Targeting Src SH3 domain-mediated glycolysis of HSC suppresses transcriptome, myofibroblastic activation, and colorectal liver metastasis.

BACKGROUND AND AIMS: Transforming growth factor-beta 1 (TGFß1) induces HSC activation into metastasis-promoting cancer-associated fibroblasts (CAFs), but how the process is fueled remains incompletely understood. We studied metabolic reprogramming induced by TGFß1 in HSCs. APPROACHES AND RESULTS: Activation of cultured primary human HSCs was assessed by the expression of myofibroblast markers. Glucose transporter 1 (Glut1) of murine HSC was disrupted by Cre recombinase/LoxP sequence derived from bacteriophage P1 recombination (Cre/LoxP). Plasma membrane (PM) Glut1 and glycolysis were studied by biotinylation assay and the Angilent Seahorse XFe96 Analyzer. S.c. HSC/tumor co-implantation and portal vein injection of MC38 colorectal cancer cells into HSC-specific Glut1 knockout mice were performed to determine in vivo relevance. Transcriptome was obtained by RNA sequencing of HSCs and spatialomics with MC38 liver metastases. TGFß1-induced CAF activation of HSCs was accompanied by elevation of PM Glut1, glucose uptake, and glycolysis. Targeting Glut1 or Src by short hairpin RNA, pharmacologic inhibition, or a Src SH3 domain deletion mutant abrogated TGFß1-stimulated PM accumulation of Glut1, glycolysis, and CAF activation. Mechanistically, binding of the Src SH3 domain to SH3 domain-binding protein 5 led to a Src/SH3 domain-binding protein 5/Rab11/Glut1 complex that activated Rab11-dependent Glut1 PM transport under TGFß1 stimulation. Deleting the Src SH3 domain or targeting Glut1 of HSCs by short hairpin RNA or Cre recombinase/LoxP sequence derived from bacteriophage P1 recombination suppressed CAF activation in mice and MC38 colorectal liver metastasis. Multi-omics revealed that Glut1 deficiency in HSCs/CAFs suppressed HSC expression of tumor-promoting factors and altered MC38 transcriptome, contributing to reduced MC38 liver metastases. CONCLUSION: The Src SH3 domain-facilitated metabolic reprogramming induced by TGFß1 represents a target to inhibit CAF activation and the pro-metastatic liver microenvironment.

Metabolic reprogramming and its clinical implication for liver cancer.

Cancer cells often encounter hypoxic and hypo-nutrient conditions, which force them to make adaptive changes to meet their high demands for energy and various biomaterials for biomass synthesis. As a result, enhanced catabolism (breakdown of macromolecules for energy production) and anabolism (macromolecule synthesis from bio-precursors) are induced in cancer. This phenomenon is called "metabolic reprogramming," a cancer hallmark contributing to cancer development, metastasis, and drug resistance. HCC and cholangiocarcinoma (CCA) are 2 different liver cancers with high intertumoral heterogeneity in terms of etiologies, mutational landscapes, transcriptomes, and histological representations. In agreement, metabolism in HCC or CCA is remarkably heterogeneous, although changes in the glycolytic pathways and an increase in the generation of lactate (the Warburg effect) have been frequently detected in those tumors. For example, HCC tumors with activated ß-catenin are addicted to fatty acid catabolism, whereas HCC tumors derived from fatty liver avoid using fatty acids. In this review, we describe common metabolic alterations in HCC and CCA as well as metabolic features unique for their subsets. We discuss metabolism of NAFLD as well, because NAFLD will likely become a leading etiology of liver cancer in the coming years due to the obesity epidemic in the Western world. Furthermore, we outline the clinical implication of liver cancer metabolism and highlight the computation and systems biology approaches, such as genome-wide metabolic models, as a valuable tool allowing us to identify therapeutic targets and develop personalized treatments for liver cancer patients.

Improving Image Quality by Deconvolution Recovery Filter in Ultrasound Imaging.

Due to the advantages of non-radiation and real-time performance, ultrasound imaging is essential in medical imaging. Image quality is affected by the performance of the transducer in an ultrasound imaging system. For example, the bandwidth controls the pulse length, resulting in different axial resolutions. Therefore, a transducer with a large bandwidth helps to improve imaging quality. However, large bandwidths lead to increased system cost and sometimes a loss of sensitivity and lateral resolution in attenuating media. In this paper, a deconvolution recovery method combined with a frequency-domain filtering technique (DRF) is proposed to improve the imaging quality, especially for the axial resolution. In this method, the received low-bandwidth echo signals are converted into high-bandwidth signals, which is similar to the echo signals produced by a high-bandwidth transducer, and the imaging quality is improved. Simulation and experiment results show that, compared with Delay-and-sum (DAS) method, the DRF method improved axial resolution from 0.60 to 0.41 mm in simulation and from 0.62 to 0.47 mm in the tissue-mimicking phantom experiment. The contrast ratio performance is improved to some extent compared with the DAS in experimental and in-vivo images. Besides, the proposed method has the potential to further improve image quality by combining it with adaptive weightings, such as the minimum variance method.

