Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide because of rapid progression and high incidence of metastasis or recurrence. Accumulating evidence shows that CD73-expressing tumor cell is implicated in development of several types of cancer. However, the role of CD73 in HCC cell has not been systematically investigated and its underlying mechanism remains elusive. CD73 expression in HCC cell was determined by RT-PCR, Western blot, and immunohistochemistry staining. Clinical significance of CD73 was evaluated by Cox regression analysis. Cell counting kit-8 and colony formation assays were used for proliferation evaluation. Transwell assays were used for motility evaluations. Co-immunoprecipitation, cytosolic and plasma membrane fractionation separation, and ELISA were applied for evaluating membrane localization of P110β and its catalytic activity. NOD/SCID/γc(null) (NOG) mice model was used to investigate the in vivo functions of CD73. In the present study, we demonstrate that CD73 was crucial for epithelial-mesenchymal transition (EMT), progression and metastasis in HCC. CD73 expression is increased in HCC cells and correlated with aggressive clinicopathological characteristics. Clinically, CD73 is identified as an independent poor prognostic indicator for both time to recurrence and overall survival. CD73 knockdown dramatically inhibits HCC cells proliferation, migration, invasion, and EMT in vitro and hinders tumor growth and metastasis in vivo. Opposite results could be observed when CD73 is overexpressed. Mechanistically, adenosine produced by CD73 binds to adenosine A2A receptor (A2AR) and activates Rap1, which recruits P110β to the plasma membrane and triggers PIP3 production, thereby promoting AKT phosphorylation in HCC cells. Notably, a combination of anti-CD73 and anti-A2AR achieves synergistic depression effects on HCC growth and metastasis than single agent alone. CD73 promotes progression and metastasis through activating PI3K/AKT signaling, indicating a novel prognostic biomarker for HCC. Our data demonstrate the importance of CD73 in HCC in addition to its immunosuppressive functions and revealed that co-targeting CD73 and A2AR strategy may be a promising novel therapeutic strategy for future HCC management.

The role of sphere-forming culture in enriching subpopulations with stem-cell properties in hepatocellular carcinoma (HCC) is unclear. The present study investigates its value in enriching cancer stem cells (CSCs) subpopulations and the mechanism by which HCC CSCs are maintained. HCC cell lines and fresh primary tumor cells were cultured in serum-free and ultra-low attachment conditions to allow formation of HCC spheres. In vitro and in vivo experiments were performed to evaluate CSC characteristics. Expression levels of CSC-related genes were assessed by qRT-PCR and the correlation between sphere formation and clinical characteristics was investigated. Finally, gene expression profiling was performed to explore the molecular mechanism underlying HCC CSC maintenance. We found that both cell lines and primary tumor cells formed spheres. HCC spheres possessed the capacity for self-renewal, proliferation, drug resistance, and contained different subpopulations of CSCs. Of interest, 500 sphere-forming Huh7 cells or 200 primary tumor cells could generate tumors in immunodeficient animals. Sphere formation correlated with size, multiple tumors, satellite lesions, and advanced stage. Further investigation identified that the PPARα-SCD1 axis plays an important role in maintenance of the CSC properties of HCC sphere cells by promoting nuclear accumulation of β-Catenin. Inhibition of SCD1 interfered with sphere formation, down-regulated expression of CSC-related markers, and reduced β-Catenin nuclear accumulation. Sphere-forming culture can effectively enrich subpopulations with stem-cell properties, which are maintained through activation of the PPARα-SCD1 axis. Therefore, we suggest that targeting the SCD1-related CSC machinery might provide a novel insight into HCC treatment.

Lymph node metastasis (LNM) facilitates distant tumor colonization and leads to the high mortality in patients with intrahepatic cholangiocarcinoma (ICC). However, it remains elusive how ICC cells subvert immune surveillance within the primary tumor immune microenvironment (TIME) and subsequently metastasize to lymph nodes (LNs). In this study, scRNA-seq and bulk RNA-seq analyses identified decreased infiltration of dendritic cells (DCs) into primary tumor sites of ICC with LNM, which was further validated via dual-color immunofluorescence staining of 219 surgically resected ICC samples. Tumor-infiltrating DCs correlated with increased CD8⁺ T cell infiltration and better prognoses in ICC patients. Mechanistically, β-catenin-mediated CXCL12 suppression accounted for the impaired DC recruitment in ICC with LNM. Two mouse ICC cell lines MuCCA1 and mIC-23 cells were established from AKT/NICD or AKT/YAP-induced murine ICCs respectively and were utilized to construct the footpad tumor LNM model. We found that expansion and activation of conventional DCs (cDCs) by combined Flt3L and poly(I:C) (FL-pIC) therapy markedly suppressed the metastasis of mIC-23 cells to popliteal LNs. Moreover, β-catenin inhibition restored the defective DC infiltration into primary tumor sites and reduced the incidence of LNM in ICC. Collectively, our findings identify tumor cell intrinsic β-catenin activation as a key mechanism for subverting DC-mediated anti-tumor immunity in ICC with LNM. FL-pIC therapy or β-catenin inhibitor could merit exploration as a potential regimen for mitigating ICC cell metastasis to LNs and achieving effective tumor immune control.

