcohol and sterol metabolites were rapidly quaternized by the single-cell on-probe derivatization method. The reaction products were directly ionized with no postreaction processing. Furthermore, a new ionization source for noncontact nanocarbon fiber ionization was developed to show good compatibility with dichloromethane, a low-polarity solvent used in on-probe derivatization. The quaternized fatty alcohols and sterols exhibited evidently enhanced ionization efficiency in mass spectra. In applications of the developed method, seven kinds of even-numbered-carbon fatty alcohols (C12-C22) and five kinds of sterols were detected in single L-02 and HepG2 cells. Then the L-02 and HepG2 cells were readily discriminated through principal component analysis. Additionally, a rough quantitative analysis of the detected fatty alcohols and sterols in single cells was performed. The mass intensities of fatty alcohols show a significant difference between L-02 and HepG2 cells while those of sterols remain stable.

Photodynamic Therapy for ras-Driven Cancers: Targeting G-Quadruplex RNA Structures with Bifunctional Alkyl-Modified Porphyrins.

Designing small molecules able to break down G4 structures in mRNA (RG4s) offers an interesting approach to cancer therapy. Here, we have studied cationic porphyrins (CPs) bearing an alkyl chain up to 12 carbons, as they bind to RG4s while generating reactive oxygen species upon photoirradiation. Fluorescence-activated cell sorting (FACS) and confocal microscopy showed that the designed alkyl CPs strongly penetrate cell membranes, binding to KRAS and NRAS mRNAs under low-abundance cell conditions. In Panc-1 cells, alkyl CPs at nanomolar concentrations promote a dramatic downregulation of KRAS and NRAS expression, but only if photoactivated. Alkyl CPs also reduce the metabolic activity of pancreatic cancer cells and the growth of a Panc-1 xenograft in SCID mice. Propidium iodide/annexin assays and caspase 3, caspase 7, and PARP-1 analyses show that these compounds activate apoptosis. All these data demonstrate that the designed alkyl CPs are efficient photosensitizers for the photodynamic therapy of ras-driven cancers.