The Dedifferentiation Mechanism And Clinical Significance Of High Endothelial Venules In The Draining Lymph Nodes Of Lung Cancer

Chapter Ⅰ The phenomenon,mechanism and clinical significance of HEVs dedifferentiation in TDLNs of lung cancerObject:To explore whether the dedifferentiation of high endothelial venules(HEVs)exists in tumor-draining lymph nodes(TDLNs)of non-small cell lung cancer(NSCLC)patients,as well as the mechanism and clinical significance of HEVs dedifferentiation.Method:1.Study population:Patients with non-neoplastic benign lumps/lesions(Control group)and NSCLC patients(Lung cancer group)who underwent surgery in the same surgical treatment group of the Second Affiliated Hospital of Nanchang University from September 2017 to December 2020 were collected.The Lung cancer group was then divided into lung adenocarcinoma group(LUAD group)and lung squamous cell carcinoma group(LUSC group).The research subjects were screened according to inclusion and exclusion criteria.2.Collection of clinical data:The patients’gender,age,body mass index,smoking history,tumor diameter,lymph node metastasis and distant metastasis,pathological grade,p TNM stage,clinical stage,and adjuvant therapy were collected.3.Follow up:The combination of inpatient/outpatient review records and telephone follow-up was carried out to follow up with patients.The content of follow-up included the postoperative adjuvant treatment,recurrence and survival of the patients.4.Group:Normal lymph node(NLN),Non-metastatic lymph node(NMLN),Metastatic lymph node(MLN).5.By comparing the density of total HEVs,the density of dilated HEVs,the composition ratio of dilated HEVs,and the number of CD3+T cells around HEVs,we explored the dedifferentiation in the structure and function of HEVs.By comparing the number of CD11c-positive dendritic cells(CD11c+DCs)around HEVs in each group of lymph nodes,we initially explored the mechanism of HEVs dedifferentiation.The survival curve(K-M curve)was plotted using total HEVs density,dilated HEVs density,and the value of dilated HEVs density/total HEVs density as prognostic indicators,also the Cox regression analysis was carried out to explore the clinical significance of HEVs dedifferentiation.Result:1.Basic characteristics of the study population:A total of 72 subjects were included in this study,including 52 patients in the Lung cancer group and 20 patients in the Control group.In the study population,79.2%were male,with an average age of 57.83±8.03 years,an average BMI of 20.41±4.94kg/m~2,and a smoking rate of51.4%.The smoking rate in the Lung cancer group was significantly higher than that in the Control group(59.6%VS 30.0%,P=0.024),and there were no significant differences among other general data(P>0.05).2.Clinicopathological parameters of LUAD and LUSC patients:Ki-67 index in LUSC patients was significantly higher than those in LUAD patients(50.63±16.04VS 27.04±18.14,P<0.05),while the number of metastasis lymph nodes was significantly lower in patients with LUAD[2.00(1.00,3.00)VS 4.00(2.00,8.00),P<0.05].There was no difference among other indicators(P>0.05).3.Structural changes in HEVs dedifferentiation in TDLNs of lung cancer:The density of HEVs in NMLN was significantly higher than that in NLN and MLN(P<0.05),but there was no significant difference between NLN and MLN(P>0.05).The density of dilated HEVs in both NMLN and MLN was significantly higher than that in NLN(P<0.05),and that in MLN was lower than that in NMLN,but the difference was not statistically significant(P>0.05).The composition ratio of dilated HEVs in NLN,NMLN and MLN gradually increased,which was significantly different between the three groups(P<0.05).Subgroup analysis showed that in LUAD patients,HEVs density in NMLN was significantly higher than that in NLN(P<0.05),while there was no significant difference between MLN and NLN(P>0.05).The density of dilated HEVs in NMLN and MLN was significantly higher than that in NLN(P<0.05),and the density of MLN was lower than that in NMLN,but the difference was not statistically significant(P>0.05).The composition ratio of dilated HEVs in NLN,NMLN and MLN gradually increased,which was significantly different between the three groups(P<0.05).In LUSC patients,HEVs density in NMLN was significantly higher than that in NLN(P<0.05),while there was no significant difference between MLN and NLN(P>0.05).The density of dilated HEVs in NMLN was significantly higher than that in NLN(P<0.05),but there was no significant difference between MLN and NLN(P>0.05).There was no significant difference in the composition ratio of dilated HEVs in NLN,NMLN and MLN(P=0.054).4.Altered function of HEVs dedifferentiation:The number of CD3+T cells around dedifferentiated HEVs in MLN was significantly lower than that around normal HEVs in NLN(P<0.05).Although NMLN was also lower than NLN,this change was not statistically significant(P>0.05).5.Dedifferentiated HEVs provides exit route to circulation for tumor cells in lymph