Clinical value of serum soluble receptor for advanced glycation end products in evaluating the condition of patients with bronchial asthma.

Objective: This study focused on the clinical value of serum soluble receptor for advanced glycation end products (sRAGE) levels in evaluating the severity of bronchial asthma (BA). Methods: Serum sRAGE expression was measured by using enzyme-linked immunosorbent assay, eosinophils (EOS) count was measured by using an automatic blood cell counter, and forced expiratory volume in 1 second (FEV1) was measured by pulmonary function analyzer in 120 patients with BA, 40 patients with non-BA pulmonary disease, and 40 healthy controls. Receiver operating characteristic curves were used to analyze the clinical value of sRAGE expression levels, EOS counts, and FEV1 level to assess the severity of illness in the patients with BA. Results: Compared with the healthy controls and the patients without BA, the patients with BA had the lowest serum sRAGE expression level (47.36 ± 6.3 ng/L versus 75.3 ± 6.3 ng/L versus 67.5 ± 5.06 ng/L; p < 0.05), the highest EOS count (231.2 ± 18.3 106/L versus 175.9 ± 15.6 106/L versus 197.8 ± 19.6 106/L; p < 0.05), and the lowest FEV1 level (1.19 ± 0.15 L versus 1.57 ± 0.2 L versus 1.3 ± 0.17 L; p < 0.05). Correlation analysis revealed that the serum sRAGE expression levels were notably negatively correlated with the EOS counts (r value of -0.471, p < 0.05) but significantly positively linked to FEV1 levels (r value of 0.362, p < 0.05). Serum sRAGE expression levels could help in accurately diagnosing patients with severe BA (area under the receiver operating characteristic curve (AUC) = 0.904), whereas prediction in the patients with mild BA was achieved by EOS counts (AUC = 0.857). Conclusion: The serum sRAGE level has potential value in diagnosing the severity of BA, which is conducive to identifying patients with severe BA and guiding in development of new therapeutic strategies.

Single-cell quantification and dose-response of cytosolic siRNA delivery.

Endosomal escape and subsequent cytosolic delivery of small interfering RNA (siRNA) therapeutics is believed to be highly inefficient. Since it has not been possible to quantify cytosolic amounts of delivered siRNA at therapeutic doses, determining delivery bottlenecks and total efficiency has been difficult. Here, we present a confocal microscopy-based method to quantify cytosolic delivery of fluorescently labeled siRNA during lipid-mediated delivery. This method enables detection and quantification of sub-nanomolar cytosolic siRNA release amounts from individual release events with measures of quantitation confidence for each event. Single-cell kinetics of siRNA-mediated knockdown in cells expressing destabilized eGFP unveiled a dose-response relationship with respect to knockdown induction, depth and duration in the range from several hundred to thousands of cytosolic siRNA molecules. Accurate quantification of cytosolic siRNA, and the establishment of the intracellular dose-response relationships, will aid the development and characterization of novel delivery strategies for nucleic acid therapeutics.

Liver cancer cells as the model for developing liver-targeted RNAi therapeutics.

RNAi is a sequence-specific gene regulation mechanism that involves small interfering RNAs (siRNAs). RNAi therapeutic has become a new class of precision medicine and has shown great potential in treating liver-associated diseases, especially metabolic diseases. To facilitate the development of liver-targeted RNAi therapeutics in cell model, we surveyed a panel of liver cancer cell lines for the expression of genes implicated in RNAi therapeutics including the asialoglycoprotein receptor (ASGR) and metabolic disease associated genes PCSK9, ANGPTL3, CIDEB, and LDLR. A high-content screen assay based on lipid droplet staining confirmed the involvement of PCSK9, ANGPTL3, and CIDEB in lipid metabolism in selected liver cancer cell lines. Several liver cancer cell lines have high levels of ASGR1 expression, which is required for liver-specific uptake of GalNAc-conjugated siRNA, a clinically approved siRNA delivery platform. Using an EGFP reporter system, we demonstrated Hep G2 can be used to evaluate gene knockdown efficiency of GalNAc-siRNA. Our findings pave the way for using liver cancer cells as a convenient model system for the identification and testing of siRNA drug candidate genes and for studying ASGR-mediated GalNAc-siRNA delivery in liver.

