Early postoperative endoscopy for predicting anastomotic leakage after minimally invasive esophagectomy: A large-volume retrospective study.

BACKGROUND: Anastomotic leakage is one of the most severe adverse events of minimally invasive esophagectomy for esophageal cancer. Early postoperative endoscopy is considered to be the most objective means to diagnose anastomotic leakage, but its safety is questioned by clinicians. This study aimed to evaluate the safety and effectiveness of early postoperative endoscopy in predicting anastomotic leakage. METHODS: Patients who underwent minimally invasive esophagectomy (from January 2017 to June 2021) in our center were identified and divided into early postoperative endoscopy and control groups according to whether they underwent early postoperative endoscopy within 72 hours after surgery. Propensity score matching was used to balance baseline characteristics. The incidence of postoperative adverse events was compared between the 2 groups, risk variables for anastomotic leakage were identified using logistic regression, and abnormal endoscopic findings related to anastomotic leakage occurrence were explored. RESULTS: A total of 436 patients were enrolled, of whom 134 underwent early postoperative endoscopy. One hundred and thirty-two pairs were matched by propensity score matching, and baseline characteristics were well-balanced. Both before and after propensity score matching, early postoperative endoscopy did not increase the incidence of postoperative adverse events (chyle leak, hypoproteinemia, pneumonia, etc) and in-hospital mortality. Notably, the incidence of anastomotic leakage (9.8% vs 22.7%) and the length of mean postoperative hospital stay (17.6 vs 20.9 days) was significantly decreased in the early postoperative endoscopy group. Finally, based on the findings under early postoperative endoscopy, we found that gastric graft ischemia is related to a higher incidence of anastomotic leakage (P = .023). CONCLUSION: Early postoperative endoscopy does not increase postoperative adverse events after minimally invasive esophagectomy and may guide early prediction and intervention strategies for anastomotic leakage in patients undergoing minimally invasive esophagectomy.

Impact of previous extra-pulmonary malignancies on surgical outcomes of sequential primary non-small cell lung cancer.

Reduced cancer deaths have led to an increase in the number of cancer survivors and the risk of the second primary tumor. This study explored the surgical outcomes of patients with non-small cell lung cancer as the second primary tumor and the impact of previous extra-pulmonary malignancies. Patients' data were obtained from Surveillance, Epidemiology and End Results database. The patients were divided into lung surgery and non-surgery groups. Propensity-score matching was used to balance potential confounders. Kaplan-Meier curves were generated to test the overall survival and lung-cancer-specific survival. Cox regression analysis was performed to calculate death risk. In total 3054 lung surgery and 1094 non-surgery patients with stage I-II non-small cell lung cancer as the second primary tumor were included. The surgery group showed longer overall survival (68 vs. 22 months) and lung cancer-specific survival (not reached vs. 37 months) than those of non-surgery groups (both P < 0.001). Patients with previous hormone-dependent malignancies had similar survival rates (overall survival: 22 vs. 20 months, P = 0.666; lung cancer-specific survival: 38 vs. 37 months, P = 0.292) as those with non-hormone dependent malignancies in the non-surgery group. Significantly longer overall survival (90 vs. 60 months, P = 0.001) was observed in patients with hormone-dependent malignancies in the surgery group; however, there was no difference in lung cancer-specific survival (P = 0.225). Competing risk analysis showed that for patients undergoing lung surgery, there was higher previous malignancy-induced mortality in patients with non-hormone dependent malignancies than in patients with hormone-dependent malignancies. However, there was no difference in lung cancer-induced mortality between the two groups. Patients who underwent lobectomy showed longer survival than those who underwent pneumonectomy and other resection types (89, 27.5 and 65 months, P < 0.001). In summary, lung surgery is beneficial for patients with stage I-II non-small cell lung cancer as the second primary tumor after hormone-dependent malignancy resection.

Therapeutic potential of alkaloid extract from Codonopsis Radix in alleviating hepatic lipid accumulation: insights into mitochondrial energy metabolism and endoplasmic reticulum stress regulation in NAFLD mice.

