Tumor-associated Macrophages Induce Lymphangiogenesis In Cervical Cancer Via Interaction With Tumor Cells

Part Ⅰ The presence and distribution characters of TAMs in cervical cancer tissuesObjectiveTo explore the presence and distribution characters of tumor-associated macrophages (TAMs) in cervical cancer tissues.MethodsImmunohistochemistry was used for detection of D2-40-positive lymphatic endothelial cells and CD68-positive macrophages in117cervix tissues including sixty-one invasive carcinomas of uterine cervix, twenty-seven cervical intraepithelial neoplasms (CIN), and twenty-nine normal cervix. And the relationship between the number of TAMs and the clinicopathological features in61human cervical cancers were analyzed.Results1. Macrophages in cervical cancer were enriched in tumor nests and stroma when compared with normal cervix and CIN (P<0.05), whereas no significant differences among peritumor, paratumor and normal cervix were found (P>0.05).2. Lymphatic vessels in cervical cancer were mainly located in the tumor stroma and peritumor, but rarely observed in cancer nests. LVD in tumor stroma was apparently higher than that in normal cervix and CIN (all P<0.05).3. Pearson tests showed there was a significant positive correlation between stromal TAMs and stromal LVD (P=0.0002), and between stromal TAMs and peritumor LVD (P=0.0322). Significant correlation was not found between tumor nests TAMs and stromal LVD, between peritumor TAMs and peritumor LVD, or between paratumor TAMs and paratumor LVD (P>0.05).4. Stromal MD in cases with lymphatic metastasis were increased, when compared with those without lymphatic metastasis (P=0.033). And there were increased stromal TAMs in tumors2cm or greater in diameter, when compared with that in those smaller than2cm in diameter (P=0.006). There was no significant correlation between TAMs and age, FIGO stage, invasion depth, histological differentiation, or histological type.ConclusionIncreased stromal TAMs are significantly correlated with LVD and lymphatic metastasis in cervical cancer. Part Ⅱ The source of TAMs in cervical cancer microenviromentObjectiveTo explore the source of TAMs in the tumor microenviroment of cervical cancer.Methods1. The influence of cervical cancer cells (Hela, Siha or C33A) on migration of PMA-treated THP-1macrophages was examed by transwell assays, which imitated the macrophages recruitment in vitro.2. The proliferation activity of macrophages in cervical cancer tissues was evaluated by double staining with CD68antibody and Ki67antibody by immunohistochemistry (IHC) and immunofluorescence (IF) stains.Results1. Cervical cancer cells were more potential to promote the migration of macrophages towards them compared with blank control and normal cervix epithelium cell line CRL2614(all P<0.001).2. Both IHC and IF showed that macrophages marked by CD68in cervical cancer tissues were Ki67negative. ConclusionCervical cancer cells promote the directional migration of existed macrophages, and part of the increased TAMs in tumor nests and stroma might be owned to recruitment of the preexisting macrophages in the surrounding. Part Ⅲ The influence of TAMs on the invasion and migration of cervical cancer cellsObjectiveTo explore the influence of TAMs on the invasion and migration of cervical cancer cells.Methods1. The influence of TAMs on invasion of cervical cancer cells (Hela, Siha or C33A) was assessed by transwell assays with Matrigel supplied.2. The influence of TAMs on migration of cervical cancer cells (Hela, Siha or C33A) was assesses by transwell assays.Results1. The number of invading cervical cancer cells increased in presence of TAMs, compared with blank (P<0.001).2. The number of migrating cervical cancer cells increased in presence of TAMs, compared with blank (P<0.001).ConclusionTAMs promote the invasion and migration of cervical cancer cells. Part IV The effect of TAMs and human cervical cancer cells on lymphangiogenesisObjectiveTo detect the effect of TAMs and human cervical cancer cells on lymphangiogenesis.Methods1. The tube formation assays in vitro of HLEC were performed in different culture supernatants, including supernatants of CRL2614, cervical cancer cells, macrophages, and macrophage-cervical cancer cell coculture.2. ELISA assays were performed to determine the levels of IL-1β and IL-8in the conditioned media from macrophage-cervical cancer cell coculture.3.The mRNA expression of imflammatory factors (IL-1β and IL-8) and VEGFs (VEGF-C, VEGF-A and VEGF-D) in macrophages and cervical cancer cells cultured alone or cocultured were analyzed by RT-PCR.4. The mRNA expressions of VEGFR-3in HLEC isolated from "macrophage-cervical cancer cell-HLEC model" or "macrophage-CRL2614-HLEC model" was analyzed by RT-PCR.Results1. There was no significant difference in the number of tube-like structures formed by HLEC between cells cultured in common LEC medium and those cultured in conditioned media from CRL2614, macrophages, Hela, Siha or C33A (P>0.05). Only the conditioned medium from cervical cancer cells-macrophages cocluture resulted in a obvious increase in number of tube-like structures of HLEC (P<0.001).2. The levels of IL-1β and IL-8were obviously increased in the macrophage-cervical cancer cell coculture supernatants, when compared with that in culture supernatants of CRL2614, macrophages, or cervical cancer cells(all P<0.001).3.RT-PCR showed IL-1β and IL-8increased obviously in cervical cancer cells (Hela, Siha or C33A) cocultured with macrophages, respectively compared with Hela, Siha or C33A (all P<0.05). VEGF-C and VEGF-A increased in macrophages cocultured with cervical cancer cells (Hela, Siha or C33A) compared with macrophages cultured alone. VEGF-D showed increased in Hela group and IL-1β also was found increased in Hela or Siha groups (all P<0.05).4. The mRNA expression of VEGFR-3and PDPN were increased in HLEC cocultured with macrophages and Hela/Siha/C33A compared with those cocultured with macrophages and CRL2614(P<0.05).ConclusionMacrophages and tumor cells act synergistically to promote lymphangiogenesis in cervical cancer.