Exosomal PD-L1 predicts response with immunotherapy in NSCLC patients.

Immune Check-Point Inhibitors (ICIs) have shown remarkable promise in treating tumors, including non-small cell lung cancer (NSCLC). Nevertheless, the treatment response rate is low. Studies have found that the high expression of exosomal PD-L1 is one of the reasons for the low treatment response. Therefore, this study focused on the relationship between the exosomal PD-L1 and the clinical response to immunotherapy in NSCLC patients to evaluate whether it could be used as a biomarker to predict the efficacy of ICIs. In this study, clinical information and blood samples of 149 NSCLC patients receiving ICIs were collected. The expression level of exosomal PD-L1 was detected by enzyme-linked immunosorbent assay method, and the relationship between exosomal PD-L1 and the efficacy of ICIs was explored. Overall, our study found that the expression level of exosomal PD-L1 was lower at pre-treatment, or the max fold increasing change higher at 3-6 weeks had a higher disease control rate and longer progression-free survival. It revealed that the exosomal PD-L1 was associated with the treatment response of patients using ICIs and provided a new tool for the evaluation of clinical efficacy of lung cancer immunotherapy.

Regulation of exosomes secretion by low-intensity pulsed ultrasound in lung cancer cells.

Low-intensity pulsed ultrasound (LIPUS) is a noninvasive therapeutic method which gradually being used in clinic including cancers. Exosomes mediate intercellular communication functions in disease development and the potential clinical applications in diagnosis and therapy. However, few studies have discussed the relationship between LIPUS and exosomes. Herein, we show that low intensity (0.6-2.1 W/cm2 or 0.6-3.4 W/cm2) LIPUS promoted exosomes secretion whereas higher intensity (3.4-5.0 W/cm2 or 5.0 W/cm2) LIPUS inhibited exosomes secretion, and this phenomenon is associated with autophagy. Pretreatment with 3-MA or down-regulation of LC3 potentiated low intensity LIPUS's promotion of exosomes secretion and conferred resistance to higher intensity LIPUS's effects on exosomes secretion. Furthermore, pretreatment with PP242 attenuated LIPUS-influenced exosomes secretion while expression of constitutively active Akt (Ad-myr-Akt) elevated LIPUS-influenced exosomes secretion, implying mTOR-dependent mechanism involved. The findings indicate that LIPUS influences exosomes secretion by targeting mTOR-mediated LC3 signaling in SPC-A1 and SPC-A1-BM cells. Our data provided initial evidence to connect LIPUS and secretion of exosomes, and highlight that LIPUS may be exploited in exosome-related diseases.

Exosomal miR-499a-5p promotes cell proliferation, migration and EMT via mTOR signaling pathway in lung adenocarcinoma.

Tumor-derived exosomes contain informative microRNAs involved in carcinogenesis, cell migration, invasion and epithelial-mesenchymal transition (EMT), eventually contributing to metastasis of cancers. This study aims to clarify which and how exosomal miRNA affects tumor carcinogenesis and metastasis. Among them, miR-499a-5p was upregulated in both highly metastatic lung cancer cell line and their exosomes. MiR-499a-5p overexpression promoted cell proliferation, migration and EMT, while miR-499a-5p knockdown suppressed these processes in vitro. Inhibition of miR-499a-5p by antagomirs administration restrained tumor growth in vivo. Consequently, miR499a-sufficient exosomes, derived from highly metastatic cell line, enhanced cell proliferation, migration and EMT via mTOR pathway, and the effect could be inhibited by miR-499a-5p inhibitor. The study reveals the potential diagnostic and therapeutic value of cancer-derived exosomal miR-499a-5p, and sheds a new insight on a novel molecular mechanism which modulates metastasis.

Klotho-beta attenuates Rab8a-mediated exosome regulation and promotes prostate cancer progression.