Alkaloids are a class of naturally occurring bioactive compounds that are widely distributed in various food sources and Traditional Chinese Medicine. This study aimed to investigate the therapeutic effects and underlying mechanisms of alkaloid extract from Codonopsis Radix (ACR) in ameliorating hepatic lipid accumulation in a mouse model of non-alcoholic fatty liver disease (NAFLD) induced by a high-fat diet (HFD). The results revealed that ACR treatment effectively mitigated the abnormal weight gain and hepatic injury associated with HFD. Furthermore, ACR ameliorated the dysregulated lipid metabolism in NAFLD mice, as evidenced by reductions in serum triglyceride, total cholesterol, and low-density lipoprotein levels, accompanied by a concomitant increase in the high-density lipoprotein level. ACR treatment also demonstrated a profound anti-oxidative effect, effectively alleviating HFD-induced oxidative stress and promoting ATP production. These effects were achieved through the up-regulation of the activities of mitochondrial electron transfer chain complexes I, II, IV, and V, in addition to the activation of the AMPK/PGC-1α pathway, suggesting that ACR exhibits therapeutic potential in alleviating the HFD-induced dysregulation of mitochondrial energy metabolism. Moreover, ACR administration mitigated HFD-induced endoplasmic reticulum (ER) stress and suppressed the overexpression of ubiquitin-specific protease 14 (USP14) in NAFLD mice. In summary, the present study provides compelling evidence supporting the hepatoprotective role of ACR in alleviating lipid deposition in NAFLD by improving energy metabolism and reducing oxidative stress and ER stress. These findings warrant further investigation and merit the development of ACR as a potential therapeutic agent for NAFLD.

Advanced data post-processing method for rapid identification and classification of the major triterpenoids of Alismatis rhizoma by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

INTRODUCTION: Alismatis rhizoma (AR), a distinguished diuretic traditional Chinese herbal medicine, is widely used for the treatment of diarrhea, edema, nephropathy, hyperlipidemia, and tumors in clinical settings. Most beneficial effects of AR are attributed to the major triterpenoids, whose contents are relatively high in AR. To date, only 25 triterpenoids in AR have been characterized by LC-MS because the low-mass diagnostic ions are hardly triggered in MS, impeding structural identification. Herein, we developed an advanced data post-processing method with abundant characteristic fragments (CFs) and neutral losses (NLs) for rapid identification and classification of the major triterpenoids in AR by UPLC-Q-TOF-MSE . OBJECTIVE: We aimed to establish a systematic method for rapid identification and classification of the major triterpenoids of AR. METHODS: UPLC-Q-TOF-MSE coupled with an advanced data post-processing method was established to characterize the major triterpenoids of AR. The abundant CFs and NLs of different types of triterpenoids were discovered and systematically summarized. The rapid identification and classification of the major triterpenoids of AR were realized by processing the data and comparing with information described in the literature. RESULTS: In this study, a total of 44 triterpenoids were identified from AR, including three potentially new compounds and 41 known ones, which were classified into six types. CONCLUSION: The newly established approach is suitable for the chemical profiling of the major triterpenoids in AR, which could provide useful information about chemical constituents and a basis for further exploration of its active ingredients in vivo.

MiR-302c-5p affects the stemness and cisplatin resistance of nasopharyngeal carcinoma cells by regulating HSP90AA1.

Nasopharyngeal carcinoma (NPC) is one of the most frequent malignant tumors diagnosed in China. Cisplatin is one of the most commonly used anticancer drugs containing platinum in combined chemotherapy. The molecular mechanism of NPC is still largely unknown, and we aim to spare no effort to elucidate it. Normal human nasopharyngeal epithelial cells and NPC cell lines were cultured. The expression levels of miR-302c-5p and HSP90AA1 were detected with quantitative real-time PCR. Western blotting was used to analyze levels of the HSP90AA1, protein kinase B (AKT), p-AKT, CD44 and SOX2 proteins. The interaction between miR-302c-5p and HSP90AA1 was detected using a luciferase reporter assay. The bicinchoninic acid assay was used to observe cisplatin resistance in NPC cells. Our records confirmed that the expression of miR-302c-5p was substantially reduced and HSP90AA1 was increased in NPC cells. Additionally, miR-302c-5p inhibited cisplatin resistance and the traits of stem cells in NPC. A luciferase assay confirmed that miR-302c-5p is bound to HSP90AA1. Overexpression of HSP90AA1 may reverse the effects of overexpressed miR-302c-5p and inhibit cisplatin resistance and stem cell traits of NPC. This study investigated whether miR-302c-5p inhibited the AKT pathway by regulating HSP90AA1 expression and altered the resistance of NPC cells to cisplatin and the traits of tumor stem cells, which has not yet been reported.

CD155 Cooperates with PD-1/PD-L1 to Promote Proliferation of Esophageal Squamous Cancer Cells via PI3K/Akt and MAPK Signaling Pathways.