Background: Predicting the efficacy of immune-based therapy in patients with unresectable hepatocellular carcinoma (HCC) remains a clinical challenge. This study aims to evaluate the prognostic value of the systemic immune-inflammation index (SII) in forecasting treatment response and survival outcomes for HCC patients undergoing immune-based therapy. Methods: We analyzed a cohort of 268 HCC patients treated with immune-based therapy from January 2019 to March 2023. A training cohort of 93 patients received atezolizumab plus bevacizumab (T + A), while a validation cohort of 175 patients underwent treatment with tyrosine kinase inhibitors (TKIs) combined with anti-PD-(L)1 therapy. The SII cutoff value, determined using X-tile analysis based on overall survival (OS) in the training cohort, divided patients into high (> 752*10⁹) and low (≤ 752*10⁹) SII groups. Prognostic factors were identified through univariate and multivariate logistic and Cox regression analyses, and survival outcomes were assessed using Kaplan–Meier methods. The predictive accuracy of SII was evaluated using receiver operating characteristic (ROC) curves. Results: An optimal SII cutoff of 752*10⁹ stratified patients into high and low SII groups. Univariate and multivariate logistic regression indicated that SII was a significant predictor of the objective response rate (ORR), which was markedly different between the low and high SII subgroups (34.72% vs. 9.52%, P = 0.019). This finding was consistent in the validation cohort (34.09% vs. 16.28%, P = 0.026). SII also demonstrated prognostic value in Cox regression and Kaplan–Meier analyses. ROC curves confirmed that SII had superior predictive accuracy compared to common clinical indicators, with predictive relevance even in AFP-negative patients. Furthermore, a lower SII was associated with a higher T cell ratio and an increased number of CD8⁺ T cells and Granzyme B⁺ CD8⁺ T cells in peripheral blood. Conclusion: SII is a promising predictor of both therapeutic efficacy and prognosis in HCC patients undergoing immune-based treatments. Its application may enhance clinical decision-making, thereby improving patient outcomes from immune-based therapy.

Background & aims: Patients with intrahepatic cholangiocarcinoma (iCCA) respond poorly to immune checkpoint blockades (ICBs). In this study, we aimed to dissect the potential mechanisms underlying poor response to ICBs and explore a rational ICB-based combination therapy in iCCA. Methods: scRNA-seq dataset GSE151530 was analyzed to investigate the differentially expressed genes in malignant cells following ICBs therapy. RNA-seq analysis and western blot assays were performed to examine the upstream and downstream signaling pathways of CD73. Subcutaneous tumor xenograft models were utilized to investigate the impact of CD73 on iCCA growth. Plasmid AKT/NICD-induced spontaneous murine iCCAs were used to explore the therapeutic efficacy of CD73 enzymatic inhibitor AB680 combined with PD-1 blockade. Time-of-flight mass cytometry (CyTOF) was conducted to identify the tumor-infiltrating immune cell populations and their functional changes in murine iCCAs treated with AB680 in combination with PD-1 antibody. Results: scRNA-seq analysis identified elevated CD73 expression in malignant cells in response to ICBs therapy. Mechanistically, ICBs therapy upregulated CD73 expression in malignant cells via TNF-α/NF-κB signaling pathway. In vivo studies revealed that CD73 inhibition suppressed the growth of subcutaneous tumors, and achieved synergistic depression effects with gemcitabine and cisplatin (GC). Adenosine produced by CD73 activates AKT/GSK3β/β-catenin signaling axis in iCCA cells. CD73 inhibitor AB680 potentiates anti-tumor efficacy of PD-1 antibody in murine iCCAs. CyTOF analysis showed that AB680 combined with anti-PD-1 therapy promoted the infiltration of CD8⁺ T, CD4⁺ T cells, and NK cells in murine iCCAs, while simultaneously decreased the proportions of macrophages and neutrophils. Moreover, AB680 combined with anti-PD-1 significantly upregulated the expression of Granzyme B, Tbet and co-stimulatory molecule ICOS in infiltrating CD8⁺ T cells. Conclusions: CD73 inhibitor AB680 limits tumor progression and potentiates therapeutic efficacy of GC chemotherapy or anti-PD-1 treatment in iCCA. AB680 combined with anti-PD-1 therapy effectively elicits anti-tumor immune response.

Immune checkpoint inhibitors (ICI) benefit some cancer patients but de novo resistance remains poorly understood. Analyzing transcriptional data from two clinical trial cohorts, GO30140 and IMbrave150, we find B cell lymphoma 9 (BCL9), a Wnt/β-catenin co-factor, associated with resistance. We develop a BCL9-targeting peptide, hsBCL9_Z96, which suppresses tumor growth in combination with anti-PD-L1 ab in preclinical hepatocellular carcinoma (HCC) mouse models. Multi-omics analyses implicate targeting BCL9 inhibits BMP4 secretion and downregulates CD24 on tumor cells, reprogramming macrophages toward a tumor-suppressive phenotype and promoting macrophage phagocytosis. This in turn rejuvenates T cell immunity via enhanced macrophage-mediated antigen presentation. Our data extend our understanding of how tumor-derived Wnt/β-catenin signaling impedes the innate and adaptive immune responses in the tumor microenvironment and provide preliminary evidence that targeting BCL9 is a promising preclinical strategy to mitigate ICI resistance in HCC.