nodes:The process of tumor cells in TDLNs migrating to dedifferentiated HEVs,approaching the wall of dedifferentiated HEVs,passing through the wall,and entering the HEVs lumen was observed for the first time in human lymph nodes.6.The decreased numbers of CD11c+DCs is related to HEVs dedifferentiation in TDLNs:The number of CD11c+DCs around dedifferentiated HEVss in NMLN and MLN was significantly lower than that around normal HEVs in NLN(P<0.05).7.HEVs dedifferentiation in TDLNs is a risk factor for poor prognosis in patients with lung cancer:the K-M curve was plotted using the dilated HEVs density as a prognostic indicator,and it was found that the survival time of patients with high values was significantly reduced,and the difference was statistically significant(P=0.030).Multivariate Cox regression analysis showed that the value of dilated HEVs/total HEVs was an independent risk factor for poor prognosis in lung cancer patients(P=0.024).Conclusion:1.HEVs dedifferentiation was present in TDLNs in lung cancer patients,regardless of the presence or absence of metastases.HEVs dedifferentiation leads to a decrease in the number of HEVs in TDLNs and is related to the decreased homing function of their recruitment of T cells,which is a risk factor for poor prognosis in lung cancer patients.2.For the first time,it was confirmed that dedifferentiated HEVs in human TDLNs could provide exit routes for tumor cells in lymph nodes and promote the systemic dissemination of tumors.3.The decrease in the number of CD11c+DCs around HEVs in TDLNs is associated with HEVs dedifferentiation.Chapter Ⅱ HEVs dedifferentiation in TDLNs of animal modelObject:An animal model of lymph node metastasis was constructed to further explore the phenomenon of HEVs dedifferentiation in TDLNs and its immunological effects.Method:1.BALB/c Nude nude mice were used to establish tumor-bearing animal models under the footpad,including a Normal Control group(NC group,25 mice)and a Tumor group(T group,15 mice).2.Intervention:NC group:inject 50μL PBS under the right footpad;T group:inject 50μL A549 cell suspension(cell concentration:1×10~8/ml)under the right footpad.3.Lymph node grouping:Normal lymph node(NLN):right lower limb lymph node of NC group mice;Non-draining lymph node(NDLN):lymph nodes on the non-tumor drainage side of tumor-bearing mice;TDLN:tumor draining lymph nodes of tumor-bearing mice.NLN,NDLN,and TDLN all include popliteal lymph nodes(PLN)and inguinal lymph nodes(ILN).4.The density of total HEVs,the number of total HEVs,and the composition ratio of dilated HEVs in PLN and ILN were compared to explore the dedifferentiation of HEVs in the first and second draining lymph nodes.Multiplex immunofluorescence staining was used to explore HEVs neogenesis in lymph nodes in blank nude mice and tumor-bearing nude mice.Result:1.Tumorigenesis rate under the footpad and lymph node metastasis:The tumorigenesis rate rate was 100%.At 4 weeks of tumor bearing,the metastasis rate of TDLN(PLN)was 40%,and that of TDLN(ILN)was 0%.At 6 weeks of tumor bearing,the metastasis rate of TDLN(PLN)was 100%,and that of TDLN(ILN)was0%.2.Changes in the density of HEVs in the first draining lymph node TDLN(PLN):HEVs density in NLN was significantly higher than that in NDLN at 6 weeks of tumor bearing(P<0.05),and there was no difference in HEVs density among the other groups(P>0.05).3.Changes in the total number of HEVs in TDLN(PLN):the number of HEVs in TDLN was significantly higher than that in NLN and NDLN at 4 weeks of tumor bearing(P<0.05);At 6 weeks of tumor bearing,the number of HEVs in TDLN was significantly higher than that in NLN and NDLN(P<0.05),while that in NDLN was significantly lower than that in NLN(P<0.05);Compared with that at 4 weeks,the number of HEVs in NLN increased significantly at 6 weeks(P<0.05),and there was no significant difference in the total number of HEVs among the other groups(P>0.05).Structural alterations associated with significant dedifferentiation of HEVs in TDLNs were not observed.4.Changes in HEVs in the second draining lymph node TDLN(ILN):The changes of HEVs density and quantity in ILN of each group at 4 weeks and 6 weeks of tumor bearing were generally similar to those in PLN,but there was no statistical difference(P>0.05).5.HEVs neogenesis leads to increased HEVs numbers in NLN(PLN),and tumor suppresses HEVs neogenesis in tumor-bearing mice:The results of multiple immunofluorescence staining showed that there was no significant difference in the expression of pan-endothelial cell marker CD31 between NLN(PLN)and NDLN(PLN)(P>0.05).While the expressions of MECA-79 and naive HEVs marker-MAd CAM-1 were significantly decreased in NDLN(PLN)(P<0.05).The results of correlation analysis showed that the expressions of MECA-79 and MAd CAM-1 were significantly correlated,and the correlation coefficient was 0.936(P<0.05).These results suggest that tumors can inhibit HEVs neogenesis in NDLN(PLN)of tumor-bearing mice.Conclusion:1.The dedifferentiation