Adoptively transferred mesenchymal stem cells (MSCs) home to solid tumors. Biologic features within the tumor environment can be used to selectively activate transgenes in engineered MSCs after tumor invasion. One of the characteristic features of solid tumors is hypoxia. We evaluated a hypoxia-based imaging and therapy strategy to target expression of the sodium iodide symporter (NIS) gene to experimental hepatocellular carcinoma (HCC) delivered by MSCs.MSCs engineered to express transgenes driven by a hypoxia-responsive promoter showed robust transgene induction under hypoxia as demonstrated by mCherry expression in tumor cell spheroid models, or radioiodide uptake using NIS. Subcutaneous and orthotopic HCC xenograft mouse models revealed significant levels of perchlorate-sensitive NIS-mediated tumoral radioiodide accumulation by tumor-recruited MSCs using 123I-scintigraphy or 124I-positron emission tomography. Functional NIS expression was further confirmed by ex vivo 123I-biodistribution analysis. Administration of a therapeutic dose of 131I in mice treated with NIS-transfected MSCs resulted in delayed tumor growth and reduced tumor perfusion, as shown by contrast-enhanced sonography, and significantly prolonged survival of mice bearing orthotopic HCC tumors. Interestingly, radioiodide uptake into subcutaneous tumors was not sufficient to induce therapeutic effects. Our results demonstrate the potential of using tumor hypoxia-based approaches to drive radioiodide therapy in non-thyroidal tumors.

Activating germline mutations of the RET proto-oncogene are found in more than 90% of families with multiple endocrine neoplasia type 2a (MEN 2a) and familial medullary thyroid carcinoma (FMTC). The majority of patients with these hereditary tumors carry germline mutations that result in the substitution of one of five cysteine residues in exon 10 and 11. Different mutations in exons 13, 14 and 15 affecting non-cysteine residues have also been described but are considered to be rare. We now for the first time report a double mutation of the RET proto-oncogene occurring in the germline of a kindred with FMTC. Both mutations occur within the tyrosine kinase domain in exon 14 and lead to the substitution of valine 804 by methionine and arginine 844 by leucine. Since the double mutated allele cosegregated with the disease and was not identified in 200 unrelated normal probands, we conclude that they represent mutations that predispose the individual to the development of FMTC with a mild phenotype.

The tumor-homing properties of mesenchymal stem cells (MSC) have led to their development as delivery vehicles for the targeted delivery of therapeutic genes such as the sodium-iodide symporter (NIS) to solid tumors. External beam radiation therapy may represent an ideal setting for the application of engineered MSC-based gene therapy, as tumor irradiation may enhance MSC recruitment into irradiated tumors through the increased production of select factors linked to MSC migration. In the present study, the irradiation of human liver cancer cells (HuH7; 1-10 Gy) showed a strong dose-dependent increase in steady-state mRNA levels of CXCL8, CXCL12, FGF2, PDGFB, TGFB1, THBS1, and VEGF (0-48 h), which was verified for most factors at the protein level (after 48 h). Radiation effects on directed MSC migration were tested in vitro using a live cell tracking migration assay and supernatants from control and irradiated HuH7 cells. A robust increase in mean forward migration index, mean center of mass, and mean directionality of MSCs toward supernatants was seen from irradiated as compared to non-irradiated tumor cells. Transferability of this effect to other tumor sources was demonstrated using the human breast adenocarcinoma cell line (MDA-MB-231), which showed a similar behavior to radiation as seen with HuH7 cells in quantitative polymerase chain reaction and migration assay. To evaluate this in a more physiologic in vivo setting, subcutaneously growing HuH7 xenograft tumors were irradiated with 0, 2, or 5 Gy followed by CMV-NIS-MSC application 24 h later. Tumoral iodide uptake was monitored using I-scintigraphy. The results showed increased tumor-specific dose-dependent accumulation of radioiodide in irradiated tumors. The results demonstrate that external beam radiation therapy enhances the migratory capacity of MSCs and may thus increase the therapeutic efficacy of MSC-mediated NIS radionuclide therapy.

Mitochondrial genomes (mtDNA) encode essential subunits of the mitochondrial respiratory chain. Mutations in mtDNA can cause a shortage in cellular energy supply, which can lead to numerous mitochondrial diseases. How cells secure mtDNA integrity over generations has remained unanswered. Here, we show that the single-celled yeast Saccharomyces cerevisiae can intracellularly distinguish between functional and defective mtDNA and promote generation of daughter cells with increasingly healthy mtDNA content. Purifying selection for functional mtDNA occurs in a continuous mitochondrial network and does not require mitochondrial fission but necessitates stable mitochondrial subdomains that depend on intact cristae morphology. Our findings support a model in which cristae-dependent proximity between mtDNA and the proteins it encodes creates a spatial "sphere of influence," which links a lack of functional fitness to clearance of defective mtDNA.

The sodium iodide symporter (NIS) as well-characterized theranostic gene represents an outstanding tool to target different cancer types allowing noninvasive imaging of functional NIS expression and therapeutic radioiodide application. Based on its overexpression on the surface of most cancer types, the cMET/hepatocyte growth factor receptor serves as ideal target for tumor-selective gene delivery. Sequence-defined polymers as nonviral gene delivery vehicles comprising polyethylene glycol (PEG) and cationic (oligoethanoamino) amide cores coupled with a cMET-binding peptide (cMBP2) were complexed with NIS-DNA and tested for receptor-specificity, transduction efficiency, and therapeutic efficacy in hepatocellular cancer cells HuH7. In vitro iodide uptake studies demonstrated high transduction efficiency and cMET-specificity of NIS-encoding polyplexes (cMBP2-PEG-Stp/NIS) compared to polyplexes without targeting ligand (Ala-PEG-Stp/NIS) and without coding DNA (cMBP2-PEG-Stp/Antisense-NIS). Tumor recruitment and vector biodistribution were investigated in vivo in a subcutaneous xenograft mouse model showing high tumor-selective iodide accumulation in cMBP2-PEG-Stp/NIS-treated mice (6.6 ± 1.6% ID/g (123)I, biological half-life 3 hours) by (123)I-scintigraphy. Therapy studies with three cycles of polyplexes and (131)I application resulted in significant delay in tumor growth and prolonged survival. These data demonstrate the enormous potential of cMET-targeted sequence-defined polymers combined with the unique theranostic function of NIS allowing for optimized transfection efficiency while eliminating toxicity.