Oleic acid-PPARγ-FABP4 loop fuels cholangiocarcinoma colonization in lymph node metastases microenvironment.

BACKGROUND AND AIMS: Lymph node metastasis is a significant risk factor for patients with cholangiocarcinoma, but the mechanisms underlying cholangiocarcinoma colonization in the lymph node microenvironment remain unclear. We aimed to determine whether metabolic reprogramming fueled the adaptation and remodeling of cholangiocarcinoma cells to the lymph node microenvironment. APPROACH AND RESULTS: Here, we applied single-cell RNA sequencing of primary tumor lesions and paired lymph node metastases from patients with cholangiocarcinoma and revealed significantly reduced intertumor heterogeneity and syntropic lipid metabolic reprogramming of cholangiocarcinoma after metastasis to lymph nodes, which was verified by pan-cancer single-cell RNA sequencing analysis, highlighting the essential role of lipid metabolism in tumor colonization in lymph nodes. Metabolomics and in vivo CRISPR/Cas9 screening identified PPARγ as a crucial regulator in fueling cholangiocarcinoma colonization in lymph nodes through the oleic acid-PPARγ-fatty acid-binding protein 4 positive feedback loop by upregulating fatty acid uptake and oxidation. Patient-derived organoids and animal models have demonstrated that blocking this loop impairs cholangiocarcinoma proliferation and colonization in the lymph node microenvironment and is superior to systemic inhibition of fatty acid oxidation. PPARγ-regulated fatty acid metabolic reprogramming in cholangiocarcinoma also contributes to the immune-suppressive niche in lymph node metastases by producing kynurenine and was found to be associated with tumor relapse, immune-suppressive lymph node microenvironment, and poor immune checkpoint blockade response. CONCLUSIONS: Our results reveal the role of the oleic acid-PPARγ-fatty acid-binding protein 4 loop in fueling cholangiocarcinoma colonization in lymph nodes and demonstrate that PPARγ-regulated lipid metabolic reprogramming is a promising therapeutic target for relieving cholangiocarcinoma lymph node metastasis burden and reducing further progression.

Cancer-Associated Fibroblasts Promote Lymphatic Metastasis in Cholangiocarcinoma via the PDGF-BB/PDGFR-ß Mediated Paracrine Signaling Network.

Patients with cholangiocarcinoma (CCA) with lymph node metastasis (LNM) have the worst prognosis, even after complete resection; however, the underlying mechanism remains unclear. Here, we established CAF-derived PDGF-BB as a regulator of LMN in CCA. Proteomics analysis revealed upregulation of PDGF-BB in CAFs derived from patients with CCA with LMN (LN+CAFs). Clinically, the expression of CAF-PDGF-BB correlated with poor prognosis and increased LMN in patients with CCA, while CAF-secreted PDGF-BB enhanced lymphatic endothelial cell (LEC)-mediated lymphangiogenesis and promoted the trans-LEC migration ability of tumor cells. Co-injection of LN+CAFs and cancer cells increased tumor growth and LMN in vivo. Mechanistically, CAF-derived PDGF-BB activated its receptor PDGFR-ß and its downstream ERK1/2-JNK signaling pathways in LECs to promote lymphoangiogenesis, while it also upregulated the PDGFR-ß-GSK-P65-mediated tumor cell migration. Finally, targeting PDGF-BB/PDGFR-ß or the GSK-P65 signaling axis prohibited CAF-mediated popliteal lymphatic metastasis (PLM) in vivo. Overall, our findings revealed that CAFs promote tumor growth and LMN via a paracrine network, identifying a promising therapeutic target for patients with advanced CCA.

Identification of a Novel Prognostic Signature Based on N-Linked Glycosylation and Its Correlation with Immunotherapy Response in Hepatocellular Carcinoma.

