Sequential CD7 CAR T-Cell Therapy and Allogeneic HSCT without GVHD Prophylaxis.

BACKGROUND: Patients with relapsed or refractory hematologic cancers have a poor prognosis. Chimeric antigen receptor (CAR) T-cell therapy as a bridge to allogeneic hematopoietic stem-cell transplantation (HSCT) has the potential for long-term tumor elimination. However, pre-HSCT myeloablation and graft-versus-host disease (GVHD) prophylaxis agents have toxic effects and could eradicate residual CAR T cells and compromise antitumor effects. Whether the integration of CAR T-cell therapy and allogeneic HSCT can preserve CAR T-cell function and improve tumor control is unclear. METHODS: We tested a novel "all-in-one" strategy consisting of sequential CD7 CAR T-cell therapy and haploidentical HSCT in 10 patients with relapsed or refractory CD7-positive leukemia or lymphoma. After CAR T-cell therapy led to complete remission with incomplete hematologic recovery, patients received haploidentical HSCT without pharmacologic myeloablation or GVHD prophylaxis drugs. Toxic effects and efficacy were closely monitored. RESULTS: After CAR T-cell therapy, all 10 patients had complete remission with incomplete hematologic recovery and grade 4 pancytopenia. After haploidentical HSCT, 1 patient died on day 13 of septic shock and encephalitis, 8 patients had full donor chimerism, and 1 patient had autologous hematopoiesis. Three patients had grade 2 HSCT-associated acute GVHD. The median follow-up was 15.1 months (range, 3.1 to 24.0) after CAR T-cell therapy. Six patients remained in minimal residual disease-negative complete remission, 2 had a relapse of CD7-negative leukemia, and 1 died of septic shock at 3.7 months. The estimated 1-year overall survival was 68% (95% confidence interval [CI], 43 to 100), and the estimated 1-year disease-free survival was 54% (95% CI, 29 to 100). CONCLUSIONS: Our findings suggest that sequential CD7 CAR T-cell therapy and haploidentical HSCT is safe and effective, with remission and serious but reversible adverse events. This strategy offers a feasible approach for patients with CD7-positive tumors who are ineligible for conventional allogeneic HSCT. (Funded by the National Natural Science Foundation of China and the Key Project of Science and Technology Department of Zhejiang Province; ClinicalTrials.gov numbers, NCT04599556 and NCT04538599.).

Single-cell transcriptomics reveals the role of Macrophage-Naïve CD4 + T cell interaction in the immunosuppressive microenvironment of primary liver carcinoma.

BACKGROUND: Liver carcinoma generally presents as an immunosuppressive microenvironment that promotes tumor evasion. The intercellular crosstalk of immune cells significantly influences the construction of an immunosuppressive microenvironment. This study aimed to investigate the important interactions between immune cells and their targeting drugs in liver carcinoma, by using single-cell and bulk transcriptomic data. METHODS: Single-cell and bulk transcriptomic data were retrieved from Gene Expression Omnibus (GSE159977, GSE136103, and GSE125449) and The Cancer Genome Atlas (TGCA-LIHC), respectively. Quality control, dimension reduction, clustering, and annotation were performed according to the Scanpy workflow based on Python. Cell-cell interactions were explored using the CellPhone database and CellChat. Trajectory analysis was executed using a partition-based graph abstraction method. The transcriptomic factors (TFs) were predicted using single-cell regulatory network inference and clustering (SCENIC). The target genes from TFs were used to establish a related score based on the TCGA cohort; this score was subsequently validated by survival, gene set enrichment, and immune cell infiltration analyses. Drug prediction was performed based on the Cancer Therapeutics Response Portal and PRISM Repurposing datasets. RESULTS: Thirty-one patients at four different states, including health, hepatitis, cirrhosis, and cancer, were enrolled in this study. After dimension reduction and clustering, twenty-two clusters were identified. Cell-cell interaction analyses indicated that macrophage-naive CD4 + T cell interaction significantly affect cancerous state. In brief, macrophages interact with naive CD4 + T cells via different pathways in different states. The results of SCENIC indicated that macrophages present in cancer cells were similar to those present during cirrhosis. A macrophage-naive CD4 + T cell (MNT) score was generated by the SCENIC-derived target genes. Based on the MNT score, five relevant drugs (inhibitor of polo-like kinase 1, inhibitor of kinesin family member 11, dabrafenib, ispinesib, and epothilone-b) were predicted. CONCLUSIONS: This study reveals the crucial role of macrophage-naive CD4 + T cell interaction in the immunosuppressive microenvironment of liver carcinoma. Tumor-associated macrophages may be derived from cirrhosis and can initiate liver carcinoma. Predictive drugs that target the macrophage-naive CD4 + T cell interaction may help to improve the immunosuppressive microenvironment and prevent immune evasion. The relevant mechanisms need to be further validated in experiments and cohort studies.