Bacterial Transcription Factors Bind to Coding Regions and Regulate Internal Cryptic Promoters.

Transcription factors (TFs) regulate transcription by binding to the specific sequences at the promoter region. However, the mechanisms and functions of TFs binding within the coding sequences (CDS) remain largely elusive in prokaryotes. To this end, we collected 409 data sets for bacterial TFs, including 104 chromatin immunoprecipitation sequencing (ChIP-seq) assays and 305 data sets from the systematic evolution of ligands by exponential enrichment (SELEX) in seven model bacteria. Interestingly, these TFs displayed the same binding capabilities for both coding and intergenic regions. Subsequent biochemical and genetic experiments demonstrated that several TFs bound to the coding regions and regulated the transcription of the binding or adjacent genes. Strand-specific RNA sequencing revealed that these CDS-binding TFs regulated the activity of the cryptic promoters, resulting in the altered transcription of the corresponding antisense RNA. TF RhpR hindered the transcriptional elongation of a subgenic transcript within a CDS. A ChIP-seq and Ribo-seq coanalysis revealed that RhpR influenced the translational efficiency of binding genes. Taken together, the present study reveals three regulatory mechanisms of CDS-bound TFs within individual genes, operons, and antisense RNAs, which demonstrate the variability of the regulatory mechanisms of TFs and expand upon the complexity of bacterial transcriptomes. IMPORTANCE Although bacterial TFs regulate transcription by binding to specific sequences at the promoter region, little is known about the mechanisms and functions of TFs binding within the CDS. In this study, we show that bacterial TFs have same binding pattern in both CDS and promoter regions, and we reveal three regulatory mechanisms of CDS-bound TF that together demonstrate the complexity of the regulatory mechanisms of bacterial TFs and the wide spread of internal cryptic promoters in CDS.

Plakoglobin and High-Mobility Group Box 1 Mediate Intestinal Epithelial Cell Apoptosis Induced by Clostridioides difficile TcdB.

Clostridioides difficile infection (CDI) is the leading cause of antibiotic-associated intestinal disease, resulting in severe diarrhea and fatal pseudomembranous colitis. TcdB, one of the essential virulence factors secreted by this bacterium, induces host cell apoptosis through a poorly understood mechanism. Here, we performed an RNA interference (RNAi) screen customized to Caco-2 cells, a cell line model of the intestinal epithelium, to discover host factors involved in TcdB-induced apoptosis. We identified plakoglobin, also known as junction plakoglobin (JUP) or γ-catenin, a member of the catenin family, as a novel host factor and a previously known cell death-related chromatin factor, high-mobility group box 1 (HMGB1). Disruption of those host factors by RNAi and CRISPR resulted in resistance of cells to TcdB-mediated and mitochondrion-dependent apoptosis. JUP was redistributed from adherens junctions to the mitochondria and colocalized with the antiapoptotic factor Bcl-XL. JUP proteins could permeabilize the mitochondrial membrane, resulting in the release of cytochrome c. Our results reveal a novel role of JUP in targeting the mitochondria to promote the mitochondrial apoptotic pathway. Treatment with glycyrrhizin, an HMGB1 inhibitor, resulted in significantly increased resistance to TcdB-induced epithelial damage in cultured cells and a mouse ligated colon loop model. These findings demonstrate the critical roles of JUP and HMGB1 in TcdB-induced epithelial cell apoptosis. IMPORTANCE Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea. Toxins, especially TcdB, cause epithelial cell apoptosis, but the underlying cell death mechanism is less clear. Through an apoptosis-focused RNAi screen using a bacterium-made small interfering (siRNA) library customized to a human colonic epithelial cell model, we found a novel host factor, plakoglobin (γ-catenin), as a key factor required for cell apoptosis induced by TcdB. Plakoglobin targets and permeabilizes mitochondria after stimulation by TcdB, demonstrating a hitherto underappreciated role of this catenin family member in the apoptosis of intestinal epithelial cells. We also found a previously known cell death-related chromatin factor, HMGB1, and explored the inhibition of HMGB1 for CDI therapy in vivo.

