Mechanistic modeling of the effects of myoferlin on tumor cell invasion.

Myoferlin (MYOF) is a member of the evolutionarily conserved ferlin family of proteins, noted for their role in a variety of membrane processes, including endocytosis, repair, and vesicular transport. Notably, ferlins are implicated in Caenorhabditis elegans sperm motility (Fer-1), mammalian skeletal muscle development and repair (MYOF and dysferlin), and presynaptic transmission in the auditory system (otoferlin). In this paper, we demonstrate that MYOF plays a previously unrecognized role in cancer cell invasion, using a combination of mathematical modeling and in vitro experiments. Using a real-time impedance-based invasion assay (xCELLigence), we have shown that lentiviral-based knockdown of MYOF significantly reduced invasion of MDA-MB-231 breast cancer cells in Matrigel bioassays. Based on these experimental data, we developed a partial differential equation model of MYOF effects on cancer cell invasion, which we used to generate mechanistic hypotheses. The mathematical model predictions revealed that matrix metalloproteinases (MMPs) may play a key role in modulating this invasive property, which was supported by experimental data using quantitative RT-PCR screens. These results suggest that MYOF may be a promising target for biomarkers or drug target for metastatic cancer diagnosis and therapy, perhaps mediated through MMPs.

Antigen targeting and anti-tumor activity of a novel anti-CD146 212Pb internalizing alpha-radioimmunoconjugate against malignant peritoneal mesothelioma.

Malignant mesothelioma, a highly aggressive cancer that primarily affects the serosal membranes, has limited therapeutic options, particularly for cavitary tumors, such as peritoneal and pleural malignant mesothelioma. Intracavitary administration of a radioimmunoconjugate to locally target mesothelioma cancer cells has been proposed as a treatment. CD146, upregulated in mesothelioma but not in healthy tissues, is a promising therapeutic target. This study characterized CD146 expression and binding/internalization kinetics of the CD146-targeting antibody OI-3 coupled with 212Pb (212Pb-TCMC-OI-3) in human mesothelioma cells. Flow cytometry showed that both chimeric (chOI-3) and murine (mOI-3) antibodies rapidly bound and internalized within 1-6 h in MSTO-211H cells. 212Pb-TCMC-chOI-3 exhibited 3.1- to 13.7-fold and 3.1- to 8.5-fold increased internalized 212Pb and 212Bi atoms per cell at 2 and 24 h, respectively, compared to isotype control, underscoring enhanced internalization efficiency. Intraperitoneal administration of 212Pb-TCMC-mOI-3 to mice with intraperitoneal MSTO-211H xenografts improved median survival by a ratio of 1.3 compared to non-binding 212Pb-TCMC-mIgG1. The ability of 212Pb-TCMC-mOI-3 to target and inhibit the growth of intraperitoneal mesothelioma xenografts supports targeted radionuclide therapy's efficacy for metastatic peritoneal mesothelioma. This study highlights the potential of localized CD146-targeted radioimmunotherapy for malignant mesothelioma, offering a new avenue for improving patient outcomes.

An Experimental Generator for Production of High-Purity 212Pb for Use in Radiopharmaceuticals.

The feasibility, performance, and radiation safety of an experimental generator were evaluated to efficiently produce 212Pb intended for radiopharmaceuticals. Methods: The generator consisted of a flask with a removable cap containing a source of 224Ra or 228Th absorbed on quartz wool. Gaseous 220Rn emanated from the decaying source, which subsequently decayed to 212Pb, which was adsorbed on the flask's interior surface. The 212Pb was collected by washing the flask with 0.5-1 mL of 0.1 M HCl. Results: The generator collector flask trapped 62%-68% of the 212Pb, of which more than 87% (tested up to 26 MBq) could be harvested. The obtained 212Pb solution had a high purity (>99.98%) and could be used for the preparation of radioconjugates with more than 97% radiochemical purity. Future designs of the generator should aim to further reduce the risk of radon and γ-energy exposure to operators. Conclusion: The presented technology is a promising method for easy and convenient 212Pb production.

A Novel Single-Step-Labeled 212Pb-CaCO3 Microparticle for Internal Alpha Therapy: Preparation, Stability, and Preclinical Data from Mice.

