Background: Recent data indicated that macrophages may mutually interact with cancer cells to promote tumor progression and chemoresistance, but the interaction in cholangiocarcinoma (CCA) is obscure. Methods: 10x Genomics single-cell sequencing technology was used to identified the role of macrophages in CCA. Then, we measured the expression and prognostic role of macrophage markers and aPKC_ɩ in 70 human CCA tissues. Moreover, we constructed monocyte-derived macrophages (MDMs) generated from peripheral blood monocytes (PBMCs) and polarized them into M1/M2 macrophages. A co-culture assay of the human CCA cell lines (TFK-1, EGI-1) and differentiated PBMCs-macrophages was established, and functional studies in vitro and in vivo was performed to explore the interaction between cancer cells and M2 macrophages. Furthermore, we established the cationic liposome-mediated co-delivery of gemcitabine and aPKC_ɩ-siRNA and detect the antitumor effects in CCA. Results: M2 macrophage showed tumor-promoting properties in CCA. High levels of aPKC_ɩ expression and M2 macrophage infiltration were associated with metastasis and poor prognosis in CCA patients. Moreover, CCA patients with low M2 macrophages infiltration or low aPKC_ɩ expression benefited from postoperative gemcitabine-based chemotherapy. Further studies showed that M2 macrophages-derived TGFβ1 induced epithelial-mesenchymal transition (EMT) and gemcitabine resistance in CCA cells through aPKC_ɩ-mediated NF-κB signaling pathway. Reciprocally, CCL5 was secreted more by CCA cells undergoing aPKC_ɩ-induced EMT and consequently modulated macrophage recruitment and polarization. Furthermore, the cationic liposome-mediated co-delivery of GEM and aPKC_ɩ-siRNA significantly inhibited macrophages infiltration and CCA progression. Conclusion: our study demonstrates the role of Macrophages-aPKC_ɩ-CCL5 Feedback Loop in CCA, and proposes a novel therapeutic strategy of aPKC_ɩ-siRNA and GEM co-delivered by liposomes for CCA.

Objective Intrahepatic cholangiocarcinoma (iCCA) is a highly lethal hepatobiliary malignancy with an increasing incidence annually. Extensive research has elucidated the existence of a reciprocal interaction between platelets and cancer cells, which promotes tumor proliferation and metastasis. This study aims to investigate the function and mechanism underlying iCCA progression driven by the interplay between platelets and tumor cells, aiming to provide novel therapeutic strategies for iCCA. Methods The associations between platelets and cancer development were investigated by analyzing the peripheral blood platelet count, degree of platelet activation and infiltration in the microenvironment of patients with iCCA. By co-culturing tumor cells with platelets, the influence of platelet stimulation on the epithelial-mesenchymal transition (EMT), proliferation, and metastasis of iCCA cells was assessed through in vitro and in vivo experiments. Quantitative proteomic profiling was conducted to identify key downstream targets that were altered in tumor cells following platelet stimulation. The RNA interference technique was utilized to investigate the impacts of gene silencing on the malignant biological behaviors of tumor cells. Results Compared with healthy adults, patients with iCCA presented significantly higher levels of peripheral blood platelet counts, platelet activation and infiltration degrees, which were also found to be correlated with patient prognosis. Platelet stimulation greatly facilitated the EMT of iCCA cells, leading to enhanced proliferative and metastatic capabilities. Mechanistically, proteomic profiling identified a total of 67 up-regulated and 40 down-regulated proteins in iCCA cells co-cultured with platelets. Among these proteins, two elevated targets ILK and ITGB3, were further demonstrated to be partially responsible for platelet-induced iCCA progression, which might depend on their regulatory effects on FAK/PI3K/AKT signaling transduction. Conclusions Our data revealed that platelet-related indices were abnormally ascendant in iCCA patients compared to healthy adults. Co-culturing with platelets enhanced the progression of EMT, and the motility and viability of iCCA cells in vitro and in vivo. Proteomic profiling discovered that platelets promoted the development of iCCA through FAK/PI3K/AKT pathway by means of elevating the expression of ILK and ITGB3, indicating that both proteins are promising therapeutic targets for iCCA with the guidance of platelet-related indices.