Controlled Localized Phase Transition of Selenium for Color-Selective Photodetectors.

Color-selective photodetectors are widely applied in several fields; however, they suffer from complex fabrication and low resolution. Herein, we propose a simple and convenient design to achieve a logical color-selective heterojunction photodetector composed of CdS and Se with a crystalline/amorphous mixed state. The as-deposited amorphous Se top layer in the heterojunction is partly transformed to trigonal crystalline Se by localized in situ phase transformation during annealing. As these two heterojunctions have different photoresponses under the same wavelength, the integrated device can accurately identify red, green, and blue light via logical judgment. Finally, the device exhibits high recognition ability in actual tests. This work provides a potential development of high-resolution color-selective photodetectors for visible light communication and logical photoelectric devices.

Spontaneous quorum-sensing hierarchy reprogramming in Pseudomonas aeruginosa laboratory strain PAO1.

Pseudomonas aeruginosa strain PAO1 has been commonly used in the laboratory, with frequent genome variations reported. Quorum sensing (QS), a cell-cell communication system, plays important role in controlling a variety of virulence factors. However, the evolution and adaptability of QS in those laboratory strains are still poorly understood. Here we used the QS reporter and whole-genome sequencing (WGS) to systematically investigate the QS phenotypes and corresponding genetic basis in collected laboratory PAO1 strains. We found that the PAO1-z strain has an inactive LasR protein, while bearing an active Rhl QS system and exhibiting QS-controlled protease-positive activity. Our study revealed that an 18-bp insertion in mexT gene gave rise to the active QS system in the PAO1-z strain. This MexT inactivation restored the QS activity caused by the inactive LasR, showing elevated production of pyocyanin, cyanide and elastase. Our results implied the evolutionary trajectory for the PAO1-z strain, with the evulutionary order from the first Las QS inactivation to the final Rhl QS activation. Our findings point out that QS homeostasis occurs in the laboratory P. aeruginosa strain, offering a potential platform for the QS study in clinical isolates.

Profiling MicroRNAs with Associated Spatial Dynamics in Acute Tissue Slices.

MicroRNAs (miRNAs) are suggested to play important roles in the pathogenesis and progress of human diseases with heterogeneous regulation in different types of cells. However, limited technique is available for profiling miRNAs with both expression and spatial dynamics. Here, we describe a platform for multiplexed in situ miRNA profiling in acute tissue slices. The technique uses diamond nanoneedles functionalized with RNA-binding proteins to directly isolate targeted miRNAs from the cytosol of a large population of cells to achieve a quasi-single-cell analysis for a tissue sample. In addition to a quantitative evaluation of the expression level of particular miRNAs, the technique also provides the relative spatial dynamics of the cellular miRNAs in associated cell populations, which was demonstrated to be useful in analyzing the susceptibility and spatial reorganization of different types of cells in the tissues from normal or diseased animals. As a proof-of-concept, in MK-801-induced schizophrenia model, we found that astrocytes, instead of neurons, are more heterogeneously affected in the hippocampus of rats that underwent repeated injection of MK-801, showing an expression fingerprint related to differentially down-regulated miRNA-135a and miRNA-143; the associated astrocyte subpopulation is also more spatially dispersed in the hippocampus, suggesting an astrocyte dysregulation in the induced schizophrenia animals.

