Single-cell transcriptomics reveals maturation of transplanted stem cell-derived retinal pigment epithelial cells toward native state.

Transplantation of stem cell-derived retinal pigment epithelial (RPE) cells is considered a viable therapeutic option for age-related macular degeneration (AMD). Several landmark Phase I/II clinical trials have demonstrated safety and tolerability of RPE transplants in AMD patients, albeit with limited efficacy. Currently, there is limited understanding of how the recipient retina regulates the survival, maturation, and fate specification of transplanted RPE cells. To address this, we transplanted stem cell-derived RPE into the subretinal space of immunocompetent rabbits for 1 mo and conducted single-cell RNA sequencing analyses on the explanted RPE monolayers, compared to their age-matched in vitro counterparts. We observed an unequivocal retention of RPE identity, and a trajectory-inferred survival of all in vitro RPE populations after transplantation. Furthermore, there was a unidirectional maturation toward the native adult human RPE state in all transplanted RPE, regardless of stem cell resource. Gene regulatory network analysis suggests that tripartite transcription factors (FOS, JUND, and MAFF) may be specifically activated in posttransplanted RPE cells, to regulate canonical RPE signature gene expression crucial for supporting host photoreceptor function, and to regulate prosurvival genes required for transplanted RPE's adaptation to the host subretinal microenvironment. These findings shed insights into the transcriptional landscape of RPE cells after subretinal transplantation, with important implications for cell-based therapy for AMD.

Integrated risk scores from N6-methyladenosine-related lncRNAs are potential biomarkers for predicting the overall survival of bladder cancer patients.

Background: N6-methyladenosine (m6A) is the most common form of mRNA- and long noncoding RNA (lncRNA)-specific internal modification encountered in eukaryotes, with important effects on mRNA stability, translation, and splicing. The role of m6A-modified lncRNAs (m6A-lncRNAs) in bladder cancer (BLCA) is rarely reported. This study aimed to evaluate an efficient prognostic model of BLCA in patients, based on m6A-lncRNAs, and to discover potential biological targets. Methods: Differentially expressed lncRNAs were investigated in 433 BLCA samples derived from The Cancer Genome Atlas (TCGA) database. Kaplan-Meier and univariate Cox regression analyses were performed to screen for m6A-lncRNAs with prognostic roles in BLCA. We implemented Pearson correlation analysis to analyze 18 potentially prognostic lncRNAs and 20 known m6A-associated genes. Next, the data were imputed using least absolute shrinkage and selection operator (LASSO) Cox regression to establish an m6A-lncRNA prognostic signature. Results: We established an integrated risk score (RS) containing five m6A-lncRNAs and constructed a nomogram that had the ability to forecast the overall survival (OS) of patients with BLCA. We showed that the predictive accuracy of the RS for BLCA prognosis was high, which was confirmed by the area under the receiver operating characteristic (ROC) curve. We analyzed the correlation between tumor immune infiltrating cells and RS in high- and low-risk patients with BLCA and used tumor immune dysfunction and exclusion to predict the effect of immunotherapy. We screened out the most relevant modules of RS through the weighted gene co-expression network analysis network and explored their potential biological functions using GO and KEGG analyses. Conclusion: Our findings demonstrate that, compared with nomograms constructed using a single prognostic factor, the integrated RS represents a superior model for predicting survival in patients with BLCA, which may improve the clinical management of BLCA.

CD90low glioma-associated mesenchymal stromal/stem cells promote temozolomide resistance by activating FOXS1-mediated epithelial-mesenchymal transition in glioma cells.

