Wang Ruonan, Hu Mengwen, Lozzi Isis, Jin Cao Zhong Jing, Ma Dou, Splith Katrin, Mengwasser Jörg, Wolf Vincent, Feldbrügge Linda, Tang Peter, Timmermann Lea, Hillebrandt Karl Herbert, Kirchner Marieluise, Mertins Philipp, Hilfenhaus Georg, Neumann Christopher Claudius Maximilian, Kammertoens Thomas, Pratschke Johann, Malinka Thomas, Sauer Igor Maximillian, Noessner Elfriede, Guo Zong Sheng, Felsenstein Matthäus
Department of Surgery, CCM, CVK, Experimental Surgery, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.
Department of General, Visceral and Transplant Surgery, Medizinische Hochschule Hannover, Hannover, Germany.
Int J Cancer. 2025 Feb 1;156(3):638-651. doi: 10.1002/ijc.35209. Epub 2024 Oct 14.
Pancreatic ductal adenocarcinoma (PDAC) remains a particularly aggressive disease with few effective treatments. The PDAC tumor immune microenvironment (TIME) is known to be immune suppressive. Oncolytic viruses can increase tumor immunogenicity via immunogenic cell death (ICD). We focused on tumor-selective (vvDD) and cytokine-armed Western-reserve vaccinia viruses (vvDD-IL2 and vvDD-IL15) and infected carcinoma cell lines as well as patient-derived primary PDAC cells. In co-culture experiments, we investigated the cytotoxic response and the activation of human natural killer (NK). Infection and virus replication were assessed by measuring virus encoded YFP. We then analyzed intracellular signaling processes and oncolysis via in-depth proteomic analysis, immunoblotting and TUNEL assay. Following the co-culture of mock or virus infected carcinoma cell lines with allogenic PBMCs or NK cell lines, CD56 NK cells were analyzed with respect to their activation, cytotoxicity and effector function. Both, dose- and time-dependent release of danger signals following infection were measured. Viruses effectively entered PDAC cells, emitted YFP signals and resulted in concomitant oncolysis. The proteome showed reprogramming of normally active core signaling pathways in PDAC (e.g., MAPK-ERK signaling). Danger-associated molecular patterns were released upon infection and stimulated co-cultured NK cells for enhanced effector cytotoxicity. NK cell subtyping revealed enhanced numbers and activation of a rare CD56CD16 population. Tumor cell killing was primarily triggered via Fas ligands rather than granule release, resulting in marked apoptosis. Overall, the cytokine-armed vaccinia viruses induced NK cell activation and enhanced cytotoxicity toward human PDAC cells in vitro. We could show that cytokine-armed virus targets the carcinoma cells and thus has great potential to modulate the TIME in PDAC.
胰腺导管腺癌（PDAC）仍然是一种侵袭性很强的疾病，有效治疗方法很少。已知PDAC肿瘤免疫微环境（TIME）具有免疫抑制作用。溶瘤病毒可通过免疫原性细胞死亡（ICD）增加肿瘤免疫原性。我们重点研究了肿瘤选择性（vvDD）和细胞因子武装的西储备痘苗病毒（vvDD-IL2和vvDD-IL15），并感染了癌细胞系以及患者来源的原发性PDAC细胞。在共培养实验中，我们研究了细胞毒性反应以及人类自然杀伤（NK）细胞的激活情况。通过测量病毒编码的YFP来评估感染和病毒复制情况。然后，我们通过深入的蛋白质组学分析、免疫印迹和TUNEL检测来分析细胞内信号传导过程和溶瘤作用。在用模拟物或病毒感染的癌细胞系与同种异体PBMC或NK细胞系共培养后，分析CD56 NK细胞的激活、细胞毒性和效应功能。测量了感染后危险信号的剂量和时间依赖性释放。病毒有效进入PDAC细胞，发出YFP信号并导致伴随的溶瘤作用。蛋白质组显示PDAC中正常活跃的核心信号通路发生了重编程（例如，MAPK-ERK信号传导）。感染后释放了危险相关分子模式，并刺激共培养的NK细胞增强效应细胞毒性。NK细胞亚型分析显示，罕见的CD56CD16群体数量增加且激活增强。肿瘤细胞杀伤主要通过Fas配体而非颗粒释放触发，导致明显的细胞凋亡。总体而言，细胞因子武装的痘苗病毒在体外诱导NK细胞激活并增强对人PDAC细胞的细胞毒性。我们可以证明，细胞因子武装的病毒靶向癌细胞，因此在调节PDAC的TIME方面具有巨大潜力。