Retinal dehydrogenase 5 (RHD5) attenuates metastasis via regulating HIPPO/YAP signaling pathway in Hepatocellular Carcinoma.

Retinal dehydrogenase 5 (RDH5) is an important enzyme in the visual cycle. Several studies have reported that the RDH family may play crucial roles in tumor prognosis. However, the role of RDH5 in tumor prognosis is still unclear. We examined the mRNA level of RDH5 by using q-PCR in hepatocellular carcinoma (HCC) and adjacent non-cancerous tissues. The proliferation rate of HCC cells was detected by MTS assay, and the invasive ability was examined by transwell and scratch wound assays. The YAP protein localization and expression were visualized by immunofluorescence in two different cell lines. CpG islands in the promoter region were predicted by using the methprimer database. Clinical characteristics of a patient cohort data came from The Cancer Genome Atlas database. RDH5 was significantly downregulated in hepatocellular carcinoma tissues, and low RDH5 expression was associated with metastasis and poor patient prognosis. Functional assays revealed that the RDH5 promoter is methylated in HCC cell lines. Moreover, overexpressing RDH5 can suppress metastasis by reversing the epithelial-mesenchymal transition (EMT) process, and RDH5 also inhibits cell proliferation in HCC cell lines. Furthermore, suppressing RDH5 can activate the Hippo/YAP signaling pathway and promote the nuclear translocation of YAP. Clinical data demonstrated that RDH5 is an independent prognostic factor in HCC. In our study, we provided the first evidence that RDH5 plays a crucial role in suppressing proliferation and metastasis, and the RDH5 promoter is methylated in hepatocellular carcinoma. And as an important regulator, RDH5 can suppress the Hippo/YAP signaling pathway. Taken together, it revealed that RDH5 might be a potential therapeutic target in HCC patients.

Comparison of three fixation modalities for unilateral open-door cervical laminoplasty: a systematic review and network meta-analysis.

Three fixation modalities including suture suspensory, anchor, and titanium plate are used extensively in unilateral open-door cervical laminoplasty. Nevertheless, up to now no systematic review and network meta-analysis have been published, and the differences in efficacy and safety of the three fixation modalities are still unclear. The purpose of this study is to compare the effectiveness and safety of the three fixation modalities including suture suspensory, anchor, and titanium plate in unilateral open-door cervical laminoplasty. Randomized controlled trials and cohort studies which compared the three interventions in unilateral open-door cervical laminoplasty were identified using the following databases: PubMed, Cochrane Library, Embase, Web of science, China National Knowledge Infrastructure, Chinese Science and Technology Periodical Database, and Wanfang data. Network meta-analysis was performed using R 3.4.3 software and STATA version 14.0. The results revealed that compared with suture suspensory, titanium plate and anchor showed the same effects in Japanese Orthopedic Association Scores, operative time, and blood loss. However, titanium plate showed superiority in postoperative range of motion of cervical spine, incidence of axial symptoms, and C5 paralysis; in terms of cervical curvature, titanium plate also showed better effectiveness than suture suspensory, but similar as anchor. Our network meta-analysis suggests that titanium plate is preferable to suture suspensory or anchor with more range of motion and lower incidence of axial symptoms and C5 paralysis. However, considering the limitations of this research, high-quality trials are needed in the future to evaluate the outcomes.

Biomechanical Comparison of Lumbar Fixed-Point Oblique Pulling Manipulation and Traditional Oblique Pulling Manipulation in Treating Lumbar Intervertebral Disk Protrusion.

OBJECTIVE: To compare the biomechanical effect of lumbar fixed-point oblique pulling manipulation and traditional oblique pulling manipulation in the treatment of protrusion of lumbar intervertebral disk, and investigate the influence of disk degeneration on the 2 manipulations. METHODS: Three finite element models including 1 normal model, 1 mild degeneration, and 1 moderate degeneration model of L3-S1 were developed to simulate 2 oblique pulling manipulations. The disk protrusion was assumed to be in the left central and subarticular zone of the L4-L5 disk, and manipulations were carried out on the right. A 15-Nm right axial rotation moment and 150-N compressive loading were imposed on the upper endplate of L3 to simulate a traditional oblique pulling manipulation. To simulate lumbar fixed-point oblique pulling manipulation, in addition to a 15-Nm moment and 150-N compressive loading imposed on the L3 upper endplate, a 50-N force was imposed on the right lateral area of the L4 spinous process in a left front direction. The displacement and stress in the left central and subarticular zone of the L4-L5 disk were calculated and compared in the 3 models. RESULTS: The average displacement and stress in the left central and subarticular zone of L4-L5 disk were higher in fixed-point oblique pulling manipulation than those in traditional oblique pulling manipulation (P < .05). In addition, the values of average stress and displacement decreased significantly with the increase of lumbar disk degeneration (P < .05). CONCLUSION: Lumbar fixed-point oblique pulling manipulation showed a better biomechanical effect than traditional oblique pulling manipulation, and lumbar disk degeneration affected the 2 manipulations adversely in the virtual treatment of protrusion of the lumbar intervertebral disk using finite element models.

