Construction and Validation of an Epigenetic Regulator Signature as A Novel Biomarker For Prognosis, Immunotherapy, And Chemotherapy In Hepatocellular Carcinoma.

Background: Epigenetic modification regulates various aspects of cancer biology, from tumor growth and invasion to immune microenvironment modulation. Whether epigenetic regulators (EGRs) can decide tumor malignant degree and risk of immune evasion in liver hepatocellular carcinoma (LIHC) remains unclear. Method: An EGR signature called "EGRscore" was constructed based on bulk RNA-seq data of EGR in hepatocellular carcinoma (HCC). The correlation between EGRscore and overall survival (OS) was validated in HCC cohorts and other tumor cohorts. Mutation profiles, copy number alterations (CNAs), enriched pathways, and response to immunotherapy and chemotherapy were compared between EGRscore-high and EGRscore-low patients. Results: We found that EGRscore was associated with OS in HCC as well as several tumors including glioma, uveal melanoma (UVM), and kidney tumors. A mechanism study demonstrated that the distinct mutation profile of TP53 was present in EGRscore-high and EGRscore-low patients. Meanwhile, EGRscore-low patients were characterized with immune cells that promote killing tumors. Furthermore, EGRscore was associated with genes regulating drug resistance in HCC. Finally, we indicated that EGRscore-low patients had higher response rates to immunotherapy and targeted therapy. Conclusions: EGRscore could be used to distinguish OS, tumor progression, mutation pattern, and immune microenvironment. The present study contributes to improving hepatocellular carcinoma patient prognosis and predicting response to immunotherapy.

IFNα Potentiates Anti-PD-1 Efficacy by Remodeling Glucose Metabolism in the Hepatocellular Carcinoma Microenvironment.

The overall response rate for anti-PD-1 therapy remains modest in hepatocellular carcinoma (HCC). We found that a combination of IFNα and anti-PD-1-based immunotherapy resulted in enhanced antitumor activity in patients with unresectable HCC. In both immunocompetent orthotopic and spontaneous HCC models, IFNα therapy synergized with anti-PD-1 and the combination treatment led to significant enrichment of cytotoxic CD27+CD8+ T cells. Mechanistically, IFNα suppressed HIF1α signaling by inhibiting FosB transcription in HCC cells, resulting in reduced glucose consumption capacity and consequentially establishing a high-glucose microenvironment that fostered transcription of the T-cell costimulatory molecule Cd27 via mTOR-FOXM1 signaling in infiltrating CD8+ T cells. Together, these data reveal that IFNα reprograms glucose metabolism within the HCC tumor microenvironment, thereby liberating T-cell cytotoxic capacities and potentiating the PD-1 blockade-induced immune response. Our findings suggest that IFNα and anti-PD-1 cotreatment is an effective novel combination strategy for patients with HCC. SIGNIFICANCE: Our study supports a role of tumor glucose metabolism in IFNα-mediated antitumor immunity in HCC, and tumor-infiltrating CD27+CD8+ T cells may be a promising biomarker for stratifying patients for anti-PD-1 therapy. See related commentary by Kao et al., p. 1615. This article is highlighted in the In This Issue feature, p. 1599.

Mucin 1 promotes tumor progression through activating WNT/ß-catenin signaling pathway in intrahepatic cholangiocarcinoma.

