Caged Luciferase Inhibitor-Based Bioluminescence Switching Strategy Enables Efficient Detection of Serum APN Activity and the Identification of Its Roles in Metastasis of Non-Small Cell Lung Cancer.

Bioluminogenic probes emerged as powerful tools for imaging and analysis of various bioanalyses, but traditional approaches would be limited to the low sensitivity during determine the low activity of protease in clinical specimens. Herein, we proposed a caged luciferase inhibitor-based bioluminescence-switching strategy (CLIBS) by using a cleavable luciferase inhibitor to modulate the activity of luciferase reporter to amplify the detective signals, which led to the enhancement of detection sensitivity, and enabled the determination of circulating Aminopeptidase N (APN) activity in thousands of times diluted serum. By applying the CLIBS to serum samples in non-small cell lung cancer (NSCLC) patients from two clinical cohorts, we revealed that, for the first time, higher circulating APN activities but not its concentration, were associated with more NSCLC metastasis or higher metastasis stages by subsequent clinical analysis, and can serve as an independent factor for forecasting NSCLC patients' risk of metastasis.

An MRI-based Radiomics Approach to Improve Breast Cancer Histological Grading.

RATIONALE AND OBJECTIVES: Nottingham histological grade (NHG) 2 breast cancer has an intermediate risk of recurrence, which is not informative for therapeutic decision-making. We sought to develop and independently validate an MRI-based radiomics signature (Rad-Grade) to improve prognostic re-stratification of NHG 2 tumors. MATERIALS AND METHODS: Nine hundred-eight subjects with invasive breast cancer and preoperative MRI scans were retrospectively obtained. The NHG 1 and 3 tumors were randomly split into training and independent test cohort, with the NHG 2 as the prognostic validation set. From MRI image features, a radiomics-based signature predictive of the histological grade was built by use of the LASSO logistic regression algorithm. The model was developed for identifying NHG 1 and 3 radiological patterns, followed with re-stratification of NHG 2 tumors into Rad-Grade (RG)2-low (NHG 1-like) and RG2-high (NHG 3-like) subtypes using the learned patterns, and the prognostic value was assessed in terms of recurrence-free survival (RFS). RESULTS: The Rad-Grade showed independent prognostic value for re-stratification of NHG 2 tumors, where RG2-high had an increased risk for recurrence (HR 2.20, 1.10-4.40, p = 0.026) compared with RG2-low after adjusting for established risk factors. RG2-low shared similar phenotypic characteristics and RFS outcomes with NHG 1, and RG2-high with NHG 3, revealing that the model captures radiomic features in NHG 2 that are associated with different aggressiveness. The prognostic value of Rad-Grade was further validated in the NHG2 ER+ (HR 2.53, 1.13-5.56, p = 0.023) and NHG 2 ER+LN- (HR 5.72, 1.24-26.44, p = 0.025) subgroups, and in specific treatment contexts. CONCLUSION: The radiomics-based re-stratification of NHG 2 tumors offers a cost-effective promising alternative to gene expression profiling for tumor grading and thus may improve clinical decisions.

Blockade of USP14 potentiates type I interferon signaling and radiation-induced antitumor immunity via preventing IRF3 deubiquitination.

PURPOSE: The adaptive immune responses induced by radiotherapy has been demonstrated to largely rely on STING-dependent type I interferons (IFNs) production. However, irradiated tumor cells often fail to induce dendritic cells (DCs) to produce type I IFNs. Hence, we aim to uncover the limitation of STING-mediated innate immune sensing following radiation, and identify efficient reagents capable to rescue the failure of type I IFNs induction for facilitating radiotherapy. METHODS: A targeted cell-based phenotypic screening was performed to search for active molecules that could elevate the production of type I IFNs. USP14 knockout or inhibition was assayed for IFN production and the activation of STING signaling in vitro. The mechanisms of USP14 were investigated by western blot and co-immunoprecipitation in vitro. Additionally, combinational treatments with PT33 and radiation in vivo and in vitro models were performed to evaluate type I IFNs responses to radiation. RESULTS: PT33 was identified as an enhancer of STING agonist elicited type I IFNs production to generate an elevated and durable STING activation profile in vitro. Mechanistically, USP14 inhibition or deletion impairs the deubiquitylation of K63-linked IRF3. Furthermore, blockade of USP14 with PT33 enhances DC sensing of irradiated-tumor cells in vitro, and synergizes with radiation to promote systemic antitumor immunity in vivo. CONCLUSION: Our findings reveal that USP14 is one of the major IFN production suppressors and impairs the activation of IRF3 by removing the K63-linked ubiquitination of IRF3. Therefore, blockage of USP14 results in the gain of STING signaling activation and radiation-induced adaptive immune responses.