Comparison Between Oblique Pulling Spinal Manipulation and Other Treatments for Lumbar Disc Herniation: A Systematic Review and Meta-Analysis.

OBJECTIVE: The purpose of this review was to compare oblique pulling spinal manipulation with other treatments for lumbar disc herniation. METHODS: Randomized controlled trials of oblique pulling manipulation versus other treatment for lumbar disc herniation were identified using the following databases: China National Knowledge Infrastructure, Wanfang Data, Chinese Science and Technology Periodical Database, PubMed, the Cochrane Library, Embase, Chinese Biological Medicine, and Web of Science. Data extraction was carried out based on inclusion and exclusion criteria, and meta-analysis were performed using RevMan 5.3 software. RESULTS: Nine relevant randomized controlled trials with a total of 887 patients were included. Meta-analysis revealed that oblique pulling manipulation was superior in effective rate to lumbar traction (risk ratio = 1.12; 95% confidence interval [CI]: 1.06-1.19; P < .01) and acupuncture (risk ratio = 1.22; 95% CI: 1.06-1.39; P < .01) and more effective in Visual Analog Scale score (mean difference = - 1.03, 95% CI: -1.32 to -0.74; P < .01) when compared to lumbar traction. It also demonstrated a favorable effect of modified oblique pulling manipulation in Japanese Orthopedic Association scores when compared with lumbar traction (mean difference = 1.66, 95% CI: 0.89 to 2.43; P < .01). CONCLUSION: In the treatment of lumbar disc herniation, oblique pulling spinal manipulation presented with a higher effective rate than acupuncture and lumbar traction. Manipulation had a favorable effect in alleviating pain, and modified oblique pulling manipulation had significant superiority in improving lumbar function when compared with lumbar traction. However, considering the low methodological quality of included studies, more rigorously designed trials should be performed in the future.

Exercise therapy versus surgery for lumbar spinal stenosis: A systematic review and meta-analysis.

OBJECTIVE: To compare the effectiveness of exercise therapy with surgery for lumbar spinal stenosis. METHODS: Five English databases PubMed, the Cochrane Library, Web of science, OVID and PEDro database were searched for randomized controlled trials comparing surgical procedures with exercise therapy for lumbar spinal stenosis. Information on patients, study design, inclusion criteria, intervention and follow-up, outcomes, treatment details and adverse events were extracted. Meta-analysis was performed using Review Manager Version 5.3. RESULTS: Two randomized controlled trials and one mixed design trial with a total of 897 patients were included. The pooled results showed a significant difference between exercise and surgery in Oswestry Disability Index at two years (MD= 3.85, 95%CI: 0.48 to 7.22; P=0.03), but no significant difference at six months (MD= 2.18, 95%CI: -2.80 to 7.17; P=0.39) and one year (MD= 4.26, 95%CI: -1.79 to 10.32; P=0.17). In terms of physical function of 36 Items Short Form Health Survey, there were no significant differences between exercise and surgery at six months (MD= -2.23, 95% CI: -7.46 to 2.99; P=0.40), one year (MD= -2.17, 95% CI: -7.44 to 3.10; P=0.42) and two years (MD= -0.67, 95% CI: -6.16 to 4.82; P=0.81). CONCLUSION: In brief, the current evidence demonstrated a trend that exercise therapy had a similar effect for lumbar spinal stenosis compared with decompressive laminectomies. However, for the small sample size and low methodology quality of the included trials, some rigorously designed and large-scaled RCTs need to be performed to confirm the conclusion.

Comparison of two types of exercises in the treatment of lumbar spinal stenosis.