Combined inhibition of RNA polymerase I and mTORC1/2 synergize to combat oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the major cause of morbidity and mortality in head and neck cancer patients worldwide. This malignant disease is challenging to treat because of the lack of effective curative strategies and the high incidence of recurrence. This study aimed to investigate the efficacy of a single and dual approach targeting ribosome biogenesis and protein translation to treat OSCC associated with the copy number variation (CNV) of ribosomal DNA (rDNA). Here, we found that primary OSCC tumors frequently exhibited a partial loss of 45S rDNA copy number and demonstrated a high susceptibility to CX5461 (a selective inhibitor of RNA polymerase I) and the coadministration of CX5461 and INK128 (a potent inhibitor of mTORC1/2). Combined treatment displayed the promising synergistic effects that induced cell apoptosis and reactive oxygen species (ROS) generation, and inhibited cell growth and proliferation. Moreover, INK128 compromised NHEJ-DNA repair pathway to reinforce the antitumor activity of CX5461. In vivo, the cotreatment synergistically suppressed tumor growth, triggered apoptosis and strikingly extended the survival time of tumor-bearing mice. Additionally, treatment with the individual compounds and coadministration appeared to reduce the incidence of enlarged inguinal lymph nodes. Our study supports that the combination of CX5461 and INK128 is a novel and efficacious therapeutic strategy that can combat this cancer and that 45S rDNA may serve as a useful indicator to predict the efficacy of this cotreatment.

Cycloastragenol Attenuates Osteoclastogenesis and Bone Loss by Targeting RANKL-Induced Nrf2/Keap1/ARE, NF-κB, Calcium, and NFATc1 Pathways.

Osteoporosis, which typically affects postmenopausal women, is an osteolytic disease due to over-activation of osteoclasts. However, current drugs targeting osteoclast inhibition face various side effects, making natural compounds with great interest as alternative treatment options. Cycloastragenol (CAG) is a triterpenoid with multiple biological activities. Previously, CAG's activity against aging-related osteoporosis was reported, but the mechanisms of actions for the activities were not understood. This study demonstrated that CAG dose-dependently inhibited osteoclast formation in receptor activator of nuclear factor-κB ligand (RANKL)-stimulated bone marrow macrophage (BMMs). Mechanism studies showed that CAG inhibited NF-κB, calcium, and nuclear factor of activated T cells 1 (NFATc1) pathways. Additionally, CAG also promoted the nuclear factor-erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1)/anti-oxidative response element (ARE) pathway that scavenges reactive oxygen species (ROS). Furthermore, CAG was also found to prevent bone loss of postmenopausal osteoporosis (PMO) in a preclinical model of ovariectomized (OVX) mice. Collectively, our research confirms that CAG inhibits the formation and function of osteoclasts by regulating RANKL-induced intracellular signaling pathways, which may represent a promising alternative for the therapy of osteoclast-related disease.

Effectiveness of fire needle combining with moist healing dressing to promote the growth of granulation tissue in chronic wounds: A case report.

In this case study, we analyzed the wound-healing process of a patient with a chronic wound who underwent fire needle treatment, and we tracked the coverage of granulation tissue and decrease of slough and exudate. An 85-year-old man had repeated right shoulder and back pain, itching, and skin festering for more than 1.5 years. A fire needle was administered combined with moist dressing once every 5 days to promote wound healing. After six rounds of fire needle treatment, granulation tissue formed over the surface of the wound base, the depth of the wound had become shallow, and the wound area was reduced. No complications occurred during the intervention. Fire needle therapy combined with a moist wound-healing dressing can be an effective alternative approach in managing chronic wounds.

Tombusvirus p19 Captures RNase III-Cleaved Double-Stranded RNAs Formed by Overlapping Sense and Antisense Transcripts in Escherichia coli.

