目的：肿瘤休眠已经成为临床肿瘤耐药、远端转移和复发的一个重要原因，免疫系统被认为参与了该过程。然而，免疫系统中I型干扰素是否参与肿瘤休眠以及相关机制并未被揭示。已有文献报道证实，进入休眠的细胞主要是肿瘤干细胞。然而体外成功分离并培养干细胞一直是一大难题，幸运的是，我们实验室早在2012年就开发了3D纤维凝胶培养体系，可以分离并大量扩增干细胞样肿瘤再生细胞。基于该项技术，本研究将以黑色素瘤为主要研究对象，探讨免疫系统中的I型干扰素，尤其是β干扰素，在肿瘤再生细胞休眠过程中所发挥的功能，阐明β干扰素诱导休眠的相关机制，并进一步寻找打破休眠的方案，为提高临床免疫治疗效果提供理论支持。

方法：（1）为了探讨β干扰素对体内肿瘤生长的影响，我们构建了B16（C57BL/6）和A375（NOD-SCID）小鼠模型，待瘤体长至5x5毫米后分组并分别给与PBS、β干扰素单用或联合中和抗体治疗，然后获得肿瘤组织并检测细胞周期、β半乳糖苷酶染色、免疫组化和H&E染色、免疫荧光共染CD133（干细胞标志物）和Ki67（增殖标志物）的表达。（2）为了研究β干扰素在体外对肿瘤再生细胞的作用，我们采用β干扰素处理B16和A375肿瘤再生细胞，然后评估细胞克隆大小、存活克隆数目、细胞周期、葡萄糖消耗量、β半乳糖苷酶染色；接着我们采用Cis/Pax/MTX处理β干扰素预处理的肿瘤再生细胞，流式细胞术检测细胞凋亡比例；此外我们还采用β干扰素处理平板培养的B16和A375细胞，分别检测细胞周期、拍照并统计细胞数目。（3）为了检测STAT1/STAT2是否参与β干扰素信号转导，我们敲除了B16细胞中STAT1或STAT2，将细胞种植在3D纤维凝胶并经β干扰素处理后，评估克隆大小和存活克隆数目。（4）为了探讨IDO1/AhR是否参与β干扰素诱导的肿瘤再生细胞休眠，我们检测β干扰素处理对IDO1或Kyn表达的影响；随后过表达IDO1，将细胞种植到3D系统，评估克隆生长能力并检测细胞周期；接着我们检测了β干扰素处理后，肿瘤再生细胞中AhR和p27转录水平，并评估p27敲除后β干扰素对肿瘤再生细胞生长的作用。（5）为了检测抑制AhR通路是否影响β干扰素功能，我们敲除IDO1/AhR或采用化学抑制剂处理肿瘤再生细胞，并检测克隆生长、凋亡比例。（6）为了探讨STAT3是否参与β干扰素联合AhR通路抑制剂对肿瘤再生细胞的作用，检测单用或联合处理后STAT3不同磷酸化位点的表达水平、对入核能力的影响、与p27或p53启动子结合能力。（7）为了研究β干扰素在体内的信号转导过程，我们构建了B16和A375小鼠模型，经β干扰素治疗后获取肿瘤组织，分别检测IDO1/AhR/p27/STAT3表达，并共定位S100和AhR。（8）为了评估β干扰素联合AhR通路抑制剂对实体瘤的治疗效果，我们分别构建了B16（C57BL/6）、A375（NOD-SCID）、H22（Balb/c）、MCF-7（NOD-SCID）等小鼠模型，并分别给与β干扰素单用或与1-MT/DMF联用，测量瘤体体积、记录生存期、称量瘤重。

