| BackgroundAblation treatment for early stage NSCLCWorldwide,lung cancer is the malignant tumor with the second incidence rate and the first mortality rate.In 2020,there will be 2.2 million new cases and 1.8 million deaths worldwide.According to histopathological classification,lung cancer can be divided into Non-Small Cell Lung Cancer,NSCLC)and Small Cell Lung Cancer,SCLC),of which NSCLC accounts for about 85%of all lung cancer cases.Early diagnosis of lung cancer,Early treatment can greatly improve the prognosis of patients.Two large-scale experiments,National Lung Cancer Screening（NLST）and Dutch-Belgian Lung Cancer Screening（NELSON）,show that low-dose spiral CT screening can reduce the mortality of lung cancer by 20%.Low-dose CT screening has become an important means for screening high-risk groups of lung cancer,and more and more early lung cancer has been found.The treatment of early stage（stage I and stage IIA with negative lymph nodes）NSCLC mainly depends on surgical resection.For patients who are unsuitable or unwilling to undergo surgery,stereotactic body radiation therapy（SBRT）is the first choice.Image-guided Thermal Ablation has also been recommended by the national comprehensive cancer network（nccn）for early NSCLC that cannot tolerate surgical resection since 2017.But,For inoperable early NSCLC,SBRT recommended priority over ablation.The results of a large number of clinical studies show that although there is no statistical difference in 1-year and 3-year survival rates between SBRT and RFA（68.2-95%vs.81-85.7%;36-87.5%vs.42.7-56%）。However,the 5-year survival rate of ablation（20.1-27%）was significantly lower than that of SBRT（47%）.Therefore,compared with SBRT,the high recurrence rate of ablation is still the bottleneck affecting its clinical application.Compared with SBRT,ablation has the advantages of good repeatability,no radiation,little influence on lung funion,short hospitalization time and low cost.If we can reduce the recurrence rate of ablation and improve the 5-year survival rate,many patients with early NSCLC will benefit.Previous studies have shown that for NSCLC with a diameter less than 3cm,the therapeutic effect of thermal ablation is equivalent to that of SBRT.However,for early stage lung cancer with diameter larger than 3cm or irregular shape,the recurrence rate of thermal ablation increased significantly,and the 5-year survival time decreased significantly.This is because the tumor has not been completely inactivated.What biological changes will happen to tumor cells that are stimulated by heat but not dead?The influence of these changes on prognosis is a problem worth exploring in prolonging ablation of early lung cancer and improving survival time.Total ablation and sublethal thermal injury.In the thermal ablation of primary liver cancer,scholars put forward the concept of Sublethal Heat Damage caused by incomplete ablation.Sublethal thermal injury is defined as 37℃ to 60℃,which can cause some tumor cells to die,while some tumor cells survive,and the surviving cells have undergone a series of biological changes.Cell and animal experiments in hepatocellular carcinoma proved that,After thermal stimulation at one or several temperatures in this temperature range,the surviving cells will undergo epithelial-mesenchymal transition（EMT）,with the enhancement of invasion and migration ability.This may be the key factor for recurrence of liver cancer after ablation.In a study,liver cancer cell lines were exposed to heat,and it was found that tumor cells formed spindle shape,tumor dryness and EMT indexes cd133,ck7 and ck19.The expression of procollagen-α1（Ⅰ）and Snail increased.Animal experiments also showed that the tumor growth induced by transplantation of heat-exposed liver cancer cells into nude mice was faster and more invasive.Tan et al.also got similar results,and at 46℃,it can enhance the invasion and migration ability of hepatoma cell lines.At the same time,the team established an incomplete ablation model of transplanted hepatocellular carcinoma cells in nude mice.It was found that the metastatic ability of residual tumor increased significantly.Yamada S et al.also confirmed that the sub-lethal temperature of 48℃ induced the up-regulation of Ncadherin and the down-regulation of E-cadherin expression in hepatoma cell lines.The above-mentioned series of experiments confirmed that the sublethal temperature can indeed lead to changes in the biological behavior of tumor cells,mainly the up-regulation of EMT phenotype and the increase of invasion and migration ability.Sublethal thermal damage and NSCLC.However,inNSCLC ablation,whether tumor recurrence and metastasis and incomplete ablation lead to increased malignant biological behavior of residual tumor cells has not been reported in the literature.No one has explored the mechanism of how lethal thermal injury leads to malignant transformation of NSCLC cells.Research purposesIn this paper,the necessity of complete ablation is put forward by reviewing clinical data,and then the