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Supplemental Figures and Tables Figure S1. The purity of the isolated fibroblasts. Figure S2. Knockdown of FAP or STAT3 impairs the inflammatory phenotype of CAFs. Figure S3. Forced FAP expression in the normal fibroblasts. Figure S4. Knockdown of STAT3 or uPAR has no effect on the expression of inflammatory genes in control fibroblasts. Figure S5. FAP knockdown abolishes the tumor-promoting effects of CAFs in Hepa1-6 tumor. Figure S6. FAP expression is dispensable for the direct effect of CAFs on the cancer stemness or the immune function of MDSCs or CD8+T cells. Figure S7. Stromal expression of p-STAT3 or CCL2 is negatively associated with the survival of ICC patients.

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Cancer-associated fibroblasts (CAF) are components of the tumor microenvironment whose contributions to malignant progression are not fully understood. Here, we show that the fibroblast activation protein (FAP) triggers induction of a CAF subset with an inflammatory phenotype directed by STAT3 activation and inflammation-associated expression signature marked by CCL2 upregulation. Enforcing FAP expression in normal fibroblasts was sufficient to endow them with an inflammatory phenotype similar to FAP+CAFs. We identified FAP as a persistent activator of fibroblastic STAT3 through a uPAR-dependent FAK–Src–JAK2 signaling pathway. In a murine liver tumor model, we found that FAP+CAFs were a major source of CCL2 and that fibroblastic STAT3–CCL2 signaling in this setting promoted tumor growth by enhancing recruitment of myeloid-derived suppressor cells (MDSC). The CCL2 receptor CCR2 was expressed on circulating MDSCs in tumor-bearing subjects and FAP+CAF-mediated tumor promotion and MDSC recruitment was abrogated in Ccr2-deficient mice. Clinically, we observed a positive correlation between stromal expression of FAP, p-STAT3, and CCL2 in human intrahepatic cholangiocarcinoma, a highly aggressive liver cancer with dense desmoplastic stroma, where elevated levels of stromal FAP predicted a poor survival outcome. Taken together, our results showed how FAP–STAT3–CCL2 signaling in CAFs was sufficient to program an inflammatory component of the tumor microenvironment, which may have particular significance in desmoplasia-associated cancers. Cancer Res; 76(14); 4124–35. ©2016 AACR.

IntroductionAlthough multiple targeted treatments have appeared, hepatocellular carcinoma (HCC) is still one of the most common causes of cancer-related deaths. The immunosuppressive tumor microenvironment (TME) is a critical factor in the oncogenesis and progression of HCC. The emerging scRNA-seq makes it possible to explore the TME at a high resolution. This study was designed to reveal the immune-metabolic crosstalk between immune cells in HCC and provide novel strategies to regulate immunosuppressive TME.MethodIn this study, we performed scRNA-seq on paired tumor and peri-tumor tissues of HCC. The composition and differentiation trajectory of the immune populations in TME were portrayed. Cellphone DB was utilized to calculate interactions between the identified clusters. Besides, flow cytometry, RT-PCR and seahorse experiments were implemented to explore potential metabolic and epigenetic mechanisms of the inter-cellular interaction.ResultA total of 19 immune cell clusters were identified and 7 were found closely related to HCC prognosis. Besides, differentiation trajectories of T cells were also presented. Moreover, a new population, CD3+C1q+ tumor-associated macrophages (TAM) were identified and found significantly interacted with CD8+ CCL4+T cells. Compared to the peri-tumor tissue, their interaction was attenuated in tumor. Additionally, the dynamic presence of this newly found cluster was also verified in the peripheral blood of patients with sepsis. Furthermore, we found that CD3+C1q+TAM affected T cell immunity through C1q signaling-induced metabolic and epigenetic reprogramming, thereby potentially affecting tumor prognosis.ConclusionOur study revealed the interaction between CD3+C1q+TAM and CD8+ CCL4+T cells and may provide implications for tackling the immunosuppressive TME in HCC.