Liver cancer is the second leading cause of tumor‑related death worldwide, and a serious threat to lives and health. Circulating tumor cells (CTCs) facilitate the progression of various cancers, including liver cancer. The relationship between CTC/circulating tumor microemboli‑related genes (CRGs) and the prognosis of liver cancer is unclear. The aim of the present study was to identify CTC/circulating tumour microemboli‑related genes (CRGs) in hepatocellular carcinoma and to investigate their clinical significance. Transcriptomic data from The Cancer Genome Atlas (International Cancer Genome Consortium (ICGC) and GSE117623 databases were combined, and differentially expressed CRGs were identified. These were subsequently analyzed via least absolute shrinkage and selection operator and multivariate Cox analyses, and a five‑gene risk signature was constructed. The signature was validated in the ICGC and GSE14520 dataset with survival analysis and receiver operating characteristic curve analysis. Immunocyte infiltration, tumor mutation burden (TMB), tumor immune dysfunction and exclusion (TIDE), and the somatic mutation rate were also compared between high‑ and low‑risk groups, based on the median predictive index, to further evaluate the immunotherapeutic value of the model. Molecular subtypes of liver cancer were characterized by the non‑negative matrix factorization method and potential therapeutic compounds were evaluated for different subtypes. Nomograms were utilized to predict the prognosis of patients, and the signature was compared with previous literature models. Additionally, the biological function of one of the CRGs, tumor protein p53 inducible protein 3 (TP53I3), in liver cancer was further explored through in vitro experiments. Analysis of the prognostic characteristics of the five CRGs led to the identification of two liver cancer subtypes. Patients in the low‑risk group had a longer survival compared with those in the high‑risk group, and patients in the latter group were associated with a higher TMB, immunocyte infiltration and somatic mutation rate, and lower TIDE scores. The prognostic profile was validated in the ICGC and GSE14520 datasets and exhibited a good predictive performance. In vitro analysis showed that the knockdown of TP53I3 suppressed liver cancer cell proliferation. In summary, CRGs were used to develop a new prognostic signature to predict the prognosis of patients with liver cancer. This signature may be used to assess the prognosis of patients and may provide new insights for clinical management strategies. In addition, TP53I3 is potentially a therapeutic target for liver cancer.

Objective Hepatocellular carcinoma (HCC) is an extremely deadly cancer with few effective therapeutic options available. Ceramide synthases (CERS), a family of enzymes that regulate sphingolipid metabolism, have been suggested to play a role in cancer initiation and progression. Whereas the specific functions of CERS in HCC pathogenesis have not yet been fully elucidated. Methods The TCGA and ICGC databases were employed to analyze the expression levels and clinical relevance of CERS genes in HCC. Functional enrichment analyses were performed to identify pathways and functions associated with CERS5. The correlation between CERS5 and the tumor immune microenvironment was investigated. The mutation landscape and immunotherapy efficacy were evaluated. Functional experiments in vitro were conducted to assess CERS5’s impact on HCC cell proliferation and invasion. Results Aberrant expression of the CERS family was detected not only in HCC but also in other cancers, and has been linked to both overall survival and disease-free survival. Among the CERS family members, CERS5 was identified as the only prognosis-related gene, with up-regulated in HCC validated in the ICGC database and clinical tissue samples. Higher expression levels of CERS5 were associated with a poorer prognosis as well as an advanced pathologic stage and grade, as confirmed by the TCGA and ICGC databases. Besides, a prognostic nomogram combining pathologic stage, tumor status, and the expression of CERS5 was established and further validated, which suggested a favorable value for prognosis prediction. Functional enrichment analyses showed that the overexpression of CERS5 resulted in enriched pathways associated with carcinogenesis, drug metabolism, the PI3K/AKT/mTOR signaling pathway, and cancer immune-related pathways. In addition, the overexpression of CERS5 correlated positively with the expression of genes associated with immunogenic cell death modulators and immune checkpoints, levels of immune cell infiltration, and immunotherapy response, which was featured in an immunologically “hot” environment in the tumor microenvironment. Finally, the functional experiments showed that CERS5 knockdown has been shown to inhibit the growth and invasion of hepatocellular carcinoma, potentially through targeting the PI3K/AKT/mTOR signaling pathway. Conclusions Based on our findings, CERS5 appears to have great potential as both a precise prognostic biomarker and a novel therapeutic target in HCC.

Background: Recent data indicated that macrophages may mutually interact with cancer cells to promote tumor progression and chemoresistance, but the interaction in cholangiocarcinoma (CCA) is obscure. Methods: 10x Genomics single-cell sequencing technology was used to identified the role of macrophages in CCA. Then, we measured the expression and prognostic role of macrophage markers and aPKCi in 70 human CCA tissues. Moreover, we constructed monocyte-derived macrophages (MDMs) generated from peripheral blood monocytes (PBMCs) and polarized them into M1/M2 macrophages. A co-culture assay of the human CCA cell lines (TFK-1, EGI-1) and differentiated PBMCs-macrophages was established, and functional studies in vitro and in vivo was performed to explore the interaction between cancer cells and M2 macrophages. Furthermore, we established the cationic liposome-mediated co-delivery of gemcitabine and aPKCi-siRNA and detect the antitumor effects in CCA. Results: M2 macrophage showed tumor-promoting properties in CCA. High levels of aPKCi expression and M2 macrophage infiltration were associated with metastasis and poor prognosis in CCA patients. Moreover, CCA patients with low M2 macrophages infiltration or low aPKCi expression benefited from postoperative gemcitabine-based chemotherapy. Further studies showed that M2 macrophages-derived TGFb1 induced epithelial-mesenchymal transition (EMT) and gemcitabine resistance in CCA cells through aPKCi-mediated NF-kB signaling pathway. Reciprocally, CCL5 was secreted more by CCA cells undergoing aPKCi-induced EMT and consequently modulated macrophage recruitment and polarization. Furthermore, the cationic liposome-mediated co-delivery of GEM and aPKCi-siRNA significantly inhibited macrophages infiltration and CCA progression. Conclusion: our study demonstrates the role of Macrophages-aPKCi-CCL5 Feedback Loop in CCA, and proposes a novel therapeutic strategy of aPKCi-siRNA and GEM co-delivered by liposomes for CCA.