Magnetic Resonance Imaging of Mitochondrial Dysfunction and Metabolic Activity, Accompanied by Overproduction of Superoxide

This study shows that a mitochondria-penetrating nitroxide probe (mito-TEMPO) allows detection of superoxide and visualization of mitochondrial dysfunction in living cells due to the effect of <i>T</i>₁ shortening in MRI. Mitochondrial dysfunction was induced by treatment of cells with rotenone and 2-methoxyestradiol (2-ME/Rot). The MRI measurements were performed on 7T MRI. The 2-ME/Rot-treated cells were characterized by overproduction of superoxide, which was confirmed by a conventional dihydroethidium test. In the presence of mito-TEMPO, the intensity of MRI signal in 2-ME/Rot-treated cells was ∼30–40% higher, in comparison with that in untreated cells or culture media. In model (cell-free) systems, we observed that superoxide, but not hydrogen peroxide, increased the intensity of <i>T</i>₁-weighted MRI signal of mito-TEMPO. Moreover, the superoxide restores the <i>T</i>₁-weighted MRI contrast of mito-TEMPOH, a noncontrast (diamagnetic) analogue of mito-TEMPO. This was also confirmed by using EPR spectroscopy. The results demonstrate that superoxide radical is involved in the enhancement of <i>T</i>₁-weighted MRI contrast in living cells, in the absence and presence of mito-TEMPO. This report gives a direction for discovering new opportunities for functional MRI, for detection of metabolic activity, accompanied by overproduction of superoxide, as well as by disturbance of the balance between superoxide and hydrogen peroxide, a very important approach to clarify the fine molecular mechanisms in the regulation of many pathologies. The visualization of mitochondrial activity in real-time can be crucial to clarify the molecular mechanism of the functional MRI in its commonly accepted definition, as a method for detection of neurovascular coupling

Giant Vesicles Containing Superparamagnetic Iron Oxide as Biodegradable Cell-Tracking MRI Probes

A major breakthrough in <i>in vivo</i> cellular imaging has been the clinical/preclinical use of magnetic resonance imaging (MRI) with contrast agent. Superparamagnetic iron oxide (SPIO) is a promising candidate for the development of smart MRI probes for cell-tracking. In the present study, we describe biodegradable probes made of giant vesicles (GVs; closed lipid membranes with diameters >1 μm) that encapsulate SPIO for use as an MRI contrast agent. These SPIO-containing GVs (SPIO-GVs) exhibited excellent contrast enhancement in the single cell of medaka fish (<i>Oryzias latipes</i>) embryos immediately after their microinjection, and this enhancement disappeared when the GV membranes were destroyed. Our results demonstrate that SPIO-GVs are useful MRI probes for single cell-tracking that have minimum cytotoxicity and will greatly improve clinical/preclinical <i>in vivo</i> cellular imaging techniques

Growth curves of tumors and body weight of mice undergoing radioimmunotherapy with ⁹⁰Y-lableled antibodies.

<p>Tumor growth curve (<b>A</b>) and body weight (<b>B</b>) for mice treated with [⁹⁰Y]12A8. Tumor growth curve (<b>C</b>) and body weight (<b>D</b>) for mice treated with [⁹⁰Y]67A2 (closed circles, untreated (PBS); open circles, unlabeled IgG alone; closed triangles, 0.74 MBq; open diamonds, 1.85 MBq; black squares, 3.7 MBq).</p

Biodistribution of [¹¹¹In]67A2 in nude mice bearing SY xenograft.

<p>Data are expressed as decay-corrected % ID/g±SD normalized to a 20-g body weight mouse.</p

Histological analysis of tumors.

<p>(<b>A</b>) H&E and TUNEL stained tumor sections one week after injection of [⁹⁰Y]12A8 and [⁹⁰Y]67A2. (<b>B</b>) Quantification of apoptotic cells in tumors treated with [⁹⁰Y]12A8 (black bars) and [⁹⁰Y]67A2 (white bars). *<i>P</i><0.01 (Student's <i>t</i>-test).</p

<i>In vitro</i> assay of [¹¹¹In]12A8, [¹¹¹In]67A2 and [¹²⁵I]67A2.

<p>(<b>A</b>) Cell binding assay for [¹¹¹In]12A8 (closed circles) and [¹¹¹In]67A2 (open circles). (<b>B</b>) Competitive inhibition assay for [¹¹¹In]12A8 (closed circles) and [¹¹¹In]67A2 (open circles). Internalization assay for [¹¹¹In]67A2 (<b>C</b>) and [¹²⁵I]67A2 (<b>D</b>). Changes in % of total radioactivity for each fraction are plotted against incubation time at 37°C (closed circles, internalized fraction; open circles, membrane-bound fraction; closed triangles, protein-bound fraction in the culture medium; cross marks, non-protein-bound fraction in the culture medium).</p

<i>In vitro</i> characterization of ¹¹¹In-TSP-A01 and ¹¹¹In-TSP-A02.

