摘要
肝细胞癌(HCC)是全球常见第六位肿瘤,并位于肿瘤死因的第三位,虽然手术仍为主要的治疗手段,但是术后高复发率成为提高患者总体生存的主要障碍,而抗肿瘤血管生成等靶向治疗有望成为防治肝癌复发转移的新手段。针对单一分子或单一通路的靶向治疗似乎成为目前解决肿瘤问题的法宝,但先天性和获得性耐药仍是阻碍分子靶向治疗的难题。此外,昂贵的价格也使绝大多数肝癌患者无缘承受。分子靶向药物的研发前景究竟是多个分子靶向药物的联合,还是发展多靶点的药物,也是尚未解决的问题。
干扰素α(IFNα)作为一个多用途的细胞因子,不仅用于治疗肝癌的背景疾病-慢性肝炎,还可直接抑制肿瘤细胞增殖、抑制肿瘤血管生成,我所的裸鼠实验以及临床随机对照研究中明确证实IFNα具备抑制肝癌生长,延缓术后复发的作用,已成为预防肝癌术后复发的常规治疗方案之一。但是临床研究发现IFNα停药后患者的复发率以及肺转移率增加,提示IFNα治疗过程中,肝癌细胞可能发生某些生物学的改变,以适应在IFNα的环境中生存。因此有必要研究:(1)IFNα长期治疗和停药对肝癌生长和转移等特性的影响;(2)IFNα治疗对肝癌细胞生物学的影响;(3)抗血管生成作用作为IFNα的主要作用机制,其在抑制肿瘤生长的同时是否诱导肿瘤缺氧,以及上调侵袭转移相关因子,继而增加肿瘤的侵袭性?
本课题是在原有工作基础上,通过我所建立的具备100%肺转移率的高转移人肝癌原位移植裸鼠模型模拟临床治疗模式,围绕上述问题进行系统探索。
第一部分
长期应用干扰素α对肝癌的生长和侵袭转移的影响
本部分应用我所建立的高转移潜能人肝癌原位移植裸鼠模型HCCLM3模拟临床治疗模式,采用形态学观察方法观察长期IFNα治疗(持续12w)对肝癌生长和肺转移的影响;应用血管生成因子的PCR芯片,RT-PCR,western-blotting和免疫组化(IHC)分析以及验证IFNα治疗过程中主要时间点对肝癌血管生成因子,肿瘤缺氧的影响;应用体外细胞学实验如肿瘤细胞的增殖,迁移以及侵袭试验等方法来研究IFNα对肝癌细胞侵袭转移的直接影响;运用流式细胞学技术检测外周血稳定转染红色荧光蛋白(RFP)的循环肝癌细胞。
1.IFNα能够显著抑制肝癌生长,提高总体生存。
12w的IFNα治疗能够显著抑制肝癌生长,IFNα治疗第2w起,治疗组和对照组(给予生理盐水,NS)的肿瘤大小开始有显著性差异,分别为1231.13±212.37 mm~3vs.2544.59±369.48 mm~3(P=0.0216),而且这种差异持续存在,提示长期使用IFNα治疗不会发生治疗失败的现象。长期IFNα能够显著改善裸鼠总体生存,持续IFNα治疗组平均生存时间为89.2±0.6天,明显长于持续NS组(58.3±9.6天)(logrank,P=-0.0067),在整个疗程,IFNα治疗并未引起任何显著的体重减轻。
2.IFNα主要通过抗血管生成作用抑制肝癌生长
为进一步研究长期IFNα治疗延缓肝癌生长的机制,我们设计了第二阶段裸鼠动物实验,设立治疗组和对照组,每组6只,设立4个观察时间点,治疗时间开始于建立模型后1w,分别为治疗1w,3w,4w,6w。研究提示肝癌生长变化和肿瘤组织的PECAM(经PCR芯片验证)和CD31(经IHC证实)的变化一致;提示肿瘤血管生成还是肝癌生长的重要基础。通过PCR array检测,和同期的对照组相比,在瘤龄第4w,IFNα能够显著下调血管生成相关因子,其中以IL-1B和STAB1的下降为最明显(下调倍数分别为3.0和2.95),其次为PDGFA(2.07),IL-6(1.73),VEGF-A(1.45),其余多数血管生成因子也呈下调,同期IFNα组的PECAM被下调2.05倍(PCR芯片),CD31密度(MVD,CD31阳性面积占总面积百分比)也被大大下调,治疗组和对照组的CD31密度分别为5.3%±0.6%vs.10.2%±0.4%(P=0.0022),提示此时IFNα能够显著下调肝癌的血管生成。此外,体外实验证实,IFNα在100 U/ml有明显的抑制HUVEC增殖的影响。表明,IFNα能够直接或间接抑制血管内皮细胞,可能和减少肿瘤细胞生长的血管生成因子有关。
3.长疗程IFNα治疗可加重肿瘤缺氧,上调侵袭转移相关因子IFNα治疗5w后,肿瘤缺氧程度较对照组开始加重,至第7w时显著加重;应用外源性缺氧探针pimonidazole进行IHC检测肿瘤缺氧情况,发现瘤龄第7w两组pimonidazole的平均IOD分别为39.8%±6.1%vs.23.0%±2.5%(P=0.030)。通过PCR芯片验证,与相同时间点对照组比较,从肿瘤生长第5w开始,IFNα治疗组的部分血管生成因子RNA水平开始上调;至第7w时,多数因子显著上调,包括c-met(上调倍数为1.75),PDGF-A(3.28),UPA(5.12),IL-8(7.26)。通过IHC以及Western blotting进一步证实了上述因子的组织和蛋白水平变化。推测这些侵袭转移因子的上调可能和治疗诱导肿瘤缺氧环境有关。
4.IFNα对肝癌细胞本身无明显直接作用
