Preserving Single Cells in Space and Time for Analytical Assays.

Analytical assays performed within clinical laboratories influence roughly 70% of all medical decisions by facilitating disease detection, diagnosis, and management. Both in clinical and academic research laboratories, single-cell assays permit measurement of cell diversity and identification of rare cells, both of which are important in the understanding of disease pathogenesis. For clinically utility, the single-cell assays must be compatible with the clinical workflow steps of sample collection, sample transportation, pre-analysis processing, and single-cell assay; therefore, it is paramount to preserve cells in a state that resembles that in vivo rather than measuring signaling behaviors initiated in response to stressors such as sample collection and processing. To address these challenges, novel cell fixation (and more broadly, cell preservation) techniques incorporate programmable fixation times, reversible bond formation and cleavage, chemoselective reactions, and improved analyte recovery. These technologies will further the development of individualized, precision therapies for patients to yield improved clinical outcomes.

Design of an automated capillary electrophoresis platform for single-cell analysis.

Single-cell analysis of cellular contents by highly sensitive analytical instruments is known as chemical cytometry. A chemical cytometer typically samples one cell at a time, quantifies the cellular contents of interest, and then processes and reports that data. Automation adds the potential to perform this entire sequence of events with minimal intervention, increasing throughput and repeatability. In this chapter, we discuss the design considerations for an automated capillary electrophoresis-based instrument for assay of enzymatic activity within single cells. We describe the key requirements of the microscope base and capillary electrophoresis platforms. We also provide detailed protocols and schematic designs of our cell isolation, lysis, sampling, and detection strategies. Additionally, we describe our signal processing and instrument automation workflows. The described automated system has demonstrated single-cell throughput at rates above 100cells/h and analyte limits of detection as low as 10-20mol.

Selection and optimization of enzyme reporters for chemical cytometry.

Chemical cytometry, sensitive analytical measurements of single cells, reveals inherent heterogeneity of cells within a population which is masked or averaged out when using bulk analysis techniques. A particular challenge of chemical cytometry is the development of a suitable reporter or probe for the desired measurement. These reporters must be sufficiently specific for measuring the desired process; possess a lifetime long enough to accomplish the measurement; and have the ability to be loaded into single cells. This chapter details our approach to rationally design and improve peptide substrates as reporters of enzyme activity utilizing chemical cytometry. This method details the iterative approach used to design, characterize, and identify a peptidase-resistant peptide reporter which acts as a kinase substrate within intact cells. Small-scale, rationally designed peptide libraries are generated to rapidly and economically screen candidate reporter peptides for substrate suitability and peptidase resistance. Also detailed are strategies to characterize and validate the designed reporters by determining kinetic parameters, intracellular substrate specificity, resistance to degradation by intracellular peptidases, and behavior within lysates and intact cells.

"Fix and assay": separating in-cellulo sphingolipid reactions from analytical assay in time and space using an aldehyde-based fixative.

Chemical cytometry using capillary electrophoresis (CE) is a powerful tool for measuring single-cell enzyme activity. However, these measurements are often confounding as dynamic processes within cells rapidly change depending on environment, meaning that cell handling, transport, and storage can affect signaling pathways and alter results. To meet these challenges, we describe a method utilizing aldehyde fixation to simultaneously terminate cellular reactions across a population, freezing reaction results in time prior to analytical analysis. Fluorescent sphingosine was loaded into cells of different lineages (leukemia and lymphoma cell lines and primary leukemia cells) and allowed to react before fixing. The remaining sphingosine and any products formed were then quantified with chemical cytometry utilizing CE. When cells were loaded with sphingosine followed by glyoxal fixation and immediate analysis, 55 ± 5% of lipid was recoverable compared to an unfixed control. Storage of fixed cells for 24 h showed no statistical differences in total amount of recoverable sphingolipid compared to samples analyzed immediately after fixation-though there was a difference in recovery of low-abundance products. Sphingosine kinase activity decreased in response to inhibitor treatment compared to treatment with a DMSO vehicle (21 ± 3% product formed in inhibitor-treated cells vs. 57 ± 2% in control cells), which was mirrored in single-cell measurements. This "fix and assay" strategy enables measurement of sphingosine kinase activity in single cells followed by subsequent analytical assay separated in space and time from reaction initiation, enabling greater temporal control over intracellular reactions and improving future compatibility with clinical workflow.

