Background: The mechanism underlying sorafenib resistance in hepatocellular carcinoma (HCC) remains unclear. Accumulating evidence suggests that tumor-initiating cells (TICs) are a pivotal driving force. Both CD44 and Hedgehog signaling play crucial roles in TIC properties in HCC. In this study, we explored the roles of CD44 and Hedgehog signaling in sorafenib resistance and evaluated the therapeutic effect of cotreatment with sorafenib and Hedgehog signaling inhibitors in HCC patient-derived organoid (PDO) models to improve treatment efficacy. Methods: We collected HCC specimens to establish PDO models. Cell viability and malignant transformation properties were investigated after treatment with different TIC-related inhibitors alone or in combination with sorafenib to evaluate the therapeutic effect in PDOs and cell lines by in vitro and in vivo experiments. Expression levels of Hedgehog signaling proteins and CD44 were monitored to reveal potential relationships. Results: We demonstrated that our HCC PDO models strongly maintained the histological features of the corresponding tumors and responded to drug treatment. Furthermore, CD44-positive HCC PDOs were obviously resistant to sorafenib, and sorafenib increased CD44 levels. A drug screen showed that compared with Notch, Hippo and Wnt signaling inhibitors, a Hedgehog signaling inhibitor (GANT61) potently suppressed HCC PDO cell viability. In addition, there was a highly synergistic effect in vitro and in vivo on the suppression of cell viability and malignant properties when sorafenib and GANT61 were added to CD44-positive HCC PDOs and cell lines, respectively. Furthermore, the upregulation of CD44 and Hedgehog signaling induced by sorafenib was reversed by GANT61. Conclusions: GANT61 significantly suppressed Hedgehog signaling to reverse sorafenib resistance in CD44-positive HCC. The combination of sorafenib and Hedgehog signaling inhibitors might be effective in HCC patients with high CD44 levels as a personalized-medicine approach.

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Numerous studies reported a small subpopulation of TCRαβ+CD4-CD8- (double-negative) T cells that exert regulatory functions in the peripheral lymphocyte population. However, the origin of these double-negative T (DNT) cells is controversial. Some researchers reported that DNT cells originated from the thymus, and others argued that these cells are derived from peripheral immune induction. We report a possible mechanism for the induction of nonregulatory CD4+ T cells to become regulatory double-negative T (iDNT) cells in vitro. We found that immature bone marrow dendritic cells (CD86+MHC-II- DCs), rather than mature DCs (CD86+MHC-II+), induced high levels of iDNT cells. The addition of an anti-MHC-II antibody to the CD86+MHC-II+ DC group significantly increased induction. These iDNT cells promoted B cell apoptosis and inhibited B cell proliferation and plasma cell formation. A subgroup of iDNT cells expressed NKG2D. Compared to NKG2D- iDNT cells, NKG2D+ iDNT cells released more granzyme B to enhance B cell regulation. This enhancement may function via NKG2D ligands expressed on B cells following lipopolysaccharide stimulation. These results demonstrate that MHC-II impedes induction, and iDNT cells may be MHC independent. NKG2D expression on iDNT cells enhanced the regulatory function of these cells. Our findings elucidate one possible mechanism of the induction of peripheral immune tolerance and provide a potential treatment for chronic allograft rejection in the future.

future studies need to carry out sub-analysis for virus etiology (HBV or HCV), viral load and antiviral therapy strategies with different methods of spleen stiffness measurement (SWE). Again, future studies need to clarify whether the combination of SSM and immunological parameters could be better for predicting late recurrence of HCC.