体外IFNα治疗(即对HCCLM3细胞的直接作用)是否能够直接影响肿瘤细胞侵袭转移的能力,以及上调上述侵袭转移相关因子;体外增殖实验结果表明,多种浓度的IFNα无法抑制LM3细胞增殖;划痕实验提示多种浓度的IFNα未能抑制LM3细胞的迁徙;侵袭实验提示体外IFNα未能抑制LM3的侵袭穿膜能力;IFNα体外作用并不上调各种侵袭转移相关因子;因此提示IFNα体外对LM3细胞的直接作用并不能影响肝癌细胞的侵袭转移能力,而体内治疗过程中,肿瘤环境的改变如肿瘤缺氧可能是肿瘤侵袭转移能力改变的主要原因。
5.IFNα治疗相对增加外周血循环肿瘤细胞。
进一步评估IFNα所诱导的肿瘤缺氧以及上调的转移相关因子是否引起原发瘤侵袭性的改变,通过流式细胞仪检测两组外周血中的循环肿瘤细胞(RFP-LM3,CTC),两组没有显著性差异(0.075%±0.020%vs.0.063%±0.018%,P=0.574),但通过肿瘤大小标化后,发现治疗组的CTC有高于对照组的趋势(0.068%±0.022%vs.0.016%±0.006%,P=0.040),提示长期IFNα治疗过程中,相对增加了肝癌细胞出血管的过程,增加了原发瘤的侵袭转移能力。进一步研究发现,在治疗6w时,两组肿瘤的增殖水平(Ki-67)无显著性差异,治疗组和对照组分别为48.60%±3.1%vs.49.40%±2.9%(P=0.962),提示IFNα治疗对CTC的诱导和对原发瘤增殖的影响无关。
第二部分
IFNα治疗抑制肝癌肺转移的相关机制
前一部分研究发现IFNα治疗能够抑制肝癌生长,但增加了肝癌细胞的侵袭性;而肺转移作为肝癌侵袭转移的重要特性,提示我们需要进一步评估IFNα治疗对肝癌肺转移的影响,虽然IFNα治疗相对增加了外周血循环肿瘤细胞,但是肿瘤转移是个多步骤的过程,肿瘤细胞入血只是众多转移步骤之一,肿瘤细胞在继发部位的生长是个低效率事件,可能决定了肺转移最终的生长和转移,其中文献报道肺组织MMP-9以及巨噬细胞是肺转移的重要影响因素,因此有必要进一步评估IFNα治疗对肺转移的影响,以及相关的分子机制。本部分通过采用稳定转染RFP的HCCLM3细胞建立肝癌的原位移植模型,获取新鲜肺组织,通过荧光成像显微镜观察肺组织中的转移灶(转移灶为标记RFP的肿瘤组织),并通过H&E染色进行确定;同时通过免疫组化以及RT-PCR检测肺组织中转移灶的血管生成因子,以及肺组织MMP-9表达和巨噬细胞的浸润。同时采用无瘤裸鼠动物模型,以及IFNα预处理3周后尾静脉注射RFPHCCLM3细胞试验组,进一步探讨IFNα对肺环境的改造作用。
1 IFNα治疗能够显著抑制肺转移的数目和大小
采用RFPHCCLM3原位肝癌模型,IFNα治疗剂量和方案同第一部分,于治疗6w结束后,获取两组的新鲜肺组织,通过荧光成像显微镜观察肺组织中的转移灶(转移灶为标记RFP的肿瘤组织),结果提示,虽然IFNα治疗组的肺转移率和对照组无显著性差异(5/6,83%vs.6/6,100%,P=1.000),但是治疗组的肺转移灶数目(1.75±1.0 vs.28.0±6.3,P=0.008)和大小(IOD:116.8±72.2 vs.和5226.4±1355.7,P=0.020)显著低于对照组,提示IFNα治疗能够抑制肺转移灶的发展。
2.IFNα治疗不抑制肺转移灶的肿瘤血管生成。
进一步探究IFNα抑制肺转移灶发展的分子机制,我们采用人源特异性引物,利用RT-PCR检测肝癌肺转移灶主要的血管生成因子mRNA表达水平;结果发现IFNα治疗组肺转移灶中VEGF-A(2.88±0.30 vs.0.02±0.01;P=0.011)、PDGF-A(3.40±0.22 vs.0.54±0.19;P=0.000)和IL-6(0.08±0.02 vs.0.02±0.01;P=0.014)的相对表达水平均显著高于对照组。提示IFNα治疗并非通过抑制转移灶的血管生成而抑制肺转移灶的生长。
3.IFNα治疗可降低裸鼠肺组织中MMP-9表达和巨噬细胞数目MMP-9是肺转移的重要因子;应用IHC检测发现治疗组肺组织中的MMP-9表达水平显著低于对照组(5.1%±1.7%vs.21.9%±0.4%,P＜0.000);进一步应用鼠源特异性引物进行RT-PCR验证,发现治疗组MMP-9的mRNA水平较对照组下调5.0倍(P=0.034)。肺部巨噬细胞数和肺内的MMP-9表达有关,可由其直接产生或与肺血管内皮细胞共同相互作用而产生;采用抗巨噬细胞抗原F4/80进行肺组织染色,结果表明,IFNα治疗组的肺巨噬细胞数目显著低于对照组,巨噬细胞密度分别为0.20%±0.04%vs.1.36%±0.21%(P=0.0058)。提示IFNα治疗可能通过下调肺内的巨噬细胞和MMP-9水平,而发挥抑制肿瘤细胞在肺内生长和转移的作用。
为进一步确定IFNα对肺内MMP-9和巨噬细胞浸润的作用是否和原发瘤的存在有关,选用无瘤裸鼠重复上述实验。与对照组相比,治疗组肺内巨噬细胞(0.12%±0.03%vs.1.13%±0.04%,P=0.0001)和MMP-9(3.8%±1.2%vs.20.8%±0.3%,P=0.0038)均显著下调.这表明IFNα能够直接抑制肺内的巨噬细胞浸润和MMP-9表达,和原发瘤的存在无关。
