Targeting CD44v6 for fluorescence-guided surgery in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is an often highly invasive tumor, infiltrating functionally important tissue areas. Achieving complete tumor resection and preserving functionally relevant tissue structures depends on precise identification of tumor-free resection margins during surgery. Fluorescence-guided surgery (FGS), by intraoperative detection of tumor cells using a fluorescent tracer, may guide surgical excision and identify tumor-positive resection margins. Using a literature survey on potential surface molecules followed by immunohistochemical validation, we identified CD44 variant 6 (CD44v6) as a constitutively expressed antigen in the invasion zone of HNSCC lesions. The monoclonal anti-CD44v6 antibody BIWA was labeled with both a near-infrared fluorescent dye (IRDye800CW) and a radioactive label (Indium-111) and dual-modality imaging was applied in a locally invasive tumor mouse model. BIWA accurately detected human HNSCC xenografts in mice with a tumor uptake of 54 ± 11% ID/g and invasion regions with an accuracy of 94%. When dissected under clinical-like conditions, tumor remnants approximately 0.7 mm in diameter consisting of a few thousand cells were identified by fluorescence imaging, resulting in reliable dissection of invasive microregions. These data indicate that CD44v6 is a suitable target for reliable near-infrared detection and FGS of invasive HNSCC lesions in vivo.

Intracellular Distribution and Nuclear Activity of Antisense Oligonucleotides After Unassisted Uptake in Myoblasts and Differentiated Myotubes In Vitro.

Clinical efficacy of antisense oligonucleotides (AONs) for the treatment of neuromuscular disorders depends on efficient cellular uptake and proper intracellular routing to the target. Selection of AONs with highest in vitro efficiencies is usually based on chemical or physical methods for forced cellular delivery. Since these methods largely bypass existing natural mechanisms for membrane passage and intracellular trafficking, spontaneous uptake and distribution of AONs in cells are still poorly understood. Here, we report on the unassisted uptake of naked AONs, so-called gymnosis, in muscle cells in culture. We found that gymnosis works similarly well for proliferating myoblasts as for terminally differentiated myotubes. Cell biological analyses combined with microscopy imaging showed that a phosphorothioate backbone promotes efficient gymnosis, that uptake is clathrin mediated and mainly results in endosomal-lysosomal accumulation. Nuclear localization occurred at a low level, but the gymnotically delivered AONs effectively modulated the expression of their nuclear RNA targets. Chloroquine treatment after gymnotic delivery helped increase nuclear AON levels. In sum, we demonstrate that gymnosis is feasible in proliferating and non-proliferating muscle cells and we confirm the relevance of AON chemistry for uptake and intracellular trafficking with this method, which provides a useful means for bio-activity screening of AONs in vitro.

Effect of calcium phosphate ceramic substrate geometry on mesenchymal stromal cell organization and osteogenic differentiation.

The composition of calcium phosphate (CaP) ceramics in combination with surface features have been shown to influence biological performance, and micro- and nano-scale topography is known to stimulate osteogenic differentiation of mesenchymal stromal cells (MSCs). In view of this, adipose tissue derived MSCs were cultured on CaP disks featuring hemispherical concavities of various sizes (440, 800 or 1800 µm diameter). It was hypothesized that (i) surface concavities would promote cell proliferation, cellular organization within the concavities, and osteogenic differentiation, as a result of a more pronounced 3D micro-environment and CaP nucleation in concavities, and (ii) MSC proliferation and osteogenic differentiation would increase with smaller concavity size due to more rapidly occurring 3D cell-cell interactions. We found that concavities indeed affect cell proliferation, with 440 µm concavities increasing cell proliferation to a larger extent compared to 800 and 1800 µm concavities as well as planar surfaces. Additionally, concavity size influenced 3D cellular organization within the concavity volume. Interestingly, concavity size promoted osteogenic differentiation of cells, as evidenced by increased osteocalcin gene expression in 440 µm concavities, and osteocalcin staining predominantly for 440 and 800 µm concavities, but not for 1800 µm concavities and only slightly for planar surface controls.

Cell membrane integrity in myotonic dystrophy type 1: implications for therapy.