结果：（1）同PBS处理相比，β干扰素可有效将瘤体控制在一定大小使其不随时间推移而生长，而加入中和抗体清除后却可逆转这种抑制现象；β干扰素处理可上调体内G1/S比例且对β半乳糖苷酶阳性细胞比例无影响，可上调干细胞（CD133）阳性比例，但是下调了干细胞中Ki67的阳性率。（2）发现β干扰素可控制B16或A375肿瘤再生细胞克隆生长，但是去除药物后恢复生长能力；体外β干扰素处理上调肿瘤再生细胞中G1/S比例并降低葡萄糖消耗量，却未增强β半乳糖苷酶阳性细胞比例，然而β干扰素并未改变分化的B16和A375细胞中G1/S比例，也未影响细胞增殖能力。采用化疗药处理β干扰素预处理的肿瘤再生细胞后发现，预处理组细胞凋亡比例显著低于未预处理组。（3）发现β干扰素无法有效抑制缺失STAT1/STAT2的B16或A375肿瘤再生细胞克隆生长，对存活克隆数目无影响。（4）β干扰素处理可显著上调B16或A375肿瘤再生细胞中IDO1和Kyn表达水平；且过表达IDO1可诱导B16或A375肿瘤再生细胞休眠、上调G1/S比例。Kyn作为转录因子AhR的激动剂，被发现可增强AhR转录活性及入核能力并上调p27表达水平；而敲除p27后，发现β干扰素无法控制B16或A375肿瘤再生细胞克隆生长。（5）β干扰素联和1-MT或DMF可增强对细胞克隆生长的抑制作用、显著降低存活克隆数目并提高凋亡细胞比例，然而敲除STAT3会逆转该现象。（6）β干扰素与1-MT或DMF联用可显著上调p-STAT3 (S、Y)与p53启动子结合能力，并升高凋亡细胞比例、减少存活克隆数目、降低6PGDH/GPDH表达并上调ROS释放能力，然而敲除p53却逆转这种现象。（7）发现β干扰素处理可上调体内肿瘤组织中IDO1/AhR/p27表达，共定位结果显示肿瘤细胞中AhR表达增强。（8）发现β干扰素与1-MT/DMF联用处理小鼠模型可有效控制瘤体生长、显著延长生存期。

结论：β干扰素可直接诱导分化的黑色素瘤细胞凋亡，此外还通过IDO1/AhR通路参与肿瘤再生细胞生命活动。一方面，IFN-β信号经STAT1/STAT2传导可激活IDO1/AhR/Kyn通路并上调p27表达进而诱导细胞休眠；另一方面，当阻断AhR通路时，IFN-β导致STAT3丝氨酸和酪氨酸位点被双磷酸化，后者通过调控p53蛋白表达而导致细胞凋亡。β干扰素联合AhR通路抑制剂可打破β干扰素诱导的休眠并增强肿瘤再生细胞凋亡，具有潜在应用价值。

ive:tumor dormancy, as a pivotal contributer to the tumor resistance, distant metastasis and recurrence, is considered to be regulated by the immune system. however, whether type i interferons involves in the induced tumor dormancy is not well illustrated. cancer stem cells, which are difficult to culture, are reported as the main subpopulation induced into dormancy. fortunately, our previous studies have confirmed that 3d fibrin gel culture could separate and spread stem cell-like tumor repopulating cells (trcs) that have the same type and function with cancer stem cells. on this basis, our study will explore the role of type i interferon, especially ifn-β, in the melanoma trcs dormancy, and try to elucidate the underlying mechanism. further, we plan to seek potential strategy to break dormant state and provide an important basis for improving the effectiveness of immunotherapy in clinic.