4.IFNα预处理能够抑制实验性肺转移
为消除循环肿瘤细胞的生物学特性对肺转移的影响,直接研究IFNα对肺组织的改造作用对肝癌肺转移的影响。两组裸鼠分别给予IFNα和生理盐水处理3w后,每组裸鼠通过尾静脉注射相同数量的RFPHCCLM3细胞,然后均给予NS治疗持续6w,评估肺转移情况。两组的肺转移发生率无显著性差异(分别为4/5与5/5);但IFNα预处理组的肺转移灶数目和大小显著减少,肺转移灶数目分别为11.8±4.2 vs.46.8±15.3(P=0.021),肺转移灶大小[IOD]分别为2489.8±838.1和12803.3±4016.1(P=0.007)。进一步研究发现IFNα预处理组的MMP-9(19.0%±0.2%vs.34.9%±0.1%,P=0.001)和巨噬细胞浸润(1.10%±0.00%vs.1.68%±0.00%,P=0.001)均显著低于对照组;IFNα预处理组的肺MMP-9的mRNA含量较对照组组下调1.9倍(P=0.032)。
第三部分
IFNα停药后对肝癌生长和肺转移的影响
已有研究报道IFN停药后会导致其他肿瘤如黑色素肿瘤侵袭性的增加;我所之前的临床试验发现,IFNα停药后会增加患者术后复发率和肺转移率;因此有必要评估IFNα停药对肝癌生长和转移的影响以及相关的分子机制。本研究通过我所建立的高转移潜能人肝癌原位移植裸鼠模型RFPHCCLM3模拟临床停药模式,采用形态学观察IFNα治疗停药后肝癌生长和肺转移;运用血管生成因子相关的PCR芯片,RT-PCR检测IFNα停药后肿瘤缺氧水平以及主要血管生成因子的变化;获取新鲜的肺组织,利用荧光显微镜检测稳定转染RFP的肺转移灶;采用RT-PCR以及IHC研究并验证IFNα停药后肺组织中MMP-9以及肺巨噬细胞的变化。
1.IFNα治疗后停药,肝癌恢复生长
IFNα治疗3w后停药(减少肝癌平台期生长的影响),肝癌重新恢复生长,显著高于继续长期治疗组;提示IFNα停药后,肝癌恢复生长。IFNα使用3w后停药组中位总体生存时间为56.2±10.5天,显著低于持续IFNα治疗组的89.2±0.6天(log-rank,P=0.011),提示IFNα治疗后停药后延缓肿瘤生长的作用逐渐消失,同时降低总体生存,提示停药的潜在危险性以及长期治疗的必要性。
2.IFNα停药对原发瘤生物学的影响
应用pimonidazole探针研究发现,IFNα停药后,肿瘤组织缺氧水平高于继续用药组(50.8%±1.3%vs.39.8%±6.1%,P=0.05)。提示IFNα停药后肿瘤恢复生长可能与肿瘤缺氧进一步加重有关;和长期用药组相比,血管生成因子PCR芯片提示IFNα停药组的IL-6显著上调2.1倍;而体外实验以及RT-PCR试验提示,多种浓度的IFNα未能直接上调HCCLM3细胞中IL-6的表达情况,之前的动物实验也发现IL-6是肝癌生长过程中和缺氧显著相关的血管生成因子,因此提示IFNα停药后加重的肿瘤缺氧可能上调了IL-6,从而促进了停药后肝癌的重新生长。
3.IFNα治疗后停药,肺转移恢复生长
通过评估肺转移灶,我们发现停药组的肺转移灶的数目和大小也远远大于持续用药组(转移灶数目:17.2±3.8 vs.1.75±1.0,P=0.011;大小[IOD]:1483.2±598.1vs.116.8±72.2;P=0.014);但两组间的CTC数目并无显著差异(0.050%±0.010%vs.0.075%±0.020%,p=0.237);RT-PCR检测发现停药组肺转移灶的主要血管生成因子(VEGF-A,PDGF-A,IL-6)的RNA水平并无显著高于长期治疗组(三者分别为0.03±0.04 vs.2.88±0.30,P=0.025;0.04±0.02 vs 3.40±0.22,P=0.004;0.02±0.02 vs 0.08±0.02,P=0.0007,2-~(ΔCT))。提示肺转移灶的血管生成并非是IFNα停药后肺转移恢复的主要原因。
4.IFNα停药上调肺组织中巨噬细胞浸润和MMP-9
IHC检测发现,停药组(治疗3w,停药3w)中肺组织的巨噬细胞和MMP-9均显著高于长期治疗组,巨噬细胞密度和MMP-9的IOD分别为16.5%±1.2%(P=0.0007),0.79%±0.13%(P=0.013),且RT-PCR发现停药组的MMP-9的RNA水平显著高于对照组(上调2.4倍,P=0.038),同时在无瘤裸鼠中,同样方法检测肺部巨噬细胞和MMP-9的表达,发现停药组两者的表达均高于持续用药组,巨噬细胞密度和MMP-9的IOD分别为(0.72%±0.03%vs.0.12%±0.03%,P=0.013)和MMP-9(14.1%±1.2%vs 3.8%±1.2%,P=0.0007),提示IFNα对肺组织的直接改造作用可随停药而逐渐减弱,为肺转移营造土壤。
结论
1长期IFNα治疗能够持续抑制肝癌生长,提高总体生存,无治疗失败的现象