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by toxic RNA from a DMPK gene carrying an expanded (CTG•CAG)n repeat. Promising strategies for treatment of DM1 patients are currently being tested. These include antisense oligonucleotides and drugs for elimination of expanded RNA or prevention of aberrant binding to RNP proteins. A significant hurdle for preclinical development along these lines is efficient systemic delivery of compounds across endothelial and target cell membranes. It has been reported that DM1 patients show elevated levels of markers of muscle damage or loss of sarcolemmal integrity in their serum and that splicing of dystrophin, an essential protein for muscle membrane structure, is abnormal. Therefore, we studied cell membrane integrity in DM1 mouse models commonly used for preclinical testing. We found that membranes in skeletal muscle, heart and brain were impermeable to Evans Blue Dye. Creatine kinase levels in serum were similar to those in wild type mice and expression of dystrophin protein was unaffected. Also in patient muscle biopsies cell surface expression of dystrophin was normal and calcium-positive fibers, indicating elevated intracellular calcium levels, were only rarely seen. Combined, our findings indicate that cells in DM1 tissues do not display compromised membrane integrity. Hence, the cell membrane is a barrier that must be overcome in future work towards effective drug delivery in DM1 therapy.

Plasmodium berghei Δp52&p36 parasites develop independent of a parasitophorous vacuole membrane in Huh-7 liver cells.

The proteins P52 and P36 are expressed in the sporozoite stage of the murine malaria parasite Plasmodium berghei. Δp52&p36 sporozoites lacking expression of both proteins are severely compromised in their capability to develop into liver stage parasites and abort development soon after invasion; presumably due to the absence of a parasitophorous vacuole membrane (PVM). However, a small proportion of P. berghei Δp52&p36 parasites is capable to fully mature in hepatocytes causing breakthrough blood stage infections. We have studied the maturation of replicating Δp52&p36 parasites in cultured Huh-7 hepatocytes. Approximately 50% of Δp52&p36 parasites developed inside the nucleus of the hepatocyte but did not complete maturation and failed to produce merosomes. In contrast cytosolic Δp52&p36 parasites were able to fully mature and produced infectious merozoites. These Δp52&p36 parasites developed into mature schizonts in the absence of an apparent parasitophorous vacuole membrane as shown by immunofluorescence and electron microscopy. Merozoites derived from these maturing Δp52&p36 liver stages were infectious for C57BL/6 mice.

PTPBR7 binding proteins in myelinating neurons of the mouse brain.

Mouse protein tyrosine phosphatase PTPBR7 is a receptor-like, transmembrane protein that is localized on the surface of neuronal cells. Its protein phosphatase activity is reduced upon multimerization, and PTPBR7-deficient mice display motor coordination defects. Extracellular molecules that may influence PTPBR7 activity, however, remain to be determined. We here show that the PTPBR7 extracellular domain binds to highly myelinated regions in mouse brain, in particular the white matter tracks in cerebellum. PTPBR7 deficiency does not alter this binding pattern, as witnessed by RAP in situ staining of Ptprr⁻/⁻ mouse brain sections. Additional in situ and in vitro experiments also suggest that sugar moieties of heparan sulphate and chondroitin sulphate glycosaminoglycans are not critical for PTPBR7 binding. Candidate binding proteins were affinity-purified exploiting the PTPBR7 extracellular domain and identified by mass spectrometric means. Results support the suggested link between PTPRR isoforms and cerebellar calcium ion homeostasis, and suggest an additional role in the process of cell-cell adhesion.

Multimodal imaging of nanovaccine carriers targeted to human dendritic cells.