OBJECTIVE: To evaluate the efficacy of core stability exercise versus conventional exercise in the treatment of lumbar spinal stenosis. METHODS: Between January 2014 and May 2017, patients with lumbar spinal stenosis were recruited and divided into group of core stability exercise or conventional exercise randomly. All the patients were treated using middle frequency electrotherapy, in addition to that, the patients in group of core stability exercise were treated using core stability exercise. The patients in group of conventional exercise were treated using conventional exercise. The outcome was evaluated using Japanese Orthopedic Association (JOA) score, self-reported walking capacity and lumbar lordosis angle at baseline and after treatment. RESULTS: In the current study, sixty-two patients with lumbar spinal stenosis met the inclusion and exclusion criteria, in which 33 patients were included in group of core stability exercise and 29 in group of conventional exercise. After treatment, both Japanese Orthopedic Association scores (p<0.05) and self-reported walking capacity (p<0.05) increased significantly in each group when compared with baseline. The self-reported walking capacity and JOA scores in the group of core stability exercise were significantly higher than those in the conventional exercise group (p<0.05). However, both the intragroup and intergroup comparison of lumbar lordosis presented with no significance (p>0.05). CONCLUSION: Core stability exercise presents with better efficacy than conventional exercise in the treatment of lumbar spinal stenosis.

Integrating single-cell and spatial transcriptomics to elucidate the crosstalk between cancer-associated fibroblasts and cancer cells in hepatocellular carcinoma with spleen-deficiency syndrome.

Background and aim: Most patients with hepatocellular carcinoma (HCC) in China have been diagnosed with spleen deficiency syndrome (SDS), which accelerates the progression of HCC by disrupting the tumor microenvironment homeostasis. This study aimed to investigate the intercellular crosstalk in HCC with SDS. Experimental procedure: An HCC-SDS mouse model was established using orthotopic HCC transplantation based on reserpine-induced SDS. Single-cell data analysis and cancer cell prediction were conducted using Seurat and CopyKAT package, respectively. Intercellular interactions were explored using CellPhoneDB and CellChat and subsequently validated using co-culture assays, ELISA and histological staining. We performed pathway activity analysis using gene set variation analysis and the Seurat package. The extracellular matrix (ECM) remodeling was assessed using a gel contraction assay, atomic force microscopy, and Sirius red staining. The deconvolution of the spatial transcriptomics data using the "CARD" package based on single-cell data. Results and conclusion: We successfully established the HCC-SDS mouse model. Twenty-nine clusters were identified. The interactions between cancer cells and cancer-associated fibroblasts (CAFs) were significantly enhanced via platelet-derived growth factor (PDGF) signaling in HCC-SDS. CAFs recruited in HCC-SDS lead to ECM remodeling and the activation of TGF-ß signaling pathway. Deconvolution of the spatial transcriptome data revealed that CAFs physically surround cancer cells in HCC-SDS. This study reveals that the crosstalk of CAFs-cancer cells is crucial for the tumor-promoting effect of SDS. CAFs recruited by HCC via PDGFA may lead to ECM remodeling through activation of the TGF-ß pathway, thereby forming a physical barrier to block immune cell infiltration under SDS.

Prognostic Role of Unfolded Protein Response-Related Genes in Hepatocellular Carcinoma.

AIMS: To reveal the prognostic role of unfolded protein response (UPR) -related genes in hepatocellular carcinoma (HCC). BACKGROUND: Hepatocellular carcinoma is a genetically heterogeneous tumor, and the prediction of its prognosis remains a challenge. Studies elucidating the molecular mechanisms of UPR have rapidly increased. However, the UPR molecular subtype characteristics of the related genes in HCC progression have yet to be thoroughly studied. OBJECTIVE: Conducting a comprehensive assessment of the prognostic signature of genes related to the UPR in patients with HCC can advance our understanding of the cellular processes contributing to the progression of HCC and offer innovative strategies in precise therapy. METHODS: Based on the gene expression profiles associated with UPR in HCC, we explored the molecular subtypes mediated by UPR-related genes and constructed a UPR-related genes signature that could precisely predict the prognosis for HCC. RESULTS: Using microarray data of HCC patients, differentially expressed UPR-related genes (DEGs) were discovered in malignancies and normal tissues. The HCC was classified into two molecular subtypes by the NMF algorithm based on DEGs modification of the UPR. Moreover, we developed a UPR-related model for predicting HCC patients' prognosis. The robustness of the UPR- related model was confirmed in external validation. Moreover, we analyzed immune responses in different risk groups. Analysis of immune functions revealed that Treg, Macrophages, aDCs, and MHC class-I were significantly up-regulated in high-risk HCC. At the same time, cytolytic activity and type I and II INF response were higher in a low-risk subgroup. CONCLUSION: This study identified two UPR molecular subtypes of HCC and developed a ten-gene HCC prognostic signature model (EXTL3, PPP2R5B, ZBTB17, CCT3, CCT4, CCT5, GRPEL2, HSP90AA1, PDRG1, and STC2), which can robustly forecast the progression of HCC.

Adaptor SH3BGRL drives autophagy-mediated chemoresistance through promoting PIK3C3 translation and ATG12 stability in breast cancers.

