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：局部肿瘤侵袭是前列腺癌男性患者预后的关键影响因素。尤为值得关注的是，精囊侵袭（seminal vesicle invasion, SVI）已被证实与更差的预后结局相关。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具有重要意义。
方法：本研究纳入超过1000名前列腺癌男性患者，明确了局部肿瘤进展对患者预后的影响。对前列腺癌标本采用双重免疫组织化学染色，分别标记增殖标志物Ki-67与衰老标志物p16INK4A。通过创伤愈合实验与针对p16INK4A的免疫荧光显微镜成像，体外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学术观点，即合并精囊侵袭的患者，其预后较仅存在前列腺外扩散的患者更差。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。前列腺内肿瘤周边区域同时是细胞增殖与p16INK4A表达的热点区域，而精囊侵袭区域的增殖活性较低，但同时是细胞核p16INK4A表达升高细胞的热点区域。体外实验证实，细胞衰老与前列腺癌细胞的迁移加速存在显著关联。
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理念开辟全新的研究方向。