Abstract 1271: Generation and optimization of humanized mice models in metastatic uveal melanoma Free

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, with poor prognosis in cases of liver metastasis. Tebentafusp, a bispecific T cell engager, has shown survival benefits in patients with metastatic uveal melanoma (MUM); however, the response rate is low (<10%), and there is an unmet need to improve the outcome of immunotherapy against MUM. A major challenge of translational research in MUM immunotherapy is the lack of suitable preclinical models. To address this, we developed a humanized mouse model using human bone marrow (BM)-derived CD34⁺ hematopoietic stem cells (HSCs).

HSCs were injected into NSG Flt3KO, hFLT3LG Tg mice (NSG-hFLT3) via the spleen or tail vein after 150 cGy whole body irradiation. Blood specimens from the mice were routinely collected to confirm the development of chimerism through human and mouse CD45 staining. Mice were sacrificed at 9 or 20 weeks, and blood specimens and various organs were collected. The distribution and differentiation of human CD45⁺ cells in BM, spleen, and blood were analyzed by flow cytometry. Furthermore, human CD45⁺ cells were isolated from the mouse spleen and BM for immune potency assays. For the liver metastasis model in humanized mice, the tdTomato-labeled human MUM cell line, UM001 (HLA-A2), was injected into the mouse liver, 3 or 9 weeks after injection of HLA-A2 matched HSCs into NSG-hFLT3 mice. MUM tumor specimens in the liver were collected 4 weeks after MUM cell line inoculation, and the interactions between human MUM tumors and human CD45⁺ cells were analyzed.

Three weeks post-injection, human CD45⁺ cells were confirmed in the blood, indicating the differentiation of injected human CD34⁺ HSCs. Flow cytometry detected human CD3⁺, CD19⁺, CD14⁺, CD11c⁺, and CD56⁺ cells in hematological organs. Monocytes and B cells predominated in the spleen at 9 weeks, while T cells were more abundant in the peripheral blood at 20 weeks. There was no difference in HSC injection routes regarding CD45⁺ cell differentiation and distribution. Human TNF-α production with LPS was detected from human CD45⁺ cells obtained from the mouse BM. IFN-γ production with phytohemagglutinin (PHA) was also confirmed in 21.1% of human CD3⁺ T cells obtained from the mouse BM. In the spleen, B cells and T cells were confirmed, but those cells were rarely seen in other organs. Interestingly, human CD45⁺ cells infiltrated the MUM tumors in the liver of humanized mice.

NSG-hFLT3 mice successfully adopted human CD34⁺ HSCs and developed the chimerism with functional human immune cells. This indicates that this mouse model can be utilized to study the interaction between MUM tumors and human immune cells and serve as a platform for investigating the efficacy and resistance mechanisms of cancer immunotherapies. This approach may also be used for an autologous humanized mouse model using CD34⁺ cells and patient-derived xenograft (PDX) from the same patients.

Sota Deguchi, Thomas Alcaro, Ryota Tanaka, Sergei Koshkin, Vitali Alexeev, Kelly Mccorkell, Usama Gergis, Takami Sato, Mizue Terai. Generation and optimization of humanized mice models in metastatic uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1271.