BACKGROUND: Esophageal cancer is still a leading cause of death among all tumors in males, with unsatisfactory responses to novel immunotherapies such as anti-PD-1 agents. Herein, we explored the role of CD155 in esophageal squamous cell cancer (ESCA) and its underlying molecular mechanisms. METHODS: Publicly available datasets were used for differential gene expression and immune infiltration analyses, and their correlation with patient survival. A total of 322 ESCA and 161 paracancer samples were collected and evaluated by performing immunohistochemistry and the H score was obtained by performing semiquantitative analysis. In vitro transfection of ESCA cell lines with lentivirus vectors targeting CD155 was performed to knockdown the protein. These cells were analyzed by conducting RNA sequencing, and the effects of CD155 knockdown on cell cycle and apoptosis were verified with flow cytometry and Western blotting. In addition, in vivo experiments using these engineered cell lines were performed to determine the role of CD155 in tumor formation. A small interfering RNA-mediated knockdown of Nectin3 was used to determine whether it phenocopied the profile of CD155 knockdown. RESULTS: CD155 is highly expressed in ESCA tissues and is positively associated with PD1, PDL1, CD4, IL2RA, and S100A9 expression. Furthermore, CD155 knockdown inhibited ESCA cells' proliferation by impairing the cell cycle and inducing cell apoptosis. Bioinformatics analysis of the gene expression profile of these engineered cells showed that CD155 mainly contributed to the regulation of PI3K/Akt and MAPK signals. The downregulation of Nectin3 expression phenocopied the profile of CD155 knockdown. DISCUSSION: CD155 may cooperate with PD-1/PD-L1 to support ESCA proliferation in ways other than regulating its underlying immune mechanisms. Indeed, CD155 downregulation can impair ESCA cell pro-cancerous behavior via the inhibition of the PI3K/Akt and MAPK signaling pathways. Moreover, Nectin3 may be a ligand of CD155 and participate in the regulation of ESCA cells' proliferation. Hence, the inhibition of CD155 may enhance the therapeutic effect of anti-PD-1 immunotherapies in ESCA.

The Proliferative Role of Immune Checkpoints in Tumors: Double Regulation.

Cancer remains a serious social health problem, and immunotherapy has become the major treatments in tumor treatment. Additionally, improving the efficiency and safety of treatment is necessary. Further, more therapy targets are warranted for future tumor treatments. In this review, in addition to examining the currently recognized role of immune regulation, we focus on the proliferative role of 15 immune checkpoints in various tumors, including PD1, PD-L1, FGL1, CD155, CD47, SIRPα, CD276, IDO1, SIGLEC-15, TIM3, Galectin-9, CD70, CD27, 4-1BBL, and HVEM. We managed to conclude that various immune checkpoints such as PD1/PD-L1, FGL1, CD155, CD47/SIRPα, CD276, and SIGLEC-15 all regulate the cell cycle, and specifically through Cyclin D1 regulation. Furthermore, a variety of signal pathways engage in proliferation regulation, such as P13K, AKT, mTOR, and NK-κB, which are also the most common pathways involved in the regulation of immune checkpoint proliferation. Currently, only PD1/PD-L1, CD47/SIRPα, TIM3/Galectin-9, and CD70/CD27 checkpoints have been shown to interact with each other to regulate tumor proliferation in pairs. However, for other immune checkpoints, the role of their receptors or ligands in tumor proliferation regulation is still unknown, and we consider the enormous potential in this area. An increasing number of studies have validated the various role of immune checkpoints in tumors, and based on this literature review, we found that most of the immune checkpoints play a dual regulatory role in immunity and proliferation. Therefore, the related pathways in proliferation regulation can served the role of therapy targets in tumor therapy. Further, great potential is displayed by IDO1, SIGLEC-15, 4-1BBL, and HVEM in tumor proliferation regulation, which may become novel therapy targets in tumor treatment.

Dual immunological and proliferative regulation of immune checkpoint FGL1 in lung adenocarcinoma: The pivotal role of the YY1-FGL1-MYH9 axis.

