Anti–PD-L1–based combination immunotherapy has become the first-line treatment for unresectable hepatocellular carcinoma (HCC). However, the objective response rate is lower than 40%, highlighting the need to identify mechanisms of tolerance to immune checkpoint inhibitors and accurate biomarkers of response. In this study, we used next-generation sequencing to analyze HCC samples from 10 patients receiving anti–PD-L1 therapy. Activation of the renin–angiotensin system was elevated in nonresponders compared with responders, and angiotensin-converting enzyme 2 (ACE2) expression was significantly downregulated in nonresponders. ACE2 deficiency promoted HCC development and anti–PD-L1 resistance, whereas ACE2 overexpression inhibited HCC progression in immune-competent mice. Mass cytometry by time of flight revealed that ACE2-deficient murine orthotopic tumor tissues featured elevated M2-like tumor-associated macrophages, displayed a CCR5+PD-L1+ immunosuppressive phenotype, and exhibited high VEGFα expression. ACE2 downregulated tumor-intrinsic chemokine (C–C motif) ligand 5 expression by suppressing NF-κB signaling through the ACE2/angiotensin-(1–7)/Mas receptor axis. The lower chemokine (C–C motif) ligand 5 levels led to reduced activation of the JAK–STAT3 pathway and suppressed PD-L1 and VEGFα expression in macrophages, blocking macrophage infiltration and M2-like polarization. Pharmacologic targeting of CCR5 using maraviroc enhanced the tumor-suppressive effect of anti–PD-L1 therapy. Together, these findings suggest that activation of the ACE2 axis overcomes the immunosuppressive microenvironment of HCC and may serve as an immunotherapeutic target and predictive biomarker of response to PD-L1 blockade. Significance: ACE2 regulates the immune landscape of hepatocellular carcinoma by abrogating M2-like macrophage polarization and sensitizes tumors to anti–PD-L1, suggesting that harnessing the ACE2 axis could be a promising strategy to improve immunotherapy efficacy.

Autophagy in tumor was also found to influence immune microenvironment. The relation between autophagy and cancer intrinsic PD1 and PD-L1 expression was not clear. With data from TCGA and GTEx databases, mRNA expression levels of autophagy-related genes were compared between tumor samples and normal tissues, which were also correlated with survival status. Expression of autophagy-related genes were also associated with clinical traits in datasets of GSE14520 and ICGC LIRI. Single sample gene set enrichment analysis (ssGSEA) was used to calculate autophagy scores in tumor samples, using signatures from MSigDB database. Lentivirus (PD1 and PD-L1), siRNA (ATG13) and plasmids (LC3A/B) were used to target specific genes in tumor cells; Western blot was used to examine protein expression accordingly. Co-immunoprecipitation was performed to find PD1 or PD-L1 interacting proteins; colony formation and EdU analysis were used to evaluate tumor cell growth abilities. mRNA levels of autophagy markers were increased in tumor and correlated with worse survival of cancer patients. In hepatocellular carcinoma (HCC), high mRNA expression of autophagy markers was related to poor clinical status; increasing LC3 expression in HCC cell lines could promote tumor growth. Tumor intrinsic PD1 or PD-L1 were related to higher autophagy levels in specific tumor types; over-expression of PD1 or PD-L1 could increase autophagy in tumor cells through ATG13 interaction. Autophagy could promote tumor growth in specific cancer types. Tumor intrinsic PD1 or PD-L1 could both increase autophagy through ATG13 interaction.

Background: Cell adhesion molecule cluster of differentiation 44 (CD44) plays a significant role in cancer cell local invasion, intravasation, migration, and the establishment of metastatic lesions. However, little is known about the underlying mechanism of how CD44 regulates hepatocellular carcinoma (HCC) extrahepatic metastasis (EHM). Methods: The expression of CD44 in HCC tissues and cell lines was detected through western blot and immunohistochemistry (IHC). Through gain- and loss-of-function assays, we examined the oncogenic roles of CD44 in regulating HCC cell growth and metastasis in vitro and in vivo. To identify the potential mechanism, we employed quantitative real-time polymerase chain reaction, and western blot. Results: In this study, CD44 was highly expressed in HCC cells and HCC-patient specimens that exhibited high malignancy potential. The overall survival (OS) was worse and the cumulative recurrence rate was higher in HCC patients with CD44 overexpression than those with low levels of CD44 expression. Our in-vitro and in-vivo experiments showed that CD44 downregulation reduced HCC cell colony formation, migration, and invasion, and HCC tumor growth and metastasis, and that the pro-metastasis effect of CD44 was mediated by the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) signaling-chemokine receptor C-X-C chemokine receptor type 4 (CXCR4) axis. The reported capacity of CD44 to induce CXCR4 expression and increase the propensity of tumors to invade and metastasize to distant organs is consistent with the aggressive clinical characteristics of HCCs. Conclusions: CD44 could represent a future therapeutic target for EHM.

Tumor-infiltrating myeloid cells (TIMs) are pivotal cell populations involved in the immunosuppressive tumor immune microenvironment (TIME). However, there has been little success in large-scale clinical trials of myeloid cell modulators. We aim to investigate potential molecular targets for TIMs and disclose the underlying mechanism. Using mass cytometry by time of flight (CyTOF), we analyzed 24 spontaneous HCC tissues from mouse. Orthotopic and subcutaneous tumor models were established with or without anti-SIRPα antibody treatment. Patient-derived tumor xenografts model (PDX) was used to identify the CD47-SIRPα axis blocked therapy. In 24 murine spontaneous HCC tissues, we observed that the proportion of myeloid-derived suppressor cells (MDSCs) plus macrophages accounts for 40–90% of TIMs and SIRPα was highly expressed in TIMs, especially in macrophages and MDSCs. Through in vivo experiments, we showed that anti-SIRPα therapy inhibited tumor growth, accompanied by increased CD8⁺ T cells infiltration and decreased TIMs including MDSCs and macrophages. We found that anti-SIRPα inhibited immunosuppressive function, migration and PD-L1 expression of myeloid cells. In a series of in vivo experiments, we demonstrated the anti-tumor and immune-active effect of SIRPα-blocked therapy. Mechanistically, anti-SIRPα inhibited the immunosuppressive function and PD-L1 expression of TIMs through downregulating PI3K/AKT signaling in myeloid cells. At last, anti-SIRPα enhanced the antitumor effect of anti-PD-L1 therapy in orthotopic and spontaneous murine models. Together, SIRPα blocked therapy reversed the immunosuppressive TIME, which provides a promising therapeutic rationale for increasing the efficacy of anti-PD-L1 therapy in treating HCC.