2 IFNα治疗虽然能够减少肺转移灶的数目和大小,但是不减少循环肿瘤细胞和肺转移率。
3 IFNα治疗能进一步加重肿瘤缺氧,通过上调一系列缺氧相关的血管生成因子,增加原发瘤的侵袭性
4 IFNα能够直接改造肺组织,降低肺内巨噬细胞浸润和MMP-9水平,可能是其抑制肺转移灶生长和转移的重要原因。
5 IFNα治疗停药后,以IL-6为主因子的上调促进肝癌生长;肺组织改造工作的减弱,恢复了肺转移的生长。
应用价值
1证实了长期IFNα治疗肝癌的可行性,谨慎停药。
2提示了IFNα治疗在肝癌术后预防或者延缓肺转移的可行性;提示可尝试治疗伴肺转移的无手术指征的肝癌患者。
3 IL-6可能是预防IFNα停药后肿瘤生长的重要靶点。
创新点
1首次揭示IFNα治疗晚期能够诱导更严重的肿瘤缺氧,且增加了肿瘤的侵袭性。
2首次揭示IFNα能够直接改造肺组织,阻碍肺转移灶的生长。
3首次提出IL-6是IFNα停药后肿瘤恢复生长的重要因子。
Hepatocellular carcinoma(HCC) is the sixth most common cancer and the third most common cause of death from cancer worldwide.Patient survival has been improved with recent advances in diagnostic and therapeutic modalities in those with resectable HCC.However,a considerable number of patients with HCC with multiple lung metastasis are not candidates for surgery and systemic chemotherapy and,thus,have an extremely poor prognosis.Moreover,lung metastasis is also a frequent site of extrahepatic recurrence after hepatectomy,which remains the major obstacle for further improving the survival of patients with HCC after surgical treatment. Therefore,potential agents are in great demand to prevent and treat lung metastasis of HCC.
IFNαhas a variety of biologic properties,including antiviral,immunomodulatory, antiproliferative,and antiangiogenic effects.Previous studies showed that IFNαexerted its inhibitory effect on HCC growth mainly through antiangiogenesis by down-regulation of VEGF-A.Several recent studies have also reported the survival benefits of IFNαmonotherapy or IFNα-based combination therapy for advanced HCC with extrahepatic lung metastasis or tumor thrombi in the major trunk.Such therapies are beneficial also in patients with HCC after hepatectomy or ablation by postponing or decreasing tumor recurrence,including lung metastasis,which mainly rely on the growth inhibition of residual tumor cells in remnant liver.However,the biologic responses of the tumor to IFNαmonotherapy are not clear.In a clinical study, we found that IFNαwithdrawal usually resulted in a higher incidence of tumor recurrence or metastasis to the lung,even though tumor recurrence was significantly decreased in patients