Tumor-secreted exosomes have a wide range of effects on the growth, metastasis, and drug resistance of cancer cells. However, whether and how the molecular mechanisms that regulate the secretion of exosomes could affect tumor progression remains poorly understood. Klotho beta (KLB) has been reported dysregulated in prostate cancer, but its function remains unknown. Herein, we first determined that KLB was upregulated in prostate cancer and its expression level was positively correlated with prostate cancer malignant phenotype both in vitro and in vivo. Intriguingly, KLB overexpression could impair the release of exosomes and cause the intracellular accumulation of multivesicular bodies (MVBs) in prostate cancer cells. Mechanistically, KLB attenuated exosomes secretion through a Rab8a-dependent pathway. Rab8a was downregulated in KLB overexpressing cells whereas overexpression of Rab8a could rescue the impaired release of exosomes and attenuate the KLB-induced malignant phenotype of prostate cancer both in vitro and in vivo. Taken together, this study has unveiled the tumor-promoting role of KLB mediated by its regulation on exosomes secretion through a Rab8a-dependent mechanism. These findings could be exploited to develop novel theranostic targets for prostate cancer.

Celery (Apium graveolens L.) Exosome-like Nanovesicles as a New-Generation Chemotherapy Drug Delivery Platform against Tumor Proliferation.

Extracellular vesicles (EVs) released from cells have shown robust efficacy in drug delivery compared with traditional synthetic carriers. Hampered by the high production cost and complex purification process, the clinical application of EVs as drug carriers is still limited. Nanoparticles isolated from plants with exosome-like morphology and similar delivery effects could be a new option for drug delivery. The celery exosome-like nanovesicles (CELNs) showed higher cellular uptake efficiency compared to the other three common plant-derived exosome-like nanovesicles, which is an essential advantage for CELNs as a drug carrier. The less toxicity and better tolerance of CELNs as biotherapeutic roles were verified in mice models. Then, doxorubicin (DOX) was encapsulated into CELNs to construct engineered CELNs (CELNs-DOX), which proved to be more efficient in treating tumors than conventional synthetic carriers like liposome both in vitro and in vivo. In conclusion, this study, for the first time, has proposed the emerging role of CELNs as a new-generation drug delivery carrier with distinct advantages.

Effects of the Extraction Solvents on Dissolution Rate and Antioxidant Capacity of Auricularia auricula (Agaricomycetes) Polysaccharides In Vitro and In Vivo.

This paper reports the effects of solvents on the dissolution rate and antioxidant capacity of Auricularia auricula polysaccharides (AAPs). The ultra-low temperature combined with microwave extraction (UME) was used to compare the dissolution rates and molecular weights of AAPs using deionized water and deep eutectic solvents (DES) as solvents, respectively. Scanning electron microscope (SEM) was used to observe the effects of water extract (AAPs-FW) and DES extract (AAPs-FD) on the cell wall of A. auricula. The antioxidant capacity of polysaccharide extracts in vitro was assessed by using various methods (DPPH, ABTS, and hydroxyl radicals). In addition, in vivo oxidative stress was assessed using Caenorhabditis elegans models. The extract yield of AAPs varied among the extracts and was 19.58% ± 0.56% in AAPs-FW. Whereas DES-UME increased the yield of polysaccharides (AAPs-FD) by 9.81% in the extraction medium containing triethylene glycol-choline chloride, under the optimum conditions of 60 min freezing time, 350 W, and 90 s microwave time. The microstructure of the cell wall shown by SEM was consistent with the results of polysaccharide yields. The molecular weights of AAPs-FW and AAPs-FD were found to be 398.107 kDa and 89.099 kDa, respectively. The results demonstrated that AAPs-FD exhibited potent radical scavenging activity against DPPH and a weaker scavenging ability for ABTS and OH radicals compared to AAPs-FW. In addition, both polysaccharide extracts increased the survival rate of C. elegans under methyl viologen induced oxidative stress at specific concentrations (p < 0.05), and the antioxidant capacity of AAPs-FD was higher than that of AAPs-FW at low concentrations (0.125 mg/mL). This indicated that both polysaccharides had a protective effect against damage induced by intracellular free radical generators (methyl viologen).

NDFIP1 limits cellular TAZ accumulation via exosomal sorting to inhibit NSCLC proliferation.