Self-renewing Monolayer of Primary Colonic or Rectal Epithelial Cells.

BACKGROUND & AIMS: Three-dimensional organoid culture has fundamentally changed the in vitro study of intestinal biology enabling novel assays; however, its use is limited because of an inaccessible luminal compartment and challenges to data gathering in a three-dimensional hydrogel matrix. Long-lived, self-renewing 2-dimensional (2-D) tissue cultured from primary colon cells has not been accomplished. METHODS: The surface matrix and chemical factors that sustain 2-D mouse colonic and human rectal epithelial cell monolayers with cell repertoires comparable to that in vivo were identified. RESULTS: The monolayers formed organoids or colonoids when placed in standard Matrigel culture. As with the colonoids, the monolayers exhibited compartmentalization of proliferative and differentiated cells, with proliferative cells located near the peripheral edges of growing monolayers and differentiated cells predominated in the central regions. Screening of 77 dietary compounds and metabolites revealed altered proliferation or differentiation of the murine colonic epithelium. When exposed to a subset of the compound library, murine organoids exhibited similar responses to that of the monolayer but with differences that were likely attributable to the inaccessible organoid lumen. The response of the human primary epithelium to a compound subset was distinct from that of both the murine primary epithelium and human tumor cells. CONCLUSIONS: This study demonstrates that a self-renewing 2-D murine and human monolayer derived from primary cells can serve as a physiologically relevant assay system for study of stem cell renewal and differentiation and for compound screening. The platform holds transformative potential for personalized and precision medicine and can be applied to emerging areas of disease modeling and microbiome studies.

Required hydrophobicity of fluorescent reporters for phosphatidylinositol family of lipid enzymes.

The phosphatidylinositol (PtdIns) family of lipids plays important roles in cell differentiation, proliferation, and migration. Abnormal expression, mutation, or regulation of their metabolic enzymes has been associated with various human diseases such as cancer, diabetes, and bipolar disorder. Recently, fluorescent derivatives have increasingly been used as chemical probes to monitor either lipid localization or enzymatic activity. However, the requirements of a good probe have not been well defined, particularly modifications on the diacylglycerol side chain partly due to challenges in generating PtdIns lipids. We have synthesized a series of fluorescent PtdIns(4,5)P2 (PIP2) and PtdIns (PI) derivatives with various lengths of side chains and tested their capacity as substrates for PI3KIα and PI4KIIα, respectively. Both capillary electrophoresis and thin-layer chromatography were used to analyze enzymatic reactions. For both enzymes, the fluorescent probe with a longer side chain functions as a better substrate than that with a shorter chain and works well in the presence of the endogenous lipid, highlighting the importance of hydrophobicity of side chains in fluorescent phosphoinositide reporters. This comparison is consistent with their interactions with lipid vesicles, suggesting that the binding of a fluorescent lipid with liposome serves as a standard for assessing its utility as a chemical probe for the corresponding endogenous lipid. These findings are likely applicable to other lipid enzymes where the catalysis takes place at the lipid-water interface.

Chemical fixation to arrest phospholipid signaling for chemical cytometry.