Acquired chemotherapy resistance is one of the main culprits in the relapse of breast cancer. But the underlying mechanism of chemotherapy resistance remains elusive. Here, we demonstrate that a small adaptor protein, SH3BGRL, is not only elevated in the majority of breast cancer patients but also has relevance with the relapse and poor prognosis of breast cancer patients. Functionally, SH3BGRL upregulation enhances the chemoresistance of breast cancer cells to the first-line doxorubicin treatment through macroautophagic/autophagic protection. Mechanistically, SH3BGRL can unexpectedly bind to ribosomal subunits to enhance PIK3C3 translation efficiency and sustain ATG12 stability. Therefore, inhibition of autophagy or silence of PIK3C3 or ATG12 can effectively block the driving effect of SH3BGRL on doxorubicin resistance of breast cancer cells in vitro and in vivo. We also validate that SH3BGRL expression is positively correlated with that of PIK3C3 or ATG12, as well as the constitutive occurrence of autophagy in clinical breast cancer tissues. Taken together, our data reveal that SH3BGRL upregulation would be a key driver to the acquired chemotherapy resistance through autophagy enhancement in breast cancer while targeting SH3BGRL could be a potential therapeutic strategy against breast cancer.Abbreviations: ABCs: ATP-binding cassette transporters; Act D: actinomycin D; ACTB/ß-actin: actin beta; ATG: autophagy-related; Baf A1: bafilomycin A1; CASP3: caspase 3; CHX: cycloheximide; CQ: chloroquine; Dox: doxorubicin; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GEO: gene expression omnibus; GFP: green fluorescent protein; G6PD: glucose-6-phosphate dehydrogenase; GSEA: gene set enrichment analysis; IHC: immunochemistry; KEGG: Kyoto Encyclopedia of Genes and Genomes; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; 3-MA: 3-methyladenine; mRNA: messenger RNA; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; SH3BGRL: SH3 domain binding glutamate-rich protein-like; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1.

A Network Pharmacology Approach to Reveal the Underlying Mechanisms of Artemisia annua on the Treatment of Hepatocellular Carcinoma.

OBJECTIVE: To investigate the potential active ingredients and underlying mechanisms of Artemisia annua (AA) on the treatment of hepatocellular carcinoma (HCC) based on network pharmacology. METHODS: In the present study, we used a network pharmacological method to predict its underlying complex mechanism of treating HCC. First, we obtained relative compounds of AA based on the traditional Chinese medicine systems pharmacology (TCMSP) database and collected potential targets of these compounds by target fishing. Then, we built HCC-related targets target by the oncogenomic database of hepatocellular carcinoma (OncoDB.HCC) and biopharmacological network (PharmDB-K) database. Based on the matching results between AA potential targets and HCC targets, we built a protein-protein interaction (PPI) network to analyze the interactions among these targets and screen the hub targets by topology. Furthermore, the function annotation and signaling pathways of key targets were performed by Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis using DAVID tools. Finally, the binding capacity between active ingredients and key targets was validated by molecular docking. RESULTS: A total of 19 main active ingredients of AA were screened as target prediction; then, 25 HCC-related common targets were seeked out via multiple HCC databases. The areas of nodes and corresponding degree values of EGFR, ESR1, CCND1, MYC, EGF, and PTGS2 were larger and could be easily found in the PPI network. Furthermore, GO and KEGG enrichment analysis showed that these key targets were significantly involved in multiple biological processes and pathways which participated in tumor cell proliferation, apoptosis, angiogenesis, tumor invasion, and metastasis to accomplish the anti-HCC activity. The molecular docking analysis showed that quercetin could stably bind to the active pocket of EGFR protein 4RJ5 via LibDock. CONCLUSION: The anticancer effects of AA on HCC were predicted to be associated with regulating tumor cell proliferation, apoptosis, angiogenesis, tumor invasion, and metastasis via various pathways such as the EGFR signaling pathway, ESR1 signaling pathway, and CCND1 signaling pathway. It is suggested that AA might be developed as a broad-spectrum antitumor drug based on its characteristics of multicomponent, multipath, and multitarget.

A Comprehensive Pan-Cancer Analysis of 33 Human Cancers Reveals the Immunotherapeutic Value of Aryl Hydrocarbon Receptor.