Rational: Lung cancer is the most common tumor worldwide, with the highest mortality rate and second highest incidence. Immunotherapy is one of the most important treatments for lung adenocarcinoma (LUAD); however, it has relatively low response rate and high incidence of adverse events. Herein, we explored the therapeutic potential of fibrinogen-like protein 1 (FGL1) for LUAD. Methods: Data from GEPIA and ACLBI databases were assessed to explore gene-gene correlations and tumor immune infiltration patterns. A total of 200 patients with LUAD were recruited. FGL1 levels in the serum and cellular supernatant were determined by enzyme-linked immunosorbent assay. In vitro and in vivo experiments were performed to assess the effect FGL1 on the proliferation of LUAD cells. Cocultures were performed to explore the effect of FGL1 knockdown in lung cancer cells on T cells, concerning cytokine secretion and viability. PROMO and hTFtarget databases were used for transcription factor prediction. Quantitative polymerase chain reaction (qPCR), chromatin immunoprecipitation, and dual luciferase reporter assays were performed to validate the identified transcription factor of FGL1. Immunoprecipitation, mass spectrometry and gene ontology analysis were performed to explore the downstream partners of FGL1. Results: FGL1 expression in LUAD was positively associated with PDL1, but not for PD1 expression. Moreover, FGL1 was positively associated with the CD3D expression and negatively associated with FOXP3, S100A9, and TPSB2 within the tumor site. FGL1 promotes the secretion of interleukin-2 by T cells in vitro, simultaneously inducing their apoptosis. Indeed, YY1 is the upstream molecule of FGL1 was found to be transcriptionally regulated by YY1 and to directly by to MYH9 to promote the proliferation of LUAD cells in vitro and in vivo. Conclusions: FGL1 is involved in the immunological and proliferative regulation of LUAD cells by controlling the secretion of important immune-related cytokines via the YY1-FGL1-MYH9 axis. Hence, targeting FGL1 in LUAD may pave the way for the development of new immunotherapies for tackling this malignancy.

IGSF11 and VISTA: a pair of promising immune checkpoints in tumor immunotherapy.

Immunotherapy has become the major treatment for tumors in clinical practice, but some intractable problems such as the low response rate and high rates of immune-related adverse events still hinder the progress of tumor immunotherapy. Hence, it is essential to explore additional immunotherapy treatment targets. In this review, we focus on the structure, expression and expression-related mechanisms, interactions, biological functions and the progress in preclinical/clinical research of IGSF11 and VISTA in tumors. We cover the progress in recent research with this pair of immune checkpoints in tumor immune regulation, proliferation, immune resistance and predictive prognosis. Both IGSF11 and VISTA are highly expressed in tumors and are modulated by various factors. They co-participate in the functional regulation of immune cells and the inhibition of cytokine production. Besides, in the downregulation of IGSF11 and VISTA, both inhibit the growth of some tumors. Preclinical and clinical trials all emphasize the predictive role of IGSF11 and VISTA in the prognosis of tumors, and that the predictive role of the same gene varies from tumor to tumor. At present, further research is proving the enormous potential of IGSF11 and VISTA in tumors, and especially the role of VISTA in tumor immune resistance. This may prove to be a breakthrough to solve the current clinical immune resistance, and most importantly, since research has focused on VISTA but less on IGSF11, IGSF11 may be the next candidate for tumor immunotherapy.

Chemical screen identifies shikonin as a broad DNA damage response inhibitor that enhances chemotherapy through inhibiting ATM and ATR.

DNA damage response (DDR) is a highly conserved genome surveillance mechanism that preserves cell viability in the presence of chemotherapeutic drugs. Hence, small molecules that inhibit DDR are expected to enhance the anti-cancer effect of chemotherapy. Through a recent chemical library screen, we identified shikonin as an inhibitor that strongly suppressed DDR activated by various chemotherapeutic drugs in cancer cell lines derived from different origins. Mechanistically, shikonin inhibited the activation of ataxia telangiectasia mutated (ATM), and to a lesser degree ATM and RAD3-related (ATR), two master upstream regulators of the DDR signal, through inducing degradation of ATM and ATR-interacting protein (ATRIP), an obligate associating protein of ATR, respectively. As a result of DDR inhibition, shikonin enhanced the anti-cancer effect of chemotherapeutic drugs in both cell cultures and in mouse models. While degradation of ATRIP is proteasome dependent, that of ATM depends on caspase- and lysosome-, but not proteasome. Overexpression of ATM significantly mitigated DDR inhibition and cell death induced by shikonin and chemotherapeutic drugs. These novel findings reveal shikonin as a pan DDR inhibitor and identify ATM as a primary factor in determining the chemo sensitizing effect of shikonin. Our data may facilitate the development of shikonin and its derivatives as potential chemotherapy sensitizers through inducing ATM degradation.

