Analysis of 13 C and 14 C labeling in pyruvate and lactate in tumor and blood of lymphoma-bearing mice injected with 13 C- and 14 C-labeled pyruvate.

Measurements of hyperpolarized 13 C label exchange between injected [1-13 C]pyruvate and the endogenous tumor lactate pool can give an apparent first-order rate constant for the exchange. The determination of the isotope flux, however, requires an estimate of the labeled pyruvate concentration in the tumor. This was achieved here by measurement of the tumor uptake of [1-14 C]pyruvate, which showed that <2% of the injected pyruvate reached the tumor site. Multiplication of this estimated labeled pyruvate concentration in the tumor with the apparent first-order rate constant for hyperpolarized 13 C label exchange gave an isotope flux that showed good agreement with a flux determined directly by the injection of non-polarized [3-13 C]pyruvate, rapid excision of the tumor after 30 s and measurement of 13 C-labeled lactate concentrations in tumor extracts. The distribution of labeled lactate between intra- and extracellular compartments and the blood pool was investigated by imaging, by measurement of the labeled lactate concentration in blood and tumor, and by examination of the effects of a gadolinium contrast agent and a lactate transport inhibitor on the intensity of the hyperpolarized [1-13 C]lactate signal. These measurements showed that there was significant export of labeled lactate from the tumor, but that labeled lactate in the blood pool produced by the injection of hyperpolarized [1-13 C]pyruvate showed only relatively low levels of polarization. This study shows that measurements of hyperpolarized 13 C label exchange between pyruvate and lactate in a murine tumor model can provide an estimate of the true isotope flux if the concentration of labeled pyruvate that reaches the tumor can be determined.

Detecting treatment response in a model of human breast adenocarcinoma using hyperpolarised [1-13C]pyruvate and [1,4-13C2]fumarate.

BACKGROUND: The recent introduction of a dynamic nuclear polarisation technique has permitted noninvasive imaging of tumour cell metabolism in vivo following intravenous administration of (13)C-labelled cell substrates. METHODS: Changes in hyperpolarised [1-(13)C]pyruvate and [1,4-(13)C(2)]fumarate metabolism were evaluated in both MDA-MB-231 cells and in implanted MDA-MB-231 tumours following doxorubicin treatment. RESULTS: Treatment of MDA-MB-231 cells resulted in the induction of apoptosis, which was accompanied by a decrease in hyperpolarised (13)C label flux between [1-(13)C]pyruvate and lactate, which was correlated with a decrease in the cellular NAD(H) coenzyme pool. There was also an increase in the rate of fumarate conversion to malate, which accompanied the onset of cellular necrosis. In vivo, the decrease in (13)C label exchange between pyruvate and lactate and the increased flux between fumarate and malate, following drug treatment, were shown to occur in the absence of any detectable change in tumour size. CONCLUSION: We show here that the early responses of a human breast adenocarcinoma tumour model to drug treatment can be followed by administration of both hyperpolarised [1-(13)C]pyruvate and [1,4-(13)C(2)]fumarate. These techniques could be used, therefore, in the clinic to detect the early responses of breast tumours to treatment.

A model for gene therapy of human hereditary lymphedema.

Primary human lymphedema (Milroy's disease), characterized by a chronic and disfiguring swelling of the extremities, is associated with heterozygous inactivating missense mutations of the gene encoding vascular endothelial growth factor C/D receptor (VEGFR-3). Here, we describe a mouse model and a possible treatment for primary lymphedema. Like the human patients, the lymphedema (Chy) mice have an inactivating Vegfr3 mutation in their germ line, and swelling of the limbs because of hypoplastic cutaneous, but not visceral, lymphatic vessels. Neuropilin (NRP)-2 bound VEGF-C and was expressed in the visceral, but not in the cutaneous, lymphatic endothelia, suggesting that it may participate in the pathogenesis of lymphedema. By using virus-mediated VEGF-C gene therapy, we were able to generate functional lymphatic vessels in the lymphedema mice. Our results suggest that growth factor gene therapy is applicable to human lymphedema and provide a paradigm for other diseases associated with mutant receptors.