methods:(1) to investigate the effect of ifn-β in vivo, we constructed b16 (c57bl/6) and a375 (nod-scid) mouse models. when the tumor volume reaching 5×5 mm, mice were grouped randomly and recieved ifn-β monotherapy or combining with neutralizing antibody. then mice were sacrificed and tumor tissues were obtained for detection of cell cycle, β-galactosidase staining, ihc and h&e staining, colocalization of cd133 (stem cell marker) and ki67 (proliferation marker). (2) treating b16 and a375 trcs with ifn-β, then we evaluated the size of clones, counted number of surviving clones, detected cell cycle/glucose consumption, and performed β-galactosidase staining to examine the role of ifn-βin vitro. we also detected the cell apoptosis of trcs treated with cis/pax/mtx with or without ifn-β-pretreatment. besides, we treated the b16 and a375 cells cultured in plate with ifn-β, followed by detecting cell cycle and taking photographs for counting cell numbers. (3) to study whether stat1/stat2 participate in ifn-β signaling, we treated wild type and knocking out stat1/stat2 b16 trcs with ifn-β, and then the clone size and surviving clone number were examined. (4) to investigate whether ido1/ahr is involved in ifn-β-induced trcs dormancy, we examined the expressions of ido1 or kyn after ifn-β treatment; then ido1-overexpressng b16 cells were cultured in 3d system, followed by assessing clone growth capacity and detecting cell cycle. also, we examined the tranional capacity of ahr and p27 in trcs in the presence or absence of ifn-β. and we evaluated the effect of ifn-β on the growth of p27 knocking out trcs. (5) to detect whether blocking ahr pathway affects ifn-β function on trcs, we knocked out ido1/ahr or treated trcs with inhibitors, then detected the clone size and cell apoptosis. (6) to investigate whether stat3 participate in the ifn-β combining with ahr pathway inhibitors induced trcs apoptosis, we treated trcs with ifn-β monotherapy or combined with 1-mt/dmf, and then detected the level of different sites of p-stat3, the ability of nuclear translocation, and its ability to bind p27 or p53 promoter. (7) to study the signal transduction of ifn-β induced dormancy in vivo, we constructed mouse models bearing b16 and a375 cells, and obtained tumor tissue after ifn-β treatment. we detected ido1/ahr/p27/stat3 in the protein level. also, we colocalized s100 and ahr in tumor tissues. (8) to evaluate the therapeutic effect of ifn-β combined with ahr pathway inhibitorin vivo, we constructed b16 (c57bl/6), a375 (nod-scid), h22 (balb/c), and mcf-7 (nod-scid) mouse models. after the tumor volume reaching 7x7 mm, the mice were divided randomly and received ifn-β monotherapy or combining with 1-mt/dmf, we measured tumor volume, recorded death date, and weighed tumor tissues.

results:(1) ifn-β could effectively control the tumor growth in a small volume as time lapse, however, adding neutralizing antibody reversed the phenomenon. ifn-β could increase g1/s ratio while did not affect the proportion of β-galactosidase-positive cells. co-localization revealed that ifn-β treatment up-regulated the proportion of cancer stem cells (cd133), but decreased ki67 expression in cancer stem cells. (2) ifn-β could control the growth of b16 or a375 trcs in a reversible way, however, ifn-β had no effect on g1/s ratio or proliferation ability of differentiated b16 and a375 cells. ifn-β treatment increased the g1/s ratio in trcs and decreased glucose consumption, while had no effect on the proportion of β-galactosidase-positive cells. and ifn-β-pretreatment reduced trcs apoptosis in the presence of chemotherapeutic drugs. (3) ifn-β could not effectively inhibit the growth of stat1/stat2 knocking out b16 or a375 trcs, and had no effect on the number of surviving clones. (4) ifn-β treatment could significantly increase the expression of ido1 and kyn in b16 or a375 trcs. and overexpression of ido1 induced b16 or a375 trcs into dormancy as well as up-regulated g1/s ratio. as an agonist of ahr, kyn enhanced the ability of ahr translocating to nuclear and increased the expression of p27. and knocking down p27, ifn-β could not control b16 or a375 trcs clone growth. (5) ifn-β combining with 1-mt or dmf could significantly reduce the number of surviving clones and increase the cell apoptosis, while knocking out stat3 reversed this phenomenon. (6) ifn-β combining with 1-mt or dmf could significantly up-regulate the binding ability of p-stat3 (s, y) to p53, increase the cell apoptosis, control the number of clones, decrease 6pgdh/gpdh ratio and up-regulate ros release, however, knocking out p53 reversed the phenomenon. (7) ifn-β could up-regulate the expression of ido1/ahr/p27 in tumor tissues and enhanced the nuclear transduction of ahr. (8) the combination of ifn-β and 1-mt/dmf could effectively control tumor growth and significantly prolong survival time.

conclusion:ifn-β can directly induce apoptosis of differentiated melanoma cells, and induce trcs dormancy through ido1/ahr pathway. on the one hand, ifn-β can up-regulate p27 via ido1/ahr/kyn pathway to induce trcs dormancy. on the other hand, when blocking ahr pathway, ifn-β induced trcs apoptosis via p-stat3 (s, y)/p53 pathway. combining ifn-β with ahr pathway inhibitors can break the ifn-β-induced dormancy and enhance the apoptosis of trcs, which may open a new venue for cancer immunotherapy.