The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC-target genes.

Background: The mechanisms involved in the malignant progression of lung adenocarcinoma (LUAD) are still inconclusive. Fibrinogen-like protein 1 (FGL1) and LAG3 are a pair of immune checkpoints that create an inhibitory immune microenvironment in tumors. However, other roles of FGL1 in LUAD have not been extensively studied. Our study aims to explore the role of FGL1 in the malignant progression of LUAD and to provide new therapeutic targets and strategies for LUAD treatment. Methods: Differential gene expression of FGL1 was analyzed using the Gene Expression Profiling Interactive Analysis (GEPIA), Oncomine, UALCAN, and Gene Expression Omnibus (GEO) databases. A pan-cancer analysis was conducted using the Oncomine, TIMER, and UALCAN databases. A total of 140 tumor tissues and paired normal tissues were collected, IHC and immunofluorescence staining were used to explore the expression of FGL1. GeneMANIA database and STRING database were used to analyze gene-gene interaction and protein-protein interaction, respectively. A mutation analysis was conducted using the cBioPortal database, and an immune infiltration analysis was conducted using the TIMER database. A survival analysis was carried out using the GEPIA and PrognoScan database. The knockdown of FGL1 was confirmed by western blot (WB) and immunofluorescence staining. Cell proliferation was tested by cell cycle analysis and real-time cell analysis. RNA sequencing (RNA-seq) was used to explore the differential genes of FGL1 knockdown in LUAD cells. Results: Multiple databases showed that FGL1 was highly expressed in LUAD. The results of IHC indicated that FGL1 was highly expressed in the cytoplasm of LUAD cells. FGL1 was negatively associated with immune infiltration in LUAD. The main mutation of FGL1 is deep deletion, the altered group and high expression group indicated poor prognosis. The downregulation of FGL1 lead to a significantly decreased percentage of PC9 cells in S phase, but had little effect on the proliferation of Jurkat T cells. RNA-seq and GSEA analysis indicated that the differential genes were mainly enriched in MYC-target genes, which suggested that the downregulation of FGL1 inhibited cell proliferation by regulating MYC-target genes. Conclusions: FGL1 exerts in LUAD proliferation in addition to immune regulation. The downregulation of FGL1 inhibits the proliferation of LUAD cells by regulating MYC-target genes. Thus, FGL1 may be a novel therapeutic target in LUAD.

CircCRIM1 promotes nasopharyngeal carcinoma progression via the miR-34c-5p/FOSL1 axis.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a rare malignancy with multiple risk factors (Epstein-Barr virus, etc.) that seriously threatens the health of people. CircRNAs are known to regulate the tumorigenesis of malignant tumours, including NPC. Moreover, circCRIM1 expression is reported to be upregulated in NPC. Nevertheless, the impact of circCRIM1 on NPC progression is not clear. METHODS: An MTT assay was performed to assess cell viability. In addition, cell invasion and migration were assessed by the transwell assay. Dual luciferase assays were performed to assess the association among circCRIM1, miR-34c-5p and FOSL1. Moreover, RT-qPCR was applied to assess mRNA levels, and protein levels were determined by Western blot. RESULTS: CircCRIM1 and FOSL1 were upregulated in NPC cells, while miR-34c-5p was downregulated. Knockdown of circCRIM1 significantly decreased the invasion, viability and migration of NPC cells. The miR-34c-5p inhibitor notably promoted the malignant behaviour of NPC cells, while miR-34c-5p mimics exerted the opposite effect. Moreover, circCRIM1 could bind with miR-34c-5p, and FOSL1 was identified to be downstream of miR-34c-5p. Furthermore, circCRIM1 downregulation notably inhibited the proliferation and invasion of NPC cells, while this phenomenon was significantly reversed by FOSL1 overexpression. CONCLUSION: Silencing circCRIM1 inhibited the tumorigenesis of NPC. Thus, circCRIM1 might be a novel target for NPC.

Surgical treatment of primary tracheobronchial tumors: 16-year experience in a single center.