BACKGROUND: The tumour microenvironment contributes to chemotherapy resistance in gliomas, and glioma-associated mesenchymal stromal/stem cells (gaMSCs) are important stromal cell components that play multiple roles in tumour progression. However, whether gaMSCs affect chemotherapy resistance to the first-line agent temozolomide (TMZ) remains unclear. Herein, we explored the effect and mechanism of gaMSCs on resistance to TMZ in glioma cells. METHODS: Human glioma cells (cell line U87MG and primary glioblastoma cell line GBM-1) were cultured in conditioned media of gaMSCs and further treated with TMZ. The proliferation, apoptosis and migration of glioma cells were detected by Cell Counting Kit-8 (CCK-8), flow cytometry and wound-healing assays. The expression of FOXS1 in glioma cells was analysed by gene microarray, PCR and Western blotting. Then, FOXS1 expression in glioma cells was up- and downregulated by lentivirus transfection, and markers of the epithelial-mesenchymal transformation (EMT) process were detected. Tumour-bearing nude mice were established with different glioma cells and treated with TMZ to measure tumour size, survival time and Ki-67 expression. Finally, the expression of IL-6 in gaMSC subpopulations and its effects on FOXS1 expression in glioma cells were also investigated. RESULTS: Conditioned media of gaMSCs promoted the proliferation, migration and chemotherapy resistance of glioma cells. The increased expression of FOXS1 and activation of the EMT process in glioma cells under gaMSC-conditioned media were detected. The relationship of FOXS1, EMT and chemotherapy resistance in glioma cells was demonstrated through the regulation of FOXS1 expression in vitro and in vivo. Moreover, FOXS1 expression in glioma cells was increased by secretion of IL-6 mainly from the CD90low gaMSC subpopulation. CONCLUSIONS: CD90low gaMSCs could increase FOXS1 expression in glioma cells by IL-6 secretion, thereby activating epithelial-mesenchymal transition and resistance to TMZ in glioma cells. These results indicate a new role of gaMSCs in chemotherapy resistance and provide novel therapeutic targets.

Cell fusion in cancer hallmarks: Current research status and future indications.

Cell fusion is involved in several physiological processes, such as reproduction, development and immunity. Although cell fusion in tumors was reported 130 years ago, it has recently attracted great interest, with recent progress in tumorigenesis research. However, the role of cell fusion in tumor progression remains unclear. The pattern of cell fusion and its role under physiological conditions are the basis for our understanding of the pathological role of cell fusion. However, the role of cell fusion in tumors and its functions are complicated. Cell fusion can directly increase tumor heterogeneity by forming polyploids or aneuploidies. Several studies have reported that cell fusion is associated with tumorigenesis, metastasis, recurrence, drug resistance and the formation of cancer stem cells. Given the diverse roles cell fusion plays in different tumor phenotypes, methods based on targeted cell fusion have been designed to treat tumors. Research on cell fusion in tumors may provide novel ideas for further treatment.

Roles of long non-coding RNAs in the hallmarks of glioma.

Glioma is one of the most common types of tumor of the central nervous system. Due to the aggressiveness and invasiveness of high-level gliomas, the survival time of patients with these tumors is short, at ~15 months, even after combined treatment with surgery, radiotherapy and/or chemotherapy. Recently, a number of studies have demonstrated that long non-coding RNA (lncRNAs) serve crucial roles in the multistep development of human gliomas. Gliomas acquire numerous biological abilities during multistep development that collectively constitute the hallmarks of glioma. Thus, in this review, the roles of lncRNAs associated with glioma hallmarks and the current and future prospects for their development are summarized.

Tumor-Microenvironment-on-a-Chip for Evaluating Nanoparticle-Loaded Macrophages for Drug Delivery.

Targeted drug delivery remains attractive but challenging for cancer therapy. Cell-mediated drug delivery has emerged as a promising strategy to improve targeted drug delivery to tumors due to the intrinsic ability of certain types of cells (e.g., macrophage) to pass through physiological barriers and specifically home to tumors. To fundamentally understand how macrophage-based drug carriers transport and interact with the tumor microenvironment, we developed a tumor-microenvironment-on-a-chip (TMOC) model that enables the coculture of tumor spheroids and macrophages in a three-dimensional (3D) gel matrix. By introducing drug-loaded macrophages, the TMOC model allows real-time observation of macrophage migration toward the tumor, infiltration into tumor spheroids, and subsequent response of tumor to drugs. Our results demonstrated the superior capability of macrophages migrating toward the tumor and infiltrate tumor spheroids. Drug loading in macrophages had minimum effect on their cell viability, and drug-carrying macrophages exhibited greater tumor cell cytotoxicity compared to their nanoparticle counterparts. Our work highlighted the great potential of macrophages as novel drug carriers for targeted drug delivery, and the TMOC model serves as a versatile platform to enable quick evaluation of such cell-mediated drug delivery systems.

A Microfluidic Tumor-on-a-Chip for Assessing Multifunctional Liposomes' Tumor Targeting and Anticancer Efficacy.

