【作者】 陈璐；

【导师】 沈关心； 张智红；

【作者基本信息】 华中科技大学 ， 免疫学， 2020， 博士

【摘要】 【背景与目的】近年来研究发现GRP78可以分泌型蛋白的形式释放到细胞微环境中,课题组前期研究发现分泌型GRP78(sGRP78)通过模式受体调控DC、B细胞等免疫细胞功能,诱导负性免疫调节细胞因子的分泌。本课题研究发现GRP78在肝脏中的表达水平高于其他组织和器官。然而,GRP78是否可以作为肝脏肿瘤细胞定植和转移的调控分子尚不清楚。为了进一步探索GRP78在肿瘤转移中的作用,我们建立了sGRP78高表达细胞株及其实验性肝转移模型小鼠。旨在探讨sGRP78在肿瘤肝转移生态位中的作用,肝脏肿瘤转移能力是否与sGRP78表达相关,肿瘤来源的sGRP78是否可以作用于肝DCs,调节NK活性,促进促转移生态位的形成。数据显示,sGRP78与DC细胞和F4/80+巨噬细胞相互作用,抑制DC活化,促进F4/80+巨噬细胞M2样极化。在肝组织内,sGRP78上调TGF-β的表达。免疫细胞和分泌的TGF-β共同构成一个适应肿瘤生长的耐受微环境。sGRP78在促转移生态位中的作用为靶向GRP78实现肿瘤治疗提供了理论依据。【实验方法】1.真核表达PB-m Cherry-sGRP78质粒的构建及其单克隆E0771-m CherrysGRP78肿瘤细胞株的筛选使用含有CMV启动子驱动的m Cherry或m Cherry-sGRP78编码序列的PB转座子分别转染E0771细胞,并将其命名为E0771-m Cherry细胞和E0771-m Cherry-sGRP78细胞;将m Cherry-sg GRP78、m Cherry或m Cherry-sGRP78编码序列分别转染B16F10细胞,获得B16-m Cherry-sg GRP78、B16-m Cherry和B16-m Cherry-sGRP78细胞。2.Western blot与ELISA检测野生型小鼠组织以及肿瘤细胞GRP78的表达取8周龄C57BL/6小鼠的组织和器官,测量其重量。湿重相同的组织样品在含有蛋白酶抑制剂的NP-40裂解缓冲液(5μl/mg)中裂解获取组织匀浆。分离组织匀浆并将其上清保存在-80℃备用。106细胞接种于平板,在无血清培养基中培养24小时,收集上清,使用RIPA裂解液裂解细胞沉淀。以纯化后的GRP78蛋白作为标准样品,分别采用Bi P抗体western blot检测细胞中GRP78蛋白表达,ELISA检测上清和组织样本中GRP78表达水平。3.划痕法分析sGRP78表达上调对肿瘤细胞增殖以及迁移的影响复苏肿瘤细胞株(E0771、E0771-m Cherry、E0771-m Cherry-sGRP78),细胞状态良好后在6孔板中按照1×104细胞/孔均匀铺肿瘤细胞,并记为第1天,连续7天细胞计数。绘制细胞增殖曲线并与E0771组比较分析肿瘤增殖。6孔板接种4×105个E0771肿瘤细胞,细胞贴壁后0小时划痕。CCD照片记录伤口在0和24小时内的愈合情况,分析肿瘤细胞迁移情况。4.sGRP78对乳腺癌原位瘤生长的影响复苏E0771-m Cherry与E0771-m Cherry-sGRP78肿瘤细胞株,扩大培养,将细胞活率大于95%的肿瘤细胞原位接种C57/BL6鼠(106细胞/只),从第6天开始每两天测量一次肿瘤体积,肿瘤体积大于104 cm3时记小鼠死亡。5.sGRP78表达上调对肿瘤肝转移瘤的影响C57BL/6小鼠腹腔注射10 mg/kg的甲苯噻嗪和100 mg/kg盐酸氯胺酮混合物麻醉。复苏E0771-m Cherry与E0771-m Cherry-sGRP78肿瘤细胞株,扩大培养,在麻醉过程中,使用电热板将小鼠体温维持在37℃。腹部切口暴露小鼠脾脏,将细胞活率大于95%的肿瘤细胞经脾注射1×106 E0771或5×105 B16F10细胞,7分钟后将注射肿瘤细胞的一半脾脏切除,手术缝合。记实验小鼠的生存率。取实验小鼠E0771肿瘤细胞肝转移后第21天和B16F10肿瘤细胞肝转移后第15天的肝脏,称重,并对肝转移负荷进行评估。苏木精和伊红(H&E)染色肝脏病理切片,尼康Ni-E显微镜成像切片。使用NIS-Elements软件获取图像,使用Image J进行分析。转移灶所占面积(mm2)除以肝脏表面总面积(mm2)计算转移负担。6.多色荧光抗体标记流式细胞术检测sGRP78对肝脏APC的激活作用采用颈椎脱位法处死肝转移雌性C57BL/6小鼠(7~8周龄)。肝脏被切成1mm厚的组织块,用胶原酶IV(Worthington)和DNAase II(Sigma)在37℃下消化30分钟。将消化后的肝提取物用70μm细胞滤网过滤,500×g离心5分钟。将得到的细胞重悬于含有100 U/m L肝素的10 m L 35%Percoll中,室温下700×g离心15分钟。3 m L红细胞裂解液重悬收集含白细胞的细胞沉淀,室温孵育3分钟后,用含5%胎牛血清的RPMI 1640洗涤细胞2次。根据制造商的说明,用抗CD45(104),CD146(ME-9F1),CD11b(M1/70),F4/80(BM8),CD11c(N418),MHC II(M5/114.15.2)和CD86(GL-1)荧光标记抗体对肝细胞进行染色。使用可固定活性染料e Fluor506(e Bioscience)评估细胞存活率。使用Cyto FLEX流式细胞仪(Beckman Coulter,USA)分析细胞。流式细胞术数据采用Flow Jo软件(Flow Jo,Ashland,OR,USA)进行分析。7.转盘共聚焦成像检测sGRP78与DCs以及巨噬细胞相互作用取小鼠肝脏组织浸泡在4%多聚甲醛中4℃固定12 h,然后在30%蔗糖溶液中脱水24 h。脱水处理后的肝组织用OCT包埋并使用冰冻切片机切片成20μm厚的组织切片。组织切片在PBS中洗涤三次,Alexa Fluor 647抗小鼠F4/80、Alexa Fluor 647抗小鼠CD11c或Alexa Fluor 647抗小鼠NK1.1抗体按1:200稀释免疫染色。所有切片均使用配备了Ultra VIEW Vo X 3D活细胞成像系统(Perkin Elmer)的Olympus IX83共焦显微镜进行成像。图像分析使用Image J软件(美国国立卫生研究院)。8.活体检测sGRP78对髓源性细胞的募集将1×106个E0771-m Cherry或E0771-m Cherry-sGRP78细胞(第0天)接种于6~10周龄的CX3CR1-GFP C57BL/6小鼠。在第4天和第7天以10mg/kg的甲苯噻嗪和100mg/kg的盐酸氯胺酮混合液麻醉小鼠。麻醉小鼠放入定制的成像盒中,并在成像过程中使用异氟醚(1.5~2%(v/v)异氟醚在氧气中)保持麻醉。在整个成像过程中,小鼠被放置在一个加热垫上以保持体温37℃。使用奥林巴斯IX83共聚焦显微镜活体成像,该显微镜配备了Ultra VIEW Vo X 3D活细胞成像系统(Perkin Elmer)。使用20/0.75 NA的objective和Volocity 6.3(Perkin Elmer)软件获取图像。图像分析使用Image J软件(美国国立卫生研究院)。9.