The diagnostic performance of ultrasound computer-aided diagnosis system for distinguishing breast masses: a prospective multicenter study.

OBJECTIVES: To evaluate the diagnostic value of computer-aided diagnosis (CAD) software on ultrasound in distinguishing benign and malignant breast masses and avoiding unnecessary biopsy. METHODS: This prospective, multicenter study included patients who were scheduled for pathological diagnosis of breast masses between April 2019 and November 2020. Ultrasound images, videos, CAD analysis, and BI-RADS were obtained. The AUC, accuracy, sensitivity, specificity, PPV, and NPV were calculated and compared with radiologists. RESULTS: Overall, 901 breast masses in 901 patients were enrolled in this study. The accuracy, sensitivity, specificity, PPV and NPV of CAD software were 89.6%, 94.2%, 87.0%, 80.4%, and 96.3, respectively, in the long-axis section; 89.0%, 91.4%, 87.7%, 80.8%, and 94.7%, respectively, in the short-axis section. With BI-RADS 4a as the cut-off value, CAD software has a higher AUC (0.906 vs 0.734 vs 0.696, all p < 0.001) than both experienced and less experienced radiologists. With BI-RADS 4b as the cut-off value, CAD software showed better AUC than less experienced radiologists (0.906 vs 0.874, p < 0.001), but not superior to experienced radiologists (0.906 vs 0.883, p = 0.057). After the application of CAD software, the unnecessary biopsy rate of BI-RADS categories 4 and 5 was significantly decreased (33.0% vs 11.9%, 37.8% vs 14.5%), and the malignant rate of biopsy in category 4a was significantly increased (11.6% vs 40.7%, 7.4% vs 34.9%, all p < 0.001). CONCLUSIONS: CAD software on ultrasound can be used as an effective auxiliary diagnostic tool for differential diagnosis of benign and malignant breast masses and reducing unnecessary biopsy. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov (NCT03887598) KEY POINTS: • Prospective multicenter study showed that computer-aided diagnosis software provides greater diagnostic confidence for differentiating benign and malignant breast masses. • Computer-aided diagnosis software can help radiologists reduce unnecessary biopsy. • The management of patients with breast masses becomes more appropriate.

Management of breast lesions seen on US images: dual-model radiomics including shear-wave elastography may match performance of expert radiologists.

PURPOSE: To develop a nomogram incorporating B-mode ultrasound (BMUS) and shear-wave elastography (SWE) radiomics to predict malignant status of breast lesions seen on US non-invasively. METHODS: Data on 278 consecutive patients from Hospital #1 (training cohort) and 123 cases from Hospital #2 (external validation cohort) referred for breast US with subsequent histopathologic analysis between May 2017 and October 2019 were retrospectively collected. Using their BMUS and SWE images, we built a radiomics nomogram to improve radiology workflow for management of breast lesions. The performance of the algorithm was compared with a consensus of three ACR BI-RADS committee experts and four individual radiologists, all of whom interpreted breast US images in clinical practice. RESULTS: Twelve features from BMUS and three from SWE were selected finally to construct the respective radiomic signature. The nomogram based on the dual-modal US radiomics achieved good diagnostic performance in the training (AUC 0.96; 95% confidence intervals [CI], 0.94-0.98) and the validation set (AUC 0.92; 95% CI, 0.87-0.97). For the 123 test lesions, the algorithm achieved 105 of 123 (85%) accuracy, comparable to the expert consensus (104 of 123 [85%], P =  0.86) and four individual radiologists (93, 99, 95 and 97 of 123, with P value of 0.05, 0.31, 0.10 and 0.18 respectively). Furthermore, the model also performed well in the BI-RADS 4 and 5 categories. CONCLUSIONS: Performance of a dual-model US radiomics nomogram based on SWE for breast lesion classification may comparable to that of expert radiologists who used ACR BI-RADS guideline.

Polysaccharide Isolated From Tetrastigma hemsleyanum Activates TLR4 in Macrophage Cell Lines and Enhances Immune Responses in OVA-Immunized and LLC-Bearing Mouse Models.

