DataSheet2_Spatial distribution of tumor-associated macrophages in an orthotopic prostate cancer mouse model.docx

Mounting evidence suggests that the immune landscape within prostate tumors influences progression, metastasis, treatment response, and patient outcomes. In this study, we investigated the spatial density of innate immune cell populations within NOD.SCID orthotopic prostate cancer xenografts following microinjection of human DU145 prostate cancer cells. Our laboratory has previously developed nanoscale liposomes that attach to leukocytes via conjugated E-selectin (ES) and kill cancer cells via TNF-related apoptosis inducing ligand (TRAIL). Immunohistochemistry (IHC) staining was performed on tumor samples to identify and quantify leukocyte infiltration for different periods of tumor growth and E-selectin/TRAIL (EST) liposome treatments. We examined the spatial-temporal dynamics of three different immune cell types infiltrating tumors using QuPath image analysis software. IHC staining revealed that F4/80+ tumor-associated macrophages (TAMs) were the most abundant immune cells in all groups, irrespective of time or treatment. The density of TAMs decreased over the course of tumor growth and decreased in response to EST liposome treatments. Intratumoral versus marginal analysis showed a greater presence of TAMs in the marginal regions at 3 weeks of tumor growth which became more evenly distributed over time and in tumors treated with EST liposomes. TUNEL staining indicated that EST liposomes significantly increased cell apoptosis in treated tumors. Additionally, confocal microscopy identified liposome-coated TAMs in both the core and periphery of tumors, highlighting the ability of liposomes to infiltrate tumors by “piggybacking” on macrophages. The results of this study indicate that TAMs represent the majority of innate immune cells within NOD.SCID orthotopic prostate tumors, and spatial density varies widely as a function of tumor size, duration of tumor growth, and treatment of EST liposomes.

日益增多的证据表明，前列腺肿瘤内部的免疫景观影响着肿瘤的进展、转移、治疗反应以及患者的预后。在本研究中，我们调查了在人类DU145前列腺癌细胞微注射后，NOD.SCID原位前列腺癌异种移植中先天免疫细胞群体的空间密度。我们的实验室先前已开发出纳米级脂质体，通过偶联E选择素（ES）附着于白细胞，并通过TNF相关凋亡诱导配体（TRAIL）杀伤癌细胞。通过对肿瘤样本进行免疫组化（IHC）染色，我们识别并量化了不同肿瘤生长时期及E选择素/TRAIL（EST）脂质体治疗期间的淋巴细胞浸润。利用QuPath图像分析软件，我们研究了三种不同免疫细胞类型浸润肿瘤的空间-时间动态。IHC染色显示，F4/80+肿瘤相关巨噬细胞（TAMs）在所有组别中均为最丰富的免疫细胞，无论时间或治疗方式。TAMs的密度随着肿瘤的生长而减少，并且在EST脂质体治疗下也有所减少。肿瘤内与边缘的比较分析表明，在肿瘤生长3周时，边缘区域存在更多的TAMs，但随着时间的推移和治疗EST脂质体的肿瘤中，分布变得更加均匀。TUNEL染色表明，EST脂质体显著增加了治疗肿瘤中的细胞凋亡。此外，共聚焦显微镜识别出肿瘤核心和边缘都存在脂质体包裹的TAMs，突显了脂质体能够通过“搭便车”的方式浸润肿瘤的能力。本研究的结果表明，TAMs构成了NOD.SCID原位前列腺肿瘤中大多数先天免疫细胞，其空间密度因肿瘤大小、肿瘤生长持续时间以及EST脂质体治疗而有很大差异。