Chemical cytometry is a powerful tool for measuring biological processes such as enzymatic signaling at the single cell level. Among these technologies, single-cell capillary zone electrophoresis (CZE) has emerged as a powerful tool to assay a wide range of cellular metabolites. However, analysis of dynamic processes within cells remains challenging as signaling pathways are rapidly altered in response to changes in the cellular environment, including cell manipulation and storage. To address these limitations, we describe a method for chemical fixation of cells to stop the cellular reactions to preserve the integrity of key signaling molecules or reporters within the cell and to enable the cell to act as a storage reservoir for the reporter and its metabolites prior to assay by single-cell CZE. Fluorescent phosphatidylinositol 4,5-bisphosphate reporters were loaded into cells and the cells were chemically fixed and stored prior to analysis. The reporter and its metabolites were electrophoretically separated by single-cell CZE. Chemical fixation parameters such as fixative, fixation time, storage solution, storage duration, and extraction solution were optimized. When cells were loaded with a fluorescent C6- or C16-PIP2 followed by glutaraldehyde fixation and immediate analysis, 24±2% and 139±12% of the lipid was recoverable, respectively, when compared to an unfixed control. Storage of the cells for 24h yielded recoverable lipid of 61±3% (C6-PIP2) and 55±5% (C16-PIP2) when compared to cells analyzed immediately after fixation. The metabolites observed with and without fixation were identical. Measurement of phospholipase C activity in single leukemic cells in response to an agonist demonstrated the capability of chemical fixation coupled to single-cell CZE to yield an accurate snapshot of cellular reactions with the probe. This methodology enables cell assay with the reporter to be separated in space and time from reporter metabolite quantification while preserving assay integrity.

Development of a protease-resistant reporter to quantify BCR-ABL activity in intact cells.

A peptidase-resistant ABL kinase substrate was developed by identifying protease-susceptible bonds on an ABL substrate peptide and replacing flanking amino acids with non-native amino acids. After an iterative design process, the lead, or designed, peptide X-A possesses a six-fold longer life in a cytosolic lysate than that of the starting peptide. The catalytic efficiency (kcat/KM) of purified ABL kinase for the lead peptide (125 s-1 µM-1) is similar to that of the starting peptide (103 s-1 µM-1) demonstrating preservation of the peptide's ability to serve as a kinase substrate. When incubated in cytosolic lysates, the lead peptide is slowly degraded into 4 fragments over time. In contrast, when loaded into intact cells, the peptide is metabolized into 5 fragments, with only 2 of the fragments corresponding to those in the lysate. Thus the two environments possess differing peptidase activities, which must be accounted for when designing peptidase-resistant peptides. In both settings, the substrate is phosphorylated by BCR-ABL providing a readout of BCR-ABL activity. A small panel of tyrosine kinase inhibitors verified the substrate's specificity for BCR-ABL/ABL kinase activity in both lysates and cells in spite of the multitude of other kinases present. The designed peptide X-A acts as a long-lived BCR-ABL kinase reporter in the leukemic cells possessing the BCR-ABL mutation.

Rational Design of a Dephosphorylation-Resistant Reporter Enables Single-Cell Measurement of Tyrosine Kinase Activity.

Although peptide-based reporters of protein tyrosine kinase (PTK) activity have been used to study PTK enzymology in vitro, the application of these reporters to intracellular conditions is compromised by their dephosphorylation, preventing PTK activity measurements. Nonproteinogenic amino acids may be utilized to rationally design selective peptidic ligands by accessing greater chemical and structural diversity than is available using the native amino acids. We describe a peptidic reporter that, upon phosphorylation by the epidermal growth factor receptor (EGFR), is resistant to dephosphorylation both in vitro and in cellulo. The reporter contains a conformationally constrained phosphorylatable moiety (7-(S)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) in the place of L-tyrosine and is efficiently phosphorylated in A431 epidermoid carcinoma cells. Dephosphorylation of the reporter occurs 3 orders of magnitude more slowly compared with that of the conventional tyrosine-containing reporter.

Co-fabrication of chitosan and epoxy photoresist to form microwell arrays with permeable hydrogel bottoms.