Background: Primary tracheobronchial tumor (TBT) is a rare disease, and the prognostic factors of surgical treatment have not been well identified. Methods: Patients with primary TBT and accepted surgical treatment between January 2004 and January 2020 at our institution were retrospectively analyzed. The univariate analysis and multivariate analysis were conducted on the malignant cases. The overall survival (OS) was analyzed using Kaplan-Meier method, and potential prognostic factors were analyzed using Cox regression analysis. Results: A total of 69 patients (29 males and 40 females) were included. The median follow-up duration was 75.7 months (1.2-177.4 months). The most common histology was adenoid cystic carcinoma (ACC) (37.7%) followed by squamous cell carcinoma (SCC) (23.2%). For patients with malignant tumors, the estimated 5-year OS of the overall population was 77.2% and the estimated 5-year OS of SCC patients was 73.8% especially. The univariate Cox regression analysis identified that age and tumor size had significant effects on OS. The multivariate analysis showed that age (≤50 or >50 years) was independent prognostic factor for OS (P<0.05). Conclusions: Age is independent factor affecting the OS of primary TBT treated by surgery. And patients of TBT with younger age should be much more referred for surgery.

Evaluation of local aggressive lung therapy versus systemic therapy in oligometastatic non-small cell lung cancer: a systematic review and meta-analysis.

BACKGROUND: Previous studies have shown the feasibility and effectiveness of local aggressive thoracic therapy (surgery and radiotherapy) for oligometastatic non-small cell lung cancer compared with systemic therapy, but with small sample. This study aims to perform a pooled analysis to explore whether LT could improve outcomes of oligometastatic patients with non-small cell lung cancer. METHODS: Protocol of present study was registered on PROSPERO as number: CRD42021233095. PubMed, Embase and Web of knowledge were searched, and eligible studies investigating local therapy for non-small cell lung cancer with 1-5 metastases regardless of organs were included. Linear regression between survival and clinical characteristics were conducted. Hazard ratios of survival and adverse effects were merged. Pooled survival curves were carried out. RESULTS: Three randomized controlled trials and 5 cohort studies enrolling 499 patients were included. There was a trend that median overall survival declined with the increasing proportion of N2-3 positive patients in local therapy group, but with no statistical difference (P=0.09, R2=0.98). Undergoing local therapy for oligometastatic non-small cell lung cancer achieved reduction of 47% and 60% in the risk of death and cancer progression (P<0.001), respectively. In subgroup analysis, patients receiving local therapy including surgery showed hazard ratio of 0.33 on progression-free survival and 0.55 of these excluding surgery. Patients receiving consolidative local therapy (local therapy after systemic therapy) obtained hazard ratios 0.33 and 0.45 on progression-free and overall survival vs. systemic therapy, respectively. Hazard ratios of those receiving upfront local therapy (local therapy first) were 0.62 and 0.68 on progression-free and overall survival vs. systemic therapy. Pooled survival analysis showed median overall and progression-free survival of local therapy (21.6 and 14 months) group were both longer than systemic one (14.3 and 6.5 months). Odds ratio of adverse effects were no difference between 2 groups (P=0.16). CONCLUSIONS: Local aggressive thoracic therapy could prolong 7 months overall and progression-free survival compared with systemic therapy in patients with oligometastatic non-small cell lung cancer. Consolidative local therapy might be a more favorable choice of local therapy. Benefits of local therapy for N2-3 positive patients should explored further.

Clinical Research on the Mechanisms Underlying Immune Checkpoints and Tumor Metastasis.

This study highlights aspects of the latest clinical research conducted on the relationship between immune checkpoints and tumor metastasis. The overview of each immune checkpoint is divided into the following three sections: 1) structure and expression; 2) immune mechanism related to tumor metastasis; and 3) clinical research related to tumor metastasis. This review expands on the immunological mechanisms of 17 immune checkpoints, including TIM-3, CD47, and OX-40L, that mediate tumor metastasis; evidence shows that most of these immune checkpoints are expressed on the surface of T cells, which mainly exert immunomodulatory effects. Additionally, we have summarized the roles of these immune checkpoints in the diagnosis and treatment of metastatic tumors, as these checkpoints are considered common predictors of metastasis in various cancers such as prostate cancer, non-Hodgkin lymphoma, and melanoma. Moreover, certain immune checkpoints can be used in synergy with PD-1 and CTLA-4, along with the implementation of combination therapies such as LIGHT-VTR and anti-PD-1 antibodies. Presently, most monoclonal antibodies generated against immune checkpoints are under investigation as part of ongoing preclinical or clinical trials conducted to evaluate their efficacy and safety to establish a better combination treatment strategy; however, no significant progress has been made regarding monoclonal antibody targeting of CD28, VISTA, or VTCN1. The application of immune checkpoint inhibitors in early stage tumors to prevent tumor metastasis warrants further evidence; the immune-related adverse events should be considered before combination therapy. This review aims to elucidate the mechanisms of immune checkpoint and the clinical progress on their use in metastatic tumors reported over the last 5 years, which may provide insights into the development of novel therapeutic strategies that will assist with the utilization of various immune checkpoint inhibitors.

