Liming Guan,1 Xuexiang Xu,1 Junhao Xu,2 Gang Xu,3 Yunzhu Zhang,4 Haitao Xia1 

1Department of Obstetrics and Gynecology, Zhabei Centrial Hospital, Jing’an District, Shanghai, 200070, People’s Republic of China; 2Clinical Medicine2020, Capital University of Medical Sciences, Fengtai District, Beijing, 100069, People’s Republic of China; 3Department of Radiation Oncology, Cancer Hospital & Institute, Haidian District, Beijing, 100142, People’s Republic of China; 4Department of Obstetrics and Gynecology, Pudong New Area People’s Hospital, Shanghai, 201299, People’s Republic of China

Correspondence: Liming Guan, Email guanliming1@126.com

Background: Exact detection of lymphovascular infiltration (LVI) status can guide accurate surgical operation scope in cervical cancer, but LVI reduces the overall survival (OS) of patients and is not easily detected by hematoxylin–eosin (H&E) staining. The role of tumor-associated macrophages (TAMs) in this process is not well defined.
Methods: Early-stage cervical cancer patients received carbon nanoparticles for sentinel lymph-node mapping, laparotomy pelvic lymph-node dissection, and radical hysterectomy. The excised specimens were analyzed using ultrastaging, double immunohistochemical (IHC) staining, flow cytometry, and Western blot analysis. Single-cell data from the Gene Expression Omnibus for cervical cancer were obtained and analyzed.
Results: The integration of carbon nanoparticle mapping, ultrastaging, and double IHC staining enhanced the detection of tumor LVI over H&E staining (41.8% [41/98] vs. 20.4% [20/98], P=0.046). When the number of vascular invasion foci was greater than two, there was a negative correlation with OS (P< 0.05). More M2 TAMs emerged surrounding the tumor vasculature labeled by double IHC staining, accompanied by a higher M2:M1 ratio detected with flow cytometry (P< 0.05). M2 TAM numbers were positively correlation with the degree of tumor LVI (P=0.0024), combined with higher protein expression of MMP2, SPARC, and GNLY in the tumor LVI-positive group on Western blot analysis, and the OS of the patients decreased accordingly. Single-cell data showed that the M1:M2 ratio decreased significantly, accompanied by higher M2 TAM-related gene expression. Immunosurveillance and anti-immunoescape scores for M1 were obviously higher than for M2 TAMs. GO and KEGG analysis showed M2 TAM activity increased from precancerous lesions to cervical cancer.
Conclusion: Combining different methods may accurately determine tumor LVI status, guide exact surgical operation scope, and improve cervical cancer patient outcomes. M2 TAM activity increased in cervical cancer, forming an immunosuppressive environment, TAM-related genes could be good markers in determining cervical cancer LVI and serve as potential targets for immunotherapy.

Keywords: cervical cancer, double immunohistochemical staining, lymphovascular infiltration, LVI, tumor-associated macrophages, TAMs, single-cell data analysis