Background: One major etiology of hepatic sinusoidal obstruction syndrome (HSOS) in China is the intake of pyrrolizidine alkaloids (PAs). Since PAs-induced HSOS is a rare disease that has not been clearly characterized until now, the aim of this study was to investigate clinical characteristics, CT features, and pathological findings of PA-induced HSOS. Methods: This retrospective cohort study included 116 patients with PAs-induced HSOS and 68 patients with Budd-Chiari syndrome from Jan 2006 to Sep 2016. We collected medical records of the patients, and reviewed image features of CT, and analyzed pathological findings. Results: Common clinical manifestations of PAs-induced HSOS were abdominal distention (98.26%), ascites (100%), jaundice (52.94%), abdominal pain (36.36%). Abnormal liver function was observed in most of PAs-induced HSOS. On CT scan, common findings included: ascites, hepatomegaly, the thickening of gallbladder wall, pleural effusion, patchy liver enhancement, and heterogeneous hypoattenuation. Most of the patients had a low ascitic total protein (< 25 g/L) and a high SAAG (≥ 11.0 g/L). In acute stage, pathologic features were massive sinusoidal dilatation, sinusoidal congestion, the extravasation of erythrocytes, hepatocellular necrosis, the accumulation of macrophages, the deposition of hemosiderin. In subacute stage, complete loss of pericentral hepatocytes, sinusoidal dilatation, the deposition of pigment granules were observed. Conclusions: The PAs-induced HSOS patients displayed distinct clinical characteristics, imaging features, and pathological findings, which provided some evidences for the diagnosis of PAs-induced HSOS. Trial registration: ChiCTR-DRD-17010709.

Objective: To investigate the efficacy of an injectable hydrogel loaded with lysed OK-432 (lyOK-432) and doxorubicin (DOX) for residual liver cancer after incomplete radiofrequency ablation (iRFA) of hepatocellular carcinoma (HCC), and explore the underlying mechanism. Materials and Methods: The effect of OK-432 and lyOK-432 was compared in activating dendritic cells (DCs). RADA16-I (R) peptide was dissolved in a mixture of lyOK-432 (O) and DOX (D) to develop an ROD hydrogel. The characteristics of ROD hydrogel were evaluated. Tumor response and mice survival were measured after different treatments. The number of immune cells and cytokine levels were measured, and the activation of cGAS/STING/IFN-I signaling pathway in DC was evaluated both in vitro and in vivo. Results: LyOK-432 was more effective than OK-432 in promoting DC maturation and activating the IFN-I pathway. ROD was an injectable hydrogel for effectively loading lyOK-432 and DOX, and presented the controlled-release property. ROD treatment achieved the highest tumor necrosis rate (p < 0.001) and the longest survival time (p < 0.001) compared with the other therapies. The ROD group also displayed the highest percentages of DCs, CD4⁺ T cells and CD8⁺ T cells (p < 0.001), the lowest level of Treg cells (p < 0.001), and the highest expression levels of IFN-γ and TNF-α (p < 0.001) compared with the other groups. The expression levels of pSTING, pIRF3, and IFN-β in DCs were obviously higher after treatment of lyOK-432 in combination with DOX than the other therapies. The surviving mice in the ROD group showed a growth inhibition of rechallenged subcutaneous tumor. Conclusion: The novel ROD peptide hydrogel induced an antitumor immunity by activating the STING pathway, which was effective for treating residual liver cancer after iRFA of HCC.