Lead-212 is recognized as a promising radionuclide for targeted alpha therapy for tumors. Many studies of 212Pb-labeling of various biomolecules through bifunctional chelators have been conducted. Another approach to exploiting the cytotoxic effect is coupling the radionuclide to a microparticle acting as a carrier vehicle, which could be used for treating disseminated cancers in body cavities. Calcium carbonate may represent a suitable material, as it is biocompatible, biodegradable, and easy to synthesize. In this work, we explored 212Pb-labeling of various CaCO3 microparticles and developed a protocol that can be straightforwardly implemented by clinicians. Vaterite microparticles stabilized by pamidronate were effective as 212Pb carriers; labeling yields of ≥98% were achieved, and 212Pb was strongly retained by the particles in an in vitro stability assessment. Moreover, the amounts of 212Pb reaching the kidneys, liver, spleen, and skeleton of mice following intraperitoneal (i.p.) administration were very low compared to i.p. injection of unbound 212Pb2+, indicating that CaCO3-bound 212Pb exhibited stability when administered intraperitoneally. Therapeutic efficacy was observed in a model of i.p. ovarian cancer for all the tested doses, ranging from 63 to 430 kBq per mouse. Lead-212-labeled CaCO3 microparticles represent a promising candidate for treating intracavitary cancers.

Improved Formulation of 224Ra-Labeled Calcium Carbonate Microparticles by Surface Layer Encapsulation and Addition of EDTMP.

Radium-224-labeled CaCO3 microparticles have been developed to treat peritoneal carcinomatosis. The microparticles function as carriers of 224Ra, facilitating intraperitoneal retention of the alpha-emitting radionuclide. It was necessary to control the size of microparticles in suspension over time and introduce a sterilization process for the clinical use of the radiopharmaceutical. Ethylenediamine tetra(methylene phosphonic acid) (EDTMP) was investigated as a stabilizing additive. The possibility of encapsulating the radiolabeled microparticles with an outer surface layer of CaCO3 for the improved retention of radioactivity by the carrier was studied. This work evaluated these steps of optimization and their effect on radiochemical purity, the biodistribution of radionuclides, and therapeutic efficacy. An EDTMP concentration of >1% (w/w) relative to CaCO3 stabilized the particle size for at least one week. Without EDTMP, the median particle size increased from ~5 µm to ~25 µm immediately after sterilization by autoclaving, and the larger microparticles sedimented rapidly in suspension. The percentage of adsorbed 224Ra progeny 212Pb increased from 56% to 94% at 2.4-2.5% (w/w) EDTMP when the 224Ra-labeled microparticles were layer-encapsulated. The improved formulation also resulted in a suitable biodistribution of radionuclides in mice, as well as a survival benefit for mice with intraperitoneal ovarian or colorectal tumors.

Calcium Carbonate Microparticles as Carriers of 224Ra: Impact of Specific Activity in Mice with Intraperitoneal Ovarian Cancer.

BACKGROUND: Patients with advanced-stage ovarian cancer face a poor prognosis because of recurrent peritoneal cavity metastases following surgery and chemotherapy. Alpha-emitters may enable the efficient treatment of such disseminated diseases because of their short range and highly energetic radiation. Radium-224 is a candidate α-emitter due to its convenient 3.6-day half-life, with more than 90% of the decay energy originating from α-particles. However, its inherent skeletal accumulation must be overcome to facilitate intraperitoneal delivery of the radiation dose. Therefore, 224Ra-labeled CaCO3 microparticles have been developed. OBJECTIVE: The antitumor effect of CaCO3 microparticles as a carrier for 224Ra was investigated, with an emphasis on the ratio of activity to mass dose of CaCO3, that is, specific activity. METHODS: Nude athymic mice were inoculated intraperitoneally with human ovarian cancer cells (ES-2) and treated with a single intraperitoneal injection of 224Ra-labeled CaCO3 microparticles with varying combinations of mass and activity dose, or cationic 224Ra in solution. Survival and ascites volume at sacrifice were evaluated. RESULTS: Significant therapeutic effect was achieved for all tested specific activities ranging from 0.4 to 4.6 kBq/mg. Although treatment with a mean activity dose of 1305 kBq/kg of cationic 224Ra prolonged the survival compared with the control, equivalent median survival could be achieved with 224Ra-labeled microparticles with a mean dose of only 420 kBq/kg. The best outcome was achieved with the highest specific activities (2.6 and 4.6 kBq/mg). CONCLUSION: Radium-224-labeled CaCO3 microparticles present a promising therapy against cancer dissemination in body cavities.

hnRNPDL extensively regulates transcription and alternative splicing.