NDFIP1 has been previously reported as a tumor suppressor in multiple solid tumors, but the function of NDFIP1 in NSCLC and the underlying mechanism are still unknown. Besides, the WW domain containing proteins can be recognized by NDFIP1, resulted in the loading of the target proteins into exosomes. However, whether WW domain-containing transcription regulator 1 (WWTR1, also known as TAZ) can be packaged into exosomes by NDFIP1 and if so, whether the release of this oncogenic protein via exosomes has an effect on tumor development has not been investigated to any extent. Here, we first found that NDFIP1 was low expressed in NSCLC samples and cell lines, which is associated with shorter OS. Then, we confirmed the interaction between TAZ and NDFIP1, and the existence of TAZ in exosomes, which requires NDFIP1. Critically, knockout of NDFIP1 led to TAZ accumulation with no change in its mRNA level and degradation rate. And the cellular TAZ level could be altered by exosome secretion. Furthermore, NDFIP1 inhibited proliferation in vitro and in vivo, and silencing TAZ eliminated the increase of proliferation caused by NDFIP1 knockout. Moreover, TAZ was negatively correlated with NDFIP1 in subcutaneous xenograft model and clinical samples, and the serum exosomal TAZ level was lower in NSCLC patients. In summary, our data uncover a new tumor suppressor, NDFIP1 in NSCLC, and a new exosome-related regulatory mechanism of TAZ.

Preparation of Auricularia auricula polysaccharides and their protective effect on acute oxidative stress injury of Caenorhabditis elegans.

This research enhanced the extraction procedure for Auricularia auricula crude polysaccharides by utilizing a modified Fenton reagent as a solvent, and obtained A. auricula polysaccharides (AAPs-VH) via alcohol precipitation and deproteinization. The HPLC profile revealed that the purified AAPs-VH using Sepharose 6FF was mainly a heteropolysaccharide, consisting primarily of mannose, glucuronic acid, glucose, and xylose. The Mw and Mn of the purified AAPs-VH were 87.646 kDa and 48.854 kDa, respectively. The FT-IR and NMR spectra revealed that the purified AAPs-VH belonged to pyranose and were mainly formed by (1 â†’ 3)-linked-ß-D glucan formation. In vivo experiments conducted with Caenorhabditis elegans, AAPs-VH was found to notably influence the lifespan, improve the antioxidant system, and decrease the level of cell apoptosis. This might be achieved by up-regulating the expression of genes in the IIS and TOR pathways. The study concludes that the modified Fenton reagent can increase Auricularia auricula polysaccharide solubleness and active sites, which may be an essential prompt for future studies.

FGF19 Is Coamplified With CCND1 to Promote Proliferation in Lung Squamous Cell Carcinoma and Their Combined Inhibition Shows Improved Efficacy.

Lung squamous cell carcinoma (LUSC) remains as a major cause of cancer-associated mortality with few therapeutic options. Continued research on new driver genes is particularly important. FGF19, a fibroblast growth factor, is frequently observed as amplified in human LUSC, which is also associated with multiple genomic gains and losses. However, the importance of these associated changes is largely unknown. In this study, we aimed to clarify a novel mechanism that link neighboring oncogene co-amplification in the development of LUSC. We found that FGF19 was co-amplified and co-expressed with its neighboring gene CCND1 in a subset of LUSC patients and associated with poor prognosis. Moreover, FGF19 combined with CCND1 promoted the cell cycle progression of LUSC cells. Mechanistically, FGF19 also enhanced CCND1 expression by activating FGFR4-ERK1/2 signaling and strengthening CCND1-induced phosphorylation and inactivation of retinoblastoma (RB). In a murine model of lung orthotopic cancer, knockdown of CCND1 was found to prolong survival by attenuating FGF19-induced cell proliferation. Furthermore, the combination treatment of the FGFR4 inhibitor BLU9931 and the CDK4/6 inhibitor palbociclib potentiated the growth inhibition and arrested cells in G1 phase. In vivo, co-targeting FGFR4 and CDK4/6 also showed marked inhibition of tumor growth than single agent treatment. These findings further elucidate the oncogenic role of FGF19 in LUSC and provide insights into how the co-amplification of neighboring genes synergistically function to promote cancer growth, and combined inhibition against both FGF19 and CCND1 is more effective.

Targeted inhibition of SIRT6 via engineered exosomes impairs tumorigenesis and metastasis in prostate cancer.