多色荧光抗体标记流式细胞术分析sGRP78重塑肝转移肿瘤微环境细胞构成采用颈椎脱位法处死肝转移雌性C57BL/6小鼠(7-8周龄)。肝脏被切成1mm厚的组合块,用胶原酶IV(Worthington)和DNAase II(Sigma)在37℃下消化30分钟。将消化后的肝提取物用70μm细胞滤网过滤,500×g离心5分钟。将得到的细胞沉淀重悬于含有100 U/m L肝素钠的10 m L 35%Percoll中,室温下700×g离心15分钟。3 mL红细胞裂解液重悬收集的含细胞的细胞沉淀,室温孵育3分钟后,用含5%胎牛血清的RPMI 1640洗涤细胞2次。抗体购自Bio Legend公司。固定活性染料e Fluor506购自e Bioscience公司。根据制造商的说明,用抗CD45(104),CD3(17A2),NK1.1(PK136),CD19(6D5),CD69(H1.2F3),CD4(RM4-4),CD8(53-6.7),Ki-67(11F6),Ly6G(1A8),PD-1(RMP1-14)和Fox P3(MF-14)的荧光标记抗体对肝细胞进行染色。使用可固定活性染料e Fluor506(e Bioscience)评估细胞存活率。随后,使用破膜缓冲液(Biolegend)对细胞进行通透性处理,并使用Ki-67和Fox P3抗体染色。使用Cyto FLEX流式细胞仪(Beckman Coulter,USA)分析细胞。流式细胞术数据采用Flow Jo软件(Flow Jo,Ashland,OR,USA)进行分析。10.ELISA检测试剂盒检测肿瘤微环境中细胞因子的表达收集小鼠在肝转移后第4天和第7天的肝脏并测量其质量。组织样本在含有蛋白酶抑制剂的NP-40裂解缓冲液(5μl/mg)中裂解并获取组织匀浆。分离组织匀浆,并将其上清保存在-80℃备用。使用LEGENDplex TM小鼠细胞因子板阵列(Bio Legend)根据厂家说明书对样品进行检测。数据采用Legendplex软件(Bio Legend)进行分析。采用小鼠TGF-beta ELISA试剂盒(DAKEWE)检测小鼠TGF-β水平。【实验结果】1.GRP78在不同组织以及不同肿瘤细胞中的表达1)ELISA检测结果显示肝脏匀浆上清(SN)中GRP78的水平是脾脏和肺中的两倍,与脑和肾相比虽然没有显著性差异,但仍然表现出更高的表达。这些结果表明组织器官之间GRP78的表达亦存在差异性。2)收集不同肿瘤细胞株的培养上清,浓缩后ELISA检测上清中GRP78浓度。结果表明在报道中具有高增殖与高转移能力的肿瘤细胞株B16以及4T1的sGRP78表达显著高于其他肿瘤细胞株,提示不同肿瘤细胞株GRP78分泌表达的差异可能与其增殖转移能力相关。2.高表达sGRP78肿瘤细胞株的建立。1)为了检测肿瘤细胞分泌sGRP78对肿瘤生长的影响,我们选择sGRP78相对低表达的乳腺癌E0771细胞株,转染构建的PB-m Cherry-sGRP78质粒,根据培养上清中sGRP78的检测水平,筛选出单克隆高GRP78分泌的肿瘤细胞株。2)WB检测显示E0771-m Cherry-sGRP78细胞在100Kb左右有蛋白表达条带,且能被GRP78抗体标记,提示具有融合蛋白m Cherry-sGRP78的表达。ELISA检测发现E0771-m Cherry-sGRP78细胞株分泌的GRP78是单荧光转染E0771-m Cherry细胞株的4倍以上。结果表明E0771-sGRP78-m Cherry肿瘤细胞株具有显著性的sGRP78高表达。3.sGRP78对肿瘤细胞体外生长的影响对筛选的单克隆肿瘤细胞株进行体外培养,检测其侵袭以及增殖能力。划痕实验结果表明sGRP78在肿瘤细胞中高表达可促进肿瘤细胞的侵袭能力。连续7天的细胞计数体外细胞增殖曲线显示,上调的sGRP78不影响E0771细胞的增殖。实验结果表明E0771-m Cherry-sGRP78细胞与E0771-m Cherry对照组细胞比较,sGRP78的上调其迁移能力增强,而增殖能力无差异。4.sGRP78对原位瘤生长的影响在原位接种肿瘤实验中,E0771-m Cherry-sGRP78与E0771-m Cherry细胞组间肿瘤生长曲线没有显著性差异。在接种肿瘤40天内,sGRP78对E0771肿瘤生长的影响并不明显且无小鼠死亡。表明肿瘤来源的sGRP78在活体内影响肿瘤细胞在原位的生长不明显。5.sGRP78对转移瘤生长的影响经脾注射E0771肿瘤细胞20天后,通过测量肝脏重量和组织病理切片评估过表达sGRP78对肝转移的影响。E0771-m Cherry-sGRP78荷瘤小鼠的肝脏重量几乎是E0771-m Cherry荷瘤小鼠肝脏的两倍。同时,病理检测结果证实E0771-m Cherry-sGRP78荷瘤小鼠转移负担更高,这些结果表明肿瘤来源的sGRP78在体内可以促进肿瘤细胞的扩散和定植,加速肝转移。6.sGRP78对DC与巨噬细胞的共定位信息以及功能影响1)采用流式细胞术评估不同的APCs在肝脏中的比例,并定量包括CD86和MHC II在内的细胞活性蛋白表达水平。结果发现肿瘤分泌的sGRP78通过诱导DC和F4/80+巨噬细胞的耐受表型影响肝脏免疫微环境,但不影响LSEC的活化。2)对实验性肝转移小鼠模型的肝组织切片进行免疫荧光染色,以评估sGRP78结合CD11c+DCs和F4/80+巨噬细胞的能力。F4/80+巨噬细胞的密度在E0771-mCherry-sGRP78荷瘤小鼠的转移病灶中是E0771-mCherry荷瘤小鼠中的1.3倍。在此基础上,m Cherry阳性F4/80+巨噬细胞的密度在E0771-m Cherry-sGRP78荷瘤小鼠的转移病灶中是E0771-m Cherry荷瘤小鼠的3倍,同时m Cherry阳性CD11c+DCs的密度是E0771-m Cherry荷瘤小鼠的5.4倍。7.GRP78对髓源性DC以及巨噬细胞的CX3CR1+细胞运动行为模式的影响1)在CX3CR1-GFP肝转移小鼠模型中发现肿瘤分泌的sGRP78被F4/80+CX3CR1+和F4/80-CX3CR1+细胞摄取。与CX3CR1-F4/80+细胞相比,m Cherry阳性CX3CR1+F4/80-和CX3CR1+F4/80+细胞分别高了5倍和3.5倍。这些结果证实,CX3CR1-GFP转基因小鼠是一种有前途的用于分析DC和巨噬细胞的运动行为的动物模型。2)肝脏活体成像数据表明,在转移病灶中,肿瘤分泌GRP78水平与CX3CR1+细胞迁移水平之间存在关联,特别是在癌细胞定殖的早期阶段。接种肿瘤细胞4天后,在sGRP78的作用下荷瘤小鼠体内CX3CR1+细胞的运动能力明显提高。7天后,sGRP78对CX3CR1+细胞活力的影响不明显,说明sGRP78通过招募CX3CR1+细胞,在转移前的生态位建立过程中可促进早期的免疫耐受。8.sGRP78抑制NK细胞的募集与激活通过流式细胞术测试肝转移过程中sGRP78过表达对NK细胞和其他效应淋巴细胞的影响,包括自然杀伤性T(NKT)细胞、B细胞、CD4+和CD8+T细胞以及巨噬细胞和嗜中性粒细胞。结果表明,sGRP78的表达对T细胞,B细胞和其他淋巴细胞的影响不明显,而NK细胞的募集与激活显著的受sGRP78表达抑制。