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The prognosis of metastatic uveal melanoma (UM) is extremely poor. Approximately 90% of patients with metastatic UM die from hepatic metastasis. Controlling hepatic metastases has been a rational approach to extend survival. This raises an urgent need to develop a novel mouse model for further investigation. Patient-derived tumor xenograft (PDX) mouse model provides potential benefit through a personalized medicine approach. This model may be useful for predictions of clinical outcomes, drug efficacy and biomarker identification. Most PDX models, including UM PDX models, are made by ectopic subcutaneous implantation. Subcutaneous PDX models have some obstacles in translational research due to differences in anatomic microenvironment from tumor origin, low engraftment rate, and slow tumor growth. Our goal was to develop an orthotopic PDX model of liver metastatic UM which would overcome these difficulties. In developing our model, we first compared the differences in growth in a liver with a subcutaneous site using two UM cell lines (UM001 and UM004) in NSG mice, to ascertain which site is a more suitable tumor microenvironment. Second, we investigated the feasibility of surgical orthotopic implantation (SOI) of tumor masses to develop a practical liver tumor xenograft mouse model with UM cell lines in 20 mice. Finally, we tried to establish an orthotopic PDX mouse model of liver metastatic UM in 5 mice. We also evaluated concordance for tumor characteristics between pre-implant and post-implant tumors. Our results showed that the liver was a more suitable microenvironment to grow the two UM cell lines compared to the subcutaneous site. UM001 engrafted in the liver, whereas it did not engraft in the subcutaneous site. UM004 displayed more rapid growth in the liver compared to the subcutaneous site. A new SOI of tumor chunk was created to develop a practical liver tumor xenograft model. Donor tumors, which were generated in the liver after injecting UM cell lines, were surgically transplanted into the liver of recipient mice. The new SOI was successfully performed in all 20 mice without adverse events. Recipient mice formed tumors in the liver after SOI. For comparison, the tumor chunks of UM cell lines were also implanted subcutaneously, in which we were able to show that the growth was more rapid in the liver. Both donor and recipient tumors had matching tumor characteristics in histological and proteomic analysis. We established an orthotopic PDX mouse model of liver metastatic UM with a new SOI method. The success rate of engraftment was 80% (4/5) within 6 months. Tumor characteristics of both patient's original tumors and xenograft mouse tumors matched in histological, genomic and proteomic analysis. This new SOI method can establish a patient-derived liver metastatic uveal melanoma xenograft model in NSG mouse at a highly successful rate. Citation Format: Ken Kageyama, Masahiro Ohara, Kengo Saito, Shinji Ozaki, Mizue Terai, Andrew E. Aplin, Michael J. Mastrangelo, Takami Sato. Feasibility of surgical orthotopic implantation for establishment of patient-derived liver metastatic uveal melanoma xenograft model in NSG mouse. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 623.

Circulating tumor cells (CTCs) have been shown to be a prognostic marker in breast cancer1. We hypothesize that circulating melanoma cell (CMC) detection could be utilized in the management of uveal melanoma, including early intervention. Prior methodologies for circulating uveal melanoma cell (CUMC) detection have been fraught with poor sensitivity, limiting their clinical utility2. Development of an improved method is necessary to establish the clinical utility of CUMC monitoring. Photoacoustics, also referred to as laser-induced ultrasound, is a novel platform for the detection and capture of CMCs. Photoacoustics uses short duration pulsed light to create ultrasonic acoustic waves in an optically absorbing medium, in this case melanin within melanoma3. As light is absorbed by irradiated chromophores, the optical energy gets converted into kinetic thermal energy trapped within the chromophore and subsequent thermal expansion ensues. Transient thermoelastic expansion of the absorbent cell results in the propagation of ultrasonic acoustic waves which can be detected and analyzed using a piezoelectric response mechanism. In addition, detected CMCs can be isolated by a two-phase flow cell separation technique4. Due to the low cost and melanoma specific capabilities of photoacoustics, we evaluated this technology for the purpose of CUMC detection. Methods: Cells from uveal melanoma cell line UM002B, established at Thomas Jefferson, were titrated to various cell concentrations and analyzed in a neutral density solution utilizing the photoacoustic method. Uveal melanoma cells of differing concentrations were spiked into isolated healthy donor peripheral blood mononuclear cells (PBMCs) and healthy whole blood samples. PBMC isolates were analyzed for CUMCs. Results: CUMCs were successfully quantified by the photoacoustic method including single cell detection. Recovery rates of cultured cells in a neutral density solution approached 25%. Recovery rates for CUMCs in whole blood averaged 10% of expected cell yield (56/540 cells detected) with a higher detection rate at lower cell concentrations. Photoacoustics offers a viable method for the detection of CUMCs with an accuracy that meets or exceeds previously reported CUMC yields. Studies analyzing CUMCs from patients with metastatic disease are ongoing. 1. Cristofanilli, M, et al., Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med, 2004. 351(8): 781-91. 2. Bidard FC, et al., Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma. Int J Cancer. 2014 Mar 1;134(5):1207-13. 3. Weight RM, et al. Photoacoustic detection of metastatic melanoma cells in the human circulatory system. Opt Lett. 2006 Oct 15;31(20):2998-3000. 4. O’Brien CM, et al. Capture of circulating tumor cells using photoacoustic flowmetry and two phase flow. J Biomed Opt. 2012 Jun;17(6):061221. Citation Format: Ryan M. Weight, Shingo Sato, Masahiro Ohara, Mizue Terai, Michael Mastrangelo, Marlana Orloff, Benjamin Goldschmidt, John Viator, Takami Sato. Detection of circulating tumor cells in high-risk primary uveal melanoma by the photoacoustic method. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 471.