Downregulation of HMGB1 in thymoma cells affects T cell differentiation.

Introduction: Thymoma is the most common anterior mediastinal tumor and is closely associated with myasthenia gravis (MG). Our previous study showed that the expression of Th17 cells increased and the expression of Treg decreased in MG-associated thymoma tissues and peripheral blood. High mobility group box 1 (HMGB1) is an inflammatory mediator and participates in the pathogenesis of various autoimmune diseases. However, its function in thymoma is still unclear. Material and methods: We first analyzed immune indices in peripheral blood of patients with MG-associated thymoma and patients with thymoma alone. Next, we explored the expression of HMGB1 in MG-associated thymoma and thymoma alone tissues. Furthermore, we transfected si-HMGB1 in thymoma cell line Thy0517 and co-cultured Thy0517 with peripheral blood mononuclear cells (PBMC). Results: In this study, the levels of IgG, C3, C4, CRP and globulins in peripheral blood of patients with MG-associated thymoma were different from those of patients with thymoma alone (p < 0.05). The expression of HMGB1 in MG-associated thymoma tissues was higher than thymoma alone. Co-culture of Thy0517 and PBMC showed that the percentage of Th17 cells in PBMC was lower than that in the control group, and the percentage of Treg cells was higher than that in the control group. Conclusions: These findings demonstrate that HMGB1 is involved in the mechanism of abnormal Th17/Treg cell differentiation in thymoma and affects the occurrence of immune abnormalities in MG-associated thymoma.

Minimum variance beamforming combined with covariance matrix-based adaptive weighting for medical ultrasound imaging.

BACKGROUND: The minimum variance (MV) beamformer can significantly improve the image resolution in ultrasound imaging, but it has limited performance in noise reduction. We recently proposed the covariance matrix-based statistical beamforming (CMSB) for medical ultrasound imaging to reduce sidelobes and incoherent clutter. METHODS: In this paper, we aim to improve the imaging performance of the MV beamformer by introducing a new pixel-based adaptive weighting approach based on CMSB, which is named as covariance matrix-based adaptive weighting (CMSAW). The proposed CMSAW estimates the mean-to-standard-deviation ratio (MSR) of a modified covariance matrix reconstructed by adaptive spatial smoothing, rotary averaging, and diagonal reducing. Moreover, adaptive diagonal reducing based on the aperture coherence is introduced in CMSAW to enhance the performance in speckle preservation. RESULTS: The proposed CMSAW-weighted MV (CMSAW-MV) was validated through simulation, phantom experiments, and in vivo studies. The phantom experimental results show that CMSAW-MV obtains resolution improvement of 21.3% and simultaneously achieves average improvements of 96.4% and 71.8% in average contrast and generalized contrast-to-noise ratio (gCNR) for anechoic cyst, respectively, compared with MV. in vivo studies indicate that CMSAW-MV improves the noise reduction performance of MV beamformer. CONCLUSION: Simulation, experimental, and in vivo results all show that CMSAW-MV can improve resolution and suppress sidelobes and incoherent clutter and noise. These results demonstrate the effectiveness of CMSAW in improving the imaging performance of MV beamformer. Moreover, the proposed CMSAW with a computational complexity of [Formula: see text] has the potential to be implemented in real time using the graphics processing unit.

Protein diaphanous homolog 1 (Diaph1) promotes myofibroblastic activation of hepatic stellate cells by regulating Rab5a activity and TGFß receptor endocytosis.

TGFß induces the differentiation of hepatic stellate cells (HSCs) into tumor-promoting myofibroblasts but underlying mechanisms remain incompletely understood. Because endocytosis of TGFß receptor II (TßRII), in response to TGFß stimulation, is a prerequisite for TGF signaling, we investigated the role of protein diaphanous homolog 1 (known as Diaph1 or mDia1) for the myofibroblastic activation of HSCs. Using shRNA to knockdown Diaph1 or SMIFH2 to target Diaph1 activity of HSCs, we found that the inactivation of Diaph1 blocked internalization and intracellular trafficking of TßRII and reduced SMAD3 phosphorylation induced by TGFß1. Mechanistic studies revealed that the N-terminal portion of Diaph1 interacted with both TßRII and Rab5a directly and that Rab5a activity of HSCs was increased by Diaph1 overexpression and decreased by Diaph1 knockdown. Additionally, expression of Rab5aQ79L (active Rab5a mutant) increased whereas the expression of Rab5aS34N (inactive mutant) reduced the endosomal localization of TßRII in HSCs compared to the expression of wild-type Rab5a. Functionally, TGFß stimulation promoted HSCs to express tumor-promoting factors, and α-smooth muscle actin, fibronection, and CTGF, markers of myofibroblastic activation of HSCs. Targeting Diaph1 or Rab5a suppressed HSC activation and limited tumor growth in a tumor implantation mouse model. Thus, Dipah1 and Rab5a represent targets for inhibiting HSC activation and the hepatic tumor microenvironment.

