A method for isolating and culturing ectopic epithelial and stromal cells to study human adenomyosis.

PURPOSE: Although adenomyosis is a common and benign gynecological disease, the specific pathogenesis of this condition is yet to be fully elucidated. It is difficult to culture primary cells of the ectopic endometrial epithelia and stroma from human adenomyosis lesions. Most of the previous of studies on adenomyosis were based on primary eutopic endometrium cells. However, as yet, no efficient protocols have been developed for the isolation, culture or purification of primary ectopic epithelial and stromal cells from human adenomyosis lesions. Therefore, the present study aimed to develop an efficient protocol for the isolation and culture of primary ectopic epithelial and stromal cells from human adenomyosis lesions. METHODS: In the present study, we aimed to obtain ectopic endometrium tissue from human adenomyosis foci and use a simple and operable type I collagenase digestion method for primary culture. Cells were isolated by sterile cell strainer filtration and flow cytometry was performed to identify, purify, and evaluate the viability of isolated ectopic endometrial cells. RESULTS: Using our method, we successfully isolated and cultured highly purified and active ectopic endometrial epithelial and stromal cells from human adenomyosis foci. Ep-CAM was expressed in ectopic epithelial cells of human adenomyosis with a purity of 93.74% and a viability of 80.58%. In addition, CD10 were robustly expressed by ectopic stromal cells in human adenomyosis. Cellular purity and viability were determined to be 96.37 and 93.49%, respectively. CONCLUSION: Our method provides a new experimental model for studying the molecular pathogenesis of human adenomyosis.

Investigation of the Therapeutic Effect of Salbutamol on Endometriosis in a Mouse Model.

Endometriosis is an immune chronic inflammatory disease, and there are currently no more effective drugs for treating endometriosis due to its unknown etiology. Salbutamol is a ß2-adrenergic receptor (ß2AR) agonist commonly used to treat asthma by selectively activating ß2 receptors on airway smooth muscle and leukocytes, exerting bronchial dilation and synergistic anti-inflammatory effects. In recent years, ß2AR agonists have been used in endometriosis studies, and we speculate that salbutamol may have a therapeutic effect on endometriosis. The purpose of this research was to explore the therapeutic effect of salbutamol on endometriosis mice. The mouse endometriosis model was established and treated with different doses of salbutamol. Endometrial lesions were harvested for pathological diagnosis, immunohistochemistry (IHC), Masson staining, and toluidine blue analysis. We found that the number and size of endometriotic lesions were all significantly decreased after 3 weeks of treatment with different doses of salbutamol on endometriosis model mice (P < 0.05). After Salbutamol treatment, the amount of mast cells (toluidine blue) and macrophages (F4/80) in the lesions as well as the expressions of interleukin (IL)-1ß, tumor necrosis factor (TNF)-ɑ, platelet-derived growth factor subunit B (PDGFB), CD31, transforming growth factor (TGF)-ß, Masson staining, BCL2, TUBB3, substance P (SP), and nerve growth factor (NGF) were significantly reduced (P < 0.05). These results suggested that salbutamol could effectively treat endometriosis in mice by reducing immune inflammatory cells and factors, angiogenesis, and fibrosis, increasing apoptosis of endometriotic lesions, and decreasing neurogenesis.

Long non coding RNA, C8orf49, a novel diagnostic and prognostic biomarker, enhances PTEN/FZD4-mediated cell growth and metastasis by sponging miR-1323 in endometriosis.

Lack of sensitive biomarkers in the early stages of endometriosis (EMs) results in delayed diagnosis and intervention. Long non-coding RNAs (lncRNAs) have prognostic and diagnostic values in various diseases. However, the prognostic and diagnostic effects of lncRNAs on EMs have rarely been discussed in EMs. In this study, we found that lncRNA C8orf49 was stably overexpressed in EMs tissues/plasma, and its expression greatly influenced dysmenorrhea (p = 2.2605E-9) and the revised American Society for Reproductive Medicine stage (p = 0.040765) of EMs. Multivariate logistic regression results revealed that C8orf49 expression was an independent risk factor for EMs [p = 6.4997E-17, 95% confidence interval (CI) = 0.000559-0.023853]. In primary endometrial stromal cells (ESCs), inhibition of C8orf49 could impede the proliferation and metastasis of ESCs. C8orf49 influenced the expression of PTEN/FZD4 by absorbing miR-1323, thus controlling ESCs activity. The results of a subcutaneous endometriosis animal model showed that the inhibition of C8orf49 restrained endometrial growth. Overall, C8orf49 functioned as an activator of EMs pathogenesis via the C8orf49/miR-1323/PTEN/FZD4 axis.