Background: Current treatment options for intrahepatic cholangiocarcinoma (ICC) are limited by the lack of understanding of the disease pathogenesis. It has been known that mucin 1 (MUC1) is a cell surface mucin that highly expressed in various cancer tissues. However, its role in ICC has not been well studied. The purpose of this study was to investigate the clinical significance and biological function of MUC1 in ICC. Methods: qRT-PCR and western blot assays were performed to examine MUC1 expression. RNA-Seq (RNA Sequencing) s conducted to explore the RNA expression. A tissue microarray study including 214 ICC cases was also conducted to evaluate the clinical relevance and prognostic significance of MUC1. The role and underlying mechanisms of MUC1 in regulating cell growth and invasion were further explored both in vitro and in vivo models. Results: The mRNA and protein levels of MUC1 were significantly up-regulated in ICC compared to paired non-tumor tissues. Depletion of MUC1 in HCCC9810 cells significantly inhibited cell proliferation, migration and invasion in vitro and overexpression of MUC1 in RBE cells resulted in increased cell proliferation, migration and invasion. Both univariate and multivariate analysis revealed that the protein expression of MUC1 was associated with overall survival and relapse-free survival after tumor resection. Clinically, high MUC1 expression was more commonly observed in aggressive tumors. Further studies indicated that MUC1 exerted its function through activating Wnt/ ß-catenin pathway. Conclusions: Our data suggests that MUC1 promoted ICC progression via activating Wnt / ß-catenin pathway. This study not only deciphered the role of MUC in ICC pathogenesis, but also shed light upon identifying novel potential therapeutic targets.

Circulating tumor cell detection and single-cell analysis using an integrated workflow based on ChimeraX® -i120 Platform: A prospective study.

Circulating tumor cell (CTC) analysis holds great potential to be a noninvasive solution for clinical cancer management. A complete workflow that combined CTC detection and single-cell molecular analysis is required. We developed the ChimeraX® -i120 platform to facilitate negative enrichment, immunofluorescent labeling, and machine learning-based identification of CTCs. Analytical performances were evaluated, and a total of 477 participants were enrolled to validate the clinical feasibility of ChimeraX® -i120 CTC detection. We analyzed copy number alteration profiles of isolated single cells. The ChimeraX® -i120 platform had high sensitivity, accuracy, and reproducibility for CTC detection. In clinical samples, an average value of > 60% CTC-positive rate was found for five cancer types (i.e., liver, biliary duct, breast, colorectal, and lung), while CTCs were rarely identified in blood from healthy donors. In hepatocellular carcinoma patients treated with curative resection, CTC status was significantly associated with tumor characteristics, prognosis, and treatment response (all P < 0.05). Single-cell sequencing analysis revealed that heterogeneous genomic alteration patterns resided in different cells, patients, and cancers. Our results suggest that the use of this ChimeraX® -i120 platform and the integrated workflow has validity as a tool for CTC detection and downstream genomic profiling in the clinical setting.

Detection of circulating tumour cells enables early recurrence prediction in hepatocellular carcinoma patients undergoing liver transplantation.

BACKGROUND & AIMS: Liver transplantation (LTx) is one of the most effective treatments for hepatocellular carcinoma (HCC); however, tumour recurrence after LTx often leads to poor outcomes. This study investigated the value of circulating tumour cells (CTCs) as a predictor of recurrence following LTx in patients with HCC. METHODS: This analysis included 193 patients with HCC who underwent LTx at our institute and accepted pre- and post-operative CTC detection; 38 were selected for serial CTC monitoring. The predictive value of CTCs for tumour recurrence in patients with HCC following LTx was evaluated. Single-cell whole genome sequencing was used to characterize CTCs. RESULTS: Overall, the CTC burden decreased after LTx (P < .05). Post-operative CTC count ≥ 1 per 5 mL peripheral blood was identified as a potential biomarker for predicting tumour recurrence after LTx, especially in patients with no detectable CTCs prior to LTx and negative tumour serological biomarkers. The predictive value of post-operative CTC count ≥ 1 per 5 mL blood was retained in patients who did not meet the Milan criteria, University of California San Francisco (UCSF) criteria, or Fudan criteria (all P < .05). Furthermore, post-operative serial CTC detection may be useful in post-surgical surveillance for HCC recurrence. CONCLUSIONS: CTCs may be a useful biomarker to evaluate recurrence risk following LTx in patients with HCC. Evaluation based on CTC detection may enhance the post-transplant management of HCC, and improve the therapeutic efficacy of LTx.

CD13 promotes hepatocellular carcinogenesis and sorafenib resistance by activating HDAC5-LSD1-NF-κB oncogenic signaling.