p300 Acetyltransferase Is a Cytoplasm-to-Nucleus Shuttle for SMAD2/3 and TAZ Nuclear Transport in Transforming Growth Factor ß-Stimulated Hepatic Stellate Cells.

Nuclear translocation of mothers against decapentaplegic homolog 2/3 (SMAD2/3), core transcription factors of transforming growth factor ß (TGF-ß) signaling, is critical for hepatic stellate cell (HSC) differentiation into metastasis-promoting myofibroblasts. SMAD2/3 have multiple coactivators, including WW domain-containing transcription regulator protein 1 (WWTR1 or TAZ) and p300 acetyltransferase. In the nucleus, TAZ binds to SMAD2/3 to prevent SMAD2/3 nuclear export. However, how TAZ and SMAD2/3 enter the nucleus remains poorly understood because neither contains a nuclear localization signal (NLS), an amino acid sequence tagging proteins for nuclear transport. p300 is an NLS-containing large scaffold protein, so we hypothesized that SMAD2/3 and TAZ may undergo nuclear import through complexing with p300. Coimmunoprecipitation, immunofluorescence, and nuclear fractionation assays revealed that TGF-ß1 promoted binding of SMAD2/3 and TAZ to p300 and that p300 inactivation disrupted TGF-ß1-mediated SMAD2/3 and TAZ nuclear accumulation. Deleting the p300 NLS blocked TGF-ß1-induced SMAD2/3 and TAZ nuclear transport. Consistently, p300 inactivation suppressed TGF-ß1-mediated HSC activation and transcription of genes encoding tumor-promoting factors, such as connective tissue growth factor, Tenascin C, Periostin, platelet-derived growth factor C, and fibroblast growth factor 2, as revealed by microarray analysis. Chromatin immunoprecipitation-real-time quantitative PCR showed that canonical p300-mediated acetylation of histones also facilitated transcription in response to TGF-ß1 stimulation. Interestingly, although both TGF-ß1-mediated and stiffness-mediated HSC activation require p300, comparison of gene expression data sets revealed that transcriptional targets of TGF-ß1 were distinct from those of stiffness-p300 mechanosignaling. Lastly, in tumor/HSC coinjection and intrasplenic tumor injection models, targeting p300 of activated-HSC/myofibroblasts by C646, short hairpin RNA, or cre-mediated gene disruption reduced tumor and liver metastatic growth in mice. Conclusion: p300 facilitates TGF-ß1-stimulated HSC activation by both noncanonical (cytoplasm-to-nucleus shuttle for SMAD2/3 and TAZ) and canonical (histone acetylation) mechanisms. p300 is an attractive target for inhibiting HSC activation and the prometastatic liver microenvironment.