Background: Circulating tumor cells (CTCs) represent a surrogate biomarker for hematogenous metastases. The detection of CTCs has gained increasing interest for prediction of clinical outcome. However, it remains to be determined whether uveal melanoma cells circulating in a peripheral vein predict systemic recurrence and poorer overall prognosis. Uveal melanoma, the most common primary cancer of the eye in adults, is unique in that the uveal tract is devoid of lymphatics. Since systemic recurrence develops exclusively via hematogenous spread, patients with uveal melanomas present a special opportunity to explore the prognostic potential of CTCs. Although the lung is the first organ through which venous drainage from the affected eye passes and indeed the only organ through which all venous blood must pass, 80-95% of systemic metastases are found first in the liver without development of lung metastases. To address these issues, we investigated the numbers of CTCs in paired arterial (femoral) and venous (antecubital) blood specimens obtained from uveal melanoma patients with hepatic metastases. Methods: CTCs in blood specimens were measured in 17 uveal melanoma patients with multiple hepatic metastases, including 10 patients with liver-only metastases and 7 patients with hepatic and extra-hepatic metastases. Peripheral arterial and venous blood specimens were collected at the same time prior to liver directed treatment. CTCs were analyzed using CellTracks Circulating melanoma Cell Kit by CellSearch System. The clinical information and sources of blood specimens were blinded when CTCs were analyzed. Result: CTCs were detectable from all 17 arterial blood specimens (100%) (median 5, range 1 to 168). In contrast, much smaller numbers of CTCs were detectable in 52.9% (9/17) of venous blood specimens (median 1, range 0 to 8) from the same patients. In terms of degree of tumor burden, patients who have hepatic as well as extra-hepatic metastasis showed higher numbers of arterial CTCs (median 12, range 5 to 168), compared to patients who have liver-only metastasis (median 4, range 1 to 11). More importantly, there is no significant correlation between numbers of arterial CTCs and the degree of tumor volume in the liver in patients who have liver-only metastases. Conclusions: Using this technology, the detection of uveal melanoma cells in peripheral blood is feasible. Peripheral capillary beds effectively filter CTCs from venous blood; thus venous blood might not be the best source for measurement of CTCs in patients with metastatic uveal melanoma. The paucity of clinically evident non-hepatic metastases despite the effective filtration by peripheral capillary beds remains unexplained and may be a demonstration of the “seed and soil” hypothesis. CTCs are a non-invasive source of uveal melanoma cells for evaluating tumor biology. Further investigation is warranted. Citation Format: Mizue Terai, Zhaomei Mu, David Eschelman, Carin Gonsalves, Ken Kageyama, Michael J. Mastrangelo, Marlana Orloff, Ryan Weight, Massimo Cristofanilli, Takami Sato. Detection of circulating melanoma cells in paired arterial and venous specimens from uveal melanoma patients with hepatic metastatic. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 384. doi:10.1158/1538-7445.AM2015-384

Background: The survival of patients with metastatic uveal melanoma remains poor; most patients ultimately die of hepatic metastasis. Tumor cell expression of insulin-like growth factor-I receptor (IGF-1R) is a marker of poor prognosis in primary uveal melanoma. IGF-1 is produced mainly in the liver, which may account for the hepatic tropism of uveal melanoma. Therefore, we investigated the importance of IGF-1/IGF-1R interaction in hepatic metastases. Methods: The expression pattern of IGF-1R in hepatic metastases of uveal melanoma was analyzed by immunohistochemistry in a series of archival samples from patients treated at Thomas Jefferson University Hospital. To investigate the biological role of exogenous and endogenous IGF-1 on the growth of a uveal melanoma, a long-term, cell line obtained from a hepatic metastasis (TJU-UM001) was evaluated concurrently. Results: All 24 metastases stained positive by IGF-1R. Tumors showed 80-95% of cellular staining while adjacent normal liver showed negative staining. IGF-1R was expressed in both the cytoplasm and the exterior membrane of uveal melanoma cells. As previously reported (AACR 2010, Abstract #359), more than 90% of TJU-UM001 cells expressed IGF-1R on their surface and did so at approximately 1.2 x 104 IGF-1R per TJU-UM001 cell. The cell proliferation assay (Quick Cell Proliferation Assay Kit II; BioVision, Mountain View, CA) showed that quiescent TJU-UM001 was induced to proliferate by 2.6-fold (p Conclusion: Although the strong