RNA binding proteins (RBPs) are key players of genome regulation. Here we report the transcriptome study of HnRNP D-Like protein, which belongs to the hnRNP family. We used RNA-seq to analyze the global transcript level and alternative splicing on hnRNPDL shRNA-treated cells and control. Sh-hnRNPDL extensively increased in the expression of genes involved in female pregnancy, cell apoptosis, cell proliferation and cell migration. HnRNPDL regulated alternative splicing of hundreds of genes enriched in transcription regulation and signaling pathways including NOD-like receptor signaling, Notch signaling, and TNF signaling. This study provides the first transcriptome-wide analysis of hnRNPDL regulation of gene expression, which adds to the understanding of critical hnRNPDL functions.

Expression of zinc finger E-box-binding homeobox factor 1 in epithelial ovarian cancer: A clinicopathological analysis of 238 patients.

A growing body of evidence indicates that aberrant activation of epithelial-to-mesenchymal transition (EMT) plays a key role in tumor cell invasion and metastasis. Zinc finger E-box-binding homeobox factor 1 (ZEB1), as a crucial mediator of EMT, contributes to the malignant progression of various epithelial tumors. To determine whether ZEB1 is involved in the progression of ovarian cancer, we immunohistochemically evaluated the expression of ZEB1 in 238 cases of epithelial ovarian cancer (EOC) and analyzed its associations with clinicopathological parameters. Positive expression of ZEB1 was observed in 32.8% (78/238) of EOCs and it was found to be significantly associated with advanced tumor stage (P=0.001). The survival analysis indicated that the expression of ZEB1 was associated with a poor 5-year progression-free survival (PFS) (P=0.021). A similar tendency was also observed between the expression of ZEB1 and 5-year overall survival, although it did not reach statistical significance (P=0.118). Moreover, the multivariate analysis demonstrated that ZEB1 expression was an independent risk factor for 5-year PFS in ovarian cancer. Taken together, our data provide evidence that ZEB1 may play a crucial role in promoting aggressive ovarian carcinoma progression. Therefore, ZEB1 may serve as an effectively predictive marker and a potential target for therapeutic intervention in EOC.

Loss of myoferlin redirects breast cancer cell motility towards collective migration.

Cell migration plays a central role in the invasion and metastasis of tumors. As cells leave the primary tumor, they undergo an epithelial to mesenchymal transition (EMT) and migrate as single cells. Epithelial tumor cells may also migrate in a highly directional manner as a collective group in some settings. We previously discovered that myoferlin (MYOF) is overexpressed in breast cancer cells and depletion of MYOF results in a mesenchymal to epithelial transition (MET) and reduced invasion through extracellular matrix (ECM). However, the biomechanical mechanisms governing cell motility during MYOF depletion are poorly understood. We first demonstrated that lentivirus-driven shRNA-induced MYOF loss in MDA-MB-231 breast cancer cells (MDA-231(MYOF-KD)) leads to an epithelial morphology compared to the mesenchymal morphology observed in control (MDA-231(LTVC)) and wild-type cells. Knockdown of MYOF led to significant reductions in cell migration velocity and MDA-231(MYOF-KD) cells migrated directionally and collectively, while MDA-231(LTVC) cells exhibited single cell migration. Decreased migration velocity and collective migration were accompanied by significant changes in cell mechanics. MDA-231(MYOF-KD) cells exhibited a 2-fold decrease in cell stiffness, a 2-fold increase in cell-substrate adhesion and a 1.5-fold decrease in traction force generation. In vivo studies demonstrated that when immunocompromised mice were implanted with MDA-231(MYOF-KD) cells, tumors were smaller and demonstrated lower tumor burden. Moreover, MDA-231(MYOF-KD) tumors were highly circularized and did not invade locally into the adventia in contrast to MDA-231(LTVC)-injected animals. Thus MYOF loss is associated with a change in tumor formation in xenografts and leads to smaller, less invasive tumors. These data indicate that MYOF, a previously unrecognized protein in cancer, is involved in MDA-MB-231 cell migration and contributes to biomechanical alterations. Our results indicate that changes in biomechanical properties following loss of this protein may be an effective way to alter the invasive capacity of cancer cells.