Preventing tumor recurrence after radiofrequency ablation (RFA) of malignant solid tumors with large size or in high-risk locations represents a great challenge. In this study, we explored the feasibility of using oncolytic peptide LTX-315 plus an anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) antibody for inhibiting residual tumors after RFA of hepatocellular carcinoma (HCC). In in vitro experiment, the CD8⁺T cells from Hepa1-6 tumors, after being subjected to three different treatments (control, iRFA, iRFA + LTX-315), were extracted and were then co-cultured with Hepa1-6 cells and an anti-CTLA-4 antibody. The enzyme-linked immunospot, flow cytometry, and cell counting kit-8 assay were employed to assess the cytotoxicity of extracted CD8⁺T cells on Hepa1-6 cells. In in vivo experiment, different murine orthotopic HCC models were variously treated by: (1) pseudo iRFA + phosphate-buffered saline (PBS); (2) iRFA + PBS; (3) iRFA + LTX-315; (4) iRFA + anti-CTLA-4 antibody; and (5) iRFA + LTX-315 + anti-CTLA-4 antibody. The treatment effects were compared among different groups and were pathologically confirmed. The possible mechanisms of the combination treatment (LTX-315+anti-CTLA-4 antibody) for residual tumors after iRFA of HCC were explored. LTX-315 significantly reduced the PD-1 expression and significantly increased CTLA-4 expression of CD8⁺T cells in residual tumors, and additional treatment of anti-CTLA-4 antibody could significantly enhance the cytotoxicity of CD8⁺T cells for Hepa1-6 cells in vitro experiments. Compared with the other treatments, the combined treatment of LTX-315 with anti-CTLA-4 antibody achieved a better tumor response and longer survival, and it could synergistically activate the cGAS-STING pathway and elicit an immunogenic cell death, leading to a strong anti-tumor immunity after iRFA of HCC. The immunosuppressive microenvironment of residual tumors was significantly improved by the combination therapy with a significantly increased ratio of M1-like tumor-associated macrophages to M2-like tumor-associated macrophages, a significantly decreased infiltration of regulatory T cells and myeloid-derived suppressor cells, and a significantly lower expression of PD-1 and CTLA-4. Overall, the results of this study demonstrated that LTX-315 plus anti-CTLA-4 antibody could synergistically improve the immunosuppressive microenvironment of residual tumors and induce a strong anti-tumor immunity after iRFA of HCC. This combination treatment strategy may offer a new alternative to reduce the tumor recurrence after RFA of malignant solid tumors with large sizes or in high-risk locations.

Radiofrequency ablation (RFA) has been used as an alternative to surgical management of early-stage hepatocellular carcinoma (HCC). However, when large and irregular HCCs are subjected to RFA, a safety margin is usually difficult to obtain, thus causing a sublethal radiofrequency hyperthermia (RFH) at the ablated tumor margin. This study investigated the feasibility of using RFH to enhance the effect of OK-432 on HCC, with the aim to generate a tumor-free margin during RFA of HCC. Our results showed OK-432 could activate the cGAS-STING pathway, and RFH could further enhance the activation. Meanwhile, RFH could induce a high expression of TLR4, and TLR4 might be an upstream molecular of the cGAS-STING pathway. The combined therapy of RFH with OK-432 resulted in a better tumor response, and a prolonged survival compared to the other three treatments. In conclusion, RFH in combination with OK-432 might reduce the residual and recurrent tumor after RFA of large and irregular HCCs, and serve as a new option for other solid malignancies treated by RFA.

Radiofrequency ablation (RFA) often results in incomplete ablation for medium-to-large and irregular tumors. To solve this clinical problem, we proposed a new treatment strategy of OK-432 in combination with an anti-programmed cell death protein 1 (αPD-1) antibody for residual tumors after incomplete RFA (iRFA) of hepatocellular carcinoma (HCC). The effect of OK-432 on immature dendritic cells (iDCs) was evaluated in vitro. A CCK-8 kit and ELISPOT were used to assess the killing effect of OK-432-induced CD8⁺ T cells in combination with an αPD-1 antibody on Hepa1-6 cells. We found that OK-432 significantly increased the maturation level of DCs, and OK-432-induced CD8⁺ T cells in combination with αPD-1 antibody significantly enhanced the function of CD8⁺ T cells. In the in vivo experiment, HCC model mice were treated with (1) pseudo iRFA + phosphate-buffered saline (PBS); (2) iRFA + PBS; (3) iRFA + OK-432; (4) iRFA + αPD-1; or (5) iRFA + OK-432 + αPD-1. We found that the combined therapy of OK-432 with αPD-1 antibody significantly increased the infiltration and function of CD8⁺ T cells and significantly decreased the number of FoxP3⁺ regulatory T cells in residual tumors after iRFA of HCC. Moreover, the smallest tumor volumes and the longest survival were observed in the triple combination treatment (iRFA+OK-432 +αPD-1 antibody) group compared with the other four groups. The combined therapy of OK-432 with αPD-1 antibody induced a strong antitumor immune response, which significantly inhibited the residual tumors after iRFA of HCC. This concept may provide a new treatment strategy to increase the curative efficacy of RFA for medium-to-large and irregular HCCs. The data of this study are available from the corresponding author on reasonable request.