Transformation Of Lung Microenvironment Counteracts Tumor Hypoxia-enhanced Metastasis Capacity Of Hepatocellular Carcinoma Treated With Interferon α

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.PARTⅠIFNαinhibited HCC growth,however accelerated the metastasis capacityIn 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).PARTⅡDirect transformation of lung micro-environment by IFNαcounteracted growth of lung metastasisBecause 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 sizeIn 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 metastasisWe 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 tumorTo 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).PARTⅢIFNαwithdrawal induced the recovery of tumor growth and lung metastasisSince 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 IFNαwithdrawalIn 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-metImmunohistochemistry 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 metastasisWe 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 IFNαwithdrawalWe 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).Conclusion1.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 work1.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 work1 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.