and extensive expression of IGF-1R in archival tissue specimens suggests paracrine stimulation via IGF-1R as a major mechanism of the growth of uveal melanoma in the liver, cell proliferation in SFM and the endogenous production of IGF-1 by tumor cells indicate an autocrine mechanism via IGF-1R is also operative. The results underscore a key role of IGF-1 in promoting the growth of uveal melanoma. The present study shows autocrine and paracrine mechanisms underlay IGF-1-induced uveal melanoma growth and indicate how these mechanisms can be targeted to suppress metastatic growth and improve clinical outcome. Citation Format: Makoto Yoshida, Senthamil Selvan, Peter A. McCue, Tiziana DeAngelis, Ami Fujii, Hallgeir Rui, Renato Baserga, Michael Mastrangelo, Takami Sato. Expression of insulin-like growth factor-1 receptor in metastatic uveal melanoma and implications for potential autocrine and paracrine tumor cell growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2815. doi:10.1158/1538-7445.AM2013-2815

Background: The survival of patients with metastatic uveal melanoma remains poor due to lack of effective treatment. Uveal melanoma patients preferentially develop hepatic metastasis, which is the major cause of death. Therefore, a suitable animal model of hepatic metastasis with human uveal melanoma is critical for the investigation of its biological behavior and designing an effective therapy. Methods: Human hepatic metastatic uveal melanoma cell line (UM001), established in our laboratory, and NOD scid gamma mice (NSG mice) were used for orthotopic hepatic metastasis model. The UM001 cell line has typical characters of human melanoma, including a GNAQ mutation and expression of IGF-1 receptors. 1.00 -1.25 x 106 of UM001 cells in 15-20μl suspension of RPMI1640 medium were implanted using 27G needles under the capsule of the liver through a 1.5 cm abdominal incision. After the development of liver tumors, the NSG mice were sacrificed and investigated the behavior of the tumor cells in the liver both macro- and microscopically. Immunohistochemical analysis was performed to confirm the expression of human melanoma markers (HMB45 and Melan-A), IGF-1R. Tumor specimens were also stained for mouse CD31 (constituent of the endothelial intercellular junction of the vessels) and human laminin (major basement component of the vessels) to investigate neovascularization and vasculogenic mimicry patterns in the tumor. In addition, we performed Western blot analysis and immunohistochemistry to investigate the activation of PI3K-Akt and MAPK signaling pathways. Results: We achieved 91% (10/11cases) success rate of tumor formation using the method described in the present report. UM001 cells formed localized macroscopic nodules in the mouse liver within 4 weeks and then developed intrahepatic and peritoneal metastases within 12 weeks after implantation. Immunohistochemical analysis showed the expression of melanoma markers, IGF-1R, neovascularization positive for mouse CD31, vasculogenic mimicry patterns composed of human laminin in the hepatic tumors. In addition, activation of phosphorylated Akt and Erk1/2 in the hepatic tumor specimens was also noted. Conclusion: This orthotopic mouse model of human uveal melanoma liver metastasis showed similar histological findings as those observed in human liver. Moreover, PI3K-Akt and MAPK signaling pathway are activated in the hepatic tumor tissue. Taken together, this model is a useful tool to investigate the biological behavior of metastatic uveal melanoma in vivo, and to test novel therapies for the treatment of human uveal melanoma metastasizing to liver. Citation Format: Shinji Ozaki, Makoto Yoshida, Mizue Terai, Senthamil Selvan, Michael J. Mastrangelo, Takami Sato. Establishment and characterization of an orthotopic mouse model for human uveal melanoma hepatic metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1564. doi:10.1158/1538-7445.AM2013-1564

Uveal melanoma is the most common primary intraocular malignant tumor in adults; 30% to 50% of patients ultimately succumb to metastatic disease, predominantly to hepatic metastasis. Despite new diagnostic and therapeutic tools, the survival rate for patients with metastatic uveal melanoma remains poor. The reason for the hepato-tropism of metastatic uveal melanoma is largely unknown. It has been reported that the type 1 insulin-like growth factor receptor (IGF-1R) is expressed in more than 70% of primary uveal melanoma tissue specimens; expression of IGF-1R is correlated to poor prognosis. IGF-1 which binds to IGF-1R is produced mainly in the liver and this may account for the hepato-tropism of uveal melanoma. In this study, we investigated the effect of IGF-1 on the in vitro growth of metastatic uveal melanoma cells. The expression of IGF-1R