Development of Multiplexed Bead-Based Immunoassays for Profiling Soluble Cytokines and CD163 Using Mass Cytometry.

Bead-based immunoassays are multiparametric analysis allowing for the simultaneous quantification of a large number of biomarkers within a single sample. Mass cytometry is an emerging cytometric technique that offers a high multiplexing capacity in a high-throughput setting but has not yet been applied to bead-based assays. In this study, we developed a multiplex bead-based immunoassay of cytokines and CD163 designed for mass cytometry (MC). A set of 11 types of lanthanide-encoded microbeads were synthesized by two-stage dispersion polymerization as classifier candidates for the assay. These beads were then decorated with different Abs on the surface to capture the target cytokines in solution. Gold nanoparticles were employed as reporters to identify the binding of target cytokines on the classifier surface. As a proof-of-concept study, we first developed four-plex and nine-plex assays of mixtures of cytokines in standard solutions. The MC signal intensities of these immunoassays were responsive to the concentration differences in the standard solutions with high detection sensitivities at low analyte concentrations. Finally, we examined a sample of peripheral blood mononuclear cells (PBMCs) with the nine-plex assay, comparing an unstimulated sample with a sample stimulated to promote cytokine secretion.

Biotinylated Lipid-Coated NaLnF4 Nanoparticles: Demonstrating the Use of Lanthanide Nanoparticle-Based Reporters in Suspension and Imaging Mass Cytometry.

Lanthanide nanoparticles (LnNPs) have the potential to be used as high-sensitivity mass tag reporters in mass cytometry immunoassays. For this application, however, the LnNPs must be made colloidally stable in aqueous buffers, demonstrate minimal non-specific binding to cells, and have functional groups to attach antibodies or other targeting agents. One possible approach to address these requirements is by using lipid coating to modify the surface of the LnNPs. In this work, 39 nm diameter NaYF4:Yb, Er NPs (LnNPs) were coated with a lipid formulation consisting of egg sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-3-trimethylammonium propane, cholesterol-(polyethylene glycol-600), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000]. The resulting biotinylated lipid-coated LnNPs were characterized by dynamic light scattering to determine the hydrodynamic size and stability in phosphate buffered saline, and the composition of the lipid coatings was quantified by liquid chromatography-tandem mass spectrometry. The specific and non-specific binding of the biotinylated lipid-coated LnNPs to a model system of functionalized polystyrene microbeads were then tested by both suspension and imaging mass cytometry. We found that targeted binding with minimal non-specific binding can be achieved with the lipid-coated LnNPs and that the lipid composition of the coating has an impact on the performance of the LnNPs as mass cytometry reporters. These results additionally establish the importance of quantifying the composition of lipid-coated nanomaterials to optimize them more effectively for their desired application.

Cadherin 11 Promotes Immunosuppression and Extracellular Matrix Deposition to Support Growth of Pancreatic Tumors and Resistance to Gemcitabine in Mice.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDACs) are characterized by fibrosis and an abundance of cancer-associated fibroblasts (CAFs). We investigated strategies to disrupt interactions among CAFs, the immune system, and cancer cells, focusing on adhesion molecule CDH11, which has been associated with other fibrotic disorders and is expressed by activated fibroblasts. METHODS: We compared levels of CDH11 messenger RNA in human pancreatitis and pancreatic cancer tissues and cells with normal pancreas, and measured levels of CDH11 protein in human and mouse pancreatic lesions and normal tissues. We crossed p48-Cre;LSL-KrasG12D/+;LSL-Trp53R172H/+ (KPC) mice with CDH11-knockout mice and measured survival times of offspring. Pancreata were collected and analyzed by histology, immunohistochemistry, and (single-cell) RNA sequencing; RNA and proteins were identified by imaging mass cytometry. Some mice were given injections of PD1 antibody or gemcitabine and survival was monitored. Pancreatic cancer cells from KPC mice were subcutaneously injected into Cdh11+/+ and Cdh11-/- mice and tumor growth was monitored. Pancreatic cancer cells (mT3) from KPC mice (C57BL/6), were subcutaneously injected into Cdh11+/+ (C57BL/6J) mice and mice were given injections of antibody against CDH11, gemcitabine, or small molecule inhibitor of CDH11 (SD133) and tumor growth was monitored. RESULTS: Levels of CDH11 messenger RNA and protein were significantly higher in CAFs than in pancreatic cancer epithelial cells, human or mouse pancreatic cancer cell lines, or immune cells. KPC/Cdh11+/- and KPC/Cdh11-/- mice survived significantly longer than KPC/Cdh11+/+ mice. Markers of stromal activation entirely surrounded pancreatic intraepithelial neoplasias in KPC/Cdh11+/+ mice and incompletely in KPC/Cdh11+/- and KPC/Cdh11-/- mice, whose lesions also contained fewer FOXP3+ cells in the tumor center. Compared with pancreatic tumors in KPC/Cdh11+/+ mice, tumors of KPC/Cdh11+/- mice had increased markers of antigen processing and presentation; more lymphocytes and associated cytokines; decreased extracellular matrix components; and reductions in markers and cytokines associated with immunosuppression. Administration of the PD1 antibody did not prolong survival of KPC mice with 0, 1, or 2 alleles of Cdh11. Gemcitabine extended survival of KPC/Cdh11+/- and KPC/Cdh11-/- mice only or reduced subcutaneous tumor growth in mT3 engrafted Cdh11+/+ mice when given in combination with the CDH11 antibody. A small molecule inhibitor of CDH11 reduced growth of pre-established mT3 subcutaneous tumors only if T and B cells were present in mice. CONCLUSIONS: Knockout or inhibition of CDH11, which is expressed by CAFs in the pancreatic tumor stroma, reduces growth of pancreatic tumors, increases their response to gemcitabine, and significantly extends survival of mice. CDH11 promotes immunosuppression and extracellular matrix deposition, and might be developed as a therapeutic target for pancreatic cancer.