RATIONALE: CD13 is a new marker for liver cancer stem cells (CSCs) that contributes to sorafenib resistance in hepatocellular carcinoma (HCC). However, the underlying mechanism of CD13 in HCC sorafenib resistance remains enigmatic. METHODS: The expression of CD13 in HCC cell lines and tissues was assayed by RT-PCR, western-blot, and immunohistochemistry staining. Athymic BALB/c nu/nu mice model was used to study the in vivo functions of CD13. Clinical significance of CD13 was evaluated by Kaplan-Meier methods. Cellular proliferation rate was evaluated by cell counting kit-8 cell proliferation assay and colony formation assay. Tunel assay was used to detect cell death ratio. Transwell assay was used to evaluate the motility of cells. Immunoprecipitation (IP), liquid chromatography-mass spectrometry (LC-MS)/MS, and co-IP were applied to investigate potential protein interactions of CD13. RESULTS: In this research, we found that CD13 expression was higher in metastatic HCC samples, and its overexpression was predicted worse prognosis for patients after surgical resection. Functionally, CD13 promoted HCC proliferation, invasion, cell cycle progression as well as sorafenib resistance. Mechanistically, CD13 interacted with histone deacetylase5 (HDAC5) to promote its protein stability, thus resulting in HDAC5-mediated lysine-specific demethylase 1 (LSD1) deacetylation and protein stabilization. Consequently, LSD1 decreased the NF-κB catalytic unit p65 methylation that led to p65 protein stability. A CD13 inhibitor ubenimex in combination with sorafenib, suppressed the tumor growth and attenuated the resistance of HCC cells toward sorafenib in patient-derived xenograft models. CONCLUSIONS: CD13 promotes HCC progression and induces sorafenib resistance, mainly via interacting with HDAC5 to prevent the degradation of p65 and activate NF-kB signaling pathway. CD13 is a prognostic indicator for HCC patients underwent curative resection as well as a predictor of response to treatment with sorafenib. Our study establishes the new therapeutic potential of targeting CD13-HDAC5-LSD1-NF-κB in HCC.

Occurrence of substance P and neurokinin receptors during the early phase of spinal fusion.

Spinal fusion is widely used for patients with spinal disorders; however, patients often suffer from back pain following fusion surgery. Substance P (SP) acts as a pain neurotransmitter via the sensory nerve afferent fibres up to the spinal cord, and is involved in the conduction and modulation of pain. The use of specific SP neurokinin receptor (NKR) antagonists may decrease postoperative pain. In the present study, the effects of alterations in the quantity of SP and NKRs in the early spinal fusion process were investigated. The results of the present study revealed that SP and NKRs began to appear 1 week post­surgery in fibrous tissues. The abundance of SP and NKRs peaked at 3 weeks post­surgery; the majority of SP and NKRs were distributed around the allograft and the new microvessels. In conclusion, SP and NKRs are involved in early spinal fusion, a finding that may facilitate the development of novel strategies to promote spinal fusion from a neurogenesis perspective.

Low-intensity pulsed ultrasound promotes spinal fusion and enhances migration and proliferation of MG63s through sonic hedgehog signaling pathway.

Low-intensity pulsed ultrasound (LIPUS) has been found to accelerate the healing process of spinal fusion via a process closely related to osteoblast differentiation and migration. Sonic hedgehog (Shh) signaling plays an important role in development and homeostasis, including a critical function in bone formation. However, its role in spinal fusion during LIPUS treatment is still unknown. This study showed that LIPUS treatment after spinal fusion surgery increased bone formation. The increased bone mass under LIPUS treatment appeared to result from the increased migration and proliferation of osteoblasts, resulting from upregulation of the Shh signaling pathway. In contrast, inhibition of Shh reduced the migratory and proliferative ability of osteoblast-like MG63 cells and blocked the efficacy of LIPUS treatment.

Low-Intensity Pulsed Ultrasound Accelerates Traumatic Vertebral Fracture Healing by Coupling Proliferation of Type H Microvessels.