Antisense transcription is widespread in bacteria. By base pairing with overlapping sense RNAs, antisense RNAs (asRNA) can form double-stranded RNAs (dsRNA), which are cleaved by RNase III, a dsRNA endoribonuclease. The ectopic expression of plant Tombusvirus p19 in Escherichia coli stabilizes ∼21-nucleotide (nt) dsRNA RNase III decay intermediates, which enabled us to characterize otherwise highly unstable asRNA by deep sequencing of p19-captured dsRNA. RNase III-produced small dsRNA were formed at most bacterial genes in the bacterial genome and in a plasmid. We classified the types of asRNA in genomic clusters producing the most abundant p19-captured dsRNA and confirmed RNase III regulation of asRNA and sense RNA decay at three type I toxin-antitoxin loci and at a coding gene, rsd Furthermore, we provide potential evidence for the RNase III-dependent regulation of CspD protein by asRNA. The analysis of p19-captured dsRNA revealed an RNase III sequence preference for AU-rich sequences 3 nucleotides on either side of the cleavage sites and for GC-rich sequences in the 2-nt overhangs. Unexpectedly, GC-rich sequences were enriched in the middle section of p19-captured dsRNA, suggesting some unexpected sequence bias in p19 protein binding. Nonetheless, the ectopic expression of p19 is a sensitive method for identifying antisense transcripts and RNase III cleavage sites in dsRNA formed by overlapping sense and antisense transcripts in bacteria.

High-Efficiency Cellular Reprogramming by Nanoscale Puncturing.

Induced pluripotent stem cells (iPSCs) bear great potential for disease modeling, drug discovery, and regenerative medicine; however, the wide adoption of iPSC for clinically relevant applications has been hindered by the extremely low reprogramming efficiency. Here, we describe a high-efficiency cellular reprogramming strategy by puncturing cells with an array of diamond nanoneedles, which is applied to temporally disrupt the cell membrane in a reversible and minimally invasive format. This method enables high-efficiency cytoplasmic delivery of mini-intronic plasmid vectors to initiate the conversion of human fibroblast cells to either primed iPSCs or naïve iPSCs. The nanopuncturing operation is directly performed on cells in adherent culture without any cell lift-off and is completed within just 5 min. The treated cells are then cultured in feeder-free medium to achieve a reprogramming efficiency of 1.17 ± 0.28%, which is more than 2 orders of magnitude higher than the typical results from common methods involving plasmid delivery.

High-throughput intracellular biopsy of microRNAs for dissecting the temporal dynamics of cellular heterogeneity.

The capability to analyze small RNAs responsible for post-transcriptional regulation of genes expression is essential for characterizing cellular phenotypes. Here, we describe an intracellular biopsy technique (inCell-Biopsy) for fast, multiplexed, and highly sensitive profiling of microRNAs (miRNAs). The technique uses an array of diamond nanoneedles that are functionalized with size-dependent RNA binding proteins, working as "fishing rods" to directly pull miRNAs out of cytoplasm while keeping the cells alive, thus enabling quasi-single-cell miRNA analysis. Each nanoneedle works as a reaction chamber for parallel in situ amplification, visualization, and quantification of miRNAs as low as femtomolar, which is sufficient to detect miRNAs of a single-copy intracellular abundance with specificity to single-nucleotide variation. Using inCell-Biopsy, we analyze the temporal miRNA transcriptome over the differentiation of embryonic stem cells (ESCs). The combinatorial miRNA expression patterns derived by inCell-Biopsy identify emerging cell subpopulations differentiated from ESCs and reveal the dynamic evolution of cellular heterogeneity.

Microvesicles (MIVs) secreted from adipose-derived stem cells (ADSCs) contain multiple microRNAs and promote the migration and invasion of endothelial cells.

Extracellular vesicles (EVs) such as microvesicles (MIVs) play an important role in intercellular communications. MIVs are small membrane vesicles sized 100-1000 nm in diameter that are released by many types of cells, such as mesenchymal stem cells (MSCs), tumor cells and adipose-derived stem cells (ADSC). As EVs can carry out autocrine and paracrine functions by controlling multiple cell processes, it is conceivable that EVs can be used as delivery vehicles for treating several clinical conditions, such as to improve cardiac angiogenesis after myocardial infarction (MI). Here, we seek to investigate whether ADSC-derived MIVs contain microRNAs that regulate angiogenesis and affect cell migration of endothelial cells. We first characterized the ADSC-derived MIVs and found that the MIVs had a size range of 100-300 nm, and expressed the MIV marker protein Alix. We then analyzed the microRNAs in ADSCs and ADSC-derived MIVs and demonstrated that ADSC-derived MIVs selectively released a panel of microRNAs, several of which were related to angiogenesis, including two members of the let-7 family. Furthermore, we demonstrated that ADSC-derived MIVs promoted the cell migration and invasion of the HUVEC endothelial cells. The PKH26-labeled ADSC-derived MIVs were effectively uptaken into the cytoplasm of HUVEC cells. Collectively, our results demonstrate that the ADSC-derived MIVs can promote migration and invasion abilities of endothelial cells, suggesting pro-angiogenetic potential. Future studies should focus on investigating the roles and mechanisms through which ADSC-derived MIVs regulate angiogenesis.

