Stable Isotope-Resolved Metabolomics Shows Metabolic Resistance to Anti-Cancer Selenite in 3D Spheroids Versus 2D Cell Cultures

Conventional two-dimensional (2D) cell cultures are grown on rigid plastic substrates with unrealistic concentration gradients of O2, nutrients, and treatment agents. More importantly, 2D cultures lack cell–cell and cell–extracellular matrix (ECM) interactions, which are critical for regulating cell behavior and functions. There are several three-dimensional (3D) cell culture systems such as Matrigel, hydrogels, micropatterned plates, and hanging drop that overcome these drawbacks but they suffer from technical challenges including long spheroid formation times, difficult handling for high throughput assays, and/or matrix contamination for metabolic studies. Magnetic 3D bioprinting (M3DB) can circumvent these issues by utilizing nanoparticles that enable spheroid formation and growth via magnetizing cells. M3DB spheroids have been shown to emulate tissue and tumor microenvironments while exhibiting higher resistance to toxic agents than their 2D counterparts. It is, however, unclear if and how such 3D systems impact cellular metabolic networks, which may determine altered toxic responses in cells. We employed a Stable Isotope-Resolved Metabolomics (SIRM) approach with 13C6-glucose as tracer to map central metabolic networks both in 2D cells and M3DB spheroids formed from lung (A549) and pancreatic (PANC1) adenocarcinoma cells without or with an anti-cancer agent (sodium selenite). We found that the extent of 13C-label incorporation into metabolites of glycolysis, the Krebs cycle, the pentose phosphate pathway, and purine/pyrimidine nucleotide synthesis was largely comparable between 2D and M3DB culture systems for both cell lines. The exceptions were the reduced capacity for de novo synthesis of pyrimidine and sugar nucleotides in M3DB than 2D cultures of A549 and PANC1 cells as well as the presence of gluconeogenic activity in M3DB spheroids of PANC1 cells but not in the 2D counterpart. More strikingly, selenite induced much less perturbation of these pathways in the spheroids relative to the 2D counterparts in both cell lines, which is consistent with the corresponding lesser effects on morphology and growth. Thus, the increased resistance of cancer cell spheroids to selenite may be linked to the reduced capacity of selenite to perturb these metabolic pathways necessary for growth and survival

Getting the Most: Enhancing Efficacy by Promoting Erythropoiesis and Thrombopoiesis after Gene Therapy in Mice with Hurler Syndrome

Novel strategies are needed to solve the conundrum of achieving clinical efficacy with high vector copy numbers (VCNs) in hematopoietic stem cells (HSCs) while attempting to minimize the potential risk of oncogenesis in lentiviral vector (LV)-mediated gene therapy clinical trials. We previously reported the benefits of reprogramming erythroid-megakaryocytic (EMK) cells for high-level lysosomal enzyme production with less risk of activating oncogenes in HSCs. Herein, using a murine model of mucopolysaccharidosis type I (MPS I) with a deficiency of α-L-iduronidase (IDUA), we sought to determine the transgene minimum effective doses (MEDs) in major organs, and if a transient increase of IDUA-containing red blood cells and platelets by repeated phlebotomy would provide further therapeutic benefits in diseased mice after EMK-restricted LV-mediated gene therapy. The MEDs for complete metabolic correction ranged from 0.1 to 2 VCNs in major visceral organs, which were dramatically reduced to 0.005–0.1 VCN by one cycle of stress induction and were associated with a further reduction of pathological deficits in mice with 0.005 VCN. This work provides a proof of concept that transiently stimulating erythropoiesis and thrombopoiesis can further improve therapeutic benefits in HSC-mediated gene therapy for MPS I, a repeatable and reversible approach to enhance clinical efficacy in the treatment of lysosomal storage diseases. Keywords: preclinical efficacy, hematopoietic stems cells, gene therapy, stress erythropoiesis and thrombopoiesis, erythroid and megakaryocytic lineages, lysosomal storage disease, minimum effective transgene dosages, targeted expression, mucopolysaccharidose

Platelets are efficient and protective depots for storage, distribution, and delivery of lysosomal enzyme in mice with Hurler Syndrome