B cell acute lymphoblastic leukemia cells mediate RANK-RANKL-dependent bone destruction.

Although most children survive B cell acute lymphoblastic leukemia (B-ALL), they frequently experience long-term, treatment-related health problems, including osteopenia and osteonecrosis. Because some children present with fractures at ALL diagnosis, we considered the possibility that leukemic B cells contribute directly to bone pathology. To identify potential mechanisms of B-ALL-driven bone destruction, we examined the p53 -/-; Rag2 -/-; Prkdcscid/scid triple mutant (TM) mice and p53 -/-; Prkdcscid/scid double mutant (DM) mouse models of spontaneous B-ALL. In contrast to DM animals, leukemic TM mice displayed brittle bones, and the TM leukemic cells overexpressed Rankl, encoding receptor activator of nuclear factor κB ligand. RANKL is a key regulator of osteoclast differentiation and bone loss. Transfer of TM leukemic cells into immunodeficient recipient mice caused trabecular bone loss. To determine whether human B-ALL can exert similar effects, we evaluated primary human B-ALL blasts isolated at diagnosis for RANKL expression and their impact on bone pathology after their transplantation into NOD.Prkdcscid/scidIl2rgtm1Wjl /SzJ (NSG) recipient mice. Primary B-ALL cells conferred bone destruction evident in increased multinucleated osteoclasts, trabecular bone loss, destruction of the metaphyseal growth plate, and reduction in adipocyte mass in these patient-derived xenografts (PDXs). Treating PDX mice with the RANKL antagonist recombinant osteoprotegerin-Fc (rOPG-Fc) protected the bone from B-ALL-induced destruction even under conditions of heavy tumor burden. Our data demonstrate a critical role of the RANK-RANKL axis in causing B-ALL-mediated bone pathology and provide preclinical support for RANKL-targeted therapy trials to reduce acute and long-term bone destruction in these patients.

Clickable and High-Sensitivity Metal-Containing Tags for Mass Cytometry.