kept on the treatment.These results suggest that tumor cells,as the seeds of recurrence and metastasis,may survive beyond the IFNαtreatment by acquiring additional molecular and biologic changes as responses to the pressure of IFNαtreatment,which may be associated with the tumor hypoxia induced by the impaired tumor angiogenesis.
At the secondary metastatic organ,the interaction between tumor cells and the lung microenvironment may be the key factor in determining the fate of lung metastasis. Recent studies have recognized that macrophages and MMP-9 play a critical role in the growth of metastatic lesions in lung tissue.However,the impact of IFNαtreatment on the tumor microenvironment has not been studied,especially the interaction between metastatic tumor cells and the lung microenvironment in long-term IFNαtreatment,which may be responsible for the remarkable suppression of lung metastasis by IFNαtreatment.
Here,using an orthotopic implantation HCC-LM3 model of human HCC with a 100% incidence of lung metastasis,we found that up to 12 wk of IFNαtreatment persistently retarded tumor growth without inducing resistance and improved survival. Interestingly,although IFNαtreatment might enhance tumor metastasis by inducing severe tumor hypoxia and increasing circulating tumor cells(CTCs),it directly modulated the lung microenvironment by reducing macrophage infiltration and MMP-9 expression,which made it resistant to the growth of disseminated HCC cells and inhibited the lung metastasis of HCC.
PARTⅠ
IFNαinhibited HCC growth,however accelerated the metastasis capacity
In this part of research,using an orthotopic implantation HCC-LM3 model of human HCC with a 100%incidence of lung metastasis,we conducted the IFNαtreatment for 12 weeks to investigated the impact on the tumor growth and lung metastasis;using the angiogenesis PCR array,real-time PCR,western-blotting and immunohistochemistry staining,we studied the time-course expression of angiogenesis factors and tumor hypoxia;fluorescence microscopy was used to detect the lung metastasis and flow cytometry was used to detect the peripheral circulating tumor cells.