incomplete ablation of NSCLC（sub-lethal thermal injury）will lead to enhanced invasion and migration ability of residual tumor cells and EMT.Transcriptome sequencing was used to screen differentially expressed genes and signal pathways in NSCLC after sublethal heat injury,and to verify the role of differentially expressed molecules in malignant transformation.Objective To discover the mechanism of malignant transformation caused by incomplete ablation,and to suggest that complete ablation is the key factor to reduce recurrence.Research method1.Thirty-six patients with stage I NSCLC undergoing Microwave ablation,MWA)were analyzed retrospectively,and the relationship between the minimum ablation boundary and local tumor progression,LTP)interval,distant metastasis,DM)interval and disease-free survival,DFS)was analyzed.Patients were divided into two groups:sufficient ablation boundary group and insufficient ablation boundary group.Kaplan-Meier method was used to calculate the survival time of the two groups and log-rank method was used to test.2.Cox proportional hazards model was used to screen 7 factors that may affect the local progression interval and disease-free survival of patients,including gender,age,ECOG scores,pathological classification,T stage,tumor diameter and minimum ablation boundary.The mouse lung cancer xenograft model was established,and the tumor was formed to a certain volume,and then incomplete ablation was performed.Ten days after ablation,the mice were killed,and the tumor was dissected.Hematoxylin-eosin（H-E）staining was used to observe the morphological changes of the cells,and immunohistochemistry（IHC）was used to detect the phenotype expression related to epithelial-mesenchymal transition.3.In vitro,NSCLC cell lines were heated at 37℃-50℃,and trypan blue staining was used to determine the sublethal temperature range in vitro.4.After heat treatment of two NSCLC cell lines at sub-lethal temperature,CCK-8 method and clone formation experiment were used to detect the changes of cell proliferation ability.The changes of cell cycle and apoptosis rate after heat stimulation were detected by flow cytometry.5.Through cell migration test,the effects of sublethal heat stimulation on the migration ability of two kinds of NSCLC cells in vitro were analyzed.6.After heat treatment of two NSCLC cell lines at sub-lethal temperature,the migration ability of the cells was detected by cell scratch test.Transwell chamber test was used to detect the changes of invasion and migration ability.BALB/C Nude nude mice were selected and injected into tail vein to detect the in vivo metastasis ability of tumor cells after sublethal heat stimulation.7.RT-PCR and Western Blot were used to detect the changes of EMT-related phenotypes after heat treatment at sublethal temperature.8.Transcriptome sequencing（RNA-Seq）was used to detect the expression of differential genes in NSCLC cells after heat stimulation,and the differentially expressed genes（CPA4）and signal pathways were screened out.9.Two NSCLC cell lines were stimulated by sublethal heat,and the changes of CPA4 with time were detected by RT-PCR and Western blot.10.Collect NSCLC cases,and detect the expression of CPA4 in tumor tissues and adjacent tissues by IHC.According to the results of immunohistochemical staining,the correlation between CPA4 expression level and clinicopathological features of NSCLC patients was statistically analyzed.The follow-up data of NSCLC patients were collected,the survival curve was drawn by Kaplan-Meier method,and the relationship between the expression of CPA4 and the survival time of patients was analyzed by log-rank test.11.Establishment of stable NSCLC cell lines with high and low expression by transfection.RT-PCR and Western blot were used to detect the expression of CPA4 in normal lung epithelial cell line BEAS-2B and 4 NSCLC cell lines,and the endogenous cell lines with high CPA4 expression and low CPA4 expression were screened out.Selecting cell line H1299 with low expression of CPA4 to transfect CPA4 plasmid to up-regulate CPA4 expression,and establishing cell line with stable overexpression of CPA4 by lentivirus infection;Selecting A549 cell line with high expression of CPA4,transfecting CPA4shRNA to interfere with CPA4 expression,and establishing stable interfering CPA4 cell line by lentivirus infection.12.CCK-8 and plate clone formation experiments were used to detect the effect of CPA4 overexpression and silencing on the proliferation of NSCLC.The cell cycle distribution of NSCLC was detected by flow cytometry after CPA4 was overexpressed and silenced.Cell scratch test and Transwell chamber were used to detect the invasion and migration ability of tumor cells after CPA4 was overexpressed and silenced.13.Select BALB/c Nude nude mice to subcutaneously inject NSCLC cells that have expressed CPA4 and their control cells,and detect the tumorigenicity of NSCLC.Intravenous injection of overexpressed/interfered CPA4 cells to detect metastasis ability of tumor cells in vivo;14.RT-PCR