P300 Acetyltransferase Mediates Stiffness-Induced Activation of Hepatic Stellate Cells Into Tumor-Promoting Myofibroblasts.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) contribute to desmoplasia and stiffness of liver metastases by differentiating into matrix-producing myofibroblasts. We investigated whether stiffness due to the presence of tumors increases activation of HSCs into myofibroblasts and their tumor-promoting effects, as well as the role of E1A binding protein p300, a histone acetyltransferase that regulates transcription, in these processes. METHODS: HSCs were isolated from liver tissues of patients, mice in which the p300 gene was flanked by 2 loxP sites (p300F/F mice), and p300+/+ mice (controls). The HSCs were placed on polyacrylamide gels with precisely defined stiffness, and their activation (differentiation into myofibroblasts) was assessed by immunofluorescence and immunoblot analyses for alpha-smooth muscle actin. In HSCs from mice, the p300 gene was disrupted by cre recombinase. In human HSCs, levels of p300 were knocked down with small hairpin RNAs or a mutant form of p300 that is not phosphorylated by AKT (p300S1834A) was overexpressed. Human HSCs were also cultured with inhibitors of p300 (C646), PI3K signaling to AKT (LY294002), or RHOA (C3 transferase) and effects on stiffness-induced activation were measured. RNA sequencing and chromatin immunoprecipitation-quantitative polymerase chain reaction were used to identify HSC genes that changed expression levels in response to stiffness. We measured effects of HSC-conditioned media on proliferation of HT29 colon cancer cells and growth of tumors following subcutaneous injection of these cells into mice. MC38 colon cancer cells were injected into portal veins of p300F/Fcre and control mice, and liver metastases were measured. p300F/Fcre and control mice were given intraperitoneal injections of CCl4 to induce liver fibrosis. Liver tissues were collected and analyzed by immunofluorescence, immunoblot, and histology. RESULTS: Substrate stiffness was sufficient to activate HSCs, leading to nuclear accumulation of p300. Disrupting p300 level or activity blocked stiffness-induced activation of HSCs. In HSCs, substrate stiffness activated AKT signaling via RHOA to induce phosphorylation of p300 at serine 1834; this caused p300 to translocate to the nucleus, where it up-regulated transcription of genes that increase activation of HSCs and metastasis, including CXCL12. MC38 cells, injected into portal veins, formed fewer metastases in livers of p300F/Fcre mice than control mice. Expression of p300 was increased in livers of mice following injection of CCl4; HSC activation and collagen deposition were reduced in livers of p300F/Fcre mice compared with control mice. CONCLUSIONS: In studies of mice, we found liver stiffness to activate HSC differentiation into myofibroblasts, which required nuclear accumulation of p300. p300 increases HSC expression of genes that promote metastasis.

TPL2 Is a Key Regulator of Intestinal Inflammation in Clostridium difficile Infection.

Tumor progression locus 2 (TPL2), a serine/threonine protein kinase, is a major inflammatory mediator in immune cells. The predominant inflammatory actions of TPL2 depend on the activation of mitogen-activated protein kinases (MAPK) and the upregulated production of the cytokines tumor necrosis factor alpha (TNF-α) and interleukin 1ß (IL-1ß) in macrophages and dendritic cells in response to lipopolysaccharide (LPS). Significant increases in TNF-α, IL-6, IL-ß, and IL-8 levels in patients with Clostridium difficile infection (CDI) have been reported. Both TNF-α and IL-6 have been postulated to play key roles in the systemic inflammatory response in CDI, and IL-8 is essential for the development of local intestinal inflammatory responses in CDI. The objective of this study was to elucidate the role of TPL2 in the pathogenesis of CDI. We found that TPL2 was significantly activated in human and mouse intestinal tissues upon C. difficile toxin exposure or CDI. We further demonstrated that TPL2 knockout (TPL2-KO) mice were significantly more resistant to CDI than wild-type mice, with significantly reduced production of TNF-α, IL-6, IL-1ß, KC (a mouse homologue of IL-8), and myeloperoxidase (MPO) in the ceca and colons of TPL2-KO mice. Finally, we found that TPL2 inhibition by a specific inhibitor or TPL2 gene ablation significantly reduced TcdB-induced production of TNF-α, IL-6, IL-ß, and KC by inhibiting the activation of p38, extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinase (JNK). Taken together, our data suggest that TPL2 represents a potential therapeutic target for CDI treatment.

Oral Immunization with Nontoxigenic Clostridium difficile Strains Expressing Chimeric Fragments of TcdA and TcdB Elicits Protective Immunity against C. difficile Infection in Both Mice and Hamsters.

The symptoms of Clostridium difficile infection (CDI) are attributed largely to two C. difficile toxins, TcdA and TcdB. Significant efforts have been devoted to developing vaccines targeting both toxins through parenteral immunization routes. However, C. difficile is an enteric pathogen, and mucosal/oral immunization would be particularly useful to protect the host against CDI, considering that the gut is the main site of disease onset and progression. Moreover, vaccines directed only against toxins do not target the cells and spores that transmit the disease. Previously, we constructed a chimeric vaccine candidate, mTcd138, comprised of the glucosyltransferase and cysteine proteinase domains of TcdB and the receptor binding domain of TcdA. In this study, to develop an oral vaccine that can target both C. difficile toxins and colonization/adhesion factors, we expressed mTcd138 in a nontoxigenic C. difficile (NTCD) strain, resulting in strain NTCD_mTcd138. Oral immunization with spores of NTCD_mTcd138 provided mice full protection against infection with a hypervirulent C. difficile strain, UK6 (ribotype 027). The protective strength and efficacy of NTCD_mTcd138 were further evaluated in the acute CDI hamster model. Oral immunization with spores of NTCD_mTcd138 also provided hamsters significant protection against infection with 2 × 104 UK6 spores, a dose 200-fold higher than the lethal dose of UK6 in hamsters. These results imply that the genetically modified, nontoxigenic C. difficile strain expressing mTcd138 may represent a novel mucosal vaccine candidate against CDI.