OBJECTIVES: Patients with traumatic vertebral fractures often have major associated postoperative morbidities such as healing failure and kyphosis. Low-intensity pulsed ultrasound (US) has been found to promote bone fracture healing. The objectives of our study were to determine whether low-intensity pulsed US could promote traumatic vertebral fracture healing and to explore its inner mechanisms. METHODS: A rat model of traumatic vertebral fracture was created and treated with low-intensity pulsed US after surgery. At 4 weeks after surgery, radiographic, micro-computed tomography, and 3-dimensional reconstruction were used to assess the radiologic healing status; a histologic analysis was performed to evaluate the pathologic process and relationship between osteogenesis and type H microvessels. RESULTS: Well-remodeled trabecular meshworks were found in the low-intensity pulsed US treatment group compared to the control group. Micro-computed tomography and 3-dimensional reconstruction revealed more and thicker trabeculae after low-intensity pulsed US treatment. Abundant chondrocytes, a newly formed bone marrow cavity, trabeculae, and microvessels were formed at the fracture sites. More osterix-positive osteoblasts were circling the newly formed bone meshwork and were situated at the interface of chondrocytes in the low-intensity pulsed US treatment group. Type H microvessels were spreading around the newly formed trabecula, bone marrow cavity, osteoblasts, and interface of chondrocytes, with a larger mean vascular density in the low-intensity pulsed US group. CONCLUSIONS: Low-intensity pulsed US could accelerate traumatic vertebral fracture healing by temporally and spatially increasing chondrogenesis and osteoblast-induced osteogenesis coupled with angiogenesis of type H microvessels in a rat model of traumatic vertebral fracture.

Low-intensity pulsed ultrasound promotes endothelial cell-mediated osteogenesis in a conditioned medium coculture system with osteoblasts.

Angiogenesis plays an important role during bone regeneration. Low-intensity pulsed ultrasound (LIPUS) has been proven to accelerate the process of bone fracture healing. However, the mechanism of the effect of LIPUS on bone regeneration is still unclear. In the present study, we used human umbilical vein endothelial cell (HUVEC) and human osteosarcoma cell (MG-63) to investigate the effect of LIPUS stimulation in an endothelial cell-osteoblast coculture system. At the same time, we used transwell and in vitro angiogenesis assay to observe how LIPUS affects endothelial cells. The results demonstrated that LIPUS could significantly increase the migratory ability and promote tube formation in angiogenesis of HUVECs. Furthermore, LIPUS could significantly elevate the expression of osteogenesis-related genes on osteoblasts such as Runt-related transcription factor 2, alkaline phosphatase, Osteorix, and Cyclin-D1, indicating the pro-osteogenesis effect of LIPUS in our coculture system. In conclusion, endothelial cell is involved in LIPUS-accelerated bone regeneration, the positive effect of LIPUS may be transferred via endothelial cells surrounding fracture healing site.

Cross-cultural validation of simplified Chinese version of spine functional index.

BACKGROUND: No effective constructs were available in mainland China to assess the whole spine function. The SFI was developed to evaluate spinal function based on the concept of a single kinetic chain concept for whole spine. The SFI has been translated to Spanish and Turkish with accepted psychometric properties. It is imperative to introduce the SFI in mainland China and further to explore the measurement properties. METHODS: The English versions of the SFI was cross-culturally translated according to international guidelines. Measurement properties (content validity, construct validity and reliability) were tested in accordance with the COSMIN checklists. A total of 271 patients were included in this study, and 61 participants with neck pain and 64 participants with back pain paid a second visit three to seven days later. Confirmatory factor analysis (CFA) and principal factor analysis (PCA) were applied to test the factor structure. The Functional Rating Index (FRI), Neck Disability Index (NDI), Oswestry Disability Index (ODI), SF-12 and a Visual Analogue Scale (VAS) were employed to evaluate the construct validity. Cronbach's alpha and an intra-class correlation coefficient (ICC) were calculated for internal consistency and reproducibility. RESULTS: The means score of SC-SFI was 63.60 in patients with spinal musculoskeletal disorders. A high response rate was acquired (265/271). No item was removed due to abnormal distribution or low item-total correlation. Results of CFA did not support that one-factor structure was in goodness of fit (CMIN/DF = 3.306, NNFI = 0.687, CFI = 0.756, GFI = 0.771 and RMSEA = 0.092). Yet, PCA suggested a one-factor structure was the best, accounting for 32% of the total variance. For structural validity, the SC-SFI correlated highly with the FRI, NDI, ODI, and PF, BP in SF-12 (r = 0.661, 0.610, 0.750, 0.709, 0.605, respectively). All the a priori hypotheses were verified. The Cronbach's alpha for the SC-SFI was 0.91, and ICC was 0.96 (95% CI, 0.94-0.98). Bland-Altman plot also confirmed excellent test-retest reliability. CONCLUSIONS: The SFI has been culturally adapted into SC-SFI with remarkable clinical acceptance, excellent internal consistency, reproducibility, and construct validity when applied to patients with spinal musculoskeletal disorders. The results of current study suggest that SC-SFI can be applied by physicians and researchers to measure whole-spine functional status in mainland China.