Background: Previous studies have reported the potential of aryl hydrocarbon receptor (AhR) in cancer immunotherapy. However, the mechanisms underpinning its therapeutic value have yet to be comprehensively investigated. Thus, this research aimed to explore the underlying association between AhR and cancer immunotherapy in 33 human cancers. Methods: The gene expression data and clinical characteristics of 33 cancers were retrieved from The Cancer Genome Atlas database. The immunotherapeutic cohorts included GSE67501 and GSE78220 as well as IMvigor210, which were obtained from the Gene Expression Omnibus database and included in a previously published study respectively. Clinical parameters, including patient age, gender, survival, and tumor stage were analyzed to assess the prognostic value of AhR. The activity of AhR was generated by single sample gene set enrichment analysis and used to evaluate the difference between the AhR transcriptome and protein expression level. To better understand the role of AhR in cancer immunotherapy, the correlation between AhR and tumor microenvironment, as well as its relation to immune processes/elements, such as immune cell infiltration, immune inhibitors and stimulators, and the major histocompatibility complex were analyzed. The relevant underlying pathways associated with AhR signaling in cancer were also explored. Furthermore, the correlation between AhR and two immunotherapeutic biomarkers (tumor mutational burden and microsatellite instability) was investigated. Finally, the relationship between AhR and immunotherapeutic response was explored using three independent immunotherapeutic cohorts. Results: Although AhR was not closely associated with age (5/33), gender (3/33), or tumor stage (3/21) in any of the studied human cancers, it exhibited potential prognostic value for predicting patient survival. Consistency has been observed between AhR activity and expression in some cancers (7/33). Generally, AhR presented a robust correlation with immune cell infiltration, immune modulators, and immunotherapeutic markers. Moreover, high AhR expression was significantly related to immune-relevant pathways. However, no significant correlation was observed between AhR and the immunotherapeutic response. Conclusions: This research investigated the immunotherapeutic value of AhR in 33 human cancers, providing evidence regarding the function of AhR and its role in clinical treatment. However, considering that a bioinformatics approach was adopted, the current results are preliminary and require further validation.

Hypoxic Characteristic in the Immunosuppressive Microenvironment of Hepatocellular Carcinoma.

Background: Generally, hepatocellular carcinoma (HCC) exists in an immunosuppressive microenvironment that promotes tumor evasion. Hypoxia can impact intercellular crosstalk in the tumor microenvironment. This study aimed to explore and elucidate the underlying relationship between hypoxia and immunotherapy in patients with HCC. Methods: HCC genomic and clinicopathological datasets were obtained from The Cancer Genome Atlas (TCGA-LIHC), Gene Expression Omnibus databases (GSE14520) and International Cancer Genome Consortium (ICGC-LIRI). The TCGA-LIHC cases were divided into clusters based on single sample gene set enrichment analysis and hierarchical clustering. After identifying patients with immunosuppressive microenvironment with different hypoxic conditions, correlations between immunological characteristics and hypoxia clusters were investigated. Subsequently, a hypoxia-associated score was established by differential expression, univariable Cox regression, and lasso regression analyses. The score was verified by survival and receiver operating characteristic curve analyses. The GSE14520 cohort was used to validate the findings of immune cell infiltration and immune checkpoints expression, while the ICGC-LIRI cohort was employed to verify the hypoxia-associated score. Results: We identified hypoxic patients with immunosuppressive HCC. This cluster exhibited higher immune cell infiltration and immune checkpoint expression in the TCGA cohort, while similar significant differences were observed in the GEO cohort. The hypoxia-associated score was composed of five genes (ephrin A3, dihydropyrimidinase like 4, solute carrier family 2 member 5, stanniocalcin 2, and lysyl oxidase). In both two cohorts, survival analysis revealed significant differences between the high-risk and low-risk groups. In addition, compared to other clinical parameters, the established score had the highest predictive performance at both 3 and 5 years in two cohorts. Conclusion: This study provides further evidence of the link between hypoxic signals in patients and immunosuppression in HCC. Defining hypoxia-associated HCC subtypes may help reveal potential regulatory mechanisms between hypoxia and the immunosuppressive microenvironment, and our hypoxia-associated score could exhibit potential implications for future predictive models.

Synergetic delivery of triptolide and Ce6 with light-activatable liposomes for efficient hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC) has been known as the second common leading cancer worldwide, as it responds poorly to both chemotherapy and medication. Triptolide (TP), a diterpenoid triepoxide, is a promising treatment agent for its effective anticancer effect on multiple cancers including HCC. However, its clinical application has been limited owing to its severe systemic toxicities, low solubility, and fast elimination in the body. Therefore, to overcome the above obstacles, photo-activatable liposomes (LP) integrated with both photosensitizer Ce6 and chemotherapeutic drug TP (TP/Ce6-LP) was designed in the pursuit of controlled drug release and synergetic photodynamic therapy in HCC therapy. The TP encapsulated in liposomes accumulated to the tumor site due to the enhanced permeability and retention (EPR) effect. Under laser irradiation, the photosensitizer Ce6 generated reactive oxygen species (ROS) and further oxidized the unsaturated phospholipids. In this way, the liposomes were destroyed to release TP. TP/Ce6-LP with NIR laser irradiation (TP/Ce6-LP+L) showed the best anti-tumor effect both in vitro and in vivo on a patient derived tumor xenograft of HCC (PDXHCC). TP/Ce6-LP significantly reduced the side effects of TP. Furthermore, TP/Ce6-LP+L induced apoptosis through a caspase-3/PARP signaling pathway. Overall, TP/Ce6-LP+L is a novel potential treatment option in halting HCC progression with attenuated toxicity.