9.sGRP78调控细胞因子的分泌表达1)多因子检测试剂盒检测E0771-m Cherry与E0771-m Cherry-sGRP78肿瘤细胞肝转移第4天以及第7天肝脏组织中的细胞因子表达,多因子检测试剂盒检测结果表明,高表达sGRP78的荷瘤小鼠肝脏在第4天和第7天均显示出与对照小鼠相比更高的IL-10水平。而在IFN-γ,TNF-α,IL-2或IL-6的水平上未观察到差异。2)ELISA检测结果提示sGRP78高表达可诱导肝脏中TGF-β表达上调,其中第4天E0771-m Cherry-sGRP78荷瘤小鼠肝脏的TGF-β表达水平是E0771-m Cherry荷瘤小鼠的1.12倍。当到达第7天TGF-β的表达虽然统计学没有显著差异,但具有上调趋势。【结论】为了研究sGRP78对肝转移前生态位建立的影响,本课题首先检测了不同组织器官和不同肿瘤细胞GRP78的表达,结果表明GRP78在肝脏中的表达水平显著高于其他组织器官,提示其可能在促进肿瘤细胞定植中发挥作用。对包括6种癌症类型的肿瘤细胞在线数据对比和对其分泌上清中的sGRP78表达检测,结果显示sGRP78的表达水平在不同肿瘤细胞间存在差异,且与其恶性程度相关,在报道中具有高转移与侵袭能力的B16以及4T1肿瘤细胞检测结果显示其分泌型GRP78高表达。E0771乳腺癌细胞株相对于B16细胞系恶性程度低,检测结果显示其GRP78表达水平低于B16细胞。为了阐明sGRP78在肝转移前生态位中的作用,建立了过表达sGRP78的鼠乳腺癌细胞株及其实验性肝转移模型。研究发现,肝脏中sGRP78的高表达可促进免疫耐受,肿瘤分泌的sGRP78通过募集CX3CR1+髓样细胞来重塑肝微环境。sGRP78通过下调MHC II抑制DC激活,并诱导F4/80+巨噬细胞M2型极化。此外,sGRP78通过调节细胞因子的产生影响肝转移灶中NK细胞的浸润和活化。更多还原

【Abstract】 【Background】 The liver is an immunologically tolerant organ and a common site of distant metastasis for various cancers.The liver is rich in NK cells,which can be activated by DC cells and cytokines as the guardian of the liver.The expression levels of glucose-regulated protein 78（GRP78）have been associated with tumor malignancy.GRP78 has recently been described to be released into extracellular microenvironment as having immunomodulatory properties.We previously demonstrated that secretory GRP78（sGRP78）released from tumor cells contributes to the establishment of an immunosuppressive tumor microenvironment by regulating cytokine production in macrophages and dendritic cells（DCs）.However,the role of sGRP78 on tumor cell colonization and metastasis in the liver remains unclear.Herein,we found that GRP78 was expressed at higher levels in the liver compared to other tissues and organs.We performed intravital imaging using an sGRP78-overexpressing breast cancer cell line（E0771）and found that sGRP78 interacted with DCs and F4/80+ macrophages in the liver.Importantly,sGRP78 overexpression inhibited DC activation and induced M2-like polarization in F4/80+ macrophages.Moreover,sGRP78 overexpression enhanced TGF-β production in the liver.In conclusion,sGRP78 promotes tumor cell colonization in the liver by remodeling the tumor microenvironment and promoting immune tolerance.The ability of sGRP78-targeting strategies to prevent or treat liver metastasis should be further examined.【Methods】 1.The eukaryotic expression of PB-m Cherry-sGRP78 plasmid was constructed and monoclonal E0771-m Cherry-sGRP78 tumor cell line was screened E0771 cells were stably transfected with the PB transposon system,which contained a CMV promoter-driven m Cherry or m Cherry-sGRP78（GRP78 Gene Bank No: NM₀01163434.1）coding sequence,and named as E0771-m Cherry/E0771-m Cherry-sGRP78 cells.B16F10 cells were stably transfected with the PB transposon system,which contained the m Cherry-sg GRP78,m Cherry or m Cherry-sGRP78 coding sequence（B16-m Cherry-sg GRP78,B16-m Cherry and B16-m Cherry-sGRP78 cells）.2.Detection of GRP78 expression in wild-type mouse tissues and tumor cells Tissues and organs of C57BL/6 mice at 8 weeks