Mixed lineage kinase domain-like (MLKL) is widely accepted as an executioner of necroptosis, in which MLKL mediates necroptotic signaling and triggers cell death in a receptor-interacting protein kinase 3 (RIPK3)-dependent manner. Recently, it is increasingly noted that RIPK3 is intrinsically silenced in hepatocytes, raising a question about the role of MLKL in hepatocellular carcinoma (HCC). This study reports a previously unrecognized role of MLKL in regulating parthanatos, a programmed cell death distinct from necroptosis. In HCC cells with intrinsic RIPK3 deficiency, knockout of MLKL impedes the orthotopic tumor growth, activates the anti-tumor immune response and enhances the therapeutic effect of immune checkpoint blockade in syngeneic HCC tumor models. Mechanistically, MLKL is required for maintaining the endoplasmic reticulum (ER)-mitochondrial Mg2+ dynamics in HCC cells. MLKL deficiency restricts ER Mg2+ release and mitochondrial Mg2+ uptake, leading to ER dysfunction and mitochondrial oxidative stress, which together confer increased susceptibility to metabolic stress-induced parthanatos. Importantly, pharmacological inhibition of poly(ADP-ribose) polymerase to block parthanatos restores the tumor growth and immune evasion in MLKL-knockout HCC tumors. Together, our data demonstrate a new RIPK3-independent role of MLKL in regulating parthanatos and highlight the role of MLKL in facilitating immune evasion in HCC.

The complement cascade plays a "complementing" role in human immunity. However, the potential roles of complement system in impacting molecular and clinical features of hepatocellular carcinoma (HCC) remain unclear. In this study, eleven public datasets are analyzed to compare the complement status between normal and cancerous samples based on 18 classical complement-associated genes. The complement scores are constructed to quantify complement signatures of individual tumors. HCC patients in the The Cancer Genome Atlas (TCGA) cohort are focused to perform systematical analyses between complement status and immune infiltration, miRNA expression, DNA methylation, clinicopathological features, and drug response. The results show that the complement scores in normal tissues are dramatically higher than those of tumor tissues. Tumor samples in the TCGA cohort are classified into complement score-low and score-high groups. Pathway analysis reveals that tumor-promoting pathways are typically inhibited in complement score-high group. This study also shows that tumor-killing immune cells, such as CD8

Monocarboxylate transporter (MCT) 4 is a high-affinity lactate transporter that is primarily involved in the maintenance of intracellular pH homeostasis and highly expressed in different tumors. However, the role of MCT4 in modulating immune responses against HCC remains unknown.In this study, we demonstrated that MCT4 was overexpressed in HCC, which was associated with poor prognosis in patients. Genetic or pharmacological inhibition of MCT4 using VB124 (a highly potent MCT4 inhibitor) suppressed HCC tumor growth in immunocompetent mice model by enhancing CD8Our results revealed that lactate exportation by MCT4 has a tumor-intrinsic function in generating an immunosuppressive HCC environment and demonstrated the proof of the concept of targeting MCT4 in tailoring HCC immunotherapeutic approaches.

