| BackgroundAt the diagnosis or during the development of colorectal cancer(CRC),liver metastasis will occur in about 50%-60%of patients,and the local treatment of liver metastases has been the major factor of prognosis.As just 15%-20%of patients with colorectal liver metastasis(CRLM)has the opportunity to accept surgical resection,radiofrequency ablation(RFA)becomes the favoured replacement therapy for patients with unresectable CRLM,and has been widely utilized in clinic.A prospective study reports that compared with chemotherapy alone,RFA combined with chemotherapy can significantly prolong the overall survival of CRLM patients.As affected by tumor size(>5 cm),shape(irregular)and position(closed to large vessels or located in the caudate lobe),commonly it is difficult for RFA to completely ablated all the tumor cells of liver metastases,leading to incomplete radiofrequency ablation.Although incomplete ablation reduces the tumor burden,the residual tumor cells are the culprits of intrahepatic recurrence and extrahepatic metastasis,severely damaging the prognosis of patients.Currently,the studies on the specific mechanism of how incomplete ablation promotes the extrahepatic metastasis of CRLM are still in their infancy.Effectively supressing tumor metastasis after incomplete ablation still challenges the clinic,and becomes the key for improving prognosis.In this study,we construct the CRLM mouse model treated with incomplete ablation to explore the effect of incomplete ablation on the occurrence of extrahepatic metastasis.Combined with sequencing analysis,molecular biology experiments and in-vitro cellular and in-vivo animal studies,we deeply investigate the specific mechanism.Therefore,strong evidence from preclinical study will be provided for optimizing RFA treatment.Hypothesis1.Incomplete ablation of the colorectal liver metastases promotes the occurrence of extrahepatic metastasis.2.Incomplete ablation induces lipid metabolism reprogramming in residual tumor cells.3.PPAR signalling pathway participates in the regulation of lipid metabolism reprogramming in residual tumor cells.4.Lipid metabolism reprogramming affects the malignant biological behaviors of residual tumor cells mediated by the relevant signalling pathway.Methods and Materials1.The effect of incomplete ablation of CRLM on lung metastasisFirst,we construct the CRLM mouse(BALB/c and C57BL/6)models by CT26-Luc/eGFP and MC38 hepatic capsule implantation,or CT26 subcutaneous tumor capsule transplantation.Seven days after implantation or transplantation,we perform RFA on liver tumors,and the mice are classified into sham-operated,complete ablation(the ablation edge is about 1-2 mm outside the tumor edge with no visible tumor tissues remained)and incomplete ablation(part of tumor tissues are still remained on the edge)groups.The in vivo imaging system is utilized for dynamically observation.And 14 d after RFA treatment,the lung tissues are collected for photographing,weighting and HE staining to evaluate the extent of lung metastasis in different treated mice.We construct the bilaterally tumor-implanted(left lobe,CT26/CT26-Luc/eGFP;right lobe:CT26-mCherry)mouse model,and perform incomplete ablation treatment on left/right liver tumor.Immunofluorescent(GFP and mCherry)and immunochemistry(mCherry)staining are performed on the lung tissues collected on days 1,3,5 and 7 after RFA to ensure the cell components of lung metastases and evaluate the time point of the residual tumor cells for lung colonization.Furtherly,we perform hepatectomy on days 1 and 3 after incomplete ablation,and then observe the effect on lung metastasis.Finally,we construct the in vitro heat stressed tumor cells-45℃-CT26.The migration and invasion potentials of tumor cells are evaluated by Transwell migration and metastasis assays,respectively.2.The lipid metabolism changes in the residual tumor cells after incomplete ablationFirst,the CT26 tumor tissues from the sham-operated group and the residual CT26 tumor tissues from the incomplete ablation group are collected on day 1 after RFA for transcriptome sequencing and single cell RNA sequencing,which finds that lipid metabolism changes related to PPAR signalling pathway occur in the residual tumor cells.Then,lipid metabonomics is conducted in the in vitro 37℃-CT26 and 45℃-CT26 cells to ensure the level changes in major components of lipid metabolism,and the levels of fatty acid(FA)and triglyceride(TG)in the tumor cells and tissues are determined by quantitative measurement,tissue oil red O staining and Nile red cell staining,respectively.Finally,oxygen consumption rate(OCR)and fatty acid oxidation(FAO)rate are both tested to ensure the levels of FAO in different treated CT26 and DLD1 cells.3.The effect of PPAR signalling pathway on the lipid metabolism of the residual tumor