of HEVs in the TDLN of tumor-bearing mice is mainly manifested by the reduction of the number,rather than the change of morphology.2.The influence of xenograft tumors on HEVs in TDLNs has a sequence or degree relationship.3.Xenograft tumors can inhibit HEVs neogenesis in NDLN of tumor-bearing mice.Chapter Ⅲ Transcriptomic analysis of TDLN and NLN in nude miceObject:To investigate the difference in transcriptome levels between the first draining lymph nodes TDLN of tumor-bearing mice and NLN of normal control mice at 6weeks.Method:1.Sample preparation:The tumor-draining lymph nodes in the right popliteal of6 week tumor-bearing mice(TDLN,5 samples)and the right popliteal lymph nodes from normal control mice(NLN,15 samples,every 3 samples are mixed into one sequencing sample,a total of 5 sequencing NLN samples)were collected for transcriptomic sequencing.2.Screen differentially expressed genes(DEGs):Screen DEGs with FDR<0.05and|log2Fold Change|>1 as thresholds.3.GO and KEGG enrichment analysis:GO and KEGG enrichment analysis was performed on the obtained DEGs to explore the enriched items and pathways related to the immune status and HEVs phenotype and function.Result:1.DEGs:A total of 1122 DEGs were screened out from the transcriptomic analysis of TDLN and NLN,including 883 up-regulated DEGs and 239 down-regulated DEGs.2.GO enrichment analysis of DEGs:up-regulated DEGs enriched to immune response-related items include responses to other organisms,responses to external biological stimuli,biological stimuli responses,immune responses,and biological processes of defense responses,while down-regulated DEGs mainly enriched collection of items related to hormones,drugs,and lipid responses,indicating that the immune response to tumor metastases in TDLN is still strong at this time.The items related to HEVs phenotype and function enriched by up-regulated DEGs include regulating cell migration,leukocyte migration,regulating cell movement,cell surface composition,and mucopolysaccharide binding,etc.,indicating that the function of HEVs in TDLN is also significantly enhanced compared with NLN at this time.Venn diagrams showed that most of the upregulated DEGs involved in immune responses were also upregulated DEGs enriched into items related to HEVs function.The above results indicated that the immune response of TDLN to tumor metastases was still strong at 6 weeks after tumor bearing,and the function of HEVs was significantly stronger than that of NLN at this time,suggesting that the intensity of immune response was related to HEVs function.3.The HEVs maturation differentiation gene Chst4 and dendritic cell marker CD11c were significantly upregulated in TDLN(FDR<0.05,|log2Fold Change|>1),and the expressions of the two were significantly correlated(r=0.818,P<0.05),indicating that CD11c was significantly correlated with HEVs maturation and differentiation status,which is consistent with the results of Chapter I.MAd CAM-1was significantly down-regulated in TDLN(FDR<0.05,|log2Fold Change|>1),indicating that the xenograft tumor at this time inhibited the neogenesis of HEVs in TDLN,which is consistent with the results of Chapter II.4.KEGG enrichment analysis of DEGs:Up-regulated DEGs are enriched in tumor-related signaling pathways such as proteoglycan and focal adhesions in cancer,while down-regulated DEGs are mainly enriched in metabolism,biosynthesis,secretion and other related pathways.Among the top 20 enriched pathways of up-regulated DEGs,4 pathways were related to HEVs function,namely TNF signaling pathway,chemokine signaling pathway,RAP1 signaling pathway and cytokine-cytokine receptor interaction.Conclusion:1.At 6 weeks of tumor-bearing,the immune response-related genes and HEVs function-related genes in TDLN were significantly upregulated,indicating that the enhancement of immune response was related to the enhancement of HEVs function and the increment of HEVs quantity.2.By inhibiting HEVs neogenesis in tumor-bearing mice,the xenograft tumors may cause immunosuppression,enhancing the body’s immune tolerance to xenograft tumors,and promoting immune escape of xenograft tumors.Research Conclusion1.There is dedifferentiation of HEVs in lung cancer TDLNs,which includes structural dedifferentiation,functional dedifferentiation,and reduction of HEV quantity;There is a sequential/degree relationship between the effect of lung cancer on the dedifferentiation of HEVs in TDLNs.2.Lung cancer not only dedifferentiates HEVs by affecting the number of CD11c+DCs in TDLNs,but also inhibits the neogenesis of HEVs,which in turn may inhibit the body’s anti-tumor immune response,improve the body’s immune tolerance to tumors,and promote immune escape.3.For the first time,we observed the dedifferentiated HEVs in human TDLNs to provide exit routes for tumor cells,promote the systemic spread of tumors,and are risk factors for poor prognosis in lung cancer patients,which also provide theoretical support for non-sequential metastasis of tumors.