Impact of deviated balance of regulatory and cytotoxic T cells on release and reseeding of circulating tumor cells in hepatocellular carcinoma.

Abstract

e22133 Background: Our previous data showed that a preoperative CTC ≥2 was significantly related with relapse of hepatocellular carcinoma (HCC) after surgery. However, how CTCs managed to evade immune surveillance, invade into circulation and reseed in the original organ still remains largely unclear. In this study, we illustrated the pro-tumor effect of Treg infiltration in the context of CTC releasing and reseeding. Methods: Using CellSearch system, 115 HCC patients were tested for CTCs prior to resection. Foxp3+, CD8+ and Granzyme B-positive (GrB+) tumor -infiltrating lymphocytes were evaluated by immunohistochemistry (IHC) in tissue microarrays. For IHC markers, the cutoff for stratification subsets was the median value. The pro-invasiveness properties of Tregs were analyzed in both in vitro and vivo assays. Results: Twenty-two patients with high Tregs and low activated CTLs in tumoral tissues had increased number of CTCs compared to 22 patients with low Tregs and high activated CTLs (6.68 vs. 0.91, P<0.001). In 77 patients with detectable CTC, high Tregs and low activated CTLs in peritumoral tissues was significantly associated with an increased incidence of HCC recurrence (80.5% vs. 19.4%, P<0.001), and was an independent prognostic factor for HCC early recurrence (P=0.007). Further in vitro study showed Tregs-derived TGF-β promoted HCC cell migration and invasion; down-regulated E-cadherin and up-regulated N-cadherin, vimentin, fibronectin and snail. The data in human tumor sample also showed that the TGF-β level had positive relationships with Treg count, N-cadherin level and CTC count; and had inverse relationship with E-cadherin (all P values <0.05). In mice, depletion of Tregs by neutralizing antibody abolished intrahepatic colonization and lung metastasis. Conclusions: Local immune contexture in HCC orchestrates with biological behavior of CTCs. High intratumoral Tregs may promote the release of CTCs through inducing tumor cells EMT transition via TGF-β secretion. High peritumoral Tregs created an immune-subversive microenvironment by suppressing CTL to favor colonization of CTCs, which led to recurrence after HCC resection.