<p>Cell binding assay of ¹¹¹In-TSP-A01 (A) and ¹¹¹In-TSP-A02 (B) to AsPC-1 (black circles), BxPC-3 (white triangles), and MIAPaCa-2 cells (white circles). Correlation analysis of cell binding at 6.25 × 10⁵ cells with TfR protein expression for ¹¹¹In-TSP-A01 (<b>C</b>) and ¹¹¹In-TSP-A02 (<b>D</b>). The a.u. means arbitrary unit. Competitive inhibition assay for TSP-A01 (<b>E</b>) and TSP-A02 (<b>F</b>) using the corresponding intact (black circles, solid line) and DOTA-conjugated (white circles, dashed line) antibodies.</p

TfR protein expression analysis.

<p>The expression in pancreatic cell lines (AsPC-1, BxPC-3, and MIAPaCa-2) was determined by immunofluorescence staining with the anti-TfR antibody (red). DAPI stained nuclei (blue).</p

Evaluation of Efficacy of Radioimmunotherapy with ⁹⁰Y-Labeled Fully Human Anti-Transferrin Receptor Monoclonal Antibody in Pancreatic Cancer Mouse Models

<div><p>Objective</p><p>Pancreatic cancer is an aggressive tumor and the prognosis remains poor. Therefore, development of more effective therapy is needed. We previously reported that ⁸⁹Zr-labeled TSP-A01, an antibody against transferrin receptor (TfR), is highly accumulated in a pancreatic cancer xenograft, but not in major normal organs. In the present study, we evaluated the efficacy of radioimmunotherapy (RIT) with ⁹⁰Y-TSP-A01 in pancreatic cancer mouse models.</p><p>Methods</p><p>TfR expression in pancreatic cancer cell lines (AsPC-1, BxPC-3, MIAPaCa-2) was evaluated by immunofluorescence staining. ¹¹¹In-labeled anti-TfR antibodies (TSP-A01, TSP-A02) were evaluated <i>in vitro</i> by cell binding assay with the three cell lines and by competitive inhibition assay with MIAPaCa-2. <i>In vivo</i> biodistribution was evaluated in mice bearing BxPC-3 and MIAPaCa-2 xenografts. Tumor volumes of BxPC-3 and MIAPaCa-2 were sequentially measured after ⁹⁰Y-TSP-A01 injection and histological analysis of tumors was conducted.</p><p>Results</p><p>MIAPaCa-2 cells showed the highest TfR expression, followed by AsPC-1 and BxPC-3 cells. ¹¹¹In-TSP-A01 and ¹¹¹In-TSP-A02 bound specifically to the three cell lines according to TfR expression. The dissociation constants for TSP-A01, DOTA-TSP-A01, TSP-A02, and DOTA-TSP-A02 were 0.22, 0.28, 0.17, and 0.22 nM, respectively. ¹¹¹In-TSP-A01 was highly accumulated in tumors, especially in MIAPaCa-2, but this was not true of ¹¹¹In-TSP-A02. The absorbed dose for ⁹⁰Y-TSP-A01 was estimated to be 8.3 Gy/MBq to BxPC-3 and 12.4 Gy/MBq to MIAPaCa-2. MIAPaCa-2 tumors treated with 3.7 MBq of ⁹⁰Y-TSP-A01 had almost completely disappeared around 3 weeks after injection and regrowth was not observed. Growth of BxPC-3 tumors was inhibited by 3.7 MBq of ⁹⁰Y-TSP-A01, but the tumor size was not reduced.</p><p>Conclusion</p><p>⁹⁰Y-TSP-A01 treatment achieved an almost complete response in MIAPaCa-2 tumors, whereas it merely inhibited the growth of BxPC-3 tumors. ⁹⁰Y-TSP-A01 is a promising RIT agent for pancreatic cancer, although further investigation is necessary to improve the efficacy for the radioresistant types like BxPC-3.</p></div

Evaluation of Efficacy of Radioimmunotherapy with ^{90 - Fig 4}Y-Labeled Fully Human Anti-Transferrin Receptor Monoclonal Antibody in Pancreatic Cancer Mouse Models

<p>Growth curves of BxPC-3 <b>(A)</b> and MIAPaCa-2 <b>(B)</b> tumors in mice treated with 0 MBq (black circles), 0.74 MBq (white squares), 1.85 MBq (black triangles), and 3.7 MBq (white diamonds) of ⁹⁰Y-TSP-A01. *<i>P</i> < 0.05, **<i>P</i> < 0.01 (vs. 0 MBq). Growth curves of BxPC-3 (<b>C</b>) and MIAPaCa-2 (<b>D</b>) tumors of mice treated with 0 Gy (black circles), 15 Gy (white squares), 30 Gy (black triangles), and 60 Gy (white diamonds) of X-rays. **<i>P</i> < 0.01 (vs. 0 Gy).</p