[Short- and mid-term effectiveness of impaction bone allograft with acetabular components in treatment of severe acetabular defects].

OBJECTIVE: To investigate the short- and mid-term effectiveness of revision hip arthroplasty by using impaction bone allograft and acetabular components in treatment of severe acetabular defects. METHODS: A clinical data of 42 patients (44 hips) with severe acetabular defects between February 2011 and May 2018 were retrospectively analyzed. All patients underwent revision hip arthroplasty by using impaction bone allograft and acetabular components. Cemented cup (24 cases, 24 hips) and non-cemented cup (18 cases, 20 hips) were used in the revision surgery. There were 17 males and 25 females with an average age of 62.8 years (range, 22-84 years). The interval between the first total hip arthroplasty and revision was 2.5-12.0 years (mean, 8.3 years). The patients were accepted revision surgery for prosthesis aseptic loosening in 32 hips (31 cases) and the periprosthetic infection in 12 hips (11 cases). Twenty-nine hips (28 cases) were Paprosky type ⅢA and 15 hips (14 cases) were type ⅢB. The preoperative Harris score was 22.25±10.31 and the height of hip rotation center was (3.67±0.63) cm and the length difference of lower limbs was (3.41±0.64) cm. RESULTS: The operation time was 130-245 minutes (mean, 186 minutes) and the intraoperative blood loss was 600-2 400 mL (mean, 840 mL). The postoperative drainage volume was 250-1 450 mL (mean, 556 mL). Superficial infection of the incision occurred in 1 case, and the incisions healed by first intention in the other patients. All patients were followed up 6-87 months, with an average of 48.6 months. At last follow-up, the Harris score was 85.85±9.31, which was significantly different from the preoperative score ( t=18.563, P=0.000). Imaging examination revealed that the allogeneic bone gradually fused with the host bone, and no obvious bone resorption was observed. At last follow-up, the height of the hip rotation center was (1.01±0.21) cm, which was significantly different from the preoperative level ( t=17.549, P=0.000); the length difference of lower limbs was (0.62±0.51) cm, which was significantly different from the preoperative level ( t=14.211, P=0.000). The Harris score in the cemented group and non-cemented group increased significantly at last follow-up. The height of the hip rotation center decreased, and the hip rotation centers of both groups were within the Ranawat triangle zone. The length difference of the lower limbs also decreased, and the differences in all indexes were significant between pre- and post-operation ( P<0.05). There was significant difference in the height of the hip rotation center between groups ( t=2.095, P=0.042), but there was no significant difference in the Harris score and the length difference of lower limbs between groups ( P>0.05). CONCLUSION: For severe acetabular defect (Paprosky type Ⅲ), the hip can be reconstructed with the impaction bone allograft and cemented or non-cemented components in revision hip arthroplsty. The short- and mid-term effectiveness are satisfactory.

Lipid-based Vehicles for siRNA Delivery in Biomedical Field.

BACKGROUND: Genetic drugs have aroused much attention in the past twenty years. RNA interference (RNAi) offers novel insights into discovering potential gene functions and therapies targeting genetic diseases. Small interference RNA (siRNA), typically 21-23 nucleotides in length, can specifically degrade complementary mRNA. However, targeted delivery and controlled release of siRNA remain a great challenge. METHODS: Different types of lipid-based delivery vehicles have been synthesized, such as liposomes, lipidoids, micelles, lipoplexes and lipid nanoparticles. These carriers commonly have a core-shell structure. For active targeting, ligands may be conjugated to the surface of lipid particles. RESULTS: Lipid-based drug delivery vehicles can be utilized in anti-viral or anti-tumor therapies. They can also be used to tackle genetic diseases or discover novel druggable genes. CONCLUSION: In this review, the structures of lipid-based vehicles and possible surface modifications are described, and applications of delivery vehicles in biomedical field are discussed.