Microfabrication technology offers the potential to create biological platforms with customizable patterns and surface chemistries, allowing precise control over the biochemical microenvironment to which a cell or group of cells is exposed. However, most microfabricated platforms grow cells on impermeable surfaces. This report describes the co-fabrication of a micropatterned epoxy photoresist film with a chitosan film to create a freestanding array of permeable, hydrogel-bottomed microwells. These films possess optical properties ideal for microscopy applications, and the chitosan layers are semi-permeable with a molecular exclusion of 9.9 ± 2.1 kDa. By seeding cells into the microwells, overlaying inert mineral oil, and supplying media via the bottom surface, this hybrid film permits cells to be physically isolated from one another but maintained in culture for at least 4 days. Arrays co-fabricated using these materials reduce both large-molecular-weight biochemical crosstalk between cells and mixing of different clonal populations, and will enable high-throughput studies of cellular heterogeneity with increased ability to customize dynamic interrogations compared to materials in currently available technologies.

Measurement of protein kinase B activity in single primary human pancreatic cancer cells.

An optimized peptide substrate was used to measure protein kinase B (PKB) activity in single cells. The peptide substrate was introduced into single cells, and capillary electrophoresis was used to separate and quantify nonphosphorylated and phosphorylated peptide. The system was validated in three model pancreatic cancer cell lines before being applied to primary cells from human pancreatic adenocarcinomas propagated in nude mice. As measured by phosphorylation of peptide substrate, each tumor cell line exhibited statistically different median levels of PKB activity (65%, 21%, and 4% phosphorylation in PANC-1 (human pancreatic carcinoma), CFPAC-1 (human metastatic ductal pancreatic adenocarcinoma), and HPAF-II cells (human pancreatic adenocarcinoma), respectively) with CFPAC-1 cells demonstrating two populations of cells or bimodal behavior in PKB activation levels. The primary cells exhibited highly variable PKB activity at the single cell level, with some cells displaying little to no activity and others possessing very high levels of activity. This system also enabled simultaneous characterization of peptidase action in single cells by measuring the amount of cleaved peptide substrate in each cell. The tumor cell lines displayed degradation rates statistically similar to one another (0.02, 0.06, and 0.1 zmol pg(-1) s(-1), for PANC-1, CFPAC-1, and HPAF-II cells, respectively) while the degradation rate in primary cells was 10-fold slower. The peptide cleavage sites also varied between tissue-cultured and primary cells, with 5- and 8-residue fragments formed in tumor cell lines and only the 8-residue fragment formed in primary cells. These results demonstrate the ability of chemical cytometry to identify important differences in enzymatic behavior between primary cells and tissue-cultured cell lines.

Fluorous enzymatic synthesis of phosphatidylinositides.

A fluorous tagging strategy coupled with enzymatic synthesis is introduced to efficiently synthesize multiple phosphatidylinositides, which are then directly immobilized on a fluorous polytetrafluoroethylene (PTFE) membrane to probe protein-lipid interactions.

ß-Turn sequences promote stability of peptide substrates for kinases within the cytosolic environment.

A strategy was developed to extend the lifetime of an peptide-based substrate for Abl kinase in the cytosolic environment. Small ß-turn structures were added to the peptide's N-terminus to block entry into peptidase catalytic sites. The influence of the size of the ß-turn and two covalent cross-linking strategies on the rate of hydrolysis was assessed. The most peptidase-resistant substrate was degraded at a rate of 0.6 pmol mg(-1) s(-1) and possessed a half-life of 20.3 ± 1.7 min in a Baf/BCR-ABL cytosolic lysate, representing 16- and 40-fold improvements, respectively, over that of a control peptide lacking the ß-turn structure. Furthermore, the kcat/KM value of this peptide was 432 µM(-1) min(-1), a 1.25× increase over the unmodified control, verifying that the added ß-turn did not hinder the substrate properties of the peptide. This improved peptide was microinjected into single Baf/BCR-ABL cells and substrate phosphorylation measured. Zero to forty percent of the peptide was phosphorylated in the single cells. In contrast, when the control peptide without a ß-turn was loaded into cells, the peptide was too rapidly degraded to detect phosphorylation. This work demonstrates that small ß-turn structures can render peptides more resistant to hydrolysis while retaining substrate efficacy and shows that these stabilized peptides have the potential to be of high utility in single-cell enzyme assays.