cellsFirst,the three subunits of PPAR,PPARα,PPARβ/δ and PPARγ,are selected by qPCR assay,PPAR responsive element(PPRE)combining assay and western blot.And combined with the western blot performed on the tumor tissues collected from the sham-operated and incomplete ablation groups,we determine the activation of PPARγ in the residual tumor cells.Then,the target genes regulated by PPARγ are confirmed by qPCR assay and western blot.Finally,the levels of FA and TG in tumor cells treated by PPARγ interference or PPARγ inhibitor are determined by quantitative measurement and Nile red cell staining to explore the regulation of PPARγ on the lipid metabolism of the residual tumor cells.4.The effect of lipid metabolism changes on the malignant biological behaviors of residual tumor cellsFirst,the proliferation and invasion potentials of different treated CT26 and DLD1 cells are evaluated by EdU and Transwell assays.Then,we perform western blot and analyze the sequencing data to select the related signalling pathways,and confirm the activation of P38 MAPK pathway.With the treatment of PPAR inhibitor and TG inhibitor,we explore the effect of lipid metabolism changes on the protein expression of p-P38.And the effect of P38 MAPK signalling pathway on the proliferation and migration potentials of CT26 and DLD1 cells is determined with the treatment of P38 inhibitors.Furtherly,after treated with P38 inhibitors,the nuclear expression of PPARγ and the protein expression of PPARγ target genes in CT26 and DLD1 cells are detected by western blot.And through FA quantification measurement and Nile red cell staining,we study whether P38 MAPK signalling pathway will affect the expression and function of PPARγ.Finally,we treat the CRLM mice with P38 inhibitor-Ralimetinib to explore the effect of P38 MAPK pathway on lung metastasis of CRLM mice after incomplete ablation.Results1.Incomplete ablation of CRLM promotes lung metastasisIn the CRLM mice(CT26-Luc/eGFP/MC38 capsule implantation or CT26 subcutaneous tumor capsule transplantation)with sham-operation or incompleteablation,we found that on day 14 after RFA,although the liver tumor burden was reduced by incomplete ablation,the mice in the incomplete ablation group showed the severer lung metastasis compared with the sham-operated group.And as the liver tumors were all ablated by complete ablation,there was no visible lung metastasis in the mice treated with complete ablation.For the bilateral tumor-implanted mouse model,when we performed incomplete ablation on the left lobe tumor(CT26-Luc/eGFP),only the GFP positive tumor cells could be observed in the lung tissues on days 3 and 5 after RFA;while when we performed incomplete ablation on the right lobe tumor(CT26-mCherry),only the mCherry positive tumor cells could be observed in the lung tissues.Also,in another bilateral tumor-implanted mouse model(left lobe,CT26;right lobe,CT26-mCherry),we found that lung metastases were both formed in the two groups on day 7 after RFA,and for incomplete ablation of left lobe,the lung metastases were without mCherry expression;while for incomplete ablation of right lobe,the lung metastases were mCherry positive.Finally,we found that there was no lung metastasis for the mice with hepatectomy on day 1 after incomplete ablation,while for the mice with hepatectomy on day 3 after incomplete ablation,lung metastases were observed,but the numbers were less than the mice without hepatectomy.The results from Trans well assay showed that compared with 37℃-CT26 cells,the 45℃-CT26 cells presented the enhanced migration and invasion potentials.2.Lipid metabolism reprogramming occurs in the residual tumor cells after incomplete ablationTranscriptome sequencing showed that the upregulation of PPAR signalling pathway was most significant in the residual tumor tissues after incomplete ablation,and there were 14 differential expressed genes between the sham-operated and incomplete ablation groups,most of which were related to lipid metabolism.Single cell RNA sequencing further confirmed that the PPAR signalling pathway related genes were significantly enriched in the residual tumor cells.Lipid metabonomics found that the level of TG was most significantly increased in the 45℃-CT26 cells,and the content of oleic acid(OA)and myristic acid in TG were most abundant.Then we tested the levels of FA and TG in tissues,finding that the level of TG was highest in the residual tumor tissues,and necrotic ablation area presented the increased level of FA.The oil red O staining showed that compared with the sham-operated group,the lipid level of the residual tumor tissues was obviously increased,and the lipids were mainly distributed in the residual tumor tissues on the ablation edge.Heat stress and/or OA were used to treat the CT26 and DLD1 cells in vitro,and then the