Mass cytometry is a highly multiplexed single-cell analysis platform that uses metal-tagged reagents to identify multiple cellular biomarkers. The current metal-tagged reagent preparation employs thiol-maleimide chemistry to covalently couple maleimide-functionalized metal-chelating polymers (MCPs) with antibodies (Abs), a process that requires partial reduction of the Ab to form reactive thiol groups. However, some classes of Abs (for example, IgM) as well as biomolecules lacking cysteine residues have been challenging to label using this method. This inherent limitation led us to develop a new conjugation strategy for labeling a wide range of biomolecules and affinity reagents. In this report, we present a metal tagging approach using a new class of azide- or transcyclooctene-terminated MCPs with copper(I)-free strain-promoted alkyne-azide cycloaddition or tetrazine-alkene click chemistry reactions, in which biomolecules with -NH2 functional groups are selectively activated with a dibenzocyclooctyne or tetrazine moiety, respectively. This approach enabled us to generate highly sensitive and specific metal-tagged IgGs, IgMs, small peptides, and lectins for applications in immunophenotyping and glycobiology. We also created dual-tagged reagents for simultaneous detection of markers by immunofluorescence, mass cytometry, and imaging mass cytometry using a two-step conjugation process. The Helios mass cytometer was used to test the functionality of reagents on suspension human leukemia cell lines and primary cells. The dual-tagged Abs, metal-tagged lectins, and phalloidin staining reagent were used to visualize target proteins and glycans on adherent cell lines and frozen/FFPE tissue sections using the Hyperion Imaging System. In some instances, reagents produced by click conjugation showed superior sensitivity and specificity compared to those of reagents produced by thiol-maleimide chemistry. In general, the click chemistry-based conjugation with new MCPs could be instrumental in developing a wide range of highly sensitive metal-containing reagents for proteomics and glycomics applications.

Simultaneous Detection of Protein and mRNA in Jurkat and KG-1a Cells by Mass Cytometry.

Mass cytometry uniquely enables high-dimensional single-cell analysis of complex populations. This recently developed technology is based on inductively coupled time-of-flight mass spectrometry for multiplex proteomic analysis of more than 40 markers per cell. The ability to characterize the transcriptome is critical for the understanding of disease pathophysiology, medical diagnostics, and drug discovery. Current techniques allowing the in situ detection of transcripts in single cells are limited to a small number of simultaneous targets and are generally tedious and labor-intensive. In this report, we present the development of a multiplex method for targeted RNA detection by combining the mass cytometry and RNAscope® platforms. This novel assay, called Metal In Situ Hybridization (MISH), includes the hybridization of RNA-specific target probes followed by signal amplification achieved through a cascade of hybridization events, ending with the binding of amplifier-specific detector probes. The detector probes are tagged with isotopically pure metal atoms used for detection by mass cytometry. Proof-of-principle experiments show the simultaneous detection of three mRNA targets in Jurkat cells in suspension cell assay mode. The localization of transcripts was also investigated using the imaging mass cytometry platform in Jurkat and KG-1a cells. In addition, we optimized the antibody staining procedure to allow the co-detection of mRNA and cell surface markers. Our data demonstrate that MISH can be used to complement protein detection by mass cytometry as well as to investigate gene transcription and translation in single cells. © 2017 International Society for Advancement of Cytometry.

Calcium polyphosphate particulates for bone void filler applications.

This study investigates the characteristics of porous calcium polyphosphate particulates (CPPp) formed using two different processing treatments as bone void fillers in non- or minimally load-bearing sites. The two calcium polyphosphate particulate variants (grades) were formed using different annealing conditions during particulate preparation to yield either more slowly degrading calcium polyphosphate particulates (SD-CPPp) or faster degrading particulates (FD-CPPp) as suggested by a previous degradation study conducted in vitro (Hu et al., Submitted for publication 2016). The two CPPp grades were compared as bone void fillers in vivo by implanting particulates in defects created in rabbit femoral condyle sites (critical size defects). The SD-CPPp and FD-CPPp were implanted for 4- and 16-week periods. The in vivo study indicated a significant difference in amount of new bone formed in the prepared sites with SD-CPPp resulting in more new bone formation compared with FD-CPPp. The lower bone formation characteristic of the FD-CPPp was attributed to its faster degradation rate and resulting higher local concentration of released polyphosphate degradation products. The study results indicate the importance of processing conditions on preparing calcium polyphosphate particulates for potential use as bone void fillers in nonload-bearing sites. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 874-884, 2017.

Adynamic Bone Decreases Bone Toughness During Aging by Affecting Mineral and Matrix.