Background: The complex tumor microenvironment of hepatocellular carcinoma (HCC) has led to a low response to immune checkpoints inhibitors (ICIs) and a poor prognosis. PD-L1, as one of the indications for ICIs, is rich in glycosylation modifications, which result in untimely ICIs. Our study constructed a prognostic model based on N-linked glycosylation related genes for predicting the prognosis and the response to ICIs. Methods: The list of N-linked glycosylation related genes is from the AmiGO2 database. The patients in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) cohorts were enrolled. The Cox regression was performed to develop a prognostic model and patients were divided into a low- and high-risk subgroups. The role of signature in HCC was well investigated by prognostic analysis, gene set enrichment analysis, and immune infiltration analysis. 21 recurrent HCC patients who received postoperative adjuvant ICIs were recruited to evaluate the relationship between immunotherapy response and the signature. In vitro studies were conducted to investigate the oncogenic effects of DDOST, STT3A and TMEM165 in HCC. Results: 59 N-linked glycosylation related differentially expressed genes were screened from HCC and normal tissues in the TCGA cohort. The prognostic model was developed with DDOST, STT3A and TMEM165. The risk score could be an independent prognostic factor. Patients in the high-risk subgroup showed a worse prognosis than patients in the low-risk one. ssGSEA showed that patients in the low-risk subgroup tended to be in the immune-activated state, with higher levels of B cell and macrophage cell infiltrations and lower levels of regulatory T cell (Treg) infiltrations in both TCGC and GEO cohorts. Immunohistochemistry studies showed that DDOST, STT3A and TMEM165 are highly expressed in tumor tissues and patients with a high-risk score correlated with poor progression free survival and worse immunotherapeutic response. Furthermore, the proliferation of HCC cells was reduced after the knockdown of DDOST, as well as upon the knockdown of STT3A and TMEM165. Conclusion: In this study, we establish that the risk model based on N-linked glycosylation related genes could efficiently predict the prognosis and tumor microenvironment immune state of HCC patients, and the risk score could serve as a novel indicator of immunotherapy.

Bioinformatic Analysis and In Vitro and In Vivo Experiments Reveal That Fibrillarin Participates in the Promotion of Lung Metastasis in Hepatocellular Carcinoma.

Lung metastasis, the most frequent metastatic pattern in hepatocellular carcinoma, is an important contributor to poor prognosis. However, the mechanisms responsible for lung metastasis in hepatocellular carcinoma remain unknown. Aiming to explore these mechanisms, weighted gene coexpression network analysis (WGCNA) was firstly used to find hub genes related to lung metastasis. Then, we obtained 67 genes related to lung metastasis in hepatocellular carcinoma which were mainly related to ribosomal pathways and functions, and a protein interaction network analysis identified that fibrillarin (FBL) might be an important hub gene. Furthermore, we found that FBL is highly expressed in hepatocellular carcinoma and that its high expression increases the rate of lung metastasis and indicates a poor prognosis. Knockdown of FBL could significantly reduce proliferation and stemness as well as inhibiting the migration and invasion of hepatocellular carcinoma cells. Moreover, we found that FBL might be involved in the regulation of MYC and E2F pathways in hepatocellular carcinoma. Finally, we demonstrated that the knockdown of FBL could suppress hepatocellular carcinoma cell growth in vivo. In conclusion, ribosome-biogenesis-related proteins, especially Fibrillarin, play important roles in lung metastasis from hepatocellular carcinoma.

A Co-word Analysis of Selected Science Education Literature: Identifying Research Trends of Scaffolding in Two Decades (2000-2019).

This study aims to identify research trends of scaffolding in the field of science education. To this end, both descriptive analysis and co-word analysis were conducted to examine the selected articles published in the Social Science Citation Index journals from 2000 to 2019. A total of 637 papers were retrieved as research samples through rounds of searching in Web of Science database. Overall, this study reveals a growing trend of science educators' academic publications about scaffolding in the recent two decades. In these sample papers, from 1,487 non-repeated keywords, we extracted 286 author-defined keywords shared by at least two studies as a benchmark dictionary. A series of co-word analyses were then conducted based on the dictionary to reveal the underlying co-occurring relationships of the words in title and abstract of the sample papers. Results showed that "scaffolding," "support," and "design" were the top three most frequently used keywords during 2000 and 2019. Visualization of co-word networks in each 5-year period further helps clarify both educators' common research foci and relevant research trends. Derived discussion and potential research directions are also provided.

Cirsilineol attenuates LPS-induced inflammation in both in vivo and in vitro models via inhibiting TLR-4/NFkB/IKK signaling pathway.

The anti-inflammatory activity of cirsilineol in in vivo condition was assessed by measuring the relative organ weight, lung dry/wet weight ratio, protein concentration, and infiltration of inflammatory cells in bronchoalveolar lavage fluid. We estimated the myeloperoxidase activity and levels of cytokines, chemokines, and inflammatory markers to analyze the efficacy of cirsilineol against lipopolysaccharide (LPS)-induced lung inflammation. Furthermore, we quantified the gene expression of NFkB/IKK signaling molecules in cirsilineol-treated and untreated acute lung injury mice to confirm the anti-inflammatory property of cirsilineol. The lung histology was assessed with hematoxylin and eosin staining. Apart from in vivo experiments, in vitro tests with LPS-stimulated RAW 264.7 macrophages were also performed. Cell viability assay was performed in the presence and absence of LPS in RAW 264.7 macrophages to determine the cytotoxic effect of cirsilineol against macrophages. Reverse-transcription polymerase chain reaction (RT-PCR) analysis was done to analyze the gene expression of inflammatory markers in LPS-treated RAW 264.7 macrophages to prove that cirsilineol effectively inhibits inflammation in vitro. The results of our study prove that cirsilineol effectively inhibits inflammation in both in vivo and in vitro conditions. RT-PCR analysis results of NFkB/IKK signaling molecules clearly illustrate that cirsilineol inhibited the expression of NFkB/IKK signaling protein and thereby prevented inflammation in in vivo condition, and it is further confirmed with the results of inflammatory protein expression in vitro model. The lung histopathological studies authentically confirm that cirsilineol potentially prevented the mice from LPS-induced lung inflammation.