1 IFNαinhibited HCC growth and improved survival The IFNαtreatment started at the end of the first week(1 wk) after implantation (average tumor volume,100 mm~3) and lasted for 12 wk.IFNαtreatment significantly inhibited tumor growth during the entire treatment course,but did not induce any significant loss of body weight.Moreover,IFNαtreatment significantly improved the survival of nude mice models(median survival time,12 wk) compared with the controls(10 wk,log-rank,P=0.0067).
2 IFNαinhibited HCC growth by anti-angiogenesis effect.
we performed another experiment with the same treatment schedule as described above and harvested HCC tissues after 1,3,4,and 6 wk of treatment(the tumor ages were 2,4,5,and 7 wk,respectively).VEGF-A expression detected by PCR array was reduced by 1.53-fold at 4 wk of tumor age;several other angiogenesis factors were also remarkably reduced,which included IL-1B(reduced by 3.0-fold),STAB1(by 2.95-fold),PDGFA(by 2.07-fold),and IL-6(by 1.73-fold).PECAM(reduced by 2.05-fold measured by PCR array,Figure 1A) and CD31 density(5.3%±0.6%versus 10.2%±0.4%,P=0.0022) was also significantly reduced at the 4-wk tumor age. Moreover,IFNαat≥100 U/mL had an obvious inhibitory effect on HUVEC proliferation in vitro.These results suggested that IFNαinhibited HCC growth by anti-angiogenesis effect.
3 IFNαtreatment induced more severe tumor hypoxia and up-regulation of metastasis-related genes.
At the 7wk tumor age,more severe tumor hypoxia was induced in the IFNα-treated group compared with the controls(measured by immunohistochemistry staining of pimonidazole,mean integrated optical density[IOD]:39.8%±6.1%versus 23.0%±2.5%,P=0.030).Compared with HCC tissues of the controls,several metastasis-related genes in the IFNαtreated group were up-regulated by＞1.5-fold, including HIF-1α(increased by 1.78-fold),c-met(by 1.75-fold),u-PA(by 5.12-fold), PDGF-A(by 3.28-fold),and IL-8(by 7.26-fold).VEGF-A was also up-regulated by a marginal 1.45-fold;Figure 5A).The protein levels of these genes were also confirmed by western blotting and immunohistochemical assay.These findings indicated that IFNαtreatment induced more severe tumor hypoxia and up-regulation of metastasis-related genes at a later stage of tumor age.
4 IFNαhad no direct effect on migration and invasion of HCC cells.
Both wound healing and transwell invasion assays studies showed that IFNαtreatment at doses of 100,1000,5000,20,000,and 100,000 U/mL for 24 or 48 h did not have any significant promoting effect on the migration and invasion of HCC-LM3 cells(Figure 3B,C).Moreover,no significant difference in the mRNA expression level of MMP-9,c-met,IL-8 detected by real-time RT-PCR was found between IFNα-treated and control HCC cells at both time points.
5 IFNαrelatively increased the circulating tumor cells.
When normalized by tumor size,the number of CTCs in the IFNα-treated mice models(0.068%±0.022%) was much higher than that in the controls(0.016%±0.006%,P=0.040).These results suggested that IFN-αtreatment at least did not reduce the tumor cell dissemination from primary tumor.Moreover,no significant difference in the Ki-67 expression level was found between IFNα-treated and control groups(48.60%±3.1%versus 49.40%±2.9%,P=0.962).
PARTⅡ
Direct transformation of lung micro-environment by IFNαcounteracted growth of lung metastasis
Because of the similar incidence of lung metastasis may be due to the similar arrest of CTC in lung tissue,early after the CTC arrested in the lung tissue,tumor angiogenesis in lung metastasis foci may do contribution to its progression as well as the environment they encountered in the lung tissue.We tested this hypothesis by examining the tumor angiogenesis in lung metastasis by RT-PCR and expression of MMP-9 and its associated macophages by immunohistochemistry staining in lung tissue which may participated in lung metastasis.