Adynamic bone is the most frequent type of bone lesion in patients with chronic kidney disease; long-term use of antiresorptive therapy may also lead to the adynamic bone condition. The hallmark of adynamic bone is a loss of bone turnover, and a major clinical concern of adynamic bone is diminished bone quality and an increase in fracture risk. Our current study aims to investigate how bone quality changes with age in our previously established mouse model of adynamic bone. Young and old mice (4 months old and 16 months old, respectively) were used in this study. Col2.3Δtk (DTK) mice were treated with ganciclovir and pamidronate to create the adynamic bone condition. Bone quality was evaluated using established techniques including bone histomorphometry, microcomputed tomography, quantitative backscattered electron imaging, and biomechanical testing. Changes in mineral and matrix properties were examined by powder X-ray diffraction and Raman spectroscopy. Aging controls had a natural decline in bone formation and resorption with a corresponding deterioration in trabecular bone structure. Bone turnover was severely blunted at all ages in adynamic animals, which preserved trabecular bone loss normally associated with aging. However, the preservation of trabecular bone mass and structure in old adynamic mice did not rescue deterioration of bone mechanical properties. There was also a decrease in cortical bone toughness in old adynamic mice that was accompanied by a more mature collagen matrix and longer bone crystals. Little is known about the effects of metabolic bone disease on bone fracture resistance. We observed an age-related decrease in bone toughness that was worsened by the adynamic condition, and this decrease may be due to material level changes at the tissue level. Our mouse model may be useful in the investigation of the mechanisms involved in fractures occurring in elderly patients on antiresorptive therapy who have very low bone turnover.

Role of bioactive 3D hybrid fibrous scaffolds on mechanical behavior and spatiotemporal osteoblast gene expression.

Three-dimensional (3D) bioactive organic-inorganic (O/I) hybrid fibrous scaffolds are attractive extracellular matrix (ECM) surrogates for bone tissue engineering. With the aim of regulating osteoblast gene expression in 3D, a new class of hybrid fibrous scaffolds with two distinct fiber diameters (260 and 600 nm) and excellent physico-mechanical properties were fabricated from tertiary (SiO2-CaO-P2O5) bioactive glass (BG) and poly (ε-caprolactone) (PCL) by in situ sol-gel and electrospinning process. The PCL/BG hybrid fibrous scaffolds exhibited accelerated wetting properties, enhanced pore sizes and porosity, and superior mechanical properties that were dependent on fiber diameter. Contrary to control PCL fibrous scaffolds that were devoid of bonelike apatite particles, incubating PCL/BG hybrid fibrous scaffolds in simulated body fluid (SBF) revealed bonelike apatite deposition. Osteoblast cells cultured on PCL/BG hybrid fibrous scaffolds spread with multiple attachments and actively proliferated suggesting that the low temperature in situ sol-gel and electrospinning process did not have a detrimental effect. Targeted bone-associated gene expressions by rat calvarial osteoblasts seeded on these hybrid scaffolds demonstrated remarkable spatiotemporal gene activation. Transcriptional-level gene expressions for alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OCN) were significantly higher on the hybrid fibrous scaffolds (p < 0.001) that were largely dependent on fiber diameter compared. Taken together, our results suggest that PCL/BG fibrous scaffolds may accelerate bone formation by providing a favorable microenvironment.

Hydroxyapatite formation on sol-gel derived poly(ε-caprolactone)/bioactive glass hybrid biomaterials.

Investigation of novel biomaterials for bone regeneration is based on the development of scaffolds that exhibit bone-bonding ability, biocompatibility, and sufficient mechanical strength. In this study, using novel poly (ε-caprolactone)/bioactive glass (PCL/BG) hybrids with different organic/inorganic ratios, the effects of BG contents on the in vitro bone-like hydroxyapatite (HA) formation, mechanical properties, and biocompatibility were investigated. Rapid precipitation of HA on the PCL/BG hybrid surfaces were observed after incubating in simulated body fluid (SBF) for only 6 h, as confirmed by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), Fourier-transform infrared spectroscopy (FTIR), and inductively coupled plasma atomic emission spectroscopy (ICPS). The ICPS elemental analysis results were further analyzed in terms of the Ca(2+) and PO4(3-) which were consumed to form the apatite layer. The results revealed that the rate and total amount of HA deposition decreased with an increase in PCL content. The compressive modulus and strength of the PCL/BG hybrids increased with the decrease in PCL content. The highest values were achieved at the lowest PCL content (10 wt %) and were around, 90 MPa and 1.4 GPa, respectively. To evaluate the cytotoxicity of PCL/BG bioactive hybrids, MC3T3-E1 osteoblast-like cells were cultured for up to 72 h. Our data indicated that whereas initial cell attachment was marginally lower than the control tissue culture poly styrene (TCPS) surface, the hybrid materials promoted cell growth in a time-dependent manner. Cell viability within the different PCL/BG hybrid samples appeared to be influenced by compositional differences whereby higher PCL contents correlated with slight reduction in cell viability. Taken together, this study adds important new information to our knowledge on hydroxyapatite formation, mechanical properties, and cytotoxic effects of PCL/BG hybrids prepared by the sol-gel process using a tertiary glass composition and may have considerable potential for bone tissue regeneration applications.