Comprehensive Analysis of RNA-Seq in Endometriosis Reveals Competing Endogenous RNA Network Composed of circRNA, lncRNA and mRNA.

Although long non coding RNAs (lncRNAs) and circular RNAs (circRNAs) play important roles in the pathogenesis of diseases, endometriosis related lncRNAs and circRNAs are still rarely reported. This study focused on the potential molecular mechanism of endometriosis related competitive endogenous RNA (ceRNA) composed of lncRNAs and circRNAs. We performed high-throughout sequencing of six normal endometria, six eutopic endometria and six ectopic endometria for the first time to describe and analyze the expression profile of lncRNA, circRNA and mRNA. Our results showed that 140 lncRNAs, 107 circRNAs and 1,206 mRNAs were differentially expressed in the ectopic group, compared with the normal and eutopic groups. We established an lncRNA/circRNA-mRNA co-expression network using pearson correlation test. Meanwhile, the results of Gene set enrichment analysis analysis showed that the 569 up-regulated differentially expressed mRNA (DEmRNA) were mainly related to the epithelial-mesenchymal transition, regulation of immune system process and immune effector process. Subsequently, we established a DElncRNA-miRNA and DEcircRNA-miRNA network using the starbase database, identified the common miRNAs and constructed DElncRNA/DEcircRNA-miRNA pairs. miRDB, Targetscan, miRwalk and circRNA/lncRNA-mRNA pairs jointly determined the miRNA-mRNA portion of the circRNA/lncRNA-miRNA-mRNA co-expression network. RT-qPCR results of 15 control samples and 25 ectopic samples confirmed that circGLIS2, circFN1, LINC02381, IGFL2-AS1, CD84, LYPD1 and FAM163A were significantly overexpressed in ectopic tissues. In conclusion, this is the first study to illustrate ceRNA composed of differentially expressed circRNA, lncRNA and mRNA in endometriosis. We also found that lncRNA and circRNA exerted a pivotal function on the pathogenesis of endometriosis, which can provide new insights for further exploring the pathogenesis of endometriosis and identifying new targets.

A chimeric switch-receptor PD1-DAP10-41BB augments NK92-cell activation and killing for human lung Cancer H1299 Cell.

Engineered natural killer (NK) cell-based therapies have been potentially broadly applicable and exhibited promising results in clinical trials, particularly in the fight against cancers. NK cell immunotherapy however always remains variable. One major obstacle is the inhibitory pathway including PD1/PDL1, providing tumor cells an escape mechanism from immunosurveillance. In this regard, we rationally designed a chimeric switch-receptor (CSR) PD1-DAP10-41BB, which comprising the ectodomain of PD1 fused to the co-stimulatory receptor DAP10 and 41BB. Therefore, by exchanging the transmembrane and cytoplasmic tail of PD1 with positive costimulatory molecules DAP10 and 41BB signaling domains, the negative PD1/PDL1 signal pathway was thus converted into a positive one. This CSR-expressing NK92 cells showed a typical parental NK92 phenotype and improved cytotoxicity against human lung cancer H1299 cells. Besides, the expression of CSR elicited a significant increase of effector molecules such as perforin and granzymes, which can induce apoptosis of H1299 cells. More importantly, in the solid tumor cell H1299-bearing mice model, the CSR-modified NK92 cells significantly inhibited tumor growth. Collectively, we demonstrated that expression of PD1-DAP10-41BB augmented NK92-cell activation and killing in vitro and in vivo, which provides a considerable avenue of using NK-tailored chimeric receptor engineered NK92 cells to treat a wide range of solid tumors.

ROMA Index Is an Effective Predictor for Advanced Endometrial Cancer before Surgery.

