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.

HER-2/neu tolerant and non-tolerant mice for fine assessment of antimetastatic potency of dendritic cell-tumor cell hybrid vaccines.

Main obstacles to cancer vaccine efficacy are pre-existing antigenic load and immunoescape mechanisms, including tolerance against self tumor-associated antigens. Here we explored the role of tolerance in an antimetastatic vaccine approach based on dendritic cell-tumor cell (DC-TC) hybrids, thanks to the comparison between BALB-neuT mice, transgenic for and tolerant to rat HER-2/neu, with their non-tolerant strain of origin BALB/c. Allogeneic DC-TC hybrid vaccine displayed a high antimetastatic activity in non-tolerant mice, but was far less effective in tolerant mice, even with intensified vaccine schedule. Tolerant BALB-neuT mice revealed a reduced ability to mount polarized Th1 responses. A further attempt to increase the antimetastatic activity by using LPS-matured DC hybrids failed. Allogeneic LPS-matured DC-TC hybrids induced high IFN-γ levels, but concomitantly also the highest production of IL-4 and IL-10 suggesting activation of mechanisms sustaining regulatory cells able to blunt vaccine efficacy. Our data in tolerant versus non-tolerant hosts suggest that clinical translation of effective DC-based strategies could benefit from more extensive investigations in tolerant transgenic models.

In silico modeling and in vivo efficacy of cancer-preventive vaccinations.

Cancer vaccine feasibility would benefit from reducing the number and duration of vaccinations without diminishing efficacy. However, the duration of in vivo studies and the huge number of possible variations in vaccination protocols have discouraged their optimization. In this study, we employed an established mouse model of preventive vaccination using HER-2/neu transgenic mice (BALB-neuT) to validate in silico-designed protocols that reduce the number of vaccinations and optimize efficacy. With biological training, the in silico model captured the overall in vivo behavior and highlighted certain critical issues. First, although vaccinations could be reduced in number without sacrificing efficacy, the intensity of early vaccinations was a key determinant of long-term tumor prevention needed for predictive utility in the model. Second, after vaccinations ended, older mice exhibited more rapid tumor onset and sharper decline in antibody levels than young mice, emphasizing immune aging as a key variable in models of vaccine protocols for elderly individuals. Long-term studies confirmed predictions of in silico modeling in which an immune plateau phase, once reached, could be maintained with a reduced number of vaccinations. Furthermore, that rapid priming in young mice is required for long-term antitumor protection, and that the accuracy of mathematical modeling of early immune responses is critical. Finally, that the design and modeling of cancer vaccines and vaccination protocols must take into account the progressive aging of the immune system, by striving to boost immune responses in elderly hosts. Our results show that an integrated in vivo-in silico approach could improve both mathematical and biological models of cancer immunoprevention.

High metastatic efficiency of human sarcoma cells in Rag2/gammac double knockout mice provides a powerful test system for antimetastatic targeted therapy.

Immunodeficient animal models are invaluable tools to investigate the metastatic propensity of human tumours. However residual immune responses, in particular natural killer (NK) cells, severely hamper the traffic and growth of human tumour cells. We studied whether a genetically modified mouse host lacking T, B and NK immunity allowed an improved expression of the metastatic phenotype of malignant human tumours. Metastatic spread of a panel of human sarcoma cell lines was studied in double knockout Rag2(-/-);gammac(-/-) mice in comparison with NK-depleted nude mice. Rag2(-/-);gammac(-/-) mice receiving intravenous (i.v.) or subcutaneous (s.c.) human sarcoma cell lines developed extensive multiorgan metastases. Metastatic efficiency in Rag2(-/-);gammac(-/-) was superior than in nude mice in terms of both metastatic sites and metastasis number. Metastatic growth in Rag2(-/-);gammac(-/-) mice was faster than that in nude mice, thus allowing an earlier metastasis evaluation. Most human sarcomas metastasised in the liver of Rag2(-/-);gammac(-/-) mice, a kind of organ preference undetectable in nude mice and specific of sarcomas, as several carcinoma cell lines failed to colonise the liver of Rag2(-/-);gammac(-/-) mice, independently of their metastatic spread to other sites. In vitro analysis of the molecular mechanisms of liver metastasis of sarcomas implicated liver-produced growth and motility factors, in particular the insulin-like growth factor (IGF) axis. NVP-BEZ235, a specific inhibitor of downstream signal transduction targeting PI3K and mTOR, strongly inhibited liver metastasis of human sarcoma cells. In conclusion, the Rag2(-/-);gammac(-/-) mouse model allowed the expression of human metastatic phenotypes inapparent in conventional immunodeficient mice and the preclinical testing of appropriate targeted therapies.

Tamoxifen combined to anti-HER-2/neu cell vaccine does not hamper cancer immunopreventive efficacy.

The possible interference of tamoxifen with anti-tumor vaccines was studied in a translational view of combined preventive approaches. Tamoxifen treatment of HER-2/neu transgenic mice combined to anti-HER-2/neu cell vaccine did not hamper the efficacy of cancer immunoprevention, and caused a significantly increased production of interferon-gamma. These data suggest that tamoxifen could even have a positive impact on the efficacy of cancer immunoprevention.

Opposing control of rhabdomyosarcoma growth and differentiation by myogenin and interleukin 4.

Rhabdomyosarcoma is a tumor of striated muscle origin that displays defective myogenic differentiation. Terminal myogenesis switches off cell proliferation and migration, hence, the promotion of rhabdomyosarcoma differentiation should antagonize tumor growth and metastasis. Terminal myogenesis is controlled by cell-intrinsic myogenic transcription factors like myogenin and environmental mediators like interleukin 4 (IL-4). We studied whether the expression of myogenin or exposure to IL-4 could promote the myogenesis of poorly differentiating human rhabdomyosarcoma cells RD/12. Forced expression of myogenin amplified myosin expression and the formation of myotube-like elements, inhibited cell migration, and reduced the growth of local tumors and liver metastases in immunodepressed mice. In contrast, exposure to IL-4 promoted cell proliferation and survival, especially at high cell density, inhibited myogenin expression, and myogenesis. Moreover, IL-4 stimulated the directed migration of cells with low myogenin levels, but not of cells with higher (spontaneous or forced) levels. Thus, IL-4, which was known to promote late stages of normal myogenesis, favors growth and migration, and inhibits further differentiation of the myogenic stages attained by rhabdomyosarcoma cells. Strategies to increase myogenin expression and block IL-4 could simultaneously reduce growth and migration, and enhance terminal differentiation of rhabdomyosarcoma, thus contributing to the control of tumor growth and metastatic spread.

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.