and Western Blot were used to detect the changes of EMT-related phenotype in NSCLC cell lines with stable high expression and low expression.15.After interfering with CPA4,the invasion and migration ability of NSCLC cells were tested by Transwell chamber invasion and migration test,the influence of thermal stimulation on apoptosis of NSCLC was tested by Annexin V-FITC/PI flow cytometry,and the influence of thermal stimulation on the phenotype of EMT related molecules after interfering with CPA4 was tested by Western blot.Research results:1.The minimum ablation boundary is closely related to the prognosis of early NSCLC ablation.The local recurrence-free survival（LTP）and disease-free survival（DFS）of patients with minimum ablation boundary≥5mm were significantly longer than those with minimum ablation boundary<5mm.Univariate and multivariate analysis showed that tumor size（hazard ratio[HR]=1.91,P<0.01;HR=2.41,P=0.01）and minimum ablation boundary（HR=0.13,P<0.01;HR=0.11,P<0.01）was an independent prognostic factor of LTP interval.Meanwhile,the tumor size（HR=1.96,P<0.01;HR=2.35,P<0.01）and the minimum ablation margin（HR=0.17,P<0.01;HR=0.13,P<0.01）is an independent prognostic factor of DFS.In our retrospective analysis,50%（18/36）patients had ablation boundaries less than 5 mm These patients are considered to be at high risk of recurrence.2.In animal experiments,incomplete ablation can lead to increased expression of EMT-related phenotype in residual tumor lesions.The tumor volume in incomplete ablation group was significantly smaller than that in control group.In tHE core area of ablation,he staining showed that the cells were necrotic,most of the cell structures were destroyed,and only the tissue outline,nucleus fragmentation and nuclear pyknosis were found.However,the cells around the core area are spindle-shaped and loose,which may lead to EMT process.Immunohistochemistry detected the molecular phenotype changes related to EMT,Compared with the control group,the expression of E-cadherin,an epithelial related molecule,was significantly decreased,while the expression of N-cadherin and Vimentin,an interstitial related molecule,was up-regulated.3.The survival rate after thermal stimulation decreases with the increase of temperature.According to the results of trypan blue staining,we defined the sublethal temperature range in vitro as 37℃-4℃.4.Sublethal heat stimulation at 46℃ and 48℃ can inhibit proliferation and increase apoptosis of NSCLC cells in vitro.In vitro,CCK-8 and plate cloning experiments showed that sub-lethal heat stimulation at 46℃ and 48℃ could inhibit the proliferation of NSCLC cells,while below 46℃ had no significant effect on the proliferation.Annexin V-FITC/PIflow cytometry showed that 46℃ and 48℃ increased apoptosis,while 42℃ and 44℃ had no significant effect on apoptosis of NSCLC.5.Sub-lethal heat stimulation at 46℃ and 48 ℃ will lead to G0/G1 arrest.Cell cycle test showed that 42℃ and 44℃ had no significant effect on cell cycle of lung cancer cells.However,46℃ and 48℃ caused cell cycle arrest of NSCLC cells in G0/G1 phase.6.Sublethal heat stimulation at 44℃ and 46℃ can enhance the invasion and migration ability of NSCLC cells in vitro.The cell scratch test showed that the migration ability of NSCLC was significantly enhanced after 72h heat stimulation at 44℃ and 46℃.Transwell cell invasion and migration experiments also confirmed that the invasion and migration ability of NSCLC cells was significantly enhanced after 72 hours of heat stimulation at 44℃ and 46℃.7.After heat stimulation at 44℃ and 46℃,the metastasis ability of lung cancer cell lines in vivo was enhanced.The tail injection experiment verified that the number of liver and lung metastases in lung cancer cell mice stimulated by heat at 44℃ and 46℃ was significantly higher than that in the control group.8.In vitro,heat stimulation at 44℃ and 46℃ induced an increase in EMT-related phenotype expression in non-small cell lung cancer.RT-PCR showed that after heat stimulation at 44℃ and 46℃,the expression of transcription factors began to rise at 48 hours,and reached the highest level at 72 hours.Among them,the increase in 44℃ group was the most significant.After heat stimulation at 44℃ and 46℃,western blot showed that the expression of E-caherin protein was significantly down-regulated,while the expression of N-cadherin and Vimentin was significantly up-regulated.At 44℃,the expression of E-caherin decreased with time,while the expression of N-cadherin and Vimentin increased with time.The expression increased most significantly at 72h.9.RNA-Seq showed that CPA4 expression increased after heat stimulation.Transcriptome sequencing showed that CPA4 was significantly differentially expressed（P<0.01）,and EMT-related genes were significantly enriched.RT-PCR and Western Blot verified that heat stimulation at 44℃ could increase CPA4 in a time-dependent manner.After heat stimulation,the expression of CPA4 was up-regulated from 8h,reached its peak at 48h,and began to decrease at 72 h.10.CPA4 