Exosome CTLA-4 Regulates PTEN/CD44 Signal Pathway in Spleen Deficiency Internal Environment to Promote Invasion and Metastasis of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common primary cancers, and its pathogenesis is complicated and difficult to screen. Currently, there is no effective treatment. In traditional Chinese medicine, a large proportion of patients with HCC have been diagnosed with spleen deficiency (SD) syndrome and treated with tonifying traditional Chinese medicine, which has significant clinical efficacy. However, the role and molecular mechanism of SD in HCC remain unclear. In this study, 40 mice were randomly divided into four groups: control, SD, HCC, and SD-HCC groups. The liver cancer model of SD was established by reserpine induction and orthotopic transplantation. The effects of SD on the proliferation, apoptosis, invasion, and metastasis of HCC cells were studied by cell proliferation, cell apoptosis, cell scratch, and transwell assay. We found that compared with the HCC group, the protein expressions of cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), phosphatase and tensin homolog (PTEN), and AKT (also known as protein kinase B or PKB) in the exosomes of the SD-HCC group were upregulated. In addition, the metastases and self-renewal of exosomes in the SD-HCC group were more aggressive than those in the HCC group, which could be partially reversed with the addition of CTLA-4 inhibitors. Further studies showed that in the internal environment of SD, CTLA-4 promoted tumor invasion and metastasis by regulating the PTEN/CD44 pathway. In conclusion, our findings suggest that during SD in the internal environment, exosome CTLA-4 regulates the PTEN/CD44 signal pathway to promote the proliferation, self-renewal, and metastasis of liver cancer.

Comparative Efficacy of Graft Options in Anterior Cruciate Ligament Reconstruction: A Systematic Review and Network Meta-Analysis.

PURPOSE: To evaluate the effectiveness of various graft options for anterior cruciate ligament reconstruction using network meta-analysis. METHODS: A medical literature search was conducted of PubMed, the Cochrane Library, Embase, SCOPUS, and Web of Science from their inception through March 1, 2019. The outcomes, including International Knee Documentation Committee (IKDC) form, Lachman test, Lysholm score, Pivot shift test, and Tegner score, were evaluated among graft options. Data extraction was carried out according to inclusion and exclusion criteria, and a network meta-analysis was performed using STATA 14.0. RESULTS: A total of 45 trials with 3992 patients were included. The forest plots revealed no significant differences in IKDC, Lysholm, or Tegner score among the grafts. In Lachman score, a significant difference was found in the comparisons of hamstring tendon allograft (HT-AL) versus patellar tendon autograft (PT-AU) and HT-AL versus hamstring tendon autograft (HT-AU). In pivot shift test, PT-AU was superior to all the other grafts, and quadriceps tendon autograft (QT-AU) was superior to HT-AL and artificial ligament (Art-L) in the number of cases with negative results. According to surface under the cumulative ranking area (SUCRA), PT-AU had the highest probability to be the best intervention in Lachman test and Tegner score; tibialis anterior tendon allograft (TA-AL) in IKDC and Lysholm score; and QT-AU in pivot shift test. Based on the cluster analysis of SUCRA, PT-AU was considered the most appropriate intervention by IKDC and Lachman test. CONCLUSION: This study suggests that PT-AU may be the most appropriate graft for ACL reconstruction according to IKDC and Lachman test results. LEVEL OF EVIDENCE: Level I, network meta-analysis of randomized controlled trials.

A Novel Signature Based on mTORC1 Pathway in Hepatocellular Carcinoma.