There is a high rate of inconformity between clinical staging and surgical-pathologic staging in endometrial cancer. Many patients with advanced endometrial cancer are preoperatively understaged and thereby do not receive the optimal therapy. Here, we aimed to develop a predictive model or biomarker for preoperative diagnosis of advanced endometrial cancer via multivariate logistic regression analysis. In this study, 259 eligible patients were included, and 195 patients were assigned to the training dataset and 64 patients to validation dataset. Age, menopause status, sterilization situation, parity, body mass index, hypertension, diabetes mellitus, tumor size, and ovarian malignancy algorithm (ROMA) index were included as predictive variables, and the binary outcome was advanced endometrial cancer or not. When the P value was set as less than 0.01 in forward stepwise regression, only ROMA index was retained. The odds ratio of being positive ROMA index was 15.531 times that of negative value. The area under receiver operating characteristic curve was 0.790 in the training dataset and 0.776 in the validation dataset. The decision curve analysis curve showed that the prediction by ROMA index added more net benefits for almost all threshold probabilities. Therefore, ROMA index is an effective predictor for advanced endometrial cancer before surgery. Since ROMA index is a standard, measurable, and reliable laboratory test, it can be used as a reference tool for gynecologists to design the appropriate therapeutic schedule for patients with high-stage endometrial cancer before surgery.

Genetical engineering for NK and T cell immunotherapy with CRISPR/Cas9 technology: Implications and challenges.

Immunotherapy has become one of the most promising strategies in cancer therapies. Among the therapeutic alternatives, genetically engineered NK/T cell therapies have emerged as powerful and innovative therapeutic modalities for cancer patients with precise targeting and impressive efficacy. Nonetheless, this approach still faces multiple challenges, such as immunosuppressive tumor microenvironment, exhaustion of immune effector cells in tumors, off-target effects manufacturing complexity, and poor infiltration of effector cells, all of which need to be overcome for further utilization to cancers. Recently, CRISPR/Cas9 genome editing technology, with the goal of enhancing the efficacy and increasing the availability of engineered effector cell therapies, has shown considerable potential in the novel strategies and options to overcome these limitations. Here we review the current progress of the applications of CRISPR in cancer immunotherapy. Furthermore, we discuss issues related to the NK/T cell applications, gene delivery methods, efficiency, challenges, and implications of CRISPR/Cas9.

Chimeric antigen receptor-modified macrophages trigger systemic anti-tumour immunity.

Preliminary results and emerging data have shown that lipid droplet high (LDhi ) immunosuppressive cells accumulate in tumour tissues. By tracking and phenotypic profiling of LDhi cells, we find that LDhi CD19+ , LDhi CD11b+ , and LDhi Ly6G+ immune cell populations appear in the spleen, thymus, and tumour tissues in a syngeneic tumour model. Using a contact-dependent reporter system, we discover a LDhi CCR7hi immunosuppressive cell population that migrates from tumour tissues to the spleen and thymus. Hence, we engineered a family of chimeric antigen receptor-modified macrophages (CAR-Ms) that direct macrophages to CCR7-positive cells and show that the cytosolic domain from Mer receptor tyrosine kinase (MerTK) triggers tumour cell cytotoxicity by the CAR-Ms. In vivo, CCR7-targeted CAR-Ms suppressed tumour growth and prolonged survival by preventing metastasis and by inducing systemic anti-tumour immunity through retarding the migration of LDhi CCR7hi immunosuppressive cells from tumour tissues to distal immune organs, indicating an important role for CCR7 in tumour cell-induced immune tolerance. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

FGF13 interaction with SHCBP1 activates AKT-GSK3α/ß signaling and promotes the proliferation of A549 cells.

FGF13, a member of the FGF subfamily, has been found to be highly expressed in cancer cells such as prostate cancer, melanoma, glioma and multiple myeloma. However, the mechanism of FGF13 function during cancer cell proliferation remains to be unexplored, especially Non-small cell lung cancer (NSCLC). In this study, the cell proliferation effect of FGF13 on A549 cells was checked by CCK-8, clone formation, Ki67 immunofluorescence staining and Flow Cytometry assay. Localization of FGF13 within A549 cells was performed with confocal laser scanning microscope. The protein variations and interaction were measured by western blotting and co-immunoprecipitation analysis. It showed that FGF13 was mainly distributed in the cytoplasm and exhibited a high expression level in A549 cells. High expression of FGF13 activated AKT-GSK3 signaling pathway, and inhibited the activity of p21 and p27. Thus, FGF13 enhanced the process of transition from G1 to S phase and promoted A549 cells proliferation. Furthermore, the interaction between FGF13 and SHCBP1 was confirmed. Meanwhile, FGF13 and SHCBP1 had a cooperative effect to accelerate the cell cycle progression, especially the ability to promote cell proliferation is significantly enhanced via protein interaction. Hence, we conclude that FGF13 played a positive regulation role during A549 cells proliferation. FGF13 interacted with SHCBP1 to facilitate cell cycle progression, providing new insights into deep understanding of non-small cell lung cancer mechanisms of proliferation and regulation function of FGF13.