Two principal methods for cancer drug testing are widely used, namely, in vitro 2D cell monolayers and in vivo animal models. In vitro 2D culture systems are simple and convenient but are unable to capture the complexity of biological processes. Animal models are costly, time-consuming, and often fail to replicate human activity. Here a microfluidic tumor-on-a-chip (TOC) model designed for assessing multifunctional liposome cancer targeting and efficacy is presented. The TOC device contains three sets of hemispheric wells with different sizes for tumor spheroid formation and evaluation of liposomes under a controlled flow condition. There is good agreement between time-elapsed tumor targeting of fluorescent liposomes in the TOC model and in in vivo mouse models. Evaluation of the anticancer efficacy of four PTX-loaded liposome formulations shows that compared to 2D cell monolayers and 3D tumor spheroid models, the TOC model better predicts the in vivo anticancer efficacy of targeted liposomes. Lastly, the TOC model is used to assess the effects of flow rates and tumor size on treatment outcome. This study demonstrates that the TOC model provides a convenient and powerful platform for rapid and reliable cancer drug evaluation.

Tumor-Vasculature-on-a-Chip for Investigating Nanoparticle Extravasation and Tumor Accumulation.

Nanoparticle tumor accumulation relies on a key mechanism, the enhanced permeability and retention (EPR) effect, but it remains challenging to decipher the exact impact of the EPR effect. Animal models in combination with imaging modalities are useful, but it is impossible to delineate the roles of multiple biological barriers involved in nanoparticle tumor accumulation. Here we report a microfluidic tumor-vasculature-on-a-chip (TVOC) mimicking two key biological barriers, namely, tumor leaky vasculature and 3D tumor tissue with dense extracellular matrix (ECM), to study nanoparticle extravasation through leaky vasculature and the following accumulation in tumor tissues. Intact 3D tumor vasculature was developed with selective permeability of small molecules (20 kDa) but not large ones (70 kDa). The permeability was further tuned by cytokine stimulation, demonstrating the independent control of the leaky tumor vasculature. Combined with tumor spheroids in dense ECM, our TVOC model is capable of predicting nanoparticles' in vivo tumor accumulation, thus providing a powerful platform for nanoparticle evaluation.

Synergetic Combinations of Dual-Targeting Ligands for Enhanced In Vitro and In Vivo Tumor Targeting.

The concept of dual-ligand targeting has been around for quite some time, but remains controversial due to the intricate interplay between so many different factors such as the choice of dual ligands, their densities, ratios and length matching, etc. Herein, the synthesis of a combinatorial library of single and dual-ligand nanoparticles with systematically varied properties (ligand densities, ligand ratios, and lengths) for tumor targeting is reported. Folic acid (FA) and hyaluronic acid (HA) are used as two model targeting ligands. It is found that the length matching and ligand ratio play critical roles in achieving the synergetic effect of the dual-ligand targeting. When FA is presented on the nanoparticle surface through a 5K polyethylene glycol (PEG) chain, the dual ligand formulations using the HA with either 5K or 10K length do not show any targeting effect, but the right length of HA (7K) with a careful selection of the right ligand ratio do enhance the targeting efficiency and specificity significantly. Further in vitro 3D tumor spheroid models and in vivo xenograft mice models confirm the synergetic targeting efficiency of the optimal dual-ligand formulation (5F2H7K ). This work provides a valuable insight into the design of dual-ligand targeting nanosystems.

Understanding the Effects of Nanocapsular Mechanical Property on Passive and Active Tumor Targeting.

The physicochemical properties of nanoparticles (size, charge, and surface chemistry, etc.) influence their biological functions often in complex and poorly understood ways. This complexity is compounded when the nanostructures involved have variable mechanical properties. Here, we report the synthesis of liquid-filled silica nanocapsules (SNCs, ∼ 150 nm) having a wide range of stiffness (with Young's moduli ranging from 704 kPa to 9.7 GPa). We demonstrate a complex trade-off between nanoparticle stiffness and the efficiencies of both immune evasion and passive/active tumor targeting. Soft SNCs showed 3 times less uptake by macrophages than stiff SNCs, while the uptake of PEGylated SNCs by cancer cells was independent of stiffness. In addition, the functionalization of stiff SNCs with folic acid significantly enhanced their receptor-mediated cellular uptake, whereas little improvement for the soft SNCs was conferred. Further in vivo experiments confirmed these findings and demonstrated the critical role of nanoparticle mechanical properties in regulating their interactions with biological systems.

Design of Modular Peptide Surfactants and Their Surface Activity.