Background Early-stage hepatocellular carcinoma (HCC) is the ideal indication for liver resection. High recurrence rate limits the radical possibility. Current clinicopathological determinants are insufficient to reliably evaluate the recurrence risk after surgery. To address this global issue, we aimed to use deep learning to explore novel pathological signatures based on histological slides for predicting early-stage HCC recurrence and to evaluate the relationship between histological features and multi-omics information. Methods 576 pathological images collected from 547 patients with BCLC stage 0-A HCC who underwent hepatectomy from 2006 to 2015 were randomly divided into the training cohort and validation cohort. The external validation cohort was composed of 147 TNM I patients from TCGA database. Weakly supervised convolutional neural networks were used to identify six classes of HCC tissues. Pathological signatures were extracted and two novel risk scores were constructed by LASSO Cox to predict recurrence. The forecast performance of the scores and patients' prognosis were evaluated. The relationship between histological score (HS) and immune infiltrating cells was estimated by clustering analysis. Results The classification accuracy of HCC tissue was 94.17%. The C-indexes of histological score in the training, validation and TCGA cohorts were 0.804, 0.739 and 0.708, respectively. Multivariate analysis showed that microvascular invasion (HR = 1.46, 95% CI: 1.09-1.95) and HS (HR = 4.05, 95% CI: 3.40-4.84) were independent risk factors for recurrence-free survival. Patients in HS high-risk group had elevated alpha fetoprotein, worse tumor differentiation and higher proportion of microvascular invasion. HS was positively correlated with the expression of CD14 in adjacent normal liver tissue (P = 0.013), and negatively correlated with the expression of CD8 in tumor (P Conclusions This study established and validated two novel risk scores based on the histological slides using deep learning. HS performed well in recurrence prediction for early-stage HCC patients and indication of important clinicopathological features.

The liver is a metabolically active organ and is also 'tolerogenic', exhibiting sophisticated mechanisms of immune regulation that prevent pathogen attacks and tumorigenesis. How metabolism impacts the tumor microenvironment (TME) in hepatocellular carcinoma (HCC) remains understudied.We investigated the role of the metabolic regulator SIRT5 in HCC development by conducting metabolomic analysis, gene expression profiling, flow cytometry and immunohistochemistry analyses in oncogene-induced HCC mouse models and human HCC samples.We show that SIRT5 is downregulated in human primary HCC samples and that Sirt5 deficiency in mice synergizes with oncogenes to increase bile acid (BA) production, via hypersuccinylation and increased BA biosynthesis in the peroxisomes of hepatocytes. BAs act as a signaling mediator to stimulate their nuclear receptor and promote M2-like macrophage polarization, creating an immunosuppressive TME that favors tumor-initiating cells (TICs). Accordingly, high serum levels of taurocholic acid correlate with low SIRT5 expression and increased M2-like tumor-associated macrophages (TAMs) in HCC patient samples. Finally, administration of cholestyramine, a BA sequestrant and FDA-approved medication for hyperlipemia, reverses the effect of Sirt5 deficiency in promoting M2-like polarized TAMs and liver tumor growth.This study uncovers a novel function of SIRT5 in orchestrating BA metabolism to prevent tumor immune evasion and suppress HCC development. Our results also suggest a potential strategy of using clinically proven BA sequestrants for the treatment of patients with HCC, especially those with decreased SIRT5 and abnormally high BAs.Hepatocellular caricinoma (HCC) development is closely linked to metabolic dysregulation and an altered tumor microenvironment. Herein, we show that loss of the metabolic regulator Sirt5 promotes hepatocarcinogenesis, which is associated with abnormally elevated bile acids and subsequently an immunosuppressive microenvironment that favors HCC development. Targeting this mechanism could be a promising clinical strategy for HCC.

Intrahepatic cholangiocarcinoma (ICC) remains a highly heterogeneous disease with poor prognosis. Tumor‐infiltrating lymphocytes were predictive in various cancers, but their prognostic value in ICC is less clear. A total of 168 ICC patients who had received liver resection were enrolled and assigned to the derivation cohort. Sixteen immune markers in tumor and peritumor regions were examined by immunohistochemistry. A least absolute shrinkage and selection operator model was used to identify prognostic markers and to establish an immune signature for ICC (ISICC). An ISICC‐applied prediction model was built and validated in another independent dataset. Five immune features, including CD3peritumor (P), CD57P, CD45RAP, CD66bintratumoral (T) and PD‐L1P, were identified and integrated into an individualized ISICC for each patient. Seven prognostic predictors, including total bilirubin, tumor numbers, CEA, CA19‐9, GGT, HBsAg and ISICC, were integrated into the final model. The C‐index of the ISICC‐applied prediction model was 0.719 (95% CI, 0.660‐0.777) in the derivation cohort and 0.667 (95% CI, 0.581‐0.732) in the validation cohort. Compared with the conventional staging systems, the new model presented better homogeneity and a lower Akaike information criteria value in ICC. The ISICC‐applied prediction model may provide a better prediction performance for the overall survival of patients with resectable ICC in clinical practice., Using tissue microarray, we examined the density of 16 immune biomarkers in 280 ICC patients who underwent hepatectomy, and established a novel ISICC‐based prediction model (IPM) to predict patients’ overall survival with bilirubin, tumor numbers, CEA, CA19‐9, γ‐glutamyl transferase (GGT), HBsAg and ISICC. The new model may provide a better prediction performance for the overall survival of patients with resectable ICC in clinical practice.