is highly expressed in NSCLC and is closely related to poor prognosis.The expression of CPA4 in NSCLC tissues is higher than that in adjacent tissues,and the prognosis of NSCLC patients with high expression of CPA4 is worse.The survival time of patients with positive CPA4 expression was significantly shorter than that of patients with negative CPA4 expression（14.4 months VS.22.2 months）.In addition,the expression of CPA4 was closely related to the degree of tumor differentiation（P=03）,lymph node metastasis（P=0.01）and clinical stage（=0.02）.11.CPA4 promotes proliferation,invasion and migration of NSCLC cells.RT-PCR and Western blot showed that CPA4 was expressed in four NSCLC cell lines（A549,H1299,H1975 and PC9）,among which H1299 was the lowest and A549 was the highest.The ability of proliferation,invasion and migration of H1299 cells was significantly enhanced after overexpression of CPA4.The ability of proliferation,invasion and migration of A549 cells was significantly inhibited after CPA4 interference.12.CPA4 promotes tumor formation and metastasis of NSCLC cells in vivo.The establishment of subcutaneous transplanted tumor model showed that after overexpression of CPA4,the growth rate of tumor was significantly faster than that of the control group,and the tumor volume was larger than that of the control group.The tail injection model showed that after overexpression of CPA4,the number of metastatic foci in lung tissue of nude mice was significantly higher than that of the control group.After interfering with CPA4,the number of lung metastases in nude mice was less than that in control group.13.Overexpression of CPA4 induces EMT in NSCLC cells.H1299 cells overexpressing CPA4 were detected by RT-PCR and Western blot.the expression of E-cadherin in epithelial cells decreased significantly,while the expression of N-cadherin and Vimentin in stroma increased significantly.The expression of E-cadherin in A549 cells after low CPA4 knockdown increased significantly,while the expression of interstitial phenotype N-cadherin and Vimentin decreased.Immunohistochemistry showed that the subcutaneous tumor tissue of H1299 cells expressing CPA4 was significantly lower than that of the control group.The expression of N-cadherin and Vimentin increased.14.Silence of CPA4 can partially reverse the malignant biological behavior and EMT of NSCLC induced by sublethal heat stimulation.Lung cancer cell lines were stimulated by heat at 44℃ after knocking down CPA4.compared with blank NSCLC cell lines,the invasion and migration ability of lung cancer cell lines subjected to heat stimulation after knocking down CPA4 still increased,but the degree of increase was significantly lower than that of lung cancer cells without knocking down CPA4 but subjected to heat stimulation.It is suggested that silencing CPA4 can partially reverse the invasion and migration enhancement caused by sublethal thermal injury.Low CPA4 was knocked out,and then 44 ℃ heat was used to stimulate lung cancer cell lines.the results showed that compared with the blank group without heat stimulation,the expression of E-cadherin decreased significantly,while the expression of N-cadherin and Vimentin increased significantly,while the expression of N-cadherin and Vimentin decreased compared with the heat stimulation group.Therefore,silencing CPA4 can partially reverse EMT caused by sublethal thermal injury.15.Silence of CPA4 can increase apoptosis of A549 and H1975 cells after heat stimulation.With the interference of CPA4,A549 and H1975 cells were more sensitive to heat injury and the apoptosis rate increased.Conclusion1.The ablation boundary is closely related to the prognosis of early NSCLC,and the ablation of early NSCLC must reach a safe ablation boundary,with the minimum boundary not less than 5mm.2.Sublethal thermal stimulation caused by insufficient ablation can promote EMT in NSCLC cells and enhance their metastatic ability.The results of in vitro studies show that two sub-lethal temperatures,44 ℃ and 46℃,are trigger temperature for malignant transformation,and the phenotype induced by 44℃ is more significant.3.Inadequate ablation can induce increased expression of CPA4.CPA4,as an oncogene,plays a key role in malignant transformation of tumor cells caused by sublethal thermal injury.Low CPA4 knockdown can reduce the invasion and migration ability of NSCLC,and overexpression of CPA4 can induce epithelial-mesenchymal transition.4.Patients with tumor overexpressing CPA4 have shorter survival time,and NSCLC cell lines overexpressing CPA4 have stronger proliferation and invasion ability.Silence of CPA4 can partially reverse EMT and enhance invasion and migration ability caused by sublethal heat stimulation.Therefore,whether CPA4 is used as a tumor marker to monitor whether ablation is complete or as an index for follow-up after ablation,Or targeting CPA4 to treat the recurrence caused by incomplete ablation,all of which provide ideas for our future work. |
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