Tetrastigma hemsleyanum Diels et Gilg is a valuable Chinese medicinal herb with a long history of clinical application. Our previous study isolated and characterized a purified polysaccharide from the aerial part of Tetrastigma hemsleyanum (SYQP) and found it having antipyretic and antitumor effects in mice. A preliminary mechanistic study suggests these effects may be related to the binding of toll-like receptor (TLR4). The objective of this study is to further explore the detailed stimulating characteristics of SYQP on TLR4 signaling pathway and its in vivo immune regulating effect. We use HEK-BLUE hTLR4, mouse and human macrophage cell lines, as research tools. In vitro results show SYQP activated HEK-BLUE hTLR4 instead of HEK-BLUE Null cells. The secretion and the mRNA expression of cytokines related to TLR4 signaling significantly increased after SYQP treatment in both PMA-induced THP-1 and RAW264.7 macrophage cell lines. The TLR4 antagonist TAK-242 can almost completely abolish this activation. Furthermore, molecules such as IRAK1, NF-κB, MAPKs, and IRF3 in both the MyD88 and TRIF branches were all activated without pathway selection. In vivo results show SYQP enhanced antigen-specific spleen lymphocyte proliferation and serum IgG levels in OVA-immunized C57BL/6 mice. Orally administered 200 mg/kg SYQP induced obvious tumor regression, spleen weight increase, and the upregulation of the mRNA expression of TLR4-related cytokines in Lewis lung carcinoma-bearing mice. These results indicate SYQP can act as both a human and mouse TLR4 agonist and enhance immune responses in mice (p < 0.05). This study provides a basis for the development and utilization of SYQP as a new type of TLR4 agonist in the future.

Tussilagone inhibits allergic responses in OVA-induced allergic rhinitis guinea pigs and IgE-stimulated RBL-2H3 cells.

Farfarae Flos is the dried flower buds of Tussilago farfara L. which is widely used to treat allergic and inflammatory diseases in Chinese folk. Tussilagone (TSL), a sesquiterpene compound purified from Farfarae Flos, has been confirmed the main active component in the plant. However, its anti-allergic activity hasn't been reported yet. The purpose of this study is to investigate the anti-allergic effect of TSL in ovalbumin (OVA)-induced allergic rhinitis (AR) guinea pigs and immunoglobulin E (IgE)-stimulated RBL-2H3 cells. The AR symptoms such as nasal scratching, sneezing and runny nose were scored and the histological changes of nasal mucosa were observed by H&E staining. The levels of histamine, OVA-specific IgE, IL-6 and TNF-α in the serum were measured by ELISA. In IgE-stimulated RBL-2H3 cells, the phosphoryration of Lyn, Syk, Akt, NF-κB p65, ERK and p38 MAPK were investigated by western blot analysis. The results showed that intraperitoneal injection of TSL at doses of 25 and 50 mg/kg significantly alleviated the allergic symptoms and the histological changes of nasal mucosa in OVA-induced allergic rhinitis guinea pigs. Moreover, the levels of histamine, IgE and IL-6 in the serum decreased significantly (p < .05). In vitro, TSL suppressed the phosphorylation of Lyn, Syk, Akt, NF-κB p65, ERK and p38 MAPK in IgE-stimulated RBL-2H3 cells. These results indicate TSL has therapeutic effect on allergic rhinitis in guinea pigs. The anti-allergic mechanism may be through the inhibition of allergic and inflammatory related pathways in mast cells.

Polysaccharide from Phellinus Igniarius activates TLR4-mediated signaling pathways in macrophages and shows immune adjuvant activity in mice.

Polysaccharide from Phellinus igniarius (PPI) is known for its immune-regulating effect with low toxicity. Toll like receptor 4 (TLR4) is important in both innate and adaptive immune responses and considered to be a promising target for new immune adjuvants. In this study, PPI was investigated for its effect on activating TLR4 in RAW264.7 and peritoneal macrophages. The adjuvant potential of PPI was evaluated in OVA-immunized mice. The results showed PPI treatment significantly increased the secretion and the mRNA expression of both MyD88 dependent and TRIF dependent cytokines. IRAK-1, a key molecule on the downstream of MyD88, was polyubiquitinated while IRF-3, another key molecule on the downstream of TRIF, was phosphorylated obviously after the treatment of PPI. The phosphorylation of molecules involved in both NF-κB pathway and MAPK pathway were significantly up-regulated after PPI treatment. In addition, the effects of PPI on the macrophages almost completely disappeared after treating the cells with the TLR4 antagonist TAK-242. Further in vivo results showed PPI significantly increased the serum OVA-specific antibody and the OVA-specific spleen cell proliferation. Taken together, PPI can specifically stimulate TLR4 and activate both MyD88 and TRIF pathways. PPI has immune adjuvant activity and may become a new potential immune adjuvant.