Mesenchymal Stem Cells and Cancer: Clinical Challenges and Opportunities.

Stem cell-based therapies exhibit profound therapeutic potential for treating various human diseases, including cancer. Among the cell types that can be used for this purpose, mesenchymal stem cells (MSCs) are considered as promising source of stem cells in personalized cell-based therapies. The inherent tumor-tropic property of MSCs can be used to target cancer cells. Although the impacts of MSCs on tumor progression remain elusive, they have been genetically modified or engineered as targeted anticancer agents which could inhibit tumor growth by blocking different processes of tumor. In addition, there are close interactions between MSCs and cancer stem cells (CSCs). MSCs can regulate the growth of CSCs through paracrine mechanisms. This review aims to focus on the current knowledge about MSCs-based tumor therapies, the opportunities and challenges, as well as the prospective of its further clinical implications.

Tumor-secreted extracellular vesicles promote the activation of cancer-associated fibroblasts via the transfer of microRNA-125b.

Tumour cells release large quantities of extracellular vesicles (EVs) to mediate their interactions with other cells in the tumour microenvironment. To identify host cells that naturally take up EVs from tumour cells, we created breast cancer cell lines secreting fluorescent EVs. These fluorescent EVs are taken up most robustly by fibroblasts within the tumour microenvironment. RNA sequencing indicated that miR-125b is one of the most abundant microRNAs secreted by mouse triple-negative breast cancer 4T1 and 4TO7 cells. Treatment with 4T1 EVs leads to an increase in fibroblast activation in isogenic 4TO7 tumours, which is reversed by blocking miR-125b in 4T1 EVs; hence, miR-125b delivery by EVs is responsible for fibroblast activation in mouse tumour models. miR-125b is also secreted by human breast cancer cells and the uptake of EVs from these cells significantly increases cellular levels of miR-125b and expression of multiple cancer-associated fibroblast markers in resident fibroblasts. Overexpression of miR-125b in both mouse and human fibroblasts leads to an activated phenotype similar to the knockdown of established miR-125b target mRNAs. These data indicate that miR-125b is transferred through EVs from breast cancer cells to normal fibroblasts within the tumour microenvironment and contributes to their development into cancer-associated fibroblasts.

Self-assembled Lipid Nanoparticles for Ratiometric Codelivery of Cisplatin and siRNA Targeting XPF to Combat Drug Resistance in Lung Cancer.

DNA damage repair through the nucleotide excision repair (NER) pathway is one of the major reasons for the decreased antitumor efficacy of platinum-based anticancer drugs that have been widely applied in the clinic. Inhibiting the intrinsic NER function may enhance the antitumor activity of cisplatin and conquer cisplatin resistance. Herein, we report the design, optimization, and application of a self-assembled lipid nanoparticle (LNP) system to simultaneously deliver a cisplatin prodrug together with siRNA targeting endonuclease xeroderma pigmentosum group F (XPF), a crucial component in the NER pathway. The LNP is able to efficiently encapsulate both the platinum prodrug and siRNA molecules with a tuned ratio. Both platinum prodrug and XPF-targeted siRNA are efficiently carried into cells and released; the former damages DNA and the latter specifically downregulates both mRNA and protein levels of XPF to potentiate the platinum drug, leading to enhanced expression levels of apoptosis markers and improved cytotoxicity in both cisplatin-sensitive and -resistant human lung cancer cells. Our results demonstrate an effective approach to utilize a multi-targeted nanoparticle system that can specifically silence an NER-related gene to promote apoptosis induced by cisplatin, especially in cisplatin-refractory tumors.