BACKGROUND: mTORC1 signal pathway plays a role in the initiation and progression of hepatocellular carcinoma (HCC), but no relevant gene signature was developed. This research aimed to explore the potential correlation between the mTORC1 signal pathway and HCC and establish the related gene signature. METHODS: HCC cases were retrieved from The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), and Gene Expression Omnibus (GEO) databases. The genes included in mTORC1-associated signature were selected by performing univariate and multivariate Cox regression analyses and lasso regression analysis. The protein expression level of included genes was verified by The Human Protein Altas. Then, the signature was verified by survival analysis and multiple receiver operating characteristic (ROC) curve. Moreover, the correlation between signature and immune cells infiltration was investigated. Furthermore, a nomogram was established and evaluated by C-index and calibration plot. RESULTS: The signature was established with the six genes (ETF1, GSR, SKAP2, HSPD1, CACYBP, and PNP). Three genes (ETF1, GSR, and HSPD1) have verified their protein expression level in HCC. Under the grouping from signature, patients in the high-risk group showed worse survival than those in the low-risk group in both three datasets. The signature was found to be significantly associated with the infiltration of B cells, CD4+ T-cells, CD8+ T-cells, dendritic cells, macrophages, and neutrophils. The univariate and multivariate Cox regression analysis indicated that mTORC1-related signature could be the potential independent prognostic factor in HCC. Finally, the nomogram involving age, gender, stage, and signature has been established and verified. CONCLUSION: The mTORC1-associated gene signature established and validated in our research could be used as a potential prognostic factor in HCC.

Novel Molecular Subtypes Associated With 5mC Methylation and Their Role in Hepatocellular Carcinoma Immunotherapy.

BACKGROUND: 5-methylcytosine (5mC) has been reported in the prognosis of a variety of cancers, however, its role in hepatocellular carcinoma (HCC) has not been investigated yet. This study aimed at identifying the molecular subtypes associated with 5mC and establishing a relevant score to predict its prognosis in HCC. METHODS: Somatic gene mutation data and gene expression data were retrieved from The Cancer Genome Atlas database. Molecular subtypes were identified by unsupervised clustering based on the expression of 5mC regulators, and the molecular features of each subtype were investigated by survival, mutation, gene set variation, and immune cell infiltration analyses. Next, we performed a differentially expressed analysis based on the new subtypes and selected the overlapping genes for further analysis. We undertook univariate Cox analysis to analyze these genes and constructed a prognostic model by lasso regression analysis. Meanwhile, survival and gene set enrichment analyses were used to explore the prognosis and the relevant pathways, respectively. The LIRI cohort from the International Cancer Genome Consortium database was used as a reference to validate the 5mC subtypes and 5mC score. RESULTS: Twenty-one types of 5mC regulators were employed in this study, and three 5mC-associated molecular subtypes were identified. These three subtypes presented significant differences in prognosis, immune cell infiltration, immune checkpoint inhibitors, signaling pathways, and mutational features. Compared with cluster 3, cluster 2 exhibited significantly increased expression of PD-L1, TIM3, Galectin9, CTLA4, and CD80, while PD-L1, TIM3, and CD80 were higher in cluster 2 than in cluster 1. Furthermore, a 5mC-related score, composed of seven genes (SGPP2, SALL4, B3GNT7, ROR1, MYBL2, SLC7A1, and CAND2), was proven to be significantly associated with prognosis. The established subtypes and scores were thus successfully verified by the validated cohort. CONCLUSION: To the best of our knowledge, this is the first study to identify a novel molecular subtype based on 5mC regulators. The identification of the 5mC-associated subtype may help reveal the potential relation between 5mC and immunity and provide novel insights for the development of individualized therapy for HCC.

An Integrative Analysis Reveals the Underlying Association Between CTNNB1 Mutation and Immunotherapy in Hepatocellular Carcinoma.

Background: Tumor mutational burden (TMB) was verified to be closely associated with immune checkpoint inhibitors, but it is unclear whether gene mutation has an effect on immunotherapy of hepatocellular carcinoma (HCC). This research aimed to investigate the underlying correlation between gene mutation and immunotherapy in HCC. Methods: The somatic gene mutation data and gene expression data were retrieved from International Cancer Genome Consortium database and The Cancer Genome Atlas (TCGA) database. The mutational genes were selected by the intersection of three cohorts and further identified using survival analysis and TMB correlation analysis. After the identification of key mutational gene, we explored the correlation between gene mutation and both the immune cell infiltration and immune inhibitors. The signaling pathways associated with gene mutation were confirmed through gene set enrichment analysis. Furthermore, the survival analysis and mutational analysis based on TCGA cohort were performed for the validation of included gene. Results: As one of the frequently mutational genes in HCC, CTNNB1 was finally included in our research, for which it showed the significant result in survival analysis and the positive association with TMB of the three cohorts. Meanwhile, the validation of TCGA showed the significant results. Furthermore, natural killer (NK) cells and neutrophil were found to significantly infiltrate CTNNB1 mutation group from two cohorts. Besides, further analysis demonstrated that four types of immune inhibitors (CD96, HAVCR2, LGALS9, and TGFB1) were downregulated in CTNNB1 mutation group. Conclusion: Our research firstly revealed the underlying association between CTNNB1 mutation and immunotherapy, and we speculated that CTNNB1 mutation may modulate NK cells by affecting CD96. However, more functional experiments should be performed for verification.