The Role of Endoglin in Hepatocellular Carcinoma.

Endoglin (CD105) is a type-1 integral transmembrane glycoprotein and coreceptor for transforming growth factor-ß (TGF-ß) ligands. The endoglin/TGF-ß signaling pathway regulates hemostasis, cell proliferation/migration, extracellular matrix (ECM) synthesis and angiogenesis. Angiogenesis contributes to early progression, invasion, postoperative recurrence, and metastasis in hepatocellular carcinoma (HCC), one of the most widespread malignancies globally. Endoglin is overexpressed in newly formed HCC microvessels. It increases microvessel density in cirrhotic and regenerative HCC nodules. In addition, circulating endoglin is present in HCC patients, suggesting potential for use as a diagnostic or prognostic factor. HCC angiogenesis is dynamic and endoglin expression varies by stage. TRC105 (carotuximab) is an antibody against endoglin, and three of its clinical trials were related to liver diseases. A partial response was achieved when combining TRC105 with sorafenib. Although antiangiogenic therapy still carries some risks, combination therapy with endoglin inhibitors or other targeted therapies holds promise.

Sonic Hedgehog Signaling in Organogenesis, Tumors, and Tumor Microenvironments.

During mammalian embryonic development, primary cilia transduce and regulate several signaling pathways. Among the various pathways, Sonic hedgehog (SHH) is one of the most significant. SHH signaling remains quiescent in adult mammalian tissues. However, in multiple adult tissues, it becomes active during differentiation, proliferation, and maintenance. Moreover, aberrant activation of SHH signaling occurs in cancers of the skin, brain, liver, gallbladder, pancreas, stomach, colon, breast, lung, prostate, and hematological malignancies. Recent studies have shown that the tumor microenvironment or stroma could affect tumor development and metastasis. One hypothesis has been proposed, claiming that the pancreatic epithelia secretes SHH that is essential in establishing and regulating the pancreatic tumor microenvironment in promoting cancer progression. The SHH signaling pathway is also activated in the cancer stem cells (CSC) of several neoplasms. The self-renewal of CSC is regulated by the SHH/Smoothened receptor (SMO)/Glioma-associated oncogene homolog I (GLI) signaling pathway. Combined use of SHH signaling inhibitors and chemotherapy/radiation therapy/immunotherapy is therefore key in targeting CSCs.

Sperm quality assessment via separation and sedimentation in a microfluidic device.

A major reason for infertility is due to male factors, including the quality of spermatozoa, which is a primary factor and often difficult to assess, particularly the total sperm concentration and its motile percentage. This work presents a simple microfluidic device to assess sperm quality by quantifying both total and motile sperm counts. The key design feature of the microfluidic device is two channels separated by a permeative phase-guide structure, where one channel is filled with raw semen and the other with pure buffer. The semen sample was allowed to reach equilibrium in both chambers, whereas non-motile sperms remained in the original channel, and roughly half of the motile sperms would swim across the phase-guide barrier into the buffer channel. Sperms in each channel agglomerated into pellets after centrifugation, with the corresponding area representing total and motile sperm concentrations. Total sperm concentration up to 10(8) sperms per ml and motile percentage in the range of 10-70% were tested, encompassing the cutoff value of 40% stated by World Health Organization standards. Results from patient samples show compact and robust pellets after centrifugation. Comparison of total sperm concentration between the microfluidic device and the Makler chamber reveal they agree within 5% and show strong correlation, with a coefficient of determination of R(2) = 0.97. Motile sperm count between the microfluidic device and the Makler chamber agrees within 5%, with a coefficient of determination of R(2) = 0.84. Comparison of results from the Makler Chamber, sperm quality analyzer, and the microfluidic device revealed that results from the microfluidic device agree well with the Makler chamber. The sperm microfluidic chip analyzes both total and motile sperm concentrations in one spin, is accurate and easy to use, and should enable sperm quality analysis with ease.