were harvested and their mass was measured.Tissue samples with the same wet weight were lysed in NP-40 lysis buffer（5 μL/mg）containing a protease inhibitor cocktail（Sigma-Aldrich）.Lysates were separated and stored at-80 °C until further use.1 × 106 cells were seeded in the plates and cultured in serum-free culture media for 24 h.The supernatants were collected and the cell pellets were lysed with RIPA lysis buffer.Then cell pellets were assayed by western blot using the Bi P（C50B12）Rabbit m Ab（CST）.Supernatants and tissue samples were assayed by ELISA using the Bi P（C50B12）Rabbit m Ab（CST）.The purified GRP78 protein was used as the standard sample.3.The effect of sGRP78 up-regulation on tumor cell proliferation and migration E0771 tumor cell lines（E0771/E0771-m Cherry/E0771-m Cherry-sGRP78）were resuscitated and expanded.The 6-well plates were seeded with 104 E0771 tumor cells on day 0,and then the cells were counted for 7 consecutive days.Data were analyzed by Welch’s ANOVA（versus E0771 group）.The 6-well plates were seeded with 4 × 105 E0771 tumor cells.After the cells adhere to the wall,the wound was scratched as the 0 hour.And CCD photographs record the wound healing at 0 and 24 hours.4.The effect of sGRP78 on the growth of breast cancer in situ The E0771-m Cherry and E0771-m Cherry-sGRP78 tumor cell lines were resuscitated and expanded.C57BL/6 mice（106 cells/mouse）were inoculated in situ with tumor cells with cell viability greater than 95%.The tumor volume was measured every two days from the 6th day.The mouse is considered dead when the tumor volume is larger than 104 cm3.5.The effect of sGRP78 up-regulation on liver metastases of breast cancer The mice were anesthetized by intraperitoneal（i.p.）injection of a mixture of 10 kg xylazine and 100 mg/kg ketamine hydrochloride（Sigma,St.Louis,Missouri,USA）.During anesthesia,body temperature was maintained at 37°C using a warm plate.Mouse spleens were exposed by a small upper abdomen incision,followed by injection of 1 × 106 E0771 or 5 × 105 B16F10 cells.Seven minutes later,half of the spleen containing the tumor cell injection site was resected.The hepatic metastatic burden was assessed on day 21 for E0771 and day 15 for B16F10 after tumor cell inoculation.Hematoxylin and eosin（H&E）stain was purchased from Servicebio Biotechnology（Wuhan,China）and slides were scanned using a Nikon Ni-E.Images were acquired using the NIS-Elements software and analyzed using Image J.The metastatic burden was calculated by dividing the area occupied by metastatic foci（mm2）by the total surface liver area（mm2）.6.Detection of sGRP78 activation of liver APC cells Female C57BL/6 mice（7-8 weeks old）were sacrificed by cervical dislocation.The liver was dissected into 1 mm pieces and digested using collagenase IV（Worthington）and DNAase II（Sigma）for 30 minutes at 37°C.The digested liver extracts were filtered through a 70 μm cell strainer and centrifuged at 500 × g for 5 minutes.The resulting cell pellet was resuspended in 10 m L 35% Percoll containing 100 U/m L heparin and