Use of megakaryocytes/platelets for transgene expression may take advantage of their rapid turnover and protective storage in platelets and reduce the risk of activating oncogenes in hematopoietic stem and progenitor cells (HSCs). Here, we show that human megakaryocytic cells could overexpress the lysosomal enzyme, α-l-iduronidase (IDUA), which is deficient in patients with mucopolysaccharidosis type I (MPS I). Upon megakaryocytic differentiation, the amount of released enzyme increased rapidly and steadily by 30-fold. Using a murine MPS I model, we demonstrated that megakaryocyte/platelets were capable of producing, packaging, and storing large amounts of IDUA with proper catalytic activity, lysosomal trafficking, and receptor-mediated uptake. IDUA can be released directly into extracellular space or within microparticles during megakaryocyte maturation or platelet activation, while retaining the capacity for cross-correction in patient’s cells. Gene transfer into 1.7% of HSCs led to long-term normalization of plasma IDUA and preferential distribution of enzyme in liver and spleen with complete metabolic correction in MPS I mice. Detection of GFP (coexpressed with IDUA) in Kupffer cells and hepatocytes suggested liver delivery of platelet-derived IDUA possibly via the clearance pathway for senile platelets. These findings provide proof of concept that cells from megakaryocytic lineage and platelets are capable of generating and storing fully functional lysosomal enzymes and can also lead to efficient delivery of both the enzymes released into the circulation and those protected within platelets/microparticles. This study opens a door for use of the megakaryocytes/platelets as a depot for efficient production, delivery, and effective tissue distribution of lysosomal enzymes

Comprehensive evaluation of blood-brain barrier-forming micro-vasculatures: Reference and marker genes with cellular composition

<div><p>Primary brain microvessels (BrMV) maintain the cellular characters and molecular signatures as displayed in vivo, and serve as a vital tool for biomedical research of the blood-brain barrier (BBB) and the development/optimization of brain drug delivery. The variations of relative purities or cellular composition among different BrMV samples may have significant consequences in data interpretation and research outcome, especially for experiments with high-throughput genomics and proteomics technologies. In this study, we aimed to identify suitable reference gene (RG) for accurate normalization of real-time RT-qPCR analysis, and determine the proper marker genes (MG) for relative purity assessment in BrMV samples. Out of five housekeeping genes, β-actin was selected as the most suitable RG that was validated by quantifying mRNA levels of alpha-L-iduronidase in BrMV isolated from mice with one or two expressing alleles. Four marker genes highly/selectively expressed in BBB-forming capillary endothelial cells were evaluated by RT-qPCR for purity assessment, resulting in <i>Cldn</i>5 and <i>Pecam1</i> as most suitable MGs that were further confirmed by immunofluorescent analysis of cellular components. <i>Plvap</i> proved to be an indicator gene for the presence of fenestrated vessels in BrMV samples. This study may contribute to the building blocks toward overarching research needs on the blood-brain barrier.</p></div

Verification of <i>Actb</i> as the best reference gene by <i>Idua</i> expression in BrMV of WT mice and Het mice.

<p><b>(A)</b> Standard curve for absolute quantification of <i>Idua</i> mRNA. A plasmid containing <i>Idua</i> cDNA was used for generating standard curve with copy numbers by qPCR. Data was derived from 2 sets of standard samples, each amplified three times in duplicate. Error bars, standard deviation. (B, C) <i>Idua</i> expression in BrMV isolated from either wild-type C57/Bl6 mice (WT) or littermates of heterozygous for <i>Idua</i> knock-out (Het) with normalization by RG candidates. Total RNA from 4 WT and 4 Het samples were examined by RT-qPCR and calculated either by absolute <i>Idua</i> standard curve for copy numbers per ng RNA (B), or by ΔΔCt method for relative <i>Idua</i> fold changes (C).</p

Quantification of RNA input using RG standard curves derived from 3T3 or primary CDB samples.

<p>Total RNA samples isolated from 3T3 cell line or CDB samples of C57/Bl6 mice were serially diluted, applied for reverse transcription, and followed by qPCR of 4 reference genes. (A) Standard curves of RNA amounts generated by qPCR of 4 RGs. Data were derived from 3 dilution sets with each amplified in triplicates. E, amplification efficiency for a combination of RT and qPCR steps calculated from the slope of each standard curve; R² range from 0.986 to 0.998. (B) Quantification of total RNA inputs of BrMV and CDB isolates from 10 isolation experiments using different standard curves. Reverse transcription was conducted at 25 ng/ul (by NanoDrop) for all RNA samples, and real-time qPCR was performed using 25 ng/reaction (and indicated as dashed line). Each symbol represents mean of calculated RNA amount derived from Ct value of triplicate qPCR reactions of one sample. Short lines represent mean ± SD of RNA amounts calculated using different standard curves from each of RGs.</p

Cellular components of BrMV isolates determined by immunostaining.

