Multiorgan Metastasis of Human HER-2+ Breast Cancer in Rag2−/−;Il2rg−/− Mice and Treatment with PI3K Inhibitor

In vivo studies of the metastatic process are severely hampered by the fact that most human tumor cell lines derived from highly metastatic tumors fail to consistently metastasize in immunodeficient mice like nude mice. We describe a model system based on a highly immunodeficient double knockout mouse, Rag2−/−;Il2rg−/−, which lacks T, B and NK cell activity. In this model human metastatic HER-2+ breast cancer cells displayed their full multiorgan metastatic potential, without the need for selections or additional manipulations of the system. Human HER-2+ breast cancer cell lines MDA-MB-453 and BT-474 injected into Rag2−/−;Il2rg−/− mice faithfully reproduced human cancer dissemination, with multiple metastatic sites that included lungs, bones, brain, liver, ovaries, and others. Multiorgan metastatic spread was obtained both from local tumors, growing orthotopically or subcutaneously, and from cells injected intravenously. The problem of brain recurrencies is acutely felt in HER-2+ breast cancer, because monoclonal antibodies against HER-2 penetrate poorly the blood-brain barrier. We studied whether a novel oral small molecule inhibitor of downstream PI3K, selected for its penetration of the blood-brain barrier, could affect multiorgan metastatic spread in Rag2−/−; Il2rg−/− mice. NVP-BKM120 effectively controlled metastatic growth in multiple organs, and resulted in a significant proportion of mice free from brain and bone metastases. Human HER-2+ human breast cancer cells in Rag2−/−;Il2rg−/− mice faithfully reproduced the multiorgan metastatic pattern observed in patients, thus allowing the investigation of metastatic mechanisms and the preclinical study of novel antimetastatic agents

Cell-based imaging of sodium iodide symporter activity with the yellow fluorescent protein variant YFP-H148Q/I152L

The sodium iodide symporter (NIS) mediates iodide (I(-)) transport in the thyroid gland and other tissues and is of increasing importance as a therapeutic target and nuclear imaging reporter. NIS activity in vitro is currently measured with radiotracers and electrophysiological techniques. We report on the development of a novel live cell imaging assay of NIS activity using the I(-)-sensitive and genetically encodable yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In FRTL-5 thyrocytes stably expressing YFP-H148Q/I152L, I(-) induced a rapid and reversible decrease in cellular fluorescence characterized by 1) high affinity for extracellular I(-) (35 muM), 2) inhibition by the NIS inhibitor perchlorate, 3) extracellular Na(+) dependence, and 4) TSH dependence, suggesting that fluorescence changes are due to I(-) influx via NIS. Individual cells within a population of FRTL-5 cells exhibited a 3.5-fold variation in the rate of NIS-mediated I(-) influx, illustrating the utility of YFP-H148Q/I152L to detect cell-to-cell difference in NIS activity. I(-) also caused a perchlorate-sensitive decrease in YFP-H148Q/I152L fluorescence in COS-7 cells expressing NIS but not in cells lacking NIS. These results demonstrate that YFP-H148Q/I152L is a sensitive biosensor of NIS-mediated I(-) uptake in thyroid cells and in nonthyroidal cells following gene transfer and suggest that fluorescence detection of cellular I(-) may be a useful tool by which to study the pathophysiology and pharmacology of NIS

Tumor growth of human breast cancer cell lines in Rag2^−/−;Il2rg^−/−, but not in nude mice.

<p>Abbreviations: wk, weeks; SEM, standard error of the mean.</p>a<p>Tumor volumes at 12–13 weeks after cell injection, except 474-EGFP at 23 weeks.</p>b<p>All nude mice were tumor-free 15 weeks after cell injection.</p

Quantitative analysis of antimetastatic activity of oral NVP-BKM120.

<p>Each bar represents the mean and SEM of groups of 6–9 mice treated i.v. with 453-EGFP cells, percentage inhibition is shown in each graph above NVP-BKM120 bar. (A) Metastatic burden in the brain as evaluated by Real-Time PCR, <i>see </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039626#s4" target="_blank">Materials and Methods</a> for calculations; (B, C) Cytofluorometric determination of HER-2⁺ metastatic cells in the dissociated brain (B) and femural bone marrow (C); (D, E, F) Visual count of metastatic sites per mouse, <i>see </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039626#pone-0039626-g001" target="_blank">Fig. 1</a> for representative pictures. Statistical evaluation of metastasis inhibition by NVP-BKM120: panels A, B, D, F, p<0.05 at least by the Student's <i>t</i> test.</p

Multiorgan inhibition of 453-EGFP metastatic growth by NVP-BKM120.

<p>Incidence of metastases in different sites (A) and local tumor growth (B) after intramammary cell injection. Treatment with NVP-BKM120 started seven days after cell injection. Vehicle, n = 6; NVP-BKM120, n = 5. (C) Incidence of metastases in different sites after intravenous cell injection. Vehicle, n = 9; NVP-BKM120, n = 6. Treatment with NVP-BKM120 started one day after cell injection. A significant inhibition of metastasis by NVP-BKM120 was recorded in the brain, bone marrow and liver, p<0.05 at least, Fisher's exact test. (D) Representative samples from groups of panel C of dissected control and treated mouse brains (ventral view) and lungs showing reduction in metastatic burden by NVP-BKM120. Bars correspond to 2.5 mm (brain) or 7.5 mm (lungs).</p

Representative images of metastatic dissemination of human breast cancer cells in Rag2^−/−;Il2rg^−/− mice.

<p>In each picture metastases are bright green, clearly distinct from either brownish internal organs or yellow autofluorescence of mouse skin and fur. Panels A-H, 453-EGFP cells; panels I-K, 474-EGFP cells. Routes of injection: intravenous (A, B, G); subcutaneous (D, E, H); intramammary (C, F, I, J, K). (A) Ventral view of a mouse showing multiple metastases prevalently affecting bones. Bar corresponds to 10 mm. (B) Dorsal view of a mouse showing bone and brain metastases. Bar corresponds to 10 mm. (C) Dissected lungs showing widespread metastatic dissemination. Bar corresponds to 3.3 mm. Dorsal (D) and ventral (E) views of a dissected brain with multiple metastases. Bar corresponds to 3.3 mm. (F) Dissected ovaries and uterus with one large ovarian metastasis. Bar corresponds to 5 mm. (G) Dissected liver with metastases. Bar corresponds to 10 mm. (H) Dissected kidneys and adrenal glands with multiple bilateral metastases. Bar corresponds to 10 mm. (I) Dissected primary tumor disseminating from the abdominal mammary gland to an axillary lymph node. Bar corresponds to 5 mm. (J) Dissected femur with metastasis. Bar corresponds to 5 mm. (K) Dissected liver with metastases. Bar corresponds to 9.2 mm.</p

Multiorgan metastatic ability of human breast cancer cell lines in Rag2^−/−;Il2rg^−/− mice.

a<p>Table includes only those mice for which all indicated metastatic sites were homogeneously evaluable, excluding, for example, those cases in which selected organs were separately processed for morphologic or immunohistochemical studies (<i>see</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039626#pone-0039626-g002" target="_blank">Fig. 2</a>).</p>b<p>Other metastatic sites include salivary glands, snout, uterus, interscapular space.</p>c<p>Mice were sacrificed when they showed signs of distress; mean ± standard error of the mean survival times in weeks were: MDA-MB-453: 9.1±0.32; 453-EGFP: 8.4±0.57; BT-474: 28.9±3.36; 474-EGFP: 59.3±4.92.</p