This multicenter retrospective study investigated the clinical utility of patient-derived tumor organoids (PDOs) in guiding adjuvant chemotherapy for intrahepatic cholangiocarcinoma (ICC) patients. A total of 82 ICC patients at the Shanghai Eastern Hepatobiliary Surgery Hospital and the Shanghai Tenth People’s Hospital between 2021 and 2022 were included in the study. All of these patients underwent surgical resection and were pathologically confirmed to have high-risk factors for recurrence. Therefore, they received adjuvant chemotherapy post-surgery. Sixty-one PDOs were successfully established from resected tumor tissues and tested against four common regimens: Gemcitabine + cisplatin, gemcitabine + oxaliplatin, capecitabine, and S-1. A novel fluorescence-based method was developed for drug sensitivity testing, showing significant concordance with conventional half-maximal inhibitory concentration quantification. Patients were stratified into matched (n=26) and unmatched (n=35) groups based on alignment between PDOs-based drug sensitivity results and actual chemotherapy received. The matched group demonstrated significantly improved recurrence-free survival (RFS) (hazard ratio = 2.52, p=0.007), with 63% versus 17% experiencing recurrence events. Through eXtreme Gradient Boosting machine learning and Cox regression analyses, we identified PDOs-based drug sensitivity, tumor multiplicity, and vascular invasion as key predictors of 2-year recurrence. A clinical nomogram incorporating these factors showed that patients with matched PDO-guided therapy had 46% 12-month RFS probability versus 28% in unmatched cases. This study provides a clinically translatable platform to guide adjuvant chemotherapy decisions and predict the prognosis of ICC patients with a high recurrence risk after surgery.

Background: Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with limited treatment options in advanced stages. While c-Met is a promising therapeutic target in HCC, identifying patients who will benefit from c-Met inhibitors remains a significant challenge. This study aimed to investigate the role of HHLA2, a B7 family member, in HCC and its potential as a liquid biopsy marker for c-Met inhibitor therapy. Methods: HHLA2 expression was analyzed in clinical HCC samples and public databases. In vitro studies using HCC cell lines assessed HHLA2’s impact on proliferation, migration, invasion, and angiogenesis. In vivo studies using mouse models (orthotopic xenografts and hydrodynamic tail vein injection) evaluated HHLA2’s role in tumor growth and metastasis. Mass spectrometry, co-immunoprecipitation, split-luciferase, and ELISA assays were used to investigate HHLA2-c-Met interactions. Patient-derived organoids (PDOs) were used to assess drug response. Statistical analyses included Student’s t-tests, ANOVA, and Cox regression. Results: HHLA2 was found to be upregulated in HCC and associated with advanced disease, aggressive clinicopathological features, and poor prognosis. HHLA2 interacted with and constitutively activated c-Met, leading to increased expression of MMP9 and VEGFA, enhancing HCC cell proliferation, invasion, and angiogenesis. HHLA2 also suppressed hepatic natural killer cell infiltration in vivo. Inhibition of c-Met with PHA665752 effectively reversed HHLA2-mediated tumor-promoting effects in vitro and in vivo. HHLA2 expression in HCC tissues correlated with c-Met phosphorylation, and HHLA2 could be detected in the serum of patients with high tumor HHLA2 levels. PDOs with high HHLA2 expression exhibited increased sensitivity to c-Met inhibition. Conclusions: HHLA2 acts as an oncogene in HCC by activating c-Met, promoting tumor progression and metastasis. HHLA2 expression correlates with c-Met activation and predicts poor prognosis in HCC patients. Importantly, HHLA2 can serve as a stratification marker for c-Met inhibitor therapy, potentially enabling a personalized approach to improve therapeutic outcomes in this challenging disease.

Introduction: Colorectal cancer liver metastasis (CRLM) develops in a hepatic niche where tumor cells encounter organ‑specific microbial, metabolic, and immune constraints. The role of intratumoral bacteria in this metastatic ecosystem remains incompletely defined. Methods: We analyzed tumor and peritumoral tissues from 20 patients with non‑metastatic colorectal cancer (CRC) and 20 patients with CRLM by 5R 16S rDNA sequencing, untargeted metabolomics, and functional assays. Public datasets were used for single‑cell analysis of the tumor immune microenvironment. Results: Primary colorectal lesions and paired liver metastases showed distinct microbial structures. Liver metastatic lesions had lower microbial diversity, and 73 genera differed between CRLM primary tumors and liver metastases. Bacillus was enriched in metastatic tumors and was associated with fatty‑acid‑related microbial pathways, serum CEA and CA19‑9 levels, and lipid‑related metabolites that accumulated in liver metastases. Bacillus supernatant increased SW480 and SW620 cell migration and proliferation in vitro , with the strongest activity at a multiplicity of infection (MOI)‑equivalent of 20. In an intrasplenic CRLM model, viable Bacillus and Bacillus supernatant increased metastatic tumor growth, whereas antibiotic intervention reduced this effect. Metabolomic profiling detected enrichment of lipids and lipid‑like molecules, including carnitine‑ and bile‑acid‑related metabolites, in liver metastatic lesions. Single‑cell analysis of public datasets showed remodeling of the tumor immune microenvironment (TIME) and identified APOE⁺ macrophages as lipid‑associated myeloid cells with broad ligand‑receptor communication with epithelial, immune, and stromal compartments. Discussion: Together, these findings provide evidence that Bacillus promotes metastatic phenotypes and reveal associations among intratumoral Bacillus , lipid‑related metabolic alterations, and APOE ⁺ macrophage‑associated communication signatures in CRLM.