HSV-tk gene therapy for human renal cell carcinoma in nude mice.

We have treated Caki-2 human renal cell carcinoma in vivo using herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Both stably transduced Caki-2 tumors, generated using retrovirus-mediated ex vivo HSV-tk gene transfer and direct intratumoral adenovirus-mediated HSV-tk gene transfer of wild type tumors, were tested. Similar treatments with LacZ containing retro- and adenoviruses were used as controls. The outcome was evaluated by imaging the tumors before and after the treatment with magnetic resonance imaging, and using histology, immunocytochemistry, and survival analysis. When implanted orthotopically into nude mouse kidneys, Caki-2 cells formed reproducible cystic papillary kidney carcinomas. In vivo magnetic resonance imaging provided an important tool for the evaluation of tumor growth. Transduction efficiency of wild-type tumors in vivo with adeno-LacZ was 22+/-14%. Significant tumor regression was achieved with direct intratumoral adeno-HSV-tk transduction followed by intraperitoneal ganciclovir (GCV) (P<.001). Also, the treatment of stably transduced Caki-2 tumors with intraperitoneal GCV resulted in a significant treatment response in the HSV-tk group as compared to the LacZ group (P<.009). Increased apoptosis and macrophage infiltrations, reduced proliferation, and degenerative changes were observed in the tumors treated with HSV-tk and GCV. Also, significant prolongation in survival was achieved with adeno-HSV-tk- and GCV-treated mice as compared to the controls. It is concluded that adeno-HSV-tk gene therapy may be useful for the treatment of renal cell carcinoma in vivo.

Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3.

The lymphatic vasculature transports extravasated tissue fluid, macromolecules and cells back into the blood circulation. Recent reports have focused on the molecular mechanisms regulating the lymphatic vessels. Vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to stimulate lymphangiogenesis and their receptor, VEGFR-3, has been linked to human hereditary lymphedema. Here we show that a soluble form of VEGFR-3 is a potent inhibitor of VEGF-C/VEGF-D signaling, and when expressed in the skin of transgenic mice, it inhibits fetal lymphangiogenesis and induces a regression of already formed lymphatic vessels, though the blood vasculature remains normal. Transgenic mice develop a lymphedema-like phenotype characterized by swelling of feet, edema and dermal fibrosis. They survive the neonatal period in spite of a virtually complete lack of lymphatic vessels in several tissues, and later show regeneration of the lymphatic vasculature, indicating that induction of lymphatic regeneration may also be possible in humans.

Characterization of a new animal model for human renal cell carcinoma.

BACKGROUND: Human renal cell carcinoma (RCC) is the most common kidney malignancy with significant mortality. Human tumor xenograft models are important tools for cancer research. MATERIALS AND METHODS: We have established and characterized a new animal model for human RCC using Caki-2 cells implanted into the renal subcapsule (RSC) of nude mice. Histology, immunocytochemistry, in situ hybridization and magnetic resonance imaging (MRI) were used to analyze the tumors. RESULTS: The implantations generated reproducible carcinomas which closely resemble human RCC. The tumors showed cystic-papillary structures, rich capillary network and fibro-septa formations. Proliferation varied from 0-5% and from 1-60% in cystic and solid areas, respectively. Apoptosis was less than 1%. Macrophages and other inflammatory cell infiltrations were detected in the tumors. VEGF-A and angiopoietin I were expressed in a small number of cells in large tumors. Tumors did not metastasize outside peritoneal cavity. Survival of the tumor bearing animals was 23 +/- 3 weeks. CONCLUSIONS: It is concluded that Caki-2 carcinomas implanted into renal subcapsule of nude mice resemble human RCC in several aspects and represent a good animal model for studies regarding the pathogenesis and treatment of human RCC.