<p>(A) Representative pictures of immunofluorescence analysis for BrMV isolates. The brain BrMV isolates were stained with fluorescein-labeled (green) lectin for BBB-forming endothelial cells, and Alexa 568-conjugated (red) anti-mouse CD68 for brain macrophages, anti-mouse GFAP for astrocytes, anti-mouse NeuN for neurons, or anti-mouse PDGFR-β for pericytes, and followed by counter-staining with DAPI (blue) for nucleic. Scale bar represents 50 μm for all views. (B) Semi-quantifications of cellular composition for nine BrMV samples. Each sample was evaluated with a total of >500 nuclei. Pearson correlation coefficient between Lectin⁺ and other cell types are -0.862 for CD68⁺ cells, -0.837 for GFAP⁺ cells, -0.776 for NeuN⁺ and -0.190 for PDGFR+ cells. (C) Semi-quantification of main cell types in BrMV isolates. Data are derived from 4 BrMV samples with similar purities (ranging of 64–69%) as determined by immunostaining; error bars represent SD. CV, coefficient of variation.</p

The expression stability of RG candidates among endothelial cell lines and brain samples.

<p>Total RNA was extracted and converted into cDNA by reverse-transcription at 25 ng/ul, and followed by real-time qPCR with 25 ng/reaction. (A) Distribution of cycle threshold (Ct) values for five RG candidates by quantitative RT-PCR in BMEC and bEnd3 cell lines. The experiments were repeated 3 times in duplicate reactions. Boxes showed the range of Ct values for each candidate gene. The central line indicated the median Ct; the extended upper and lower indicate 75 and 25 percentiles. (B) The average expression stability (M value) of RG candidates in two endothelial cell lines analyzed by geNorm. RG candidates were ranked from the least stable to the most stable (left to right). (C) Ct values for four RG candidates in BrMV and CDB. Data were derived from 10 BrMV isolation experiments with 4 from WT mice, 2 from Het and 4 from <i>Idua</i> knock-out mice. Each sample was tested 3 times in duplicate. **, p<0.01, and ***, p<0.001. (D) The expression stability of four reference genes among all BrMV and CDB samples analyzed by geNorm. (E) RG candidates were ranked in the order of their expression stability evaluated by Bestkeeper based on coefficient of variation (CV%) and SD.</p

Correlation of purities measured by both qPCR and immunofluorescent microscopy.

<p>The relative purities of BrMV samples were determined by real-time RT-qPCR with relative comparison of mRNA abundances using standard curves of different marker genes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197379#pone.0197379.g004" target="_blank">Fig 4</a>, as well as by immunofluorescence analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197379#pone.0197379.g005" target="_blank">Fig 5</a>. Each symbol represents relative purity of individual BrMV sample derived from 3 RT-qPCR experiments in duplicate reactions for qPCR, as well as from evaluation of >200 DAPI⁺ nuclei from cytospin slides by immunostaining analysis.</p

Engineering a lysosomal enzyme with a derivative of receptor-binding domain of apoE enables delivery across the blood-brain barrier

To realize the potential of large molecular weight substances to treat neurological disorders, novel approaches are required to surmount the blood–brain barrier (BBB). We investigated whether fusion of a receptor-binding peptide from apolipoprotein E (apoE) with a potentially therapeutic protein can bind to LDL receptors on the BBB and be transcytosed into the CNS. A lysosomal enzyme, α-L-iduronidase (IDUA), was used for biological and therapeutic evaluation in a mouse model of mucopolysaccharidosis (MPS) type I, one of the most common lysosomal storage disorders with CNS deficits. We identified two fusion candidates, IDUAe1 and IDUAe2, by in vitro screening, that exhibited desirable receptor-mediated binding, endocytosis, and transendothelial transport as well as appropriate lysosomal enzyme trafficking and biological function. Robust peripheral IDUAe1 or IDUAe2 generated by transient hepatic expression led to elevated enzyme levels in capillary-depleted, enzyme-deficient brain tissues and protein delivery into nonendothelium perivascular cells, neurons, and astrocytes within 2 d of treatment. Moreover, 5 mo after long-term delivery of moderate levels of IDUAe1 derived from maturing red blood cells, 2% to 3% of normal brain IDUA activities were obtained in MPS I mice, and IDUAe1 protein was detected in neurons and astrocytes throughout the brain. The therapeutic potential was demonstrated by normalization of brain glycosaminoglycan and β-hexosaminidase in MPS I mice 5 mo after moderate yet sustained delivery of IDUAe1. These findings provide a noninvasive and BBB-targeted procedure for the delivery of large-molecule therapeutic agents to treat neurological lysosomal storage disorders and potentially other diseases that involve the brain