Myoferlin depletion in breast cancer cells promotes mesenchymal to epithelial shape change and stalls invasion.

Myoferlin (MYOF) is a mammalian ferlin protein with homology to ancestral Fer-1, a nematode protein that regulates spermatic membrane fusion, which underlies the amoeboid-like movements of its sperm. Studies in muscle and endothelial cells have reported on the role of myoferlin in membrane repair, endocytosis, myoblast fusion, and the proper expression of various plasma membrane receptors. In this study, using an in vitro human breast cancer cell model, we demonstrate that myoferlin is abundantly expressed in invasive breast tumor cells. Depletion of MYOF using lentiviral-driven shRNA expression revealed that MDA-MB-231 cells reverted to an epithelial morphology, suggesting at least some features of mesenchymal to epithelial transition (MET). These observations were confirmed by the down-regulation of some mesenchymal cell markers (e.g., fibronectin and vimentin) and coordinate up-regulation of the E-cadherin epithelial marker. Cell invasion assays using Boyden chambers showed that loss of MYOF led to a significant diminution in invasion through Matrigel or type I collagen, while cell migration was unaffected. PCR array and screening of serum-free culture supernatants from shRNA(MYOF) transduced MDA-MB-231 cells indicated a significant reduction in the steady-state levels of several matrix metalloproteinases. These data when considered in toto suggest a novel role of MYOF in breast tumor cell invasion and a potential reversion to an epithelial phenotype upon loss of MYOF.

Inflammatory gene regulatory networks in amnion cells following cytokine stimulation: translational systems approach to modeling human parturition.

A majority of the studies examining the molecular regulation of human labor have been conducted using single gene approaches. While the technology to produce multi-dimensional datasets is readily available, the means for facile analysis of such data are limited. The objective of this study was to develop a systems approach to infer regulatory mechanisms governing global gene expression in cytokine-challenged cells in vitro, and to apply these methods to predict gene regulatory networks (GRNs) in intrauterine tissues during term parturition. To this end, microarray analysis was applied to human amnion mesenchymal cells (AMCs) stimulated with interleukin-1ß, and differentially expressed transcripts were subjected to hierarchical clustering, temporal expression profiling, and motif enrichment analysis, from which a GRN was constructed. These methods were then applied to fetal membrane specimens collected in the absence or presence of spontaneous term labor. Analysis of cytokine-responsive genes in AMCs revealed a sterile immune response signature, with promoters enriched in response elements for several inflammation-associated transcription factors. In comparison to the fetal membrane dataset, there were 34 genes commonly upregulated, many of which were part of an acute inflammation gene expression signature. Binding motifs for nuclear factor-κB were prominent in the gene interaction and regulatory networks for both datasets; however, we found little evidence to support the utilization of pathogen-associated molecular pattern (PAMP) signaling. The tissue specimens were also enriched for transcripts governed by hypoxia-inducible factor. The approach presented here provides an uncomplicated means to infer global relationships among gene clusters involved in cellular responses to labor-associated signals.

Bioassembly of three-dimensional embryonic stem cell-scaffold complexes using compressed gases.

Tissues are composed of multiple cell types in a well-organized three-dimensional (3D) microenvironment. To faithfully mimic the tissue in vivo, tissue-engineered constructs should have well-defined 3D chemical and spatial control over cell behavior to recapitulate developmental processes in tissue- and organ-specific differentiation and morphogenesis. It is a challenge to build a 3D complex from two-dimensional (2D) patterned structures with the presence of cells. In this study, embryonic stem (ES) cells grown on polymeric scaffolds with well-defined microstructure were constructed into a multilayer cell-scaffold complex using low pressure carbon dioxide (CO(2)) and nitrogen (N(2)). The mouse ES cells in the assembled constructs were viable, retained the ES cell-specific gene expression of Oct-4, and maintained the formation of embryoid bodies (EBs). In particular, cell viability was increased from 80% to 90% when CO(2) was replaced with N(2). The compressed gas-assisted bioassembly of stem cell-polymer constructs opens up a new avenue for tissue engineering and cell therapy.