Cervical cancer lymphovascular infiltration determination and tumor-associated macrophage enhancing immune escape microenvironment analysis

Abstract

Abstract Background:Lymphovascular infiltration(LVI) reduced cervical cancer patients overall survival(OS),while it was not easily discovered by routine HE staining,while the role of tumor-associated macrophages(TAMs) in this course need define. Methods: Early stage cervical cancer patients were received carbon nanoparticles(CNP) for sentinel lymph nodes(SLNs) mapping,laparotomy pelvic lymph node dissection and radical hysterectomy.The samples were detected for ultra staging,cocktail double immunohistochemical(IHC) staining,Western blot anlysis.Single cell data from(GEO) for cervical cancer were obtained and analysis. Results: The combine of CNP mapping,ultra staging and double IHC staining enhance determining ratio for tumor LVI than that of HE staining alone(41.8% (41/98) vs. 20.4% (20/98), P=0.046)). When the number of vascular invasion foci>2.0,or the number of cancer emboli cells ≥5.0, there was negative correlation with the OS for patients (p < 0.05). More M2 macrophage emerged surrounding the tumor vasculature than surroungding normal cervix(P<0.05).Western blot analysis,TAMs related genes MMP2,SPARC and GNLY expessed higher level in tumor, while the OS of the patients decreased accordingly. Single cell data were screened, reduced dimensions, and constructed pseudo-trajectories. Differentially expressed genes (DEGs) analysis showed, M2 macrophage clusters related genes expession level MMP2,SPARC and GNLY were obviosely higher than that of M1 clusters in cervical cancer; M1/M2 ratio decreased significantly. MMP2,GNLY could destroy tumor vasculauture basement membrane; SPARC anti-adhesion, played a key role in EMT and tumor invasion. M1 macrophages cluster anti-inflammatory, immune surveillance and anti-immune escape scores were obviously higher than that of M2 macrophages cluster. Gene Ontology(GO) analysis showed innate immune response gene expression for M2 macrophage clusters decreased obviously than that of M1 macrophages.Kyoto Encyclopedia of Genes and Genomes(KEGG) showed that normal cervix and precancerous lesions higher enrichement of M1 macrophage clusters, presenting positive regulation of response stimulus signaling pathway,while cervical cancer and metastatic lymph nodes enriched M2 clusters presenting negative regulation to these signaling pathway. Conclusions:CNP mapping could be as a SLN tracer in early cervical cancer LVI screening.Cocktail double IHC staining could enhance diagnostic efficiency than HE staining in determining LVI. M2 TAMs activity increased from precancerous lesions to cervical cancer forming a immunosuppressive envioment gradually, TAMs related genes could play a key role in this process.