centrifuged at 700 × g for 15 minutes at room temperature.The cell pellet containing leukocytes was collected and resuspended in 3 m L red blood cell lysis solution（155 mmol/L NH4 CL,10 mmol/L KHCO3,1 mmol/L EDTA,170 mmol/L Tris;p H 7.3）.After incubation for 3 minutes at room temperature,cells were washed twice with RPMI 1640 containing 5% FBS.Antibodies against CD45（104）,CD146（ME-9F1）,CD11b（M1/70）,F4/80（BM8）,CD11c（N418）,MHC II（M5/114.15.2）and CD86（GL-1）were purchased from Bio Legend.The fixable viability dye e Fluor506 was purchased from e Bioscience.Liver cell suspensions were subjected to surface staining with fluorescently labeled antibodies according to the manufacturer’s instructions.Cell viability was assessed using the fixable viability dye e Fluor506（e Bioscience）.Cells were analyzed on a Cyto FLEX flow cytometer（Beckman Coulter,USA）.Flow cytometry data were analyzed using Flow Jo software（Flow Jo,Ashland,OR,USA）.7.Interaction detection of sGRP78 with DCs and macrophages For the immunofluorescence analysis,liver tissues were fixed in 4% paraformaldehyde for 12 h at 4 °C and then dehydrated in 30% sucrose solution.The tissues were then frozen in OCT compound and sectioned into 20 μm slices using a freezing microtome.OCT was removed by washing three times in PBS,and the sections were immunostained with Alexa Fluor 647 anti-mouse F4/80,Alexa Fluor 647 anti-mouse CD11 c or Alexa Fluor 647 anti-mouse NK1.1 at 1:200 dilution.All the sections were imaged with Olympus IX83 confocal microscope outfitted with an Ultra VIEW Vo X 3D live cell imaging system（Perkin Elmer）.Images were analyzed with Image J software（National Institutes of Health）.9.In vivo detection of recruitment of myeloid cells by sGRP78 CX3CR1-GFP C57BL/6 mice（6-10 weeks old）were inoculated with 1 × 106 E0771-m Cherry or E0771-m Cherry-sGRP78 cells（day 0）.On days 4 and 7,mice were anesthetized by i.p.injection of a mixture of 10 mg/kg xylazine and 100 mg/kg ketamine hydrochloride（Sigma,St.Louis,Missouri,USA）.Mice were maintained anesthesia with isoflurane inhalation [1.5 to 2%（v/v）isoflurane in O2] and placed within a custom-designed imaging box.Throughout the imaging process,mice were placed on a heating pad to maintain a body temperature of 37°C（Thermo Plate,TOKAI HIT,Shizuoka-ken,Japan）.Intravital imaging was performed using an Olympus IX83 confocal microscope outfitted with an Ultra VIEW Vo X 3D live cell imaging system（Perkin Elmer）.Images were acquired using a 20×/0.75 NA objective and Volocity 6.3（Perkin Elmer）software.Images were analyzed with Image J software（National Institutes of Health）.10.GRP78 remodels the cellular structure of liver metastasis tumor microenvironment Female C57BL/6 mice（7-8 weeks old）were sacrificed by cervical dislocation.The liver was dissected into 1 mm pieces and digested using collagenase IV（Worthington）and DNAase II（Sigma）for 30 minutes at 37°C.The digested liver extracts were filtered through a 70 μm cell strainer and centrifuged at 500 × g for 5 minutes.The resulting cell pellet was resuspended in 10 m