on a metastatic uveal melanoma cell line (UM001), established from a hepatic metastasis, was demonstrated by flow cytometry and western blot analyses. The effect of IGF-1 on tumor-cell growth was then tested by adding IGF-1 (R&D Systems, Minneapolis, MN) to the culture wells of UM001 cells with serum-free medium (SFM) containing RPMI 1640 supplemented with 50 μg/ml transferrin and 0.1% bovine serum albumin. MTT and BrdU assays were used to measure proliferative activity. Furthermore, anti-IGF-1R antibody (MAB391; R&D Systems) was added to a serum-containing culture medium to investigate the role of IGF-1 on the growth of metastatic uveal melanoma cells. IGF-1R negative (R-) and positive (R600) cell lines were used as controls in our experiments. The R- cell line was derived from a 3T3-like fibroblasts isolated from mouse embryos with a targeted disruption of the IGF-IR gene (IGF-1R negative). The R600 cell line is a stable cell line which was derived from the R- cell line and expresses 30,000 molecules of human IGF-IR/cell. Flow cytometric analysis showed that UM001 cells express IGF-1R on their surface (>90%). In western blotting analysis, UM001 cells have approximately 11,700 molecules of human IGF-IR/cell. In proliferation assays, UM001 cells in SFM were exposed to IGF-I in concentration ranging from 0 to 300 ng/ml for 72 hours. The UM001 cells proliferated in response to exogenous IGF-1 and increased BrdU uptake by 49.1% at IGF-1 level of 300 ng/ml compared to 0 ng/ml (p=0.039). In a blocking assay, UM001 cells were cultured for 24 hours in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibody against either IGF-1R (30 μg/ml) or mouse IgG1 as an isotope control. We found that 30 μg/ml of anti-IGF-1R antibody blocks the proliferative activity of UM001 cells by 70%. These results suggest that IGF-1 is an important growth factor for metastatic uveal melanoma cells. Further investigation on the efficacy of IGF-1R blockade on the growth of metastatic uveal melanoma may open a new treatment approach for this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 359.

Cytokine production in the tumor microenvironment is a key regulatory mechanism for tumor progression. We reported that IL-10 is produced in more than 85% of freshly isolated metastasic melanoma cell suspensions. That IL-10 production decreases when melanoma cells are maintained in vitro suggested that an interaction between melanoma cells and the tumor microenvironment is important for IL-10 production by melanoma cells. We have demonstrated that IL-10 production by both fresh and established metastatic melanoma cells is increased in response to recombinant human IL-6 (rhIL-6) (Invitrogen). The stimulative effect of rhIL-6 is completely blocked by anti-IL-6 antibody (BD bioscience) and anti-IL-6 receptor antibody (BD bioscience). We concluded that a pro-inflammatory cytokine, IL-6 has a key role on the induction of IL-10 production by melanoma cells. In this study, we investigated the signal pathways that transduce IL-6 receptor activation into IL-10 production in a metastatic human melanoma cell line (JB) using pharmacological inhibitors or siRNAs for various kinases. Inhibitors were added to quiescent JB cells, 1 hour prior to a submaximum dose of IL-6 (2 ng/ml). After 24 hour stimulation, aliquots of culture media were assayed for IL-10 by ELISA. It was found that a JAK inhibitor, JAK inh1, suppressed 90% of IL-6-induced IL-10 production by JB cells compare to control. In parallel, cells were fixed with TCA, and the total proteins were subjected to phospho-immunoblotting assay. IL6 stimulation induced a 96-fold increase in STAT3 phosphorylation within 30 min, and the phosphorylation was sustained for 24 hours leading IL-10 production. JAK inhibitor-1 eliminated IL-10 production and STAT3 phosphorylation, suggesting a dominant role of JAK/STAT3 in IL-6-induced signals. ERK1/2 phosphorylation was spontaneously high in quiescent JB cells, and elevated 37% in response to IL-6 stimulation. Inhibition of ERK phosphorylation with inhibitors for c-Raf (GW5074) and MEK (U0126) eliminated IL-6-induced IL-10 production. A PI3K inhibitor (LY294002) suppressed IL-10 production without the inhibition of STAT3 or ERK phosphorylation. Rapamycin, an mTOR inhibitor, showed only a marginal effect on IL-10 production, indicating a minimum contribution of mTOR to the PI3K-mediated IL-10 production in melanoma. Thus, the spontaneous ERK phosphorylation and PI3K activity are necessary for STAT3-mediated IL-10 production in melanoma. IL-10 is purported to surpress anti-tumor cellular immune responses. Therefore, inhibitors that block IL-6-induced IL-10 production are expected to potentiate an anti-tumor immune response at tumor sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1913.