Development of a peptidase-resistant substrate for single-cell measurement of protein kinase B activation.

An iterative design strategy using three criteria was utilized to develop a peptidase-resistant substrate peptide for protein kinase B. Libraries of peptides possessing non-native amino acids were screened for time to 50% phosphorylation, degradation half-life within a lysate, and appearance of a dominant fragment. The lead peptide possessed a half-life of 92 ± 7 and 16 ± 2 min in HeLa and LNCaP cytosolic lysates, respectively, representing a 4.6- and 2.7-fold lifetime improvement over that of the starting peptide. The redesigned peptide possessed a 4.5-fold improvement in phosphorylation efficiency compared to the starting peptide. The same peptide fragments were formed when the lead peptide was incubated in a lysate or loaded into single cells although the fragments formed in significantly different ratios suggesting that distinct peptidases metabolized the peptide in the two preparations. The rate of peptide degradation and phosphorylation was on average 0.1 ± 0.2 zmol pg(-1) s(-1) and 0.04 ± 0.08 zmol pg(-1) s(-1), respectively, for single LNCaP cells loaded with 4 ± 8 µM of peptide. Peptidase-resistant kinase substrates should find widespread utility in both lysate-based and single-cell assays of kinase activity.

Metabolism of peptide reporters in cell lysates and single cells.

The stability of an Abl kinase substrate peptide in a cytosolic lysate and in single cells was characterized. In the cytosolic lysate, the starting peptide was metabolized at an average initial rate of 1.7 ± 0.3 zmol pg(-1) s(-1) with a t(1/2) of 1.3 min. Five different fragments formed over time; however, a dominant cleavage site was identified. Multiple rational design cycles were utilized to develop a lead peptide with a phenylalanine and alanine replaced by an (N-methyl)phenylalanine and isoleucine, respectively, to attain cytosolic peptidase resistance while maintaining Abl substrate efficacy. This lead peptide possessed a 15-fold greater lifetime in the cytosolic lysate while attaining a 7-fold improvement in k(cat) as an Abl kinase substrate compared to the starting peptide. However, when loaded into single cells, the starting peptide and lead peptide possessed nearly identical degradation rates and an altered pattern of fragmentation relative to that in cell lysates. Preferential accumulation of a fragment with cleavage at an Ala-Ala bond in single cells suggested that dissimilar peptidases act on the peptides in the lysate versus single cells. A design strategy for peptide stabilization, analogous to that demonstrated for the lysate, should be effective for stabilization in single cells.

Effects of elevated temperature and mobile phase composition on a novel C18 silica column.

A novel polydentate C18 silica column was evaluated at an elevated temperature under acidic, basic, and neutral mobile phase conditions using ACN and methanol as the mobile phase organic modifier. The temperature range was 40-200 degrees C. The mobile phase compositions were from 0 to 80% organic-aqueous v/v and the mobile phase pH levels were between 2 and 12. The maximum operating temperature of the column was affected by the amount and type of organic modifier used in the mobile phase. Under neutral conditions, the column showed good column thermal stability at temperatures ranging between 120 and 200 degrees C in methanol-water and ACN-water solvent systems. At pH 2 and 3, the column performed well up to about 160 degrees C at two fixed ACN-buffer compositions. Under basic conditions at elevated temperatures, the column material deteriorated more quickly, but still remained stable up to 100 degrees C at pH 9 and 60 degrees C at pH 10. The results of this study indicate that this novel C18 silica-based column represents a significant advancement in RPLC column technology with enhanced thermal and pH stability when compared to traditional bonded phase silica columns.