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Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice.

ABSTRACT: In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

SUBMITTER: Kageyama K

PROVIDER: S-EPMC7445893 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice.

Kageyama Ken K Ozaki Shinji S Sato Takami T

Journal of visualized experiments : JoVE 20191106 153

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful can ...[more]

PMID: 31762467

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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.

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:The profound disparity in response to immune checkpoint blockade (ICB) by cutaneous melanoma (CM) and uveal melanoma (UM) patients is not well understood. Therefore, we characterized metastases of CM and UM from the same metastatic site (liver), in order to dissect the potential underlying mechanism in differential response on ICB. Tumor liver samples from CM (n=38) and UM (n=28) patients were analyzed at the genomic (whole exome sequencing), transcriptional (RNA sequencing) and protein (immunohistochemistry and GeoMx Digital Spatial Profiling) level. Comparison of CM and UM metastases from the same metastatic site revealed that, although originating from the same melanocyte lineage, CM and UM differed in somatic mutation profile, copy number profile, tumor mutational burden (TMB) and consequently predicted neoantigens. A higher melanin content and higher expression of the melanoma differentiation antigen MelanA was observed in liver metastases of UM patients. No difference in B2M and human leukocyte antigen-DR (HLA-DR) expression was observed. A higher expression of programmed cell death ligand 1 (PD-L1) was found in CM compared with UM liver metastases, although the majority of CM and UM liver metastases lacked PD-L1 expression. There was no difference in the extent of immune infiltration observed between CM and UM metastases, with the exception of a higher expression of CD163 (p<0.0001) in CM liver samples. While the extent of immune infiltration was similar for CM and UM metastases, the ratio of exhausted CD8 T cells to cytotoxic T cells, to total CD8 T cells and to Th1 cells, was significantly higher in UM metastases. While TMB was different between CM and UM metastases, tumor immune infiltration was similar. The greater dependency on PD-L1 as an immune checkpoint in CM and the identification of higher exhaustion ratios in UM may both serve as explanations for the difference in response to ICB. Consequently, in order to improve current treatment for metastatic UM, reversal of T cell exhaustion beyond programmed cell death 1 blockade should be considered.

Dataset Information

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice.

Publications

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice.

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