A new strategy for discovering effective substances and mechanisms of traditional Chinese medicine based on standardized drug containing plasma and the absorbed ingredients composition, a case study of Xian-Ling-Gu-Bao capsules.

ETHNOPHARMACOLOGICAL RELEVANCE: The overall therapeutic effect of traditional Chinese medicine formulae (TCMF) was achieved by the interactions of multiple components with multiple targets. However, current pharmacology research strategies have struggled to identify effective substance groups and encountered challenges in elucidating the underlying mechanisms of TCMF. AIM: In this study, a comprehensive strategy was proposed and applied to elucidate the interactions of the multiple components that underlie the functions of the famous TCMF: Xian-Ling-Gu-Bao (XLGB) capsule on bone metabolism in vivo and to elucidate the molecular mechanisms underlying the effects of XLGB on bone cells, especially on osteoblasts. METHODS: The efficacy of XLGB in the protection against bones loss in ovariectomized (OVX) rats was confirmed by Micro-CT analysis. The anti-osteoporosis mechanism involved in the systemic regulatory actions of XLGB was elucidated by transcriptome sequencing analysis on bone marrow mesenchymal stem cells isolated from OVX rats. Moreover, the components absorbed in XLGB-treated plasma were characterized by mass spectrometry analysis, and subsequently, a standardized preparation process of drug-containing plasma was established. The synergistic osteogenic effect of the multiple components in plasma was investigated by a combination and then knockout of components using pre-osteoblast MC3T3-E1 cells. In order to decipher the underlying mechanism of XLGB, the targets of the absorbed components on bone were predicted by target prediction and network pharmacology analysis, then several interactions were validated by biochemical and cell-based assay. RESULTS: A total of 18 genes, including HDC, CXCL1/2, TNF, IL6 and Il1b, were newly found to be the major target genes regulated by XLGB. Interestingly, we found that a combination of the three absorbed components, i.e. MSP, rather than their single form at the same concentration, stimulated the formation of calcified nodules in MC3T3-E1 cells, suggesting a synergistic effect of these components. Besides, target prediction and experimental validation confirmed the binding affinity of corylin and icaritin for estrogen receptor α and ß, the inhibitory activity of isobavachin and isobavachalcone on glycogen synthase kinase-3ß, and the inhibitory activity of isobavachalcone on cathepsin K. The cell-based assay further confirmed the result of the biochemical assay. A network that integrated absorbed components of XLGB-targets-perturbation genes-pathways against osteoporosis was established. CONCLUSION: Our current study provides a new systemic strategy for discovering active ingredient groups of TCM formulae and understanding their underlying mechanisms.

PD-L1 expression and immune cells infiltration in primary tracheobronchial neoplasm.

BACKGROUND: Primary tracheobronchial neoplasm is rare yet poses a serious threat to life. Due to its low incidence, the immune microenvironment of such tumors remained unclear. This study aimed to clarify the expression of programmed death-ligand 1 (PD-L1) and infiltration of immune cells in primary tracheobronchial neoplasm, which might be useful for guiding treatment and evaluating clinical outcome. METHODS: We assessed retrospectively the expression of PD-L1 and infiltration in cells expressing CD8, CD16, CD68, CD163 and FOXP3 in 21 patients with primary tracheobronchial neoplasm who underwent surgery in Tangdu Hospital from January 2016 to July 2021. The expression of PD-L1 was assessed based on the tumor proportion score system. The density of immune cells was analyzed by automatic image analysis software. RESULTS: In this study, all of 16 participants with adenoid cystic carcinoma (ACC) had no expression of PD-L1, whereas 4/5 (80%) of those with squamous cell carcinomas (SCC) were positive for PD-L1 expression. Compared with ACC, the density of FOXP3+ cells in both the intratumoral region and peritumoral region was higher in SCC (P<0.01). The density of FOXP3+ cells was significantly higher than that of CD8+, CD16+, and CD163+ cells in SCC in the intratumoral region (P<0.01). In contrast, the density of FOXP3+ cells was significantly lower than that of CD8+, CD16+, and CD68+ cells in ACC in both the intratumoral region and peritumoral regions. The density of CD68+ cells was significantly higher than that of CD8+ cells (P<0.05) and CD163+ cells (P<0.01) in ACC in the intratumoral region. Furthermore, the tumors of patients with metastasis more commonly of immune-excluded status, in which the CD8+ cells accumulated in peritumoral region. CONCLUSIONS: This study demonstrated that the expression of PD-L1 in primary tracheobronchial neoplasm was mainly concentrated in patients with SCC. In the immune microenvironment of SCC, FOXP3+ cells were the dominant immune cells, while in the immune microenvironment of ACC, CD68+ cells were the main immune cells. Therefore, the immune microenvironment was significantly different in primary tracheobronchial neoplasm according to histology.