L 35% Percoll containing 100 U/m L heparin and centrifuged at 700 × g for 15 minutes at room temperature.The cell pellet containing leukocytes was collected and resuspended in 3 m L red blood cell lysis solution（155 mmol/L NH4 CL,10 mmol/L KHCO3,1 mmol/L EDTA,170 mmol/L Tris;p H 7.3）.After incubation for 3 minutes at room temperature,cells were washed twice with RPMI 1640 containing 5% FBS.Antibodies against CD45（104）,CD3（17A2）,NK1.1（PK136）,CD19（6D5）,CD69（H1.2F3）,CD4（RM4-4）,CD8（53-6.7）,Ki-67（11F6）,Ly6G（1A8）,PD-1（RMP1-14）,and Fox P3（MF-14）were purchased from Bio Legend.The fixable viability dye e Fluor506 was purchased from e Bioscience.Liver cell suspensions were subjected to surface staining with fluorescently labeled antibodies according to the manufacturer’s instructions.Cell viability was assessed using the fixable viability dye e Fluor506（e Bioscience）.Subsequently,cells were permeabilized using the Transcription Factor Buffer Set（Biolegend）and stained for Ki-67,Fox P3.Cells were analyzed on a Cyto FLEX flow cytometer（Beckman Coulter,USA）.Flow cytometry data were analyzed using Flow Jo software（Flow Jo,Ashland,OR,USA）.11.GRP78 changes the expression of cytokines in the tumor microenvironment Livers were harvested and their mass was measured at days 4 and 7 after injection.Tissue samples were lysed in NP-40 lysis buffer（5 μL/mg）containing a protease inhibitor cocktail（Sigma-Aldrich）.Lysates were separated and stored at-80°C until further use.Samples were assayed using the LEGENDplex TM Mouse Cytokine Panel array（Bio Legend）according to the manufacturer’s instructions.Data were analyzed with Legendplex software（Bio Legend）.Mouse TGF-beta ELISA kit（DAKEWE）was used to detect TGF-β levels in the samples.【Results】 1.GRP78 is routinely expressed in normal cells and tumor cells.1)ELISA result suggested that the level of GRP78 in the supernatant（SN）of liver homogenate was two times higher than in the NS from spleen and lung,and on significant with Higher brain and kidney.Those suggested that highly expressed GRP78 provides a basis for tumor liver metastasis 2)Higher sGRP78 expression was found in tumor cells with stronger proliferation and higher metastasis,such as B16 and 4T1.This suggested that sGRP78 maight be related to the proliferation and metastasis ability of tumor cells.2.Obtain high expression tumor cell lines.1)E0771 cells were selected for liver metastasis experiments because of the low expression of GRP78.E0771 cells were stably transfected with the PB transposon system,which contained the m Cherry or m Cherry-sGRP78 coding sequence（E0771-m Cherry/E0771-m Cherry-sGRP78 cells）.2)Wasten blot result displayed that E0771-m Cherry-sGRP78 cells have a protein band labeled with GRP78 antibody at about 100 Kb,suggesting that m Cherry-sGRP78 was successfully expressed.ELISA assay indicated that the level of sGRP78 in the supernatant of E0771-m Cherry-sGRP78 cells was four times higher than that in E0771-m Cherry or E0771 control cells.3.The effect of sGRP78 on the growth of tumor cells in vitro Cell