1 IFNαinhibited lung metastasis number and size
In the orthotopical HCC model using HCC-LM3 cells transfected with red-fluorescent protein(RFP) gene,we detected the metastatic lesions of HCC in the lung tissues harvested at 7 wk of tumor age under fluorescence microscopy.Both the number and size of the lung metastatic lesions of the IFNα-treated mice were much smaller than those of the controls(number:1.75±1.0 versus 28.0±6.3,P=0.008;metastasis size [pixels]:116.8±72.2 versus 5226.4±1355.7,P=0.020).
2 IFNαtreatment had no significant inhibiton on tumor angiogenesis in the lung metastasis
We used the human-specific primers to detect the expression of several angiogenesis factors that are prominently reduced in primary tumor,such as VEGF-A,PDGF-A, and IL-6 in the lung tissue hosting metastatic loci,and found a higher expression of these factors in lung metastatic foci in the IFNα-treated group compared with the controls(2.88±0.30 versus 0.02±0.01,P=0.011 for VEGF-A;3.40±0.22 versus 0.54±0.19,P=0.000 for PDGF-A;0.08±0.02 versus 0.02±0.01,P=0.014 for IL-6).These indicated that the tumor cells in the metastatic foci were not sensitive to IFNαtreatment.
3 IFNαinhibited macrophage infiltration and MMP-9 expression in the lung tissues.
The expression of MMP-9,one of the key players involved in tumor metastases and "premetastatic niche",was examined using immunohistochemistry.The results showed that MMP-9 expression in the lung tissues was much lower in the IFNαtreated mice compared with the untreated mice(mean IOD:5.1%±1.7%versus 21.9%±0.4%,P＜0.000);Real-time PCR using the mouse-specific primer also confirmed the lower MMP-9 RNA level in the lung tissue in the IFNα-treated mice (5.0-fold lower than the untreated mice,P=0.034).Macrophage infiltration in the lung tissue may be responsible for MMP-9 expression.The results showed that the number of macrophages in IFNαtreated mice was significantly lower compared with that in the untreated mice(macrophages density:0.20%±0.04%versus 1.36%±0.21%,P= 0.0058).
4 IFNαinhibited macrophage infiltration and MMP-9 expression in the lung tissues independent of primary tumor
To ascertain whether IFNαhad direct impact on expression of MMP-9 and macrophage infiltration,mice without tumor were treated with IFNα(1.5×10~7 U/kg/d, n=5) and NS(n=5) for 6 wk.Compared with NS treated mice,both macrophages (0.12%±0.03%versus 1.13%±0.04%,P=0.0001) and MMP-9(3.8%±1.2% versus 20.8%±0.3%,P=0.0038) were significantly reduced in IFNαtreated mice, which suggested that macrophages and MMP-9 in the lung were directly affected by IFNαirrespective of the presence of the primary tumor.
5 Pretreatment with IFNαinhibited experimental lung metastasis.
After pretreatment by IFNα(1.5×10~7 U/kg/d) or NS for 3 wk,mice(5/group) received a tail vein injection of RFP-LM3 cells(1.0×10~6).Both groups then received NS for another 6 wk.We found the incidence of lung metastasis in IFNα-pretreated mice was similar compared with the NS-pretreated mice(4/5 versus 5/5);however, the number and size of metastatic foci were remarkably smaller in IFNα-pretreated mice(number:11.8±4.2 versus 46.8±15.3,P=0.021;size[pixels]:2489.8±838.1 versus 12803.3±4016.1,P=0.007 for IFNαand NS pretreated groups,respectively). Next,the immunohistochemistry study showed that less MMP-9(19.0%±0.2% versus 34.9%±0.1%,P=0.001) and macrophage infiltration(1.10%±0.00%versus 1.68%±0.00%,P=0.001) in IFNα-pretreated group,so was the MMP-9 RNA level in the lung(1.9-fold lower than NS-pretreated group,P=0.032).
PARTⅢ
IFNαwithdrawal induced the recovery of tumor growth and lung metastasis
Since IFNαadministration led to the relative much higher hypoxia level in later phase of tumor age as compared the control group,thus we conduct the drug withdrawal as to ascertain whether IFNαwithdrawal had some impact on the tumor hypoxia and pertinent hypoxia induced factors and related tumor behavior.
1 Recovery of tumor growth after IFNαwithdrawal
In the aforementioned orthotopic HCC model,the withdrawal group of 6 mice was treated by IFNα(1.5×10~7 U/kg/d) for 3 wk(stopped at 4 wk of tumor age) and followed by NS for another 3 wk,whereas the other 6 mice were received IFNα(1.5×10~7 U/kg/d) for a continuous 6 wk.In withdrawal group,tumor quickly resumed the dynamic growth.Furthermore,IFNαwithdraw could also reduce overall survival in nude mice,which had much lower than the IFNαgroup(9w versus 12w long-rank, P=0.011).