Dendritic cells (DCs) are key players in the initiation of adaptive immune responses and are currently exploited in immunotherapy against cancer and infectious diseases. The targeted delivery of nanovaccine particles (NPs) to DCs in vivo is a promising strategy to enhance immune responses. Here, targeted nanovaccine carriers were generated that allow multimodal imaging of nanocarrier-DC interactions from the subcellular to the organism level. These carriers were made of biodegradable poly(D,L-lactide-co-glycolide) harboring superparamagnetic iron oxide particles (SPIO) and fluorescently labeled antigen in a single particle. Targeted delivery was facilitated by coating the NPs with antibodies recognizing the DC-specific receptor DC-SIGN. The fluorescent label allowed for rapid analysis and quantification of specific versus nonspecific uptake of targeted NPs by DCs compared to other blood cells. In addition, it showed that part of the encapsulated antigen reached the lysosomal compartment of DCs within 24 h. Moreover, the presence of fluorescent label did not prevent the antigen from being presented to antigen-specific T cells. The incorporated SPIO was applied to track the NPs at subcellular cell organel level using transmission electron microscopy (TEM). NPs were found within endolysosomal compartments, where part of the SPIO was already released within 24 h. Furthermore, part of the NPs seemed to localize within the cytoplasm. Ex vivo loading of DCs with NPs resulted in efficient labeling and detection by MRI and did not abolish cell migration within collagen scaffolds. In conclusion, incorporation of two imaging agents within a single carrier allows tracking of targeted nanovaccines on a subcellular, cellular and possibly organism level, thereby facilitating rational design of in vivo targeted vaccination strategies.

Abnormal actomyosin assembly in proliferating and differentiating myoblasts upon expression of a cytosolic DMPK isoform.

DMPK, the product of the mutated gene in myotonic dystrophy type 1, belongs to the subfamily of Rho-associated serine-threonine protein kinases, whose members play a role in actin-based cell morphodynamics. Not much is known about the physiological role of differentially localized individual DMPK splice isoforms. We report here that prominent stellar-shaped stress fibers are formed during early and late steps of differentiation in DMPK-deficient myoblast-myotubes upon complementation with the short cytosolic DMPK E isoform. Expression of DMPK E led to an increased phosphorylation status of MLC2. We found no such effects with vectors that encode a mutant DMPK E which was rendered enzymatically inactive or any of the long C-terminally anchored DMPK isoforms. Presence of stellar structures appears associated with changes in cell shape and motility and a delay in myogenesis. Our data strongly suggest that cytosolic DMPK participates in remodeling of the actomyosin cytoskeleton in developing skeletal muscle cells. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Triplet-repeat oligonucleotide-mediated reversal of RNA toxicity in myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is caused by toxicity of an expanded, noncoding (CUG)n tract in DM protein kinase (DMPK) transcripts. According to current evidence the long (CUG)n segment is involved in entrapment of muscleblind (Mbnl) proteins in ribonuclear aggregates and stabilized expression of CUG binding protein 1 (CUGBP1), causing aberrant premRNA splicing and associated pathogenesis in DM1 patients. Here, we report on the use of antisense oligonucleotides (AONs) in a therapeutic strategy for reversal of RNA-gain-of-function toxicity. Using a previously undescribed mouse DM1 myoblast-myotube cell model and DM1 patient cells as screening tools, we have identified a fully 2'-O-methyl-phosphorothioate-modified (CAG)7 AON that silences mutant DMPK RNA expression and reduces the number of ribonuclear aggregates in a selective and (CUG)n-length-dependent manner. Direct administration of this AON in muscle of DM1 mouse models in vivo caused a significant reduction in the level of toxic (CUG)n RNA and a normalizing effect on aberrant premRNA splicing. Our data demonstrate proof of principle for therapeutic use of simple sequence AONs in DM1 and potentially other unstable microsatellite diseases.

Cysts of PRKCSH mutated polycystic liver disease patients lack hepatocystin but express Sec63p.

Polycystic liver disease (PCLD) is an inherited disorder caused by mutations in either PRKCSH (hepatocystin) or SEC63 (Sec63p). However, expression patterns of the implicated proteins in diseased and normal liver are unknown. We analyzed subcellular and cellular localization of hepatocystin and Sec63p using cell fractionation, immunofluorescence, and immunohistochemical methods. Expression patterns were assessed in fetal liver, PCLD liver, and normal adult liver. We found hepatocystin and Sec63p expression predominantly in the endoplasmic reticulum. In fetal tissue, there was intense expression of hepatocystin in ductal plate, bile ducts, and hepatocytes. However, Sec63p staining was prominent in early hepatocytes only and weak in bile ducts throughout development. In PCLD tissue, hepatocystin was expressed in hepatocytes, bile ducts, and in cyst epithelium of patients negative for PRKCSH mutation. In contrast, the majority of cysts from PRKCSH mutation carriers did not express hepatocystin. Sec63p expression was observed in all cyst epithelia regardless of mutational state. We conclude that hepatocystin is probably required for development of bile ducts and does not interact with Sec63p. The results support the hypothesis that cyst formation in PCLD results from a cellular recessive mechanism involving loss of hepatocystin. Cystogenesis in SEC63-associated PCLD occurs via a different mechanism.

