Epithelial, fibroblast, myeloid, T cell, primary prostate cancer

The collection and use of tissue for this study had Melbourne Health institutional review board approval and patients provided written informed consent (Melbourne Health Local Project Number: 2016.087). Following the prostatectomy of 13 patients, ranging from 52 to 78 years of age and from CAPRA-S risk score of 0 (attributed to benign tissue samples, harvested from a site far from a low grade, low volume cancer) to 7 (Supplementary file 2), a four millimeter tissue core was collected from the prostate tumour site, conditional to histopathological verification66,67. If not otherwise specified, all procedures were carried out at 4 °C. Tissue blocks were washed in Phosphate-buffered saline (PBS) solution for 2 minutes and minced for 2 minutes with a scalpel. Homogenised tissue was added to a solution (total volume of 7 ml) composed by of 1 mg/ml collagenase IV (Worthington Biochemical Corp, USA), 0.02 mg/ml DNase 1 (New England Biolabs, USA), 0.2 mg/ml dispase (Merck, USA). The tissue homogenised was serially digested at 37 °C at 180 rpm, through three steps of 5, 10 and 10 minutes of duration, with the final 3 minutes dedicated to sedimentation at 0 rpm. After each digestion step, the supernatant was aspirated and filtered through a 70 μm strainer into a pre-chilled tube, diluting the solution with 15 ml of 2% bovine serum PBS to quench the enzymatic reaction. The resulting cumulative solution was then centrifuged at 1500 rpm for five minutes, with the supernatant collected and the cell pellet resuspended into 1 ml 2% PBS-serum prior to labelling (Fig. S1).

本研究的组织采集与使用已获得墨尔本健康机构审查委员会的批准，患者已提供书面知情同意（墨尔本健康地方项目编号：2016.087）。在13名年龄介于52至78岁之间、CAPRA-S风险评分从0（归因于良性组织样本，采集自远离低级别、低体积癌症的部位）至7（见补充文件2）的患者进行前列腺切除术之后，从前列腺肿瘤部位采集了直径四毫米的组织芯，并依据组织病理学验证（66,67）。除非另有说明，所有操作均在4°C下进行。组织块用磷酸盐缓冲盐水（PBS）溶液清洗2分钟，并用手术刀切碎2分钟。将匀质化组织加入由1 mg/ml的胶原蛋白酶IV（Worthington生化公司，美国）、0.02 mg/ml的DNase 1（新英格兰生物实验室，美国）、0.2 mg/ml的dispase（默克，美国）组成的溶液（总体积7 ml）中。组织匀质化在37°C、180 rpm的条件下进行连续消化，分三步进行，每步持续5、10和10分钟，最后3分钟以0 rpm的速度进行沉降。在每次消化步骤之后，吸取上清液并通过70 μm的滤网过滤到预冷的试管中，用15 ml的2%牛血清白蛋白PBS溶液稀释溶液以终止酶促反应。所得的累积溶液随后以1500 rpm的速度离心五分钟，收集上清液并将细胞沉淀重新悬浮于1 ml的2% PBS-serum中，以便进行标记（图S1）。