levels of FA and TG in tumor cells were tested.It was found that the levels of FA and TG in 45℃+OA-CT26/DLD1 cells were higher than that of 37℃-CT26/DLD1,45℃-CT26/DLD1,and 37℃+OA-CT26/DLD1 cells.And the FA uptake rate of 45℃+OA-CT26/DLD1 was also the highest.The OCR of different treated cells were detected by Seahorse assay.The results showed that compared with 37℃-CT26/DLD1 cells,the basal respiration,maximal respiration,spare respiratory capacity and ATP production of 37℃+OA-CT26/DLD1 cells were significantly increased,while were decreased in 45℃-CT26/DLD1 cells.After combined with the treatment of OA,the basal respiration,maximal respiration,spare respiratory capacity and ATP production of 37℃+OA-CT26/DLD1 cells showed no significant difference compared with 37℃-CT26/DLD1 cells.The same changes as the OCR were shown in the four groups in FAO rate test.3.Lipid accumulation in the residual tumor cells is induced by the activation of PPARγAccording to the qPCR,the mRNA expressions of PPARa and PPARγ were higher in 45℃+OA-CT26/DLD1 cells compared with the other three groups,while the expression of PPARβ/δ showed no significant difference.The results of PPRE combining assay showed that the combining abilities of PPARa and PPARγ were both increased in 45℃+OA-CT26/DLD1 cells,and PPARγ were the highest.It was further found by western blot that PPARγ was mainly expressed in nuclear,and with the highest nuclear expression in 45℃+OA-CT26/DLD1 cells.Confirmed by the tissue western blot,the nuclear expression of PPARγ in the residual tumor tissues was indeed much higher than that in the sham-operated group.Then,we selected the PPARγ target genes by qPCR,and found that the expression levels of CD36 and FABP4/5 were most significantly increased in 45℃-OA-CT26/DLD1 cells.Western blot also showed the upregulated protein expressions of CD36 and FABP4/5.With the treatment of PPARγ interference or PPARγ inhibitor,it was found that when the expression and function of PPARγ were inhibited,the mRNA and protein expressions of CD3 6 and FABP4/5 were both decreased,and the levels of FA and TG were also reduced.The results of EdU and Transwell assays showed that the proliferation and invasion potentials of 45℃+OA-CT26/DLD1 cells were enhanced compared with the other three groups.While with the treatment of PPARγ and TG inhibitors,the proliferation and invasion of CT26 and DLD1 cells were both inhibited.4.The activation of PPARγ-TG-P38 MAPK-PPARγ positive feedback pathway facilitates tumor metastasisThe results of western blot showed that the expression level of p-P38 was significantly increased in 45℃+OA-CT26/DLD1 cells compared with the other three groups,while the expression levels of p-mTOR,p-SAPK/JNK,p-ERK1/2,p-PI3K and p-AKT showed no significant differences.The tissues western blot further confirmed that the expression of p-P38 in the residual tumor tissues was higher than that in the sham-operated group.Combined with the GSEA analysis of single cells RNA sequencing,we found that the MAPK signalling pathway related genes were significantly enriched in the residual tumor cells.After treated by PPARγ and TG inhibitors,or PPARγ interference,the expression level of p-P38 was reduced.The results of EdU and Transwell showed that the proliferation and invasion of CT26 and DLD1 cells were inhibited by the P38 inhibitors.In addition,after treated with P38 inhibitors,the protein expressions of PPARγ,CD36 and FABP4/5 were all decreased,and the levels of FA and TG were also reduced.Finally,the CRLM mice after sham-operation or incomplete ablation were treated with PBS and P38 inhibitor-Ralimetinib,respectively.It was found that compared with the sham-operated groups with PBS or Ralimetinib treatment,the incomplete ablation group with PBS treatment showed severer lung metastasis,while after combined with Ralimetinib,the lung metastasis was significantly inhibited.Conclusion1.Incomplete ablation of CRLM promotes the occurrence of lung metastasis,and the residual tumor cells are the major origin of lung metastases.2.The residual tumor cells after incomplete ablation show lipid metabolism reprograming,presenting as increased extracellular uptake of FA,enhanced biosynthesis of TG,and FAO haemostasis.3.The activated PPARγ in the residual tumor cells upregulates the expression of CD36 and FABP4/5,and further promotes the extracellular uptake of FA and biosynthesis of TG.4.Increased biosynthesis of TG enhances proliferation and invasion of the residual tumor cells via the activation of P38 MAPK signalling pathway.5.A PPARy-TG-P38 MAPK-PPARy positive feedback pathway is formed in the residual tumor cells.6.The lung metastasis is significantly inhibited by P38 inhibitor-Ralimetinib combined treatment in CRLM mice after incomplete ablation. |
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