Hepatic radiofrequency ablation: markedly reduced systemic effects by modulating periablational inflammation via cyclooxygenase-2 inhibition.

OBJECTIVES: To determine whether celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, can potentiate hepatic radiofrequency ablation (RFA)-induced local cellular infiltration and distant tumour growth. METHODS: First, COX-2 expression was evaluated using immunohistochemistry in the local periablational rim 24 h after hepatic RFA without/with intraperitoneal celecoxib in normal Fisher 344 rat liver. Next, local cellular infiltration of macrophages, stellate cells, and hepatocyte proliferation were quantified in C57BL6 mice 3-7d after RFA without/with celecoxib. c-Met, HGF, and VEGF levels after RFA were also measured. Finally, distant tumour growth and proliferation (Ki67 and CD34) were observed in subcutaneous R3230 tumours after hepatic RFA with/without celecoxib. RESULTS: Hepatic RFA-induced local activation of COX-2 was significantly suppressed using celecoxib. Celecoxib also reduced RFA-associated a) increased c-Met expression at 24 h, b) HGF and VEGF levels at 72 h, c) periablational macrophage and stellate cells at 3d, and d) hepatocyte proliferation at 7d. Similarly, celecoxib with RFA reduced distant tumour growth, tumour cell proliferation, and tumour microvascular density to sham levels, compared to increases observed with hepatic RFA alone. CONCLUSIONS: Increased activation of COX-2 after hepatic RFA contributes to periablational cellular infiltration and inflammation-mediated distant tumour growth, which can be successfully suppressed with a COX-2 inhibitor. KEY POINTS: • Thermal ablation of liver tissue can increase local inflammation and COX-2 expression. • Ablation-induced local inflammation can contribute to stimulation of distant tumour growth. • Local COX-2 inhibition with celecoxib can block ablation-induced distant tumour growth.

Hepatic Radiofrequency Ablation-induced Stimulation of Distant Tumor Growth Is Suppressed by c-Met Inhibition.

PURPOSE: To elucidate how hepatic radiofrequency (RF) ablation affects distant extrahepatic tumor growth by means of two key molecular pathways. MATERIALS AND METHODS: Rats were used in this institutional animal care and use committee-approved study. First, the effect of hepatic RF ablation on distant subcutaneous in situ R3230 and MATBIII breast tumors was evaluated. Animals were randomly assigned to standardized RF ablation, sham procedure, or no treatment. Tumor growth rate was measured for 3½ to 7 days. Then, tissue was harvested for Ki-67 proliferative indexes and CD34 microvascular density. Second, hepatic RF ablation was performed for hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and c-Met receptor expression measurement in periablational rim, serum, and distant tumor 24 hours to 7 days after ablation. Third, hepatic RF ablation was combined with either a c-Met inhibitor (PHA-665752) or VEGF receptor inhibitor (semaxanib) and compared with sham or drug alone arms to assess distant tumor growth and growth factor levels. Finally, hepatic RF ablation was performed in rats with c-Met-negative R3230 tumors for comparison with the native c-Met-positive line. Tumor size and immunohistochemical quantification at day 0 and at sacrifice were compared with analysis of variance and the two-tailed Student t test. Tumor growth curves before and after treatment were analyzed with linear regression analysis to determine mean slopes of pre- and posttreatment growth curves on a per-tumor basis and were compared with analysis of variance and paired two-tailed t tests. RESULTS: After RF ablation of normal liver, distant R3230 tumors were substantially larger at 7 days compared with tumors treated with the sham procedure and untreated tumors, with higher growth rates and tumor cell proliferation. Similar findings were observed in MATBIII tumors. Hepatic RF ablation predominantly increased periablational and serum HGF and downstream distant tumor VEGF levels. Compared with RF ablation alone, RF ablation combined with adjuvant PHA-665752 or semaxanib reduced distant tumor growth, proliferation, and microvascular density. For c-Met-negative tumors, hepatic RF ablation did not increase distant tumor growth, proliferation, or microvascular density compared with sham treatment. CONCLUSION: RF ablation of normal liver can stimulate distant subcutaneous tumor growth mediated by HGF/c-Met pathway and VEGF activation. This effect was not observed in c-Met-negative tumors and can be blocked with adjuvant c-Met and VEGF inhibitors.

Hepatic Thermal Ablation: Effect of Device and Heating Parameters on Local Tissue Reactions and Distant Tumor Growth.

