Expanding the prostate cancer cell line repertoire with ACRJ-PC28, an AR-negative neuroendocrine cell line derived from an African-Caribbean patient

Prostate cell lines from diverse backgrounds are important to addressing disparities in prostate cancer (PCa) incidence and mortality rates among Black men. ACRJ-PC28 was developed from a transrectal needle biopsy and established via inactivation of the CDKN2A locus and simultaneous expression of human telomerase. Characterization assays included growth curve analysis, immunoblots, IHC, 3D cultures, immunofluorescence imaging, confocal microscopy, flow cytometry, WGS, and RNA-Seq. ACRJ-PC28 has been passaged more than 40 times in vitro over 10 months with a doubling time of 45 hours. STR profiling confirmed the novelty and human origin of the cell line. RNA-Seq confirmed the expression of prostate specific genes alpha-methylacyl-CoA racemase (AMACR) and NKX3.1 and Neuroendocrine specific markers synaptophysin (SYP) and enolase 2 (ENO2) and IHC confirmed the presence of AMACR. Immunoblots indicated the cell line is of basal-luminal type; expresses p53 and pRB and is AR negative. WGS confirmed the absence of exonic mutations and the presence of intronic variants that appear to not affect function of AR, p53, and pRB. RNA-Seq data revealed numerous TP53 and RB1 mRNA splice variants and the lack of AR mRNA expression. This is consistent with retention of p53 function in response to DNA damage and pRB function in response to contact inhibition. Soft agar anchorage-independent analysis indicated that the cells are transformed, confirmed by principal component analysis (PCA) where ACRJ-PC28 cells cluster alongside other PCa tumor tissues, yet was distinct. The novel methodology described should advance prostate cell line development, addressing the disparity in PCa among Black men.

不同遗传背景的前列腺细胞系，对于破解黑人男性群体前列腺癌（PCa）发病率与死亡率的健康差异具有重要价值。ACRJ-PC28细胞系源自经直肠针吸活检组织，通过敲除CDKN2A位点并同时表达人端粒酶构建获得。本研究对该细胞系开展了系统性表征实验，涵盖生长曲线分析、免疫印迹、免疫组织化学（immunohistochemistry, IHC）、三维培养、免疫荧光成像、共聚焦显微镜检测、流式细胞术、全基因组测序（whole genome sequencing, WGS）及转录组测序（RNA-Seq）。ACRJ-PC28细胞系在体外连续传代超过40次，历时10个月，群体倍增时间为45小时。短串联重复序列（short tandem repeat, STR）分型结果证实，该细胞系具有新颖性且来源于人类。RNA-Seq检测结果显示，该细胞系表达前列腺特异性基因α-甲基酰基辅酶A消旋酶（alpha-methylacyl-CoA racemase, AMACR）与NKX3.1，同时表达神经内分泌特异性标志物突触素（synaptophysin, SYP）及烯醇化酶2（enolase 2, ENO2）；免疫组织化学实验进一步验证了AMACR的蛋白表达。免疫印迹结果表明，ACRJ-PC28属于基底-腔上皮型细胞，表达p53与pRB蛋白，且雄激素受体（androgen receptor, AR）表达呈阴性。WGS分析证实，该细胞系未检测到外显子突变，但存在内含子变异，且此类变异似乎不会影响AR、p53及pRB的功能。RNA-Seq数据还显示，细胞内存在大量TP53与RB1的mRNA剪接变体，且无AR mRNA的表达。这一结果与p53在DNA损伤应答中功能保留、pRB在接触抑制应答中功能保留的现象相符。软琼脂非锚定依赖性生长实验结果表明，该细胞已发生恶性转化；主成分分析（principal component analysis, PCA）显示，ACRJ-PC28细胞与其他前列腺癌肿瘤组织聚类为同一类群，但仍存在显著差异。本研究报道的新型细胞系构建方法，将有助于推动前列腺细胞系的开发进程，助力解决黑人男性群体前列腺癌的健康差异问题。