Purpose The purpose of this work was to assess the potential of deep convolutional neural networks in automated measurement of cerebellum tracer uptake in F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) scans. Methods Three different three-dimensional (3D) convolutional neural network architectures (U-Net, V-Net, and modified U-Net) were implemented and compared regarding their performance in 3D cerebellum segmentation in FDG PET scans. For network training and testing, 134 PET scans with corresponding manual volumetric segmentations were utilized. For segmentation performance assessment, a fivefold cross-validation was used, and the Dice coefficient as well as signed and unsigned distance errors were calculated. In addition, standardized uptake value (SUV) uptake measurement performance was assessed by means of a statistical comparison to an independent reference standard. Furthermore, a comparison to a previously reported active-shape-model-based approach was performed. Results Out of the three convolutional neural networks investigated, the modified U-Net showed significantly better segmentation performance. It achieved a Dice coefficient of 0.911 ± 0.026, a signed distance error of 0.220 ± 0.103 mm, and an unsigned distance error of 1.048 ± 0.340 mm. When compared to the independent reference standard, SUV uptake measurements produced with the modified U-Net showed no significant error in slope and intercept. The estimated reduction in total SUV measurement error was 95.1%. Conclusions The presented work demonstrates the potential of deep convolutional neural networks in automated SUV measurement of reference regions. While it focuses on the cerebellum, utilized methods can be generalized to other reference regions like the liver or aortic arch. Future work will focus on combining lesion and reference region analysis into one approach.

Production of TGF-β by T cells is key to various aspects of immune homeostasis, with defects in this process causing or aggravating immune-mediated disorders. The molecular mechanisms that lead to TGF-β generation by T cells remain largely unknown. To address this issue, we take advantage of the fact that intestinal helminths stimulate Th2 cells besides triggering TGF-β generation by T lymphocytes and regulate immune-mediated disorders. We show that the Th2 cell–inducing transcription factor STAT6 is necessary and sufficient for the expression of TGF-β propeptide in T cells. STAT6 is also necessary for several helminth-triggered events in mice, such as TGF-β–dependent suppression of alloreactive inflammation in graft-versus-host disease. Besides STAT6, helminth-induced secretion of active TGF-β requires cleavage of propeptide by the endopeptidase furin. Thus, for the immune regulatory pathway necessary for TGF-β production by T cells, our results support a two-step model, composed of STAT6 and furin.

Helminths stimulate the secretion of Th2 cytokines, like IL-4, and suppress lethal graft-versus-host disease (GVHD) after bone marrow transplantation. This suppression depends on the production of immune-modulatory TGF-β and is associated with TGF-β–dependent in vivo expansion of Foxp3+ regulatory T cells (Treg). In vivo expansion of Tregs is under investigation for its potential as a therapy for GVHD. Nonetheless, the mechanism of induced and TGF-β–dependent in vivo expansion of Tregs, in a Th2 polarized environment after helminth infection, is unknown. In this study, we show that helminth-induced IL-4 production by host cells is critical to the induction and maintenance of TGF-β secretion, TGF-β–dependent expansion of Foxp3+ Tregs, and the suppression of GVHD. In mice with GVHD, the expanding donor Tregs express the Th2-driving transcription factor, GATA3, which is required for helminth-induced production of IL-4 and TGF-β. In contrast, TGF-β is not necessary for GATA3 expression by Foxp3+ Tregs or by Foxp3− CD4 T cells. Various cell types of innate or adaptive immune compartments produce high quantities of IL-4 after helminth infection. As a result, IL-4–mediated suppression of GVHD does not require invariant NKT cells of the host, a cell type known to produce IL-4 and suppress GVHD in other models. Thus, TGF-β generation, in a manner dependent on IL-4 secretion by host cells and GATA3 expression, constitutes a critical effector arm of helminthic immune modulation that promotes the in vivo expansion of Tregs and suppresses GVHD.

