Supplementary Material for: Senescent tumor cells are frequently present at the invasion front - implications for improving disease control in patients with locally advanced prostate cancer

Introduction: Local tumor invasion is a critical factor for the outcome of men with prostate cancer. In particular seminal vesicle invasion (SVI) has been reported to be associated with a more unfavorable prognosis. A better understanding of the functional state of invading prostate cancer cells is crucial to develop novel therapeutic strategies for patients with locally advanced disease. Methods: The prognostic impact of local tumor progression was ascertained in over 1,000 men with prostate cancer. Prostate cancer specimens were stained by double-immunohistochemistry for the proliferation marker Ki-67 and the senescence marker p16INK4A. The migratory properties of senescent prostate cancer cells were analyzed in vitro using a wound healing assay and immunofluorescence microscopy for p16INK4A. Results: We confirm the notion that patients with SVI have a more unfavorable prognosis than patients with extraprostatic extension alone. Surprisingly, we found that the tumor invasion front frequently harbors p16INK4A positive and Ki-67 negative i.e., senescent, tumor cells. While the intraprostatic tumor periphery was a hotspot for both proliferation and expression of p16INK4A, the area of SVI showed less proliferative activity but was at the same time a hotspot of cells with increased nuclear p16INK4A expression. Senescence was associated with an accelerated migration of prostate cancer cells in vitro. Discussion/Conclusion: This proof-of-concept study shows that invading prostate cancer cells frequently show signs of cellular senescence. This finding may open new avenues for neoadjuvant and adjuvant treatment concepts in men with locally advanced prostate cancer.

引言：局部肿瘤侵袭是影响前列腺癌男性患者预后的关键因素。其中，精囊侵犯（seminal vesicle invasion, SVI）已被证实与更差的预后密切相关。深入解析侵袭性前列腺癌细胞的功能状态，对于开发局部晚期前列腺癌患者的新型治疗策略至关重要。
方法：本研究对1000余名前列腺癌男性患者的局部肿瘤进展的预后影响进行了分析确认。我们采用双重免疫组织化学染色法，对前列腺癌标本的增殖标志物Ki-67与衰老标志物p16INK4A进行标记。同时，通过体外划痕愈合实验及针对p16INK4A的免疫荧光显微镜成像，分析了衰老前列腺癌细胞的迁移特性。
结果：本研究证实了此前的观点：相较于仅存在前列腺外扩散的患者，伴有精囊侵犯（SVI）的患者预后更差。令人意外的是，我们发现肿瘤侵袭前沿常存在p16INK4A阳性且Ki-67阴性的肿瘤细胞，即衰老肿瘤细胞。尽管前列腺内肿瘤边缘同时是细胞增殖与p16INK4A表达的活跃区域，但精囊侵犯区域的增殖活性较低，却同时是细胞核p16INK4A高表达细胞的聚集热点。体外实验证实，细胞衰老与前列腺癌细胞的迁移加速密切相关。
讨论与结论：本概念验证研究表明，侵袭性前列腺癌细胞常表现出细胞衰老的特征。这一发现可为局部晚期前列腺癌男性患者的新辅助与辅助治疗方案提供全新的研究方向。