Inoue Tomoko, Adachi Katsuyuki, Kawana Kei, Taguchi Ayumi, Nagamatsu Takeshi, Fujimoto Asaha, Tomio Kensuke, Yamashita Aki, Eguchi Satoko, Nishida Haruka, Nakamura Hiroe, Sato Masakazu, Yoshida Mitsuyo, Arimoto Takahide, Wada-Hiraike Osamu, Oda Katsutoshi, Osuga Yutaka, Fujii Tomoyuki
Department of Obstetrics and Gynecology, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan.
Int J Oncol. 2016 Oct;49(4):1297-304. doi: 10.3892/ijo.2016.3631. Epub 2016 Jul 26.
Cancer-associated fibroblasts (CAFs) play an important role in cancer expansion and progression in tumor microenvironment (TME), via both direct and indirect interactions. Natural killer (NK) cells play a crucial role in anticancer immunity. We investigated the inhibitory effects of CAFs on NK cell activity. CAFs were isolated from endometrial cancer tissue, while normal endometrial fibroblasts (NEFs) were obtained from normal endometrium with no pathological abnormality. NK cells were obtained from allogenic healthy volunteers. CAFs or NEFs were co-cultured at an NK/fibroblast ratio of 1:1 with or without inserted membrane. For NK cell activity, K562 cells were cultured as target cells. NK cell-killing activity was determined by calculating the ratio of PI-positive K562 cells in the presence of NK cells co-cultured with fibroblasts versus NK cells alone. To examine whether NK cell activity was suppressed by IDO pathway, we inhibited IDO activity using the IDO inhibitor 1-MT. We demonstrated that CAFs derived from endometrial cancer induced greater suppression of the killing activity of allogenic NK cells compared with normal endometrial fibroblasts (NEFs). The suppression of NK cell activity by CAFs was inhibited when a membrane was inserted between the CAFs and NK cells, but not by 1-MT, an inhibitor of IDO. We focused on receptor-ligand interactions between CAFs and NK cell and found that cell-surface poliovirus receptor (PVR/CD155), a ligand of activating NK receptor DNAM-1, was downregulated in the CAFs compared with NEFs. To confirm whether PVR downregulation results in the decrease of NK cell-killing activity, PVR expression in NEFs was knocked down using siRNA against PVR (PVRsi). NK cell activity was suppressed by co-culture with PVR-knockdown NEFs, to a similar extent than CAF-induced suppression. CAFs showed increased suppression of NK cell-killing activity compared with NEFs, due to decreased PVR cell surface expression, a ligand of an NK activating receptor. This study demonstrated a novel mechanism of suppression of NK cell activity by CAFs in the TME.
癌症相关成纤维细胞（CAFs）通过直接和间接相互作用在肿瘤微环境（TME）中的癌症扩展和进展中发挥重要作用。自然杀伤（NK）细胞在抗癌免疫中起关键作用。我们研究了CAFs对NK细胞活性的抑制作用。CAFs从子宫内膜癌组织中分离出来，而正常子宫内膜成纤维细胞（NEFs）则取自无病理异常的正常子宫内膜。NK细胞取自同种异体健康志愿者。CAFs或NEFs以1:1的NK/成纤维细胞比例与或不插入膜的情况下共培养。对于NK细胞活性，将K562细胞作为靶细胞进行培养。通过计算与成纤维细胞共培养的NK细胞存在下PI阳性K562细胞与单独NK细胞的比例来确定NK细胞杀伤活性。为了研究NK细胞活性是否被吲哚胺2,3-双加氧酶（IDO）途径抑制，我们使用IDO抑制剂1-甲基色氨酸（1-MT）抑制IDO活性。我们证明，与正常子宫内膜成纤维细胞（NEFs）相比，源自子宫内膜癌的CAFs对同种异体NK细胞杀伤活性的抑制作用更强。当在CAFs和NK细胞之间插入膜时，CAFs对NK细胞活性的抑制作用受到抑制，但IDO抑制剂1-MT则没有这种作用。我们关注CAFs与NK细胞之间的受体-配体相互作用，发现与NEFs相比，CAFs中作为激活NK受体DNAX辅助分子-1（DNAM-1）配体的细胞表面脊髓灰质炎病毒受体（PVR/CD155）下调。为了确认PVR下调是否导致NK细胞杀伤活性降低，使用针对PVR的小干扰RNA（PVRsi）敲低NEFs中的PVR表达。与敲低PVR的NEFs共培养可抑制NK细胞活性，其程度与CAF诱导的抑制作用相似。与NEFs相比，CAFs对NK细胞杀伤活性的抑制作用增强，这是由于NK激活受体的配体PVR细胞表面表达降低所致。本研究证明了TME中CAFs抑制NK细胞活性的一种新机制。
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