Lysyl oxidase like 4 (LOXL4), a member of the secreted copper-dependent amine oxidases that contribute to the assemble and maintenance of the extracellular matrix (ECM), was found to be up-regulated or down-regulated in different cancer types, suggesting its paradoxical roles in cancer. The specific role of LOXL4 in hepatocellular carcinoma (HCC), however, is still yet to be defined. Twenty-eight pairs of HCC specimens were used for LOXL4 mRNA expression analysis. The mRNA expression in HCC cell lines was examined, and HepG2 was selected for LOXL4 small interfering RNA (siRNA) interference to investigate the biological function of LOXL4, LOXL4 immunohistochemical staining was performed using a tissue microarray containing 298 HCC patients. The prognostic and diagnostic value of LOXL4 was evaluated using Cox regression and Kaplan-Meier analysis. LOXL4 mRNA or protein expression was significantly lower in HCC tissues than peritumoral tissues (LOXL4 mRNA expression, P = 0.018; LOXL4 protein expression, P < 0.001). Low LOXL4 expression was associated with lower overall survival (OS) rates and higher cumulative recurrence rates. Multivariate analysis indicated that LOXL4 was an independent prognostic indicator for OS and time to recurrence (TTR). Our results revealed that LOXL4 was down-regulated in HCC and correlated with aggressive tumors and a worse clinical outcome. LOXL4 may be a potential biomarker to identify the HCC patients with a higher risk of recurrence.

The study of interactions between proteins and nanoparticles is important to advancing applications of nanoparticles in biology, medicine, and materials science. Here, we report the encapsulation of a 5-nm diameter gold nanoparticle (AuNP) by thermophilic ferritin (tF), achieved in nearly quantitative yield under mild conditions that preserved the secondary structure, ferroxidase activity, and thermal stability of the native, 4-helix bundle protein subunits. Chromatography-based assays determined that stable protein assembly around AuNPs occurred on long time scales (~48h) and was reversible. Apparent association constants were determined at 25°C for equilibrated tF-BSPP-capped AuNP samples (KA=(2.1±0.4)×10(78)M(-11)) and compared favorably to salt-assembled tF samples (KA=(2.2±0.5)×10(68)M(-11)) at the same protein concentration (0.3mg/mL). Finally, addition of gold ions and mild reducing agent to the tF-AuNP assembly produced 8-nm diameter AuNPs with surface plasmon resonance band unchanged at 520nm, indicative of templating by the protein shell.

The Beijing FEL is a rf linac based facility designed to work in the IR spectra range. In this paper, the design consideration and system layout are briefly reviewed first, and then various measures adopted to optimize the electron beam quality to satisfy the lasing requirement are described. Spontaneous emission, stored spontaneous emission and lasing were observed in sequence. The optical spectrum, detuning curve and wavelength shift were measured to confirm first lasing. Further work to be performed is indicated.

An intense current RF-linac for the far-infrared FEL is now under construction at CIAE. The normalized brightness of 3.4 x 10{sup 9} A/(m-rad) was obtained from the injector of the linac. An acceleration section with 9 cells will be connected with the injector to provide an electron beam for the 200 {mu}m FEL oscillator. In this paper, the late results from the injector beam test will be reported. The physical design and research progress in the acceleration section, beam transport, undulator as well as optical cavity will be introduced respectively.