KNDC1 Is a Predictive Marker of Malignant Transformation in Borderline Ovarian Tumors.

BACKGROUND: Few screening markers for malignant transformation in borderline ovarian tumors (BOT) have been clearly established. The kinase noncatalytic C-lobe domain containing 1 (KNDC1), a brain-specific Ras guanine nucleotide exchange factor, negatively regulates dendrite growth. However, the biological role and underlying mechanism of KNDC1 in human cancers, including ovarian cancer (OC), remain unknown. METHODS: Gene chip screening was used to detect the expression of KNDC1 mRNA in normal ovarian tissues, BOT tissues, and OC tissues. And results were further validated by RT-qPCR, Western blotting and immunohistochemistry. KNDC1 overexpression and knockdown ovarian cancer cells were established to study the possible pathways that KNDC1 was involved. The effects of KNDC1 on the malignant behaviors of ovarian tumors were also investigated both in vitro and in vivo. RESULTS: We observed that the expression of KNDC1 mRNA and KNDC1 protein in OC was significantly downregulated compared with BOT. Subsequent investigation revealed that knockdown of KNDC1 enhanced the proliferation of ovarian cancer cells in vitro via induction of ERK1/2 phosphorylation, whereas reinforcing the expression of KNDC1 attenuated the ERK1/2 activity. Similarly, knockdown of KNDC1 also promoted cell proliferation in vivo. Survival analysis showed that lower KNDC1 predicted a poor progression-free survival (PFS) for patients. CONCLUSION: Collectively, we conclude that KNDC1 might function as a tumor suppressor in ovarian tumors, inhibiting the proliferation of ovarian cells by suppressing ERK1/2 activity and hindering the malignant transformation of BOT.

A novel bispecific chimeric PD1-DAP10/NKG2D receptor augments NK92-cell therapy efficacy for human gastric cancer SGC-7901 cell.

Cell-based immunotherapy continues to be a promising avenue for cancers that standard therapy has failed. Although the specificity, avidity, and efficacy of infused cells have improved, immunocytotherapy still faces substantial hurdles. To this end, we developed a structure-based rational design approach and constructed a novel Dual Targeting Chimeric Receptor (DTCR) PD1-DAP10/NKG2D comprising the truncated ectodomain of PD1 fused to a key co-stimulatory receptor DAP10, and subsequently harnessed the activating receptor NKG2D, which evaluated the capacity of solid tumor cell killing. Retroviral transduction of DTCR dramatically increased NK92 cell surface expression of PD1 and NKG2D, which boosted robust cytotoxicity against human gastric cell SGC-7901. Chimeric receptor DTCR stimulation elicited a significant increase of TNF-α and TRAIL, which can trigger apoptosis of SGC-7901 cells. More importantly, DTCR-NK92 cells had considerable antitumor activity in the solid tumor cell SGC-7901-bearing mice model. Collectively, we demonstrated that expression of DTCR markedly augmented the cytotoxic potential of NK92 cells against solid tumor cells, and this potentially promising treatment modality will facilitate clinical translation of potent NK-tailored chimeric receptor strategy for a generalized cellular therapy that may be conducive to treat a wide range of solid tumors.

Tumor-Associated Macrophages (TAMs): A Critical Activator In Ovarian Cancer Metastasis.