Purpose To determine whether variable hepatic microwave ablation (MWA) can induce local inflammation and distant pro-oncogenic effects compared with hepatic radiofrequency ablation (RFA) in an animal model. Materials and Methods In this institutional Animal Care and Use Committee-approved study, F344 rats (150 gm, n = 96) with subcutaneous R3230 breast adenocarcinoma tumors had normal non-tumor-bearing liver treated with RFA (70°C × 5 minutes), rapid higher-power MWA (20 W × 15 seconds), slower lower-power MWA (5 W × 2 minutes), or a sham procedure (needle placement without energy) and were sacrificed at 6 hours to 7 days (four time points; six animals per arm per time point). Ablation settings produced 11.4 mm ± 0.8 of coagulation for all groups. Distant tumor growth rates were determined to 7 days after treatment. Liver heat shock protein (HSP) 70 levels (at 72 hours) and macrophages (CD68 at 7 days), tumor proliferative indexes (Ki-67 and CD34 at 7 days), and serum and tissue levels of interleukin 6 (IL-6) at 6 hours, hepatocyte growth factor (HGF) at 72 hours, and vascular endothelial growth factor (VEGF) at 72 hours after ablation were assessed. All data were expressed as means ± standard deviations and were compared by using two-tailed t tests and analysis of variance for selected group comparisons. Linear regression analysis of tumor growth curves was used to determine pre- and posttreatment growth curves on a per-tumor basis. Results At 7 days, hepatic ablations with 5-W MWA and RFA increased distant tumor size compared with 20-W MWA and the sham procedure (5-W MWA: 16.3 mm ± 1.1 and RFA: 16.3 mm ± 0.9 vs sham: 13.6 mm ± 1.3, P < .01, and 20-W MWA: 14.6 mm ± 0.9, P < .05). RFA and 5-W MWA increased postablation tumor growth rates compared with the 20-W MWA and sham arms (preablation growth rates range for all arms: 0.60-0.64 mm/d; postablation: RFA: 0.91 mm/d ± 0.11, 5-W MWA: 0.91 mm/d ± 0.14, P < .01 vs pretreatment; 20-W MWA: 0.69 mm/d ± 0.07, sham: 0.56 mm/d ± 1.15; P = .48 and .65, respectively). Tumor proliferation (Ki-67 percentage) was increased for 5-W MWA (82% ± 5) and RFA (79% ± 5), followed by 20-W MWA (65% ± 2), compared with sham (49% ± 5, P < .01). Likewise, distant tumor microvascular density was greater for 5-W MWA and RFA (P < .01 vs 20-W MWA and sham). Lower-energy MWA and RFA also resulted in increased HSP 70 expression and macrophages in the periablational rim (P < .05). Last, IL-6, HGF, and VEGF elevations were seen in 5-W MWA and RFA compared with 20-W MWA and sham (P < .05). Conclusion Although hepatic MWA can incite periablational inflammation and increased distant tumor growth similar to RFA in an animal tumor model, higher-power, faster heating protocols may potentially mitigate such undesired effects. © RSNA, 2016.

Far-focus compound ultrasound imaging with lag-one coherence-based zero-cross factor.

BACKGROUND: Ultrasound imaging has been widely used in clinical examination because of portability, safety, and low cost. However, there are still some main challenges of imaging quality that remain in conventional ultrasound systems. OBJECTIVE: Improving image quality of SA-based methods using an improved imaging mode named far-focus compound (FSC) imaging. METHODS: A far-focus compound (FSC) imaging based on full-aperture transmission and full-aperture reception is proposed in this paper. In transmission, it uses the full aperture to transmit the focused beam to ensure image resolution and emission of sound field energy. In reception, the full aperture is used to receive the reflected beam to ensure the image quality. A lag-one coherence-based zero-cross factor (LOCZF) is then implemented in FSC for improvement of contrast ratio (CR). The LOCZF uses lag-one coherence as zero-cross factorâs adaptive coefficient. Comparisons were made with several other weighting techniques by performing simulations and experiments for performance evaluation. RESULTS: Results confirm that LOCZF applied to FSC offers a good image contrast and simultaneously the speckle pattern. For simulated cysts, CR improvement of LOCZF reaches 194.1%. For experimental cysts, CR improvement of LOCZF reaches 220%. From the in-vivo result, compared with FSC, CR improvement of LOCZF reaches 112.7%. CONCLUSION: Proved gCNR performance. In addition, the LOCZF method shows good performance in experiments. The proposed method can be used as an effective weighting technique for improvement of image quality in ultrasound imaging.

Distribution of multi-level B cell subsets in thymoma and thymoma-associated myasthenia gravis.