Identification of a rat model for usher syndrome type 1B by N-ethyl-N-nitrosourea mutagenesis-driven forward genetics.

The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis to increase genetic variation in laboratory rats and identified a recessive mutant, named tornado, showing aberrant circling behavior, hyperactivity, and stereotypic head shaking. More detailed analysis revealed profound deafness due to disorganization and degeneration of the organ of Corti that already manifests at the onset of hearing. We set up a single nucleotide polymorphism (SNP)-based mapping strategy to identify the affected gene, revealing strong linkage to the central region of chromosome 1. Candidate gene resequencing identified a point mutation that introduces a premature stopcodon in Myo7a. Mutations in human MYO7A result in Usher syndrome type 1B, a severe autosomal inherited recessive disease that involves deafness and vestibular dysfunction. Here, we present the first characterized rat model for this disease. In addition, we demonstrate proof of principle for the generation and cloning of human disease models in rat using ENU mutagenesis, providing good perspectives for systematic phenotypic screens in the rat.

Characterization of multiple transcripts and isoforms derived from the mouse protein tyrosine phosphatase gene Ptprr.

The use of alternative splice sites, promoters and translation start sites considerably adds to the complexity of organisms. Four mouse cDNAs (PTPBR7, PTP-SL, PTPPBSgamma+ and PTPPBSgamma-) have been cloned that contain different 5' parts but encode identical protein tyrosine phosphatase PTPRR catalytic domains. We investigated the genomic origin and coding potential of these transcripts to elucidate their interrelationship. Mouse gene Ptprr exons were identified within a 260 kbp segment on chromosome 10, revealing PTP-SL- and PTPPBSgamma-specific transcription start sites within introns two and four, respectively, relative to the 14 PTPBR7 exons. Northern and RT-PCR analyses demonstrated differential expression patterns for these promoters. Furthermore, transfection studies and AUG codon mutagenesis demonstrated that in PTP-SL and PTPPBSgamma messengers multiple translation initiation sites are being used. Resulting 72, 60, 42 and 37 kDa PTPRR protein isoforms differ not only in the length of their N-terminal part but also in their subcellular localization, covering all major PTP subtypes; receptor-like, membrane associated and cytosolic. In summary, mouse gene Ptprr gives rise to multiple isoforms through the use of distinct promoters, alternative splicing and differential translation starts. These results set the stage for further investigations on the physiological roles of PTPRR proteins.

Cloning and characterization of mCRIP2, a mouse LIM-only protein that interacts with PDZ domain IV of PTP-BL.

BACKGROUND: In the mouse submembranous protein tyrosine phosphatase PTP-BL five PDZ domains are present in between the N-terminal FERM domain, which directs the protein to the cell cortex, and the C-terminal catalytic phosphatase domain. To understand more on the physical role of PTP-BL in this microenvironment, we started to search for PTP-BL PDZ domain-interacting proteins. RESULTS: Yeast two-hybrid screening for PTP-BL targets resulted in the identification of a novel mouse LIM-only protein termed CRIP2 that is highly homologous to rat ESP1 and human CRP2 sequences. Mouse CRIP2 has a predicted molecular weight of 23 kD and consists of two LIM domains spaced by 68 amino acids. The fourth PDZ domain of PTP-BL is responsible for the binding of CRIP2 protein. Both PTP-BL and CRIP2 mRNAs display a wide, overlapping tissue distribution. Western blot analysis revealed a more restricted expression pattern for CRIP2 with high expression in lung, heart and brain. CRIP2 protein is localized at cell cortical, actin-rich structures, which is concurrent with the subcellular localization of PTP-BL. CONCLUSIONS: The observed characteristics of the LIM domain-containing adaptor protein CRIP2 are consistent with a potential role of PTP-BL in the dynamics of the cortical actin cytoskeleton.