Tumor-associated macrophages (TAMs) that appear in every stage of cancer progression are usually tumor-promoting cells and are present abundantly in the tumor-associated microenvironment. In ovarian cancer, the overall and intratumoral M1/M2 ratio is a relatively efficient TAM parameter for predicting the prognosis of patients, especially for serous tissue type cancer. TAMs exhibit immunological checkpoint modulators, such as the B7 family and programmed death-ligand 1 (PD-L1), and play a key role in the development, metastasis and invasion of ovarian cancer, but the underlying mechanism is barely understood. Ovarian cancer is a severe gynecological malignancy with high mortality. Ovarian cancer-associated death can primarily be attributed to cancer metastasis. The majority of patients are diagnosed with wide dissemination in the peritoneum and omentum, limiting the effectiveness of surgery and chemotherapy. In addition, unlike other well-documented cancers, metastasis through vasculature is not a usual dissemination pathway in ovarian cancer. This review sheds light on TAMs and the main process and mechanism of ovarian cancer metastasis.

Identification of key biomarkers associated with development and prognosis in patients with ovarian carcinoma: evidence from bioinformatic analysis.

BACKGROUND: Ovarian cancer (OC) is the deadliest cause in the gynecological malignancies. Most OC patients are diagnosed in advanced stages with less than 40% of women cured. However, the possible mechanism underlying tumorigenesis and candidate biomarkers remain to be further elucidated. RESULTS: Gene expression profiles of GSE18520, GSE54388, and GSE27651 were available from Gene Expression Omnibus (GEO) database with a total of 91 OC samples and 22 normal ovarian (OV) tissues. Three hundred forty-nine differentially expressed genes (DEGs) were screened between OC tissues and OV tissues via GEO2R and online Venn software, followed by KEGG pathway and gene ontology (GO) enrichment analysis. The enriched functions and pathways of these DEGs contain male gonad development, cellular response to transforming growth factor beta stimulus, positive regulation of transcription from RNA polymerase II promoter, calcium independent cell-cell adhesion via plasma membrane cell adhesion molecules, extracellular matrix organization, pathways in cancer, cell cycle, cell adhesion molecules, PI3K-AKT signaling pathway, and progesterone mediated oocyte maturation. The protein-protein network (PPI) was established and module analysis was carried out using STRING and Cytoscape. Next, with PPI network analyzed by four topological methods in Cytohubba plugin of Cytoscape, 6 overlapping genes (DTL, DLGAP5, KIF15, NUSAP1, RRM2, and TOP2A) were eventually selected. GEPIA and Oncomine were implemented for validating the gene expression and all the six hub genes were highly expressed in OC specimens compared to normal OV tissues. Furthermore, 5 of 6 genes except for DTL were associated with worse prognosis using Kaplan Meier-plotter online tool and 3 of 6 genes were significantly related to clinical stages, including RRM2, DTL, and KIF15. Additionally, cBioPortal showed that TOP2A and RRM2 were the targets of cancer drugs in patients with OC, indicating the other four genes may also be potential drug targets. CONCLUSION: Six hub genes (DTL, DLGAP5, KIF15, NUSAP1, RRM2, and TOP2A) present promising predictive value for the development and prognosis of OC and may be used as candidate targets for diagnosis and treatment of OC.

Specific expression pattern of IMP metabolism related-genes in chicken muscle between cage and free range conditions.

Inosine monophosphate (IMP) is a key factor affecting the fleshy flavor of meat; meanwhile, the free-range mode is an efficient strategy to improve muscular IMP content. To assess expression differences in IMP metabolism-related genes under different feeding patterns, Illumina Nextseq 500 sequencing was used to catalog the global gene expression profiles of muscle samples from Lueyang black-bone chicken under free-range and caging conditions. A total of 15510 unigenes were assembled, with 13423 (86.54%) and 6088 (39.25%) unigenes correctly annotated in the GO and KOG databases, respectively. Next, the "purine metabolism" pathway in the "nucleotide metabolism group" was assessed in depth. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, we retrieved 172 nucleotide- and 5 purine- metabolism related genes that were differentially expressed in muscle samples from free-range and caged chickens. At 60-day-old, AMPD1, NT5C1A and ENTPD8 showed higher levels in the free-range group, while only ENTPD8 was upregulated in 120-day-old chickens. In addition, GART, GARS and ADSL in free-range chickens showed higher levels compared with caged animals. Furthermore, IMPDH levels in free-range chicken were lower than those of caged chicken. Real-time quantitative polymerase chain reaction (qPCR) was used to validate the above findings. These results revealed a set of differentially expressed genes potentially related to IMP metabolism in chicken under different breeding modes, providing novel insights into controlling IMP levels in chicken meat.