The treatment for metastatic castration-resistant prostate cancer patients remains a great challenge in the clinic and continuously demands discoveries of new targets and therapies. Here, we assess the function and therapeutic value of SIRT6 in metastatic castration-resistant prostate cancer. Methods: The expression of SIRT6 was examined in prostate cancer tissue microarray by immunohistochemistry staining. The functions of SIRT6 and underlying mechanisms were elucidated by in vitro and in vivo experiments. We also developed an efficient method to silence SIRT6 by aptamer-modified exosomes carrying small interfering RNA and tested the therapeutic effect in the xenograft mice models. Results: SIRT6 expression is positively correlated with prostate cancer progression. Loss of SIRT6 significantly suppressed proliferation and metastasis of prostate cancer cell lines both in vitro and in vivo. SIRT6-driven prostate cancer displays activation of multiple cancer-related signaling pathways, especially the Notch pathway. Silencing SIRT6 by siRNA delivered through engineered exosomes inhibited tumor growth and metastasis. Conclusions: SIRT6 is identified as a driver and therapeutic target for metastatic prostate cancer in our findings, and inhibition of SIRT6 by engineered exosomes can serve as a promising therapeutic tool for clinical application.

Enhanced autocrine FGF19/FGFR4 signaling drives the progression of lung squamous cell carcinoma, which responds to mTOR inhibitor AZD2104.

Lung cancer occurrence and associated mortality ranks top in all countries. Despite the rapid development of targeted and immune therapies, many patients experience relapse within a few years. It is urgent to uncover the mechanisms that drive lung cancer progression and identify novel molecular targets. Our group has previously identified FGF19 as a prognostic marker and potential driver gene of lung squamous cell carcinomas (LSQ) in Chinese smoking patients. However, the underlying mechanism of how FGF19 promotes the progression of LSQ remains unclear. In this study, we characterized and confirmed that FGF19 serves as an oncogenic driver in LSQ development and progression, and reported that the amplification and high expression of FGF19 in LSQ was significantly associated with poor overall and progression-free survival. A higher serum level of FGF19 was found in lung cancer patients, which could also serve as a novel diagnostic index to screen lung cancer. Overproduction of FGF19 in LSQ cells markedly promoted cell growth, progression and metastasis, while downregulating FGF19 effectively inhibited LSQ progression in vitro and in vivo. Moreover, downregulating the receptor FGFR4 was also effective to suppress the growth and migration of LSQ cells. Since FGF19 could be induced by smoking or endoplasmic reticulum stress, to tackle the more malignant FGF19-overproducing LSQ, we reported for the first time that inhibiting mTOR pathway by using AZD2014 was effective and feasible. These findings have offered a new strategy by using anti-FGF19/FGFR4 therapy or mTOR-based therapy in FGF19-driven LSQ.

Comparison of serum exosome isolation methods on co-precipitated free microRNAs.

BACKGROUND: Exosomes are nano-sized extracellular vesicles containing different biomolecules such as proteins and microRNAs (miRNAs) that mediate intercellular communication. Recently, numerous studies have reported the important functions of exosomal miRNAs in disease development and the potential clinical application as diagnostic biomarkers. Up to now, the most commonly used methods to extract exosomes are ultracentrifugation (UC) and precipitation-based commercial kit (e.g., ExoQuick). Generally, both UC and ExoQuick method could co-isolate contaminating proteins along with exosomes, with the UC method yielding even purer exosomes than ExoQuick. However, the comparison of these two methods on co-precipitated free miRNAs is still unknown. METHODS: In this study, we isolated exosomes from the human serum with exogenously added cel-miR-39 by UC and ExoQuick and compared the proportion of cel-miR-39 co-precipitated with exosomes extracted by these two methods. RESULTS: Using exogenous cel-miR-39 as free miRNAs in serum, we concluded that ExoQuick co-isolates a small proportion of free miRNAs while UC hardly precipitates any free miRNAs. We also found that incubation at 37 °C for 1 h could decrease the proportion of free miRNAs, and exosomal miRNAs like miR-126 and miR-152 also decreased when RNase A was used. In conclusion, our findings provide essential information about the details of serum exosome isolation methods for further research on exosomal miRNAs.