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Tetrac Delayed the Onset of Ocular Melanoma in an Orthotopic Mouse Model.

ABSTRACT: Ocular melanoma research, the most common primary intraocular malignancy in adults, is hindered by limited in vivo models. In a series of experiments using melanoma cells injected intraocularly into mouse eyes, we developed a model for ocular melanoma. Inoculation of 5 × 10⁵ B16F10 cells led to rapid tumor growth, extensive lung metastasis, and limited animal survival, while injection of 10² cells was sufficient for intraocular tumors to grow with extended survival. In order to improve tumor visualization, 10² melanoma cells (B16F10 or B16LS9) were inoculated into Balb/C albino mouse eyes. These mice developed intraocular tumors that did not metastasize and exhibited extended survival. Next, we studied the therapeutic potential of inhibitor of the thyroid hormones-?v?3 integrin signaling pathway in ocular melanoma. By utilizing tetraiodothyroacetic acid (tetrac), a thyroid hormone derivative, a delay in tumor onset in the B16F10 (integrin+) arm was observed, compared to the untreated group, while in the B16LS9 cells (integrin-) a similar rate of tumor onset was noticed in both experimental and control groups. In summary, following an optimization process, the mouse ocular melanoma model was developed. The models exhibited an extended therapeutic window and can be utilized as a platform for investigating various drugs and other treatment modalities.

SUBMITTER: Ashur-Fabian O

PROVIDER: S-EPMC6331424 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Tetrac Delayed the Onset of Ocular Melanoma in an Orthotopic Mouse Model.

Ashur-Fabian Osnat O Zloto Ofira O Fabian Ina I Tsarfaty Galya G Ellis Martin M Steinberg David M DM Hercbergs Aleck A Davis Paul J PJ Fabian Ido Didi ID

Frontiers in endocrinology 20190108

Ocular melanoma research, the most common primary intraocular malignancy in adults, is hindered by limited in vivo models. In a series of experiments using melanoma cells injected intraocularly into mouse eyes, we developed a model for ocular melanoma. Inoculation of 5 × 105 B16F10 cells led to rapid tumor growth, extensive lung metastasis, and limited animal survival, while injection of 102 cells was sufficient for intraocular tumors to grow with extended survival. ...[more]

PMID: 30671022

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Project description:BackgroundMetastatic uveal melanoma is a highly fatal disease; most patients die from their hepatic metastasis within 1 year. A major drawback in the development of new treatments for metastatic uveal melanoma is the difficulty in obtaining appropriate cell lines and the lack of appropriate animal models. Patient-derived xenograft (PDX) tumor models, bearing ectopically implanted tumors at a subcutaneous site, have been developed. However, these ectopically implanted PDX models have obstacles to translational research, including a low engraftment rate, slow tumor growth, and biological changes after multiple passages due to the different microenvironment. To overcome these limitations, we developed a new method to directly transplant biopsy specimens to the liver of immunocompromised mice.ResultsBy using two metastatic uveal melanoma cell lines, we demonstrated that the liver provides a more suitable microenvironment for tumor growth compared to subcutaneous sites and that surgical orthotopic implantation (SOI) of tumor pieces allows the creation of a liver tumor in immunocompromised mice. Subsequently, 10 of 12 hepatic metastasis specimens from patients were successfully xenografted into the immunocompromised mice (83.3% success rate) using SOI, including 8 of 10 needle biopsy specimens (80%). Additionally, four cryopreserved PDX tumors were re-implanted to new mice and re-establishment of PDX tumors was confirmed in all four mice. The serially passaged xenograft tumors as well as the re-implanted tumors after cryopreservation were similar to the original patient tumors in histologic, genomic, and proteomic expression profiles. CT imaging was effective for detecting and monitoring PDX tumors in the liver of living mice. The expression of Ki67 in original patient tumors was a predictive factor for implanted tumor growth and the success of serial passages in PDX mice.ConclusionsSurgical orthotopic implantation of hepatic metastasis from uveal melanoma is highly successful in the establishment of orthotopic PDX models, enhancing their practical utility for research applications. By using CT scan, tumor growth can be monitored, which is beneficial to evaluate treatment effects in interventional studies.

