| Purpose:The aim of this study was to investigate the expression of angiopoietin-2(Ang-2)and its correlation with the clinical and biological characteristics of cutaneous squamous cell carcinoma(cSCC),as well as to explore the role of Ang-2 through the exosomal pathway in lymphatic vessel generation.Methods:Bioinformatics analysis using the GEO database was performed to determine the differential expression of Ang-2 in cSCC and normal tissues.Cell experiments were conducted to investigate how cSCC-derived exosomes(cSCC-ex)regulate the proliferation,migration,and capillary formation of lymphatic endothelial cells(LECs)through Ang-2 protein.A low Ang-2 expression cSCC cell line(cSCCsh Ang-2)was constructed by lentivirus-mediated knockdown of Ang-2 gene,and its exosomes were extracted,observed by transmission electron microscopy for morphology and measured for size distribution using nanoparticle tracking analysis(NTA).Western blot was used to detect exosome-specific markers and Ang-2 expression.Exosomes secreted by ang-2-knockdown cSCC cells and untreated cSCC cells were co-cultured with lymphocytes,with PBS group as control.The biological effects of the two groups of exosomes on LECs were compared through cell migration and tube formation assays.Clinical studies were conducted to validate the expression of Ang-2 in cSCC and its correlation with tumor lymphatic vessel regeneration.A total of 44 primary cSCC patients admitted to Jiangsu University Affiliated Hospital from September 2013 to February 2021 were enrolled.The expression of Ang-2 and LYVE-1 in cSCC specimens was detected by immunohistochemistry(IHC),and LYVE-1 expression was represented by microlymphatic vessel density(MLVD).Statistical analysis was performed using SPSS26.0 software.Results:Bioinformatics analysis showed that Ang-2 was significantly upregulated in cSCC compared to normal tissues.In cell experiments,exosomes showed irregular spherical morphology under transmission electron microscopy.NTA revealed that the exosomes had a size distribution peak at around 115 nm.Exosomes containing Ang-2 promoted the migration and tube formation of LECs.When the expression level of Ang-2 in exosomes was reduced,the migration and tube formation of LECs were weakened but still stronger than those controls wi thout exosome co-culture.In clinical studies,the high expression rate of Ang-2 in cSCC without lymph node metastasis was 44%(15/34),significantly lower than that in lymph node metastasis group(90%,9/10)(P<0.05).The high expression rate of Ang-2 in well-differentiated cSCC was 35%(9/26),significantly lower than that in moderately and poorly differentiated cSCC(83%,15/18)(P<0.05).LYVE-1 expression in cSCC without lymph node metastasis was 8.49 ± 4.26,significantly lower than that in lymph node metastasis group(13.48±5.91)(P<0.05).LYVE-1expression in well-differentiated cSCC was 6.39 ± 2.09,significantly lower than that in moderately and poorly differentiated cSCC(14.28±4.42)(P<0.05).Gender,age,and tumor size had no significant effect on the expression of Ang-2 and LYVE-1 in cSCC(all P>0.05).Moreover,the expression level of Ang-2 in cSCC was positively correlated with LYVE-1(r=0.598,P<0.01).Conclusion:Experimental results have shown that the expression of Ang-2 is elevated in cSCC tumor tissues compared to normal skin tissues.Moreover,exosomes released by cSCC cells can promote the proliferation,migration,and tube formation of LECs through the Ang-2 protein pathway.Ang-2 plays a crucial role in driving the occurrence and development of cSCC,and it is also involved in the lymph node metastasis of cSCC.These findings provide strong support for exploring new directions for the treatment of cSCC and offer a basis for the feasibility of targeted drug therapy for cSCC. |
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