B-cell subsets in peripheral blood (PB) and tumor microenvironment (TME) were evaluated to determine myasthenia gravis (MG) severity in patients with thymoma-associated MG (TMG) and the distribution of B cells in type B TMG. The distribution of mature B cells, including Bm1-Bm5, CD19+ and CD20+ B cells and non-switched (NSMBCs) and switched (SMBCs) memory B cells, were determined in 79 patients with thymoma or TMG. Quantitative relationships between the T and TMG groups and the TMG-low and TMG-high subgroups were determined. NSMBCs and SMBCs were compared in TME and PB. Type B thymoma was more likely to develop into MG, with types B2 and B3 being especially associated with MG worsening. The percentage of CD19+ B cells in PB gradually increased, whereas the percentage of CD20+ B cells and the CD19/CD20 ratio were not altered. The (Bm2 + Bm2')/(eBm5 + Bm5) index was significantly higher in the TMG-high than in thymoma group. The difference between SMBC/CD19+ and NSMBC/CD19+ B cell ratios was significantly lower in the thymoma than TMG group. NSMBCs assembled around tertiary lymphoid tissue in thymomas of patients with TMG. Few NSMBCs were observed in patients with thymoma alone, with these cells being diffusely distributed. MG severity in patients with TMG can be determined by measuring CD19+ B cells and Bm1-Bm5 in PB. The CD19/CD20 ratio is a marker of disease severity in TMG patients. Differences between NSMBCs and SMBCs in PB and TME of thymomas can synergistically determine MG severity in patients with TMG.

Construction of PR domain eukaryotic expression vector and its inhibitory effect on esophageal cancer cells.

OBJECTIVE: PR domain is responsible for the tumor suppressing activity of RIZ1. The study aimed to construct human PR domain eukaryotic expression vectors, transfect human esophageal cancer cells (TE13), and evaluate the anticancer activity of PR domain on human esophageal cancer TE13 cells. METHODS: First, mRNA was extracted from human esophageal cancer tissue by RT-PCR, then reverse-transcribed to cDNA. After amplifying from the DNA template, PR domain was linked to T vector. Second, after extraction, PR domain was cut using enzyme and linked to pcDNA3.1(+). Then, the plasmid was transfered to Trans1-T1 phage resistant competent cells, following by extracting the ultrapure plasmid, and transfecting into TE13 cells. In the end, the protein expression of pcDNA3.1(+)/PR domain in TE13 was detected by Western blot, and the apoptosis of TE13 by technique of flow cytometry. RESULTS: More than 5,000 bp purposed band of pcDNA3.1(+)/PR domain plasmid was found by agarose gel electrophoresis. After transfection, the PR domain (molecular weight of about 28 Da) was found only in 3, 4 and 5 groups by Western blot. Flow cytometry assay showed apoptosis in experimental group was significantly more than that in the control group (P<0.05). CONCLUSIONS: The PR domain eukaryotic expression vector was constructed successfully. The protein of the PR domain could be expressed in esophageal cancer TE13 cells firmly after transfection, and a single PR domain could promote apoptosis of TE13 cells.

An adaptive beamformer based on dynamic phase coherence factor for pixel-based medical ultrasound imaging.

BACKGROUND: Pixel-based beamforming realizes dynamic focusing at the pixel level with a focused beam by assuming that the received signals are composed of spherical pulses. Far-focused pixel-based (FPB) imaging was proposed to avoid artifacts around the focal depth. However, the contrast improvement is limited. OBJECTIVE: We propose an adaptive weighting method based on dynamic phase coherence factor (DPCF) to improve the image contrast while preserving the speckle pattern. METHODS: The phase variation is dynamically estimated based on the noise energy proportion of echo signals and it is used to calculate phase coherence weights for suppressing interference and preserving desired signals. A depth-dependent parameter is designed for DPCF to enhance the performance of noise and clutter suppression in the far-field region. We further use the subarray averaging technique to smooth the speckle texture. RESULTS: The proposed method was evaluated on simulated, phantom experimental, and in vivo data. Results show that, compared with the phase coherence factor (PCF) based method, DPCF respectively leads to average CR improvements by more than 60% and 24% in simulation and experiment, while obtaining an improved speckle signal-to-noise ratio. CONCLUSIONS: The proposed method is a potentially valuable approach to obtaining high-quality ultrasound images in clinical applications.

Improving axial resolution based on the deconvolution recovery method combined with adaptive weighting techniques for ultrasound imaging.