Designed peptide surfactants offer a number of advanced properties over conventional petrochemical surfactants, including biocompatibility, sustainability, and tailorability of the chemical and physical properties through peptide design. Their biocompatibility and degradability make them attractive for various applications, particularly for food and pharmaceutical applications. In this work, two new peptide surfactants derived from an amphiphilic peptide surfactant (AM1) were designed (AM-S and C8-AM) to better understand links between structure, interfacial activity, and emulsification. Based on AM1, which has an interfacial α-helical structure, AM-S and C8-AM were designed to have two modules, that is, the α-helical AM1 module and an additional hydrophobic moiety to provide for better anchoring at the oil-water interface. Both AM-S and C8-AM at low bulk concentration of 20 µM were able to adsorb rapidly at the oil-water interface and reduced interfacial tension to equilibrium values of 17.0 and 8.4 mN/m within 400 s, respectively. Their relatively quick adsorption kinetics allowed the formation of nanoemulsions with smaller droplet sizes and narrower size distribution. AM-S and C8-AM at 800 µM bulk concentration could make nanoemulsions of average diameters 180 and 147 nm, respectively, by simple sonication. With respect to the long-term stability, a minimum peptide concentration of 400 µM for AM-S and a lower concentration of 100 µM for C8-AM were demonstrated to effectively stabilize nanoemulsions over 3 weeks. Compared to AM1, the AM-S nanoemulsion retained its stimuli-responsive function triggered by metal ions, whereas the C8-AM nanoemulsions did not respond to the stimuli as efficiently as AM-S because of the strong anchoring ability of the hydrophobic C8 module. The two-module design of AM-S and C8-AM represents a new strategy in tuning the surface activity of peptide surfactants, offering useful information and guidance of future designs.

Systematic identification of factors for provirus silencing in embryonic stem cells.

Embryonic stem cells (ESCs) repress the expression of exogenous proviruses and endogenous retroviruses (ERVs). Here, we systematically dissected the cellular factors involved in provirus repression in embryonic carcinomas (ECs) and ESCs by a genome-wide siRNA screen. Histone chaperones (Chaf1a/b), sumoylation factors (Sumo2/Ube2i/Sae1/Uba2/Senp6), and chromatin modifiers (Trim28/Eset/Atf7ip) are key determinants that establish provirus silencing. RNA-seq analysis uncovered the roles of Chaf1a/b and sumoylation modifiers in the repression of ERVs. ChIP-seq analysis demonstrates direct recruitment of Chaf1a and Sumo2 to ERVs. Chaf1a reinforces transcriptional repression via its interaction with members of the NuRD complex (Kdm1a, Hdac1/2) and Eset, while Sumo2 orchestrates the provirus repressive function of the canonical Zfp809/Trim28/Eset machinery by sumoylation of Trim28. Our study reports a genome-wide atlas of functional nodes that mediate proviral silencing in ESCs and illuminates the comprehensive, interconnected, and multi-layered genetic and epigenetic mechanisms by which ESCs repress retroviruses within the genome.

Right-Crossed, Fused Renal Ectopia L-Shaped Kidney Type with Urinary Chyluria.

Crossed fused renal ectopia combined with chyluria is extremely rare. Here we report the case of a patient who was admitted to our institution since milky urine and was finally found to have an L-shaped fused kidney and renal pelvis fistula. The patient was cured by renal pelvic instillation sclerotherapy.

Retroperitoneal adrenal-sparing surgery for the treatment of Cushing's syndrome caused by adrenocortical adenoma: 8-year experience with 87 patients.

BACKGROUND: The objective of this study was to present our 8-year experience with partial adrenalectomy via the retroperitoneal approach for the treatment of Cushing's adenoma. METHODS: A total of 93 patients who underwent adrenal surgery for Cushing's adenoma from March 2003 to December 2010 were enrolled in this study. Preoperative, intraoperative, and postoperative variables were reviewed from the database. Student's t test was used to analyze the continuous data, and the χ(2) test was used to analyze the categoric data. A value of p < 0.05 was considered statistically significant. RESULTS: Adrenal-sparing surgery was performed in 87 cases (31 by open surgery, 56 by retroperitoneal laparoscopy). Six patients underwent open/laparoscopic total adrenalectomy because of recurrent disease or a large size. The cure rate in our series was 97.8%. Hypertension resolved in 34 of 64 patients (53.1%), diabetes in 7 of 27 patients (25.9%) and obesity in 28 of 48 patients (58.3%). One patient died during the postoperative period. The intraoperative complication rate for the open surgery group was significantly higher than that for the retroperitoneal laparoscopy group (9.1 vs. 1.7%). CONCLUSIONS: The retroperitoneal approach is reliable and safe for treating Cushing's syndrome. The laparoscopic technique can decrease the prevalence of intraoperative complications. Retroperitoneal laparoscopic partial adrenalectomy can be performed with extremely low morbidity and achieves an excellent outcome, although death may occur during the postoperative period in high-risk patients. Postoperative management plays an important role in the surgical treatment of Cushing's syndrome.