proliferation assay revealed that overexpression of sGRP78 did not affect the proliferation of E0771 cells though the cells were counted for 7 consecutive day in vitro.To investigate the effect of sGRP78 overexpression on cancer cell migration,we performed a scratch wound healing assay.E0771-m Cherry-sGRP78 cells were more migrative than E0771-m Cherry control cells.Those results indicated that Tumor secreted GRP78 affected invasion rather proliferation.4.The effect of sGRP78 on the growth of tumor in situ In the tumor in situ experiment,there was no significant difference in tumor growth curve between E0771-m Cherry-sGRP78 and E0771-m Cherry cell groups.The effect of sGRP78 on the growth of E0771 tumor cells was not obvious within 40 days of tumor inoculation,and no mice died.It shows that sGRP78 derived from tumor does not affect the growth of tumor cells in vivo.5.The effect of sGRP78 on the growth of metastatic tumors Twenty days after splenic injection of tumor cells,we evaluated the effect of sGRP78 overexpression on liver metastasis by measuring the weight of the liver and histology.Liver weight was higher nearly by a two-fold in E0771-m CherrysGRP78-bearing mice compared with E0771-m Cherry-bearing mice.Histology confirmed the higher metastatic burden in E0771-m Cherry-sGRP78-bearing mice.These findings suggest that tumor-derived sGRP78 promotes tumor cell dissemination and colonization in vivo,accelerating liver metastasis.6.GRP78 affects the co-localization information and function of DC and macrophages 1)Immunofluorescence staining on liver sections from our experimental liver metastasis mouse model was performed to evaluate the ability of sGRP78 to bind to CD11c+ DCs and F4/80+ macrophages.Although the density of F4/80+ macrophages in metastatic lesions was higher by a 1.3-fold in E0771-m Cherry-sGRP78-bearing mice compared with control mice,the numbers of m Cherry-positive CD11c+ DCs and F4/80+ macrophages were considerably higher in E0771-m Cherry-sGRP78-bearing mice（5.4-fold for CD11c+ cells and 3-fold for F4/80+ cells）.These findings suggest that DCs and macrophages directly bind sGRP78.2)We performed flow cytometry evaluating the frequencies of different hepatic APCs,as well as quantifying the expression levels of CD86 and MHC II.These results suggest that tumor-secreted GRP78 affects the hepatic immune microenvironment by inducing tolerogenic phenotypes in DC and F4/80+ macrophages,but not in LSEC.7.The role of GRP78 to characterize the motor behavior of myeloid CX3CR1+ cells of DC and macrophages 1)In our CX3CR1-GFP liver metastasis mouse model,tumor-secreted GRP78 was taken up by both F4/80+CX3CR1+ and F4/80-CX3CR1+ cells.Compared to CX3CR1-F4/80+ cells,the numbers of m Cherry-positive CX3CR1+F4/80-and CX3CR1+F4/80+ cells were 5 times and 3.5 times higher,respectively.These results confirm that CX3CR1 transgenic mice are a promising model for analyzing the behavior of DCs and macrophages during the establishment of the pre-metastatic niche in the liver.2)Imaging data suggest an