BACKGROUND: A fundamental challenge in medical ultrasound imaging is to improve the resolution accurately. Adaptive beamforming is often used to improve lateral resolution, such as minimum variance (MV) and phase coherence factor (PCF). However, it is difficult to improve the axial resolution due to the limitation of the spatial pulse length (SPL) of the transmitted signal. OBJECTIVE: A deconvolution recovery method combines two adaptive weighting techniques to improve axial resolution. METHODS: A deconvolution recovery (DR) technique is used to improve axial resolution with a shorter SPL. Then, the DR is combined with MV and PCF (DR-MVPCF) to suppress the sidelobe. The influence of different transmission modes, regularization parameters, and the estimation of point spread function are discussed on the proposed algorithm. RESULTS: In simulation, DR-MVPCF improved axial resolution from 0.41 mm (0.98 λ) to 0.09 mm (0.21 λ) compared with MV-PCF. In the water bath experiment, DR-MVPCF provided improvement of axial resolution from 0.39 mm (0.93 λ) to 0.07 mm (0.17 λ) compared with MV-PCF. In-vivo data experiment, the DR-MVPCF method increased the speckle signal-to-noise ratio and visibility of the structure while the contrast ratio and contrast-noise ratio decreased. CONCLUSIONS: The proposed method can improve the axial resolution significantly.

Decreased expression of retinoblastoma protein-interacting zinc-finger gene 1 in human esophageal squamous cell cancer by DNA methylation.

BACKGROUND: To study the expression of the RIZ1 (Retinoblastoma protein-interacting zinc-finger gene 1) gene and investigate the promoter region methylation status of RIZ1 gene in the human esophageal squamous cell carcinoma (ESCC) cell lines of KYSE150, KYSE510, TE13, EC9706, CaEsl7, and EC109. To investigate the influence of DNMT (DNA methyltransferase) 5-aza-CdR(5-aza-2'-deoxycytidine) on the transcription of the RIZ1 gene in one cell line whose RIZ1 gene promoter region methylation was detected, and to investigate its influence on the cell proliferation. METHODS: Real-time PCR (Real-time quantitative PCR) and an immunohistochemistry technique was used to get the expression of RIZ1 in specimens from 6 human ESCC cell lines and 28 ESCC patients (tumor tissues and adjacent non-cancerous tissues). MSP (Methylation-specific PCR) was used to investigate the promoter region methylation status of the RIZ1 gene in the 6 ESCC cell lines. One cell line, whose RIZ1 gene promoter region methylation was detected, was chosen for the next studies in which it was treated it by with 5-aza-CdR. Real-time PCR was used to investigate its influence on the transcription of RIZ1 gene and MTT (methyl thiazolyl tetrazolium) was used to detect if 5-aza-CdR inhibits the proliferation of the cell line. RESULTS: In the 28 ESCC patient samples, RIZ1 expression was significantly lower in the tumor tissues than that in their adjacent non-cancerous tissues (p < 0.05). Consistently, immunohistochemistry analyses of RIZ1 protein expression showed that in the ESCC tissues RIZ1 protein expression was also significantly lower than in the adjacent tissues. In the human ESCC tissues the rate of expression accounts for 0% (0/12), and in the adjacent noncancerous tissues the rate of expression was 66.7% (8/12), the correlation was highly significant (chi2 = 12.000, p < 0.05). Promoter methylation of the RIZ1 gene was detected in TE13, CaEsl7, EC109. The cell line TE13 was chosen for the next studies. The expression of RIZ1 mRNA in TE-13 was up-regulated after having been treated with 5-aza-CdR. 5-aza-CdR inhibited cell proliferation of TE-13 in a time and concentration-dependent manner. CONCLUSIONS: Promoter methylation may play an important role in the epigenetic silencing of RIZ1 gene expression. Methylation of the RIZ1 promoter and loss of RIZ1 expression in human ESCC are independent biomarkers. Their determination may offer guidance for selecting appropriate diagnoses and treatments. RIZ1 may be a potential tumor suppressor in human ESCC.

Covariance Matrix-Based Statistical Beamforming for Medical Ultrasound Imaging.

Medical ultrasound image quality is often limited by clutter, which is the dominant mechanism of image degradation. A variety of beamforming methods have been extensively studied to reduce clutter and, thus, enhance ultrasound image quality. This article introduces a new beamforming approach, called covariance matrix-based statistical beamforming (CMSB), to improve the image contrast and preserve the background speckle pattern while simultaneously achieving a high-resolution performance. In CMSB, adaptive selection of subarray length, diagonal reducing, and mean-to-standard-deviation ratio-based subarray averaging are inherently combined to differentiate and reduce off-axis energy effectively. Moreover, rotary averaging prior to diagonal reducing is introduced to preserve speckle statistics. Simulated, experimental, and in vivo datasets were used to evaluate the imaging performance of the proposed method. The quantitative results indicate that, compared with delay-and-sum (DAS) beamforming, CMSB leads to average improvements of 44.5% and 97.3% in lateral resolution and contrast, respectively, in phantom experiments. Our work shows that CMSB is capable of improving image resolution and contrast while maintaining the speckle reliably. Preliminary in vivo study also demonstrates that the CMSB can enhance image contrast and lesion detection.