[Therapeutic effect of tracheal anastomosis versus interventional bronchoscopy in the treatment of airway stenosis].

OBJECTIVE: To compare the therapeutic effects of tracheal anastomosis and interventional bronchoscopy in the treatment of airway stenosis. METHODS: From February, 1990 to February 2008, 53 patients with airway stenosis including 32 male and 21 female patients aged 2 to 80 years (mean 38 years) received end-to-end anastomosis or bronchoscopic stent-graft treatment in our hospital. End-to-end anastomosis of the narrow sleeves resected was performed in 26 cases, balloon dilatation in 14 cases, and stent implantation in 13 cases. The clinical and follow-up data of the patients were analyzed retrospectively and the surgical outcomes were evaluated. RESULTS: No perioperative deaths occurred in these cases. Eight patients (5 with anastomosis, 2 with balloon dilation, and 1 with stent implantation) were lost to the follow-up ranging from 1 to 204 (mean 44.94-/+54.56) months. Of the patients available to the follow-up, 12 had airway restenosis, including 2 receiving end-to-end anastomosis (9.5%), 6 (50%) receiving balloon angioplasty, and 6 with granulation hyperplasia following stent implantation (50%). Of the 3 treatment, end-to-end anastomosis was associated with the lowest restenosis rate (Chi2=8.869, P=0.012). CONCLUSION: End-to-end anastomosis, with a low postoperative restenosis rate and better prognosis, should be the primary choice for treatment of tracheal airway stenosis.

[Clinical analysis of primary thymic lymphoma: a report of 27 cases].

OBJECTIVE: To investigate clinicopathologic features and optimal treatment of primary thymic lymphoma. METHODS: Clinical records of 27 primary thymic lymphoma patients treated from 1990 to 2007 were reviewed. RESULTS: Of the 27 patients, 8 received mastectomy and chemotherapy, 12 received excision of the thymic lesion and chemotherapy, 5 received chemotherapy alone, and 2 received lesion excision alone. 24 achieved complete remission after scheduled treatment, 1 achieved partial remission, and 2 patients had progressive disease. With a follow-up of 10 years and median 38 months, the 5-year overall and disease-free survival rates of the 27 patients were 47% and 23% respectively. CONCLUSION: The main subtypes of primary thymic lymphoma are diffuse large B-cell lymphoma and peripheral T-cell lymphoma. The optimal sequence is radical operation followed by standard anthracycline-based regimens and radiotherapy.

[Surgical treatment of 486 cases of adrenal gland diseases: a retrospective study].

OBJECTIVE: To evaluate the roles of laparoscopic adrenalectomy (LA) and open adrenalectomy (GA) for treatment of adrenal gland diseases. METHODS: The data of 486 patients with adrenal gland diseases was analyzed retrospectively during 5 years. A total of 478 patients received surgical treatments including 318 GAs and 160 LAs. The operation time, bleeding volume during operation, intestine function recovery time, pain postoperatively, hospital stay time postoperatively and postoperative complications in group GA and group LA respectively were compared. RESULTS: All cases in group GA were successful. A total of 154 cases in group LA were successful, and 6 cases were converted to open surgery. In group LA, there were 9 cases whose tumor diameter exceeded 6 cm. There were 3 malignant cases in group LA, and no recurrence and metastasis were observed during 3-20 months follow-up. The average operation time was (112 +/- 16) mmn and (69 +/- 10) min in group GA and LA respectively. The average bleeding volume during operation was (286 +/- 23) ml, (56 +/- 10) ml in group GA and LA respectively. The average intestine function recovery time was (66 +/- 7) h, (24 +/- 7) h in group GA and LA respectively. The average frequency of treatment of pain was 1.9 +/- 0.4 and 0.5 +/- 0.1 in group GA and LA respectively. The average hospital stay time postoperatively was (10.3 +/- 1.1) d and (7.2 +/- 0.7) d in group GA and LA respectively. The rate of postoperative complications was 40.3% and 7.5% in group GA and LA respectively. All differences were significant (P = 0.023, 0.007, 0.039, 0.003, 0.029 and 0.001). CONCLUSIONS: LA has the added benefit of shorter convalescent times, improving patients satisfaction and less associated complications, as it has proved to be as effective as OA.