Immune Checkpoint LAG3 and Its Ligand FGL1 in Cancer.

LAG3 is the most promising immune checkpoint next to PD-1 and CTLA-4. High LAG3 and FGL1 expression boosts tumor growth by inhibiting the immune microenvironment. This review comprises four sections presenting the structure/expression, interaction, biological effects, and clinical application of LAG3/FGL1. D1 and D2 of LAG3 and FD of FGL1 are the LAG3-FGL1 interaction domains. LAG3 accumulates on the surface of lymphocytes in various tumors, but is also found in the cytoplasm in non-small cell lung cancer (NSCLC) cells. FGL1 is found in the cytoplasm in NSCLC cells and on the surface of breast cancer cells. The LAG3-FGL1 interaction mechanism remains unclear, and the intracellular signals require elucidation. LAG3/FGL1 activity is associated with immune cell infiltration, proliferation, and secretion. Cytokine production is enhanced when LAG3/FGL1 are co-expressed with PD-1. IMP321 and relatlimab are promising monoclonal antibodies targeting LAG3 in melanoma. The clinical use of anti-FGL1 antibodies has not been reported. Finally, high FGL1 and LAG3 expression induces EGFR-TKI and gefitinib resistance, and anti-PD-1 therapy resistance, respectively. We present a comprehensive overview of the role of LAG3/FGL1 in cancer, suggesting novel anti-tumor therapy strategies.

Sweroside promotes osteoblastic differentiation and mineralization via interaction of membrane estrogen receptor-α and GPR30 mediated p38 signalling pathway on MC3T3-E1 cells.

BACKGROUND: Dipsaci Radix has been clinically used for thousands of years in China for strengthening muscles and bones. Sweroside is the major active iridoid glycoside isolated from Dipsaci Radix. It has been reported that sweroside can promote alkaline phosphatase (ALP) activity in both the human osteosarcoma cell line MG-63 and rat osteoblasts. However, the underlying mechanism involved in these osteoblastic processes is poorly understood. PURPOSE: This study aimed to characterize the bone protective effects of sweroside and to investigate the signaling pathway that is involved in its actions in MC3T3-E1 cells. METHODS: Cell proliferation, differentiation and mineralization were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, ALP test and Alizarin Red S staining, respectively. The concentration of sweroside in intracellular and extracellular fluids was determined by ultra-performance liquid chromatography coupled to triple quadrupole xevo-mass spectrometry (UPLC/TQ-XS-MS). Proteins associated with the osteoblastic signaling pathway were analysed by western blot and immunofluorescence methods. RESULTS: Sweroside did not obviously affect the proliferation but significantly promoted the ALP activity and mineralization of MC3T3-E1 cells. The maximal absorption amount 0.465 ng/ml (1.3 × 10-9 M) of sweroside was extremely lower than the tested concentration of 358.340 ng/ml (10-6 M), indicating an extremely low absorption rate by MC3T3-E1 cells. Moreover, the ALP activity, the protein expression of ER-α and G protein-coupled receptor 30 (GPR30) induced by sweroside were markedly blocked by both the ER antagonist ICI 182780 and the GPR30 antagonist G15. In addition, sweroside also activated the phosphorylation of p38 kinase (p-p38), while the phosphorylation effects together with ALP and mineralization activities were completely blocked by a p38 antagonist, SB203580. Additionally, the phosphorylation of p38 induced by sweroside were markedly blocked by both the ER antagonist ICI 182780 and the GPR30 antagonist G15. CONCLUSIONS: The present study indicated that sweroside, as a potential agent in treatment of osteoporosis, might exert beneficial effects on MC3T3-E1 cells by interaction with the membrane estrogen receptor-α and GPR30 that then activates the p38 signaling pathway. This is the first study to report the specific mechanism of the effects of sweroside on osteoblastic differentiation and mineralization of MC3T3-E1 cells.