Low-Intensity Pulsed Ultrasound-Induced Spinal Fusion is Coupled with Enhanced Calcitonin Gene-Related Peptide Expression in Rat Model.

Low-intensity pulsed ultrasound (LIPUS) has been found to accelerate fracture healing. In this study, we analyzed the role of calcitonin gene-related peptide (CGRP) in a rat spinal fusion model treated with LIPUS. The results revealed that LIPUS significantly increases bone formation, and the process was coupled with elevated CGRP innervation. CGRP was located in fibrous tissue, closely surrounding the allograft and newly formed cartilage. The density of CGRP peaked at week 3 after surgery in both the control (non-LIPUS-treated) and LIPUS-treated groups. These results suggest that LIPUS might accelerate spinal fusion by promoting sensory nerve fiber innervation.

Validations and psychological properties of a simplified Chinese version of pain anxiety symptoms scale (SC-PASS).

The Pain Anxiety Symptoms Scale (PASS) has been developed to evaluate pain anxiety, which leads to avoidance of daily activities and normal movements. However, a simplified Chinese version of PASS is still not available. Physicians are not aware of which patients are prone to anxiety, and what the risk factors are.To cross-culturally adapt the PASS into a simplified Chinese version and test the reliability and validity. Factors affecting pain anxiety were also explored.The PASS was first translated into a simplified Chinese version according to a forward-backward method. Then, validations were tested including content validity, construct validity, and reliability. Content validity was analyzed by response trend. Construct validity was analyzed by confirmatory factor analysis (CFA), exploratory factor analysis, and priori hypotheses testing. Reliability was analyzed by internal consistency and test-retest reliability. Risk factors of catastrophizing were analyzed by performing multivariate liner regression.A total of 219 patients were included in the study. The scores of items were well distributed. Both CFA and exploratory factor analysis suggested a 2nd-order, 4-factor model, accounting for 65.42% of the total variance according to principle component analysis. SC-PASS obtained good reliability with a Cronbach α = 0.92 and ICC = 0.90. College education, long pain duration, and both married and divorced status were risk factors. Factors reduced pain-related anxiety were no medication assumption, female sex, widowed status, non-Han ethnicity, and having no religious belief.The SC-PASS was applicable in Chinese patients and it was suitable for the clinical uses in mainland China.

Spatiotemporal Changes of Calcitonin Gene-Related Peptide Innervation in Spinal Fusion.

Few studies have investigated the role calcitonin gene-related peptide (CGRP) plays in the process of spinal fusion. The aim of the present study is to observe the temporal and spatial changes of CGRP induced by experimental fusion surgery in rats and elucidate the role of CGRP in spinal fusion. Male Sprague-Dawley rats were used in the study and the specimens were collected on the 7th, 14th, 21st, and 28th day, respectively. Then, histological and immunohistochemical analysis were applied to evaluate the fusion mass and spatiotemporal changes of CGRP chronologically. The results demonstrated that density of CGRP reached peak on the 21st day after surgery and most of the CGRP expression located surrounding the interface of allograft and fibrous tissue where the cells differentiate into osteoblasts, indicating that CGRP might be involved in the process of bone formation and absorption.