Supplementary Material for: Dynamic observation: Immune-privileged microenvironment limited the effectiveness of immunotherapy in an intraocular metastasis mouse model

Introduction: Intraocular metastasis (IM) occurred in approximately 8-10% of patients with metastatic malignancy, for whom oncological immunotherapies showed poor visual potential. However, the mechanism for that inefficiency remains unclear and requires further exploration. Methods: we established a novel mouse model of intraocular metastasis by intracarotid injection of cutaneous melanoma cells. We investigated disease progression using ophthalmic and histological examinations. We used combined anti-PD-1 and anti-CTLA4 antibodies for immunotherapy and evaluate the therapeutic effects in mice model. In addition, we characterized the immune microenvironment of tumor infiltrating CD8+ T by fluorescence staining and assessed their cytotoxicity by flow cytometry. Results: All mice presented IM in the left eye, while the right eye was healthy. Uveal tissues with rich vascularity (e.g., the iris, ciliary body, and choroid) initiated intraocular metastasis at an early stage, and intraocular metastasis development resulted in several secondary changes, including corneal swelling, retinal detachment, and intratumoral haemorrhage. Immunotherapy could inhibit intraocular metastasis, prolong the time to eye rupture but did not prevent rupture ending. This inefficiency might be attributed to ocular tissues specificities that inhibited CD8+ T cells infiltration via PD-L1 expression. PD-L1low corneal tissue resisted tumor invasion with high levels of CD8+ T cells infiltration, whereas CD8+ T cells were deficient in PD-L1high uveal metastasis. Furthermore, we found a significantly increased PD-1+/- CD4+ and PD-1+/- CD8+ T cells infiltrating the intratumoral haemorrhage area. Although these CD8+ T cells in the IM were not exhausted and had a higher capacity of cytotoxicity (higher Interferon-γ ratio) than CD8+ T cells in the blood, FasL+ PD-L1+ ocular tissue can strongly inhibit these IM infiltrating T cells. Conclusions: Immunotherapy can inhibit the disease progression of intraocular metastasis. Enhancing the effects of tumor-infiltrating CD8+ T cells should be one of the highest potentials to improve the visual potential.

引言：眼内转移瘤（Intraocular Metastasis, IM）在转移性恶性肿瘤患者中占比约8%~10%，此类患者接受肿瘤免疫治疗后的视力预后不佳。然而，该治疗低效的具体机制尚未明确，仍需进一步探索。方法：本研究通过颈动脉注射皮肤黑色素瘤细胞，构建了一种新型眼内转移瘤小鼠模型。采用眼科检查与组织病理学检查追踪疾病进展；联合使用抗PD-1抗体（anti-PD-1 antibodies）与抗CTLA4抗体（anti-CTLA4 antibodies）开展免疫治疗，并评估其在小鼠模型中的治疗效果。此外，通过荧光染色（fluorescence staining）对肿瘤浸润CD8+ T细胞的免疫微环境（immune microenvironment）进行表征，并借助流式细胞术（flow cytometry）检测其细胞毒性（cytotoxicity）。结果：所有小鼠均在左眼出现眼内转移瘤，右眼则保持健康状态。血管丰富的葡萄膜组织（如虹膜、睫状体与脉络膜）在早期即可启动眼内转移进程；眼内转移瘤的进展可引发多种继发性改变，包括角膜水肿、视网膜脱离以及瘤内出血。免疫治疗可抑制眼内转移瘤的进展，延长眼球破裂的发生时间，但无法阻止眼球破裂的最终结局。该治疗低效现象可能与眼部组织的特异性相关：眼部组织通过表达PD-L1（Programmed Death-Ligand 1）抑制CD8+ T细胞的浸润。PD-L1低表达的角膜组织可抵抗肿瘤侵袭，伴随高水平CD8+ T细胞浸润；而在PD-L1高表达的葡萄膜转移灶中，CD8+ T细胞则存在浸润不足的情况。此外，本研究发现瘤内出血区域浸润的PD-1± CD4+与PD-1± CD8+ T细胞数量显著增加。尽管眼内转移瘤中的CD8+ T细胞并未耗竭，且相较于血液中的CD8+ T细胞具备更强的细胞毒性（干扰素-γ（Interferon-γ）分泌比例更高），但表达FasL与PD-L1的眼部组织可强力抑制这些浸润至眼内转移灶的T细胞。结论：免疫治疗可抑制眼内转移瘤的疾病进展。增强肿瘤浸润CD8+ T细胞的功能，有望成为改善该类患者视力预后的最具潜力的策略之一。