Tracking the fates of iron-labeled tumor cells in vivo using magnetic particle imaging.

The use of imaging to detect and monitor the movement and accumulation of cells in living subjects can provide significant insights that can improve our understanding of metastasis and guide therapeutic development. For cell tracking using Magnetic Resonance Imaging (MRI), cells are labeled with iron oxides and the effects of the iron on water provides contrast. However, due to low specificity and difficulties in quantification with MRI, other modalities and approaches need to be developed. Magnetic Particle Imaging (MPI) is an emerging imaging technique which directly detects iron, allowing for a specific, quantitative and sensitive readout. Here, we use MPI to image iron-labeled tumor cells longitudinally, from implantation and growth at a primary site to movement to distant anatomic sites. In vivo bioluminescent imaging (BLI) was used to localize tumor metastases and computed tomography (CT) allowed for correlation of these signals to anatomic locations. These three imaging modalities provide information on immune escape and metastasis of iron-labeled, and unlabeled, tumor cells, and the accumulation of cell-free iron contrast over time. We localized iron signals by MPI and tumor cells via BLI, and correlated these positive contrast images with CT scans to reveal the anatomic sites with cancer cells; histologic analysis confirmed the presence of iron-labeled tumor cells in the tissues, suggesting that the metastatic cells retained enough iron for MPI detection. The use of multi-modality cell tracking reveals the movement, accumulation and fates of labeled cells that will be helpful understanding cancer progression and guiding the development of targeted therapies.

Magnetic Particle Imaging of Magnetotactic Bacteria as Living Contrast Agents Is Improved by Altering Magnetosome Arrangement.

Superparamagnetic iron oxide nanoparticles (SPIONs) can be used as imaging agents to differentiate between normal and diseased tissue or track cell movement. Magnetic particle imaging (MPI) detects the magnetic properties of SPIONs, providing quantitative and sensitive image data. MPI performance depends on the size, structure, and composition of nanoparticles. Magnetotactic bacteria produce magnetosomes with properties similar to those of synthetic nanoparticles, and these can be modified by mutating biosynthetic genes. The use of Magnetospirillum gryphiswaldense, MSR-1 with a mamJ deletion, containing clustered magnetosomes instead of typical linear chains, resulted in improved MPI signal and resolution. Bioluminescent MSR-1 with the mamJ deletion were administered into tumor-bearing and healthy mice. In vivo bioluminescence imaging revealed the viability of MSR-1, and MPI detected signals in livers and tumors. The development of living contrast agents offers opportunities for imaging and therapy with multimodality imaging guiding development of these agents by tracking the location, viability, and resulting biological effects.

Pathways of calcium regulation, electron transport, and mitochondrial protein translation are molecular signatures of susceptibility to recurrent exertional rhabdomyolysis in Thoroughbred racehorses.

Recurrent exertional rhabdomyolysis (RER) is a chronic muscle disorder of unknown etiology in racehorses. A potential role of intramuscular calcium (Ca2+) dysregulation in RER has led to the use of dantrolene to prevent episodes of rhabdomyolysis. We examined differentially expressed proteins (DEP) and gene transcripts (DEG) in gluteal muscle of Thoroughbred race-trained mares after exercise among three groups of 5 horses each; 1) horses susceptible to, but not currently experiencing rhabdomyolysis, 2) healthy horses with no history of RER (control), 3) RER-susceptible horses treated with dantrolene pre-exercise (RER-D). Tandem mass tag LC/MS/MS quantitative proteomics and RNA-seq analysis (FDR <0.05) was followed by gene ontology (GO) and semantic similarity of enrichment terms. Of the 375 proteins expressed, 125 were DEP in RER-susceptible versus control, with 52 ↑DEP mainly involving Ca2+ regulation (N = 11) (e.g. RYR1, calmodulin, calsequestrin, calpain), protein degradation (N = 6), antioxidants (N = 4), plasma membranes (N = 3), glyco(geno)lysis (N = 3) and 21 DEP being blood-borne. ↓DEP (N = 73) were largely mitochondrial (N = 45) impacting the electron transport system (28), enzymes (6), heat shock proteins (4), and contractile proteins (12) including Ca2+ binding proteins. There were 812 DEG in RER-susceptible versus control involving the electron transfer system, the mitochondrial transcription/translational response and notably the pro-apoptotic Ca2+-activated mitochondrial membrane transition pore (SLC25A27, BAX, ATP5 subunits). Upregulated mitochondrial DEG frequently had downregulation of their encoded DEP with semantic similarities highlighting signaling mechanisms regulating mitochondrial protein translation. RER-susceptible horses treated with dantrolene, which slows sarcoplasmic reticulum Ca2+ release, showed no DEG compared to control horses. We conclude that RER-susceptibility is associated with alterations in proteins, genes and pathways impacting myoplasmic Ca2+ regulation, the mitochondrion and protein degradation with opposing effects on mitochondrial transcriptional/translational responses and mitochondrial protein content. RER could potentially arise from excessive sarcoplasmic reticulum Ca2+ release and subsequent mitochondrial buffering of excessive myoplasmic Ca2+.

Commercial genetic testing for type 2 polysaccharide storage myopathy and myofibrillar myopathy does not correspond to a histopathological diagnosis.