association between the level of tumor-secreted GRP78 and the level of CX3CR1+ cell migration in metastatic lesions,particularly during the early stages of cancer cell colonization.Four days after tumor cell inoculation,the motility of CX3CR1+ cells in the E0771-m Cherry-sGRP78-bearing mice was significantly increased under the action of GRP78.The effect of sGRP78 on CX3CR1+ cell motility was less profound seven days after cell inoculation,suggesting that sGRP78 promotes immune tolerance early during pre-metastatic niche establishment by recruiting CX3CR1+ cells.8.GRP78 inhibits the recruitment and activation of NK cells We tested the effects of sGRP78 overexpression on NK cells and other effector lymphocytes,including natural killer T（NKT）cells,B cells,CD4+,and CD8+ T cells,as well as on macrophages and neutrophils,during liver metastasis by Flow cytometry.These results suggest that T cells,B cells,and other lymphocytes were not influenced by sGRP78.And tumor-secreted GRP78 facilitates tumor metastasis by inhibiting NK activity.9.GRP78 regulates the secretory expression of cytokines 1)The effects of sGRP78 on the recruitment and activation of immune cells was reflected on cytokines secretion.Compared with control mice,sGRP78-expressing mice exhibited higher levels of IL-10 at both time points.Nevertheless,no differences were observed on the levels of IFN-γ,TNF-α,IL-2,or IL-6.2)ELISA detection found that TGF-β levels were increased by 1.12-fold in sGRP78-expressing mice compared with control mice;however,TGF-β upregulation was not observed before day 7.【Conclusion】 In order to study the effect of sGRP78 on the establishment of liver pro-metastatic niche,we tested the expression of GRP78 in different organs and tumor cells.The results showed that the expression level of GRP78 in livers is significantly higher than other tissues and organs,suggesting that GRP78 might play a role in reshaping the liver microenvironment and promoting liver metastasis.The online data and the secretory GRP78 expression in their supernatants of tumors including 6 cancer types showed that the expression level of GRP78 varies among different tumor cells and was related to the degree of malignancy.Higher sGRP78 expression was found in tumor cells with stronger proliferation and higher metastasis,such as B16 and 4T1.The E0771 breast cancer cell line was less malignant and the lower GRP78 expression level than that of B16 cell;To elucidate the role of sGRP78 in the hepatic pre-metastatic niche,we established an experimental liver metastasis model using sGRP78-overexpressing murine breast cancer cells.Results suggested that higher expression of GRP78 in the liver can promote immune tolerance.We found that tumor-secreted GRP78 reshaping the hepatic microenvironment by recruiting CX3CR1+ myeloid cells.sGRP78 inhibited DC activation by downregulating MHC II,as well as induced F4/80+ M2-type polarization.Furthermore,sGRP78 affected NK cell infiltration and activation in hepatic metastatic lesions by regulating cytokine production.更多还原