Control of surface topography in biomimetic calcium phosphate coatings.

The behavior of cells responsible for bone formation, osseointegration, and bone bonding in vivo are governed by both the surface chemistry and topography of scaffold matrices. Bone-like apatite coatings represent a promising method to improve the osteoconductivity and bonding of synthetic scaffold materials to mineralized tissues for regenerative procedures in orthopedics and dentistry. Polycaprolactone (PCL) films were coated with calcium phosphates (CaP) by incubation in simulated body fluid (SBF). We investigated the effect of SBF ion concentration and soaking time on the surface properties of the resulting apatite coatings. CaP coatings were examined by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectrometry (FTIR), and energy dispersive X-ray spectrometry (EDX). Young's modulus (E(s)) was determined by nanoindentation, and surface roughness was assessed by atomic force microscopy (AFM) and mechanical stylus profilometry. CaP such as carbonate-substituted apatite were deposited onto PCL films. SEM and AFM images of the apatite coatings revealed an increase in topographical complexity and surface roughness with increasing ion concentration of SBF solutions. Young's moduli (E(s)) of various CaP coatings were not significantly different, regardless of the CaP phase or surface roughness. Thus, SBF with high ion concentrations may be used to coat synthetic polymers with CaP layers of different surface topography and roughness to improve the osteoconductivity and bone-bonding ability of the scaffold.

Bioactive and biodegradable nanocomposites and hybrid biomaterials for bone regeneration.

Strategies for bone tissue engineering and regeneration rely on bioactive scaffolds to mimic the natural extracellular matrix and act as templates onto which cells attach, multiply, migrate and function. Of particular interest are nanocomposites and organic-inorganic (O/I) hybrid biomaterials based on selective combinations of biodegradable polymers and bioactive inorganic materials. In this paper, we review the current state of bioactive and biodegradable nanocomposite and O/I hybrid biomaterials and their applications in bone regeneration. We focus specifically on nanocomposites based on nano-sized hydroxyapatite (HA) and bioactive glass (BG) fillers in combination with biodegradable polyesters and their hybrid counterparts. Topics include 3D scaffold design, materials that are widely used in bone regeneration, and recent trends in next generation biomaterials. We conclude with a perspective on the future application of nanocomposites and O/I hybrid biomaterials for regeneration of bone.

Synthesis and electrospinning of ε-polycaprolactone-bioactive glass hybrid biomaterials via a sol-gel process.

Strategies of bone tissue engineering and regeneration rely on bioactive scaffolds to mimic the natural extracellular matrix (ECM) as templates onto which cells attach, multiply, migrate, and function. For this purpose, hybrid biomaterials based on smart combinations of biodegradable polymers and bioactive glasses (BGs) are of particular interest, since they exhibit tailored physical, biological, and mechanical properties, as well as predictable degradation behavior. In this study, hybrid biomaterials with different organic-inorganic ratios were successfully synthesized via a sol-gel process. Poly(ε-caprolactone) (PCL) and tertiary bioactive glass (BG) with a glass composition of 70 mol % SiO(2), 26 mol % CaO, and 4 mol % of P(2)O(5) were used as the polymer and inorganic phases, respectively. The polymer chains were successfully introduced into the inorganic sol while the networks were formed. Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analyses (TGA), scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX) were used to investigate the presence of different chemical groups, structural crystallinity, thermal property, elemental composition, and homogeneity of the synthesized hybrid biomaterials. Identification of chemical groups and the presence of molecular interaction by hydrogen bonding between the organic and inorganic phases was confirmed by FTIR. The XRD patterns showed that all PCL/BG hybrids (up to 60% polymer content) were amorphous. The TGA study revealed that the PCL/BG hybrid biomaterials were thermally stable, and good agreement was observed between the experimental and theoretical organic-inorganic ratios. The SEM/EDX results also revealed a homogeneous elemental distribution and demonstrated the successful incorporation of all the elements in the hybrid system. Finally, these synthesized hybrid biomaterials were successfully electrospun into 3D scaffolds. The resultant fibers have potential use as scaffolds for bone regeneration.