BACKGROUND: Commercial genetic tests for type 2 polysaccharide storage myopathy (PSSM2) and myofibrillar myopathy (MFM) have not been validated by peer-review, and formal regulation of veterinary genetic testing is lacking. OBJECTIVES: To compare genotype and allele frequencies of commercial test variants (P variants) in MYOT (P2; rs1138656462), FLNC (P3a; rs1139799323), FLNC (P3b; rs1142918816) and MYOZ3 (P4; rs1142544043) between Warmblood (WB) and Arabian (AR) horses diagnosed with PSSM2/MFM by muscle histopathology, and phenotyped breed-matched controls. To quantify variant frequency in public repositories of ancient and modern horse breeds. STUDY DESIGN: Cross sectional using archived clinical material and publicly available data. METHODS: We studied 54 control-WB, 68 PSSM2/MFM-WB, 30 control-AR, 30 PSSM2/MFM-AR and 205 public genotypes. Variants were genotyped by pyrosequencing archived DNA. Genotype and allele frequency, and number of variant alleles or loci were compared within breed between controls, PSSM2/MFM combined and MFM or PSSM2 horses considered separately using additive/genotypic and dominant models (Fisher's exact tests). Variant frequencies in modern, early domestic and Przewalski horses were determined from a public data repository. RESULTS: There was no significant association between any P locus and a histopathological diagnosis of PSSM2/MFM, and no difference between control and myopathic horses in total loci with alternative alleles, or total alternate alleles when PSSM2/MFM was considered combined or separately as PSSM2 or MFM. For all tests, sensitivity was <0.33. Allele frequencies in WB (controls/cases) were: 8%/15% (P2), 5%/6% (P3a/b) and 9%/13% (P4); in AR, frequencies were: 12%/17% (P2), 2%/2% (P3a/b) and 7%/12% (P4). All P variants were present in early domestic (400- to 5500-year-old) horses and P2 present in the Przewalski. CONCLUSIONS: Because of the lack of significant association between a histopathological diagnosis of PSSM2 or MFM and the commercial genetic test variants P2, P3 and P4 in WB and AR, we cannot recommend the use of these variant genotypes for selection and breeding, prepurchase examination or diagnosis of a myopathy.

Magnetic Particle Imaging of Macrophages Associated with Cancer: Filling the Voids Left by Iron-Based Magnetic Resonance Imaging.

PURPOSE: Magnetic particle imaging (MPI) is an emerging molecular imaging technique that directly detects iron nanoparticles distributed in living subjects. Compared with imaging iron with magnetic resonance imaging (MRI), MPI signal can be measured to determine iron content in specific regions. In this paper, the detection of iron-labeled macrophages associated with cancer by MRI and MPI was compared. PROCEDURES: Imaging was performed on 4T1 tumor-bearing mice 16-21 days post-cancer cell implantation, 24 h after intravenous injection of Ferucarbotran, a superparamagnetic iron oxide (SPIO) or Ferumoxytol, an ultra-small SPIO. Images of living mice were acquired on a 3T clinical MRI (General Electric, n = 6) or MPI (Magnetic Insight, n = 10) system. After imaging, tumors and lungs were removed, imaged by MPI and examined by histology. RESULTS: MRI signal voids were observed within all tumors. In vivo, MPI signals were observed in the tumors of 4 of 5 mice after the administration of each contrast agent and in all excised tumors. Signal voids visualized by MRI were more apparent in tumors of mice injected with Ferumoxytol than those that received Ferucarbotran; this was consistent with iron content measured by MPI. Signal voids relating to macrophage uptake of iron were not detected in lungs by MRI, since air also appears hypointense. In vivo, MPI could not differentiate between iron in the lungs vs the high signal from iron in the liver. However, once the lungs were excised, MPI signal was detectable and quantifiable. Histologic examination confirmed iron within macrophages present in the tumors. CONCLUSIONS: MPI provides quantitative information on in vivo iron labeling of macrophages that is not attainable with MRI. The optimal iron nanoparticle for MPI in general is still under investigation; however, for MPI imaging of macrophages labeled in vivo by intravenous administration, Ferumoxytol nanoparticles were superior to Ferucarbotran.

Coding sequences of sarcoplasmic reticulum calcium ATPase regulatory peptides and expression of calcium regulatory genes in recurrent exertional rhabdomyolysis.

BACKGROUND: Sarcolipin (SLN), myoregulin (MRLN), and dwarf open reading frame (DWORF) are transmembrane regulators of the sarcoplasmic reticulum calcium transporting ATPase (SERCA) that we hypothesized played a role in recurrent exertional rhabdomyolysis (RER). OBJECTIVES: Compare coding sequences of SLN, MRLN, DWORF across species and between RER and control horses. Compare expression of muscle Ca2+ regulatory genes between RER and control horses. ANIMALS: Twenty Thoroughbreds (TB), 5 Standardbreds (STD), 6 Quarter Horses (QH) with RER and 39 breed-matched controls. METHODS: Sanger sequencing of SERCA regulatory genes with comparison of amino acid (AA) sequences among control, RER horses, human, mouse, and rabbit reference genomes. In RER and control gluteal muscle, quantitative real-time polymerase chain reaction of SERCA regulatory peptides, the calcium release channel (RYR1), and its accessory proteins calsequestrin (CASQ1), and calstabin (FKBP1A). RESULTS: The SLN gene was the highest expressed horse SERCA regulatory gene with a uniquely truncated AA sequence (29 versus 31) versus other species. Coding sequences of SLN, MRLN, and DWORF were identical in RER and control horses. A sex-by-phenotype effect occurred with lower CASQ1 expression in RER males versus control males (P < .001) and RER females (P = .05) and higher FKBP1A (P = .01) expression in RER males versus control males. CONCLUSIONS AND CLINICAL IMPORTANCE: The SLN gene encodes a uniquely truncated peptide in the horse versus other species. Variants in the coding sequence of SLN, MLRN, or DWORF were not associated with RER. Males with RER have differential gene expression that could reflect adaptations to stabilize RYR1.