Project description:A spontaneous frameshift mutation, c.3481delC, in the Crb1 gene is the underlying cause of dysplasia and retinal degeneration in rd8 mice. The rd8 mutation is found in C57BL/6N but not in C57BL/6J mouse sub-strains. The development of ocular pathology in single knockout Ccl2-/-, Cx3cr1-/- and in double knockout Ccl2-/-, Cx3cr1-/- mice raised on a C57BL/6 background has been reported to depend on the presence of a rd8 mutation. In this study, we investigated the influence of the rd8 mutation on the retinal pathology that we previously described in the late-onset retinal degeneration (L-ORD) mouse model with a heterozygous S163R mutation in the C1q-tumor necrosis factor-related protein-5Ctrp5+/- gene that was generated on a C57BL/6J background.Mouse lines carrying the Ctrp5 S163R and rd8 mutations (Ctrp5+/-;rd8/rd8), corresponding controls without the rd8 mutation (Ctrp5+/-;wt/wt), and wild-type mice with and without the rd8 mutation (Wtrd8/rd8 and Wtwt/wt, respectively) were generated by systematic breeding of mice in our L-ORD mouse colony. Genotyping the mice for the rd8 (del C at nt3481 in Crb1) and Ctrp5 S163R mutations was performed with allelic PCR or sequencing. Retinal morphology was studied with fundus imaging, histology, light microscopy, electron microscopy, and immunohistochemistry.Genotype analysis of the mice in L-ORD mouse colony detected the rd8 mutation in the homozygous and heterozygous state. Fundus imaging of wild-type mice without the rd8 mutation (Wtwt/wt) revealed no autofluorescence (AF) spots up to 6-8 months and few AF spots at 21 months. However, the accumulation of AF lesions accelerated with age in the Ctrp5+/- mice that lack the rd8 mutation (Ctrp5+/-;wt/wt). The number of AF lesions was significantly increased (p<0.001), and they were small and uniformly distributed throughout the retina in the 21-month-old Ctrp5+/-;wt/wt mice when compared to the age-matched controls. Wild-type and Ctrp5+/- mice with the rd8 mutation (Wtrd8/rd8 and Ctrp5+/-;rd8/rd8, respectively) revealed an integrated retinal architecture with well-defined outer segments/inner segments (OS/IS), outer nuclear layer (ONL), outer plexiform layer (OPL), and inner nuclear layer (INL). The presence of pseudorosette structures reported in the rd8 mice between the ONL and the INL in the ventral quadrant of the retina was not observed in all genotypes studied. Further, the external limiting membrane was continuous in the Ctrp5+/-;rd8/rd8 and Wtrd8/rd8 mice. Evaluation of the retinal phenotype revealed that the Ctrp5+/-;wt/wt mice developed characteristic L-ORD pathology including age-dependent accumulation of AF spots, development of sub-retinal, sub-RPE, and basal laminar deposits, and Bruch's membrane abnormalities at older age, while these changes were not observed in the age-matched littermate WTwt/wt mice.The Wtrd8/rd8 and Ctrp5+/-;rd8/rd8 mice raised on C57BL/6J did not develop early onset retinal changes that are characteristic of the rd8 phenotype, supporting the hypothesis that manifestation of rd8-associated pathology depends on the genetic background. The retinal pathology observed in mice with the Ctrp5+/-;wt/wt genotype is consistent with the L-ORD phenotype observed in patients and with the phenotype we described previously. The lack of rd8-associated retinal pathology in the Ctrp5+/-;wt/wt mouse model raised on the C57BL/6J background and the development of the L-ORD phenotype in these mice in the presence and absence of the rd8 mutation suggests that the pathology observed in the Ctrp5+/-;wt/wt mice is primarily associated with the S163R mutation in the Ctrp5 gene.

Project description:BACKGROUND:Patients with metastatic uveal melanoma (MUM) in the liver usually die within 1 year. The development of new treatments for MUM has been limited by the lack of diverse MUM cell lines and appropriate animal models. We previously reported that orthotopic xenograft mouse models established by direct injection of MUM cells into the liver were useful for the analysis associated with tumor microenvironment in the liver. However, considering that patients with UM metastasize to the liver hematogenously, direct liver injection model might not be suitable for investigation on various mechanisms of liver metastasis. Here, we aim to establish new orthotopic xenograft models via hematogenous dissemination of tumor cells to the liver, and to compare their characteristics with the hepatic injection model. We also determine if hepatic tumors could be effectively monitored with non-invasive live imaging. METHODS:tdtTomate-labeled, patient-derived MUM cells were injected into the liver, spleen or tail vein of immunodeficient NSG mice. Tumor growth was serially assessed with In Vivo Imaging System (IVIS) images once every week. Established hepatic tumors were evaluated with CT scan and then analyzed histologically. RESULTS:We found that splenic injection could consistently establish hepatic tumors. Non-invasive imaging showed that the splenic injection model had more consistent and stronger fluorescent intensity compared to the hepatic injection model. There were no significant differences in tumor growth between splenic injection with splenectomy and without splenectomy. The splenic injection established hepatic tumors diffusely throughout the liver, while the hepatic injection of tumor cells established a single localized tumor. Long-term monitoring of tumor development showed that tumor growth, tumor distribution in the liver, and overall survival depended on the number of tumor cells injected to the spleen. CONCLUSION:We established a new orthotopic hepatic metastatic xenograft mouse model by splenic injection of MUM cells. The growth of orthotopic hepatic tumors could be monitored with non-invasive IVIS imaging. Moreover, we evaluated the therapeutic effect of a MEK inhibitor by using this model. Our findings suggest that our new orthotopic liver metastatic mouse model may be useful for preclinical drug screening experiments and for the analysis of liver metastasis mechanisms.

Dataset Information

Tetrac Delayed the Onset of Ocular Melanoma in an Orthotopic Mouse Model.

Publications

Tetrac Delayed the Onset of Ocular Melanoma in an Orthotopic Mouse Model.

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