2 IFNαwithdrawal upregulated tumor hypoxia however not HIF-1αand c-met
Immunohistochemistry staining of exogenous hypoxia probe(Pimonidazole) showed that much higher tumor hypoxia level in withdrawal group than continuous administration of IFNαgroup(50.8%±1.3%versus 39.8%±6.1%,P=0.05),however the main hypoxia induced factors such as HIF-1αand c-met were not up-regulated by IFNαwithdrawal,which was confirmed by RT-PCR and immunohistochemistry staining,which indicated that HIF-1αand c-met may not participated in the signal pathway regulated by withdrawal induced hypoxia.
3 IL-6 was associated with up-regulation of tumor hypoxia by IFNαwithdrawal Once IFNαwithdrawal,with the up-regulation of IL-6 level was also up-regulated confirmed by RT-PCR,with an increase as 2.1 when compared with continuous IFNαadministration;For IL-6 was the important factor participating in the tumor angiogenesis associated with tumor hypoxia in vivo,however in vitro assay detected by RT-PCR,IL-6 showed no difference between IFNαgroup and control group, suggesting IL-6 up-regulation may be associated with withdrawal upregulated tumor hypoxia and contributed to the resumption of tumor growth.
4 IFNαwithdrawal resume lung metastasis
We found that IFNαwithdrawal resulted in an increased mumber and size of lung metastases compared with continuous IFNαtreatment for 6 wk(number:17.2±3.8 versus 1.75±1.0,P=0.011;size[pixels]:1483.2±598.1 versus 116.8±72.2;P= 0.014).However,the number of CTCs was comparable between the continuous treatment group and withdrawal group(0.050%±0.010%versus 0.075%±0.020%,P =0.237).Next,tumor angiogenesis indicated by mRNA expression of VEGF-A, PDGF-A,and IL-6 in the lung detected by RT-PCR using the human-specific primers, were still much less in the IFN-αwithdrawal group than the continuous group which indicated that the recovery of lung metastasis may not be associated with tumor angiogenesis in the lung.
5 Recovery of macrophages,and MMP-9 in the lung after IFNαwithdrawal
We found that MMP-9 expression and macrophage infiltration in the lung tissue in the IFNαwithdrawal group was higher compared with that in the continuous IFN-αgroup (immunohistochemistry staining,MMP-9 expression,16.5%±1.2%,P=0.0007; macrophage,0.79%±0.13%,P=0.013).Real-time PCR using the mouse-specific primer also detected an increased MMP-9 RNA level derived from lung tissue in the IFNαwithdrawal group compared with the continuous IFNαgroup(2.40-fold higher, P=0.038).
Conclusion
1.IFNαtreatment could persistently retard HCC growth and suppress lung metastasis.
2.IFNαinhibited growth of lung metastasis,however not the incidence of lung metastasis and the circulating tumor cells.
3.IFNαtreatment accelerated tumor hypoxia related metastasis capacity of hepatocellular carcinoma.
4.IFNαinhibited macrophage infiltration and MMP-9 expression in the lung tissue independent of primary tumor.
5.IL-6 upregulation was responsible for recovery of tumor growth after IFNαwithdrawal;reversary of MMP-9 and macrophages infiltration in lung was responsible for the lung metastasis recovery.
The potential application of the work
1.Long-term IFNαadministration could be consistently effective without therapy failure,and the drug withdrawal was not advisable for its induced recovery of tumor growth and lung metastasis.
2.IFNαadministration could be applicated in treatment for inoperable HCC patients with multiple lung metastasis and prevention on lung metastasis in patients after hepatecomy
3.IL-6 was the potential therapy target for patients with IFNαwithdrawal.
The novelty of the work
1 Analysed and confirmed,for the first time,that IFNαcould induce the severe tumor hypoxia and its enhanced tumor metastasis behavior,and it could transform the lung microenvironment independent of primary tumor which was hostile for the sencondary metastasis,therefore,IFNαcould counteract the hypoxia-enhanced invasion phenotype by its self-rescuing ability in lung metastasis suppression.
2 IL-6 was the prominent factor for the growth recovery after IFNαwithdrawal.
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